Plasma membrane calcium pumps in smooth muscle: from fictional molecules to novel inhibitors

Plasma membrane Ca2+ pumps (PMCA pumps) are Ca2+-Mg2+ ATPases that expel Ca2+ from the cytosol to extracellular space and are pivotal to cell survival and function. PMCA pumps are encoded by the genes PMCA1, -2, -3, and -4. Alternative splicing results in a large number of isoforms that differ in their kinetics and activation by calmodulin and protein kinases A and C. Expression by 4 genes and a multifactorial regulation provide redundancy to allow for animal survival despite genetic defects. Heterozygous mice with ablation of any of the PMCA genes survive and only the homozygous mice with PMCA1 ablation are embryolethal. Some PMCA isoforms may also be involved in other cell functions. Biochemical and biophysical studies of PMCA pumps have been limited by their low levels of expression. Delineation of the exact physiological roles of PMCA pumps has been difficult since most cells also express sarco/endoplasmic reticulum Ca2+ pumps and a Na+-Ca2+-exchanger, both of which can lower cytosolic Ca2+. A major limitation in the field has been the lack of specific inhibitors of PMCA pumps. More recently, a class of inhibitors named caloxins have emerged, and these may aid in delineating the roles of PMCA pumps.     

STAT-1 mediates the stimulatory effect of IL-10 on CD14 expression in human monocytic cells

IL-10, an anti-inflammatory cytokine, has been shown to exhibit stimulatory functions including CD14 up-regulation on human monocytic cells. CD14-mediated signaling following LPS stimulation of monocytic cells results in the synthesis of proinflammatory cytokines. Our results show that LPS-induced CD14 expression on monocytic cells may be mediated by endogenously produced IL-10. To investigate the molecular mechanism by which IL-10 enhances CD14 expression, both human monocytes and the promyelocytic HL-60 cells were used as model systems. IL-10 induced the phosphorylation of PI3K and p42/44 ERK MAPK. By using specific inhibitors for PI3K (LY294002) and ERK MAPKs (PD98059), we demonstrate that LY294002 either alone or in conjunction with PD98059 inhibited IL-10-induced phosphorylation of STAT-1 and consequently CD14 expression. However, IL-10-induced STAT-3 phosphorylation remained unaffected under these conditions. Finally, STAT-1 interfering RNA inhibited IL-10-induced CD14 expression. Taken together, these results suggest that IL-10-induced CD14 up-regulation in human monocytic cells may be mediated by STAT-1 activation through the activation of PI3K either alone or in concert with the ERK MAPK.     

Nodulation competitiveness between contrasting phage phenotypes of pigeonpea rhizobial strains

Competitiveness between (I) lysogenic vs. phage-indicator strains, (II) phage-resistant vs phage-sensitive strains, and (III) large plaque vs. small plaque developing strains was examined under laboratory and field conditions in order to study the involvement of these crucial phage sensitivity patterns in the competition for nodule occupancy of pigeonpea rhizobia. The phage-indicator strain (A039) exhibited higher competitiveness over the lysogenic strain (A025 Sm(r)); the phage sensitive strain (IHP-195) over the phage resistant strain (IHP 195 Sm(r)V(r)); and the large plaque developing strain (A059) over the small plaque developing strain (IHP195 Sm(r)) in association with pigeonpea cv. bahar both under laboratory and field conditions. Dual inoculation of A025 Sm(r) + A039 and A059 + IHP195 Sm(r) (mixed in equal proportion just before treatment) improved the nodule occupancy by inoculant strains against native rhizobia and resulted into higher plant dry weight and yield as compared to their application as single inoculum. The phage-resistant mutant IHP195 Sm(r)V(r) showed reduced competitiveness against native rhizobia, compared to its parental strain. The dual inoculation of parental strain and phage-resistant mutant gave the same result as the inoculation of parental strain alone.     

Evidences showing ultraviolet-B radiation-induced damage of DNA in cyanobacteria and its detection by PCR assay

Impact of ultraviolet-B radiation in causing the damages to the DNA of the cyanobacterium, Anabaena strain BT2 has been investigated. Exposure of genomic DNA (in vitro) to UV-B radiation for 1 h did not cause any shift in the absorption peak (lambda(max)) but more than 30% increase in absorbance was noticed in comparison to untreated control DNA (no exposure to UV-B). This increase in absorbance in a way may be comparable to typical hypochromic effect but there was no decrease in absorbance following transfer of UV-B-treated DNA to fluorescent light or in the dark. That the damaging effect of UV-B radiation on native structure of DNA is indeed real was also evident from the PCR-based assay such as RAPD, rDNA amplification, and ARDRA. Template activity of UV-B-treated genomic DNA was drastically inhibited, there was no amplification in RAPD assay after prior exposure of DNA to UV-B for 60 min. Only one band of approximately 400 bp was observed even after 60 min of exposure which suggests that certain segment of DNA strand is resistant to UV-B effects. Similar to the effects on RAPD profile, amplification of rDNA was significantly inhibited following exposure of genomic DNA to UV-B. Our findings clearly demonstrate that UV-B does affect the DNA of cyanobacteria and the killings of these microbes might be due to the irreversible damages caused to DNA by this high energy radiation. It is felt that PCR assay may be conveniently used for screening the damages caused to DNA by UV-B radiation in cyanobacteria and other microorganisms.     

