Structural features of a polysaccharide from the mucin of water hyacinth

The mucin found in the nodal region of the weed, water hyacinth (Eichhornia crassipes), is a heteropolysaccharide composed of d-xylose, l-galactose and l-arabinose in the mol ratio of 1.3:1.2:1.0. Partial hydrolysis with acid gave four oligosaccharides which were characterized as: d-Xylp-(1 → 3)-l-Ara, l-Galp-(1 -→ 2)-l-Ara, d-Xylp-(1 → 3)-l-Galp-(1 → 2)-l-Ara, and d-Xylp-(1 → 2)-d-Xylp-(1 → 3)-l-Galp-(1 → 2)-l-Ara. These, together with the results of methylation analysis using GC and GC/MS and periodate oxidation, indicated that the trisaccharide repeating unit, → 4)-d-Xylp-(1 → 3)-l-Galp-(1 → 2)-l-Araf-(1 →, constitutes the backbone of the polysaccharide. Further, all the d-xylopyranosyl residues of the backbone are substituted at O-2 and, in addition, one out of seven such residues is also substituted at O-3; the substituents being l-Araf-(1 →, d-Xylp-(1 →, l-Galp-(1 →, d-Xylp-(1 → 3)-l-Araf-(1 →, residues.     

Mitochondrial Complex II Prevents Hypoxic but Not Calcium- and Proapoptotic Bcl-2 Protein-induced Mitochondrial Membrane Potential Loss

Mitochondrial membrane potential loss has severe bioenergetic consequences and contributes to many human diseases including myocardial infarction, stroke, cancer, and neurodegeneration. However, despite its prominence and importance in cellular energy production, the basic mechanism whereby the mitochondrial membrane potential is established remains unclear. Our studies elucidate that complex II-driven electron flow is the primary means by which the mitochondrial membrane is polarized under hypoxic conditions and that lack of the complex II substrate succinate resulted in reversible membrane potential loss that could be restored rapidly by succinate supplementation. Inhibition of mitochondrial complex I and F₀F₁-ATP synthase induced mitochondrial depolarization that was independent of the mitochondrial permeability transition pore, Bcl-2 (B-cell lymphoma 2) family proteins, or high amplitude swelling and could not be reversed by succinate. Importantly, succinate metabolism under hypoxic conditions restores membrane potential and ATP levels. Furthermore, a reliance on complex II-mediated electron flow allows cells from mitochondrial disease patients devoid of a functional complex I to maintain a mitochondrial membrane potential that conveys both a mitochondrial structure and the ability to sequester agonist-induced calcium similar to that of normal cells. This finding is important as it sets the stage for complex II functional preservation as an attractive therapy to maintain mitochondrial function during hypoxia.     

Control of apoptosis by IP(3) and ryanodine receptor driven calcium signals

Intracellular calcium signals mediated by IP(3)and ryanodine receptors (IP(3)R/RyR) play a central role in cell survival, but emerging evidence suggests that IP(3)R/RyR are also important in apoptotic cell death. Switch from the life program to the death program may involve coincident detection of proapoptotic stimuli and calcium signals or changes in the spatiotemporal pattern of the calcium signal or changes at the level of effectors activated by the calcium signal (e.g. calpain, calcineurin). The fate of the cell is often determined in the mitochondria, where calcium spikes may support cell survival through stimulation of ATP production or initiate apoptosis v ia opening of the permeability transition pore and release of apoptotic factors such as cytochrome c. The functional importance of these mitochondrial calcium signalling pathways has been underscored by the elucidation of a highly effective, local Ca(2+)coupling between IP(3)R/RyR and mitochondrial Ca(2+)uptake sites. This article will focus on the IP(3)R/RyR-dependent pathways to apoptosis, particularly on the mitochondrial phase of the death cascade.     

