Sulfhydryl groups in photosynthetic energy conservation. VI. Subunit distribution of sulfhydryl groups and disulfide bonds in chloroplast coupling factor and ATPase activity

The subunit distribution of sulfhydryl groups and disulfide bonds of spinach chloroplasts coupling factor I has been determined. Native coupling factor I with a latent ATPase activity has eight sulfhydryl groups distributed 4 : 2 : 0 : 0 : 2 in the alpha, beta, gamma, delta and epsilon subunits, respectively. Heat treatment of coupling factor I, in addition to the activation of its ATPase activity, induces a dithiol-disulfide interchange between the gamma and the alpha subunit, changing the sulfhydryl groups' distribution to 2 : 2 : 2 : 0 : 2. Reduction of disulfide bonds of coupling factor I by dithioerythritol during heat treatment gives a subunit distribution of 4 : 4 : 4 : 0 : 2, suggesting that native coupling factor I has three disulfide bonds, two in the gamma subunit and one in one of the beta subunits. The results suggest an asymmetric redox state of some of the subunits of coupling factor I and an asymmetric positioning of some of them in the molecular structure of coupling factor I.     

Regulation of Vascular Tone,Angiogenesis and Cellular Bioenergetics by the 3-Mercaptopyruvate Sulfurtransferase/H2S Pathway: Functional Impairment by Hyperglycemia and Restoration by dl-α-Lipoic Acid

Hydrogen sulfide (H₂S), as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important biological source of H₂S. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have an adverse effect on cellular homeostasis. In the current study, given the importance of H₂S as a vasorelaxant, angiogenesis stimulator and cellular bioenergetic mediator, we first determined whether the 3-MST/H₂S system plays a physiological regulatory role in endothelial cells. Next, we tested whether a dysfunction of this pathway develops during the development of hyperglycemia and μmol/L to diabetes-associated vascular complications. Intraperitoneal (IP) 3-MP (1 mg/kg) raised plasma H₂S levels in rats. 3-MP (10 1 mmol/L) promoted angiogenesis in vitro in bEnd3 microvascular endothelial cells and in vivo in a Matrigel assay in mice (0.3–1 mg/kg). In vitro studies with bEnd3 cell homogenates demonstrated that the 3-MP-induced increases in H₂S production depended on enzymatic activity, although at higher concentrations (1–3 mmol/L) there was also evidence for an additional nonenzymatic H₂S production by 3-MP. In vivo, 3-MP facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased mitochondrial bioenergetic function. In vitro hyperglycemia or in vivo streptozotocin diabetes impaired angiogenesis, attenuated mitochondrial function and delayed wound healing; all of these responses were associated with an impairment of the proangiogenic and bioenergetic effects of 3-MP. The antioxidants dl-α-lipoic acid (LA) in vivo, or dihydrolipoic acid (DHLA) in vitro restored the ability of 3-MP to stimulate angiogenesis, cellular bioenergetics and wound healing in hyperglycemia and diabetes. We conclude that diabetes leads to an impairment of the 3-MST/H₂S pathway, and speculate that this may contribute to the pathogenesis of hyperglycemic endothelial cell dysfunction. We also suggest that therapy with H₂S donors, or treatment with the combination of 3-MP and lipoic acid may be beneficial in improving angiogenesis and bioenergetics in hyperglycemia.     

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (V_max) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (B_max) and affinity (K_d) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.     

Glutamate (mGluR-5) gene expression in brain regions of streptozotocin induced diabetic rats as a function of age: role in regulation of calcium release from the pancreatic islets in vitro

Metastatic renal cell carcinoma (RCC) is highly resistant to conventional systemic treatments, including chemotherapy, radiotherapy and hormonal therapies. Previous studies have shown over-expression of EGFR is associated with high grade tumors and a worse prognosis. Recent studies suggest anticancer therapies targeting the EGFR pathway have shown promising results in clinical trials of RCC patients. Therefore, characterization of the level and localization of EGFR expression in RCC is important for target-dependent therapy. In this study, we investigated the clinical significance of cellular localization of EGFR in human normal renal cortex and RCC. RCC and adjacent normal kidney tissues of 63 patients were obtained for characterization of EGFR expression. EGFR protein expression was assessed by immunohistochemistry on a scale from 0 to 300 (percentage of positive cells × staining intensity) and Western blotting. EGFR membranous staining was significantly stronger in RCC tumors than in normal tissues (P < 0.001). In contrast, EGFR cytoplasmic staining was significantly higher in normal than in tumor tissues (P < 0.001). The levels of membranous or cytoplasmic EGFR expression in RCC tissues were not correlated with sex, tumor grade, TNM stage or overall survival (P > 0.05). These results showed abundant expression of membranous EGFR in RCC, and abundant expression of cytoplasmic EGFR in normal tissues. EGFR expression in RCC was mostly located in the cell membrane, whereas the EGFR expression in normal renal tissues was chiefly seen in cytoplasm. Our results suggest different locations of EGFR expression may be associated with human renal tumorigenesis.     

