Lack of significant association of an insertion/deletion polymorphism in the angiotensin converting enzyme (ACE) gene with tropical calcific pancreatitis

BACKGROUND: The genetic basis of tropical calcific pancreatitis (TCP) is different and is explained by mutations in the pancreatic secretory trypsin inhibitor (SPINK1) gene. However, mutated SPINK1 does not account for the disease in all the patients, neither does it explain the phenotypic heterogeneity between TCP and fibro-calculous pancreatic diabetes (FCPD). Recent studies suggest a crucial role for pancreatic renin-angiotensin system during chronic hypoxia in acute pancreatitis and for angiotensin converting enzyme (ACE) inhibitors in reducing pancreatic fibrosis in experimental models. We investigated the association of ACE gene insertion/deletion (I/D) polymorphism in TCP patients using a case-control approach. Since SPINK1 mutations are proposed a modifier role, we also investigated its interaction with the ACE gene variant. METHODS: We analyzed the I/D polymorphism in the ACE gene (g.11417_11704del287) in 171 subjects comprising 91 TCP and 80 FCPD patients and compared the allelic and genotypic frequency in them with 99 healthy ethnically matched control subjects. RESULTS: We found 46% and 21% of TCP patients, 56% and 19.6% of FCPD patients and 54.5% and 19.2% of the healthy controls carrying the I/D and D/D genotypes respectively (P>0.05). No significant difference in the clinical picture was observed between patients with and without the del allele at the ACE in/del polymorphism in both categories. No association was observed with the presence or absence of N34S SPINK1 mutation in these patients. CONCLUSION: We conclude that the ACE insertion/deletion variant does not show any significant association with the pathogenesis, fibrosis and progression of tropical calcific pancreatitis and the fibro-calculous pancreatic diabetes

Induction of HIV Neutralizing Antibodies against the MPER of the HIV Envelope Protein by HA/gp41 Chimeric Protein-Based DNA and VLP Vaccines

Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy

Role of Transmitted Gag CTL Polymorphisms in Defining Replicative Capacity and Early HIV-1 Pathogenesis

Initial studies of 88 transmission pairs in the Zambia Emory HIV Research Project cohort demonstrated that the number of transmitted HLA-B associated polymorphisms in Gag, but not Nef, was negatively correlated to set point viral load (VL) in the newly infected partners. These results suggested that accumulation of CTL escape mutations in Gag might attenuate viral replication and provide a clinical benefit during early stages of infection. Using a novel approach, we have cloned gag sequences isolated from the earliest seroconversion plasma sample from the acutely infected recipient of 149 epidemiologically linked Zambian transmission pairs into a primary isolate, subtype C proviral vector, MJ4. We determined the replicative capacity (RC) of these Gag-MJ4 chimeras by infecting the GXR25 cell line and quantifying virion production in supernatants via a radiolabeled reverse transcriptase assay. We observed a statistically significant positive correlation between RC conferred by the transmitted Gag sequence and set point VL in newly infected individuals (p = 0.02). Furthermore, the RC of Gag-MJ4 chimeras also correlated with the VL of chronically infected donors near the estimated date of infection (p = 0.01), demonstrating that virus replication contributes to VL in both acute and chronic infection. These studies also allowed for the elucidation of novel sites in Gag associated with changes in RC, where rare mutations had the greatest effect on fitness. Although we observed both advantageous and deleterious rare mutations, the latter could point to vulnerable targets in the HIV-1 genome. Importantly, RC correlated significantly (p = 0.029) with the rate of CD4+ T cell decline over the first 3 years of infection in a manner that is partially independent of VL, suggesting that the replication capacity of HIV-1 during the earliest stages of infection is a determinant of pathogenesis beyond what might be expected based on set point VL alone. © 2012 Prince et al

Large Changes In The Crac Segment Of Gp41 Of Hiv Do Not Destroy Fusion Activity If The Segment Interacts With Cholesterol

peer reviewedThe membrane-proximal external region (MPER) of the gp41 fusion protein of HIV is highly conserved among isolates of this virus and is considered a target for vaccine development. This region also appears to play a role in membrane fusion as well as localization of the virus to cholesterol-rich domains in membranes. The carboxyl terminus of MPER has the sequence LWYIK and appears to have an important role in cholesterol interactions. We have tested how amino acid substitutions that would affect the conformational flexibility of this segment could alter its interaction with cholesterol. We studied a family of peptides (all peptides as N-acetyl-peptide amides) with P, G, or A substituting for W and I of the LWYIK sequence. The peptide having the greatest effect on cholesterol distribution in membranes was the most flexible one, LGYGK. The corresponding mutation in gp41 resulted in a protein retaining 72% of the fusion activity of the wild-type protein. Two other peptides were synthesized, also containing two Gly residues, GWGIK and LWGIG, and did not have the ability to sequester cholesterol as efficiently as LGYGK did. Making the corresponding mutants of gp41 showed that these other two double Gly substitutions resulted in proteins that were much less fusogenic, although they were equally well expressed at the cell surface. The study demonstrates that drastic changes can be made in the LWYIK segment with the retention of a significant fraction of the fusogenic activity, as long as the mutant proteins interact with cholesterol

