Demand for Programs for Key Populations in Africa from Countries Receiving International Donor Assistance

There has been increasing attention in recent years to the HIV prevention; treatment; and care needs of key populations in Africa; in particular men who have sex with men (MSM); injection drug users (IDU); and female sex workers (FSW). While several major donors have undertaken efforts to prioritize these groups; it remains unclear which African countries are actively seeking donor support for these programs. For this analysis; we reviewed publicly available proposal and budget documentation from the US PEPFAR for fiscal years 2007 through 2010 and Rounds 1 through 10 of the Global Fund to Fight AIDS; Tuberculosis and Malaria for 40 countries in sub-Saharan Africa. Of the 164 searchable documents retrieved; nearly two-thirds contained at least one program serving FSW (65; 107 proposals); less than one-third contained at least one program serving MSM (29; 47 proposals); and a minority proposed programming for IDU (13; 21 proposals). Demand for these programs was highly concentrated in a subset of countries. Epidemiological data for at least one key population was included in a majority of these proposals (63; 67 proposals); but in many cases these data were not linked to programs

Docking to single-domain and multiple-domain proteins: old and new challenges.

The diverse selection of targets in the CAPRI experiments provides grounds for determining the limits of our rigid-body docking program MolFit, and for extending it. We find that the sensitivity of MolFit is high, enabling it to produce reasonably accurate docking solutions when the structures undergo moderate local conformation changes upon complex formation or when the docked molecules are modeled. Yet the ranks of these solutions are sometimes too low to meet the requirements of CAPRI assessment. This indicates that the selectivity of MolFit, which was optimized for docking of unbound X-ray structures, and which relies on the availability of external data from biochemical and bioinformatic sources, needs readjustment in order to meet the challenges presented by NMR or modeled structures. A different challenge is presented by large global conformation changes such as movements of domains. We show that such changes can be accommodated within the rigid-body approximation by employing rigid multibody multistage docking procedures. We also address the difficulty of ranking results from 2-body and multibody docking scans in cases in which there are no external data favoring one option over the other.

Effect of local shape modifications of molecular surfaces on rigid-body protein-protein docking.

Geometric complementarity is the most dominant term in protein-protein docking and therefore, a good geometric representation of the molecules, which takes into account the flexibility of surface residues, is desirable. We present a modified geometric representation of the molecular surface that down-weighs the contribution of specified parts of the surface to the complementarity score. We apply it to the mobile ends of the most flexible side chains: lysines, glutamines and arginines (trimming). The new representation systematically reduces the complementarity scores of the false-positive solutions, often more than the scores of the correct solutions, thereby improving significantly our ability to identify nearly correct solutions in rigid-body docking of unbound structures. The effect of trimming lysine residues is larger than trimming of glutamine or arginine residues. It appears to be independent of the conformations of the trimmed residues but depends on the relative abundance of such residues at the interface and on the non-interacting surface. Combining the modified geometric representation with electrostatic complementarity further improves the docking results.

Neomycin B-arginine conjugate, a novel HIV-1 Tat antagonist: synthesis and anti-HIV activities.

HIV-1 transactivating protein Tat is essential for virus replication and progression of HIV disease. HIV-1 Tat stimulates transactivation by binding to HIV-1 transactivator responsive element (TAR) RNA, and while secreted extracellularly, it acts as an immunosuppressor, an activator of quiescent T-cells for productive HIV-1 infection, and by binding to CXC chemokine receptor type 4 (CXCR4) as a chemokine analogue. Here we present a novel HIV-1 Tat antagonist, a neomycin B-hexaarginine conjugate (NeoR), which inhibits Tat transactivation and antagonizes Tat extracellular activities, such as increased viral production, induction of CXCR4 expression, suppression of CD3-activated proliferation of lymphocytes, and upregulation of the CD8 receptor. Moreover, Tat inhibits binding of fluoresceine isothiocyanate (FITC)-labeled NeoR to human peripheral blood mononuclear cells (PBMC), indicating that Tat and NeoR bind to the same cellular target. This is further substantiated by the finding that NeoR competes with the binding of monoclonal Abs to CXCR4. Furthermore, NeoR suppresses HIV-1 binding to cells. Importantly, NeoR accumulates in the cell nuclei and inhibits the replication of M- and T-tropic HIV-1 laboratory isolates (EC(50) = 0.8-5.3 microM). A putative model structure for the TAR-NeoR complex, which complies with available experimental data, is presented. We conclude that NeoR is a multitarget HIV-1 inhibitor; the structure, and molecular modeling and dynamics, suggest its binding to TAR RNA. NeoR inhibits HIV-1 binding to cells, partially by blocking the CXCR4 HIV-1 coreceptor, and it antagonizes Tat functions. NeoR is therefore an attractive lead compound, capable of interfering with different stages of HIV infection and AIDS pathogenesis.

