INK4a deletion results in improved kidney regeneration and decreased capillary rarefaction after ischemia-reperfusion injury

The molecular mechanisms that lead to tubular atrophy, capillary loss, and fibrosis following acute kidney injury are not very clear but may involve cell cycle inhibition by increased expression of cyclin kinase inhibitors. The INK4a/ARF locus encodes overlapping genes for two proteins, a cyclin kinase inhibitor, p16(INK4a), and a p53 stabilizer, p19(ARF), from independent promoters. To determine if decreased INK4a gene expression results in improved kidney regeneration, INK4a knockout (KO) and wild-type (WT) mice were subjected to ischemia-reperfusion injury (IRI). p16(INK4a) and p19(ARF) levels were increased markedly in WT mice at 1-28 days after injury. Kidneys were examined to determine the localization and levels of p16(INK4a), apoptosis, cell proliferation, and capillary rarefaction. KO mice displayed decreased tubular cell apoptosis, increased cell proliferation, and lower creatinine levels after injury. KO mice had significantly higher capillary density compared with WT mice at 14-42 days after IRI. Plasma granulocyte colony-stimulating factor (G-CSF) increased after ischemia in both WT and KO mice and was elevated markedly in KO compared with WT mice. KO kidney digests contained higher counts of Gr-1(+)/Cd11b(+) myeloid cells by flow cytometry. KO mice treated with a Gr-1-depleting antibody displayed reduced vascular endothelial growth factor mRNA, plasma G-CSF, and capillary density, and an increase in serum creatinine and medullary myofibroblasts, compared with untreated KO mice 14 days after ischemia. The anti-angiogenic effect of Gr-1 depletion in KO mice was confirmed by Matrigel angiogenesis assays. These results suggest that the absence of p16(INK4a) and p19(ARF) following IRI has a protective effect on the kidney through improved epithelial and microvascular repair, in part by enhancing the mobilization of myeloid cells into the kidney.     

Gene expression alterations in Rocky Mountain elk infected with chronic wasting disease

Chronic wasting disease (CWD) is an invariably fatal neurologic disease that naturally infects mule deer, white tailed deer and elk. The understanding of CWD neurodegeneration at a molecular level is very limited. In this study, microarray analysis was performed to determine changes in the gene expression profiles in six different tissues including brain, midbrain, thalamus, spleen, RPLN and tonsil of CWD-infected elk in comparison to non-infected healthy elk, using 24,000 bovine specific oligo probes. In total, 329 genes were found to be differentially expressed (> 2.0-fold) between CWD negative and positive brain tissues, with 132 genes upregulated and 197 genes downregulated. There were 249 DE genes in the spleen (168 up- and 81 downregulated), 30 DE genes in the retropharyngeal lymph node (RPLN) (18 up- and 12 downregulated), and 55 DE genes in the tonsil (21 up- and 34 downregulated). Using Gene Ontology (GO), the DE genes were assigned to functional groups associated with cellular process, biological regulation, metabolic process, and regulation of biological process. For all brain tissues, the highest ranking networks for DE genes identified by Ingenuity Pathway Analysis (IPA) were associated with neurological disease, cell morphology, cellular assembly and organization. Quantitative real-time PCR (qRT-PCR) validated the expression of DE genes primarily involved in different regulatory pathways, including neuronal signaling and synapse function, calcium signaling, apoptosis and cell death and immune cell trafficking and inflammatory response. This is the first study to evaluate altered gene expression in multiple organs including brain from orally infected elk and the results will improve our understanding of CWD neurodegeneration at the molecular level.     

Polygalacturonase-inhibiting proteins--leucine-rich repeat proteins in plant defence

Plant polygalacturonase-inhibiting proteins (PGIPs) belong to the leucine-rich repeat (LRR) family and are known to prevent pathogen invasion by inhibiting the plant cell wall degrading enzyme, polygalacturonase. Our study reveals that these multigene-encoded defence proteins found in flowering plants only exhibit identical domain architecture with 10 tandemly-arranged LRRs. This implies that variations of PGIP inhibitory properties are not associated with the number of the repeats but with subtle changes in the sequence content of the repeats. The first and eighth repeat contain more mutations compared to the strict conservation of the plant-specific LRRs or any repeat at other positions. Each of these repeats forms a separate cluster in the phylogenetic tree, both within and across plant families, thus suggesting uniqueness with respect to their position. A study of the genes encoding PGIPs, shows the existence of two categories (i) single exon and hence no intron; and (ii) two exons with an intron in between. Analyses of the intron phase and correlation of the exon-intron structure with the compact structural modules in PGIPs support insertion of introns in the pre-existing single exon genes and thus the intron late model. Lack of conservation of phase across families and formation of individual clusters for each family in the phylogenetic tree drawn with the intron sequences illustrate the event of insertion that took place separately in each of these families.     

