Salt tolerance and activity of antioxidative enzymes of transgenic finger millet overexpressing a vacuolar H-pyrophosphatase gene (SbVPPase) from Sorghum bicolor

A vacuolar proton pyrophosphatase cDNA clone was isolated from Sorghum bicolor (SbVPPase) using end-to-end gene-specific primer amplification. It showed 80–90% homology at the nucleotide and 85–95% homology at the amino acid level with other VPPases. The gene was introduced into expression vector pCAMBIA1301 under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter and transformed into Agrobacterium tumifaciens strain LBA4404 to infect embryogenic calli of finger millet (Eleusine coracana). Successful transfer of SbVPPase was confirmed by a GUS histochemical assay and PCR analysis. Both, controls and transgenic plants were subjected to 100 and 200 mM NaCl and certain biochemical and physiological parameters were studied. Relative water content (RWC), plant height, leaf expansion, finger length and width and grain weight were severely reduced (50–70%), and the flowering period was delayed by 20% in control plants compared to transgenic plants under salinity stress. With increasing salt stress, the proline and chlorophyll contents as well as the enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) increased by 25–100% in transgenics, while malondialdehyde (MDA) showed a 2–4-fold decrease. The increased activities of antioxidant enzymes and the reduction in the MDA content suggest efficient scavenging of reactive oxygen species (ROS) in transgenics and, as a consequence, probably alleviation of salt stress. Also, the leaf tissues of the transgenics accumulated 1.5–2.5-fold higher Na⁺ and 0.4–0.8-fold higher K⁺ levels. Together, these results clearly demonstrate that overexpression of SbVPPase in transgenic finger millet enhances the plant's performance under salt stress.     

Glucotoxic and diabetic conditions induce caspase 6-mediated degradation of nuclear lamin A in human islets,rodent islets and INS-1 832/13 cells

Nuclear lamins form the lamina on the interior surface of the nuclear envelope, and regulate nuclear metabolic events, including DNA replication and organization of chromatin. The current study is aimed at understanding the role of executioner caspase 6 on lamin A integrity in islet β-cells under duress of glucotoxic (20 mM glucose; 24 h) and diabetic conditions. Under glucotoxic conditions, glucose-stimulated insulin secretion and metabolic cell viability were significantly attenuated in INS-1 832/13 cells. Further, exposure of normal human islets, rat islets and INS-1 832/13 cells to glucotoxic conditions leads to caspase 6 activation and lamin A degradation, which is also observed in islets from the Zucker diabetic fatty rat, a model for type 2 diabetes (T2D), and in islets from a human donor with T2D. Z-Val-Glu-Ile-Asp-fluoromethylketone, a specific inhibitor of caspase 6, markedly attenuated high glucose-induced caspase 6 activation and lamin A degradation, confirming that caspase 6 mediates lamin A degradation under high glucose exposure conditions. Moreover, Z-Asp-Glu-Val-Asp-fluoromethylketone, a known caspase 3 inhibitor, significantly inhibited high glucose-induced caspase 6 activation and lamin A degradation, suggesting that activation of caspase 3 might be upstream to caspase 6 activation in the islet β-cell under glucotoxic conditions. Lastly, we report expression of ZMPSTE24, a zinc metallopeptidase involved in the processing of prelamin A to mature lamin A, in INS-1 832/13 cells and human islets; was unaffected by high glucose. We conclude that caspases 3 and 6 could contribute to alterations in the integrity of nuclear lamins leading to metabolic dysregulation and failure of the islet β-cell.     

An analysis of high glucose and glucosamine-induced gene expression and oxidative stress in renal mesangial cells