The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism.

Death-associated protein kinase is a calcium/calmodulin serine/threonine kinase, which positively mediates programmed cell death in a variety of systems. Here we addressed its mode of regulation and identified a mechanism that restrains its apoptotic function in growing cells and enables its activation during cell death. It involves autophosphorylation of Ser(308) within the calmodulin (CaM)-regulatory domain, which occurs at basal state, in the absence of Ca(2+)/CaM, and is inversely correlated with substrate phosphorylation. This type of phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C(6)-ceramide. The substitution of Ser(308) to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca(2+)/CaM-independent substrate phosphorylation as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. Conversely, mutation to aspartic acid reduces the binding of the protein to CaM and abrogates almost completely the death-promoting function of the protein. These results are consistent with a molecular model in which phosphorylation on Ser(308) stabilizes a locked conformation of the CaM-regulatory domain within the catalytic cleft and simultaneously also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device, which keeps death-associated protein kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.

Women's dreams reported during first pregnancy.

This study analysed women's dreams reported during first pregnancy, a subject matter located at the crossroads of the psychology of dreams and the psychology of pregnancy. In the comparison of dreams reported by first-time pregnant women, to those reported by controls, we hypothesized that pregnant women's dreams would: (i) include more pregnancy-related content; (ii) display a higher degree of anxiety; and (iii) rate higher on a primary-process thinking (PPT) scale. As predicted, it was found that pregnancy-related contents significantly occupied pregnant women's dreams, a fact that might be attributed to an attempt to process and master the experience. Contrary to our expectations, it was found that anxiety and PPT were not significantly higher among pregnant women. An attempt to account for these findings raised methodological, as well as theoretical issues, consequently leading to a re-examination of the original hypotheses. Thus, it was claimed that the linkage of pregnancy to increased anxiety and PPT is grossly unbalanced.

Autophosphorylation restrains the apoptotic activity of DRP-1 kinase by controlling dimerization and calmodulin binding.

DRP-1 is a pro-apoptotic Ca2+/calmodulin (CaM)-regulated serine/threonine kinase, recently isolated as a novel member of the DAP-kinase family of proteins. It contains a short extra-catalytic tail required for homodimerization. Here we identify a novel regulatory mechanism that controls its pro-apoptotic functions. It comprises a single autophosphorylation event mapped to Ser308 within the CaM regulatory domain. A negative charge at this site reduces both the binding to CaM and the formation of DRP-1 homodimers. Conversely, the dephosphorylation of Ser308, which takes place in response to activated Fas or tumour necrosis factor-alpha death receptors, increases the formation of DRP-1 dimers, facilitates the binding to CaM and activates the pro-apoptotic effects of the protein. Thus, the process of enzyme activation is controlled by two unlocking steps that must work in concert, i.e. dephosphorylation, which probably weakens the electrostatic interactions between the CaM regulatory domain and the catalytic cleft, and homodimerization. This mechanism of negative autophosphorylation provides a safety barrier that restrains the killing effects of DRP-1, and a target for efficient activation of the kinase by various apoptotic stimuli.

Identification of domains and amino acids involved in GLuR7 ion channel function.

The kainate receptors GluR6 and GluR7 differ considerably in their ion channel properties, despite sharing 86% amino acid sequence identity. When expressed in Xenopus oocytes GluR6 conducts large agonist-evoked currents, whereas GluR7 lacks measurable currents. In the present study, we localized the determinants that are responsible for the functional differences between GluR6 and GluR7 to the extracellular loop domain L3. In addition, we generated several GluR7 point mutants that are able to conduct currents that can be readily measured in Xenopus oocytes. In GluR6, glutamate- and kainate-evoked maximal currents are of the same magnitude when desensitization is inhibited with the lectin concanavalin A. By contrast, all functional GluR7 mutants were found to have glutamate current amplitudes significantly larger than those evoked by kainate. We localized the domain that determines the relative agonist efficacies to the C-terminal half of the L3 domain of GluR7. Our data show that EC(50) values for glutamate (but not for kainate) in GluR7 mutants or chimeras tend to be increased in comparison to the EC(50) values in GluR6. The high EC(50) for wild-type GluR7 reported in the literature appears to be linked to the S1 portion of the agonist-binding domain. Finally, we determined the C-terminal half of the L3 domain plus the far C-terminal domain of GluR7 to be responsible for the recently reported reduction of current amplitude seen when GluR7 is coexpressed with GluR6. We conclude that coexpression of GluR6 and GluR7 leads to nonstochastical assembly of heteromeric receptor complexes.

