In Down syndrome patients several metabolic abnormalities have been reported, some involving the lipid metabolism. The level of LDL in plasma is the major determinant of the risk of vascular disease. There appear to be no studies on the LDL receptor in Down syndrome patients.Flow cytometric methods for measuring the LDL receptor in peripheral blood mononuclear cells (PBMC) can identify patients with hypercholesterolemia. We applied this method in 19 old patients with Down syndrome and 23 healthy controls.Down syndrome patients had high levels of triglycerides and low levels of HDL, and high levels of CRP. We also found a down-regulation of LDL receptor expression.Down syndrome patients show no increase in the frequency of cardiovascular disease. The low incidence in cardiovascular disease despite the low level of HDL, high levels of CRP and reduction of LDL receptor expression lead to the conclusion that either these are not risk factors in these patients or that other risks factors – not yet identified – are considerably lower. Several studies have discussed the psychological and intellectual problems, immunological deficiencies, and early aging of Down syndrome (DS) patients. Several metabolic abnormalities have been reported, some involving the lipid metabolism . Apart fIn children  and alsoMeasurements of LDL receptor expression are also necessary to fully characterize the functional status of the low-density lipoprotein (LDL) pathway which substantially influences LDL levels in plasma, and its discovery constituted a major biological advance by providing molecular explanations of hypercholesterolemia. The plasma LDL level is the major determinant of the risk of vascular disease. We analyzed, also, C reactive protein (CRP), a cardiovascular risk factors coded by genes lying on Chromosome 21. Flow cytometric methods for measurement of LDL receptor on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercholesterolemia . Data inWe therefore investigated LDL receptor expression on uninduced PBMC, particularly T lymphocytes because they express more LDL receptors than monocytes . Since tBlood samples were drawn from 19 old DS patients  and 23 healthy individuals  without dyslipidemia or any family history of coronary heart disease, no smokers or drunkers, with a Body Mass Index (BMI) < 25. Lipid measurements are given in Table None had been treated with lipid-lowering drugs before blood sampling. This study was conducted in accordance with the Declaration of Helsinki, 1975, amended in 1983.g, 20°C. The interface containing the PBMCs was isolated, and the cells were washed three times in HBSS and resuspended in RPMI-1640  with L-glutamine (290 mg/L), penicillin , streptomycin (100 mg/L) and 100 mL/L human lipoprotein-deficient serum (HLPDS) to a final concentration of 106 cells/mL.Blood, collected in tubes containing EDTA, was cooled to 20°C and diluted 1:1 with Hank's buffered saline solution . PBMCs were prepared under sterile conditions, using Ficoll-Hypaque  and diluted blood was layered in a centrifuge tube and centrifuged for 40 min at 400 6 cells/mL, and 100-μL aliquots of cell suspension were pipetted into polypropylene tubes and placed in ice-water. Cells were incubated with 1.5 μg of monoclonal mouse anti-human LDL receptor-specific antibody, clone C7 , for 30 min in the dark at 4°C. After this the cells were washed twice in ice-cold HBSS with 20 mL/L HLPDS, and incubated with 3 μL of fluorescein isothiocyanate  for 30 min in the dark at 4°C. Cells were then incubated with 1 μL of R-phycoerythrin (RPE)-conjugated monoclonal antibody CD3-RPE or IgG1isotype-RPE for T lymphocytes.Tissue culture flasks were placed in ice-water for 60 min in the dark to reduce cell adhesion. PBMCs were removed by flushing with ice-cold HBSS (4°C) and washed twice in ice-cold HBSS with 20 mL/L HLPDS. The cell number was adjusted to 0.3 × 10The flow cytometry measurements were done in a FACScan flow cytometer  equipped with a 15 mW, 488 nm, air-cooled argon laser and linked to a computer with CellQuest software. Forward scatter (FSC) and side scatter (SSC) were adjusted to exclude debris and dead cells. FITC emission was measured at 530 nm (FL1) and RPE emission at 585 nm (FL2); compensation was set using FITC-conjugated C7 (C7 FITC)-labeled cells (FL2-FL1) and CD3-RPE-labeled cells (FL1-FL2).t-test or one-way analysis of variance (ANOVA). Data are presented as means ± SD. Differences were considered statistically significant at p < 0.05.Means were compared by the unpaired p = 0.8 and p = 0.9 respectively). Blood levels of CRP were higher in DS than in controls, as illustrated in Figure Table p < 0.01 and p < 0.05). Our data also show that the expression of LDL receptor on T lymphocytes was down-regulated in DS patients  (Table Mean intensity fluorescence (MIF) of LDL receptor expression was significantly different in DS patients and healthy controls (1) Table .Patients with DS who reach adolescence nowadays have a nearly normal life expectancy thanks to better medical care. When they die at a later age, cardiovascular diseases are less common than in the general population and they have even been proposed as "an atheroma-free model" . Our resMultiple factors are responsible for atherosclerosis, such as dietary habits but still the unexplained decline of LDL receptor expression with aging contributes importantly to borderline-high levels and cannot be ignored. For example the loss of estrogen-stimulated LDL receptor synthesis after menopause is an important contributor to elevated cholesterol in postmenopausal women. In addition, several genetic defects inherited singly appear to be causes of moderate hypercholesterolemia ,12. GeneIn our series, a reduction in LDL receptor expression was not correlated with high LDL serum levels or total cholesterol. Moreover, lipophospholipid (LPC) is generated by hydrolysis of phosphatidylcholine which is present in LDL; LPC may promote the start of an immune response and atherosclerosis may be the most extreme demonstration of this immune regulation pathway . LPC mayInterestingly, DS patients show no increase in their frequency of cardiovascular disease.H1 lymphocytes responses [These conditions may be explained by mild immune defects in the syndrome, mainly involving macrophages and/or Tesponses . Alternaesponses  with – iDown syndrome (DS); high-density lipoprotein (HDL); C reactive protein (CRP); Body Mass Index (BMI); low-density lipoprotein (LDL); peripheral blood mononuclear cells (PBMC); lipophospholipid (LPC).The author(s) declare that they have no competing interests.


The nuclear transcription factor NF-κB binds to the HIV-1 long terminal repeat (LTR) and is a key regulator of HIV-1 gene expression in cells latently infected with this virus. In this report, we have analyzed the ability of Kaposi's sarcoma associate herpes virus -encoded viral FLIP (Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein) K13 to activate the HIV-1 LTR.We present evidence that vFLIP K13 activates HIV-1 LTR via the activation of the classical NF-κB pathway involving c-Rel, p65 and p50 subunits. K13-induced HIV-1 LTR transcriptional activation requires the cooperative interaction of all three components of the IKK complex and can be effectively blocked by inhibitors of the classical NF-κB pathway. K13 mutants that lacked the ability to activate the NF-κB pathway also failed to activate the HIV-1 LTR. K13 could effectively activate a HIV-1 LTR reporter construct lacking the Tat binding site but failed to activate a construct lacking the NF-κB binding sites. However, coexpression of HIV-1 Tat with K13 led to synergistic activation of HIV-1 LTR. Finally, K13 differentially activated HIV-1 LTRs derived from different strains of HIV-1, which correlated with their responsiveness to NF-κB pathway.Our results suggest that concomitant infection with KSHV/HHV8 may stimulate HIV-1 LTR via vFLIP K13-induced classical NF-κB pathway which cooperates with HIV-1 Tat protein. The human immunodeficiency virus type 1 (HIV-1) establishes latent infection following integration into the host genome . The HIVThere are five known members of the NF-κB family in mammalian cells including p50/p105 (NF-κB1), p52/p100 (NF-κB2), p65 (RelA), c-Rel, and RelB . The besKaposi's sarcoma associated herpes virus (KSHV), also known as Human herpes virus 8 (HHV8), is a γ-2 herpes virus which is frequently associated with malignancy among AIDS patients -13. In aThe K13 protein contains two homologous copies of a Death Effector Domain (DED) that is also present in the prodomains of caspase 8 , caspase 10 and cellular FLICE Inhibitory Protein  . Proteinvs alternate NF-κB signaling pathways, various subunits of the IKK complex and the HIV-1 Tat to this process.We recently demonstrated that KSHV vFLIP K13 possesses the unique ability to activate both the classical and the alternate NF-κB pathways -24. SeveWe used a luciferase reporter construct to test the effect of vFLIP K13 on HIV-1 LTR transcriptional activation. This reporter construct expresses the firefly luciferase gene downstream of the HIV-1 LTR. As shown in Fig. We have recently generated point mutants of the vFLIP K13 which differ in their ability to activate the NF-κB pathway . In ordeIn order to test the hypothesis that vFLIP K13 activates HIV-1 LTR by inducing the binding of specific transcription factors to the NF-κB binding sites present in the HIV-1 LTR, we used an electrophoretic mobility shift assay (EMSA). As shown in Fig. vs alternate NF-κB pathway to vFLIP K13-induced HIV-1 LTR activation, we used a supershift assay to analyze the nature of the protein complexes bound to HIV-1 LTR from nuclear extracts of vFLIP K13-expressing cells. This assay demonstrated that p50 and c-Rel subunits are the major components of the HIV-1 LTR-bound NF-κB complexes induced by vFLIP K13 with modest contribution from the p65 subunit  pathway of NF-κB activation, which involves proteasome-mediated processing of p100/NF-κB2 into p52 subunit, has been described . We havenit Fig. . As the We have previously demonstrated that vFLIP K13 activates the classical NF-κB pathway via phosphorylation of IκBα, which leads to its ubiquitination and subsequent degradation via proteasome . We usedWe used siRNA-mediated downregulation of key subunits of the classical and alternate NF-κB pathways to test their involvement in K13-induced HIV-1 LTR activation. As shown in Fig. K13 is known to associate with a 700 kDa multi-subunit IKK complex, which consists of two catalytic subunits, IKK1/IKKα and IKK2/IKKβ and a regulatory subunit, NEMO/IKKγ . We testNext we sought to determine whether pharmacological inhibitors of the NF-κB pathway may be used to block vFLIP K13-induced HIV-1 LTR reporter activation. Lactacystin and MG132 are inhibitors of proteasome and block the NF-κB pathway by preventing the degradation of IκB. On the other hand, arsenic acid is believed to block the NF-κB pathway by inhibiting the IKK complex . As showMurr1 is a gene product that has been previously implicated in copper regulation ,31. A reHIV-1 Tat is a viral nuclear protein that plays an essential role in HIV-1 gene expression at the transcriptional level ,3. Tat hTranscriptional activation of genes is usually regulated by multiple transcription factors acting in concert. Thus, while NF-κB has been shown to play a major role in the activation of the HIV-1 LTR, it fails to do so when acting alone ,34-36. AThere is considerable sequence diversity among the HIV-1 