Inorganic nitrate ingestion improves vascular compliance but does not alter flow-mediated dilatation in healthy volunteers

Ingestion of inorganic nitrate elevates blood and tissue levels of nitrite via bioconversion in the entero-salivary circulation. Nitrite is converted to NO in the circulation, and it is this phenomenon that is thought to underlie the beneficial effects of inorganic nitrate in humans. Our previous studies have demonstrated that oral ingestion of inorganic nitrate decreases blood pressure and inhibits the transient endothelial dysfunction caused by ischaemia-reperfusion injury in healthy volunteers. However, whether inorganic nitrate might improve endothelial function per se in the absence of a pathogenic stimulus and whether this might contribute to the blood pressure lowering effects is yet unknown. We conducted a randomised, double-blind, crossover study in 14 healthy volunteers to determine the effects of oral inorganic nitrate (8 mmol KNO(3)) vs. placebo (8 mmol KCl) on endothelial function, measured by flow-mediated dilatation (FMD) of the brachial artery, prior to and 3h following capsule ingestion. In addition, blood pressure (BP) was measured and aortic pulse wave velocity (aPWV) determined. Finally, blood, saliva and urine samples were collected for chemiluminescence analysis of [nitrite] and [nitrate] prior to and 3h following interventions. Inorganic nitrate supplementation had no effect on endothelial function in healthy volunteers (6.9±1.1% pre- to 7.1±1.1% post-KNO(3)). Despite this, there was a significant elevation of plasma [nitrite] (0.4±0.1 μM pre- to 0.7±0.2 μM post-KNO(3), p<0.001). In addition these changes in [nitrite] were associated with a decrease in systolic BP (116.9±3.8mm Hg pre- vs. 112.1±3.4 mm Hg post-KNO(3), p<0.05) and aPWV (6.5±0.1 m/s pre- to 6.2±0.1 post-KNO(3), p<0.01). In contrast KCl capsules had no effect on any of the parameters measured. These findings demonstrate that although inorganic nitrate ingestion does not alter endothelial function per se, it does appear to improve blood flow, in combination with a reduction in blood pressure. It is likely that these changes are due to the intra-vascular production of NO.     

Everolimus and Sirolimus in Combination with Cyclosporine Have Different Effects on Renal Metabolism in the Rat

Enhancement of calcineurin inhibitor nephrotoxicity by sirolimus (SRL) is limiting the clinical use of this drug combination. We compared the dose-dependent effects of the structurally related everolimus (EVL) and sirolimus (SRL) alone, and in combination with cyclosporine (CsA), on the rat kidney. Lewis rats were treated by oral gavage for 28 days using a checkerboard dosing format (0, 3.0, 6.0 and 10.0 CsA and 0, 0.5, 1.5 and 3.0 mg/kg/day SRL or EVL, n = 4/dose combination). After 28 days, oxidative stress, energy charge, kidney histologies, glomerular filtration rates, and concentrations of the immunosuppressants were measured along with ¹H-magnetic resonance spectroscopy (MRS) and gas chromatography- mass spectrometry profiles of cellular metabolites in urine. The combination of CsA with SRL led to higher urinary glucose concentrations and decreased levels of urinary Krebs cycle metabolites when compared to controls, suggesting that CsA+SRL negatively impacted proximal tubule metabolism. Unsupervised principal component analysis of MRS spectra distinguished unique urine metabolite patterns of rats treated with CsA+SRL from those treated with CsA+EVL and the controls. SRL, but not EVL blood concentrations were inversely correlated with urine Krebs cycle metabolite concentrations. Interestingly, the higher the EVL concentration, the closer urine metabolite patterns resembled those of controls, while in contrast, the combination of the highest doses of CsA+SRL showed the most significant differences in metabolite patterns. Surprisingly in this rat model, EVL and SRL in combination with CsA had different effects on kidney biochemistry, suggesting that further exploration of EVL in combination with low dose calcineurin inhibitors may be of potential benefit.     