Juxtamembrane Protein Segments That Contribute To Recruitment Of Cholesterol Into Domains

We investigated the properties of several peptides with sequences related to LWYIK, a segment found in the gp41 protein of HIV and believed to play a role in sequestering this protein to a cholesterol-rich domain in the membrane. This segment fulfills the requirements to be classified as a CRAC motif that has been suggested to predict those proteins that will partition into cholesterol-rich regions of the membrane. All of the peptides were studied with the terminal amino and carboxyl groups blocked, i.e., as N-acetyl-peptide-amides. Effects of cholesterol on the intensity of W emission generally parallel DSC evidence of sequestration of cholesterol. Modeling studies indicate that all of these peptides tend to partition with their mass center at the membrane interface at the level of the hydroxyl of cholesterol. Interaction with cholesterol is dual: van der Waals interactions between mainly hydrophobic surfaces and electrostatic stabilization of the cholesterol OH group. Thus, both experiments and modeling studies indicate that the preference of CRAC motifs for cholesterol-rich domains might be related to a membrane interfacial preference of the motif, to a capacity to wrap and block the cholesterol polar OH group by H-bond interactions, and to a capacity for peptide aromatic side chains to stack with cholesterol. These results were supported by studies of single mutations in the gp41 protein of HIV-1, in which L(679) is replaced with I. Despite the similarity of the properties of these amino acid residues, this single substitution resulted in a marked attenuation of the ability of JC53-BL HeLa-based HIV-1 indicator cells to form syncytia

Sex Hormones Selectively Impact the Endocervical Mucosal Microenvironment: Implications for HIV Transmission

Several studies suggest that progesterone and estrogens may affect HIV transmission in different, possibly opposing ways. Nonetheless, a direct comparison of their effects on the mucosal immune system has never been done. We hypothesize that sex hormones might impact the availability of cells and immune factors important in early stages of mucosal transmission, and, in doing so influence the risk of HIV acquisition. To test this hypothesis, we employed 15 ovarectomized rhesus macaques: 5 were treated with Depot Medroxy Progesterone Acetate (DMPA), 6 with 17-β estradiol (E2) and 4 were left untreated. All animals were euthanized 5 weeks after the initiation of hormone treatment, a time post-DMPA injection associated with high susceptibility to SIV infection. We found that DMPA-treated macaques exhibited higher expression of integrin α4β7 (α4β7) on CD4+ T cells, the gut homing receptor and a marker of cells highly susceptible to HIV, in the endocervix than did the E2-treated animals. In contrast, the frequency of CCR5+ CD4+ T cells in DMPA-treated macaques was higher than in the E2-treated group in vaginal tissue, but lower in endocervix. α4β7 expression on dendritic cells (DCs) was higher in the DMPA-treated group in the endocervical tissue, but lower in vaginal tissue and on blood DCs compared with the E2-treated animals. Soluble MAdCAM-1, the α4β7 ligand, was present in the vaginal fluids of the control and E2-treated groups, but absent in the fluids from DMPA-treated animals. Both hormones modulated the expression and release of inflammatory factors and modified the distribution of sialomucins in the endocervix. In summary, we found that sex hormones profoundly impact mucosal immune factors that are directly implicated in HIV transmission. The effect is particularly significant in the endocervix. This may increase our understanding of the potential hormone-driven modulation of HIV susceptibility and potentially guide contraceptive policies in high-risk settings

Cholesterol-recognition motifs in membrane proteins

Abstract: The impact of cholesterol on the structure and function of membrane proteins was recognized several decades ago, but the molecular mechanisms underlying these effects have remained elusive. There appear to be multiple mechanisms by which cholesterol interacts with proteins. A complete understanding of cholesterol-sensing motifs is still undergoing refinement. Initially, cholesterol was thought to exert only non-specific effects on membrane fluidity. It was later shown that this lipid could specifically interact with membrane proteins and affect both their structure and function. In this article, we have summarized and critically analyzed our evolving understanding of the affinity, specificity and stereoselectivity of the interactions of cholesterol with membrane proteins. We review the different computational approaches that are currently used to identify cholesterol binding sites in membrane proteins and the biochemical logic that governs each type of site, including CRAC, CARC, SSD and amphipathic helix motifs. There are physiological implications of these cholesterol-recognition motifs for G-protein coupled receptors (GPCR) and ion channels, in membrane trafficking and membrane fusion (SNARE) proteins. There are also pathological implications of cholesterol binding to proteins involved in neurological disorders (Alzheimer, Parkinson, Creutzfeldt-Jakob) and HIV fusion. In each case, our discussion is focused on the key molecular aspects of the cholesterol and amino acid motifs in membrane-embedded regions of membrane proteins that define the physiologically relevant crosstalk between the two. Our understanding of the factors that determine if these motifs are functional in cholesterol binding will allow us enhanced predictive capabilities