Tyrosine Phosphorylation of Apoptotic Proteins During Hyperoxia in Mitochondria of the Cerebral Cortex of Newborn Piglets

The present study tests the hypothesis that hyperoxia results in increased tyrosine phosphorylation of apoptotic proteins Bcl-2, Bcl-xl, Bax & Bad in the mitochondrial fraction of the cerebral cortex of newborn piglets. Twelve newborn piglets were divided into normoxic [Nx, n = 6], exposed to a FiO₂ of 0.21 for 1 h and hyperoxic [Hyx, n = 6], exposed to FiO₂ of 1.0 for 1 h. PaO₂ in Hyx group was maintained at 400 mmHg while the Nx group was kept at 80 to100 mmHg. The density (O.D.x mm²) of phosphorylated Bcl2 protein on westernblot was 19.3 ± 3.6 in Nx and 41.5 ± 18.3 in Hyx, (P < 0.05). The density of phosphorylated Bcl-xl protein density was 26.9 ± 7.0 in Nx and 47.9 ± 2.5 in Hyx, (P < 0.05). Phosphorylated Bax density was 43.5 ± 5.0 in Nx and 43.3 ± 5.2 in Hyx. Phosphorylated Bad density was 23.6 ± 3.9 in Nx, 24.4 ± 4.7 in Hyx. The data show that during hyperoxia there is a significant increase in tyrosine phosphorylation of Bcl2 and Bcl-xl, while the phosphorylation of proapototic proteins Bax & Bad was not altered. We conclude that hyperoxia leads to post translational modification of anti apoptotic proteins Bcl2 and Bcl-xl in cerebral cortical mitochondria. We propose that phosphorylation of Bcl2 will result in loss of its antiapoptotic potential by preventing its dimerization with Bax leading to activation of the caspase pathway and subsequent neuronal death in the cerebral cortex of the newborn piglets.     

Analysis of transfer RNA during the early embryogenesis of the freshwater teleost,Heteropneustes fossilis

Total RNA as well as transfer RNA were quantified from mature ova apart from four different embryonic stages namely mid-cleavage, early gastrula, mid-gastrula and organogenesis of the freshwater teleostHeteropneustes fossilis. Total RNA as well as transfer RNA quantity follow a similar variation pattern, being maximum during mid-gastrulation. When analysed by total amino acid acceptance capacity, transfer RNA shows its maximum activity during mid-gastrulation. This coincides with the higher ratio of tRNA to total RNA at this stage. The relative aminoacylation capacity for Ser, Gly, Asn and Thr are found to be higher (9–34%) compared to that for other amino acids. Total tRNA, resolved into three peaks upon HPLC fractionation, shows a high cumulative peak area during mid-gastrulation and organogenesis. These results indicate a switch over of maternal to embryonic translation machinery during gastrulation.Abbreviations tRNA transfer RNA - aaRS aminoacyl tRNA synthetase - HPLC high pressure liquid chromatography - AUF absorption unit full scale     

Crystal structure studies of NADP dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

NADP⁺ dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP⁺ was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.     

Chromogranin B: intra‐ and extra‐cellular mechanisms to regulate catecholamine storage and release,in catecholaminergic cells and organisms

Chromogranin B (CHGB) is the major matrix protein in human catecholamine storage vesicles. CHGB genetic variation alters catecholamine secretion and blood pressure. Here, effective Chgb protein under‐expression was achieved by siRNA in PC12 cells, resulting in ~ 48% fewer secretory granules on electron microscopy, diminished capacity for catecholamine uptake (by ~ 79%), and a ~ 73% decline in stores available for nicotinic cholinergic‐stimulated secretion. In vivo, loss of Chgb in knockout mice resulted in a ~ 35% decline in chromaffin granule abundance and ~ 44% decline in granule diameter, accompanied by unregulated catecholamine release into plasma. Over‐expression of CHGB was achieved by transduction of a CHGB‐expressing lentivirus, resulting in ~ 127% elevation in CHGB protein, with ~ 122% greater abundance of secretory granules, but only ~ 14% increased uptake of catecholamines, and no effect on nicotinic‐triggered secretion. Human CHGB protein and its proteolytic fragments inhibited nicotinic‐stimulated catecholamine release by ~ 72%. One conserved‐region CHGB peptide inhibited nicotinic‐triggered secretion by up to ~ 41%, with partial blockade of cationic signal transduction. We conclude that bi‐directional quantitative derangements in CHGB abundance result in profound changes in vesicular storage and release of catecholamines. When processed and released extra‐cellularly, CHGB proteolytic fragments exert a feedback effect to inhibit catecholamine secretion, especially during nicotinic cholinergic stimulation.

