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Almost half of all enzymes must associate with a particular metal to function. An ambition is to understand why each metal-protein partnership arose and how it is maintained. Metal availability provides part of the explanation, and has changed over geological time and varies between habitats but is held within vital limits in cells. Such homeostasis needs metal sensors, and there is an ongoing search to discover the metal-sensing mechanisms. For metalloproteins to acquire the right metals, metal sensors must correctly distinguish between the inorganic elements.

Objective: Pregnant women with the vascular complication of preeclampsia show altered lipid metabolism characterized by elevated circulating triglycerides and nonesterified free fatty acids. We have compared the effect of maternal plasma from women with and without preeclampsia on cultured vascular endothelial cells and determined whether these plasma-induced changes were reproduced with free fatty acid solutions of palmitic, oleic and linoleic acid, representative of circulating levels reported in preeclampsia., Methods: Lipid accumulation was quantified by oil-red O staining, apoptosis by terminal dUTP nick-end labelling (TUNEL) and the measurement of mitochondrial redox capacity, and membrane potential recorded using MTT reduction and JC-1 accumulation for human umbilical vein endothelial cells (HUVECs) exposed to plasma and free fatty acids., Results: Lipid droplet accumulation was significantly increased in cultured HUVECs conditioned with maternal plasma from pregnancies with preeclampsia compared with normal uncomplicated controls. This increase was replicated following exposure to free fatty acids at the combined concentrations defined in preeclampsia. Plasma from these women also caused a significant decrease in mitochondrial dehydrogenase activity, a marked reduction in mitochondrial membrane potential and an increase in apoptosis compared with normal pregnancy. Again these effects were reproduced using free fatty acids in combination at the levels previously associated with preeclampsia., Conclusion: These findings support the concept of a circulating pathogenic factor for preeclampsia and highlight the possibility that this factor is not a single compound but perhaps the combined elevation of the free fatty acids palmitic, oleic and linoleic acid in the maternal circulation.

Purpose: To estimate the incidence of cataracts in an adult idiopathic thrombocytopenic purpura (ITP) and in a comparable non-ITP population, stratified by age, gender and medication use in order to determine whether the risk of cataracts is greater in ITP patients., Methods: Retrospective cohort of 745 newly diagnosed ITP patients and 3725 non-ITP subjects who were 18 years of age or older. The study population included patients registered on the General Practice Research Database (GPRD) during the period 1992-2005. ITP patients, identified using Read/Oxmis codes, were matched to five non-ITP patients. The exposure of interest was oral systemic steroid use and the primary outcome was cataracts., Results: The overall incidence rate of cataracts in males was 11.8 per 1000 PY (95%CI: 6.3-20.2) and in females 9.0 per 1000 PY (95%CI: 5.2-14.4). In the non-ITP population these rates were 14.0 per 1000 PY (95%CI: 11.0-17.4) and 8.1 per 1000 PY (95%CI: 6.4-10.1), respectively. In the ITP population, users of oral steroids (OS) had a cataract incidence rate of 14.0 per 1000 PY (95%CI: 8.7-21.4) and non-users 6.0 per 1000 PY (95%CI: 2.8-11.4). In the non-ITP population, these rates were 16.9 per 1000 PY (95%CI: 11.9-23.3) and 9.2 per 1000 PY (95%CI: 7.6-11.0), respectively. OS were associated with an increased risk for cataracts., Conclusions: The rates of cataract in an adult ITP population are comparable to that in a non-ITP population.

Background: Celiac disease (CD) occurs in as many as 1 in 80 pregnant women and is associated with poor pregnancy outcome, but it is not known if this is an effect on maternal nutrient absorption or, alternatively, if the placenta is an autoimmune target. The major autoantigen, tissue transglutaminase (tTG), has previously been shown to be present in the maternal-facing syncytiotrophoblast plasma membrane of the placenta., Methods: ELISA was used to demonstrate the presence of antibodies to tissue transglutaminase in a panel of CD sera. Immunohistochemistry was used to evaluate the binding of IgA autoantibodies from CD serum to term placenta. In addition, novel direct binding and activity assays were developed to mimic the in vivo exposure of the villous placenta to maternal autoantibody., Results and Discussion: CD IgA autoantibodies located to the syncytial surface of the placenta significantly more than IgA antibodies in control sera (P < 0.0001). The distribution of antigen was similar to that observed using a monoclonal antibody to tissue transglutaminase. Staining was reduced by pre-absorption of CD serum with recombinant human tissue transglutaminase. In direct binding assays, autoimmune immunoglobulin A (IgA) from the maternal compartment became associated with antigen at the syncytial surface of the placenta, as a result of which transglutaminase activity at this site was inhibited., Conclusion: These data indicate that direct immune effects in untreated CD women may compromise placental function.

A person experiencing more than one medical condition may have ambiguous clinical presentation. ITP is a serious autoimmune disease with little epidemiological evidence on its burden, risk factors, and comorbidities. Using the United Kingdom general practice research database, we conducted a 14 years population-based case control-type study to explore medical conditions more likely to cooccur with ITP and their temporal relationship in association with ITP. ITP patients were matched to non-ITP on practice, age, gender, and follow-up period. Potential comorbidities were represented by patients' medical information at the preferred term level of the MedDRA international classification. As well as death (OR = 60.0; 95% CI [4.47-806.0]) and known clinical signs and symptoms of ITP, ITP is associated with considerable number of medical conditions. The association between ITP and some of these conditions is apparent both before and after ITP diagnosis. Specific targeted studies can now be setup to reexamine observed associations.

Protein metal-coordination sites are richly varied and exquisitely attuned to their inorganic partners, yet many metalloproteins still select the wrong metals when presented with mixtures of elements. Cells have evolved elaborate mechanisms to scavenge for sufficient metal atoms to meet their needs and to adjust their needs to match supply. Metal sensors, transporters and stores have often been discovered as metal-resistance determinants, but it is emerging that they perform a broader role in microbial physiology: they allow cells to overcome inadequate protein metal affinities to populate large numbers of metalloproteins with the right metals.

Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).

Synechocystis PCC 6803 has a high demand for iron (10 times greater than Escherichia coli) to sustain photosynthesis and is unusual in possessing at least two putative iron-binding proteins of a type normally associated with ATP-binding cassette-type importers. It has been suggested that one of these, FutA2, binds ferrous iron, but herein we clearly demonstrate that this protein avidly binds Fe(III), the oxidation state preference of periplasmic iron-binding proteins. Structures of apo-FutA2 and Fe-FutA2 have been determined at 1.7 and 2.7A, respectively. The metal ion is bound in a distorted trigonal bipyramidal arrangement with no exogenous anions as ligands. The metal-binding environment, including the second coordination sphere and charge properties, is consistent with a preference for Fe(III). Atypically, FutA2 has a Tat signal peptide, and its inability to coordinate divalent cations may be crucial to prevent metals from binding to the folded protein prior to export from the cytosol. A loop containing the His(43) ligand undergoes considerable movement in apo-versus Fe-FutA2 and may control metal release to the importer. Although these data are consistent with FutA2 being the periplasmic component involved in iron uptake, deletion of another putative ferric binding protein, FutA1, has a greater effect on the accumulation of iron and is more analogous to a DeltafutA1DeltafutA2 double mutant than DeltafutA2. Here, we also discover that there is a reduced level of ferric FutA2 in the periplasm of the DeltafutA1 mutant providing an explanation for its severe iron-uptake phenotype.

