Patients accessing antiretroviral treatment (ART) programmes in sub-Saharan Africa frequently have very advanced immunodeficiency. Previous data suggest that such patients may have diminished capacity for CD4 cell count recovery.Rates of CD4 cell increase were determined over 48 weeks among ART-naïve individuals (n = 596) commencing ART in a South African community-based ART programme.The CD4 cell count increased from a median of 97 cells/μl at baseline to 261 cells/μl at 48 weeks and the proportion of patients with a CD4 cell count <100 cells/μl decreased from 51% at baseline to just 4% at 48 weeks. A rapid first phase of recovery  was followed by a slower second phase . Compared to patients with higher baseline counts, multivariate analysis showed that those with baseline CD4 counts <50 cells/μl had similar rates of phase 1 CD4 cell recovery (P = 0.42), greater rates of phase 2 recovery (P = 0.007) and a lower risk of immunological non-response (P = 0.016). Among those that achieved a CD4 cell count >500 cells/μl at 48 weeks, 19% had baseline CD4 cell counts <50 cells/μl. However, the proportion of these patients that attained a CD4 count 200 cells/μl at 48 weeks was lower than those with higher baseline CD4 cell counts.Patients in this cohort with baseline CD4 cell counts <50 cells/μl have equivalent or greater capacity for immunological recovery during 48 weeks of ART compared to those with higher baseline CD4 cell counts. However, their CD4 counts remain <200 cells/μl for a longer period, potentially increasing their risk of morbidity and mortality in the first year of ART. The World Health Organisation (WHO) estimated that in June 2005 4.7 million people living in sub-Saharan Africa were in urgent need of antiretroviral treatment (ART) . DespitePatients enrolling into ART programmes with very low CD4 cell counts have heightened risk of morbidity and mortality both before and during the initial months of ART -8. MoreoIn this study we have examined determinants of CD4 cell count recovery among patients accessing a community-based antiretroviral programme in South Africa. We focus on the hypothesis that advanced pre-treatment immunodeficiency diminishes the capacity for CD4 cell count recovery during ART as determined by rates of CD4 cell count increase and risk of immunological non-response.We studied patients accessing ART at the Gugulethu Community Health Centre, Cape Town, South Africa . The vasThe first-line ART regimen was comprised of stavudine, lamivudine plus a non-nucleoside reverse transcriptase inhibitor (efavirenz or nevirapine). The second-line regimen for those failing first-line treatment was comprised of lopinavir/ritonavir, zidovudine and didanosine. Treatment adherence exceeds 90% at one year . All trePlasma HIV-1 load was measured at baseline and 4 monthly during treatment using Versant™ HIV-1 RNA 3.0 branched chain DNA assay  with a lower limit of detection of 50 RNA copies/ml. Blood CD4 cell counts were measured at the same time-points by flow cytometry using FACSCount™ . These assays were all performed in a single nationally accredited laboratory which has rigorous quality assurance procedures.Structured clinical and laboratory records were maintained on all patients screened on entry to the ART programme and this information was transferred on a weekly basis to a computer database. Data were analysed from the start of the programme in September 2002 until data censorship in August 2005. All treatment-naive patients aged over 15 years and with at least a 16-week follow-up time point were included in the analysis. Study of these patients was in compliance with the Declaration of Helsinki and was approved by the Research Ethics Committee of the University of Cape Town; all patients enrolled gave written informed consent.Data were analysed using Stata version 9.0 . We calculated absolute responses in CD4 cell counts during three intervals , as well as rates of CD4 cell increase (cells/μl/month) during each interval. The first interval was used as an estimate of the initial (phase 1) response to ART. The CD4 cell count responses observed during the latter two intervals (16 to 32 and 32 to 48 weeks after treatment initiation) were very similar, and were combined into a single measure of the second phase of CD4 cell responses (phase 2).We further evaluated CD4 cell responses in the following ways: i) whether patients failed to attain an absolute CD4 cell count increase from baseline of at least 50 cells/μl at 48 weeks ; ii) whether patients achieved an absolute CD4 count of 200 cells/μl at the 48 week visit; iii) and whether patients had achieved an absolute CD4 cell count of 500 cells/μl at 48 weeks (super-responders).In bivariate analyses, medians were compared using Wilcoxon rank-sum and sign-rank tests; proportions were compared using chi-square tests. All statistical tests were two-sided at alpha = 0.05. Separate multiple linear regression models were used to examine factors associated with rates of CD4 cell count increase per month during the first and second phases. Baseline CD4 cell counts were categorised as follows: <50, 50–99, 100–149 and >150 cells/μl. Multiple logistic regression was used to assess factors associated with the binary outcomes of a CD4 cell increase of ≥ 50 cells/μl and ≥ 100 cells/μl during the 48 weeks of ART, as well as achieving an absolute CD4 cell count of ≥ 200 cells/μl during follow-up. Variables were included in multivariate models if they demonstrated a persistent association with the outcome of interest, or if their removal affected appreciably associations involving other variables .Of 698 individuals who commenced ART between September 2002 and April 2005, 596 (85%) had completed a 16-week follow-up visit at the time of data censorship, 34 (5%) were awaiting this appointment, 48 (7%) had died and 20 (3%) were either transferred out or were lost to follow-up. Of the 596 individuals who met the inclusion criteria for the study, 580 (97%) remained within the programme at study censorship, 11 (2%) died and 7 (1%) were lost from the programme.10 RNA copies/ml  and 58% of patients had a viral load >100,000 copies/ml. The median blood CD4 cell count was 97 cells/μl  and the proportions of patients with CD4 cell counts within the ranges <50, 50–99, 100–149 and ≥ 150 cells/μl were 25%, 26%, 23% and 26%, respectively. Eighty per cent of patients had symptomatic disease, with 53% and 27% of patients having WHO stages 3 and 4 disease, respectively. During follow-up, only 7 (1%) patients switched to the second-line drug regimen.At baseline the median age was 32 years  and 75% were female. The median plasma viral load was 4.88 logVirological responses to treatment were excellent with viral load suppressed <400 copies/ml in ≥ 94% of patients at each of the follow-up time-points Table . As a reThe rate of CD4 cell count increase in the first 16 week period greatly exceeded the rates in both the 16–32 week and 32–48 week intervals, but the rates did not differ comparing the latter two intervals. Thus, the pattern of CD4 cell count increase was divided into 2 phases: a ra–pid phase 1  and a slower phase 2 .Baseline characteristics and rates of CD4 cell count change in phase 1 and phase 2 did not differ when comparing the results of analyses of all eligible patients with those restricted to subjects who had data for every time-point (n = 292); this was also the case for all subsequent stratified analyses. Use of data from the larger cohort (n = 596) was therefore validated.10 copies/ml the phase 1 CD4 cell slope was 21.3 cells/μl/month compared to 31.0 cells/μl/month among those with baseline viral load of >5 log10 copies/ml (P < 0.001).Rates of phase 1 CD4 cell increase were similar across all CD4 cell count strata Figure . SubsequContrary to our initial hypothesis, the rate of phase 2 CD4 cell increase was greater among those with a baseline CD4 cell count <50 cells/μl compared to those with higher baseline counts Figure . MultivaIn the multivariate model predicting rates of phase 2 CD4 cell increase, factors associated with the response to ART during the first 16 weeks were also included. A lower rate of phase 1 CD4 cell increase and full suppression of viral load at 16 weeks were both strongly associated with greater rate of phase 2 CD4 cell increase. Viral load suppression <400 copies/ml at 16 weeks was associated with a subsequent rate of CD4 cell increase of 6.8 cells/μl/month compared to 0.7 cells/μl/month among those whose viral load remained >400 copies/ml (P < 0.001).We next examined whether baseline CD4 cell count was a risk factor associated with immunological non-response (defined as a CD4 cell increase of <50 cells/μl at 48 weeks). Among those followed to 48 weeks n = 311), the blood CD4 cell count increased by <50 cells/μl among 30 (9.7%) patients; of these, the viral load was suppressed <50 copies/ml among 22 patients, representing a treatment discordance rate of 7%. Contrary to our initial hypothesis, low baseline CD4 cell counts were not associated with increased risk of immunological non-response. Among patients with baseline CD4 cell counts of <50, 50–99, 100–149 and 150 cells/μl, the proportions of patients who were immunological non-responders were 5%, 4%, 11% and 19%, respectively. Furthermore, in multivariate analyses, an increment of <50 cells/μl was independently associated with higher baseline CD4 cell count as well as older age, lower baseline viral load, and a viral load >400 copies/ml at any follow-up time-point  achieved an absolute CD4 cell count of >500 cells/μl. These super-responders were principally characterised by age <40 years and by all having follow-up viral loads persistently suppressed <50 copies/ml and a CD4 cell count of 150 cells/μl after 16 weeks of ART. This group of patients had a wide distribution of baseline CD4 cell counts, and included among them were 4 (19%) who had baseline CD4 cell counts of <50 cells/μl. Thus, a low baseline CD4 cell count did not preclude patients from having an excellent immunological response.To our knowledge this is the first analysis to examine the determinants of CD4 cell count recovery among patients receiving ART in resource limited settings. These data indicate that those with baseline CD4 cell counts <50 cells/μl had similar rates of phase 1 CD4 recovery (0–16 weeks) and greater rates of phase 2 recovery 16–48 weeks) compared to rates among those with higher baseline CD4 cell counts. Moreover, those with the lowest baseline counts were least likely to be immunological non-responders to ART. Despite these observations, those with baseline CD4 cell counts <50 cells/μl were nevertheless least likely to attain a CD4 cell count 200 cells/μl at 48 weeks. Taken together, these results suggest that although patients with very low baseline CD4 cell counts retain capacity for similar or slightly greater rates of CD4 cell count recovery compared to those with higher counts, they are nevertheless likely to require a longer period of time to attain a CD4 cell count threshold of 200 cells/μl. Thus, a prolonged period below a 'safe' CD4 cell count threshold rather than a diminished rate of immunological recovery is likely to underlie the high rates of morbidity and mortality observed among those with advanced disease during the early months of ART [ weeks coThe findings of this analysis are strengthened by the relatively homogeneous study population receiving treatment at a single facility using standardised clinical protocols. Patients were all ART-naïve and received a standard triple-drug regimen with uniform follow-up time points. Quality-assured laboratory assays were all performed in a single nationally accredited laboratory. In contrast, previous studies of the determinants of CD4 cell count recovery have examined heterogeneous study populations in multiple centres and used diverse treatment regimens. Moreover, these studies included many patients with prior ART exposure -22 and set al.[et al. showed a similar but non-significant trend when comparing patients with baseline CD4 cell counts <200 cells/μl with those with higher counts [et al. found that CD4 cell increases in the first year of ART were similar comparing those with baseline counts <100 cells/μl with those with counts 100–199 cells/μl [The immunological response to ART among those with low CD4 cell counts was excellent with the proportion of patients with a CD4 cell count <100 cells/μl decreasing from 51% at baseline to just 4% at 48 weeks. However, our most important finding was that in multivariate analysis baseline CD4 cell counts <50 cells/μl were independently associated with similar rates of phase 1 CD4 cell recovery and greater rates of phase 2 CD4 cell recovery compared to individuals with higher baseline CD4 cell counts. This has not been clearly highlighted in previous publications from Europe and North America although comparison of our data with previous studies is difficult in view of differing cohort compositions. However, this overall observation is consistent with the findings of Bennett et al., and Le cells/μl .Survival bias could potentially have affected our findings. We have previously shown in this cohort that patients with the lowest baseline CD4 cell counts had a higher risk of death  and immuWe have previously shown that low baseline CD4 count at entry to an ART programme was associated with increased risks of tuberculosis and of mortality during the first year of ART ,8. Resul5 log10 copies/ml had 11-fold greater CD4 count increases than those with lower viral loads. Immune dysregulation and immune cell activation promote sequestration of CD4+CD45Ro+ memory T cells in lymphoid tissue; suppression of viral replication then triggers rapid redistribution of this cell pool and a reduction in apoptotic cell death during the initial weeks of ART [Mycobacterium tuberculosis[Cryptococcus neoformans[Rates of phase 1 and phase 2 CD4 cell increase were similar in magnitude to those previously reported from high-income countries ,20. The s of ART ,27. A poerculosis, Cryptoceoformans that is Greater phase 2 CD4 cell recovery was strongly associated with age as reported elsewhere ,24. SustPatients with the lowest CD4 counts in this setting do not have diminished capacity for immune recovery. Although patients with low baseline CD4 counts have increased risk of acute morbidity and mortality, if such patients survive the initial months of ART and fully suppress the viral load, their chances of immunological recovery are good during the first year. Future studies are required to determine the long-term prospects for immune recovery among patients treated in ART programmes in sub-Saharan Africa.The author(s) declare that they have no competing interests.SDL and RW initiated and designed the study. LM did the statistical analyses. LGB and RW established the study cohort and data collection systems. SDL wrote the manuscript, which RW, LM and LGB all helped to revise.The pre-publication history for this paper can be accessed here:


