Kinetics of iron release from ferric binding protein (FbpA): mechanistic implications in bacterial periplasm-to-cytosol Fe3+ transport

The ferric binding protein (FbpA) transports iron across the periplasmic space of certain Gram-negative bacteria and is an important component involved in iron acquisition by pathogenic Neisseria spp. (Neisseria gonorrheae and Neisseria meningitidis). Previous work has demonstrated that the synergistic anion, required for tight Fe(3+) sequestration by FbpA, also plays a key role in inserting Fe(3+) into the FbpA binding site. Here, we investigate the iron release process from various forms of holo-FbpA, Fe(3+)FbpA-X, during the course of a chelator competition reaction using EDTA and Tiron. Fe(3+)FbpA-X represents the protein assembly complex with different synergistic anions, X = PO(4)(3)(-) and NTA. Stepwise mechanisms of Fe(3+) release are proposed on the basis of kinetic profiles of these chelator competition reactions. Fe(3+)FbpA-PO(4) and Fe(3+)FbpA-NTA react differently with EDTA and Tiron during the Fe(3+)-exchange process. EDTA replaces PO(4)(3)(-) and NTA from the first coordination shell of Fe(3+) and acts as a synergistic anion to give a spectroscopically distinguishable intermediate, Fe(3+)FbpA-EDTA, prior to pulling Fe(3+) out of the protein. Tiron, on the other hand, does not act as a synergistic anion but is a more efficient competing chelator as it removes Fe(3+) from FbpA at rate much faster than EDTA. These results reaffirm the contribution of the synergistic anion to the FbpA iron transport process as the anion, in addition to playing a facilitative role in iron binding, appears to have a "gatekeeper" role, thereby modulating the Fe(3+) release process.     

Small heat shock protein alphaB-crystallin is part of cell cycle-dependent Golgi reorganization

AlphaB-crystallin is a developmentally regulated small heat shock protein known for its binding to a variety of denatured polypeptides and suppression of protein aggregation in vitro. Elevated levels of alphaB-crystallin are known to be associated with a number of neurodegenerative pathologies such as Alzheimer disease and multiple sclerosis. Mutations in alphaB-crystallin gene have been linked to desmin related cardiomyopathy and cataractogenesis. The physiological function of this protein, however, is unknown. Using discontinuous sucrose density gradient fractionation of post-nuclear supernatants, prepared from rat tissues and human glioblastoma cell line U373MG, we have identified discrete membrane-bound fractions of alphaB-crystallin, which co-sediment with the Golgi matrix protein, GM130. Confocal microscopy reveals co-localization of alphaB-crystallin with BODIPY TR ceramide and the Golgi matrix protein, GM130, in the perinuclear Golgi in human glioblastoma U373MG cells. Examination of synchronized cultures indicated that alphaB-crystallin follows disassembly of the Golgi at prometaphase and its reassembly at the completion of cytokinesis, suggesting that this small heat shock protein, with its chaperone-like activity, may have an important role in the Golgi reorganization during cell division.     

New component of ESCRT-I regulates endosomal sorting complex assembly

The endosomal sorting complex required for transport (ESCRT) complexes play a critical role in receptor down-regulation and retroviral budding. Although the crystal structures of two ESCRT complexes have been determined, the molecular mechanisms underlying the assembly and regulation of the ESCRT machinery are still poorly understood. We identify a new component of the ESCRT-I complex, multivesicular body sorting factor of 12 kD (Mvb12), and demonstrate that Mvb12 binds to the coiled-coil domain of the ESCRT-I subunit vacuolar protein sorting 23 (Vps23). We show that ESCRT-I adopts an oligomeric state in the cytosol, the formation of which requires the coiled-coil domain of Vps23, as well as Mvb12. Loss of Mvb12 results in the disassembly of the ESCRT-I oligomer and the formation of a stable complex of ESCRT-I and -II in the cytosol. We propose that Mvb12 stabilizes ESCRT-I in an oligomeric, inactive state in the cytosol to ensure that the ordered recruitment and assembly of ESCRT-I and -II is spatially and temporally restricted to the surface of the endosome after activation of the MVB sorting reaction.     