Maternal endothelial activation in pre-eclampsia is attributed to the release of unknown factors from a hypoperfused placenta. To further characterize these factors, we have used a serum-free placental villous explant culture model and investigated the effect of the liberated soluble factors produced on human endothelial cell cultures. Term placental villous explants from uncomplicated pregnancies were cultured for 4 days in 20, 6 or 1% O2 to mimic placental hyperoxia, normoxia and hypoxia. Medium collected from viable explants was applied to cultured human uterine microvascular endothelial cells. Medium conditioned by hypoxic explants caused a significant decrease in endothelial cell ATP levels and mitochondrial dehydrogenase activity, suggestive of a reduced metabolic rate. An additional reduction in mitochondrial membrane potential and increased endothelial cell death occurred as the oxygen concentration to which explants had been exposed decreased. Effects of the hypoxic explant medium were also seen ex vivo in a wire myography model of myometrial artery function, with increased vasoconstriction and attenuated vasodilation following exposure to hypoxic explant medium. These results suggest that hypoxia (1% O2) may stimulate the release of soluble factors from the placenta, which have an adverse effect on endothelial cell metabolism and mitochondrial integrity in vitro. These potentially pathogenic factors are now being characterized.

Regulatory T cells (T(reg)) play an important role in immune regulation. Their development in the thymus requires TCR activation and recognition of peptide-MHC, although the downstream signals controlling commitment to the lineage are unclear. To compare the requirements for positive selection and T(reg) development, we studied knockout and transgenic mice defective in Raf signaling and the ERK effector SRF accessory protein 1 (SAP-1), a member of the ternary complex factor family of Ets domain transcription factors. Although SAP-1 deficient mice display a severe defect in thymocyte positive selection, T(reg) development was unimpaired as assessed by expression of Foxp3 and the activation markers CD25, GITR, CTLA4, and CD103 in the CD4(+) cell population. In contrast, inhibition of Raf signaling by the interfering dominant negative Raf derivative reduced both Foxp3(+) and Foxp3(-) CD4(+) populations. In SAP-1-deficient CD4(+)CD25(+) T(reg) cells, TCR crosslinking efficiently induced ERK activation, but transcriptional induction of the immediate early gene Egr-1 was impaired. Nevertheless, neither deletion of SAP-1 nor expression of a dominant negative Raf derivative affected the ability of CD4(+)CD25(+) T(reg) cells to suppress CD4(+)CD25(-) cell proliferation in vitro. Finally the suppressive activity of CD4(+)CD25(+) T(reg) cells lacking SAP-1 in an in vivo colitis model was not significantly impaired. The signaling requirements for development of T(reg) cells in the thymus are thus distinct from those required for "conventional" T cell positive selection, and ERK signaling to SAP-1 is not required for the suppressive activity of T(reg) cells.

A novel ArsR-SmtB family transcriptional repressor, KmtR, has been characterized from mycobacteria. Mutants of Mycobacterium tuberculosis lacking kmtR show elevated expression of Rv2025c encoding a deduced CDF-family metal exporter. KmtR-dependent repression of the cdf and kmtR operator-promoters was alleviated by nickel and cobalt in minimal medium. Electrophoretic mobility shift assays and fluorescence anisotropy show binding of purified KmtR to nucleotide sequences containing a region of dyad symmetry from the cdf and kmtR operator-promoters. Incubation of KmtR with cobalt inhibits DNA complex assembly and metal-protein binding was confirmed. KmtR is the second, to NmtR, characterized ArsR-SmtB sensor of nickel and cobalt from M. tuberculosis suggesting special significance for these ions in this pathogen. KmtR-dependent expression is elevated in complete medium with no increase in response to metals, whereas NmtR retains a response to nickel and cobalt under these conditions. KmtR has tighter affinities for nickel and cobalt than NmtR consistent with basal levels of these metals being sensed by KmtR but not NmtR in complete medium. More than a thousand genes encoding ArsR-SmtB-related proteins are listed in databases. KmtR has none of the previously defined metal-sensing sites. Substitution of His88, Glu101, His102, His110, or His111 with Gln generated KmtR variants that repress the cdf and kmtR operator-promoters even in elevated nickel and cobalt, revealing a new sensory site. Importantly, ArsR-SmtB sequence groupings do not correspond with the different sensory motifs revealing that only the latter should be used to predict metal sensing.

CmtR from Mycobacterium tuberculosis is a winged helical DNA-binding repressor of the ArsR-SmtB metal-sensing family that senses cadmium and lead. Cadmium-CmtR is a dimer with the metal bound to Cys-102 from the C-terminal region of one subunit and two Cys associated with helix alphaR from the other subunit, forming a symmetrical pair of cadmium-binding sites. This is a significant novelty in the ArsR-SmtB family. The structure of the dimer could be solved at 312 K. The apoprotein at the same temperature is still a dimer, but it experiences a large conformational exchange at the dimer interface and within each monomer. This is monitored by an overall decrease of the number of nuclear Overhauser effects and by an increase of H(2)O-D(2)O exchange rates, especially at the dimeric interface, in the apo form with respect to the cadmium-bound state. The C-terminal tail region is completely unstructured in both apo and cadmium forms but becomes less mobile in the cadmium-bound protein due to the recruitment of Cys-102 as a metal-ligand. DNA binds to the apo dimer with a ratio 1:3 at millimolar concentration. Addition of cadmium to the apo-CmtR-DNA complex causes DNA detachment, restoring the NMR spectrum of free cadmium-CmtR. Cadmium binding across the dimer interface impairs DNA association by excluding the apo-conformers suited to bind DNA.

Tissue transglutaminase (TGM2; also known as TG2 or tTG) localizes to the syncytial microvillous membrane (MVM) of the human placenta, the primary interface between maternal and fetal tissue. To identify TGM2 substrates in the MVM, membrane vesicles were prepared and labeled with biotinylated acyl donor or acceptor probes. Biotinylated species were selected on an avidin affinity matrix and identified by mass spectrometry of tryptic peptides. The most abundant were cytoskeletal (actin, tubulin, and cytokeratin) and membrane-associated (annexins, integrins, and placental alkaline phosphatase) proteins. During pregnancy, apoptotic particulate material, the end product of the trophoblast life cycle, is shed from the MVM into maternal circulation. Shed material was isolated from primary trophoblast cultures in which syncytial-like masses develop by fusion. A substantial fraction of actin in the particles was in the form of covalent polymeric aggregates, in contrast to cellular actin, which dissociated completely into monomer in SDS-PAGE. When cells were cultured in the presence of transglutaminase inhibitors, actin in the shed particles remained exclusively in monomeric form, and a reduction in trophoblast intercellular fusion and differentiation was observed. These findings suggest that transglutaminase-mediated cross-linking stabilizes the particulate material shed from the placenta.