isolates that comprise the current global pandemic and these can be grouped into several distinct subtypes or clades . In part+ T lymphocytes, dendritic cells, and mononuclear phagocytes [et al, several lines of evidence suggest that the above assumption may not be completely true and HHV8 and HIV-1 may, in fact, interact in vivo [First, both HHV8 and HIV-1 can efficiently infect cells of monocyte/macrophage lineage, including dendritic cells [Second, Moir et al have shown that induction of CD4 and CXCR4 on B cells by CD40 stimulation leads to an increased susceptibility of these cells to T-trophic HIV infection [Third, HHV8-infected B cells can be infected by HIV-1 via a cell-cell pathway and such infected B cells can support productive HIV-1 replication [in vivo is unclear at the present. Therefore, it stands to reason that HHV8 and HIV-1 genomes may co-exist in the same cells in vivo and reciprocally regulate the gene expression of each other. Support for the above hypothesis is provided by a recent study which demonstrated that co-culture of HIV-1-infected CD4+ T cells with HHV8-infected B cell lines resulted in increased HIV-1 replication [Although co-infection with HHV-8 and HIV-1 is known to synergistically increase the incidence of KS, until recently intracellular interaction between HHV8 and HIV-1 has not received adequate attention under the assumption that these viruses infect distinct cell types. Thus, HHV8 is typically believed to infect B lymphocytes, epithelial cells, keratinocytes, KS tumor cells, and endothelial cells ,41, whilagocytes . Howeveric cells ,44. Seconfection . Third, lication . Finallylication .With the goal of elucidating intracellular signaling interactions which could be potentially involved in the induction of HIV-1 replication by HHV-8, we carried out a detailed analysis of the effect of HHV8 vFLIP on HIV-1 LTR activation. Consistent with an earlier report, we observed that HHV8 vFLIP strongly activates HIV-1 LTR in an NF-κB-dependent fashion . We furtFirst, our gel super-shift assay demonstrated that NF-κB complexes formed by vFLIP expression were primarily composed of c-Rel, p50 and p65 subunits. Second, siRNA-mediated downregulation of c-Rel and p65 led to near complete inhibition of K13-induced HIV-1 LTR activation whereas silencing of RelB expression was without significant effect. Third, K13-induced HIV-1 LTR activation was completely inhibited by super-repressor form of IκBα, which primarily blocks the classical NF-κB pathway. Finally, while K13 activates the alternate NF-κB pathway independent of IKK2, it failed to activate the HIV-1 LTR in IKK2-deficient MEFs.We have recently reported that vFLIP K13 can activate both the classical and alternate NF-κB pathways and, as such, we were interested in determining the relative contribution of these pathways to K13-induced HIV-1 LTR activation. Based on the following data, we believe that activation of HIV-1 LTR is mainly through the classical pathway. First, we have recently reported that K13-induced p65/50 DNA binding and NF-κB transcriptional activation is markedly reduced in IKK1-deficient MEFs [Second, recent studies suggest that IKK1 may be involved in transcriptional activation of classical NF-κB responsive genes through its ability to phosphorylate histones and p65 [Based on some early gene-knockout studies, IKK1 was believed to be not involved in cytokine-induced activation of the classical NF-κB pathway -51. In tent MEFs  Thus, th and p65 -54. ThusThe transcription of cellular and viral genes is regulated by structural and functional interactions among a number of transcriptional factors that act in concert. This is also known to be the case with HIV-1 LTR. Thus, while NF-κB plays a major role in the transcriptional activation of HIV-1, it requires synergistic interaction with a number of cellular and viral proteins for maximal stimulation of this activity . Tat hasThe human immunodeficiency virus has considerably diversified during its worldwide spread in the current pandemic and can be classified into several distinct subtypes . SubtypePlasmids containing pcDNA3-K13-Flag and pcDNA3-E8-Flag, pRSV/LacZ and 293T cells have been described previously . An exprElectrophoretic mobility shift assay was performed essentially as described previously , except 293 T cells were transfected in duplicate in a 24-well plate with the various test plasmids along with an HIV LTR/luciferase reporter construction (10 ng/well) and a pRSV/LacZ  reporter construct (75 ng/well) using calcium phosphate transfection protocol as described previously . Cells wsiRNA oligonucleotides with two thymidine residues (dTdT) at the 3'-end of the sequence were designed to p65 , c-Rel , RelB  and control , along with their corresponding antisense oligonucleotides. The RNA oligonucleotides were synthesized at RNA Oligonucleotide Synthesis Core facility, UT Southwestern Medical center. siRNA oligonucleotides (80 nM) were transfected using calcium phosphate as described previously [Western blot analysis was performed essentially as described previously . PrimaryDED, death effector domain; EMSA, electrophoretic mobility shift assay; FLICE, Fas-associated death domain-like IL-1 beta-converting enzyme; FLIP, FLICE inhibitory protein; HHV8, Human herpes virus 8; HIV-1, human immunodeficiency virus 1; KS, Kaposi's sarcoma; NEMO, NF-κB essential modulator; NIK, NF-κB-inducing kinase; NF-κB, Nuclear factor kappa B; MEF, murine embryonic fibroblast; PEL, primary effusion lymphoma, TNFR, Tumor necrosis factor receptor; IκB, inhibitor of NF-κB, IKK, IκB kinase; vFLIP, viral FLICE inhibitory protein; LTR, long terminal repeat.The author(s) declare that they have no competing interests.QS and HM carried out most of the experiments described in this manuscript. PMC conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Absence of knowledge of pregnancy-related pelvic girdle pain (PPGP) has prompted the start of a large cohort study in the Netherlands. The objective of this study was to investigate the prevalence and incidence of PPGP, to identify risk factors involved in the onset and to determine the prognosis of pregnancy-related pelvic girdle pain.7,526 pregnant women of the southeast of the Netherlands participated in a prospective cohort study. During a 2-year period, they were recruited by midwives and gynecologists at 14 weeks of pregnancy. Participants completed a questionnaire at baseline, at 30 weeks of pregnancy, at 2 weeks after delivery, at 6 months after delivery and at 1 year after delivery. The study uses extensive questionnaires with questions ranging from physical complaints, limitations in activities, restriction in participation, work situation, demographics, lifestyle, pregnancy-related factors and psychosocial factors.This large-scale prospective cohort study will provide reliable insights in incidence, prevalence and factors related to etiology and prognosis of pregnancy-related pelvic girdle pain. In the Netherlands, little information is available about prevalence, incidence, etiology and prognosis of pregnancy-related pelvic girdle pain (PPGP). It is hypothesized that during pregnancy many women (about 80%) experience some degree of pain in the pelvic region and/or the low back and that in some of these patients pain becomes chronic or recurrent. Often, symptoms impact on activities of daily life, hobbies, participation in society, planning of next pregnancies and sometimes lead to a chronic disabling condition with considerable work absenteeism in the future .Treatment and indirect costs of these chronic or recurrent patients constitute a considerable burden on health care services, health care insurers and other parties. The total health care expenditures incurred by patients with back pain in the United States in 1998 were approximately $91 billion, accounting for about 1 % of the Gross National Product . Van TulIn January 2000, the Maastricht PPGP cohort study started. It was established (I) to examine the prevalence and incidence of pregnancy-related pelvic girdle pain (PPGP) during and after pregnancy, (II) to identify risk factors involved in the onset of PPGP and to identify which factors can play an important role in the early detection of PPGP and finally (III) to determine the prognosis of PPGP and to identify prognostic factors. Furthermore, a clinical trial is embedded in this cohort, aimed at studying the effectiveness of a tailor-made treatment program in PPGP after delivery .In an observational prospective cohort study, etiology and prognosis of PPGP will be studied in 7526 pregnant women. The source population for the study comprises of pregnant women from the southeastern area of the Netherlands.Both midwives and gynecologists recruit women for the study when pregnant between 10 and 14 weeks. Women are considered eligible if they meet the following inclusion criteria: women are well versed in the Dutch language and at least 18 years. At inclusion, women receive a leaflet containing information about the research project, an informed consent form and a baseline questionnaire. After providing informed consent and filling out the first questionnaire, the women receive a second questionnaire at 30 weeks of pregnancy, a third one at 2 weeks after delivery and a fourth and fifth at 6 months and 12 months postpartum, respectively.In this study, extensive questionnaires are used with questions ranging from physical complaints (past and present), limitations in activities, restriction in participation, work situation, demographics, lifestyle, pregnancy-related factors and psychosocial factors. With the help of these questionnaires the prevalence and incidence of PPGP in the Netherlands will be described. In addition, possible risk factors and prognostic factors of PPGP will be examined.th option to the questionnaire, namely "not applicable". We also changed the phrase "because of my back " into "because of my back and/or pelvic pain" in questionnaires. Fear of movement is measured by means of the Dutch translation of the Tampa Scale for Kinesiophobia (TSK) . They concluded that there was no similarity in the selection of patients with PPGP between the authors. Most of these classification-strategies of PPGP are based on expert-opinions. Therefore, a possible reason for the lack of similarity in the selection of patients can be that they all select different small parts of the same large patient-group.Because of the relatively unknown etiology of pregnancy-related pelvic girdle pain and the lack of an all-embracing definition, we will use an extensive description of PPGP. We expect that during pregnancy almost all women experience some form of pain in the lower back, the buttocks, the symphyses, the groins and/or radiation into the legs. This pain is probably caused by hormonal and physiological changes which are considered normal during pregnancy. However, some women experience pain in a very early stage of pregnancy while others only experience pain in the final stages of pregnancy. In addition, some women are more limited in their activities (due to pain) than others. This suggests that other factors might influence the hormonal or physiological changes during pregnancy . Most woIn this study, pain during or after pregnancy is measured by using patients' self-reports. Women with pain can be identified by the question whether they experienced pain in the lower back, the buttocks, the symphyses, the groins or radiation into the legs during or after this pregnancy. To