Structure of phosphatidyl serine (PS) involved in interbilayer salt bridging and hydrogen bonding.

For understanding the experimental results indicating salt bridging and hydrogen bonding between opposite polar surfaces in planar multibilayer structures of phosphatidyl serine (PS), (1) we carried out molecular modeling of the interacting surface layers. The interacting structures in the planar multibilayers are stabilized by salt bridge arrays of the phosphates with their counterions and by hydrogen bonds of ammonia from one polar surface with carbonyls in the opposite one. In multishell liposomes, where the distances between phosphates on the facing each other surfaces are not equal and they are bound to get out of register, the interbilayer interaction cannot extend over a large enough area to form stable structures, except if the salt bridges are strong enough to break down the curved surfaces and form planar multibilayers.

Mutant cycle analysis of the active and desensitized states of an AMPA receptor induced by willardiines.

The halogenated willardiines are agonists at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptors. Although they differ only by the nature of the halogen substituent, they display marked differences in their efficacy to activate the receptor channel opening and in causing desensitization. We have studied the origin of the different agonist properties of the willardiines and in particular the nature of the structural element within the receptor binding domain that is able to distinguish between willardiines at a subatomic resolution of 0.6 A (the difference in radius between F and Br) and allow (S)-5-fluorowillardiine to cause receptor desensitization much more than (S)-5-bromowillardiine. For this purpose, we analyzed, with the thermodynamic mutant cycle method, the active and desensitized states induced by the willardiines in the GluR1 subtype of AMPA receptors and GluR1 mutants in which residues E398, Y446, L646, and S650, within the agonist binding domain, were mutated. The results were used to generate a 3D model of the willardiine docking mode. We suggest that the active and desensitized states of the AMPA-R correspond, respectively, to the open-lobe and closed-lobe conformations of the agonist binding domain.

Spectroscopic studies of inhibited alcohol dehydrogenase from Thermoanaerobacter brockii: proposed structure for the catalytic intermediate state.

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) catalyzes the reversible oxidation of secondary alcohols to the corresponding ketones using NADP(+) as the cofactor. The active site of the enzyme contains a zinc ion that is tetrahedrally coordinated by four protein residues. The enzymatic reaction leads to the formation of a ternary enzyme-cofactor-substrate complex; and catalytic hydride ion transfer is believed to take place directly between the substrate and cofactor at the ternary complex. Although crystallographic data of TbADH and other alcohol dehydrogenases as well as their complexes are available, their mode of action remains to be determined. It is firmly established that the zinc ion is essential for catalysis. However, there is no clear agreement about the coordination environment of the metal ion and the competent reaction intermediates during catalysis. We used a combination of X-ray absorption, circular dichroism (CD), and fluorescence spectroscopy, together with structural analysis and modeling studies, to investigate the ternary complexes of TbADH that are bound to a transition-state analogue inhibitor. Our structural and spectroscopic studies indicated that the coordination sphere of the catalytic zinc site in TbADH undergoes conformational changes when it binds the inhibitor and forms a pentacoordinated complex at the zinc ion. These studies provide the first active site structure of bacterial ADH bound to a substrate analogue. Here, we suggest the active site structure of the central intermediate complex and, more specifically, propose the substrate-binding site in TbADH.

The secreted glycoprotein CREG enhances differentiation of NTERA-2 human embryonal carcinoma cells.

Differentiation of the human embryonal carcinoma cell line NTERA-2 is characterized by changes in morphology, altered patterns of gene expression, reduced proliferative potential, and a loss of tumorigenicity. The cellular repressor of E1A-stimulated genes, CREG, was previously shown to antagonize transcriptional activation and cellular transformation by the Adenovirus E1A oncoprotein. These properties suggested that CREG may function to inhibit cell growth and/or promote differentiation. Here we show that CREG is a secreted glycoprotein which enhances differentiation of NTERA-2 cells. Northern blot analysis reveals that, although CREG mRNA is widely expressed in adult tissues, CREG mRNA is not significantly expressed in pluripotent mouse embryonic stem cells or NTERA-2 embryonal carcinoma cells. CREG mRNA is rapidly induced upon in vitro differentiation of both mouse embryonic stem cells and human NTERA-2 cells. We show that constitutive expression of CREG in NTERA-2 cells enhances neuronal differentiation upon treatment with retinoic acid. Media enriched in CREG was also found to promote NTERA-2 differentiation in the absence of an inducer such as retinoic acid. These studies suggest that secreted CREG protein participates in a signaling cascade important for differentiation of pluripotent stem cells such as those found in teratocarcinomas.