Periplasmic substrate binding proteins are known for iron, zinc, manganese, nickel, and molybdenum but not copper. Synechocystis PCC 6803 requires copper for thylakoid-localized plastocyanin and cytochrome oxidase. Here we show that mutants deficient in a periplasmic substrate binding protein FutA2 have low cytochrome oxidase activity and produce cytochrome c6 when grown under copper conditions (150 nm) in which wild-type cells use plastocyanin rather than cytochrome c6. Anaerobic separation of extracts by two-dimensional native liquid chromatography followed by metal analysis and peptide mass-fingerprinting establish that accumulation of copper-plastocyanin is impaired, but iron-ferredoxin is unaffected in DeltafutA2 grown in 150 nm copper. However, recombinant FutA2 binds iron in preference to copper in vitro with an apparent Fe(III) affinity similar to that of its paralog FutA1, the principal substrate binding protein for iron import. FutA2 is also associated with iron and not copper in periplasm extracts, and this Fe(III)-protein complex is absent in DeltafutA2. There are differences in the soluble protein and small-molecule complexes of copper and iron, and the total amount of both elements increases in periplasm extracts of DeltafutA2 relative to wild type. Changes in periplasm protein and small-molecule complexes for other metals are also observed in DeltafutA2. It is proposed that FutA2 contributes to metal partitioning in the periplasm by sequestering Fe(III), which limits aberrant Fe(III) associations with vital binding sites for other metals, including copper.

Objective: The objective of this study was to estimate the seroprevalence of herpes simplex virus (HSV) types 1 and 2 in male and female soldiers discharged from the Israel Defense Force (IDF) over a period of 18 years., Goal: The goal of this study was to study the secular trends of HSV-1 and HSV-2 infection rates in Israeli young adults., Study Design: Three consecutive cross-sectional studies were carried out on stored sera of systematic random samples of soldiers (median age = 20.8) discharged from the IDF in 1984-1985, 1992-1993, and 2001-2002. HSV-1 and -2 seroprevalence was analyzed using indirect IgG enzyme-linked immunosorbent assay for type-specific antibodies., Results: There was a significant decrease in HSV-1 and HSV-2 seropositivity rates at discharge from the army and an estimated 10-fold decline in the incidence rates of HSV-2 infection during the military service over the last 2 decades., Conclusions: The decline in HSV-1 seroprevalence rate is similar to that reported from other industrialized countries. The decrease in HSV-2 seroprevalence and transmission rates among Israeli young adults is steeper than that reported from Western countries.

The thylakoid compartments of plant chloroplasts are a vital destination for copper. Copper is needed to form holo-plastocyanin, which must shuttle electrons between photosystems to convert light into biologically useful chemical energy. Copper can bind tightly to proteins, so it has been hypothesized that copper partitions onto ligand-exchange pathways to reach intracellular locations without inflicting damage en route. The copper metallochaperone Atx1 of chloroplast-related cyanobacteria (ScAtx1) engages in bacterial two-hybrid interactions with N-terminal domains of copper-transporting ATPases CtaA (cell import) and PacS (thylakoid import). Here we visualize copper delivery. The N-terminal domain PacS(N) has a ferredoxin-like fold that forms copper-dependent heterodimers with ScAtx1. Removal of copper, by the addition of the cuprous-ion chelator bathocuproine disulfonate, disrupts this heterodimer, as shown from a reduction of the overall tumbling rate of the protein mixture. The NMR spectral changes of the heterodimer versus the separate proteins reveal that loops 1, 3, and 5 (the carboxyl tail) of the ScAtx1 Cu(I) site switch to an apo-like configuration in the heterodimer. NMR data ((2)J(NH) couplings in the imidazole ring of (15)N ScAtx1 His-61) also show that His-61, bound to copper(I) in [Cu(I)ScAtx1](2), is not coordinated to copper in the heterodimer. A model for the PacS(N)/Cu(I)/ScAtx1 complex is presented. Contact with PacS(N) induces change to the ScAtx1 copper-coordination sphere that drives copper release for thylakoid import. These data also elaborate on the mechanism to keep copper(I) out of the ZiaA(N) ATPase zinc sites.

Many bacterial genomes encode multiple metal-sensing ArsR-SmtB transcriptional repressors. There is interest in understanding and predicting their metal specificities. Here we analyse two arsR-smtB genes, ydeT and yozA (now aseR and czrA) from Bacillus subtilis. Purified AseR and CzrA formed complexes in gel-retardation and fluorescence-anisotropy assays with fragments of promoters that were derepressed in DeltaaseR and DeltaczrA cells. Candidate (i) partly thiolate, alpha3-helix (for AseR) and (ii) tetrahedral, non-thiolate, alpha5-helix (for CzrA) metal binding sites were predicted then tested in vitro and/or in vivo. The precedents are for such sites to sense arsenite/antimonite (alpha3) and zinc (alpha5). This correlated with the respective metal inducers of AseR and CzrA repressed promoters in B. subtilis and matched the metals that impaired formation of protein-DNA complexes in vitro. The putative sensory sites of 1024 ArsR-SmtB homologues are reported. Although AseR did not sense zinc in vivo, it bound zinc in vitro exploiting alpha3 thiols, but AseR DNA binding was not impaired by zinc. If selectivity relies on discriminatory triggering of allostery not just selective metal binding, then tight non-effector metal complexes could theoretically inhibit metal sensing. AseR remained arsenite-sensitive in equimolar zinc, while CzrA remained zinc-sensitive in equimolar arsenite in vitro. However, cupric ions did not impair CzrA-DNA complex formation but did inhibit zinc-mediated allostery in vitro and prevent zinc binding. Access to copper must be controlled in vivo to avoid formation of cupric CzrA.

Tissue transglutaminase (tTG) expression, distribution and activity were examined in human placenta and derived cells. Immunochemical techniques and RT-PCR were used to demonstrate tTG protein and mRNA in stromal cells and trophoblast in first trimester and at term, with higher levels later in pregnancy. Decidual cells also produce tTG. The data were confirmed using primary cultures of trophoblast, fibroblasts and decidual stromal cells. Substrate incorporation studies indicated tTG activity in association with fibroblast extracellular matrix and the syncytial microvillous membrane (MVM), where several target polypeptides could be observed. tTG is a major autoantigen in Coeliac disease (CoD) which is associated with poor pregnancy outcome. tTG at the placental MVM is a plausible target of maternal autoantibody action.

Each metalloprotein must somehow acquire the correct metal. We review the insights into metal specificity in cells provided by studies of ArsR-SmtB DNA binding, metal-responsive transcriptional repressors, and a bacterial copper chaperone. Cyanobacteria are the one bacterial group that have known enzymatic demand for cytoplasmic copper import. The copper chaperone and ATPases that supply cyanobacterial plastocyanin and cytochrome oxidase are reviewed, along with related ATPases for cobalt and zinc. These studies highlight the contributions of protein-protein interactions to metal speciation. Metal sensors and metallochaperones, along with metal transporters and metal-storage proteins, act in concert not only to supply the correct metals but also to withhold the wrong ones.

The amyloidogenic pathway leading to the production and deposition of Abeta peptides, major constituents of Alzheimer disease senile plaques, is linked to neuronal metal homeostasis. The amyloid precursor protein binds copper and zinc in its extracellular domain, and the Abeta peptides also bind copper, zinc, and iron. The first step in the generation of Abeta is cleavage of amyloid precursor protein by the aspartic protease BACE1. Here we show that BACE1 interacts with CCS (the copper chaperone for superoxide dismutase-1 (SOD1)) through domain I and the proteins co-immunoprecipitate from rat brain extracts. We have also been able to visualize the co-transport of membranous BACE1 and soluble CCS through axons. BACE1 expression reduces the activity of SOD1 in cells consistent with direct competition for available CCS as overexpression of CCS restores SOD1 activity. Finally, we demonstrate that the twenty-four residue C-terminal domain of BACE1 binds a single Cu(I) atom with high affinity through cysteine residues.