study etiology of PPGP, women who gave a positive answer to this question during this pregnancy were selected.For the prognosis of PPGP it is important that women have pain that started during pregnancy and persisted after delivery. At several moments during and after pregnancy, experienced pain in the lower back, the buttocks, symphyses, groins or radiation into the legs was measured. The answers to this question were assimilated into a flowchart Fig. .Based on their self-reports, the women are stratified into a case group without a history of LBP/PPGP or a case group with a history of LBP/PPGP. Some women (N = 246) were not classified into groups for the following reasons; specific disorders of the spine, rheumatism, neurological disorders and cancer. The first group consists of women without a history of LBP/PPGP, but they experience pain during pregnancy and this pain does not disappear until (at least) 2 weeks after delivery. The second group experiences pain during and after pregnancy, but they also have a history of LBP/PPGP. Both groups of cases will be analysed to study the prognosis of PPGP.Women who experience recurrent pain episodes during pregnancy, that resolve within 2 weeks after delivery, will not be considered cases in the analyses for the prognosis of PPGP. They form a miscellaneous group. However, this miscellaneous group and women who experience no pain after delivery were not excluded from follow-up.Participation rates and descriptions of baseline characteristics of the Maastricht PPGP cohort study will be presented. We calculated prevalence rates for PPGP by dividing the numbers of prevalent cases at several moments during pregnancy by the total number of subjects.Recruitment of women into the study began in November 2000 and ended in November 2002. Approximately 10,850 women were asked to participate in the study by midwives and gynecologist in the southeast of the Netherlands. The locations of the participating midwives and gynecologists are shown in Fig. In Table To examine whether the response in our study affected the determinant distributions (e.g. did primarily women in their first pregnancy respond?), a comparison of response rates was carried out. We performed a pilot-study in which data was recorded from April 2001 until November 2001 of every pregnant woman who attended one of the cooperating practices. Information about parity, PPGP during pregnancy, delivery and PPGP after delivery (until 6 weeks) was collected of 283 women (170 participants and 113 non-participants). Results of this pilot-study showed that primipara compared to multiparous women were more willing to participate in the cohort study.Furthermore, data from the responders in the cohort was compared to available data on pregnant Women in The Netherlands from the "Centraal Bureau voor Statistiek". The results of this comparison are shown in table Data are presented in 3 separate years to show possible fluctuations. It is noticeable that the mean age of pregnant women is increasing during the 3-year period in the study population and in the total Dutch population. In the study population, pregnant women are slightly older and heavier compared to the total Dutch population. Approximately 34% of all Dutch women (age 18–45 years), compared to 30% in the study population, are smoking , 30 weeks of pregnancy and 2 weeks after delivery (information about 34–40 weeks of pregnancy) were used. Almost every woman develops pain in the lower back, the buttocks, the symphyses, the groins or radiation into the legs at some time in their pregnancy. Of the 7527 women, 84% reported pain in any or all of these areas during pregnancy. Women with a history of LBP/PPGP are more likely to develop PPGP during pregnancy then women without a history of LBP/PPGP  has advantages over other epidemiological designs, it poses also a burden on the project in terms of logistics and recruitment. First of all, we are totally dependent on the cooperation and recruiting power of midwives and gynecologists. The number of pregnant women is about evenly divided between the two professions. Although professional workload for midwives in the Netherlands is extremely high, 62 % of the midwife practices took part in this study, recruiting about 90% of the patients. Whereas 58% of the hospitals in the recruitment area participate, they only recruit about 10% of the women. Coordination of the recruitment and frequent staff changes in the hospitals themselves seems to be a key problem.In etiological research there should be sufficient contrast in exposure. In this study, the study population at baseline is heterogeneous with respect to demographic variables, lifestyle characteristics and work-related factors. However, due to logistic constraints we have restricted the recruiting area to the southeast of the Netherlands, posing questions about the representation of our sample for the whole Dutch population. For instance the number of immigrants is significantly less in the southeast of the Netherlands. However, for future genetically oriented studies, this could be a major advantage. We have shown previously that our sample is slightly different from the national population of pregnant women with regards to several determinants .At baseline, 7526 pregnant women participated in our study. This is a response rate of 73.4%. Non-response and loss to follow-up might introduce selection bias in prospective studies. Loss to follow-up, especially in time series designs with repeated measurements, pose threats to representation of the sample. Especially when losses-to-follow-up are connected with negative pregnancy outcomes , co-morbidity of the mother or factors predicting for PPGP. To evaluate whether differential loss to follow-up occurs, we will compare the profile of those lost to follow-up with other participants.In this study we were able to evaluate if there are significant differences between participants and non-participants. This evaluations shows that primiparous women are more willing to participate in the study compared to multiparous women. We expect that multiparity plays an important role in the etiology and prognosis of PPGP. Therefore, an underestimation of the prevalence of PPGP reported in this study cannot be ruled out. However, the collected sample should be able to provide reliable preliminary insights in incidence and prevalence of PPGP and factors related to etiology and prognosis of PPGP in the Netherlands.PPGP is a complex syndrome and for a greater understanding of pregnancy-related pelvic girdle pain, future studies should further disentangle the multifactorial etiology and prognosis of PPGP. Future research within the framework of the Maastricht PPGP cohort study will focus on disentanglement of the complex syndrome.The author(s) declare that they have no competing interests.All authors participated in the design of the study. JMB drafted the manuscript with input from the other authors. All authors read, revised and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Retroviridae family is strong evidence that a dimerized genome is of critical importance to the viral life cycle. An obvious hypothesis is that retroviruses have evolved to preferentially package two copies of genomic RNA, and that dimerization ensures the proper packaging specificity for such a genome. However, this implies that dimerization must be a prerequisite for genome encapsidation, a notion that has been debated for many years. In this article, we review retroviral RNA dimerization and packaging, highlighting the research that has attempted to dissect the intricate relationship between these two processes in the context of HIV-1, and discuss the therapeutic potential of these putative antiretroviral targets.During virus assembly, all retroviruses specifically encapsidate two copies of full-length viral genomic RNA in the form of a non-covalently linked RNA dimer. The absolute conservation of this unique genome structure within the  However, as with many topics in retrovirology, interest in this area was heightened with the realization that the causative agent of AIDS was a retrovirus. Since then, RNA and protein sequences involved in genome dimerization have been identified for a number of retroviruses, and the dimeric nature of the retroviral genome is known to be important for various critical events in the viral life cycle. These include reverse transcription and recombination, as well as genome encapsidation. To date, a number of informative reviews have been published on retroviral RNA dimerization -3, genomd models . Anotheroviruses . In thisThe first evidence for the existence of a dimerized RNA genome came in 1967 when it was shown that viral RNA from each of Rous sarcoma virus (RSV), avian myeloblastosis virus (AMV), murine leukemia virus (MLV), and mouse mammary tumor virus (MTV) displayed sedimentation constants between 64S and 74S in sucrose gradients . Since tin vivo and in vitro approaches have been used to study retroviral RNA dimerization. The in vivo approach is that whereby RNA is isolated from virions produced in tissue culture and then analyzed by native Northern blotting [in vitro, and then studying the ability of these fragments to form dimers. The HIV-1 DLS was originally identified when it was shown that an in vitro-transcribed fragment of HIV-1 RNA could form two major bands on a native gel after incubation at 37°C for 15 min [In vivo evidence for a role of the NC protein in the dimerization process was already available [in vitro dimerization assays [Both blotting . The othr 15 min . The lowvailable , and thin assays .in vitro. The palindromic region was termed the dimerization initiation site (DIS) and it was proposed that this structural element could be exploited for targeted antiviral therapy by antisense oligonucleotides [in vitro dimerization of a 224–402 nt RNA fragment was completely blocked by an antisense oligonucleotide that targeted the palindrome [It was subsequently reported that an RNA fragment representing nt 1–311 of HIV-1 RNA  could not only form dimers, but that RNAs containing these first 311 nt could dimerize 10 times faster than RNA sequences at positions 311–415 that were previously shown to be sufficient for HIV-1 RNA dimerization . Based oleotides . These fleotides , and it lindrome . This lelindrome  or "kisslindrome  of HIV-1lindrome ,31 whichlindrome ,33. Similindrome -41.in vitro evidence supporting the above model of dimer maturation, it was not yet known where or when the RNA dimer was actually formed in vivo. However, native Northern blotting analysis of RNA from two Moloney murine leukemia virus (MuLV) protease-negative (PR-) mutants displayed dimers that migrated more slowly, and showed lower melting temperatures, than that of wild-type [Despite ample ild-type . It was ild-type . On the in vitro analysis showed that NC could convert the less thermostable dimers to a more stable conformation [in vivo, and, in agreement with earlier proposals [in vivo analysis of a panel of HIV-1 NC mutants showed that Cys-Ser substitution of amino acid residues within the second zinc finger decreased genomic RNA dimerization to the same extent as disruption of the DIS [in vivo situation.Evidence for the role of NC in this dimer maturation process came when ormation . Similarormation . Taken troposals , strongl the DIS . This fiWhy a class of viruses would evolve to have such a unique genomic structure is not entirely clear, but it is speculated that the