The 5T4 oncofetal antigen is a 72 kDa glycoprotein defined by a monoclonal antibody raised against human placental trophoblast and is expressed in many different carcinomas but detected only at low levels in some normal epithelia. Immunohistochemical analysis of the patterns of expression in colorectal carcinomas has indicated a significant association between the presence of the antigen in tumour cells and metastatic spread. The 5T4 antigen phenotype of 72 colorectal cancers has been compared with the clinical outcome of the patients in order to assess its relationship with prognosis. Forty per cent of tumours were 5T4 positive; the remainder were either unlabelled or exhibited stroma-associated labelling only. There was a significant correlation between 5T4 expression in the malignant cells and unfavourable course of disease (P < 0.001). The 5 year survival with 5T4-positive tumours was 22% compared with 75% for patients with 5T4-negative tumours; median survival was 24 versus > 90 months respectively. Stratified analysis showed that 5T4 antigen tumour positivity was acting independently of each of stage, site of tumour, age or sex. There were significant differences in survival for patients with Dukes' B and C stage carcinomas (P = 0.001 and P = 0.034). The results suggest that in colorectal cancer immunohistochemical assessment of 5T4 expression may be useful in identifying patients at high risk for tumour recurrence and for whom additional treatment strategies might be most appropriate.


Twenty five hormone manipulated patients with prostate cancer and metastatic bone disease, treated at least 6/12 previously by hormone manipulation, were given intravenous infusions of Disodium Pamidronate (APD) over a 6 month period. Patients received 30 mg weekly for 4 weeks then twice monthly for 5 months. No other treatment was administered during study. Eleven of 17 patients with pain at the start of the study were pain free at the end. Fasting morning calcium excretion and serum osteocalcin fell significantly with Pamidronate (P less than 0.0001) and urine hydroxyproline was lowered in 13/20 evaluable patients at 6 months. Alkaline phosphatase fell in a proportion of patients and five of 17 patients with previously progressive bone scans stabilised (4) or regressed (1) on treatment. Rising acid phosphatase levels were also lowered in five patients. It is concluded that Pamidronate may be effective in palliating bone pain in some patients and has a stabilising influence on abnormally high bone turnover in metastatic prostate cancer. Further controlled studies of the compound are now warranted.


The expression of 5T4, an oncotrophoblast cell surface antigen was examined in 72 colorectal and 27 gastric carcinomas, with immunoperoxidase technique, on frozen sections. Highly significant association was found between 5T4 expression in the malignant cells and metastatic spread. The results suggest that the appearance of 5T4 molecules in cancer cells reflects a change which may contribute to the development of metastatic potential.


Influx of Ca2+ in non excitable smooth muscle is believed to be predominantly through store operated channels (SOC) or receptor operated channels (ROC). Whereas agonists can activate both SOC and ROC in a range of smooth muscle types, the specific trigger for SOC activation is depletion of the sarcoplasmic reticulum Ca2+ stores. The mechanism underlying SOC activation following depletion of intracellular Ca2+ stores in smooth muscle has not been identified.Control of cytosolic calcium plays a key role in airway myocyte function. Changes in intracellular CaTo investigate the roles of the STIM homologues in SOC activation in airway myocytes, specific siRNA sequences were utilised to target and selectively suppress both STIM1 and STIM2. Quantitative real time PCR was employed to assess the efficiency and the specificity of the siRNA mediated knockdown of mRNA. Activation of SOC was investigated by both whole cell patch clamp electrophysiology and a fluorescence based calcium assay.2+ influx in response to store depletion by cyclopiazonic acid (60%) or histamine but not bradykinin. siRNA to STIM2 had no effect on these responses. In addition STIM1 suppression resulted in a more or less complete abrogation of SOC associated inward currents assessed by whole cell patch clamp.Transfection of 20 nM siRNA specific for STIM1 or 2 resulted in robust decreases (>70%) of the relevant mRNA. siRNA targeted at STIM1 resulted in a reduction of SOC associated Ca2+ store depletion or following agonist stimulation with histamine in human airway myocytes. These are the first data demonstrating a role for STIM1 in a physiologically relevant, non-transformed endogenous expression cell model.Here we show that STIM1 acts as a key signal for SOC activation following intracellular Ca The contractile/relaxant state of the airway myocyte is a key determinant of airway calibre thus contributing to bronchoconstriction in diseases such as asthma. Previous studies have demonstrated that the contractile response of airway myocytes is dependent initially upon release of intracellular Ca2+ from the sarcoplasmic reticulum  but tha2+ entry ) in cont2+ entry .2+ stores. Contractile agonists such as acetylcholine, histamine and bradykinin may vary in their ability to differentially activate ROC or SOC although all agonists are known to induce activation of phospholipase C with consequent IP3 mediated Ca2+ release from the intracellular stores. The mechanism underlying signalling for subsequent Ca2+ influx in response to store depletion, and hence refilling of the sarcoplasmic reticulum Ca2+ stores, remains unknown. In the current study we have set out to define the signals for SOC activation in human airway myocytes following both store depletion and agonist activation by spasmogens.SOC activation in many cell types including smooth muscle is known to involve depletion of the intracellular sarcoplasmic reticulum CaCRAC) in T lymphocytes via a mechanism which has been proposed to involve translocation of STIM1 from endoplasmic reticulum like sites to the cell membrane i.HASMs (passage 4–5) were plated in black walled, clear bottom 96 well plates and loaded with Fluo-4AM (Molecular probes) for 1 hour at room temperature in culture medium (DMEM) supplemented with 10% FCS and 2.5 mM probenecid . Cells were then washed with Hanks' balanced saline solution containing 10 mM Hepes, 2.5 mM probenecid, 0.1 mM CaCl2, 10 mM EGTA, 30 mM HEPES, 3.62 mM CaCl2. External Solution: 140 mM NaCl, 5 mM CsCl, 1 mM MgCl2, 10 mM D-Glucose, 10 mM HEPES, CaCl2 (as indicated). K+ was replaced by Cs+ in both external and internal solutions to block K+ currents and Cl- was replaced by an equal molar concentration of methanesulfonate to minimize Cl- currents. Nifedipine (5 μM) was included in the external solution. Pipettes were drawn from borosilicate glass and had resistances of 5–8 MΩ when filled with internal solution. HASM cells were placed directly into the cell chamber, allowed to settle and then were continuously perfused with external solution at a constant speed of 6 ml/min. Experimental drugs were delivered through a puffer pipette positioned 50 μm around the cells. Cells were held at a membrane potential of -60 mV and current-voltage relationships were analysed every 5s from voltage ramps from -100 to +100 mV at a rate of 0.5 Vs-1. Currents were filtered at 1 kHz and sampled at 4 KHz. Individual cell current densities were calculated by dividing peak current amplitude at maximum activation of inward current (at -100 mV) by cell capacitance.The conventional whole-cell patch-clamp technique  was emplHASMs grown on coverslips, transfected with either 20 nM scrambled siRNA or 20 nM STIM1 siRNA were fixed with 4% formaldehyde. Cells were permeabilized (0.5% TritonX-100) and blocked with 20% goat serum in PBS for 20 min. Cells were incubated with primary antibody   overnight at 4°C followed by labeling with Alexa fluor 488 (Molecular probes). Cells were visualized on a Zeiss LS 510 confocal microscope .Averaged data are presented as mean ± sem. Where appropriate, statistical significance was assessed by unpaired Students T tests or one-way ANOVA followed by the Dunnets test for multiple group comparisons. Data were considered significant at *P < 0.05 or **P < 0.01.Initially we assessed the expression of the two known human homologues STIM1 and STIM2 in cells relevant to airway function figure . Both ST2+]i) in monolayers of cultured human airway myocytes. SOC mediated Ca2+ influx was induced by depletion of the sarcoplasmic reticulum Ca2+ store using a combination of low external Ca2+(0.1 mM) and incubation with the SERCA inhibitor cyclopiazonic acid . Incubation of airway myocytes in low Ca2+ in the presence of CPA resulted in an initial small rise in cytosolic Ca2+ indicative of store depletion. Re-addition of extracellular Ca2+ resulted in rapid influx into the cell through SOCs. siRNA targeted at STIM1 resulted in a dose-dependent reduction of SOC associated Ca2+ influx .We next evaluated the ability of siRNA targeted to STIM1 and STIM2 to inhibit SOC using a fluorescence assay utilising Fluo-4AM, designed to measure changes in intracellular free calcium concentration . SOC currents were induced by voltage ramps at a rate of 0.5 mV·ms-1 from a holding potential of -60 mV. Inward currents (SOC currents) measured at -100 mV were then compared. Changes in current density of SOC are illustrated in Figure 2+ free buffer). The STIM1 suppressed cells showed a reduced SOC inward current compared with cells treated with scrambled or STIM2 siRNA sequences. We also noticed a change in the reversal potential in the STIM1 suppressed cells towards a more negative potential: the significance of this is uncertain but similar findings on ICRAC currents have been reported using EF-hand mutants of STIM1 in RBL cells [We next used whole cell patch clamp electrophysiology approaches to confirm that siRNA targeted at STIM1 but not STIM2 is able to inhibit SOC activation. SOC currents were activated by reducing external CaBL cells .2+ entry in human airway myocytes are shown in figure 2+ responses to be H1 receptor mediated in these cells. Under conditions of low extracellular Ca2+ the sustained rise in intracellular Ca2+ seen following agonist stimulation is reduced, an effect mimicked by a range of di and tri-valent cations including Ni2+, La3+ and Gd3+ and also by the putative ROC inhibitor SKF96365 [2+ response to agonist. Following pre-incubation with siRNA targeted at STIM1 we observed a significant reduction in the magnitude of Ca2+ influx induced by histamine  declare that they have no competing interests.Samantha Peel performed the mRNA expression and calcium studies, Bo Liu performed the electrophysiology and Ian P Hall wrote the paper. All authors were involved in the design of the studies, discussion of the results and preparation of the final manuscript.