Objective/goal: The objective of this study was to evaluate risk factors for herpes simplex virus type 2 (HSV-2) infection among men whose female partners have genital herpes (GH)., Study: Between 1998 and 2001, 717 men in heterosexual monogamous relationships, without a history of GH, completed a cross-sectional questionnaire on demographic, behavioral, and knowledge factors for GH. Their female partners were symptomatic and HSV-2-seropositive. Risk factors for HSV-2 seropositivity were assessed using logistic regression., Results: On laboratory confirmation, 25% of the men were HSV-2-seropositive. Factors significantly (P<0.01) associated with HSV-2 infection included: never using condoms (adjusted odds ratio [aOR], 1.84; 95% confidence interval [CI], 1.15-2.95), prior sexually transmitted disease (STD) (aOR, 1.80; CI, 1.27-2.58), vaginal intercourse during symptomatic episodes (aOR, 1.77; CI, 1.19-2.62), longer partnership (for each additional year aOR, 1.07; CI, 1.03-1.09), and lower knowledge of GH (aOR, 1.14; CI, 1.05-1.23)., Conclusion: Potentially modifiable risk factors for HSV-2 infection included engaging in vaginal sex during symptomatic episodes, never using condoms, and lower knowledge of GH.

Murine models of inflammatory bowel disease (IBD) are useful tools for the study of the pathogenesis and regulation of intestinal inflammation. Colitis can be induced in immune-deficient mice following transfer of populations of T cells or following infection with Helicobacter hepaticus and other intestinal pathogens. In these situations, colitis occurs as a result of the absence of a specialized population of regulatory cells, as transfer of CD4(+)CD25(+) T cells prevents disease. Importantly, from a clinical perspective, CD4(+)CD25(+) T cells can also reverse an established colitis. CD4(+)CD25(+) T cells proliferate both in the secondary lymphoid organs and at the site of inflammation, suggesting that regulation occurs both locally and systemically. CD4(+)CD25(+) T cells are not only capable of regulating other T cells but are also capable of suppressing components of the innate immune system. Control of colitis is dependent on the presence of the immunosuppressive cytokines interleukin-10 and transforming growth factor-beta, although their roles are divergent and complex. Regulatory T cells represent one of the host's mechanisms to prevent immune pathology during chronic immune stimulation. Enhancement of regulatory T-cell activity may be useful to control autoreactive T-cell responses and inhibit harmful inflammatory diseases such as asthma and IBD.

Biological strategies for interrupting transmission of human immunodeficiency virus (HIV) type 1 should be directed at reducing infectiousness of and susceptibility to HIV-1. Potential antiretroviral interventions include reducing the likelihood of transmission of HIV-1 by reducing HIV-1 load in the blood and genital tract of HIV-1--infected person, prophylaxis after high-risk exposure, and pre-exposure prophylaxis for very high risk populations. Antiviral treatment of herpes simplex virus (HSV) type 2, the most common cause of genital ulcers, should be evaluated as a strategy for HIV-1 infection prevention by reducing infectiousness of and susceptibility to HIV-1, on the basis of biological and epidemiological data indicating that HSV-2 facilitates transmission and acquisition of HIV-1. The rationale for antiretroviral and HSV-2-specific interventions and studies to test these strategies are described.

Adolescents are the focus of many interventions that aim to prevent HIV transmission. In order for these interventions to be effective, it is essential to understand adolescents' sexual behaviour. Using data collected in Yaoundé, Cameroon, in 1997, the study analysed risk exposure and HIV prevalence among 426 men and 510 women aged 15-24. Although risky behaviours seem to be more prevalent among young men, their HIV prevalence remains under 1%. In contrast, HIV prevalence is high among young women (7.5%), even those who report having had few sexual partners. Mixing patterns among sexual partners, and especially the age difference between men and women, do not seem to be sufficient to explain the large male-female discrepancy in HIV prevalence that is evident in these data. The results are therefore probably due to a greater susceptibility to infection of young women than men. This study highlights the necessity of reinforcing prevention campaigns among youth and fighting the obstacles that continue to impede the use of condoms in this population.

Zn(2+)-responsive repressor ZiaR and Co(2+)-responsive activator CoaR modulate production of P(1)-type Zn(2+)- (ZiaA) and Co(2+)- (CoaT) ATPases respectively. What dictates metal selectivity? We show that Delta ziaDeltacoa double mutants had similar Zn(2+) resistance to Deltazia single mutants and similar Co(2+) resistance to Deltacoa single mutants. Controlling either ziaA or coaT with opposing regulators restored no resistance to metals sensed by the regulators, but coincident replacement of the deduced cytosolic amino-terminal domain CoaT(N) with ZiaA(N) (in ziaR-(p) ziaA-ziaA(N)coaT) conferred Zn(2+) resistance to DeltaziaDeltacoa, Zn(2+) content was lowered and residual Co(2+) resistance lost. Metal-dependent molar absorptivity under anaerobic conditions revealed that purified ZiaA(N) binds Co(2+) in a pseudotetrahedral two-thiol site, and Co(2+) was displaced by Zn(2+). Thus, the amino-terminal domain of ZiaA inverts the metals exported by zinc-regulated CoaT from Co(2+) to Zn(2+), and this correlates simplistically with metal-binding preferences; K(ZiaAN) Zn(2+) tighter than Co(2+). However, Zn(2+) did not bleach Cu(+)-ZiaA(N), and only Cu(+) co-migrated with ZiaA(N) after competitive binding versus Zn(2+). Bacterial two-hybrid assays that detected interaction between the Cu(+)-metallochaperone Atx1 and the amino-terminal domain of Cu(+)-transporter PacS(N) detected no interaction with the analogous, deduced, ferredoxin-fold subdomain of ZiaA(N). Provided that there is no freely exchangeable cytosolic Cu(+), restricted contact with the Cu(+)-metallochaperone can impose a barrier impairing the formation of otherwise favoured Cu(+)-ZiaA(N) complexes.

The Atx1 copper metallochaperone from Synechocystis PCC 6803, ScAtx1, interacts with two P(1)-type copper ATPases to supply copper proteins within intracellular compartments, avoiding ATPases for other metals en route. Here we report NMR-derived solution structures for ScAtx1. The monomeric apo form has a betaalphabetabetaalpha fold with backbone motions largely restricted to loop 1 containing Cys-12 and Cys-15. The tumbling rate of Cu(I)ScAtx1 (0.1-0.8 mm) implies dimers. Experimental restraints are satisfied by symmetrical dimers with Cys-12 or His-61, but not Cys-15, invading the copper site of the opposing subunit. A full sequence of copper ligands from the cell surface to thylakoid compartments is proposed, considering in vitro homodimer liganding to mimic in vivo liganding in ScAtx1-ATPase heterodimers. A monomeric high resolution structure for Cu(I)ScAtx1, with Cys-12, Cys-15, and His-61 as ligands, is calculated without violations despite the rotational correlation time. (2)J(NH) couplings in the imidazole ring of His-61 establish coordination of N(epsilon2) to copper. His-61 is analogous to Lys-65 in eukaryotic metallochaperones, stabilizing Cu(I)S(2) complexes but by binding Cu(I) rather than compensating charge. Cys-Cys-His ligand sets are an emergent theme in some copper metallochaperones, although not in related Atx1, CopZ, or Hah1. Surface charge (Glu-13) close to the metal-binding site of ScAtx1 is likely to support interaction with complementary surfaces of copper-transporting ATPases (PacS-Arg-11 and CtaA-Lys-14) but to discourage interaction with zinc ATPase ZiaA and so inhibit aberrant formation of copper-ZiaA complexes.