Renal mesangial cells play an important role in the development of diabetic kidney disease. We have previously demonstrated that some of the effects of high glucose on mesangial extracellular matrix (ECM) protein expression are mediated by the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). Using Affymetrix murine expression U430 2.0 oligochips, we examined the global effects of high glucose (HG) and glucosamine (GlcN) on mRNA expression of a mouse mesangial cell line (MES-13). We sought to determine the portion of mRNA expression in MES-13 cells, which is mediated both by high glucose and glucosamine, i.e., via the HBP. Of the 34,000 genes on the chip, approximately 55.7 - 60.8% genes are detected in MES-13 cells. Culturing MES-13 cells for 48 h with HG alters the expression of approximately 389 genes at our preset threshold levels (at least 2-fold change) where 263 genes are up-regulated and 126 genes are down-regulated. GlcN also increases the expression of 106 genes and decreases 94 genes during the same period of incubation. Seventy-two genes in the chip are commonly regulated by HG and GlcN, in which 33 genes are up and 39 genes are down. The mRNA level of thioredoxin interacting protein (TXNIP), an inhibitor of thioredoxin activity, is maximally increased approximately 18.8 and 9.9-fold respectively by HG and GlcN. The differential expression of several genes found in the microarray analysis is further validated by real-time quantitative PCR. Significant biological processes commonly targeted by HG and GlcN are the TXNIP-thioredoxin system, oxidative stress, endoplasmic reticulum (ER) stress, extracellular matrix genes, and interferon-inducible genes. Stable overexpression of TXNIP in MES-13 cells increases glucose and glucosamine-mediated ECM gene expression and oxidative stress. We conclude from these results that the HBP mediates several effects of high glucose on mesangial cell metabolism, which promotes reactive oxygen species generation to cause cellular oxidative stress, ECM gene expression and apoptosis.     

Role of histone acetylation in the development of diabetic retinopathy and the metabolic memory phenomenon

Hyperglycemia is considered as one of the major determinants in the development of diabetic retinopathy, but the progression of retinopathy resists arrest after hyperglycemia is terminated, suggesting a metabolic memory phenomenon. Diabetes alters the expression of retinal genes, and this continues even after good glycemic control is re‐instituted. Since the expression of genes is affected by chromatin structure that is modulated by post‐translational modifications of histones, our objective is to investigate the role of histone acetylation in the development of diabetic retinopathy, and in the metabolic memory phenomenon. Streptozotocin‐induced rats were maintained either in poor glycemic control (PC, glycated hemoglobin, GHb >11%) or good glycemic control (GC, GHb <6%) for 12 months, or allowed to be in PC for 6 months followed by in GC for 6 months (PC‐GC). On a cellular level, retinal endothelial cells, the target of histopathology of diabetic retinopathy, were incubated in 5 or 20 mM glucose for 4 days. Activities of histone deacetylase (HDAC) and histone acetyltransferase (HAT), and histone acetylation were quantified. Hyperglycemia activated HDAC and increased HDAC1, 2, and 8 gene expressions in the retina and its capillary cells. The activity HAT was compromised and the acetylation of histone H3 was decreased. Termination of hyperglycemia failed to provide any benefits to diabetes‐induced changes in retinal HDAC and HAT, and histone H3 remained subnormal. This suggests “in principle” the role of global acetylation of retinal histone H3 in the development of diabetic retinopathy and in the metabolic memory phenomenon associated with its continued progression. J. Cell. Biochem. 110: 1306–1313, 2010. © 2010 Wiley‐Liss, Inc.     

The stimulus-secretion coupling of glucose-induced insulin release. XLIII. Na-Ca countertransport mediated by pancreatic islet native ionophores

Native ionophores extracted from isolated pancreatic islets were able to transport Ca2+ from one aqueous medium into another across an organic immiscible phase. In the presence of a K+, Na+, Li+, or H+ gradient, Ca2+ was transported against its own concentration gradient from the medium of low monovalent-cation concentration to the opposite medium. The transport of Ca2+ was abolished by the organic calcium-antagonist suloctidil. These findings provide a model for the process of Na-Ca countertransport in islet cells and its inhibition in response to the conversion of nutrient secretagogues to their acidic metabolites.     

Phospholipid N-methylation in diabetic erythrocytes: effects on membrane Na+, K+ ATPase activity.

Phospholipid methylation was quantified in non-diabetic and streptozotocin diabetic rat erythrocytes. While the total mass of methylated lipids remained the same in both groups, the relative abundance of individual methylated lipid species differed significantly in diabetic erythrocytes. Moreover, incubation of erythrocytes membranes with S-adenosyl methionine, a substrate for methyl transferases, not only increased membrane lipid methylation but also decreased Na+, K+ ATPase activity significantly. These results suggest that phospholipid methylation may cause the observed depression of erythrocyte Na+, K+ ATPase activity in diabetes and could contribute to the altered rheology of erythrocytes in diabetes.     

Acacidiol,a new nor-triterpene from the sapogenins of Acacia concinna

From the neutral fraction of the acid hydrolysate of the saponin of Acacia concinna pods, acacic acid lactone 3β-acetate and a new nor-triterpene ‘acacidiol’ have been isolated. The latter is shown to be 28-noroleana-16,18-diene-3β,21β-diol.