Accurate chromosome segregation during meiosis and mitosis is essential for the maintenance of genomic stability. Defects in the regulation of chromosome segregation during division predispose cells to undergo mitotic catastrophe or neoplastic transformation. Cohesin, a molecular glue holding sister chromatids together, is removed from chromosomes in a stepwise fashion during mitosis and meiosis. Cohesin at centromeres but not on chromosome arm remains intact until anaphase onset during early mitosis and the initiation of anaphase II during meiosis. Several recent studies indicate that the activity of protein phosphatase 2A is essential for maintaining the integrity of centromeric cohesin. Shugoshin, a guardian for sister chromatid segregation, may cooperate with and/or mediate PP2A function by suppressing the phosphorylation status of centromeric proteins including cohesin. At the molecular level, cohesin mediates sister chromatid cohesion through formation of a proposed "ring" structure that entraps chromosomes [Sister chromatid cohesion is established during DNA replication and maintained throughout Gomosomes . The cohomosomes . Upon miomosomes  that is omosomes ,5.During the past several years, the mechanism preventing centromeric cohesin from undergoing cleavage/dissociation during early mitosis as well as meiosis I and early meiosis II has been a subject of intensive investigation ,7. ThrouNature and Developmental Cell have convincingly demonstrated that enhanced dephosphorylation of centromeric proteins including cohesin may be a key mechanism responsible for centromeric cohesion of sister chromatids during meiosis and mitosis [A recent study by the Nasmyth group showed that expression of SA2 mutant resistant to Plk1 phosphorylation results in suppression of premature separation of sister chromatids in Sgo1-depleted cells . This su mitosis -17.Par1B', Ppa2C) are important for accurate chromosome segregation in fission yeast as their deletions results in moderate to severe missegregation of chromosomes; a detailed analysis revealed that chromosome missegregation occurs in meiosis II, but not in meiosis I, in fission yeast with either Par1B' or Ppa2C deletion [Sgo1 deletion. These observations thus suggest that PP2A, like Sgo1, may protect centromeric but not arm cohesion of sister chromatids. Consistent with this notion, fission yeast mutants with deletion of Par1B' or Sgo1 are impaired in retaining centromeric Rec8 following meiosis I whereas deficiency in PP2A activity does not have an effect on expression as well as centromeric localization of Rec8 before the onset of anaphase I [Using affinity protein purification followed by tandem mass spectrometry, the Nasmyth group found that protein phosphatase 2A (PP2A) subunits are the prevalent components that are co-purified in meiotic I cells of both fission and budding yeasts . PP2A isdeletion , a defecaphase I .The mechanism by which PP2A protects centromeric cohesin is fully conserved in mammalian cells. Through a combination of co-immunoprecipitation and mass spectrometry, the Watanabe group identified PP2A as a major component in Sgo1, but not in control, immunoprecipitates , suggestSimilar results regarding PP2A's physical interaction with Sgo1 in human cells are also reported by the Yu group . Co-immuDrosophila, Polo phosphorylates MEI-S332, which is essential for its removal from centromeres at anaphase [In eukaryotes, a hallmark for mitotic entry is the activation of mitotic kinases , resulting in phosphorylation of numerous mitotic targets essential for mediating mitotic progression. Early studies established that Bub1 is upstream of Sgo1 in the regulatory hierarchy ,19. Receanaphase ; (iv) Planaphase  althoughGiven the close involvement of Plk1/Polo in regulating anaphase entry and chromosome segregation, it is relevant to examine how Plk1 regulates PP2A activity, which in turn modulates centromeric cohesion. Interestingly, the Yu group showed that co-depletion of Plk1 and a key PP2A subunit through transfection of specific siRNAs causes retention of Sgo1 at centromeres in human cells, which is correlated with significant reduction of chromosomal missegregation; in contrast, co-depletion of Plk1 and Sgo1 fails to prevent missegregation of chromosomes ,15. ThusSgo1 deletion during meiosis I, implying that the localization of PP2A to the centromeric region depends on Sgo1 [The identification of PP2A as a new component in protecting centromeric cohesion of sister chromatids prompts additional studies determining its position in the regulatory hierarchy. As Sgo1 is primarily localized at centromeres during meiosis or early mitosis whereas PP2A signals are detected throughout the cell, it is possible that Sgo1 may help to recruit a specific form of PP2A to centromeres. This notion is consistent with the fact that most PP2A is not associated with Sgo1 . Support on Sgo1 . On the  on Sgo1 . Consist on Sgo1 .Although it remains not entirely clear how Sgo1 regulates PP2A or vice versa with regard to protection of centrometic coehsin/cohesion during meiosis and mitosis, a simple model is proposed to illustrate the regulatory hierarchy in animal cells (Figure


Using two monoclonal antibodies directed against placental alkaline phosphatase (H17E2 and D20L) the immunohistological staining of testicular germ cell tumours was compared with that of a wide range of normal and malignant tissues. All seminomas and malignant teratomas tested gave strong positive labelling with H17E2 but were either negative or only patchily positive with D20L. Neither antibody gave any positive reaction on the normal tissues tested. All other malignancies were negative with both antibodies apart from two cases of ovarian and one case of endometrical cancer (strongly stained by H17E2) and three cases of colonic carcinoma (weakly and patchily stained by both H17E2 and D20L). This indicates that germ cell neoplasms generally express a form of placental alkaline phosphatase recognised by antibody H17E2.


Hematological and biochemical parameters were evaluated in 31 patients receiving 150 MBq 89Strontium (89Sr) intravenously due to painful skeletal metastases from hormone resistant prostate cancer. Two and 3 months after the injection prostate specific antigen (PSA) had increased by a median of 36% and 100%, respectively, as compared to the pretreatment value whereas alkaline phosphatase (APHOS) had decreased by about 20% (median). The leucocyte and platelet counts were reduced by about 20-35%, without reaching grade greater than or equal to 2 toxicity. Pain relief was reported in 14 of 29 evaluable patients at 2 months and in 11 of 23 patients at 3 months. It is concluded that 89Sr represents a worthwhile therapeutic modality in the palliation treatment of patients with hormone resistant prostate cancer, though the biological significance of frequently increasing PSA and decreasing APHOS is not yet completely understood.


Fifteen patients with neoplastic meningitis received a single intrathecal injection of between 11 and 60 mCi of a 131I radiolabelled monoclonal antibody (MoAb), chosen for its immunoreactivity to tumour. Major toxicity was manifest as nausea, vomiting and headache (7/15 patients), reversible bone marrow suppression (3/8 patients) and seizures (2/15 patients). Nine patients were evaluable for either a tumour or clinical response. Six of these demonstrated an event-free response that was maintained for periods of between 7 and 26 months.


The MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they are significantly more abundant than other preRC components, we were interested in determining whether the entire cellular content was necessary for DNA replication in vivo.Drosophila S2 cells using dsRNA-interference. Reducing MCM2-6 levels by >95–99% had no significant effect on cell cycle distribution or viability. Depletion of MCM7 however caused an S-phase arrest. MCM2-7 depletion produced no change in the number of replication forks as measured by PCNA loading. We also depleted MCM8. This caused a 30% reduction in fork number, but no significant effect on cell cycle distribution or viability. No additive effects were observed by co-depleting MCM8 and MCM5.We performed a systematic depletion of the MCM proteins in These studies suggest that, in agreement with what has previously been observed for Xenopus in vitro, not all of the cellular content of MCM2-6 proteins is needed for normal cell cycling. They also reveal an unexpected unique role for MCM7. Finally they suggest that MCM8 has a role in DNA replication in S2 cells. The MCM (minichromosome maintenance) 2–7 proteins play an important role in DNA replication in eukaryotes. They are involved during initiation where they are needed to form the preRC (pre-Replicative Complex) reviewed . This coS. pombe active helicases have been isolated containing only MCM4/6/7 In archael species which have a single MCM protein, the active complex has been suggested to be a hexamer or double hexamer  . The mitotic index in 3MCM6 was comparable to control cells, while decreases were seen for k1214MCM6 and rl74MCM2 (20% and 26% lower)  . As repo% lower) .3MCM6 cells appeared to progress normally through mitosis (not shown). Despite rl74MCM2 and k1214MCM6 having fewer mitotic cells, no mitotic aberrations were detected, suggesting that their defect was a pre-mitotic delay. For 1dpa however the defect seemed to be due to a metaphase delay (anaphases were absent). While chromosome condensation was unaffected, mitotic chromosomes appeared broken and did not congress to a metaphase plate . We therefore decided that a more systematic approach would be to deplete each MCM protein using dsRNA-mediated interference (RNAi) in Drosophila S2 cells. A 500–600 bp dsRNA region was produced for each cDNA . The combined results of seven such experiments are shown in We also analysed the cell cycle distribution of MCM2-7 depleted cells using FACS analysis. A representative data set is shown in Drosophila MCM8 The lack of effects seen after MCM2-6 depletion could have been due to compensation by a functionally redundant protein. Since a possible candidate for this was MCM8 we targeted To determine whether MCM8 could compensate for the loss of MCM2-6 we performed RNAi simultaneously targeting MCM5 and MCM8. Lower MCM protein levels could result in less MCM at each replication fork or a reduced number of active forks. The amount of PCNA associated with chromatin has previously been used as a measure of the number of active forks in a cell Drosophila cells. It further suggests that S2 cells can tolerate a reduction in the number of active replication forks without a significant effect on the cell cycle distribution or cell viability.Depletion of MCM8 consistently reduced PCNA binding by 30–50%. This suggests a role for MCM8 protein in DNA replication in Drosophila S2 cells to efficiently deplete the MCM2-8 proteins. The data produced supports three main conclusions about MCM proteins in this system.We have used RNAi in DrosophilaFirstly, although we could demonstrate specificity of the RNAi depletions, we observed co-instability for certain combinations of MCM proteins. Some of our observations can be explained based on the composition of reported MCM sub-complexes reviewed . TherefoDrosophila S2 cells has little apparent effect on cell survival and DNA replication. This therefore suggests that the MCM paradox–originally observed in Xenopus cell free extracts Drosophila we suggest two other possibilities. Firstly, consistent with what has been suggested for Xenopus it might be that under normal circumstances most of the MCM protein in cells is redundant. We estimate that there are 50–100 MCM complexes per origin (assuming origin spacing of 40–100 kb) in S2 cells. Therefore even cells which have lost 99% of a specific MCM should have enough protein to ensure that most origins have one MCM complex. A single MCM complex per origin may therefore be sufficient to allow a full complement of activated replication forks as measured by PCNA loading. In this case our results support proposed MCM mechanisms involving single or double hexamers Secondly our data suggest that a dramatic reduction in the level of MCM2-6 and 8 in vivo in Drosophila MCM8 does play a role in replication. From our data the exact nature of its role is unclear, however the lack of an effect of the depletion on cdc45 loading suggests that unlike the MCM2-7 proteins it is unlikely to be required for the loading of downstream initiation factors. In addition MCM8 cannot be the MCM2-6 compensating protein since co-depletion of MCM5 and MCM8 does not synergistically affect cell viability or DNA replication.The second possibility is that MCM loss is compensated for by other proteins. We investigated whether MCM8 could perform this function. The decrease in PCNA loading observed on depletion of MCM8 suggests that S. cerevisiaeXenopus extracts MCM7 has also been shown to bind to the Rb protein Drosophila cells, however less efficient depletion of Drosophila MCM7 has been seen to produce the same effect (data not shown). Alternatively in addition to acting as a negative regulator of replication, MCM7 may have a positive regulatory effect on replication. In either case the effect is likely to involve MCM7 directly, rather than occurring as a secondary effect of a replication defect, since similar effects are not observed for other MCM proteins.Finally the differences observed between depletion of MCM7 and MCM2-6 suggest that not all MCM proteins are equivalent. The mechanism behind this differential behaviour is not clear. Although a complex of MCM4/6/7 has been shown to have helicase activity, there is no evidence that MCM7 acts independently. Therefore MCM7 may have additional cellular functions. A role for MCM7 as a damage sensor via the ATR pathway has been suggested by work in human rl74mcm2, 1dpa, 3mcm6, and k1214mcm6 were obtained from the bloomington stock center.all fly stocks (canton s (wild type), MCM antibodies were described previously preparation, visualisation and quantification of mitotic and s-phase indices were as described previously Proteins from SDS PAGE were blotted onto Hybond ECL (Amersham) and developed with Supersignal West Pico (Pierce). Visualisation and quantitation were carried out using an Alpha Innotech gel documentation system.6 cells) and the cells were monitored by cell count, FACS analysis, western blotting and RT-PCR over a period of 7 days.Sequence specific primers each containing a 5′ T7 RNA polymerase binding site were designed for MCM2-8 . The dsRcDNA from 500,000 S2 cells was made using the cell to cDNA kit (Ambion) as per manufacturers instructions with the following modifications: cells were lysed in 100 µl lysis buffer and the RNAase inactivated by heat treatment. 20 µl of the lysate were used for the reverse transcription reaction. The equivalent of 5,000 cells (1 µl of the reverse transcription reaction) was amplified using Megamix blue (Microzone). Amplified fragments were run on agarose gels and visualised with EtBr.Amplified regions were located at the following nucleotide positions relative to the start ATG: MCM2 1381–1550; MCM3 1701–1869; MCM4 1261–1400; MCM5 481–630; MCM6 781–922; MCM7 321-480 and MCM8 311–450.cells were harvested and fixed using ethanol. Immediately prior to use they were treated with 10ug/ml RNase/1mM EDTA and the DNA was stained with propidium iodide. Flow cytometry was carried out on an EPICS Xl (Coulter Beckman) using EXPO 32 adc software.5 cells/ml. DNAse  was added at a concentration of 100 units/ml. The incubation was for 1 hour on ice.Cell fractionation was carried out as described


In this study, 44 primary or metastatic human ovarian tumours were tested for allelic deletions on the short arm of chromosome 11. Analysis of 12 polymorphic loci by Southern blotting evidenced loss of heterozygosity (LOH) in at least one locus in 41% of cases. Moreover, two hot spots of deletions were tentatively mapped on 11p13 and 11p15.5. Our results demonstrated that LOH at 11p is a common event in ovarian carcinomas and were indicative of the possible existence in 11p of two oncosuppressor genes involved in ovarian carcinogenesis. The similarity observed with 11p allelic losses in Wilms tumours, clustered in 11p13 and 11p15.5 too, suggests that deletion and possibly inactivation of the same growth regulatory genes (WT genes) could also contribute to development of the malignant phenotype in ovarian carcinomas. Finally, a statistically significant association (P = 0.005) between 11p deletions and hepatic involvement was suggested by the analysis of distribution of 11p LOH relative to different clinical and pathological parameters of the tumour patients.


Mouse monoclonal antibodies PAb 240 and PAb 1801 which specifically immunoprecipitate p53 protein, were used to examine 27 fresh ovarian tumours . Eleven out of 16 (69%) serous adenocarcinomas and one endometrioid tumour showed positive staining with one or both antibodies and none of the mucinous or benign tumours stained with either antibody. DNA from tumour and peripheral blood leukocytes was used to identify allelic deletions on chromosome 17p in tumours. 11/12 positively staining tumours showed less of heterozygosity (LOH) on 17p at the nearest informative locus to the p53 gene. In this series of ovarian tumours, LOH on 17p correlates closely with the aberrant expression of the p53 protein in a high proportion of advanced stage serous adenocarcinomas. This observation suggests that the p53 tumour suppressor gene is involved in the evolution of epithelial ovarian cancer (EOC) and may have prognostic significance.