To determine the sensitivity and specificity of Focus HerpeSelect ELISAs, sera or plasma samples from women aged 18-55 years were collected in ten cities from eight countries and tested by HerpeSelect HSV-1 ELISA (Focus-HSV-1) and by HerpeSelect HSV-2 ELISA (Focus-HSV-2). Sera with Focus-HSV-2-positive results were retested; 94% of the 3617 samples retested were positive. A subset of sera from each site was then selected, based on the HSV-2 results, and tested by Western blot (WB). The sensitivity and specificity were determined with samples from ten sites (n = 967) for Focus-HSV-1 and from seven sites (n = 675) for Focus-HSV-2. Focus-HSV-1 and WB results were concordant (both negative or both positive) for 97% of samples, with 99% sensitivity and 77% specificity. Specimens from Songkla, Thailand had 84% concordance with WB results for HSV-1, while three other sites had 100% concordance. Concordance of Focus-HSV-2 and WB was 92%, with 97% sensitivity and 89% specificity. Ibadan, Nigeria had 78% concordance. Focus-HSV-2 sensitivity and specificity in sites other than Ibadan were 97% and 93%, respectively. Raising the positive cut-off index value for HSV-2 from 1.1 to 3.5 yielded a sensitivity of 90% and a specificity of 96%. A sensitivity of 90% and a specificity of 98% were achieved for sites other than Nigeria with the higher cut-off. In summary, the sensitivity and specificity of the Focus-HSV-1 and Focus-HSV-2 tests varied by site. Performance data generated in one area may not be applicable to other populations.

The thylakoid lumen of the cyanobacterium Synechocystis PCC 6803 is supplied with copper via two copper-transporting ATPases and a metallochaperone intermediary. We show that the copper site of this metallochaperone is unusual and consists of two cysteine residues and a histidine imidazole located on structurally dynamic loops. Substitution of this histidine residue enhances bacterial two-hybrid interaction with the cytosolic copper exporter, but not the copper importer, suggesting that the interacting surfaces are distinct, with implications for metal transfer.

We report a cadmium- and lead-detecting transcriptional repressor from Mycobacterium tuberculosis designated CmtR. Two genes were co-transcribed with cmtR, one encoding a deduced P1 type ATPase. Purified CmtR bound to the cmt operator-promoter, and repression of transcription was lost after introduction of a stop codon into cmtR. Assays of metal-dependent expression from cmt and nmt operator-promoters established that the metal specificity of CmtR in vivo was perfectly inverted relative to the nickel-cobalt sensor NmtR from the same organism, with CmtR totally insensitive to Co(II) or Ni(II) and NmtR totally insensitive to Cd(II) or Pb(II). Absorption spectroscopy of Cd(II)-, Co(II)-, and Ni(II)-substituted CmtR revealed S- to metal-charge-transfer which was absent in NmtR, providing diagnostic metal-difference spectra that discriminated between metal-binding to these two proteins. Ni(II)-binding isothermal titrations of CmtR are complex, with Kapp = 1.8 x 10(4) m(-1) for site1, three orders of magnitude weaker than KNi for NmtR. Mixing equimolar apo-NmtR and apo-CmtR with 0.9 equivalents of Cd(II) gave Cd(II)-dependent difference spectra almost identical to Cd(II)0.9-CmtR. Thus, Cd(II) bound to CmtR in preference to NmtR, whereas the converse was true for Ni(II); this correlates faithfully with and provides a simplistic basis for metal-sensing preferences. In contrast, CmtR and NmtR had similar affinities for Co(II), and alternative explanations for Co(II) sensitivities are invoked. ArsR-SmtB repressors detect metals through derivatives of one or both of two possible allosteric sites at either carboxyl-terminal alpha5 helices or helix alpha3 proximal to the DNA-binding site. Unexpectedly, neither site was required for inducer recognition by CmtR. The mutants in potential metal ligands in, or near, these regions, Cys4, Cys35, Asp79, His81, Asp97, Asp99, Glu105, Glu111, and Glu114, retained both repression and inducer recognition. Crucially, substitution of Cys57, Cys61, and Cys102 with Ser revealed that each of these three residues is obligatory for Cd(II) detection, and this defines completely new sensory sites.

CbiX is a cobaltochelatase required for the biosynthesis of vitamin B12 and is found in Archaea as a short form (CbiXS containing 120-145 amino acids) and in some bacteria as a longer version (CbiXL containing 300-350 amino acids). Purification of either recombinant Bacillus megaterium or Synechocystis CbiXL in Escherichia coli, which is facilitated by the presence of a naturally occurring histidine-rich region of the protein, results in the isolation of a dark brown protein solution. The UV/visible spectrum of the protein is consistent with the presence of a redox group, and the lack of definition within the spectrum is suggestive of a 4Fe-4S center. The presence of an iron-sulfur center was confirmed by EPR analysis of the proteins, which produces a pseudoaxial spectrum with g values at 2.04, 1.94, and 1.90. The EPR spectrum was absent at 70 K, an observation that is diagnostic of a 4Fe-4S center. Redox potentiometry coupled with optical spectroscopy allowed the midpoint potential of the redox center to be determined for the CbiXL from both B. megaterium and Synechocystis. Sequence analysis of CbiXL proteins reveals only two conserved cysteine residues within the CbiXL proteins, which are part of an MXCXXC motif. Mutagenesis of the two cysteines leads to loss of both the EPR spectrum and UV/visible spectral features of the Fe-S center in the protein, clearly indicating that these residues are involved in ligating the cofactor to the apoprotein possibly in a butterfly arrangement. The potential physiological role of the iron-sulfur center is discussed.

Background: The association between herpes simplex virus type 2 (HSV-2) and human immunodeficiency virus (HIV) and the development of HSV vaccines have increased interest in the study of HSV epidemiology., Objectives: To estimate the age and sex specific seroprevalence of HSV-1 and HSV-2 infections in selected populations in Brazil, Estonia, India, Morocco, and Sri Lanka., Methods: Serum samples were collected from various populations including children, antenatal clinic attenders, blood donors, hospital inpatients, and HIV sentinel surveillance groups. STD clinic attenders were enrolled in Sri Lanka, male military personnel in Morocco. Sera were tested using a common algorithm by type specific HSV-1 and HSV-2 antibody assay., Results: 13,986 samples were tested, 45.0% from adult females, 32.7% from adult males, and 22.3% from children. The prevalence of HSV-1 varied by site ranging from 78.5%-93.6% in adult males and from 75.5%-97.8% in adult females. In all countries HSV-1 seroprevalence increased significantly with age (p<0.001) in both men and women. The prevalence of HSV-2 infection varied between sites. Brazil had the highest age specific rates of infection for both men and women, followed by Sri Lanka for men and Estonia for women, the lowest rates being found in Estonia for men and India for women. In all countries, HSV-2 seroprevalence increased significantly with age (p<0.01) and adult females had higher rates of infection than adult males by age of infection., Conclusions: HSV-1 and HSV-2 seroprevalence was consistently higher in women than men, particularly for HSV-2. Population based data on HSV-1 and HSV-2 will be useful for designing potential HSV-2 vaccination strategies and for focusing prevention efforts for HSV-1 and HSV-2 infection.