A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity.

Death-associated protein kinase (DAP-kinase) is a Ca(+2)/calmodulin-regulated serine/threonine kinase with a multidomain structure that participates in apoptosis induced by a variety of signals. To identify regions in this protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative manner. We expressed a library of randomly fragmented DAP-kinase cDNA in HeLa cells and treated these cells with IFN-gamma to induce apoptosis. Functional cDNA fragments were recovered from cells that survived the selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-alpha-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the "linker" region, the death domain, and the C-terminal tail of DAP-kinase. Molecular modeling of the complete death domain provided a structural basis for the function of the death-domain-derived fragment by suggesting that the protective fragment constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity.

How well can molecular modelling predict the crystal structure: the case of the ligand-binding domain of glutamate receptors.

The concept that the ligand-binding domain of vertebrate glutamate receptor channels and bacterial periplasmic substrate-binding proteins (PBPs) share similar three-dimensional (3D) structures has gained increasing support in recent years. On the basis of a dual approach that included computer-assisted molecular modelling and functional studies of site-specific mutants, theoretical 3D models of this domain have been proposed. This article reviews to what extent these models could predict the crystal structure of the ligand-binding domain of an ionotropic glutamate receptor subunit recently determined at high resolution by X-ray diffraction studies.

Structural requirements of six naturally occurring isoforms of the IL-18 binding protein to inhibit IL-18.

A novel, constitutively expressed and secreted IL-18 binding protein (IL-18BP) neutralizes IL-18 and thereby suppresses the production of IFN-gamma, resulting in reduced T-helper type 1 immune responses. In the present study, four human and two mouse isoforms, resulting from mRNA splicing and found in various cDNA libraries, were expressed, purified, and assessed for binding and neutralization of IL-18 biological activities. Human IL-18BP isoform a (IL-18BPa) exhibited the greatest affinity for IL-18 with a rapid on-rate, a slow off-rate, and a dissociation constant (K(d)) of 399 pM. IL-18BPc shares the Ig domain of IL-18BPa except for the 29 C-terminal amino acids; the K(d) of IL-18BPc is 10-fold less (2.94 nM). Nevertheless, IL-18BPa and IL-18BPc neutralize IL-18 >95% at a molar excess of two. IL-18BPb and IL-18BPd isoforms lack a complete Ig domain and lack the ability to bind or neutralize IL-18. Murine IL-18BPc and IL-18BPd isoforms, possessing the identical Ig domain, also neutralize >95% murine IL-18 at a molar excess of two. However, murine IL-18BPd, which shares a common C-terminal motif with human IL-18BPa, also neutralizes human IL-18. Molecular modeling identified a large mixed electrostatic and hydrophobic binding site in the Ig domain of IL-18BP, which could account for its high affinity binding to the ligand. It is likely that preferential secretion of functional and nonfunctional isoforms of IL-18BP affect the immune response.

Oligomerization reduces heparin affinity but enhances receptor binding of fibroblast growth factor 2.

The biological response of cells to fibroblast growth factors (FGFs) depends on heparan sulphate glycosaminoglycans sharing particular structural motifs. Heparin induced FGF dimerization has been suggested to mediate receptor dimerization and activation. Here we demonstrate that heparin-derived oligosaccharides that promote receptor binding and activation specifically induce the dimerization of basic FGF (FGF2). These heparin-induced dimers of FGF2 acquire high affinity for receptor binding and are biologically active. Using biotinylated FGF2 bound to immobilized streptavidin gradually saturated with biotin, enabled a quantitative analysis of heparin-dependent and heparin-independent FGF2 monomers and oligomers. Streptavidin induced FGF2 dimers bind and activate FGF receptors only in the presence of heparin. An excess of streptavidin, forcing biotin-FGF2 into monomers, reduces receptor binding and blocks FGF-dependent cell proliferation. All these suggest predominant receptor binding and activation by heparin associated FGF2 oligomers. Unexpectedly, heparin induced dimers and higher order oligomers lose most of their affinity towards heparin. Direct binding of soluble FGF receptors (FGFRs) to either monomers or dimers of FGF2, immobilized on heparin, confirm the preferred association of FGFRs with dimers of FGF2. Computerized molecular docking predicts a cis-oriented FGF2 dimer, stabilized by heparin, which complies with all the experimental data.