Analysis of Arabidopsis with Highly Reduced Levels of Malate and Fumarate Sheds Light on the Role of These Organic Acids as Storage Carbon Molecules

While malate and fumarate participate in a multiplicity of pathways in plant metabolism, the function of these organic acids as carbon stores in C₃ plants has not been deeply addressed. Here, Arabidopsis (Arabidopsis thaliana) plants overexpressing a maize (Zea mays) plastidic NADP-malic enzyme (MEm plants) were used to analyze the consequences of sustained low malate and fumarate levels on the physiology of this C₃ plant. When grown in short days (sd), MEm plants developed a pale-green phenotype with decreased biomass and increased specific leaf area, with thin leaves having lower photosynthetic performance. These features were absent in plants growing in long days. The analysis of metabolite levels of rosettes from transgenic plants indicated similar disturbances in both sd and long days, with very low levels of malate and fumarate. Determinations of the respiratory quotient by the end of the night indicated a shift from carbohydrates to organic acids as the main substrates for respiration in the wild type, while MEm plants use more reduced compounds, like fatty acids and proteins, to fuel respiration. It is concluded that the alterations observed in sd MEm plants are a consequence of impairment in the supply of carbon skeletons during a long dark period. This carbon starvation phenotype observed at the end of the night demonstrates a physiological role of the C₄ acids, which may be a constitutive function in plants.Fumarate can accumulate to high levels in Arabidopsis (Arabidopsis thaliana) and agronomically important C₃ plants like soybean (Glycine max) and sunflower (Helianthus annuus; Chia et al., 2000; Fahnenstich et al., 2007). It is synthesized from malate through the action of fumarase (Gout et al., 1993). Malate is an intermediate of the tricarboxylic acid (TCA) cycle and a regulator of pH and nutrient uptake and stomatal function (Fernie and Martinoia, 2009). Malate also has an important role in photosynthesis in Crassulacean acid metabolism and C₄ plants (Drincovich et al., 2010). In some C₃ plants like Arabidopsis, malate and fumarate levels show diurnal changes similar to those of starch and Suc: They increase during the day and decrease during the night, suggesting that they function as transient carbon storage molecules (Fahnenstich et al., 2007). As fumarate is highly concentrated in stems (Stumpf and Burris, 1981) and phloem exudates (Chia et al., 2000), it was proposed that it might also be involved in carbon partitioning. There is variation in the extent to which C₃ plants store photosynthates in the form of sugars and organic acids in leaves during carbon assimilation (Zeeman and Ap Rees, 1999; Chia et al., 2000; Zeeman et al., 2007). In Arabidopsis, approximately half of the photoassimilates are partitioned into starch (Sun et al., 1999; Zeeman and Ap Rees, 1999). Under short days (sd), the partitioning of assimilates to the formation of starch is greater than in long days (LD; Gibon et al., 2004). Thus, the longer the night, the higher is the proportion of photoassimilates stored as starch to provide carbon skeletons during the prolonged dark period.We recently established transgenic lines of Arabidopsis with decreased malate and fumarate levels by overexpressing a maize (Zea mays) plastidic NADP-malic enzyme (MEm plants; Fahnenstich et al., 2007). This enzyme catalyzes the oxidative decarboxylation of malate rendering pyruvate, CO₂, and NADPH (Maurino et al., 1996). The MEm plants showed an accelerated dark-induced senescence that could be rescued by supplying Glc, Suc, or malate, suggesting that the lack of a readily mobilized carbon source is likely to be the initial factor leading to the premature induction of senescence in MEm plants. In line with these, malate and fumarate were the only two metabolites whose levels were significantly decreased in the MEm lines after dark incubation and whose levels recover to values similar to wild type after incubation with Glc (Fahnenstich et al., 2007).In this work we address the question whether malate and fumarate function as storage carbon molecules in the C₃ plant Arabidopsis by analyzing the consequences of sustained low levels of these organic acids on the performance of MEm plants growing in different photoperiods. We demonstrate that low malate and fumarate levels do not alter morphology, photosynthetic functions, or growth parameters in LD plants. By contrast, MEm plants suffer from a marked decrease in photosynthetic performance and show reduced biomass and a pale-green phenotype in sd. When grown in sd at the end of the night the wild type showed a shift from carbohydrates as the main substrate for respiration to organic acids, while the MEm lines used more reduced substrates (e.g. fatty acids and proteins) to fuel respiration. The alterations observed in sd point to an impairment in the supply of energy and carbon skeletons during a long night, which supports the proposed physiological roles of malate and fumarate as essential storage carbon molecules in Arabidopsis.     