Homeostatic systems for essential and non-essential metals create the cellular environments in which the correct metals are acquired by metalloproteins while the incorrect ones are somehow avoided. Cyanobacteria have metal requirements often absent from other bacteria; copper in thylakoidal plastocyanin, zinc in carboxysomal carbonic anhydrase, cobalt in cobalamin but magnesium in chlorophyll, molybdenum in heterocystous nitrogenase, manganese in thylakoidal water-splitting oxygen-evolving complex. This article reviews: an intracellular trafficking pathway for inward copper supply, the sequestration of surplus zinc by metallothionein (also present in other bacteria) and the detection and export of excess cobalt. We consider the influence of homeostatic proteins on selective metal availability.

Using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, we show that one Zn2+ ion in the Zn4 cluster of cyanobacterial metallothionein is inert to exchange with 67Zn2+. We suggest that this is zinc in site A, which together with the surrounding alpha and beta secondary structure forms a zinc-finger fold.

Herpes simplex virus type 2 (HSV-2) infection is almost always sexually transmitted, and causes genital ulceration. Significant progress in our understanding of HSV infection has occurred over the last decade, in part related to the development of accurate and sensitive laboratory tests to study HSV-2. The application of PCR and type-specific serology to individual cases and in population-based studies has enabled the identification of a potentially important role for HSV-2 infection as a cofactor in the sexual transmission of HIV. This is a particular issue in developing countries. This review describes the epidemiology of HSV-2 infection in the HIV era, the hypotheses regarding HSV-HIV interactions, and research priorities for the developing world.

This study estimated the regional and age- and gender-specific seroprevalences of herpes simplex virus type 1 (HSV-1) and HSV-2 in Ontario, Canada. Stored serum specimens from subjects aged 15 to 44 years, including men (n = 979), women not under prenatal care (n = 638), and women under prenatal care (n = 701) submitted for routine viral serology were randomly selected according to regional population size from public health laboratories. HSV-1 and HSV-2 testing was done with the MRL enzyme immunoassay (EIA) (Focus Technologies), and HSV-2 was also tested by the Gull/Meridian EIA. Specimens discordant for HSV-2 antibodies between the two EIAs were resolved by a recombinant immunoblot assay (Focus Technologies). The overall age- and gender-standardized seroprevalences of HSV-1 and HSV-2 were 51.1% (95% confidence interval [CI], 50.1 to 52.1) and 9.1% (95% CI, 8.6 to 9.7), respectively. The seroprevalence of HSV-1 antibodies increased from 26.9 to 54.7% in men between 15 to 16 and 40 to 44 years of age, from 32.0 to 88.7% in women not under prenatal care, and from 55.2 to 69.2% in women under prenatal care. The seroprevalence of HSV-2 increased from 3.8 to 21.3% in men between 15 to 16 and 40 to 44 years of age, from 0 to 18.9% in women not under prenatal care, and from 3.4 to 23.1% in women under prenatal care. HSV-2 results were discordant for 3.3% (76 of 2,318) of specimens. Both types of HSV antibodies appeared to be acquired earlier among women under prenatal care than among men and women not under prenatal care. Antibodies were more prevalent among people in northern Ontario (72.9% of subjects [range, 68.4 to 77.4%] for HSV-1 and 13.7% of subjects [95% CI, 10.2 to 17.2%] for HSV-2) than elsewhere.

Information on age- and sex-specific prevalence of herpes simplex virus (HSV) types 2 and 1 infections is essential to optimize genital herpes control strategies, which increase in importance because accumulating data indicate that HSV-2 infection may increase acquisition and transmission of human immunodeficiency virus. This review summarizes data from peer-reviewed publications of type-specific HSV seroepidemiologic surveys. HSV-2 prevalence is, in general, highest in Africa and the Americas, lower in western and southern Europe than in northern Europe and North America, and lowest in Asia. HSV-2 and -1 prevalence, overall and by age, varies markedly by country, region within country, and population subgroup. Age-specific HSV-2 prevalence is usually higher in women than men and in populations with higher risk sexual behavior. HSV-2 prevalence has increased in the United States but national data from other countries are unavailable. HSV-1 infection is acquired during childhood and adolescence and is markedly more widespread than HSV-2 infection. Further studies are needed in many geographic areas.

NmtR from Mycobacterium tuberculosis is a new member of the ArsR-SmtB family of metal sensor transcriptional repressors. NmtR binds to the operator-promoter of a gene encoding a P(1) type ATPase (NmtA), repressing transcription in vivo except in medium supplemented with nickel or, to some extent, cobalt. In a cyanobacterial host, Synechococcus PCC 7942 strain R2-PIM8(smt), NmtR-mediated repression is alleviated by cobalt but not nickel or zinc addition, while the related sensor SmtB responds exclusively to zinc. Quantification of the number of atoms of nickel per cell shows that NmtR nickel sensitivity correlates with cytosolic nickel contents. Differential metal discrimination in a common cytosol by SmtB (zinc) and NmtR (cobalt) is not simply explained by affinities at equilibrium; although NmtR does bind nickel substantially more tightly than SmtB, it has a higher affinity for zinc than for cobalt and binds cobalt more weakly than SmtB. SmtB is known to bind and sense zinc at interhelical four-coordinate, tetrahedral sites across the C-terminal alpha 5 helices, while absorption spectroscopy of Co(II)- and Ni(II)-substituted NmtR reveals five- and six-coordinate metal complexes. Site-directed mutagenesis identifies six potential cobalt/nickel ligands that are obligatory for inducer recognition but not repression by NmtR, four of which (Asp(91), His(93), His(104), His(107)) align with alpha 5 ligands of SmtB with two additional His provided by a carboxyl-terminal "extension" (designated alpha 5C). Gel retardation assays reveal that zinc does not allosterically regulate NmtR-DNA binding at concentrations where lower affinity cobalt does. These data suggest that two additional ligands form hexacoordinate metal complexes and are crucial for driving allosteric regulation of DNA binding by NmtR, thereby allowing NmtR to preferentially sense metals that favor higher coordination numbers relative to SmtB.

Zinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 7942, sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA. The dearth of other reported bacterial metallothioneins has been surprising. Here we describe related bacterial metallothioneins (BmtA) from Anabaena PCC 7120, Pseudomonas aeruginosa and Pseudomonas putida that bind multiple zinc ions with high stability towards protons. Thiol modification demonstrates that cysteine coordinates zinc in all of these proteins. Additionally, (111)Cd-NMR, and (111)Cd-edited (1)H-NMR, identified histidine ligands in Anabaena PCC 7120 BmtA, analogous to SmtA. A related Escherichia coli protein bound only a single zinc ion, via four cysteine residues, with low stability towards protons; (111)Cd-NMR and (111)Cd-edited (1)H-NMR confirmed exclusive cysteine-coordination, and these cysteine residues reacted rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid). (1)H-NMR of proteins from P. aeruginosa, Anabaena PCC 7120 and E. coli generated fingerprints diagnostic for the GATA-like zinc finger fold of SmtA. These studies reveal first the existence of multiple bacterial metallothioneins, and second proteins with SmtA-like lone zinc fingers, devoid of a cluster,and designated GatA. We have identified 12 smtA-like genes in sequence databases including four of the gatA type.