availability of two copies of the genome would be advantageous for recombination during the complex reverse transcription process, that is key to the retroviral life cycle . Indeed,gag coding region  and the rt codon ; howeverrt codon . It was rt codon ,64-68, ain vitro ,69-72. Tin vitro   [cis elements required for packaging was immediately interpreted to mean that retroviral RNA dimerization, activated by either NC or Gag precursors, should direct genomic RNA into the virion, implying that dimerization might be a prerequisite for packaging. Since HIV-1, MuLV, and RSV all contain elements involved in dimerization that were also required for packaging [Early reports on 311–415) ,88, and 311–415) -53. Thisackaging ,89,90, iackaging -27. The in vivo studies analyzing the structure of virion-associated RNA from rapid-harvest avian retroviruses showed that viral RNA appeared to be a mixture of monomers and dimers [- mutants of HIV-1 showed that substantial amounts of monomeric RNA could be detected [- dimers were shown to be less stable than wild-type dimers, it was assumed that dimers were preferentially packaged in PR- particles, but that some fragile dimeric structures had dissociated during RNA preparation. Based on these in vivo results with both MuLV and HIV-1, it was concluded that dimerization is a prerequisite for packaging and should be considered to be a general feature of retrovirus assembly.Early d dimers -93. Simid dimers ,95 and Nd dimers ,94,96,97d dimers . Based odetected . Since PFurther insights into this topic can be obtained by examination of results from a number of studies aimed at understanding the role of the DIS in HIV-1 replication. One such study, in which DIS loop palindrome sequences were mutated, found that mutation of the palindrome to shorter or longer versions of GC stretches did not have major effects on viral RNA dimerization; however, partial RNA packaging defects were observed that also corresponded to diminutions in viral replication . Based oin vivo HIV-1 RNA dimerization process. Yet, these mutant genomes could still be packaged, suggesting that HIV-1 RNAs need not be dimers for this to happen. Thus, the authors concluded that dimerization is not a prerequisite for packaging but rather serves an independent function in the retroviral life cycle. In the above-cited article, the effects of SL3 mutations on dimerization were not studied, but our group later showed that viruses containing even minor substitutions in or around SL3 could have significant effects on both dimerization and packaging [Although several groups had attempted to delineate the relationship between dimerization and packaging, the fact remains that the RNA signals that are important for both of these activities overlap in most retroviral genomes; this makes it difficult to interpret the results of mutagenesis studies. In an attempt to generate viruses that would be expected to display selective defects in dimerization or packaging, one group designed a panel of constructs containing mutations in SL1, SL3, or both . Resultsackaging ,101.in vivo studies described above commonly observed that mutations in 5' RNA sequences affected both dimerization and packaging, presumably due to the close proximity of the RNA dimerization and packaging signals.In summary, the In an attempt to separate the dimerization and packaging functions, and to characterize the DIS-DLS region without altering packaging activity, one group generated mutant constructs carrying a duplication of approximately 1000 nt from the HIV-1 5' region (termed E/DLS) including the encapsidation signal and the DIS-DLS . They foIn a follow-up study, the same group created mutant HIV-1 particles that contained only monomeric RNAs, and concluded that these mutants demonstrated the complete separation of encapsidation from physical dimerization of retroviral RNA . HoweverHowever, the above results do raise the issue of packaging specificity in mutant viruses. We and others have shown that, in COS cells, HIV-1 can incorporate significant amounts of spliced viral RNA when proper packaging of full-length viral genomic RNA is reduced ,104. DurAnother group reported similar phenotypes in the context of an HIV-1 mutant that was designed to have altered Gag/Gag-Pol ratios . AnalysiWe have also been studying the HIV-1 5' UTR and its putative interactions with Gag, and how these interactions affect dimerization and packaging activities. The DIS is known to be important for viral replication ,106-109,In a follow-up study, we created two other DIS deletions and combined them with various combinations of the previously identified compensatory point mutations. We showed that these mutant viruses, ΔLoop (lacking the loop region of SL1) and ΔDIS (lacking the complete SL1) displayed defects in replication, RNA dimerization, and packaging. Once more, all of these but dimerization were largely corrected by the compensatory point mutations in Gag . Even a The mechanism(s) whereby these compensatory point mutations functioned to restore replication had eluded us for some time. Recently, however, we employed an RNase protection assay to discriminate between genomic and spliced viral RNA packaged into virus particles. Our results showed that all of our 5' UTR mutant viruses aberrantly packaged increased levels of spliced viral RNA compared to wild-type virions. More importantly, however, the effect of one of our compensatory point mutations  was to exclude spliced viral RNA from being packaged into mutant virions . Surprisin vitro and in vivo protocols. New approaches to study dimerization and packaging within the cell will hopefully allow new hypotheses to be tested.Previous work had suggested that the packaging of spliced viral RNA is a mechanism used by packaging mutants to fill the space that would normally be occupied by genomic RNA . Were thThe packaging of spliced viral RNA and/or the exclusion of such RNA species raises the question of whether the viral RNA sequence, or possibly the RNA structure, is important in proper assembly and/or structural integrity of the virus particle itself. Evidence in support of this possibility comes from studies on the binding of NC, in the context of full-length Gag, to viral genomic RNA. This might concentrate Gag proteins onto one or more RNA molecules, thereby facilitating Gag-Gag multimerization in a template-driven manner. Hence, viral genomic RNA would be a structural element, or scaffold, on which the virion can assemble . Other rAs stated, the link between dimerization and packaging is a subject of ongoing debate ,109,110,It is clear that virus replication capacity is significantly affected whenever dimerization and/or packaging are compromised, suggesting that these activities can be exploited as anti-HIV drug targets. Indeed, the DIS was first proposed to be a potential therapeutic target at least 10 years ago, and antisense molecules were directed at this region of viral RNA ,123, wittat and rev [RNA interference (RNAi) is a novel mechanism that regulates gene expression in which small interfering RNAs direct the targeted degradation of RNA in a sequence-specific manner (reviewed in Lee and Rossi ). Althou and rev . DNA vecThe DIS might also be a good candidate for sequence-specific targeting of HIV by RNAi therapy since it is highly conserved among naturally occurring virus isolates, and, due to its position upstream of the major splice donor, is contained in all HIV-1 RNA transcripts, both spliced and unspliced. Effective DIS-directed degradation of HIV RNA should confer the same viral phenotype as observed with our ΔDIS mutant, which never showed signs of virus replication in either permissive T cell lines or blood mononuclear cells . One contat and nef genes, and the mutations that were generated blocked the effects of the RNAi without conferring any major detriment to virus replication. In contrast, RNAi may be more useful if targeted to more critical RNA elements within the genome, such as the DIS or the Ψ region, since any escape mutations that occur might result in viruses with severely impaired replication ability.Recently, the practicality of RNAi-based therapies against HIV-1 was called into question when it was shown that HIV-1 was able to escape the antiviral pressure of RNAi by generating substitutions or even deletions within RNAi target sequences ,128. ThiAll of these DIS-directed strategies rely on specifically targeting the viral RNA itself, which might not be practical given our inadequate knowledge of the overall structure of the HIV-1 5' region. The fact that RNA sequences such as SL1 and SL3 are known to form relevant RNA-protein interactions raises the possibility that the protein component of these interactions might also provide potential targets for anti-HIV therapy. Such approaches are currently being explored in research aimed at designing inhibitors of the TAR-Tat RNA-protein interaction . Similarin vitro and in vivo approaches toward more biologically relevant methods. One group has taken chemical modification protocols commonly used for in vitro RNA analysis, and adapted them for use in virus-producing cells. Hence, structural analysis of viral RNA, that would previously be carried out only in vitro on short fragments of artificially transcribed RNA, can now be performed on in vivo-generated HIV-1 genomic RNA . This method might also have application in regard to in vivo foot-printing that could allow the study of RNA-protein interactions in the context of virus-producing cells.Current HIV combination therapies have demonstrated that a multi-targeted approach against the virus results in the greatest degree of suppression of virus replication. Therefore, the identification of novel targets for anti-HIV therapy could significantly improve HIV treatment strategies. HIV-1 RNA dimerization is clearly a critical event that could be exploited as a target once its complete mechanism is elucidated. It is pleasing to see that a number of laboratories that have actively researched RNA dimerization and packaging are now moving beyond conventional ion, and ). This min vitro dimerization studies conducted on HIV-1, HIV-2, and SIV RNA [in vivo evidence directly supporting such a model in the case of retroviruses, the results of previous mutagenesis studies from several laboratories correlate with those that would be predicted from the riboswitch model, both concerning RNA packaging and RNA dimerization status [The structure of the viral RNA that exists in the cell has long been a topic of interest, and recent data suggest that different RNA sequences might be involved in higher order intrastrand structures that favor the dimerization of the two RNA molecules. Such a model has been proposed , and is  SIV RNA ,131-133. SIV RNA . Althougn status . In regaOthers have developed a fluorescence resonance energy transfer (FRET)-based system to allow visualization of RNA-Gag interactions within cells . Such a system might provide insight into the timing of genome selection and packaging. It will also be interesting to determine whether this system can be adapted to pinpoint how retroviral RNA dimerization takes place within cells, and whether dimerization indeed occurs before RNA is selected for packaging.None declared.RSR gathered the information discussed in this review, and was primary author of the manuscript. CL and MAW carefully read the manuscript and offered insightful suggestions for its revision. All authors read and approved the final version.


MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5′ to 3′ exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E) mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA.The single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB) and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut  By studying the repair of double-strand DNA breaks in vivo, evidence of a new role for the DNA-binding protein RPA has been discovered RFA1, RFA2, and RFA3 genes, respectively (Saccharomyces cerevisiae (rfa1-t11) are proficient for DNA replication, but their ability to perform mating-type (MAT) gene switching, single-strand annealing, and meiotic recombination is severely impaired (Repair of double-strand breaks (DSBs) by homologous recombination involves the search for homology to locate an intact donor sequence. The search is successful when the broken DNA molecule basepairs with the homologous template, termed synapsis, and forms strand invasion intermediates of recombination. In budding yeast and other higher eukaryotes, this process requires both the Rad51 strand exchange protein and the single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) . RPA wasectively . Deletioectively . The biorevisiae  and in oimpaired .Because RPA is essential for DNA replication, a great deal about its role in recombination has been learned from in vitro studies of Rad51-mediated strand exchange . These srfa1-t11 (Rfa1-K45E) mutation on strand exchange with Rad51. Although rfa1-t11 protein bound to ssDNA identically to wild-type and could stimulate strand exchange if Rad51 was preloaded onto ssDNA, the mutant protein exhibited delayed and less-efficient strand exchange if it was first bound to ssDNA, especially at higher concentrations, even in the presence of Rad52. This defect was explained by the slow displacement of rfa1-t11 from ssDNA by Rad51.Using the same in vitro system, S. cerevisiae as well as in higher eukaryotes to investigate the association of RPA to DSBs following γ-irradiation and during meiosis. These studies have suggested that RPA and Rad51 form subnuclear foci at sites of ssDNA after irradiation and during meiotic recombination  assays  to analychniques . The comMAT locus can be repaired by gene conversion with one of the two donor sequences, HML or HMR, or the DSB can be left unrepaired in most cells by deleting these donors .In wild-type yeast cells, a DSB created at the e donors . In ordeny hours . In thesMAT had been rendered single-stranded  at the time of cell breakage and washing with 4.7 M NaCl, although it greatly reduced background binding (data not shown). However, in formaldehyde cross-linked samples, there was no such adventitious binding of RPA to ssDNA regions, apparently because the formaldehyde-treated proteins are no longer able to bind. This was shown directly by adding 4 ng of purified single-stranded β-lactamase (AMP) gene DNA from plasmid pBR322 at the time of cell breakage. Whereas there was substantial ChIP of the AMP sequences in noncross-linked samples, there was almost no signal in cells that had first been treated with formaldehyde . Rad51 binding increased for about 1 h and then remained bound for several hours. As reported previously  mutation in the largest subunit of RPA. This mutation has little effect on DNA replication per se, but severely impairs both gene conversion (MAT switching) and single-strand annealing pathways of homologous recombination  can only amplify a strand invasion product with a primer specific for MAT-distal sequences (pB) if the 3′ end of the strand-invading DNA is extended by DNA polymerase at least 35 nucleotides  is able to allow Rad51 to bind to ssDNA, but is incapable of forming normal levels of strand invasion and primer extension products  and that the inhibition of Rad51-mediated strand exchange was impaired primarily when RPA was present in excess   plasmid  . ySL83 cectively , 2001. yectively , and ySLm OAy470 , which h Sandell  to obtai plasmid .When cells were harvested for ChIP at intervals after induction of HO (see below), a second set of DNA samples were collected for Southern blot analysis as described before . The str6 and 1 × 107 cells/ml in YP–lactate medium and HO endonuclease was induced by addition of 2% galactose. Strains undergoing DSB-induced gene conversion were treated with 2% glucose after 1 h to repress further cutting by HO. Proteins were cross-linked by addition of 1%  formaldehyde to 45 ml of culture for 10 min, followed by quenching with 125 mM glycine  for 5 min. Cells were lysed with glass beads, and the extracts were sonicated to shear the DNA to an average size of 0.5 kb. Extracts were then divided into IP and input samples (12:1 ratio). IP samples were split. Half of the extract was incubated with polyclonal anti-Rfa1 antibody (kindly provided by S. Brill) for 1 h at 4°C and bound to protein G–agarose beads for 1 h at 4°C. In the ChIP experiments described in ChIP was carried out as described previously with minor modifications . Cells wAMP) gene DNA from plasmid pBR322 was added at the time of cell breakage. IP and input samples were further subject to PCR to test the presence of the AMP sequences.In the control experiments described in MAT-specific primers were 5′-TCCCCATCGTCTTGCTCT-3′ (P1) and 5′-GCATGGGCAGTTTACCTTTAC-3′ (P2), which amplifies a PCR product of 293 bp. The HML-specific primers were 5′-TCCCCATCGTCTTGCTCT-3′ (P1) and 5′-CCCAAGGCTTAGTATACACATCC-3′ (P3), which amplifies a PCR product of 280 bp. Primers used for the amplification of the sites proximal to the DSB  on chromosome V in a multiplex experiment, by using ARG5,6 and MAT or HML primers in the same PCRs. This was accomplished by dividing each MAT or HML IP signal by the corresponding ARG5,6 IP signal to correct for differing amounts of chromatin collected at each timepoint. Then MAT or HML IP signals at later timepoints were normalized and graphed to the 0-h IP signal to measure the relative increase. For Rad51 ChIP analysis, quantification and graphing were carried out as described before  ID accession numbers for the entities discussed in this paper are ARG5,6 (S0000871), HML (L0000791), HMR (L0000792), MAT (L0001031), Rad51 (S0000897), Rad52 (S0004494), Rad55 (S0002483), Rad57 (S0002411), RFA1 (S0000065), RFA2 (S0005256), and RFA3 (S0003709).The


Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of 6). The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblast growth factor 4, HIV Tat or consisting of the (KFF)3K sequence were not required for efficient protein transduction and adversely affected enzyme solubility. Transduction of the HNC protein required 10 to 15 min for half-maximum uptake, was greatly decreased at 4°C and was inhibited by serum. Efficient recombination was observed in all cell types tested , and did not require localization of the enzyme to the nucleus.In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC) or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCHThe effects of different sequences on the delivery and/or activity of Cre in cultured cells could not be predicted in advance. Consequently, the process of developing more active cell-permeant recombinases was largely empirical. The HNC protein, with an excellent combination of activity, solubility and yield, will enhance the use of cell-permeant Cre proteins to regulate gene structure and function in living cells. The Cre recombinase from bacteriophage P1 has been widely used to induce DNA sequence-specific recombination in mammalian cells -4. HowevTo address these problems, we  and otheIn our previous report, recombinant fusion proteins bearing the 12 amino acid membrane translocation sequence (MTS) from fibroblast growth factor 4 (FGF-4) were used to deliver enzymatically active Cre proteins directly into mammalian cells. Of the four recombinant proteins tested, an enzyme containing an N-terminal 6xHis affinity tag, a nuclear localization sequence (NLS) from SV40 large T antigen, and the FGF-4 MTS (HNCM), displayed the best combination of yield, solubility, nuclear localization and enzymatic activity within cells. Recombination was observed in greater than 70% of cells treated with 10 μM HNCM for 2 hours. Widespread recombination was also observed in mice following intraperitoneal administration of HNCM, indicating that a wide variety of terminally differentiated cell types can internalize cell-permeant Cre and are competent to undergo site-specific recombination.3K sequence that was previously used to deliver peptide nucleic acid (PNA) conjugates into cells [In the present study, eleven recombinant Cre proteins were prepared in order to evaluate sequences affecting the uptake and/or activity of the enzyme in cultured cells and if possible to develop more active recombinases. Several constructs were designed to compare the activities of different PTDs, including the FGF-4 MTS , sequencto cells . Only thto cells  was alsoto cells ,10, wherto cells . We repoto cells  and the 6C have amino-terminal hexahistidine tags consisting of MGSSHHHHHHSSLVPRGSH and MHHHHHH, respectively, while CH6 is similar to H6C except the His tag is on the C-terminus. His-NLS-Cre (HNC) is similar to HC except a nuclear localization sequence (PKKKRKV) from SV40 large T antigen [7N'C is similar to HNC except the His tag contains 11 additional amino acids (MASMTGGQQMG) from the pET28a(+) polylinker and the arginine of the NLS sequence was converted to a lysine. This change, which resulted from an altered PCR primer, is unlikely to affect nuclear localization activity. HNCM, described previously [6. These consisted of the FGF-4 MTS (MCH6), an SV40 large T antigen nuclear localization sequence (NCH6), the HIV Tat sequence (TCH6), and a (KFF)3K sequence (KCH6).Eleven recombinant Cre proteins were prepared in order to evaluate sequences affecting the uptake and/or activity of cell-permeant enzymes Figure . Native  antigen  is positeviously ,19,24, wE. coli and, except for native Cre, were purified by Ni2+ affinity chromatography under non-denaturing conditions [E. coli culture  and 8% glycerol. The pH of the buffer (ranging from 7.5–8.5) had no obvious effect on protein solubility. The KCH6 protein was insoluble over a range of pH values and salt concentrations (up to 0.8 M), and the protein was not evaluated further. The remaining proteins could be prepared at final concentrations above 1 mg/ml except HNCM, which precipitated out of solution at protein concentrations above 500 μg/ml. The specific activities of the tagged fusion proteins were similar, ranging from 4.3–8.5 × 104 U/mg protein, corresponding to 47–92% of the activity of the native enzyme  plasmids in e Figure . All of 7N'C, HNCM, H6C, CH6, NCH6, and TCH6 for 2 hrs at concentrations ranging from 0 to 8 μM. The cells were then washed extensively with PBS, were cultured for 24 hrs to provide time for EGFP expression, and the percentage of EGFP-expressing cells was determined by flow cytometry. Recombination was also monitored by Southern blot hybridization  or carboxy-terminal (CH6) ends of the enzyme. HC, which contains the 6xHis tag from pET28a(+), and H6C and CH6, which contain simple 6xHis sequences, induced similar levels of recombination, ranging between 45 and 60% of cells. Activity was further increased by the addition of an SV40 large T antigen nuclear localization signal . At low concentrations, TCH6 was the most active protein tested, but higher levels of the protein were toxic to cells, presumably because of the concentration of NaCl required to maintain protein solubility during preparation of the enzyme. Finally, presence of the FGF-4 MTS sequence proved inhibitory for recombination in cultured cells. HNC, which lacks the MTS sequence, was approximately 8 times more active than HNCM as determined by the concentration of enzyme required to induce recombination in 50% of the cells. Thus, while the FGF-4 MTS has been used to transduce a variety of proteins and peptides into mammalian cells, the sequence suppressed the activity of cell-permeant Cre. This confirms the results of a earlier study, reported while this work was in progress [As shown in Figure progress .7N'C and HC) the FGF-4 MTS were therefore tested for their ability to enter cells at 4°C . These results indicate that the inhibition of protein transduction by serum is not limited to cargoes containing the FGF-4 MTS.Serum has been reported to inhibit the transduction of cargoes containing the FGF-4 MTS; whereas, transduction of proteins containing the Tat and Antennapedia PTDs appears to be unaffected by serum. The effects of serum on protein transduction were assessed by treating TexloxP.EG cells with either HC (5 μM) or HNC (2 μM) for 1 hour in the presence of varying concentrations of either fetal bovine serum Figure , FBS or 2 culture dishes and were treated with HNC (2 μM) for 1 hour  required to cover the culture dish.To assess the effects of cell density on protein transduction, TexloxP.EG cells were seeded at different concentrations in 2 cmr Figure . RecombiThe kinetics of cell-permeant Cre uptake in cultured cells was monitored by examining cells for recombination after exposure to cell-permeant Cre for different lengths of time Figure . Cells wThe uptake and localization of cell-permeant Cre in cultured cells was also monitored in living cells by using proteins labeled with the fluorescent dye, Alexa 488 Figures  and 6. TThe localization of Alexa 488-tagged HNC and HC proteins was monitored in living cells by fluorescence microscopy. The HNC protein was localized to the nuclei of Cos7 cells, but was predominately cytoplasmic in NIH3T3 cells Fig . Cre proSeveral mammalian cell types were analyzed for their