Differential effect of recombinant human leukocyte interferon on human leukemic and normal myeloid progenitor cells

Two separate clones of recombinant leukocyte interferon (IFLrA and IFLrD) inhibited the cloning efficiency in soft agar of the human leukemia cell lines HL-60 and KG-1. Inhibition of the growth in agar of normal human bone marrow myeloid progenitors was also observed, but this required considerably higher concentrations. IFLrA and IFLrD also inhibited the growth of HL-60 and KG-1 cells in suspension culture. This antiproliferative effect did not appear to be due to induction of maturation of these cells. Our results suggest that homogeneous preparations of interferon may be capable of exerting selective antiproliferative effects on malignant human myeloid progenitor cells in comparison to their normal counterparts.     

Combating iodine deficiency disorders to achieve millennium development goal 4 in India: reduction in infant mortality rate

In severe iodine deficient areas, iodine deficiency has been documented to be an important etiological factor leading to poor fetal growth and development. Iodine is essential for physical growth and development of the central nervous system of the fetus. Iodine deficiency in pregnant mothers leads to increased incidence of infertility and abortions, perinatal mortality and infant child mortality. The clinical iodine supplementation trials have documented adverse health consequences due to iodine deficiency. Evidence from observational studies concludes that prevention of iodine deficiency can lead to reduction in infant mortality rate and facilitate to achieve millennium development goal-4.     

Inhibition of mammalian ribonucleases by endogenous adenosine dinucleotides

The most potent low molecular weight inhibitors of pancreatic RNase superfamily enzymes reported to date are synthetic derivatives of adenosine 5(')-pyrophosphate. Here we have investigated the effects of six natural nucleotides that also incorporate this moiety (NADP(+), NADPH, ATP, Ap(3)A, Ap(4)A, and Ap(5)A) on the activities of RNase A and two of its homologues, eosinophil-derived neurotoxin and angiogenin. With eosinophil-derived neurotoxin and angiogenin, Ap(5)A is comparable to the tightest binding inhibitors identified previously (K(i) values at pH 5.9 are 370 nM and 100 microM, respectively); it ranks among the strongest small antagonists of RNase A as well (K(i)=230 nM). The K(i) for NADPH with angiogenin is similar to that of Ap(5)A. These findings suggest that Ap(5)A and NADPH may serve as useful new leads for inhibitor design. Examination of inhibition under physiological conditions indicates that NADPH, ATP, and Ap(5)A may suppress intracellular RNase activity significantly in vivo.     

Occurrence of a thermocline in two lakes of the semi-arid region in western Rajasthan,India

Water quality samples were obtained monthly or bimonthly 17 times from May 1974 to May 1975 at three stations in Delaware Bay. In addition, two 12-hour cruises were also conducted at one station in February and April 1975. Surface and bottom water samples were taken. Measurements and analyses included temperature, salinity, dissolved oxygen, silicate, nitrate and nitrite, orthophosphate, ammonia, chlorophylls a, b, and c, phaeopigments, and carotenoids. The annual pattern of temperature was typical of an estuary in the mid-Atlantic Bight. Salinity and dissolved oxygen ranged from 22.9 to 29.7‰ and from 4.53 to 8.53 ml/l, respectively. Nutrient and pigment values showed seasonal peaks. Silicate (30.3 μg-at/1) and orthosphate (1.59 μg-at/1) were highest in September. Highest concentrations of ammonia were commonly measured in July (6.80 μg-at/1) and September (5.13 μg-at/1), and peak concentrations of nitrate and nitrite were recorded in January (24.27 μg-at/l), February (18.2 μg-at/1), and May (16.37 μg-at/1). Peak concentrations of chlorophyll a were measured in August (17.2 μg-at/1), October (15.70 μg-at/1), and March (15.33 μg-at/1). In general, the annual pattern for chlorophylls b and c were similar to chlorophyll a. Comparison with other estuaries and bays (Narrangansett Bay, Long Island Sound, Raritan Bay, and Chesapeake Bay) indicated that concentrations of nutrients and pigments in Delaware Bay were generally similar in magnitude and seasonality, These are the first set of seasonal water quality data for lower Delaware Bay.     

New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.     

Src-dependent phosphorylation of ASAP1 regulates podosomes

Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.