     

Model class A and class L peptides increase the production of apoA-I-containing lipoproteins in HepG2 cells

Class A peptides inhibit atherosclerosis and protect cells from class L peptide-mediated lysis. Because the cytolytic process is concentration dependent, we hypothesized that at certain concentrations both classes of peptides exert similar effect(s) on cells. To test this hypothesis, we studied the effects of a class L peptide (18L = GIKKFLGSIWKFIKAFVG) and a class A peptide, 18A-Pro-18A (18A = DWLKAFYDKVAEKLKEAF) (37pA), on apolipoprotein and lipoprotein production in HepG2 cells. Secretion of (35)S-labeled apolipoprotein A-I (apoA-I) was stimulated by both 18L (110%) and 37pA (135%) at 10 and 20 nM of peptides, respectively. Both peptides enhanced the secretion of (3)H-labeled phospholipids by 140% and (14)C-labeled HDL-cholesterol (HDL-C) by 35% but had no significant effect on the total cholesterol mass or secretion. These results indicate that class L and class A peptides cause redistribution of cholesterol among lipoproteins in favor of HDL-C. Both peptides remodeled apoA-I-containing particles forming prebeta- as well as alpha-HDL. This study suggests that increased secretion of phospholipids and apoA-I and the formation of prebeta-HDL particles might contribute to the antiatherogenic properties of these peptides.     

Global Effects of Inactivation of the Pyruvate Kinase Gene in the Mycobacterium tuberculosis Complex