Homeostatic mechanisms prevent the accumulation of free zinc in the cytoplasm, raising questions regarding where surplus zinc is stored and how it is delivered to and from these stores. A genetic screen for zinc hypersensitivity in Schizosaccharomyces pombe identified a missense mutation truncating Zhf, an endoplasmic reticulum transporter. These cells were approximately 5-fold more zinc-sensitive than other independent mutants. The targeted disruption of zhf prevented growth on low zinc medium and caused hypersensitivity to elevated zinc/cobalt but resistance to cadmium. The exposure to elevated zinc but not copper also promotes the accumulation of transcripts encoding a metallothionein designated Zym1. The Sty1 pathway is required for maximal zym1 expression but is not obligatory for zinc perception. The targeted disruption of zym1 impaired cadmium tolerance but only slightly impaired zinc tolerance, whereas zym1 overexpression substantially rescued zinc hypersensitivity of zhf(-) cells. Four equivalents of zinc were displaced from Zym1 by up to 12 equivalents of p-(hydroxymercuri)phenylsulphonate. Zym1 thiols react rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) compared with bacterial zinc metallothionein (6.8 and 0.2 x 10(-4) s(-1), respectively). Zym1 is unlike known fungal metallothioneins that are induced by and sequester copper but not zinc. Less zinc but normal cadmium was accumulated by zym1Delta, consistent with zinc sequestration by Zym1 in vivo.

Background: The proportion of cases of genital ulcer disease attributable to herpes simplex virus type 2 (HSV-2) appears to be increasing in sub-Saharan Africa., Goal: To assess the contributions of HIV disease and behavioral response to the HIV epidemic to the increasing proportion of genital ulcer disease (GUD) attributable to HSV-2 in sub-Saharan Africa., Study Design: Simulations of the transmission dynamics of ulcerative sexually transmitted diseases (STDs) and HIV with use of the model STDSIM., Results: In simulations, 28% of GUD was caused by HSV-2 before a severe HIV epidemic. If HIV disease was assumed to double the duration and frequency of HSV-2 recurrences, this proportion rose to 35% by year 2000. If stronger effects of HIV were assumed, this proportion rose further, but because of increased HSV-2 transmission this would shift the peak in HSV-2 seroprevalence to an unrealistically young age. A simulated 25% reduction in partner-change rates increased the proportion of GUD caused by HSV-2 to 56%, following relatively large decreases in chancroid and syphilis., Conclusion: Behavioral change may make an important contribution to relative increases in genital herpes.

A bacterial two-hybrid assay revealed interaction between a protein now designated bacterial Atx1 and amino-terminal domains of copper-transporting ATPases CtaA (cellular import) and PacS (thylakoid import) but not the related zinc (ZiaA) or cobalt (CoaT) transporters from the same organism (Synechocystis PCC 6803). The specificity of metallochaperone interactions coincides with metal specificity. After reconstitution in a N(2) atmosphere, bacterial Atx1 bound 1 mol of copper mol(-1), and apoPacS(N) acquired copper from copper-Atx1. Copper was displaced from Atx1 by p-(hydroxymercuri)phenylsulfonate, indicative of thiol ligands, and two cysteine residues were obligatory for two-hybrid interaction with PacS(N). This organism contains compartments (thylakoids) where the copper proteins plastocyanin and cytochrome oxidase reside. In copper super-supplemented mutants, photooxidation of cytochrome c(6) was greater in Deltaatx1DeltactaA than in DeltactaA, showing that Atx1 contributes to efficient switching from iron in cytochrome c(6) to copper in plastocyanin for photosynthetic electron transport. Cytochrome oxidase activity was also less in membranes purified from low [copper]-grown Deltaatx1 or DeltapacS, compared with wild-type, but the double mutant Deltaatx1DeltapacS was non-additive, consistent with Atx1 acting via PacS. Conversely, activity in Deltaatx1DeltactaA was less than in either respective single mutant, revealing that Atx1 can function without the major copper importer and consistent with a role in recycling endogenous copper.

Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. A zinc-induced gene, smtA, is required for normal zinc-tolerance in the cyanobacterium Synechococcus PCC 7942. Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn(4)Cys(9)His(2) cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys(4) and two ZnCys(3)His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn(4)Cys(11) cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands. Also, SmtA contains elements of secondary structure not found in metallothioneins. One of the two Cys(4)-coordinated zinc ions in SmtA readily exchanges with exogenous metal ((111)Cd), whereas the other is inert. The thiolate sulfur ligands bound to zinc in this site are buried within the protein. Regions of beta-strand and alpha-helix surround the inert site to form a zinc finger resembling the zinc fingers in GATA and LIM-domain proteins. Eukaryotic zinc fingers interact specifically with other proteins or DNA and an analogous interaction can therefore be anticipated for prokaryotic zinc fingers. SmtA now provides structural proof for the existence of zinc fingers in prokaryotes, and sequences related to the zinc finger motif can be identified in several bacterial genomes.

Objectives: To estimate age- and sex-specific herpes simplex virus type-2 (HSV-2) prevalence in urban African adult populations and to identify factors associated with infection., Design and Methods: Cross-sectional, population-based samples of about 2000 adults interviewed in each of the following cities: Cotonou, Benin; Yaoundé, Cameroon; Kisumu, Kenya and Ndola, Zambia. Consenting study participants were tested for HIV, HSV-2 and other sexually transmitted infections., Results: HSV-2 prevalence was over 50% among women and over 25% among men in Yaoundé, Kisumu and Ndola, with notably high rates of infection among young women in Kisumu and Ndola (39% and 23%, respectively, among women aged 15-19 years). The prevalence in Cotonou was lower (30% in women and 12% in men). Multivariate analysis showed that HSV-2 prevalence was significantly associated with older age, ever being married, and number of lifetime sexual partners, in almost all cities and both sexes. There was also a strong, consistent association with HIV infection. Among women, the adjusted odds ratios for the association between HSV-2 and HIV infections ranged from 4.0 [95% confidence interval (CI) = 2.0-8.0] in Kisumu to 5.5 (95% CI = 1.7-18) in Yaoundé, and those among men ranged from 4.6 (95% CI = 2.7-7.7) in Ndola to 7.9 (95% CI = 4.1-15) in Kisumu., Conclusions: HSV-2 infection is highly prevalent in these populations, even at young ages, and is strongly associated with HIV at an individual level. At a population level, HSV-2 prevalence was highest in Kisumu and Ndola, the cities with the highest HIV rates, although rates were also high among women in Yaoundé, where there are high rates of partner change but relatively little HIV infection. The high prevalence of both infections among young people underlines the need for education and counselling among adolescents.