ability to undergo Cre-mediated recombination. Clones of Cos7 and NIH3T3 cells containing a single pBABE.lox.stp.EGFP provirus were treated with HNC for two hour and analyzed by Southern blot hybridization Figure . ExtensiCell permeant Cre proteins have generated considerable interest as genetic tools to regulate gene structure and function in mammalian cells . In addiWe had hoped that the native or 6xHis-tagged enzymes would lack transduction activity so that the effects of additional sequences on cell entry, nuclear transport and recombination could be compared. However, we found that the native Cre protein has an intrinsic ability to enter cells, thus confirming observations by Will, et al. . The mecin vitro. Moreover, 6xHis sequences were active when positioned on either the amino- or carboxyl-terminal ends of the enzyme. L-histidine heptamers have been shown to enter cells, although much less efficiently than arginine homopolymers [The transduction activity of the native enzyme hinders quantitative studies of sequences incorporated into recombinant Cre proteins, since structural changes associated with each modification may have varying effects on the intrinsic ability of Cre to enter cells. Even so, the 6xHis sequence appeared to facilitate cell entry, since two different 6xHis sequences enhanced Cre-mediated recombination while having little effect on the activity of the enzyme polymers ,38. Posipolymers ,40, and polymers . The trapolymers .We  and othein vitro (data not shown). Recombination was markedly suppressed at higher cell densities, possibility because binding sites in or on cells sequester the enzyme, thus lowering the effective protein concentration. Alternatively, cell density may suppress protein transduction by reducing cell size and/or available surface area [HNC, which consisted of a 6xHis tag and T antigen NLS attached to the amino-terminus of the enzyme, was highly active despite the absence of a canonical protein transduction sequence. The 6xHis tag and NLS sequences separately contributed to the transduction of the enzyme, which was 5–8 times more active in cultured cells than the HisNLSCreMTS (HNCM) protein described in our earlier study -30. Howeace area . In eithace area , illustr3K sequences (TCH6 and KCH6) were poorly soluble when dialyzed against isotonic media, and the FGF-4 MTS interfered with the activity of the enzyme in cultured cells. Since Cre is probably not unique in this regard, investigators seeking to develop other cell-permeant proteins would be advised to test a variety of sequences positioned at different places on the protein. In the present case, the HNC protein possesses an outstanding combination of activity, solubility and yield that will enhance the use of cell-permeant Cre to regulate gene structure and function in living cells.The effects of different sequences on Cre uptake or activity were difficult to predict in advance, and consequently, the process of developing a more active cell-permeant recombinase was largely empirical. Proteins containing amino-terminal Tat and (KFF)E. coli from pET28a (+)-based plasmids (Novagen). A plasmid expressing native Cre (Cre) was constructed by using the Cre #1 and Cre-stop-HindIII primers to amplify Cre coding sequences from MBP-NLS-Cre-MTS [NcoI and HindIII sites of pET28a (+). HC, His6C, HNC, and HT7C were similarly constructed by PCR amplification, using primers Cre #2, Cre #3, Cre #4 and Cre #5, respectively, together with Cre-stop-HindIII primer. The amplified DNA fragments were cloned into NheI and HindIII sites of pET28a (+). CHis6, NCHis6, MCHis6, TATCHis6, and (KFF)3KCHis6 were constructed by using primers Cre #6, Cre #7, Cre #8, Cre #9 and Cre #10, respectively, together with the Cre-XhoI primer, and then cloned into the NcoI and XhoI sites of pET28b(+).Native Cre and Cre fusion proteins were expressed in Cre #1: AGAGAGCCATGGGCTCCAATTTACTGACCGTACACCAACre #2: GTACATGCTAGCTCCAATTTACTGACCGTACACCAACre#3: AGAGAGCCATGGGCCATCATCATCATCATCACAGCTCCAATTTACTGACCGTACACCAACre#4: GTACATGCTAGCCCAAGAAGAAGAGGAAGGTGCTCCAATTTACTGACCGTACACCAACre#5: GTACATGAATTCTCCAATTTACTGACCGTACACCAACre#6: AGAGAGCCATGGGCTCCAATTTACTGACCGTACACCAACre#7: AGAGAGCCATGGGCCCCAAGAAGAAGAGGAAGGTGTCCAATTTACTGACCGTACACCAACre#8: AGAGAGCCATGGGCGCAGCCGTTCTTCTCCCTGTTCTTCTTGCCGCACCCTCCAATTTACTGACCGTACACCAACre#9: AGAGAGCCATGGGCGGTCGCAAGAAACGTCGCCAACGTCGCCGTTCCAATTTACTGACCGTACACCAACre #10: AGAGAGCCATGGGCAAATTCTTTAAGTTCTTTAAGTTCTTTAAGTCCAATTTACTGACCGTACACCAACre-stop-HindIII: GATACGAAGCTTCTACTAATCGCCATCTTCCAGCAGGCGCCre – XhoI: AGAGAGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTE. coli strain BL21 (DE3) and purified by Ni2+ affinity chromatography as described previously [600 of 0.6–1.0, were induced with 0.4 mM IPTG, and after harvesting the cells were lysed in 40 ml 50 mM Tris (pH 8.0), 50 mM sodium phosphate and 300 mM NaCl. After affinity chromatography, recombinant proteins were dialyzed against DMEM or RPMI media, except TCH6 and KCH6, which precipitated under these conditions. TCH6 was dialyzed instead against PBS supplemented with 0.3 M NaCl  and 8% glycerol. The native recombinase was similarly expressed; however, lysates were applied to a 50 ml hydroxyapatite column and step-eluted with sodium phosphate buffers (pH 8.0) of increasing concentration . The peak fraction (200 μM) was dialyzed against PBS, was concentrated by centrifugal ultra filtration to about 20 mg/ml and was fractionated by Sephacryl S-100 HR gel filtration FPLC. Peak fractions identified by SDS PAGE, were concentrated and dialyzed against PRMI-1640 medium containing 1% streptomycin/penicillin and 2% glycerol.Polyhistidine-tagged Cre fusion proteins were expressed in eviously . BacteriIn vitro assays of Cre enzyme activity measured the release of a circular plasmid inserted into a λ phage  by transformation of E. coli [4 colonies  in a 30 minute reaction containing 200 ng DNA substrate in 50 mM Tris HCl, pH 7.5, 33 mM NaCl and 10 mM MgCl2 in a total volume of 15 μl. All Cre proteins were stable for at least 6 months at -80°C without significant loss of enzymatic activity. E. coli . One uniTex.loxp.EG, 3T3.loxp.EG and Cos7.loxp.EG cells were derived from Tex (a murine thymoma line derived from p53-deficient mice), NIH3T3 and Cos7 cells, respectively, following infection with the pBABE.lox.stp.EGFP retrovirus . Cells wFluorescent HNC, HC, and HNCM proteins were prepared by using the Alexa 488 protein labeling kit , according to the manufacturer's instructions. The proteins were dialyzed against PBS, were labeled at a concentration of 2 mg/ml and purified by gel filtration through a Sephadex G50 column. Cells were treated with the fluorescent proteins at a final concentration of 1 μM.The localization of fluorescent Cre proteins was monitored in living NIH3T3 and Cos7 cells by fluorescence microscopy. The cells were cultured to 50–80% confluence in a slide chamber (NUNC), washed with PBS and then incubated with fluorescent Cre proteins at a final concentration of 1 μM for 1.5 hours. The cells were washed three times with PBS, counterstained with 1 μg/ml 4',6-diamidino-2-phenylindole (DAPI) in PBS for 20 minutes, washed three times with PBS and mounted in anti-fade fluorescence mounting medium. The stained cells were photographed with an Olympus BX60 fluorescence microscope using green and blue filters.12FDG) according to instructions provided by the manufacturer  Recombination, which activates the expression of a floxed lacZ gene, was assessed by flow cytometry.Splenocytes from ROSA-26R mice were cultured for one day in RPMI 1640 medium containing 10% FBS and were treated with HNC recombinase in serum free media for 2 hours. The cells were washed, cultured for 24 hours, and stained with a fluorescent β-galactosidase substrate (ImaGene green™, CQL developed and characterized most of the recombinant Cre proteins described in this study. DJ provided the HNCM protein and assisted with in vitro assays for Cre activity. KDGA. investigated inhibition of Cre activity by serum. ER supervised the project and drafted the final manuscript. All authors approved the final manuscript.


Fibromuscular dysplasia (FMD) is a possible cause of stroke, especially in middle-aged women. However, only few reports are available on ultrasonographic detection and monitoring.Among the 15,000 patients who underwent color Doppler imaging (CDI) of the cervicocephalic arteries during the study period, all cases fulfilling ultrasound criteria of FMD were included into the case series. Criteria of FMD were: 1. Segmental string-of-beads pattern, 2. Localization in the distal extracranial part of internal carotid artery (ICA) or vertebral artery (VA), and 3. : Direct and/or indirect criteria of stenosis.CDI detected FMD in 39 vessels (37 ICA and 2 VA segments) of 21 patients. 16 patients had bilateral manifestation on ICA, one of those also on VA, bilaterally. CDI disclosed 4 symptomatic high-grade ICA stenoses, 3 of them underwent endovascular treatment. 5 patients with moderate symptomatic ICA stenoses got medical treatment. In 6 patients FMD was the most likely cause of headache and in one patient FMD was diagnosed as a cause of vertigo.CDI may be used for detection of cervicocephalic FMD. Due to the unfavourable localisation of FMD for CDI, the sensitivity of CDI is lower in comparison to angiography. However, high-grade FMD stenoses that require invasive treatment can be recognized on the basis of indirect hemodynamic criteria. Fibromuscular dysplasia (FMD) is a non-atheromatous, non-inflammatory arteriopathy of unknown etiology with segmental manifestation on medium-sized arteries in various regions of the body . ManifesAngiography reveals in most cases the typical string-of-beads pattern fig.  with altClinical manifestations of FMD on the ICA are transitoric ischemic attacks or cerebral infarctions  as well Among the 15,000 patients who attended the neurosonography department of our clinic during the study period, 21 cases were identified fulfilling ultrasound criteria of FMD , Toshiba Powervision 6000 or Toshiba Aplio , or ATL HDI 5000 .Using the criteria of table The degree of stenosis was low in 2 patients Fig. ,4,5. 5 pFMD is an uncommon angiopathy with an incidence on the ICA of about 0.6 – 1% . HoweverUltrasound criteria of FMD correspond to those of angiography Fig. . CDI revIn comparison to angiography, the sensitivity of CDI is low: The vascular lesion can only be visualized sonographically when it is located not too far cranially on the ICA . HoweverCDI allows diagnosis of FMD in numerous cases. Due to the unfavourable localisation of FMD for CDI, the sensitivity of CDI is low in comparison to angiography. However, high-grade FMD stenoses that require invasive treatment can be recognized on the basis of indirect hemodynamic criteria.None declared.CDI Color Doppler ImagingFMD Fibromuscular DysplasiaICA Internal Carotid ArteryVA Vertebral Artery


How can you combine professional-quality research with discovery-based undergraduate education? The UCLA Undergraduate Consortium for Functional Genomics provides the answer This effort combines professional-quality research with a strategy for research-based undergraduate education.The excitement of scientific research and discovery cannot be fully conveyed by didactic lectures alone. Several recent initiatives and proposals, therefore, have supported a more participatory, discovery-based instruction for undergraduate science education ,2. In fuDrosophila eye is an intricate neurocrystalline lattice of approximately 800 individual ommatidia arrayed in a very precise order  were identified, validating the effectiveness of our screen. In addition, our genomics approach has shown that a number of novel classes of genes are involved in eye development that have not been previously described  to help them determine the identity and function of their disrupted genes. The main component of the class, however, is the laboratory portion.Drosophila eye  recombination target (FRT) chromosome, generate mutant somatic clones, and record details of the adult eye phenotype with both light and scanning electron microscopic techniques. The students then upload their data into an online database (http://www.bruinfly.ucla.edu).In the laboratory, the students perform all the necessary work to manipulate the genotypes of their stocks to determine what effect homozygous mutation of their target genes has in the adult hila eye . To accoOur database contains pictures of the mutant eyes for all of the stocks examined, as well as other information pertinent to that stock, including the gene disrupted, the exact genomic location of the P-element insertion, and whether an excision of the P-element has been performed and its results. A sample Web page of the database is shown in In summary, discovery-based experiments in functional genomics are well suited for undergraduate education: they actively engage a large number of students in research without compromising their didactic training. The sense of ownership developed from this research amplifies the students' learning experience. For the research community, the online database and the large collection of newly generated FRT-lethal lines represent a valuable resource for future experiments in eye development. Furthermore, the stocks developed can be used to create mutant clones in an investigator's tissue of choice. This novel approach for performing research, for which functional genomics is very amenable, not only encourages many students in new ways of thinking but also generates professional-quality results and resources for the scientific community.Figure S1(1.2 MB JPG).Click here for additional data file.