Bone marrow stromal cells stimulate an angiogenic program that requires endothelial MT1-MMP

Bone marrow-derived stromal/stem cells (BMSCs) have recently been characterized as mediators of tissue regeneration after injury. In addition to preventing fibrosis at the wound site, BMSCs elicit an angiogenic response within the fibrin matrix. The mechanistic interactions between BMSCs and invading endothelial cells (ECs) during this process are not fully understood. Using a three-dimensional, fibrin-based angiogenesis model, we sought to investigate the proteolytic mechanisms by which BMSCs promote vessel morphogenesis. We find that BMSC-mediated vessel formation depends on the proteolytic ability of membrane type 1-matrix metalloproteinase (MT1-MMP). Knockdown of the protease results in a small network of vessels with enlarged lumens. Contrastingly, vessel morphogenesis is unaffected by the knockdown of MMP-2 and MMP-9. Furthermore, we find that BMSC-mediated vessel morphogenesis in vivo follows mechanisms similar to what we observe in vitro. Subcutaneous, cellular fibrin implants in C.B-17/SCID mice form aberrant vasculature when MMPs are inhibited with a broad-spectrum chemical inhibitor, and a very minimal amount of vessels when MT1-MMP proteolytic activity is interrupted in ECs. Other studies have debated the necessity of MT1-MMP in the context of vessel invasion in fibrin, but this study clearly demonstrates its requirement in BMSC-mediated angiogenesis.     

αA-Crystallin and αB-Crystallin Reside in Separate Subcellular Compartments in the Developing Ocular Lens

αA-Crystallin (αA) and αB-crystallin (αB), the two prominent members of the small heat shock family of proteins are considered to be two subunits of one multimeric protein, α-crystallin, within the ocular lens. Outside of the ocular lens, however, αA and αB are known to be two independent proteins, with mutually exclusive expression in many tissues. This dichotomous view is buoyed by the high expression of αA and αB in the lens and their co-fractionation from lens extracts as one multimeric entity, α-crystallin. To understand the biological function(s) of each of these two proteins, it is important to investigate the biological basis of this perceived dichotomy; in this report, we address the question whether αA and αB exist as independent proteins in the ocular lens. Discontinuous sucrose density gradient fractionation and immunoconfocal localization reveal that in early developing rat lens αA is a membrane-associated small heat shock protein similar to αB but with remarkable differences. Employing an established protocol, we demonstrate that αB predominantly sediments with rough endoplasmic reticulum, whereas αA fractionates with smooth membranes. These biochemical observations were corroborated with immunogold labeling and transmission electron microscopy. Importantly, in the rat heart also, which does not contain αA, αB fractionates with rough endoplasmic reticulum, suggesting that αA has no influence on the distribution of αB. These data demonstrate presence of αA and αB in two separate subcellular membrane compartments, pointing to their independent existence in the developing ocular lens.     

Haptoglobin polymorphism among fourteen populations of India

Haptoglobin (HP) is a serum protein that has the capability of binding the extracorpuscular haemoglobin released during haemolysis. It plays an important role in protection of haemolytic disease by reducing the oxidative and peroxidative potential at free haemoglobin. The present study was aimed to determine the prevalence of HP polymorphism among different Indian populations, anthropologically belonging to diverse ethnicity. The polymorphism was screened among 642 unrelated individuals belonging to 14 population groups of India including both tribal and non-tribal caste groups from different geographical regions of India with distinct linguistic affiliations. An attempt is also made to understand the distribution of HP polymorphism among the studied populations. The result reveals the HP gene to be polymorphic in all the studied populations. Except the two tribal populations (Thotis of Andhra Pradesh and Patelias of Rajasthan) and one caste population (Rajput of Himachal Pradesh), all the studied populations are found to obey the Hardy-Weinberg equilibrium. The significance of the present study is elucidated with the prevalence of high mutant HP*2 allele frequency in India. Selection could be one of the most plausible explanations for this high HP frequency because of its uniformly high occurrence among all the studied populations.     

