Gorakh Mal

Distinct roles for tetraspanins CD9, CD63 and CD81 in the formation of multinucleated giant cells

Members of the tetraspanin superfamily of proteins are implicated in a variety of complex cell processes including cell fusion. However, the contribution of individual tetraspanins to these processes has proved difficult to define. Here we report the use of recombinant extracellular regions of tetraspanins to investigate the role of specific members of this family in the fusion of monocytes to form multinucleated giant cells (MGC). In contrast to their positive requirement in sperm–egg fusion, previous studies using antibodies and knockout mice have indicated a negative regulatory role for tetraspanins CD9 and CD81 in this process. In an in vitro model of fusion using human monocytes, we have confirmed observations that antibodies to CD9 and CD81 enhance MGC formation; however, in contrast to previous investigations, we found that all members of a panel of antibodies to CD63 inhibited fusion. Moreover, recombinant proteins corresponding to the large extracellular domains (EC2s) of CD63 and CD9 inhibited MGC formation, whereas the EC2s of CD81 and CD151 had no effect. The potent inhibition of fusion and binding of labelled CD63 EC2 to monocytes under fusogenic conditions suggest a direct interaction with a membrane component required for fusion. Our findings indicate that the tetraspanins CD9, CD63 and CD81 are all involved in MGC formation, but play distinct roles.

Proteomic identification of camel seminal plasma: Purification of β-nerve growth factor

The camel seminal plasma contains a diverse array of components including lipids, carbohydrates, peptides, ions and proteins. These are essential for maintaining normal physiology of spermatozoa and are secreted mainly from the prostrate, epidydimis and bulbo-urethral glands of reproductive system. The protein profiles of camel seminal plasma were resolved by two-dimensional gel electrophoresis (2D-PAGE). The majority of the protein was found in acidic regions below pI 7.0 and the 19 brightly stained proteins were identified by MALDI-TOF/MS analysis. On the basis of proteomic profiles, β-nerve growth factor (β-NGF) was purified by ion-exchange and gel filtration chromatography and identified by SDS-PAGE and MALDI-TOF/MS analysis. It was further confirmed by western blotting experiments using rabbit anti-β-NGF primary antibody.

Bioengineered probiotics as a new hope for health and diseases: an overview of potential and prospects.

Despite the use of microorganisms as therapeutics for over a century, the scientific and clinical admiration of their potential is a recent phenomenon. Genome sequencing and genetic engineering has enabled researchers to develop novel strategies, such as bioengineered probiotics or pharmabiotics, which may become a therapeutic strategy. Bioengineered probiotics with multiple immunogenic or antagonistic properties could be a viable option to improve human health. The bacteria are tailored to deliver drugs, therapeutic proteins or gene therapy vectors with precision and a higher degree of site specificity than conventional drug administration regimes. This article provides an overview of methodological concepts, thereby encouraging research and interest in this topic, with the ultimate goal of using designer probiotics as therapeutics in clinical practice.

Multiligand Specificity of Pathogen-associated Molecular Pattern-binding Site in Peptidoglycan Recognition Protein

The peptidoglycan recognition protein PGRP-S is an innate immunity molecule that specifically interacts with microbial peptidoglycans and other pathogen-associated molecular patterns. We report here two structures of the unique tetrameric camel PGRP-S (CPGRP-S) complexed with (i) muramyl dipeptide (MDP) at 2.5 Å resolution and (ii) GlcNAc and β-maltose at 1.7Å resolution. The binding studies carried out using surface plasmon resonance indicated that CPGRP-S binds to MDP with a dissociation constant of 10−7 m, whereas the binding affinities for GlcNAc and β-maltose separately are in the range of 10−4 m to 10−5 m, whereas the dissociation constant for the mixture of GlcNAc and maltose was estimated to be 10−6 m. The data from bacterial suspension culture experiments showed a significant inhibition of the growth of Staphylococcus aureus cells when CPGRP-S was added to culture medium. The ELISA experiment showed that the amount of MDP-induced production of TNF-α and IL-6 decreased considerably after the introduction of CPGRP-S. The crystal structure determinations of (i) a binary complex with MDP and (ii) a ternary complex with GlcNAc and β-maltose revealed that MDP, GlcNAc, and β-maltose bound to CPGRP-S in the ligand binding cleft, which is situated at the interface of molecules C and D of the homotetramer formed by four protein molecules A, B, C, and D. In the binary complex, the muramyl moiety of MDP is observed at the C-D interface, whereas the peptide chain protrudes into the center of tetramer. In the ternary complex, GlcNAc and β-maltose occupy distinct non-overlapping positions belonging to different subsites.