Rise of plasma ghrelin with weight loss is not sustained during weight maintenance

Objective: Ghrelin is postulated to be an orexigenic signal that promotes weight regain after weight loss (WL). However, it is not known whether this putative effect of ghrelin is sustained after weight stabilization. The objective of this study was to investigate the relationship of plasma ghrelin concentrations to active WL and weight maintenance in obese subjects. Research Methods and Procedures: This study was a randomized clinical trial, with a 12‐month follow‐up period. Obese Mexican‐American women matched for age and BMI were randomized to a 12‐month WL program (n = 25) or no intervention (controls, n = 23). Interventions included diet, exercise, and orlistat. Body weight and fasting ghrelin, leptin, insulin, and glucose concentrations were measured at baseline and 6 and 12 months. Results: The WL group lost 8.5% of body weight after 6 months and maintained the new weight for the next 6 months. Ghrelin concentrations increased significantly at 6 months but returned to baseline at 12 months. Baseline ghrelin concentrations were directly related to the degree of WL achieved after 12 months. Controls experienced no change in BMI or ghrelin levels. There were no associations between plasma ghrelin and leptin or insulin concentrations. Discussion: Consistent with previous results, ghrelin rises in response to WL, perhaps as a counterregulatory mechanism. However, the present results indicate that ghrelin concentrations return to baseline with sustained weight maintenance, suggesting that its effects are unlikely to regulate long‐term energy balance. Baseline ghrelin concentrations are related to the degree of WL that can be achieved by active weight reduction.     

Pilot study using SELDI-TOF-MS based proteomic profile for the identification of diagnostic biomarkers of thyroid proliferative diseases

Biomarkers for thyroid cancer (TCa) lack specificity. To develop TCa specific biomarkers, SELDI-TOF-MS was used to examine the proteomic profile of biopsies obtained from papillary TCa along with adjacent normal tissue. Sixty-three potential biomarkers were categorized by univariate analysis into single biomarker candidates and segregated by multivariate analysis into normal and cancerous groups. Our studies demonstrate the sensitivity and reproducibility of this approach to detect biomarkers for TCa.     

Structure–activity relationships of carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase

Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC₅₀ = 11.9 μM) in cell culture without any apparent host-toxicity.     

Analysis of unigene derived microsatellite markers in family solanaceae

The family Solanaceae is the source of several economically important plants. The aim of this study was to trace and characterize simple sequence repeat (SSR) markers from unigene sequences of Solanum lycopersicum, an important member of family Solanaceae. 18,228 unigene sequences of Solanum lycopersicum was taken in order to develop SSR markers and analyzed for the in-silico design of PCR primers. A total of 12,090 (66.32 %) unigenes containing 17,524 SSRs (microsatellites) were identified. The average frequency of microsatellites in unigenes was one in every 1.3 kb of sequence. The analysis revealed that trinucleotide motifs, coding for Glutamic acid (GAA) and AT/TA were the most frequent repeat of dinucleotide SSRs. Flanking sequences of the SSRs generated 877 primers with forward and reverse strands. Functional categorization of SSRs containing unigenes was done through gene ontology terms like Biological process, Cellular component and Molecular function.     

Transforming Growth Factor-��-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

Satellite cells/myoblasts account for the majority of muscle regenerative potential in response to injury and muscular adaptation to exercise. Although the ability to influence this process would provide valuable benefits for treating a variety of patients suffering from muscle loss, the regulatory mechanisms of myogenesis are not completely understood. We have tested the hypothesis that transforming growth factor-β-activated kinase 1 (TAK1) is an important regulator of skeletal muscle formation. TAK1 is expressed in proliferating C2C12 myoblasts, and its levels are reduced upon differentiation of myoblasts into myotubes. In vivo, TAK1 is predominantly expressed in developing skeletal muscle of young mice. However, the expression of TAK1 was significantly up-regulated in regenerating skeletal muscle of adult mice. Overexpression of a dominant negative mutant of TAK1 or knockdown of TAK1 inhibited the proliferation and differentiation of C2C12 myoblasts. TAK1 was required for the expression of myogenic regulatory factors in differentiating myoblasts. Genetic ablation of TAK1 also inhibited the MyoD-driven transformation of mouse embryonic fibroblasts into myotubes. Inhibition of TAK1 suppressed the differentiation-associated activation of p38 mitogen-activated protein kinase (MAPK) and Akt kinase. Overexpression of a constitutively active mutant of MAPK kinase 6 (MKK6, an upstream activator of p38 MAPK) but not constitutive active Akt restored the myogenic differentiation in TAK1-deficient mouse embryonic fibroblasts. Insulin growth factor 1-induced myogenic differentiation was also found to involve TAK1. Collectively, our results suggest that TAK1 is an important upstream regulator of skeletal muscle cell differentiation.     

The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice

Skeletal muscle atrophy occurs in a variety of clinical settings, including cachexia, disuse, and denervation. Inflammatory cytokines have been shown to be mediators of cancer cachexia; however, the role of cytokines in denervation- and immobilization-induced skeletal muscle loss remains unknown. In this study, we demonstrate that a single cytokine, TNF-like weak inducer of apoptosis (TWEAK), mediates skeletal muscle atrophy that occurs under denervation conditions. Transgenic expression of TWEAK induces atrophy, fibrosis, fiber-type switching, and the degradation of muscle proteins. Importantly, genetic ablation of TWEAK decreases the loss of muscle proteins and spared fiber cross-sectional area, muscle mass, and strength after denervation. Expression of the TWEAK receptor Fn14 (fibroblast growth factor–inducible receptor 14) and not the cytokine is significantly increased in muscle upon denervation, demonstrating an unexpected inside-out signaling pathway; the receptor up-regulation allows for TWEAK activation of nuclear factor κB, causing an increase in the expression of the E3 ubiquitin ligase MuRF1. This study reveals a novel mediator of skeletal muscle atrophy and indicates that the TWEAK–Fn14 system is an important target for preventing skeletal muscle wasting.