Cellular and physiological mechanisms of new-onset diabetes mellitus after solid organ transplantation

New-onset diabetes after transplantation is recognized as one of the metabolic consequences which may increase the risk of morbidity and mortality after solid organ transplantation. The pathophysiology of new-onset diabetes after transplantation has not been clearly defined and may resemble that of Type 2 diabetes, characterized by predominantly insulin resistance or defective insulin secretion, or both. This review aims to summarize the current state of knowledge regarding the prevalence, consequences, pathogenesis, and management of new-onset diabetes after transplantation, with a major focus on the possible mechanisms involved in the pathogenesis of the disorder. The aetiology of new-onset diabetes after transplantation is multifactorial, with diabetogenic immunosuppressive drugs playing a major role. Multiple cellular and physiologic mechanisms are involved in the process. Selection of an appropriate maintenance immunosuppressive regimen should involve balancing the risk of patient and graft survival vs. the potential for new-onset diabetes after transplantation.     

Assembling neurospheres: dynamics of neural progenitor/stem cell aggregation probed using an optical trap

Optical trapping (tweezing) has been used in conjunction with fluid flow technology to dissect the mechanics and spatio-temporal dynamics of how neural progenitor/stem cells (NSCs) adhere and aggregate. Hitherto unavailable information has been obtained on the most probable minimum time (～5 s) and most probable minimum distance of approach (4-6 μm) required for irreversible adhesion of proximate cells to occur. Our experiments also allow us to study and quantify the spatial characteristics of filopodial- and membrane-mediated adhesion, and to probe the functional dynamics of NSCs to quantify a lower limit of the adhesive force by which NSCs aggregate (～18 pN). Our findings, which we also validate by computational modeling, have important implications for the neurosphere assay: once aggregated, neurospheres cannot disassemble merely by being subjected to shaking or by thermal effects. Our findings provide quantitative affirmation to the notion that the neurosphere assay may not be a valid measure of clonality and "stemness". Post-adhesion dynamics were also studied and oscillatory motion in filopodia-mediated adhesion was observed. Furthermore, we have also explored the effect of the removal of calcium ions: both filopodia-mediated as well as membrane-membrane adhesion were inhibited. On the other hand, F-actin disrupted the dynamics of such adhesion events such that filopodia-mediated adhesion was inhibited but not membrane-membrane adhesion.     

Bioactive eicosanoids: Role of prostaglandin F2α and F2-isoprostanes in inflammation and oxidative stress related pathology

Oxidative stress and inflammation are supposed to be the key players of several acute and chronic diseases, and also for progressive aging process. Eicosanoids, especially prostaglandin F_2α (PGF_2α) and F₂-isoprostanes are endogenous compounds that are involved both in physiology and the above mentioned pathologies. These compounds are biosynthesized mainly from esterified arachidonic acid through both enzymatic and non-enzymatic free radical-catalysed reactions in vivo, respectively. They have shown to possess potent biological activities in addition to their application as biomarkers of oxidative stress and inflammation. Recent advancement of methodologies has made it possible to quantify these compounds more reliably and apply them in various in vivo studies successfully. Today, experimental and clinical studies have revealed that both PGF_2α and F₂-isoprostanes are involved in severe acute or chronic inflammatory conditions such as rheumatic diseases, asthma, risk factors of atherosclerosis, diabetes, ischemia-reperfusion, septic shock and many others. These evidences promote that assessment of bioactive PGF_2α and F₂-isoprostanes simultaneously in body fluids offers unique non-invasive analytical opportunity to study the function of these eicosanoids in physiology, oxidative stress-related and inflammatory diseases, and also in the determination of potency of various radical scavengers, anti-inflammatory compounds, drugs, antioxidants and diet.     

Purification and properties of insulin-activated nitric oxide synthase from human erythrocyte membranes

A membrane bound form of nitric oxide synthase of human erythrocytes that could be activated by insulin was purified to homogeneity by detergent solubilization of the purified membrane preparation of these cells. The purified enzyme (M(r) 230 KD) was found to be composed of one heavy chain (M(r) 135 KD) and one light chain (Mr 95 KD) held together by disulphide bond(s). Scatchard plot analysis of insulin binding to the purified enzyme showed the presence of 2 different populations of the binding sites and the activation were directly related to the hormone binding to the protein. Line weaver Burk plot of the purified enzyme showed that the stimulation of the enzymic activity by insulin was related to the decrease of K(m) with simultaneous increase of V(max). Treatment of the purified enzyme with anti insulin receptor antibody inhibited the activation of the enzyme and the binding of the hormone to the protein. Furthermore NO itself, at low concentration (<0.4 microM) activated the enzyme, but at higher concentration (>0.8 microM) had no effect on the activation. Incubation of the purified enzyme with insulin simultaneously stimulated the tyrosine kinase and nitric oxide synthase activities of the preparations, that could be inhibited by genistein (an inhibitor of tyrosine kinase). These results indicated that the insulin activated nitric oxide synthase could be the insulin receptor itself.     

Four identical subunits in jack fruit seed agglutinin offer only two saccharide binding sites

Gel filtration of jack fruit seed agglutinin in 6 M guanidine hydrochloride confirmed our earlier report that the native 39.5-kDa protein was a tetramer of identical noncovalently associated 10-kDa subunits. Binding studies by the fluorescence quenching method using 4-methylumbelliferyl -D-galactoside as well as equilibrium dialysis using p-nitrophenyl -D-galactoside indicated only two binding sites per tetramer. This behaviour resembles the half-of-the-sites reactivity in certain enzymes and is discussed in view of the small subunit size.