To evaluate the results of salvage conformal radiation therapy (3DC-EBRT) for patients submitted to radical prostatectomy (RP) who have achieved complete PSA response and who have never been treated with hormonal therapy (HT).To present the results of biochemical control, a period free from hormonal therapy and factors related to its prognosis.from August 2002 to December 2004, 43 prostate cancer patients submitted to RP presented biochemical failure after achieving a PSA < 0.2 ng/ml. They have never received HT and were submitted to salvage 3DC-EBRT. Median age was 62 years, median preoperative PSA was 8.8 ng/ml, median Gleason Score was 7. Any PSA rise above 0.2 was defined as biochemical failure after surgery. Median 3DC-EBRT dose was 70 Gy, biochemical failure after EBRT was defined as 3 consecutive rises in PSA or a single rise enough to trigger HT.3-year biochemical non-evidence of disease (BNED) was 71%. PSA doubling time lower than 4 months (p = 0.01) and time from recurrence to salvage EBRT (p = 0.04) were associated with worse chance of biochemical control. Biochemical control of 76% was achieved when RT had been introduced with a PSA lower than 1 ng/ml vs. 48% with a PSA higher than 1 (p = 0.19). Late toxicity was acceptable.70% of biochemical control in 3 years can be achieved with salvage radiotherapy in selected patients. The importance of PSADT was confirmed in this study and radiotherapy should be started as early as possible. Longer follow up is necessary, but it is possible to conclude that a long interval free from hormonal therapy was achieved with low rate of toxicity avoiding or at least delaying several important adverse effects related to hormonal treatment. Radical prostatectomy (RP) is an efficient method of achieving prostate cancer control. The follow-up is based on clinical history, physical exams and following the Prostate Specific Antigen (PSA) kinetics. Of the patients who fail to achieve biochemical control, the principle of selecting the best salvage therapy is based upon determining whether the disease is still on the prostate bed or if it has already spread throughout the body. Several studies are aiming at determining which variables correlate with a higher chance of detecting localized recurrences. In these cases, adopt a potentially curative salvage therapy (radiotherapy) instead of hormonal therapy alone.The problem with many of these studies is that they usually include a broad group of patients who have never reached an indetectable PSA or those who have been previously treated with some sort of hormonal therapy making assessment using PSA difficult to evaluate.The objective of this study is to evaluate the results of biochemical control and to analyze a period free from hormonal therapy after salvage radiation therapy in a selected group of patients who have never been treated before with hormonal therapy and have achieved complete PSA response after RP.The secondary objectives are to evaluate prognostic factors related to the success of the salvage radiation therapy.From August 2002 to December 2004, 79 prostate cancer patients previously treated with radical prostatectomy (RP) were submitted to salvage three-dimensional conformal external beam radiation therapy (3DC-EBRT) due to biochemical failure. Thirty-six patients were excluded from the analysis: those who have not achieved PSA nadir (<0.2 ng/ml) after RP or those who were submitted to hormonal therapy before or during salvage radiation. Forty three patients were eligible. The median age was 62 years (range 50–73) and Caucasians were predominant (88.4%). Patients and treatment characteristics are show in Table Six patients (14%) were cT1c, 3 (7%) were cT2a, 2 (4.7%) were cT2b, 1 (2.3%) was cT2c. In 31 patients (72.1%) preoperative staging was not available. Median pre-operative PSA was 8.8 ng/ml (range 3 – 62) and hormonal therapy was not administered to any of then prior to surgery. Twenty-eight patients (65%) had information on the biopsy specimen, the median Gleason score was 6 (range 4 – 8).We defined biochemical recurrence after surgery as a single PSA value greater than 0.2 ng/ml after surgery in men with no evidence of distant metastasis at the time of radiotherapy.After salvage radiotherapy, biochemical failure was based on the ASTRO (American Society of Therapeutic Radiation Oncology) criteria as three consecutive PSA rises or a single rise was high enough to trigger the initiation of hormone therapy.In surgical staging according to 2002 AJCC staging system, 11 patients (25.6%) were pT2a; 3 patients (7%) were pT2b; 7 (16.3%) were pT2c; 21 (48.8%) were pT3a and only 1 patient (2.3%) was pT3b. The median Gleason score was 7 (range 4 – 8). Surgical margin was affected in 23 patients (53.5%). Perineural invasion (PNI) was found in 30 patients (69.8%); There was no information regarding PNI, Lymphatic invasion (LI), Vascular invasion (VI) and intraepithelial neoplasia (PIN) in respectively 16.3%, 28%, 30.2% and 58% . Median PSA before salvage radiotherapy was 0.87 ng/ml (range 0.24 – 7.9). PSA doubling time (PSADT) was calculated for each patient based on logarithmic regression formula and we used at least 2 PSA values separated by 2 months in the 18 months before salvage radiation.At the time of recurrence all patients were submitted to physical examination which included a digital rectal exam, chest radiography, whole body bone scan and a trans-rectal pelvic ultrasonography. Eleven patients (25.6%) presented a nodule in the prostatic bed.®) using conformal three-dimensional technique. All patients were submitted to a pre-planning set up in a simulator (Acuity – Varian®) with retrograde urethrogram to help define the isocenter. A pelvic computed tomography (CT) was then performed to delinement of planning target volume. Twenty patients (46.6%) were treated with 2 planning tumor volumes (PTV): PTV1 included the surgical prostate bed and seminal vesicles bed with margins and the PVT2 included only the surgical prostate bed with margin. This technique was used according to the attending physician preferences based on post-surgical pathological information and pre treatment prostate image characteristics. Nineteen out of 20 patients treated this way had T3 tumors. Median dose to PTV1 was 50.4 Gy (range 46 – 54) and for all patients median dose to the prostate bed was 70 Gy (range 66 – 72). Median dose per fraction was 2 Gy (range 1.8 – 2).Patients were submitted to external beam radiation therapy with a 10 MV linear accelerator  scale.Median interval from RP to biochemical failure was 12 months (range 2 – 39) and median time after failure to salvage 3DC-EBRT was 8 months (range 1 – 52). The median follow-up after radiotherapy was 26 months (range 8 – 41). One (1) patient (2.3%) was lost to follow-up. At the end of data collection no patients had died. Distant metastasis developed in 2 (4.7%) patients and 33 patients (76.7%) were free from biochemical failure. Of these, 28 patients (85%) developed undetectable PSA (<0.1 ng/ml) after a median interval of 3 months (range 1 – 30). Actuarial Biochemical Non-Evidence of Disease (BNED) at 3 years was 70.71%  and median PSADT was 5.25 months (range 1.0 – 16.5). Univariate and multivariate analysis of selected variables are displayed on Table 1 – 7.9 aPSADT lower than 4 months was an important negative prognostic factor to BNED/3-years . Total dose to surgical bed higher than 66 Gy did not result in better BNED/3-years (p = 0.6).By multivariate analysis only a PSADT lower than 4 months was a negative predictive factor for BNED/3-years  and 1 patient (2.3%) presented grade 3 late gastrointestinal toxicity. No grade 4, acute or chronic, toxicity was seen. Table Prostate cancer is an indolent disease and the best way to evaluate disease control after radical treatment is monitoring PSA. It is estimated that about 1/3 of patients with biochemical failure following radical treatment will develop distant metastasis in a period of 8 years .After RP 70% of patients will achieve biochemical control in 10 years -6. HowevSeveral variables have been described as prognostic factors for failure after surgery: histological grade (Gleason score); capsular or seminal vesicles extension; positive lymph nodes and involvement of surgical margins -12. In oSalvage radiotherapy after RP is the only potential curative modality, but several published series have not demonstrated uniform results of BNED (18 to 68%) ,13-27.The most important issue in patients with biochemical failure is to define which patients will benefit from salvage treatment to the prostate bed. Unfortunately, an increase in PSA level after local treatment does not distinguish local recurrence from distant metastasis. Usual image exams or biopsy have not proved yet to be helpful in defining anatomical site of biochemical recurrence ,28,29. TIn our experience BNED in 3 years was 71%, a result that is compatible with results from other institutions ,16,22.Although the follow-up is somewhat short for accurately defining the effects of the salvage therapy on local control or survival, another important result of the salvage radiation treatment that should be considered is the effect on the quality of life by delaying hormonal therapy. Hormonal therapy has been shown to produce deleterious side effects on the bone mass inducing higher chance of fractures on the spine and bones that carry the body weight .The results of the present series of 70% BNED in 3 years will probably reflect on the patients' quality of life although we did not raise data to support such a conclusion.Lately, several published series have pointed out adverse factors that could define patients with lower probability of occult distant metastasis which might result in better patient selection for local salvage treatment. The worst prognostic factors related to salvage radiation up to this moment are: higher Gleason score ,18,24-26In a recent prospective trial from the European Organization for Research and Treatment of Cancer EORTC 22911), Bolla et al published results of a randomized comparison of wait-and-see after RP or immediate postoperative radiotherapy for high risk patients  and has shown that adjuvant radiotherapy results in better progression free and local-regional free survival . In smal911, BollWe performed salvage treatment using conformal three-dimensional radiotherapy with a median dose of 70 Gy (range 66 – 72 Gy) and we have not found correlation between radiation dose and BNED (p = 0.6). Small retrospective series suggest that conformal three-dimensional radiotherapy and doses higher than 64,8 Gy do correlate with better biochemical control ,27, but There is no agreement regarding which volume should be treated in prostatectomized patients. In the EORTC 22911 radiotherapy was delivered using 2 planning target volumes. The first Planning Target Volume (PTV 1) was defined by the anatomical limits of the surgical bed including those of the seminal vesicles followed by a boost in a reduced PTV (PTV2) to the prostate bed . In our Some series have described low rates of complications (RTOG > Grade 3) in patients submitted to salvage radiotherapy, but what is generally postulated is that toxicity is higher when radiotherapy is employed after surgery than with exclusive radiotherapy specially in the genitourinary tract ,27. AschOur data suggest that approximately 70% of biochemical control in 3 years can be achieved with salvage radiotherapy in selected patients and that 66 Gy may be sufficient for disease control. The importance of PSADT was confirmed in our series and radiotherapy should be started as early as possible. Longer follow up is necessary to confirm these results, but at this moment it is possible to conclude that a long interval free from hormonal therapy was achieved with low rate of toxicity avoiding, or at least, delaying several important adverse effects related to hormonal treatment.


Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to  performing mutation screening, if DNA samples of  sufficient number of family members are available, it  is worthwhile to assign the gene involved in disease  progression by the genetic linkage analysis.PKD1  and five for PKD2 . Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31–46 and PKD2 .We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients . We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2.Lod scores indicated linkage to PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course.In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in  The dis:173910) . PKD1 acPKD1, is predicted to be a large transmembrane glycoprotein [PKD2 codes for polycystin-2 which functions as a non-selective cation channel that can conduct calcium ions [Polycystin-1, the protein encoded by oprotein ,12. PKD2ium ions ,13. It hium ions . A failuPKD1 is large and most of it is reiterated in the form of homologues genes (HG) at least six times on the same chromosome [PKD1 as classic PCR amplifies both PKD1 as well as HG sequences. Several groups have already successfully screened most of the coding region of PKD1 [PKD1 and PKD2 genes with no clustering in mutation 'hot spots'. The majority of them are found to be private. It is therefore worthwhile to first assign the gene involved in a particular ADPKD family before proceeding with mutation screening. Nevertheless less than half of our ADPKD families fitted to the criteria for performing linkage analysis and only in those families could we preselect the ones suitable for PKD1 or PKD2 screening. Due to a limited budget we have screened only a part of the PKD1 gene and the entire PKD2 gene.Although presymptomatic diagnosis of ADPKD is possible by various imaging methods and is relatively reliable in adult patients, genetic diagnosis is important for pre-symptomatic diagnosis in younger individuals and in cases with no family history of the disease. Mutation screening in ADPKD is a cumbersome and expensive process as romosome . The pre of PKD1 -22. FromWe collected samples of 36 Slovene ADPKD families. Linkage studies were performed in 16 families  where samples of at least three affected members or two affected and two unaffected members were available. Ultrasound examination and confirmed history of ADPKD in the families was required to enter the study. Diagnosis of ADPKD was based on ultrasonographic criteria described by Ravine at al., 1994 . The stuPKD1 and PKD2 associated microsatellites: D16S3252 (KG8), D16S665 (SM6), D16S291 (AC2.5), D16S664 (CW3), D16S663 (CW2), D4S1534, D4S2929, D4S1563 and D4S423. We determined amplified product lengths using capillary electrophoresis. Details about primer pairs, labelling, cycling conditions and capillary electrophoresis procedures have been described previously [Genomic DNA was extracted from white blood cells by the standard salting out procedure . We devepedin.bat accordingly.In addition, we also analysed two intragenic RFLPs, p.A4058V and p.A4091A as described previously . Prior tPKD1 and 0.0001 for PKD2. For PKD1, three liability classes were assumed, corresponding to gene penetrances of 0.64, 0.92, and 0.99 for age groups 0–10, 10–30 and over 30 years respectively [PKD2, the liability classes assumed were 0.50, 0.85, and 0.95 for age groups 0–20, 20–30 and over 30 years respectively [A gene frequency of 0.001 was assumed for ectively ,29. For PKD1 exons 23 and 31–46 for mutations using direct sequencing. Primer sequences and PCR conditions are available on request. Prior to sequencing, we purified PCR products through QIAquick columns (Qiagen QIAquick PCR Purification Kit). We analysed sequences on a Perkin Elmer Thermocycler 9600 using Ready Big Dye Terminator Cycle Sequencing Kit according to the protocol of the producer (Applied Biosystems).We screened PKD2 exon amplification are available on request. We performed heteroduplex analysis (HA) using Hydrolink Mutation Detection Enhancement  gel solution. Complete PCR products (50 μl) were mixed with 0.5 μl 0.5 M EDTA, denatured at 95°C for 5 min and finally cooled at 37°C for at least 1 hour. We analysed 5:1 ratio of sample and loading dye by electrophoresis through 25 cm-1 × MDE gel with 15% urea at 250 V for 16–24 h. Then we stained gels with ethidium bromide and photographed them under UV light. DNA samples exhibiting shifted bands we amplified and sequenced in both directions using ABI Prism™ 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions. The detected changes we tested for segregation in each family by either HA or direct sequencing.Primer sequences and PCR conditions for PKD1 we further analysed for their site conservation in mouse (GenBank:U70209), rat (GenBank:AF277452), cat (GenBank:AF483210) and puffer fish (GenBank:AF013614) cDNA sequences, using BLAST [[Putative missense changes in ng BLAST . The effg BLAST [.PKD1 by only four out of the five microsatellite markers used (/PKD1/-KG8-AC2.5-CW3-CW2). We excluded SM6 from Lod score calculations as the correct determination of alleles was impossible due to stutter products and appearance of new alleles during pedigree analysis .We calculated linkage to l., 1994 ). In addPKD1  and in two families to PKD2 (pedigrees 10 and 31). Linkage to both genes was possible in seven mostly small families, where the co-segregation is likely to be due to chance . In one of the 16 families analysed (pedigree 42), Lod scores were mostly negative and linkage to neither of the two PKD loci could be assumed. [see Figure PKD loci in this pedigree. Table PKD1 to PKD2 or to either PKD1 or PKD2 were selected for further mutation screening.In six families Lod scores and segregation of alleles indicated probable linkage to PKD1 mutation screening. We selected one patient from each family where calculated Lod scores did not indicate exclusion of linkage to PKD1 (6 families with indicated linkage to PKD1 and 7 families where linkage to both genes was possible) and 20 patients from families where linkage analysis was not possible due to lack of family member samples. We screened the PKD2 gene for mutations on 9 patients from families where linkage to the PKD2 gene could not be excluded (2 families with linkage to PKD2 and 7 families where linkage to both genes was possible). We identified seven likely ADPKD causing changes and eleven polymorphisms in both genes. They are summarised in Table Altogether 33 patients were included in the PKD1. The first change is a silent polymorphism c.8509C>T that was detected in family 02. The second change p.E2771K c.8522G>A is a missense mutation already identified in 4 British families [We found 3 changes in exon 23 of families . The mutfamilies ,33 we prPKD1) we detected is the substitution from valine to methionine c.8675G>A p.V2822M. We could not analyse for segregation because no additional samples from other family members were available. The valine residue is only conserved in the cDNA sequences of the species cat, mouse and rat, but not puffer fish. Our secondary structure analysis with PHDsec [The third change in exon 23  c.11693_11697dup. The predicted consequence of this duplication is most probably termination of protein synthesis 116 amino acid residues after glutamate 3828. Patient 148 has gallstones and she started dialysis treatment at the age of 44 (rapid progressor). Patient's daughter has enlarged kidneys with multiple cysts at the age of 23 and she suffers from hypertension and proteinuria. Blood sample of the patient's daughter was not avaliable therefore we could not confirm segregation of the mutation in the family.In patient 148 of family 41 we found the duplication of five base pairs in exon 41 (PKD1) c.11745+3_5dup. The mutation was described previously in a French patient under different name 11745+2ins3 [In family 38 we found the duplication of 3 base pairs in intron 41 (45+2ins3 . We adapPKD1) c.11820_11845del. The mutation most probably results in protein termination after 80 amino acid residues. Both patients have already rather early started with dialysis treatment: patient 77 at the age of 48 and patient 161 at the age of 44. In patient 77 cysts in the liver and idiopathic dilated cardiomyopathy were detected, while sibling 161 has persistent hepatitis B.In two siblings (patients 77 and 161) from family 20 we identified the deletion of 26 base pairs in exon 42 (PKD1) c.12341A>G p.I4044V [PKD1) c.12838T>C [Previously described polymorphism in exon 44 (p.I4044V  we found12838T>C .PKD1) c.12346+22del [PKD1) c.12124C>T.In two apparently unrelated pedigrees (families 12 and 27) we found previously described intronic deletion in intron 44  we found nonsense mutation in exon 45 (PKD1 c.12772dup. The mutation most probably results in premature protein termination after 20 amino acid residues. The duplication segregates with the disease in the family 11. Patient 37 died at the age of 58 years. He was operated on kidney stones and suffered from liver cirrhosis as well as obstructive icterus. His daughter (23 years) has small cysts in kidneys that were detected at the age of 11.In family 11 [see Figure PKD2 gene we found four changes. In family 10 [see Figure (PKD2) c.916 C>T p.R306X. The mutation was already described in three Bulgarian families [In families  and in tfamilies ,42. We cPKD2 gene. In exon 1 (PKD2) we found the sequence change c.362 C>G p.G121A. This variation substitutes glycine for alanine at the N-terminal region of polycystin 2. The sequence change however does not segregate with the disease in the family 31. In the same family we also observed the presence of two already described polymorphisms c.83G>C, p.R28P in exon 1 and c.844-22 G>A in intron 3 [In family 31 we detected three changes in the intron 3 ,40.PKD1 or PKD2 prior to performing mutation analysis in ADPKD families. Of the 16 families included in linkage analysis, Lod scores and segregation of alleles indicated probable linkage to PKD1 in six families  and linkage to PKD2 was indicated in two families . Linkage to both genes was possible in seven mostly small families, where co-segregation is likely to be due to chance . For one out of the 16 families analysed (pedigree 42), Lod scores were mostly negative and linkage to neither of the two PKD loci could be assumed.As both genes associated with ADPKD are large and mutation screening is time consuming and costly, it is prudent to assess linkage to either PKD1 or PKD2 in patients from families with indicative linkage to either one or the other gene and in patients from families where linkage to both genes was possible. In addition we included patients from families where we could not perform linkage studies due to a lack of samples from family members. In total we screened 33 patients for PKD1 mutations and 9 patients for PKD2 mutations.We consequently performed mutation screening in either PKD1 has been hindered by the high (>95%) sequence identity with homologous genes, raising concerns about PCR primer specificity and hence reliability of the analysis [PKD1 (exons 33–46) as well as exons 31–32 and exon 23 of the duplicated part. Exon 23 was analysed due to its' proximity to the IVS21 polypyrimidine tract, where clustering of mutations has been proposed [PKD1, the region around the polypyrimidine tract shows a higher frequency of changes compared to other parts of the gene [Detection of mutations in the duplicated part of analysis . We haveproposed  due to fproposed ,46. Alththe gene .PKD1 and one mutation and three polymorphisms were detected in PKD2. Three mutations were described previously; p.E2771K c.8522G>A (PKD1) in four British families [PKD1) in a French patient [PKD2) in three Bulgarian [PKD1) most probably result in premature protein termination.Six mutations and eight polymorphisms were detected in families , c.11745 patient  and p.R3ulgarian  and two ulgarian ,42. FourPKD1 in a sufficient number of control chromosomes is costly, the distinction between disease causing mutations and neutral variants has to rely on family segregation studies. Segregation in family we confirmed for four out of seven identified mutations where samples at least two patients from the family were available. From the other three, one mutation was described previously (c.11745+3_5dup) [As there is no functional assay for ADPKD and analysing +3_5dup) , and froPKD1 c.12772dup, c.11820_11845del and c.11693_11697dup seem to be associated with the more severe clinical course of the disease resulting in ESRD  in the range from 44 to 48 years  as already reported by Bulgarian and Czech studies [On the basis of collected clinical data of the patients which is summarised in Table  studies -42. CompPKD1 and one mutation and three polymorphisms were detected in PKD2. Three mutations have been described previously in British, French, Czech and Bulgarian populations. Four out of 7 mutations are novel and probably private. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of the disease resulting in ESRD  in the age range of 44 to 48 years. Nonsense mutation in PKD2 seems to be associated with cysts in liver and slower progression towards ESRD.In our study group of ADPKD patients six mutations and eight polymorphisms were detected in The author(s) declare that they have no competing interest.KV carried out genotyping and linkage analysis, majority of mutation screening in PKD1, coordinated experimental work and drafted the manuscript. LS carried out part of mutation screening in PKD1 and contributed to the writing of the manuscript. JS and JR performed mutation screening in PKD2 and contributed to the writing of the manuscript. MB, TK, SJ, BL, AA, AS, RD and RH are nephrologists who were responsible for clinical examinations, contacting the family members of the patients, collecting of clinical data and blood samples. PH took part in PKD1 mutation analysis. IZP and JB carried out paternity testing in one of the families. GH wrote two subprograms that helped us to analyse linkage data. RK supervised the research activity, contributed in study design and revised the manuscript. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In the LMCE1 study using a single course of megatherapy most of the relapses occurred during the first 2 years after autologous bone marrow transplantation. A second pilot study (LMCE2) was therefore set up using a double harvest/double graft approach with two different megatherapy regimens. Objectives were to test the role of increased dose intensity on response status, relapse pattern and overall survival. Thirty-three patients  with a median age of 53 months at first megatherapy  entered this study. They were cases either with refractory disease in partial response after second line treatment for stage 4 neuroblastoma (n = 25) or after relapse from stage 4 (n = 5) or stage 3 disease (n = 3). All patients received Etoposid and/or Cisplatinum (or Carboplatin) containing treatments before megatherapy. The first megatherapy regimen was a combination of Tenoposid, Carmustine and Cisplatinum (or Carboplatin), the second applied Vincristin, Melphalan and Total Body Irradiation. The first harvest was scheduled 4 weeks after the last chemotherapy, the second 60 to 90 days after megatherapy. All marrows were purged in vitro by an immunomagnetic technique. Median follow up time since first megatherapy is 56 months. Response rates for evaluable patients were 65% (complete response rate: 16%) for megatherapy 1 and 60% (complete response rate: 25%) for megatherapy 2. Considering that only patients with delayed response or relapse were eligible for this pilot study the overall survival was encouraging with 36% at 2 years and still 32% at 5 years. The costs for these survival rates were high in terms of morbidity . Double harvesting may have the disadvantage of delayed engraftments related in part to a disturbance of marrow microenvironment by megatherapy 1. This double megatherapy approach achieved a prolonged relapse free interval  in patients reaching megatherapy 2 and justifies further evaluation of concepts with consecutive dose-escalation.