To better understand the global effects of “natural” lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis ΔpykA mutant yielded dysgonic colonies on solid medium lacking pyruvate, whereas colony morphology was eugonic on pyruvate-containing medium. Global effects of the loss of the pykA gene, possibly underlying colony morphology, were investigated by using proteomics on cultures grown in the same conditions. The levels of Icd2 increased and those of Icl and PckA decreased in the ΔpykA knockout. Proteomics suggested that the synthesis of enzymes involved in fatty acid and lipid biosynthesis were decreased, whereas those involved in β-oxidation were increased in the M. tuberculosis ΔpykA mutant, as confirmed by direct assays for these activities. Thus, the loss of pykA from M. tuberculosis results in fatty acids being used principally for energy production, in contrast to the situation in the host when carbon from fatty acids is conserved through the glyoxylate cycle and gluconeogenesis; when an active pykA gene was introduced into M. bovis, the opposite effects occurred. Proteins involved in oxidative stress—AhpC, KatG, and SodA—showed increased synthesis in the ΔpykA mutant, and iron-regulated proteins were also affected. Ald levels were decreased in the ΔpykA knockout, explaining why an M. tuberculosis ΔpykA Δald double mutant showed little additional phenotypic effect. Overall, these data show that the loss of the pykA gene has powerful, global effects on proteins associated with central metabolism.Comparison of the genome sequences of Mycobacterium bovis and Mycobacterium tuberculosis revealed >99.95% identity at the nucleotide level; however, these pathogens differ in terms of host tropism, phenotype, and virulence (16). Eleven regions of difference (RD) were observed in the M. bovis genome (2 to 12.7 kb) compared to M. tuberculosis, while one region deleted from M. tuberculosis was present in M. bovis (5, 16). In addition to the RDs, there are over 2,400 single nucleotide polymorphisms (SNPs) between M. tuberculosis and M. bovis (16). Some SNPs cosegregate with regions of deletions or other genetic markers (5); one such SNP is in the pykA gene, which cosegregates with the RD9 deletion. This SNP results in an inactive pyruvate kinase (PykA) being produced due to a Glu220Asp mutation (20). Glu220 is in the active site of the enzyme (21, 24), and its substitution results in complete loss of the enzyme activity in M. bovis (20). Thus, the pykA SNP explains one of the classic distinctions between M. bovis and M. tuberculosis, namely, the requirement for pyruvate. Neither glycerol, the preferred carbon source for isolation of tubercle bacilli, nor glucose support the growth of M. bovis when they are not supplemented with pyruvate (38), due to the inactive pyruvate kinase.On the routinely used Middlebrook 7H11 agar, containing glycerol and oleate, M. bovis shows dysgonic colony morphology, whereas M. tuberculosis, in contrast, shows eugonic colony morphology with abundant growth. Complementation of M. bovis with the active pykA gene from M. tuberculosis restored the eugonic phenotype. Thus, loss of PykA activity commits M. bovis to using nonglycolytic substrates as carbon sources, such as lipids. This in itself is of biological significance since human M. tuberculosis switches to this kind of metabolism in experimentally infected animals or in macrophages (34, 35, 39). However, even with oleate (a lipid) as a sole carbon source which allows both species to grow, there was still a difference in colony morphology (20). This led us to consider that loss of the pykA gene had wider effects since PykA is not needed for energy production on oleate and has no role in gluconeogenesis (Fig. ​(Fig.1).1). Thus, we hypothesize that the loss of the pykA gene has global effects over and above the predicted effect of determining whether or not growth can take place on glycerol. To examine our hypothesis, we created a pykA mutant of M. tuberculosis to investigate the effect of pykA deletion by using isogenic strains. This builds upon our previous study in which we had complemented M. bovis with the (active) M. tuberculosis pykA gene (20). We also created a mutant in alanine dehydrogenase (H37Rv Δald) and a H37Rv Δald ΔpykA double mutant since M. bovis naturally lacks active ald and pykA genes (16). The global effects of these knockout mutations were then examined by their on growth on a range of carbon sources and on protein expression during growth on pyruvate, a gluconeogenic carbon source. A proteomic approach was chosen since it would reveal changes in all proteins, for example, regulatory proteins, enzymes, and stress proteins; key proteins, or effects of changes in their levels, could then be assayed for directly. These approaches revealed the major metabolic consequences resulting from pykA inactivation.Open in a separate windowFIG. 1.Pathways of carbon metabolism possible in strains with or without pyruvate kinase (PykA). Boxes denote substrates and/or products where arrows are used to denote pathways. Arrows to and from boxes are pathways; other arrows show reactions catalyzed by a single enzyme. Substrates are in text with serifs; pathways and enzymes in plain text. Colored arrows are used to denote glycolysis or gluconeogenesis in red, the tricarboxylic acid cycle in blue, and the glyoxylate cycle in magenta.     

Effect of Terminalia arjuna on antioxidant defense system in cancer

Constant production of reactive oxygen species (ROS) during aerobic metabolism is balanced by antioxidant defense system of an organism. Although low level of ROS is important for various physiological functions, its accumulation has been implicated in the pathogenesis of age-related diseases such as cancer and coronary heart disease and neurodegenerative disorders such as Alzheimer’s disease. It is generally assumed that frequent consumption of phytochemicals derived from vegetables, fruits, tea and herbs may contribute to shift the balance towards an adequate antioxidant status. The present study is aimed to investigate the effect of aqueous extract of medicinal plant Terminalia arjuna on antioxidant defense system in lymphoma bearing AKR mice. Antioxidant action of T. arjuna is monitored by the activities of catalase, superoxide dismutase and glutathione S transferase which constitute major antioxidant defense system by scavenging ROS. These enzyme activities are low in lymphoma bearing mice indicating impaired antioxidant defense system. Oral administration of different doses of aqueous extract of T. arjuna causes significant elevation in the activities of catalase, superoxide dismutase and glutathione S transferase. T. arjuna is found to down regulate anaerobic metabolism by inhibiting the activity of lactate dehydrogenase in lymphoma bearing mice, which was elevated in untreated cancerous mice. The results indicate the antioxidant action of aqueous extract of T. arjuna, which may play a role in the anti carcinogenic activity by reducing the oxidative stress along with inhibition of anaerobic metabolism.