Protective effect of Premna tomentosa extract (L. verbanacae) on acetaminophen-induced mitochondrial dysfunction in rats

Allurement of herbs as health beneficial foods (physiologically functional foods) and as a source material for the development of new drugs, has led to greater furtherance in the study of herbal medicines during recent years. Plant extracts are being utilized to treat a wide variety of diseases like hepatotoxicity. Premna tomentosa is one such medicinal plant used widely in Indian ayurvedic medicine for the treatment of liver disorders. This study appraised the effectiveness of P. tomentosa leaf extract in protecting the liver against mitochondrial damage induced by acetaminophen, since mitochondrial injury has been investigated as a potential initiator of hepatotoxicity. Normal Wistar strain rats were pre-treated with P. tomentosa extract (750 mg/kg, orally) for 15 days and then intoxicated with acetaminophen (640 mg/kg, orally). Mitochondria were isolated from liver of experimental animals and assessed for the levels of lipid peroxide products, GSH and mitochondrial enzymes (isocitrate dehydrogenase, -keto glutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH dehydrogenase and cytochrome-C-oxidase). The levels of Lipid peroxidation products were increased and the levels of the other assessed parameters were significantly decreased in hepatotoxicity induced animals. Whereas, the levels were brought back to normal in P. tomentosa pre-treated rats, which shows the protective effect of the extract against mitochondrial damage. Presence of anti-oxidant compound d-limonene (58%) in P. tomentosa leaves, which is known to enhance conjugation of toxic metabolites by maintaining liver GSH concentrations may explain the hepatoprotective property of the extract.     

Effect of bacoside A on membrane-bound ATPases in the brain of rats exposed to cigarette smoke

Membrane-bound enzymes play a vital role in neuronal function through maintenance of membrane potential and impulse propagation. We have evaluated the harmful effects of chronic cigarette smoking on membrane-bound ATPases and the protective effect of Bacoside A in rat brain. Adult male albino rats were exposed to cigarette smoke for a period of 12 weeks and simultaneously administered with Bacoside A (the active principle isolated from Bacopa monniera) at a dosage of 10 mg/kg b.w/day, p.o. The levels of lipid peroxides as marker for evaluating the extent of membrane damage, the activities of Na+/K+-ATPase, Ca2+-ATPase and Mg2+-ATPase, and associated cations sodium (Na+), potassium (K+), calcium (Ca2+), and magnesium (Mg2+) were investigated in the brain. Neuronal membrane damage was evident from the elevated levels of lipid peroxides and decreased activities of membrane-bound enzymes. Disturbances in the electrolyte balance with accumulation of Na+ and Ca2+ and depletion of K+ and Mg2+ were also observed. Administration of Bacoside A inhibited lipid peroxidation, improved the activities of ATPases, and maintained the ionic equilibrium. The results of our study indicate that Bacoside A protects the brain from cigarette smoking induced membrane damage.     

Double-spanning plant viral movement protein integration into the endoplasmic reticulum membrane is signal recognition particle-dependent, translocon-mediated, and concerted

The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, but that the two transmembrane segments of the viral protein leave the translocon and enter the lipid bilayer together.     

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.