A total of 474 adult patients with malignant glioma (astrocytoma) grade 3 or 4 were randomised into an MRC study (BR2) comparing 45 Gy (in 20 fractions over 4 weeks) with 60 Gy (in 30 fractions over 6 weeks) of radiotherapy given post-operatively. Using 2:1 randomisation, 318 patients were allocated the 60 Gy course and 156 the 45 Gy course. Adjuvant chemotherapy was not given. The results show that a 60 Gy course produces a modest lengthening of progression-free and overall survival. They suggest a statistically significant prolongation of median survival from 9 months in the 45 Gy group to 12 months in the 60 Gy group . Over 80% of patients reported no morbidity from the radiotherapy, and there was no evidence of increased short-term morbidity in the higher dose group. Late morbidity was not assessed. A prognostic index defined in a previous MRC study was validated in this new cohort. It identifies a group of patients  with a 2 year survival rate of 28% . It was apparent that the survival advantage to the higher dose was maintained even in the poorest prognostic groups defined by this index.


Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated floxneoWnt4R26. Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. floxneoWnt4; Col2a1-CreR26 double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, floxneoWnt4; Col2a1-CreR26 mice had decreased expression of vascular endothelial growth factor (VEGF). These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype. Wnts have been shown to be expressed in the developing long bones, suggesting that they may have roles in endochondral bone formation. In the developing chick skeleton, Wnt4 and Wnt9a (previously known as Wnt14) are expressed in joint-forming regions, Wnt5a and Wnt11 in the perichondrium, and Wnt5b in prehypertrophic chondrocytes of the growth plate Wnt4 and Wnt5a can alter chondrogenesis and shorten limb growth, apparently by different mechanisms. Wnt4 accelerates chondrocyte differentiation, whereas Wnt5a inhibits this process Wnt9a misexpression has been shown to induce the initiation of joint formation Wnt9a knockout mice formed joints but had ectopic cartilaginous nodules that was enhanced by loss of Wnt4Wnt4/Wnt9a double mutants also had some limb bone fusions apparently because of an inability to maintain joint cell identity Wnt5b as well as Wnt5a inhibits chondrogenesis in mice, but they appear to act differently. Wnt5a inhibits the transition from resting to proliferating chondrocytes in the growth plate, whereas Wnt5b promotes this transition as well as chondrocyte proliferation Wnt signaling has been implicated in the regulation of early patterning and initial outgrowth of the vertebrate limb bud Lef1 in chondrocytes stimulated chondrocyte maturation as well as replacement of cartilage by bone LDL receptor-related protein 5 (Lrp5) gene that encodes a Wnt co-receptor, showed decreased osteoblast proliferation Lrp5-deficient mice also displayed persistent eye vascularization. These bone and eye phenotypes are similar to the abnormalities associated with osteoporosis-pseudoglioma syndrome in human, caused by mutation of LRP5Wnt signaling components have also been investigated for their roles in skeletogenesis. Frb1, a secreted form of Frizzled that is a Wnt receptor, can function as an antagonist when misexpressed in long bone, causing shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation Wnts appears to vary in different animal models. For example, in addition to the perichondrium of chick, Wnt5a expression was also found at the junction of proliferating and prehypertrophic chondrocytes in the radius and ulna of mice Most studies of Wnt signaling in skeleton development have been restricted to the chick model. However, the expression of Wnt4 expression has also been analyzed during kidney and female reproductive system development. Wnt4 homozygous mutant mice died after birth due to a failure of pretubular cell aggregation, an essential step in the formation of nephrons of the kidney Wnt4 mutant mice with an XX karyotype lacked female-specific genital ducts and developed male-specific genital ducts Wnt4 is initially expressed in joint-forming regions, and then is detected in the region of the joint capsule and surface articular chondrocytes Wnt4 expression in long bones is also detected in hypertrophic chondrocytes Wnt4 is also expressed in forming joints and mesenchyme that will form the joint capsule Wnt4 expression in chick and mouse suggest roles in joint development and chondrocyte hypertrophy. In addition, the restricted pattern of Wnt4 expression in bone-forming tissues suggests that its expression must be precisely controlled to coordinate normal bone and skeleton formation.Wnt4 during skeleton development, we created a conditional genetic system to express Wnt4 during chondrogenesis. To accomplish this, we exploited the ubiquitously expressed Rosa26 locus. The ROSA26 mouse mutant was originally produced by infection of embryonic stem (ES) cells with a ROSAβ-geo retrovirus Rosa26 heterozygotes express β-galactosidase  reporter activity ubiquitously that initiates during preimplantation development at the morula-blastocyst stage. Examination of serial sections through 9.5 days post-coitus (dpc) Rosa26 heterozygotes demonstrated β-gal activity in all cells Rosa26 homozygous mutants are viable although they are recovered at a lower than expected frequency Rosa26 locus has been used to ubiquitously or conditionally express various gene products in mice Rosa26 locus to express Wnt4 in a Cre-dependent manner. We placed a drug selection cassette flanked by loxP sites between the Rosa26 promoter and a mouse Wnt4 cDNA, blocking Wnt4 expression at the endogenous Rosa26 locus. Cre expression should delete the blocking drug selection cassette, leading to Wnt4 expression.To study the actions of Wnt4 during endochondral bone formation, we used Col2a1-Cre transgenic mice that express Cre activity in cartilage-forming tissues Wnt4 expression in chondrogenic tissues alters skeletogenesis, resulting in skull abnormalities and dwarfism. These studies indicate that alterations in Wnt4 expression can cause severe skeletal pathologies.To examine the action of Wnt4 in a Cre-dependent manner, potentially in any tissue. We modified the ubiquitously-expressed Rosa26 locus by gene targeting in ES cells  have developed in both the tibiae and femurs of controls . At 9 months of age, the tibiae of mutants (n = 2) were deficient in bone marrow and were filled with adipocytes in epiphyseal and metaphyseal regions , a member of the Hedgehog gene family, is a key molecule in endochondral ossification Ihh is expressed predominantly in prehypertrophic chondrocytes. Hybridization of Ihh showed no obvious differences in mutant tibiae relative to controls  floxneoWnt4; Col2a1-CreR26 mutants was nearly identical to wild type, although with an altered appearance. floxneoWnt4; Col2a1-CreR26 mutants have an expanded zone of hypertrophic chondrocytes and a smaller zone of proliferating chondrocytes. Overexpression of Wnt4 also causes a decrease in VEGF expression that may result in a reduction of vascularization that in turn leads to delayed formation of primary and secondary ossification centers. The Col2a1-Cre transgene is active in chondrocyte precursors of endochondral bones floxneoWnt4; Col2a1-CreR26 mutants were significantly smaller than controls. The skull is composed of elements derived from both endochondral and membranous bone formation. The alterations observed in the membranous bones of the mutant skulls may be indirect effects of altered endochondral skull bones. All of the skeletal alterations mentioned above likely contribute to the development of the dwarf phenotype.The external morphologies of the Wnt4 is expressed in the developing joint regions and a subset of hypertrophic chondrocytes Wnt4 homozygous mutant mice die within 24 hours after birth due to severe defects in kidney function Wnt4/Wnt9a double mutants show some joint cell identity abnormalities Wnt4 in the skeleton after birth. Such studies will require the generation of a Wnt4 conditional null allele Wnt4 misexpression in chick limbs accelerated chondrocyte maturation; in contrast, Wnt5a misexpression in the same model inhibited chondrocyte maturation Wnt4 misexpression, suggesting that Wnt4 influence on limb shortening may be mediated by β-catenin Wnt4 signaling may have a role in chondrocyte maturation.Wnt4 in the mouse growth plate may also influence chondrocyte maturation, as shown by reduced zones of proliferating chondrocytes and expanded zones of Col10a1 hybridization and hypertrophic chondrocytes in floxneoWnt4; Col2a1-CreR26 mutants. In addition, in growth plates of floxneoWnt4; Col2a1-CreR26 mutants, the zone of Col2a1 hybridization was significantly smaller yet the Ihh zone essentially unchanged, indicating a decrease in proliferating chondrocytes. The decrease of the zone of proliferating chondrocytes may result from lower rate of proliferation in mutants or a higher percentage of cells exiting the proliferation state. However, an in vitro micromass culture assay has shown that infection by a retroviral-delivered Wnt4 did not decrease the rate of cell proliferation Wnt5a and 5b misexpresssion using the same system. Indeed, recent studies in transgenic mice demonstrated that Wnt5b could promote proliferation of chondrocytes in vivo Wnt4 to decrease the zone of proliferating chondrocytes may represent an enhancement of the endogenous activity of Wnt4, a competition with the activity of Wnt5b that promotes proliferation of chondrocytes, or a mimicking of Wnt5a function that inhibits the transition from resting chondrocytes to proliferating chondrocytes. Moreover, since Wnt4 may accelerate the differentiation of chondrocytes, proliferating chondrocytes with overexpression of Wnt4 may exit the cell cycle rapidly, leading to narrower zone of proliferating chondrocytes.We show that overexpression of floxneoWnt4; Col2a1-CreR26 mutants also had disorganized growth plates. The organized structure of the growth plate is tightly linked between the chondrocytes and the extracellular matrix (ECM) and Wnt actions on cell adhesion have been proposed Wnt4 may interrupt such a relationship, by either changing the cell membrane structure of chondrocytes or altering components of the ECM. Wnt4 has been shown to signal canonically, non-canonically, or through neither pathway, depending upon the experimental context Wnt4-induced dwarfism documented in our study.The floxneoWnt4; Col2a1-CreR26 mutants displayed decreased VEGF expression. VEGF is a key regulator for vascularization and plays an important role during endochondral bone formation, where VEGF couples hypertrophic cartilage remodeling, ossification, and angiogenesis Vegf heterozygous mice die at early embryonic stages 120 isoform can survive to term. Vegf120/120 mutants appeared to have low angiogenesis activity floxneoWnt4; Col2a1-CreR26 mutants. These abnormalities include delayed invasion of vessels into the primary and secondary ossification centers, reduction in mineralization of mutant bone, and expansion of the zone of hypertrophic chondrocytes. Thus, these phenotypes observed in the floxneoWnt4; Col2a1-CreR26 mutants may be caused by overexpression of Wnt4, causing reduced expression of VEGF.The growth plates of floxneoWnt4; Col2a1-CreR26 mutant phenotype. However, Wnt gene family members often exert distinct functions in a particular tissue of the same stage. For instance, infection of chick limbs using a retrovirus carrying Wnt5a or Wnt4 presented distinct effects, with Wnt5a delaying chondrocyte differentiation, whereas Wnt4 accelerated it Wnt5b promoted the transition of resting chondrocytes to proliferating chondrocytes, whereas Wnt5a inhibited it Wnt genes, or endogenously, Wnt proteins may function as antagonists of each other.Although the relationship between Wnt proteins and VEGF during skeletal development has not yet been clarified, Wnt/β-catenin signaling has been suggested to play a role in activation of VEGF gene expression in benign colonic adenomas in which mutant activated Wnt/β-catenin pathway is often associated with up-regulated VEGF Wnt4 is an important regulator of female reproductive organ development in mice. The ovaries of female mice lacking Wnt4 were masculinized with indications of Leydig cell differentiation Wnt4 may repress angiogenesis in developing female gonads, blocking the testis differentiation pathway.floxneoWnt4; Col2a1-CreR26 mutants became apparent only after birth. However, skeletal changes were found earlier in development, consistent with the activity of the Col2a1-Cre transgene at the initial stages of cartilage formation Wnt4 at fetal stages may be attributable to limited expression of Wnt4 receptors or an excess of Wnt inhibitors. However, the overt dwarfism of the floxneoWnt4; Col2a1-CreR26 mutants may be the result of VEGF insufficiency. Neonatal mice homozygous for a Vegf 120 isoform allele had 10% shorter tibiae, and slight differences in bone length were detected at 16.5 dpc in comparison to controls floxneoWnt4; Col2a1-CreR26 mutants may have stronger VEGF activity than 120/120VEGF mutants because the phenotypes displayed in 120/120VEGF mutants were more severe than those of RWnt4; Col2a1-Cre mutants. For example, the expansion of the zone of hypertrophic chondrocytes was larger, and the delayed time of formation for the primary ossification center was longer in 120/120Vegf mutants. Thus, it may be reasonable to expect that the shortening of tibial length observed in floxneoWnt4; Col2a1-CreR26 mutants as in Vegf120/120 mutants becomes apparent only after birth.The dwarfism of Wnt4 is expressed during skeleton development. The studies presented here demonstrate that dysregulated Wnt4 expression in chondrogenic tissues leads to skeletal defects and dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. In addition, these studies suggest that pathologies that lead to Wnt overexpression may influence chondrogenic tissues.In summary, Wnt4 cDNA encoding the entire open reading frame was isolated by RT-PCR. RNA from 13.5 dpc gonads from mouse strain 129/EvSvTac was isolated and used to synthesize cDNA. The Wnt4 cDNA was subsequently amplified by two rounds of PCR. The first primer set was: forward 5′-CCGCGCGGCGAAAACCTG-3′ and reverse 5′-CTGTTTAAGTTATTGGCCTTC-3′. The second primer set was: forward 5′-GCCTTGGGATCCCTGCCCCGGGCTGG-3′ and reverse 5′-ACGCAGGCGGCCGCACTAGTCCTAGGCATGGTCA-3′. The final PCR product was subcloned into the BamHI and NotI sites of pBluescript KS(-) and sequenced.The mouse Wnt4 expression cassette into the Rosa26 locus. The expression cassette begins with a splice acceptor sequence (SA), followed by Pgkneo and five polyadenylation sequences flanked by loxP sites (floxneo). The mouse Wnt4 cDNA followed by a bovine growth hormone polyadenylation (bpA) sequence was placed 3′ of floxneo. The expression cassette, SA-loxP-Pgkneo-5pA-loxP-Wnt4-bpA, was inserted into the XbaI site of pR26-1, to generate the gene targeting vector (DT) is present within pR26-1 for negative selection. The targeting vector was linearized with KpnI and electroporated into AB1 ES cells and selected in G418 XbaI and analyzed by Southern blot bpA .4 and 9 month-old mice were sacrificed by COSkeletal tissues were treated with hyaluronidase  to unmask epitopes for immunohistochemistry. Immunostaining was performed using the Vectastain ABC kit , according to the manufacturer's instructions. Rabbit anti-mouse VEGF antibody  was diluted 1∶20.Mice were injected intraperitoneally with bromodeoxyuridine (BrdU) at 100 mg/g body weight and sacrificed 1 hour after injection. Histological sections of skeletal tissues were prepared and immunostained for BrdU . The proliferation rate was calculated as the number of BrdU-labeled cells divided by the total number of cells in the same microscopic field.35[S]-UTP-labeled antisense or sense RNA probes were prepared. After hybiridzation, samples were exposed for 7 to 40 days and then developed using Kodak D-19 developer and fixer, and counterstained with Hoechst dye.The procedures for RNA in situ hybridization of histological sections were adapted from those described