Retrovirology has been publishing as an Open Access online journal for approximately six months. In this editorial, I review the reasons for and the advantages of Open Access publishing, update our progress to date, and summarize where we intend to go with this journal. That morning, I awoke to an eerie silence. The normally bustling traffic outside of our apartment in a busy suburb of Benghazi was not to be heard. In fact, there was to be little or no traffic for the next several days. History will recount that day as the first day of Mohmmar Qadaffi's bloodless coup d'etat, seizing power in Libya.Thirty-five years ago, on September 1his information during time of chaos, Qadaffi held sway over the minds and the sentiments of the populace. He was thus able to pre-empt the efforts of a larger competing group of more senior army officers.What I learned that September week in 1969 was that access to and distribution of information tipped the fate of a state. On that September day, there were in fact two groups of army officers vying to overthrow the then enfeebled Libyan King Idris. Qadaffi, at the time only twenty-seven years old, headed a small group of very junior officers. He, however, had a bold and swift strategy. His first targets were not the dams, the power stations, the police stations...; but the radio stations (in 1969 television had a very minor penetrance in Libya) and newspapers. By dint of controlling the airwaves and printing presses and by freely distributing What does Qadaffi's story have to do with Open Access publishing? Imagine how the outcome might have been different in 1969 if broadcast radio was not freely and openly accessible by the masses but was an expensive subscription service limited to a few. In that setting, and perhaps in all settings, information that reaches a little audience at a steep cost is information with little effect.Curiously, the force that drives a coup d'etat is the same that motivates scientists to publish . As scientists, we want everyone who is interested to read what we have done; and ideally we want that information to get to the widest audience in the fastest, most informative, and least expensive manner. Until now, ideal and reality were two entirely different matters. Traditional publishing in print journals reaches a limited audience, is slow, bulky, and expensive. By its very nature (i.e. pages of paper are heavy to mail and expensive to print), there is a real impetus on the part of print publishers to restrict the space available to authors. We are all too familiar with the results: abbreviated presentation of methods and text, the necessity to "inadvertently" not cite many colleagues' publications, and the ubiquitous use of microscopic figure panels that provoke blurred vision and headaches. I daresay that many after having read a short "letter" or a "brief definitive" report have been left scratching heads and wondering how the experiments were done, where did the vectors come from, and why many controls were definitely missing.Retrovirology aims to change such "reality". Retrovirology is published by BioMed Central, an independent publisher committed to ensuring immediate free access to peer-reviewed biomedical research. Retrovirology is Open Access which means that it is universally and freely available online to everyone. You as the author retain copyright, and your paper is guaranteed to be archived in at least one internationally recognised repository [Retrovirology is rapid. For example, a full research article was reviewed and published in less than 20 days [Retrovirology will also give you all the space and all the colored prints (at no extra charge) to present your findings informatively and attractively. If we don't publish your paper, we will tell you honestly what is scientifically deficient about your work. I promise not to obfuscate myself using annoyingly belittling mantra that my editorial decision was guided by "intense competition for space".For the retrovirology community, pository . More im 20 days . RetroviRetrovirology readers [Retrovirology has been publishing on-line now for 6 months. While a first editorial can speak of dreams and aspirations, the burden of a second editorial is to demonstrate results and progress. Aided by an international cohort of extremely capable editors  and sixty editorial board members [Retrovirology's initial achievements. As of this writing, we have published 22 papers. These are good papers; and importantly, these papers are being widely-read. I can say with some pride that half  of US$600; there are no additional charges beyond this sum. Retrovirology will not levy additional page or colour charges on top of this fee. You are free to publish any number of colour figures and photographs, at no extra cost.Besides teaching about book covers, I think the other concept that we teach our children is that "There is no free lunch!" At the end of the day, anything worth doing has a price tag. I, my colleague editors, and all the editorial board members at Retrovirology cannot be published. I think most of you will agree based on your experience with other publishers that US$600 is more than modest and reasonable. As a point of comparison, the Public Library of Science has set up two new Open Access journals, and has set their APCs at US$1500, fully two and one-half times our charge, for each accepted article [I support the need for APC, because without this Retrovirology. More than anything else, Retrovirology is not my journal as much as it is your journal. I welcome your advice, and I look forward to hearing from you.In closing, let me thank those of you who have read, submitted and reviewed papers for APC = article-processing charge.


In 1994, the landmark "Cairo Conference" on population and development promised reproductive health for all. Ten years later, what has been achieved? In 1994, the landmark Cairo Conference promised reproductive health and rights for all. Ten years later, what has been achieved? About a decade ago, I went wandering around Cairo's City of the Dead. This might sound like a grim bit of tourism, but my connection to that vast necropolis runs deep—quite literally, as my family is buried there. After visiting their grave, I rambled through the city's dusty alleyways, past hundreds of years of history. Yet what I remember most about that day was not one of the many magnificent tombs, but a simple brick building with a sign, of all things, for a family planning clinic.I was certainly surprised by my discovery; in retrospect, I should not have been. That part of Cairo is home to hundreds of thousands of people for whom looking after the dead is a way of life. Their fertility invigorates the funereal air: the caretaker of my family's tomb, for example, had a blooming family of his own living near the grave. Where better to offer family planning than in a place so poor that reproduction seemed more a matter of fate than choice?That visit is a fitting metaphor for the field of reproductive health as a whole. Ten years ago, officials, experts, and activists from 179 countries also came to Cairo for the International Conference on Population and Development (ICPD). The conference produced a 20-year plan of action that focused on universal access to reproductive health services, including family planning and sexual health; reducing infant, child, and maternal mortality; better education, especially for girls; equality between men and women; and sustainable development.The ICPD's key achievement was to reorient thinking on reproduction away from narrowly defined, government-dictated population control to a broader appreciation of reproductive and sexual well-being within health care systems, a view driven by individual choice and rights, not official priorities. “The Cairo Conference was a peak moment,” says Sally Ethelston, vice president for communications at Population Action International, one member of a consortium of non-governmental organisations launching a report card to mark the anniversary of the Cairo Conference in early September. “There were times when people were excited that they had accomplished something, and you could see it on their faces.”Today, however, the mood is very different. While progress has been made on some of the plan's targets, effort has faltered on others. And the conference “camaraderie” that Ethelston describes has given way to conflict between faith and science, over abortion and condoms. Like signs of life in the City of the Dead, the Cairo Conference gave birth to great expectations, some of which have already expired.So, how far has the developing world come towards meeting the ICPD goals? There has certainly been progress on institutional reform in some countries, according to a recent survey of national policies by the United Nations Population Fund (UNFPA) . For exaBut translating policy into action has been difficult. Overall, the picture is one of patchy success, according to Susheela Singh, director of research at The Alan Guttmacher Institute, a nongovernmental research organisation. Official statistics, as limited as they are for many aspects of reproductive and sexual health, show mixed results. On a positive note, global population growth has slowed to roughly 77 million people a year . But whiWhile infant mortality rates have improved somewhat, maternal mortality figures have barely budged. An estimated 529,000 women still die every year from complications of pregnancy and childbirth. The highest rates are in sub-Saharan Africa, where, on average, 920 women die for every 100,000 live births, compared with 24 deaths per 100,000 live births in Europe . This isSo why hasn't more been achieved? One problem is certainly money. The 1994 Cairo Conference estimated the cost of implementing programmes for family planning, maternal health, and prevention of sexually transmitted diseases, as well as data collection and analysis in developing countries, at $18.5 billion by 2005—or $24.3 billion in today's dollars. The goal was to mobilise one-third of the money from donor nations, and the rest from developing countries themselves [Last year, global spending on reproductive health and services reached $14.7 billion, according to estimates from UNFPA, the Joint United Nations Programme on HIV/AIDS, and the Netherlands Interdisciplinary Demographic Institute . EncouraReproductive health is not alone in waiting for donors to give generously. For all the rhetoric at international summits, few rich countries have lived up to their lofty pledges of debt relief and of dedicating 0.7% of their gross domestic product to overseas development assistance. But as Steve Sinding, head of the International Planned Parenthood Federation (IPPF), points out, there are other reasons too for the shortfall. In the past donor interest was largely stimulated by fears of a population crisis. When the Cairo Conference reframed issues in terms of women's health and reproductive rights, rather than an impending population explosion, Sinding argues, the “demographic rationale” was lost, taking funding with it.Moreover, there are other issues competing for international funding, most notably AIDS. At the time of the Cairo Conference, 20 million people were infected with HIV; today the number has grown to an estimated 38 million . AIDS thRoughly half of the money spent on reproductive health last year went towards HIV/AIDS. And billions more is on the way, from the likes of the Global Fund to Fight AIDS, Tuberculosis, and Malaria and the United States President's Emergency Plan for AIDS Relief, which promises $15 billion over five years to HIV/AIDS programmes . But mucThis is a pity because it means that HIV/AIDS programmes are not making use of valuable infrastructure and expertise already on the ground in places where AIDS hits hardest. Given that 57% of HIV infections in sub-Saharan Africa are among women , and