Structural basis of the binding of fatty acids to peptidoglycan recognition protein, PGRP-S through second binding site

Short peptidoglycan recognition protein (PGRP-S) is a member of the mammalian innate immune system. PGRP-S from Camelus dromedarius (CPGRP-S) has been shown to bind to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Its structure consists of four molecules A, B, C and D with ligand binding clefts situated at A-B and C-D contacts. It has been shown that LPS, LTA and PGN bind to CPGRP-S at C-D contact. The cleft at the A-B contact indicated features that suggested a possible binding of fatty acids including mycolic acid of Mycobacterium tuberculosis. Therefore, binding studies of CPGRP-S were carried out with fatty acids, butyric acid, lauric acid, myristic acid, stearic acid and mycolic acid which showed affinities in the range of 10(-5) to 10(-8) M. Structure determinations of the complexes of CPGRP-S with above fatty acids showed that they bound to CPGRP-S in the cleft at the A-B contact. The flow cytometric studies showed that mycolic acid induced the production of pro-inflammatory cytokines, TNF-α and IFN-γ by CD3+ T cells. The concentrations of cytokines increased considerably with increasing concentrations of mycolic acid. However, their levels decreased substantially on adding CPGRP-S.

Studies on Liquefaction Time and Proteins Involved in the Improvement of Seminal Characteristics in Dromedary Camels (Camelus dromedarius)

Semen was collected from six dromedary camels using artificial vagina during rutting season. Liquefaction of the viscous semen occurred in 23.89 ± 1.49 h. During liquefaction, proteins with molecular masses of 24.55 kDa and 22.07 kDa appeared in conjunction with the disappearance of intact 26.00 kDa protein after 18–24 h. These proteins were identified as β-nerve growth factors (β-NGFs) in liquefied camel semen. Guanidine-HCL improves the rheological characteristics of dromedary camel semen along with significant (P < 0.01) increase in sperm motility. No significant differences were found in viability of spermatozoa indicating no visible detrimental effects on spermatozoa. The cause of semen viscosity, as well as proteins that are present in liquefied dromedary camel seminal plasma, is described for the first time.

Probiotics in Female Reproductive Health: Paradigms, Prospects and Challenges

Feminine reproductive health is crucial for her well-being as well as reproduction, and health of neonates. Certain microbes not only impede conception and birth of healthy baby, but can also affect maternal-fetal health, and may severely perturb the feminine reproductive health. Among the alternative strategies to prevent microbial pathogenesis, the probiotics and their metabolites have emerged as potent biotherapeutics. The probiotics have potential for preventing various genitourinary infections and consequently reduce the fetal and neonatal infections. Efforts are now underway to better understand the elemental role of human microbial communities in health and disease. While the population structure and mechanisms of human symbionts are only now being unveiled, potential health attributes are suggested by manipulating and modulating microbial functional ecology with the use of probiotics. This article reviews promising paradigms and prospects of probiotics and probiotic-metabolites in reproductive health of women. The purpose is to provide impetus to facilitate the applications of research outcomes in this important area of research into medical practice.

Epigenetics, Probiotic Metabolites and Colon Cancer Prevention: An Overview of Progress, Opportunities and Challenges

Colorectal cancer (CRC) is a common epithelial neoplasia across the globe, with about 1.2 million newly diagnosed cases and over 600,000 mortalities annually. The underlying causes are numerous including dietary ingredients, environmental factors and gut metabolome that affect colon cellular gene regulation and post-translational modifications of the proteome. Collectively, these alterations initiate epigenetic modifications leading to the inception of various metabolic diseases and malignancies. Epigenetics is at the core of modern medicine since it deals with complex processes that turn the expression of certain genes on and off, and it elucidates the mechanisms and connections between genotypes and the environment during various stages of life. However, the majority of the vertical molecular mechanisms with reference to epigenomics, epigenetic and metabolic ailments are still unidentified and need further research. This article summarizes the burgeoning role of probiotic metabolites and some prominent therapeutic dietary ingredients in the management of CRC.

In silico functional elucidation of uncharacterized proteins of Chlamydia abortus strain LLG

Aim: This study reports structural modeling, molecular dynamics profiling of hypothetical proteins in Chlamydia abortus genome database. Methodology: The hypothetical protein sequences were extracted from C. abortus LLG Genome Database for functional elucidation using in silico methods. Results: Fifty-one proteins with their roles in defense, binding and transporting other biomolecules were unraveled. Forty-five proteins were found to be nonhomologous to proteins present in hosts infected by C. abortus. Of these, 31 proteins were related to virulence. The structural modeling of two proteins, first, WP_006344020.1 (phosphorylase) and second, WP_006344325.1 (chlamydial protease/proteasome-like activity factor) were accomplished. The conserved active sites necessary for the catalytic function were analyzed. Conclusion: The finally concluded proteins are envisioned as possible targets for developing drugs to curtail chlamydial infections, however, and should be validated by molecular biological methods.