Objective: The objective of this study was to explore whether the differences in rate of spread of HIV in different regions in sub-Saharan Africa could be explained by differences in sexual behaviour and/or factors influencing the probability of HIV transmission during sexual intercourse., Methods: A cross-sectional, population-based study was conducted in two cities with a high HIV prevalence (Kisumu in Kenya and Ndola in Zambia) and two cities with a relatively low HIV prevalence (Cotonou in Benin and Yaoundé in Cameroon). In each of these cities, approximately 1000 men and 1000 women, aged 15-49 years, were randomly selected from the general population. Consenting men and women were interviewed and were tested for HIV, syphilis, herpes simplex virus type 2 (HSV-2), gonorrhoea, chlamydial infection and trichomoniasis (the latter for women only). In addition, a survey was conducted on a random sample of 300 sex workers in each city. The research instruments, including the questionnaires and the laboratory procedures, were standardized to permit comparison of results., Results: The numbers of men interviewed were 1021 in Cotonou, 973 in Yaoundé, 829 in Kisumu, and 720 in Ndola. The corresponding figures for women were 1095, 1116, 1060 and 1130. In Yaoundé, Kisumu and Ndola, the response rates for men were lower than for women due to failure to make contact with eligible men. The proportion of eligible women who were interviewed was 86% in Yaoundé, and 89% in Kisumu and Ndola. In Yaoundé, 76% of eligible men were interviewed, along with 82% in Kisumu and 75% in Ndola. The prevalence of HIV infection in men was 3.3% in Cotonou, 4.1% in Yaoundé, 19.8% in Kisumu and 23.2% in Ndola. For women, the respective figures were 3.4, 7.8, 30.1 and 31.9%. The prevalence of HIV infection among women aged 15-19 years was 23.0% in Kisumu and 15.4% in Ndola. Among women in Kisumu who had their sexual debut 5 years before the interview, the prevalence of HIV infection was 46%; in Ndola, it was 59%. Among sex workers, the prevalence of HIV infection was 57.5% in Cotonou, 34.4% in Yaoundé, 74.7% in Kisumu and 68.7% in Ndola., Conclusions: The HIV prevalence rates in the general population confirmed our preliminary assessment of the level of HIV infection in the four cities, which was based on estimates of HIV prevalence from sentinel surveillance among pregnant women. The very high prevalence of HIV infection among young women in Kisumu and Ndola calls for urgent intervention.

Objectives: To describe the epidemiology of Trichomonas vaginalis infection and its association with HIV infection, in women in four African cities with different levels of HIV infection., Design: Cross-sectional study, using standardized methods, including a standardized questionnaire and standardized laboratory tests, in four cities in sub-Saharan Africa: two with a high prevalence of HIV infection (Kisumu, Kenya and Ndola, Zambia), and two with a relatively low prevalence of HIV (Cotonou, Benin and Yaoundé, Cameroon)., Methods: In each city, a random sample of about 2000 adults aged 15-49 years was taken. Consenting men and women were interviewed about their socio-demographic characteristics and their sexual behaviour, and were tested for HIV, syphilis, herpes simplex virus type 2 (HSV-2), gonorrhoea, chlamydial infection, and (women only) T. vaginalis infection. Risk factor analyses were carried out for trichomoniasis for each city separately. Multivariate analysis, however, was only possible for Yaoundé, Kisumu and Ndola., Results: The prevalence of trichomoniasis was significantly higher in the high HIV prevalence cities (29.3% in Kisumu and 34.3% in Ndola) than in Cotonou (3.2%) and Yaoundé (17.6%). Risk of trichomoniasis was increased in women who reported more lifetime sex partners. HIV infection was an independent risk factor for trichomonas infection in Yaoundé [adjusted odds ratio (OR) = 1.8, 95% confidence interval (CI) = 0.9-3.7] and Kisumu (adjusted OR = 1.7, 95% CI = 1.1-2.7), but not in Ndola. A striking finding was the high prevalence (40%) of trichomonas infection in women in Ndola who denied that they had ever had sex., Conclusion: Trichomoniasis may have played a role in the spread of HIV in sub-Saharan Africa and may be one of the factors explaining the differences in levels of HIV infection between different regions in Africa. The differences in prevalence of trichomoniasis between the four cities remain unexplained, but we lack data on the epidemiology of trichomoniasis in men. More research is required on the interaction between trichomoniasis and HIV infection, the epidemiology of trichomoniasis in men, and trichomonas infections in women who deny sexual activity.

Synechocystis PCC 6803 contains four genes encoding polypeptides with sequence features of CPx-type ATPases, two of which are now designated pacS and ctaA. We show that CtaA and PacS (but not the related transporters, ZiaA or CoaT) facilitate switching to the use of copper (in plastocyanin) as an alternative to iron (in cytochrome c(6)) for the carriage of electrons within the thylakoid lumen. Disruption of pacS reduced copper tolerance but enhanced silver tolerance, and pacS-mediated restoration of copper tolerance was used to select transformants. Disruption of ctaA caused no change in copper tolerance but reduced the amount of copper cell(-1). In cultures supplemented with 0.2 microm copper, photooxidation of cytochrome c(6) (PetJ) was depressed in wild-type cells but remained elevated in both Synechocystis PCC 6803(ctaA) and Synechocystis PCC 6803(pacS). Conversely, plastocyanin transcripts (petE) were less abundant in both mutants at this [copper]. Synechocystis PCC 6803(ctaA) and Synechocystis PCC 6803(pacS) showed increased iron dependence with impaired growth in deferoxamine mesylate (iron chelator)-containing media. Double mutants also deficient in cytochrome c(6), Synechocystis PCC 6803(petJ,ctaA) and Synechocystis PCC 6803(petJ,pacS), were viable, but the former had increased copper dependence with severely impaired growth in the presence of bathocuproinedisulfonic acid (copper chelator). Analogous transporters are likely to supply copper to plastocyanin in chloroplasts.

Bacterial metallothioneins bind, sequester and buffer excess intracellular zinc. At present, the vast majority of the available experimental data relate to cyanobacterial metallothionein, SmtA, from Synechococcus PCC 7942. SmtA is required for normal resistance to zinc and smtA-mediated zinc resistance has been used as a selectable marker. The imidazole groups of histidine residues, in addition to the thiol groups of cysteine residues, co-ordinate zinc in bacterial metallothioneins. The structure of bacterial metallothionein must facilitate some discrimination between 'adventitious' and 'adventageous' zinc-binding sites such that under excess zinc conditions metal is predominantly scavenged from the former. It remains unclear whether or not bacterial metallothionein also acts as a zinc store that supplies zinc-requiring proteins or if under some conditions it deactivates a subset of proteins via zinc removal. Expression of smtA is induced in response to elevated concentrations of zinc via the action of SmtB. SmtB has some sequence similarity to the arsenic responsive repressor ArsR and genes encoding related proteins are present in many bacterial genomes. Metal perception by SmtB differs from ArsR. The latter contains a characteristic Cys-Val-Cys motif associated with a DNA-binding helix-turn-helix (the ArsR motif), while the former contains metal-binding motifs associated with a carboxyl-terminal alpha-helix that forms the interface between SmtB dimers (the SmtB motif). Some SmtB-ArsR family proteins, including the zinc sensor ZiaR from the cyanobacterium Synechocystis PCC 6803, have the metal-sensory motifs of both SmtB and ArsR. The mechanisms of action, and the features that allow discrimination between different metal ions by these sensors, are discussed.