Fungus mediated synthesis of gold nanoparticles and their conjugation with genomic DNA isolated from Escherichia coli and Staphylococcus aureus

Biological systems employing microorganisms have been used as an alternative to conventional chemical techniques for synthesizing gold nanoparticles. In the present study, gold nanoparticles have been synthesized from the supernatant broth (SB) and live cell filtrate (LCF) of the industrially important fungus Penicillium rugulosum. Additionally, potato dextrose broth (PDB) medium which is used for the growth of the fungus has also been able to synthesize gold nanoparticles. The size of the particles has been investigated by Bio-TEM before purification as well as after purification to find the difference in morphology pattern of the nanoparticles. Different characterization techniques like X-ray diffraction (XRD), infra-red (FTIR), X-ray photoelectron (XPS) and UV–vis spectroscopy have been used for analysis of the particles. SB of the fungus has yielded nanoparticles with better morphology and hence further optimization studies were conducted for controlling the size and shape of the above by altering pH and concentration of gold salt. A pH range of 4–6 has favored the synthesis process whereas increasing concentration of gold salt (beyond 2 mM) has resulted in the formation of bigger sized and aggregated nanoparticles. The optimized nanoparticles have been used to conjugate with isolated genomic DNA of bacteria Escherichia coli and Staphylococcus aureus. Visual observation of agarose gel electrophoresis images confirmed the binding of gold nanoparticles (4 μL and 6 μL) with isolated DNA (2 μL) fragments of both the organisms. The slight red shift of the surface plasmon (SP) band and minor aggregations noticed in Bio-TEM images for the DNA conjugated gold nanoparticles indicates that the genomic DNA could stabilize the particles against aggregation owing to negatively charged phosphate backbone.     

Metabolic effects of chronic obestatin infusion in rats

Obestatin is purported to be a peptide hormone encoded in preproghrelin. We studied the metabolic effects of continuous infusion of obestatin via subcutaneously implanted osmotic mini-pumps. Administration of up to 500nmol/kg body weight/day obestatin did not change 24h cumulative food intake or body weight in rats. Similarly, no effects were observed when obestatin was infused at 1000nmol/kg body weight/day for seven days. This dose of obestatin infused during a 24h fast did not alter weight loss, suggesting that obestatin has no effect on energy expenditure, and this dose did not alter glucose or insulin responses during an IPGTT. Obestatin was originally proposed to interact with GPR39 and subsequently the receptor for GLP-1. While both receptors are expressed in pancreatic islets, incubation with obestatin did not alter insulin release from islets in vitro. Moreover, obestatin did not bind to INS-1 beta-cells or HEK cells overexpressing GLP-1 receptors or displace GLP-1 binding to these cells. Our findings do not support the concept that obestatin is a hormone with metabolic actions.     

Some biological characteristics of lipid conjugates of protein antigen that selectively induce delayed-type hypersensitivity in mice

Covalent coupling of lipid to protein antigen, BSA, modifies the immunogenicity leading to selective induction of delayed type hypersensitivity in mice with no or very little concomitant antibody production. The mode of linkage of lipid to protein, however, controls its tissue distribution and retention in the body and cell uptake in vitro. Whereas D-BSA accumulates in the draining lymph node after foot pad inoculation, DA-BSA stays at the site. Further, DA-BSA is eliminated much more slowly and D-BSA more quickly from the body than the native antigen. Although both lipid-conjugates are taken up by lymphoid cells in vitro more than the native antigen, DA-BSA binds significantly more than D-BSA. On the basis of in vitro blastogenic response and enumeration of antigen sensitive cells and in vivo tests of delayed type hypersensitivity, DA-BSA appears superior to D-BSA but neither was as potent as BSA in CFA.