Analysis of lissencephaly-causing LIS1 mutations.

Mutations in the LIS1 gene may result in severe abnormalities of brain cortical layering known as lissencephaly. Most lissencephaly-causing LIS1 mutations are deletions that encompass the entire gene, therefore the mechanism of the disease is regarded as haploinsufficiency. So far, 13 different intragenic mutations have been reported: one point mutation, H149R; deletion of exon 9, which results in deleted acids Delta301-334; deletion of exon 4, which results in deleted amino acids Delta40-64; 10 mutations resulting in truncated proteins and one predicted to result in extra amino acids. We studied the consequences of the point mutation, deletion mutation and one of the reported truncations. In order to study LIS1 structure function, we introduced an additional point mutation and other truncations in different regions of the protein. The consequences of these mutations to protein folding were studied by gel filtration, sucrose density gradient centrifugation and measuring resistance to trypsin cleavage. On the basis of our results, we suggest that all truncation mutations and lissencephaly-causing point mutations or internal deletion result in a reduction in the amount of correctly folded LIS1 protein.

Circadian rhythm sleep disorders: toward a more precise definition and diagnosis.

The present article presents a survey of the characteristics of our case series of 322 patients suffering from circadian rhythm sleep disorders (CRSDs), a case-control study comparing a group of 50 CRSD patients and 56 age- and gender-matched normal subjects, and a proposal for new guidelines for improving the diagnosis of CRSD. The major findings were that 83.5% of our CRSD patients who seek medical help are of the delayed sleep phase syndrome (DSPS) type; 89.6% report that the onset of CRSD occurred in early childhood or adolescence; CRSD exhibits no gender differences: a familial trait exists in 44% of patients; and learning disorders (19.3%) and personality disorders (22.4%) in the DSPS-type patients are of high prevalence. The findings of this study point to the importance of clinician awareness of the clinical picture of patients presenting with CRSD so that early diagnosis and effective treatment can be achieved to prevent harmful consequences.

Structural and chemical complementarity between antibodies and the crystal surfaces they recognize.

The sequences of the variable regions of three monoclonal antibodies with different specificities to cholesterol monohydrate and 1,4-dinitrobenzene crystals were determined. The structures of their binding sites were then modeled, based on homology to other antibodies of known structure. Two of these antibodies were previously shown to specifically recognize each one well-defined face of one of the crystals, out of a number of crystal faces of closely related structure. The binding site of the antibody which recognizes the stepped (301) face of the cholesterol crystal is predicted to assume the shape of a step with one hydrophobic and one hydrophilic side, complementary to the corresponding crystal surface. Within the step, the hydroxyl groups of five tyrosines are located such that they can interact with the hydroxyl and water molecules on the cholesterol crystal face, while hydrophobic contacts are made between the cholesterol backbone and hydrophobic amino acid sidechains. In contrast, the modeled binding site of the antibody which recognizes the flat (101) face of 1,4-dinitrobenzene crystals is remarkably flat. It is lined by aromatic and polar residues, that can make favorable contacts with the aromatic ring and nitro groups of the dinitrobenzene molecules, respectively.

GTP-dependent conformational changes associated with the functional switch between Galpha and cross-linking activities in brain-derived tissue transglutaminase.

GTP and Ca2+, two well-known modulators of intracellular signaling pathways, control a structural/functional switch between two vital and mutually exclusive activities, cross-linking and Galpha activity, in the same enzyme. The enzyme, a brain-derived tissue-type transglutaminase (TGase), was recently cloned by us in two forms, one of which (s-TGN) lacks a C-terminal region that is present in the other (l-TGN). Immunoreaction with antibodies directed against a peptide present in the C-terminus of l-TGN but missing in s-TGN suggested that this site, which is located in the C-terminal fourth domain, undergoes conformational changes as a result of interaction between l-TGN and GTP. Site-directed mutagenesis suggested that the third domain is involved in mediating the inhibition of the cross-linking activity. These results were supported by molecular modeling, which further suggested that domains III and IV both participate in conformational changes leading to the functional switch between the Ca2+-dependent cross-linking activity and the Galpha activity.