thaWhere once family planning was the darling of international donors, HIV is now the cause célèbre. “There's a lot of resentment about the spotlight moving on,” says Kevin O'Reilly, a former reproductive health specialist now at the department of HIV/AIDS at the World Health Organization. However, there are now attempts to bring the two together. Meetings earlier this year in Switzerland, New York, and Bangkok have led to calls to action to strengthen links between programmes addressing HIV/AIDS and sexual and reproductive health. While this should help in the battle against AIDS, the money which flows to AIDS should also benefit reproductive health.Arguably the most formidable obstacle to that union, and indeed further progress in improving reproductive health, is ideology. Since the Cairo Conference, a fierce battle has emerged between religious conservatives who eschew abortion and condoms in favour of abstinence and fidelity, and more liberal voices who argue for a full armamentarium to tackle these problems. The clash is loudest in the field of HIV/AIDS, where the President's Emergency Plan for AIDS Relief allocates a third of its funding for disease prevention to programmes focusing on abstinence and fidelity; public health experts argue that such an approach is ineffective at best, and dangerous at worst, without an equal emphasis on the availability of condoms for all.But the clash resounds in the wider arena of reproductive health as well. Four years ago, the ICPD's central target—access to reproductive services for all by 2015—failed to make it into the Millennium Development Goals, largely because of political nervousness. But as Kofi Annan, United Nations secretary-general, has pointed out, progress on the other key targets, such as eradication of poverty and hunger, will not be achieved without a focus on women's rights, education, reproductive health, and family planning.The fight between conservatives and liberals is clearest in the case of the US, which is the world's leading bilateral donor on reproductive health, spending $429 million this year . HoweverOther strings are more obvious. In 2001, George W. Bush reinstated the Mexico City Policy, otherwise known as the “Global Gag Rule”, which denies US family planning assistance—including money and contraceptive supplies—to any non-American group unless it certifies that it neither performs nor endorses abortion. IPPF, Marie Stopes International, and their local affiliates have been hard hit by the Rule, scaling back services in Kenya, Ghana, and elsewhere that offered essential health care to thousands of women and children.Then there is the Kemp-Kasten Amendment, a piece of US legislation which prohibits US assistance to any organisation as deemed by the President that “supports or participates in the management of a program of coercive abortion or involuntary sterilization.” At the behest of conservative supporters, President Bush has used the amendment to withhold $34 million in annual congressional appropriations to the UNFPA for the past three years. The UNFPA says that the $34 million could have been used to prevent 2 million unintended pregnancies, 800,000 induced abortions, 4,700 maternal deaths, and 77,000 infant and child deaths.The White House accuses UNFPA of abetting coercive reproductive practices in China—a claim which the UNFPA strenuously denies. Moreover, a number of international delegations, including one from the US State Department in 2002, have investigated the UNFPA's activities in China and failed to find evidence to support such allegations.Fortunately, other donors are stepping in to fill the breach: earlier this year, for example, the United Kingdom announced it would raise its contribution to the UNFPA to £80 million over the next four years, as well as increase its support to IPPF by a third. But even if the shortfall is made up, the ill will such clashes have engendered cannot be so easily salved.Getting it right on reproductive health cannot wait another decade. The largest generation of young people in history—a whopping 1.2 billion aged 10–19 years—is entering adulthood [The Cairo Conference:http://www.iisd.ca/cairo.htmlPopulation Action International:www.popact.orgUNFPA:www.unfpa.orgProgram for Appropriate Technology in Health:www.path.orgThe Alan Guttmacher Institute:www.guttmacher.orgThe Joint United Nations Programme on AIDS:www.unaids.orgNetherlands Interdisciplinary Demographic Institute:www.nidi.nlIPPF:www.ippf.orgGlobal Fund to Fight AIDS, Tuberculosis, and Malaria:www.theglobalfund.orgThe World Health Organization HIV/AIDS Programme:www.who.int/hiv/en


It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway. Hepatocytes have been shown to express large levels of NO following exposure to endotoxins, such as bacterial lipopolysaccharide and/or cytokines, such as tumour necrosis factor-α (TNFα), interleukin-1. The control role of arginine in both urea and NO biosynthesis is well known, when NO is synthesized from arginine, by the NOS reaction, citrulline is produced. Thus, the urea cycle is bypassed by the NOS reaction. Many authors demonstrated in other cellular types, like cardiomyocytes, that bradykinin caused the increase in reactive oxygen species (ROS) generation. The simultaneous increase of NO and ROS levels could cause peroxynitrite synthesis, inducing damage and reducing cell viability. The aim of this research is to study the effect of bradykinin, a proinflammatory mediator, on cell viability and on urea production in cultures of rat hepatocytes.g-nitro-L-arginine methyl ester (L-NAME), to the culture medium inhibited the increase of NO production. Exposure of hepatocytes to bradykinin 0,1 mM for 2 hours resulted in a significant decrease of urea synthesis. Cell viability, instead, showed a significant decrease 24 hours after the end of bradykinin treatment as determined by 3--2,5diphenyl-2H-tetrazolium (MTT) assay. L-NAME addition recovered urea production and cell viability at control values.Hepatocytes were treated with bradykinin, that stimulates nitric oxide synthase (NOS). NO release was determined using 4,5 diaminofluorescein diacetate (DAF-2DA), as fluorescent indicator of NO. Addition of the NOS inhibitor, NThe findings suggest that the cell toxicity, after bradykinin treatment, effectively depends upon exposure to increased NO levels and the effects are prevented by L-NAME. The results show also that the increased NO synthesis induces a reduced urea production, that is another index of cell damage. NO synthase (NOS) is either a constitutive or inducible enzyme. The endothelial isoform (e-NOS) and the neuronal isoform (n-NOS) are constitutive. The inducible form of the enzyme (i-NOS), has the main property to be not regulated by intracellular calcium concentration and Ca2+-calmodulin complex, unlike the constitutive form [It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway . NO is aive form . Hepatocive form ,5. NO maive form . NO geneive form . Accordiive form . To demoive form .The amounts of released NO were measured using DAF-2DA, that specifically reacts with the oxidized form of NO, producing the fluorescent triazolofluorescein . NO dete4Cl for 2 h. Figure To evaluate urea synthesis after bradykinin treatment, the hepatocytes were treated with 1 mM NHTo determine the effects of bradykinin on cell viability, the hepatocytes were exposed to bradykinin (either 0.01 mM or 0.1 mM) for an incubation time of 2 hours. In one experimental series, the cell viability was determined by MTT test after 2 hours of incubation. In a second one, culture medium containing bradykinin was removed and replaced with the same fresh medium at 2 hours after the addition of bradykinin, and then cell viability was measured 24 hours after the end of bradykinin treatment. The MTT test after 2 hours of incubation does not indicate any significant viability difference in treated hepatocyte cultures in comparison to control figure . By MTT The role of NO as mediator of hepatic injury after endotoxic shock remains controversial . IncreasIn summary, we conclude that 0.1 mM bradykinin treatment induces an increase of NO levels and reduction of urea synthesis in the hepatocytes. This increased NO production mediates, after 24 hours, cell toxicity as shown by MTT test. In contrast, the administration of the NOS inhibitor L-NAME protects against cell damage and increases urea levels, suggesting that NO plays a key role in the bradykinin-induced liver damage.Unless otherwise specified, all chemicals were obtained from Sigma .2+-free Hanks balanced salt solution. The liver was then excised and the digestion was carried out by adding 0.05% (w/v) collagenase (type IV) in Hanks balanced salt solution supplemented with CaCl2·H2O (0.0186 g/L) at a flux rate of 40 ml/min. At this point liver was transferred to a square plate containing 100 ml of RPMI 1640 medium supplemented with 200 mM L-glutamine, 20 ml/L essential amino acid solution and 10 ml/L non-essential amino acid solution, 1% antibiotic antimycotic stabilized solution and 100 μM L-arginine (incomplete medium). The cells were dispersed by gentle distruption with a stainless steel comb. After filtration through 200 μm Nytal mesh, parenchymal cells (hepatocytes) were separated from nonparenchymal cells  by centrifugation at 50 g in Eppendorf Centrifuge 5810R at 4°C for 2 minutes and then washed twice in washing buffer [5 cells/ml with incomplete medium supplemented with 2% fetal calf serum, 0.1 U/ml insulin and 10-6 M dexamethasone (complete medium). The hepatocytes were then plated in 24 well-plates coated with rat tail collagen at the final cell density of 2.5 × 105 cells per well and incubated at 37°C in an humidified atmosphere of 5% CO2 and 95% air. After 6 hours incubation, the medium was changed and replaced with incomplete medium to remove dead cells.Hepatocytes were isolated from male rats, Wistar strain, (180 to 200 gbw), by a modification of the method of Seglen . All prog buffer . Then thg buffer . After cTo verify the isolation method efficiency, the acid fosfatase activity per mg of proteins was evaluated. According to literature data, the specific activity of acid fosfatase in nonparenchimal cells is 1,7 folds the same activity in parenchimal cells .After 24 hours of culture the hepatocytes were exposed either to bradykinin (0.01 mM and 0.1 mM) or bradykinin 0.1 mM supplemented with L-NAME 1.68 mM .DAF-2DA  was dissolved in DMSO (1 mg/0.45 ml) and diluted to 10 μM in phosphate buffer . Then the cells were either incubated in phosphate buffer containing 10 μM DAF-2DA, bradykinin (0.01 mM and 0.1 mM) and bradykinin 0.1 mM supplemented with L-NAME 1.68 mM. After 2 hours of incubation in this reaction mixture, the fluorescence from the reaction of DAF-2DA with NO released from hepatocytes was measured with Perkin-Elmer MPF-44B Spectrofluorimeter calibrated for excitation at 495 nm and emission at 515 nm. Results were expressed as a percentage of the fluorescence of the samples in comparison to control.4Cl was added. After 2 hours urea levels in the media were measured by spectrophotometric method using Urea Color 2 Kit  measuring absorbance at 600 nm and blank sample with the same NH4Cl final concentration was used. Urea synthesis was calculated as ng urea per cell per hour.To determine the effects of bradykinin on urea production, cells were treated either with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM. At the same media 1 mM NHCell viability was determined by MTT test method  and confAt least four independent determinations of each parameter were compared to control using Student's T-test. Differences were considered significant when p < 0.05 was obtained.SS: Fluorimetric analysis and overall statistical analysis of dataMG: Director of researchMS: Spectrophotometric analysisCR: Primary hepatocyte cultures and characterizationAll authors read and approved the final manuscript