Hypoglycemia induced changes in cholinergic receptor expression in the cerebellum of diabetic rats

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Hypoglycemia and hyperglycemia is found to be associated with central and peripheral nerve system dysfunction. Changes in acetylcholine receptors have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). In the present study we showed the effects of insulin induced hypoglycemia and streptozotocin induced diabetes on the cerebellar cholinergic receptors, GLUT3 and muscle cholinergic activity. Results showed enhanced binding parameters and gene expression of Muscarinic M1, M3 receptor subtypes in cerebellum of diabetic (D) and hypoglycemic group (D + IIH and C + IIH). α7nAchR gene expression showed a significant upregulation in diabetic group and showed further upregulated expression in both D + IIH and C + IIH group. AchE expression significantly upregulated in hypoglycemic and diabetic group. ChAT showed downregulation and GLUT3 expression showed a significant upregulation in D + IIH and C + IIH and diabetic group. AchE activity enhanced in the muscle of hypoglycemic and diabetic rats. Our studies demonstrated a functional disturbance in the neuronal glucose transporter GLUT3 in the cerebellum during insulin induced hypoglycemia in diabetic rats. Altered expression of muscarinic M1, M3 and α7nAchR and increased muscle AchE activity in hypoglycemic rats in cerebellum is suggested to cause cognitive and motor dysfunction. Hypoglycemia induced changes in ChAT and AchE gene expression is suggested to cause impaired acetycholine metabolism in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. The results shows that cerebellar cholinergic neurotransmission is impaired during hyperglycemia and hypoglycemia and the hypoglycemia is causing more prominent imbalance in cholinergic neurotransmission which is suggested to be a cause of cerebellar dysfunction associated with hypoglycemia.     

Systematic search for putative new domain families in Mycoplasma gallisepticum genome

Background

Stem cell factor (SCF) receptor c-Kit is recognized as a key signaling molecule, which transduces signals for the proliferation, differentiation and survival of stem cells. Binding of SCF to its receptor triggers transactivation, leading to the recruitment of kinases and phosphatases to the docking platforms of c-Kit catalytic domain. Tyrosine phosphatase-1 (Shp-1) deactivates/attenuates 'Kit' kinase activity. Whereas, Asp816Val mutation in the Kit activation loop transforms kinase domain to a constitutively activated state (switch off-to-on state), in a ligand-independent manner. This phenomenon completely abrogates negative regulation of Shp-1. To predict the possible molecular basis of interaction between c-Kit and Shp-1, we have performed an in silico protein-protein docking study between crystal structure of activated c-Kit (phosphorylated c-Kit) and full length crystal structure of Shp-2, a close structural counterpart of Shp-1.

Findings

Study revealed a stretch of conserved amino acids (Lys818 to Ser821) in the Kit activation domain, which makes decisive H-bonds with N-sh2 and phosphotyrosine binding pocket residues of the phosphatase. These H-bonds may impose an inhibitory steric hindrance to the catalytic domain of c-Kit, there by blocking further interaction of the activation loop molecules with incoming kinases. We have also predicted a phosphotyrosine binding pocket in SH2 domains of Shp-1, which is found to be predominantly closer to a catalytic groove like structure in c-Kit kinase domain.

Conclusions

This study predicts that crucial hydrogen bonding between N-sh2 domain of Shp-1 and Kit activation loop can modulate the negative regulation of c-Kit kinase by Shp-1. Thus, this finding is expected to play a significant role in designing suitable gain-of-function c-Kit mutants for inducing conditional proliferation of hematopoietic stem cells.     

In vitro inhibition of human influenza A virus replication by chloroquine

Inhibition of the human cytomegalovirus UL97 kinase by maribavir is thought to be responsible for the antiviral activity of this compound. Some mutations that confer resistance to maribavir map to UL97, however additional mutations that also confer resistance to the drug were mapped to UL27. These open reading frames share a low level of homology, yet the function of pUL27 remains unknown. A recombinant virus with a deletion in the UL27 open reading frame was reported previously to exhibit a slight replication deficit, but a more important function in vivo was hypothesized given its homology to the UL97 kinase. The potential for an important function in vivo was investigated by determining if these knockout viruses could replicate in human tissue implanted in SCID mice. None of the AD169 derived viruses replicated well in the implanted thymus/liver tissue, and is consistent with previous observations, although all of the viruses replicated to some degree in retinal tissue implants. Replication of the parent viruses was observed at 7 days post inoculation, whereas no replication was detected with any of the recombinant viruses with deletions in UL27. By day 14, replication was detected in two of the three knockout viruses and in all of the viruses by day 42. These data are consistent with minimal defects observed in cell culture, but are not consistent with an important role for UL27 in vivo. We conclude that UL27 is not required for viral replication in vivo.     

Colilargo’s occupancy and the role of native and exotic plants in hantavirus expansion and transmission risk

Mammal Research - In this work, we examined the relationship of Oligoryzomys longicaudatus’ (the Andes virus [ANDV] host, commonly known as the colilargo) occupancy and the cover of dominant...