Molecular mechanisms of anti-angiogenic effect of curcumin

Modulation of pathological angiogenesis by curcumin (diferuloylmethane), the active principle of turmeric, seems to be an important possibility meriting mechanistic investigations. In this report, we have studied the effect of curcumin on the growth of Ehrlich ascites tumor cells and endothelial cells in vitro. Further, regulation of tumor angiogenesis by modulation of angiogenic ligands and their receptor gene expression in tumor and endothelial cells, respectively, by curcumin was investigated. Curcumin, when injected intraperitoneally (i.p) into mice, effectively decreased the formation of ascites fluid by 66% in EAT bearing mice in vivo. Reduction in the number of EAT cells and human umbelical vein endothelial cells (HUVECs) in vitro by curcumin, without being cytotoxic to these cells, is attributed to induction of apoptosis by curcumin, as is evident by an increase in cells with fractional DNA content seen in our results on FACS analysis. However, curcumin had no effect on the growth of NIH3T3 cells. Curcumin proved to be a potent angioinhibitory compound, as demonstrated by inhibition of angiogenesis in two in vivo angiogenesis assay systems, viz. peritoneal angiogenesis and chorioallantoic membrane assay. The angioinhibitory effect of curcumin in vivo was corroborated by the results on down-regulation of the expression of proangiogenic genes, in EAT, NIH3T3, and endothelial cells by curcumin. Our results on Northern blot analysis clearly indicated a time-dependent (0-24h) inhibition by curcumin of VEGF, angiopoietin 1 and 2 gene expression in EAT cells, VEGF and angiopoietin 1 gene expression in NIH3T3 cells, and KDR gene expression in HUVECs. Further, decreased VEGF levels in conditioned media from cells treated with various doses of curcumin (1 microM-1mM) for various time periods (0-24h) confirm its angioinhibitory action at the level of gene expression. Because of its non-toxic nature, curcumin could be further developed to treat chronic diseases that are associated with extensive neovascularization.     

The polypeptide chain-releasing factor GSPT1/eRF3 is proteolytically processed into an IAP-binding protein

Smac/Diablo and HtrA2/Omi are inhibitors of apoptosis (IAP)-binding proteins released from the mitochondria of human cells during apoptosis and regulate apoptosis by liberating caspases from IAP inhibition. Here we describe the identification of a proteolytically processed isoform of the polypeptide chain-releasing factor GSPT1/eRF3 protein, which functions in translation, as a new IAP-binding protein. In common with other IAP-binding proteins, the processed GSPT1 protein harbors a conserved N-terminal IAP-binding motif (AKPF). Additionally, processed GSPT1 interacts biochemically with IAPs and could promote caspase activation, IAP ubiquitination and apoptosis. The IAP-binding motif of the processed GSPT1 is absolutely required for these activities. Our findings are consistent with a model whereby processing of GSPT1 into the IAP-binding isoform could potentiate apoptosis by liberating caspases from IAP inhibition, or target IAPs and the processed GSPT1 for proteasome-mediated degradation.     

SLC25A23 augments mitochondrial Ca2+ uptake,interacts with MCU,and induces oxidative stress–mediated cell death

Emerging findings suggest that two lineages of mitochondrial Ca²⁺ uptake participate during active and resting states: 1) the major eukaryotic membrane potential–dependent mitochondrial Ca²⁺ uniporter and 2) the evolutionarily conserved exchangers and solute carriers, which are also involved in ion transport. Although the influx of Ca²⁺ across the inner mitochondrial membrane maintains metabolic functions and cell death signal transduction, the mechanisms that regulate mitochondrial Ca²⁺ accumulation are unclear. Solute carriers—solute carrier 25A23 (SLC25A23), SLC25A24, and SLC25A25—represent a family of EF-hand–containing mitochondrial proteins that transport Mg-ATP/Pi across the inner membrane. RNA interference–mediated knockdown of SLC25A23 but not SLC25A24 and SLC25A25 decreases mitochondrial Ca²⁺ uptake and reduces cytosolic Ca²⁺ clearance after histamine stimulation. Ectopic expression of SLC25A23 EF-hand–domain mutants exhibits a dominant-negative phenotype of reduced mitochondrial Ca²⁺ uptake. In addition, SLC25A23 interacts with mitochondrial Ca²⁺ uniporter (MCU; CCDC109A) and MICU1 (CBARA1) while also increasing I_MCU. In addition, SLC25A23 knockdown lowers basal mROS accumulation, attenuates oxidant-induced ATP decline, and reduces cell death. Further, reconstitution with short hairpin RNA–insensitive SLC25A23 cDNA restores mitochondrial Ca²⁺ uptake and superoxide production. These findings indicate that SLC25A23 plays an important role in mitochondrial matrix Ca²⁺ influx.     

Nitric oxide prevents anoxia-induced apoptosis in colonic HT29 cells.

Apoptosis is a critical determinant of tissue mass homeostasis and may play a role in carcinogenesis. The aim of the present study was to investigate anoxia-induced cell death in colon-derived HT29 cells and the effect of nitric oxide on this phenomenon. It was found that HT29 cells subjected to anoxia undergo apoptosis in a time dependent manner, as determined by DNA fragmentation and Hoechst-33258 dye staining. Cytochrome c release from mitochondria to cytosol is a key step in this process and this release precedes DNA fragmentation. NO inhibits anoxia induced apoptosis in these cells by inhibiting the release of cytochrome c and thus may play a role in modulating the apoptotic cell death of colon-derived epithelial cells.