Here, I use two methods to estimate the anthropogenic CO2 storage and uptake for a typically large EEZ .Under the United Nations convention on the law of the sea (1982), each participating country maintains exclusive economic and environmental rights within the oceanic region extending 200 nm from its coastline, known as the Exclusive Economic Zone (EEZ). Although the ocean within each EEZ has a vast capacity to absorb anthropogenic CO2 emissions were absorbed by its own EEZ.Depending on whether the Antarctic territory is included I find that during the 1990s between 30–40% of Australia's fossil-fuel CO2 sink' could be used as a disincentive for certain nations to reduce their anthropogenic CO2 emissions, which would ultimately dampen global efforts to reduce atmospheric CO2 concentrations. Since the oceanic anthropogenic CO2 sink has limited ability to be controlled by human activities, current and future international climate change policies should have an explicit 'EEZ' clause excluding its use within national carbon accounts.This example highlights the potential significance of the EEZ carbon sink for national carbon accounts. However, this 'natural anthropogenic CO The ocean has hindered the extent of accelerated climate change and will continue to absorb about 33% of fossil-fuel emissions well into the future [2 sink is different to other carbon sinks in that it directly remediates against climate change by sequestering anthropogenic CO2 on both short and long timescales. The exclusive economic zone (EEZ) is an oceanic zone legally bound to nation states under international law [2 sink be for national carbon accounts. I use Australia as a case study to estimate the EEZ anthropogenic CO2 sink due largely to the detailed accounting information on CO2 emissions from fossil fuels and land-use changes [6 km2 [6 km2).Atmospheric COe future . The oces [6 km2 , which m2 within Australia's EEZ (including the Antarctic territory) for the 1990–1999 period to be 2.12 ± 0.7 Pg CO2 (Pg = 1 × 1015 g) with an annual increase of about 220 Mt CO2/yr , the mean anthropogenic CO2 flux for Australia's ocean is about 175 MtCO2/yr throughout the 1990s. This calculation assumes Australia's EEZ acts in proportion to the global average oceanic anthropogenic CO2 flux. Even though the ocean is relatively homogenous, most studies suggest the Southern Ocean to be the region of highest uptake [2 uptake within the Australian EEZ (175–220 MtCO2/yr) is in agreement with a recent modelling study [2/yr depending on the areal extent of the Australian EEZ.I have calculated the accumulation (storage) of anthropogenic COt uptake . Since A2 uptake is significant when comparing to Australia's CO2 emissions via fossil-fuel usage or land-use  is about 3 times the magnitude of the CO2 source due to land-use changes (655 MtCO2) and about 30–40% of the total magnitude of fossil-fuel emissions (4695 MtCO2). The implications of including Australia's EEZ or any other nations EEZ within the framework of global carbon trading would be considerable. Furthermore direct human influence of the EEZ CO2 sink through carbon runoff from land use/irrigation/agricultural practices may also significantly influence national carbon accounts for nations with large EEZs.Australia's EEZ anthropogenic CO6 km2), its EEZ anthropogenic CO2 sink only absorbs less than 3% of its annual fossil-fuel emissions[2 emissions relative to their large potential EEZ CO2 sink. Tonga, Fiji, Samoa, Soloman Islands for example have vast oceanic territories that absorb many times over their annual fossil-fuel CO2emissions. Although Australia emits near the highest amount of anthropogenic CO2 per capita in the world, its relatively low population and vast oceanic territory results in the EEZ carbon sink being highly influential to its national carbon accounting. However, this 'natural anthropogenic CO2 sink' could be used as a disincentive for certain nations to reduce their anthropogenic CO2 emissions, which would ultimately dampen global efforts to reduce atmospheric CO2 concentrations. Along with the fact that the oceanic anthropogenic CO2 sink has little ability to be controlled by human activities, it should be explicitly excluded within current or future climate change policies. The international legality of the EEZ carbon sink and its potential implications requires careful consideration in formulating an equitable future framework for climate policy that aims at reducing atmospheric CO2 levels.Nations with large EEZs (like the USA) aren't necessarily those who will benefit the most from including the EEZ carbon sink in international climate policy, as it depends on the relative amount of EEZ sink in comparison to a nations annual anthropogenic emissions. Despite the USA claiming the worlds largest oceanic territory (~10 × 10emissions. On the 2. Just as nation states have varying degrees of land coverage they also have varying degrees of EEZ extents. To demonstrate the potential implications of the EEZ anthropogenic CO2 sink, I have roughly estimated the uptake for Australias EEZ which is one of the largest in the world. By comparing the amount of anthropogenic CO2 sequestered by Australias EEZ to Australias CO2 emissions via fossil-fuel/land-use, I show that including the EEZ has significant implications for Australias national carbon accounts and any other nation who maintains a large EEZ. As the EEZ carbon sink may introduce legal grounds for nation states to possibly exploit, which would ultimately dampen efforts to reduce atmospheric CO2 concentrations, current and future international climate change policies should have an explicit 'EEZ' clause excluding its use within national carbon accounts.The global EEZ represents over a quarter of the surface area of the ocean, which undoubtedly acts as important reservoir for sequestering anthropogenic CO2 measurements within Australia's EEZ, to quantify the EEZ anthropogenic CO2 sink I use a recently developed method that exploits a purely transient tracer [2 atmospheric history [2 from 1990 to 1999. Although the method is indirect, the total uncertainty has been quantified to be between 10–20% by comparing results from direct temporal CO2 estimates [Due to the lack of temporal COt tracer . The mett tracer  to detert tracer . These w history , alkalin history  to estimstimates  and with


Responses both to hyperosmotic stress and to heat shock were compared in 3T3 cells, spontaneously transformed cells (ST3T3) and simian virus 40-transformed cells (SV3T3). Cell adaptation to these stresses was measured in terms of surviving cell viability and plating efficiency, while their induced synthesis of stress proteins was monitored in terms of the presence of mRNA for HSP70, the pattern of polypeptides synthesised and the accumulation of HSP70 detectable by monoclonal antibodies. All three types of cells responded similarly to heat shock in their expression of HSP70 and showed no clear differences in ability to recover. In contrast, both ST3T3 and SV3T3 cells adapted more poorly and much more slowly to hyperosmotic stress (0.5 osM incubation) than did normal 3T3 cells. This different pattern of adaptation to hyperosmotic stress was parallelled by the cells' different expression of a stress protein that could not be distinguished from the heat-induced HSP70 by any of the methods listed above. In view of these findings it seems possible that hyperosmotic treatment might be useful in selectively affecting the survival of tumour cells.