Active Rho kinase (ROK-alpha ) associates with insulin receptor substrate-1 and inhibits insulin signaling in vascular smooth muscle cells.

Recent studies from our laboratory have shown that insulin stimulates myosin-bound phosphatase (MBP) in vascular smooth muscle cells (VSMCs) by decreasing site-specific phosphorylation of the myosin-bound subunit (MBS) of MBP via nitric oxide/cGMP-mediated Rho/Rho kinase inactivation. Here we tested potential interactions between Rho kinase and insulin signaling pathways. In control VSMCs, insulin inactivates ROK-alpha, the major Rho kinase isoform in VSMCs, and inhibits thrombin-induced increase in ROK-alpha association with the insulin receptor substrate-1 (IRS-1). Hypertension (in spontaneous hypertensive rats) or expression of an active RhoA(V14) up-regulates Rho kinase activity and increases ROK-alpha/IRS-1 association resulting in IRS-1 serine phosphorylation that leads to inhibition of both insulin-induced IRS-1 tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activation. In contrast, expression of dominant negative RhoA or cGMP-dependent protein kinase type I alpha inactivates Rho kinase, abolishes ROK-alpha/IRS-1 association, and potentiates insulin-induced tyrosine phosphorylation and PI3-kinase activation leading to decreased MBS(T695) phosphorylation and decreased MBP inhibition. Collectively, these results suggest a novel function for ROK-alpha in insulin signal transduction at the level of IRS-1 and potential cross-talk between cGMP-dependent protein kinase type I alpha, Rho/Rho kinase signaling, and insulin signaling at the level of IRS-1/PI3-kinase.     

High prevalence of mouse mammary tumor virus-like gene sequences in breast cancer samples of Iranian women

Mouse mammary tumor virus (MMTV) is considered to be responsible for breast tumor development in mice. In several instances sequences homologous to the genome of this virus have been reported in human breast cancer samples.

Here we aimed to evaluate MMTV involvement in human breast cancer development. DNA was extracted from 118 formalin fixed and paraffin embedded malignant (n = 59) and benign (n = 59) breast lesions. Using two sets of outer and inner MMTV-like envelope specific primers, in a nested PCR setup, MMTV genome was detected in 19 samples out of 59 (%32.2) cases of breast carcinomas, while in non-malignant breast tissue samples, only 3 samples out of 59 (%5) were positive. The difference was statistically highly significant (p < 0.001). Alignment of PCR amplified sequences with BR6, GR and C3H mouse mammary tumor virus strains emerged to be around 99% identical which is indicative of tumor tissue infection by an exogenous mouse MMTV genome.     

A method for the assessment of disease associations with single-nucleotide polymorphism haplotypes and environmental variables in case-control studies

The rough draft of the human genome map has been used to identify most of the functional genes in the human genome, as well as to identify nucleotide variations, known as "single-nucleotide polymorphisms" (SNPs), in these genes. By use of advanced biotechnologies, researchers are beginning to genotype thousands of SNPs from biological samples. Among the many possible applications, one of them is the study of SNP associations with complex human diseases, such as cancers or coronary heart diseases, by using a case-control study design. Through the gathering of environmental risk factors and other lifestyle factors, such a study can be effectively used to investigate interactions between genes and environmental factors in their associations with disease phenotype. Earlier, we developed a method to statistically construct individuals' haplotypes and to estimate the distribution of haplotypes of multiple SNPs in a defined population, by use of estimating-equation techniques. Extending this idea, we describe here an analytic method for assessing the association between the constructed haplotypes along with environmental factors and the disease phenotype. This method is also robust to the model assumptions and is scalable to a large number of SNPs. Asymptotic properties of estimations in the method are proved theoretically and are tested for finite sample sizes by use of simulations. To demonstrate the use of the method, we applied it to assess the possible association between apolipoprotein CIII (six coding SNPs) and restenosis by using a case-control data set. Our analysis revealed two haplotypes that may reduce the risk of restenosis.     

Evidence for a functional interaction between the ClC-2 chloride channel and the retrograde motor dynein complex

The ClC-2 chloride channel has been implicated in essential physiological functions. Analyses of ClC-2 knock-out mice suggest that ClC-2 expression in retinal pigment epithelia and Sertoli cells normally supports the viability of photoreceptor cells and male germ cells, respectively. Further, other studies suggest that ClC-2 expression in neurons may modify inhibitory synaptic transmission via the gamma-aminobutyric acid, type A receptor. However, complete understanding of the physiological functions of ClC-2 requires elucidation of the molecular basis for its regulation. Using cell imaging and biochemical and electrophysiological techniques, we show that expression of ClC-2 at the cell surface may be regulated via an interaction with the dynein motor complex. Mass spectrometry and Western blot analysis of eluate from a ClC-2 affinity matrix showed that heavy and intermediate chains of dynein bind ClC-2 in vitro. The dynein intermediate chain co-immunoprecipitates with ClC-2 from hippocampal membranes suggesting that they also interact in vivo. Disruption of dynein motor function perturbs ClC-2 localization and increases the functional expression of ClC-2 in the plasma membranes of COS7 cells. Thus, cell surface expression of ClC-2 may be regulated by dynein motor activity. This work is the first to demonstrate an in vivo interaction between an ion channel and the dynein motor complex.     

Decreased insulin action in skeletal muscle from patients with McArdle's disease

Insulin action is decreased by high muscle glycogen concentrations in skeletal muscle. Patients with McArdle's disease have chronic high muscle glycogen levels and might therefore be at risk of developing insulin resistance. In this study, six patients with McArdle's disease and six matched control subjects were subjected to an oral glucose tolerance test and a euglycemic-hyperinsulinemic clamp. The muscle glycogen concentration was 103 +/- 45% higher in McArdle patients than in controls. Four of six McArdle patients, but none of the controls, had impaired glucose tolerance. The insulin-stimulated glucose utilization and the insulin-stimulated increase in glycogen synthase activity during the clamp were significantly lower in the patients than in controls (51.3 +/- 6.0 vs. 72.6 +/- 13.1 micromol x min(-1) x kg lean body mass(-1), P < 0.05, and 53 +/- 15 vs. 79 +/- 9%, P < 0.05, n = 6, respectively). The difference in insulin-stimulated glycogen synthase activity between the pairs was significantly correlated (r = 0.96, P < 0.002) with the difference in muscle glycogen level. The insulin-stimulated increase in Akt phosphorylation was smaller in the McArdle patients than in controls (45 +/- 13 vs. 76 +/- 13%, P < 0.05, respectively), whereas basal and insulin-stimulated glycogen synthase kinase 3alpha and protein phosphatase-1 activities were similar in the two groups. Furthermore, the ability of insulin to decrease and increase fat and carbohydrate oxidation, respectively, was blunted in the patients. In conclusion, these data show that patients with McArdle's glycogen storage disease are insulin resistant in terms of glucose uptake, glycogen synthase activation, and alterations in fuel oxidation. The data further suggest that skeletal muscle glycogen levels play an important role in the regulation of insulin-stimulated glycogen synthase activity.     

Biosynthesized silver nanoparticles induce phytotoxicity in Vigna radiata L.

With the recent developments in the field of nanotechnology, the biosynthesis of nanoparticles has increased tremendously. Silver nanoparticles (SNPs) are among the most synthesized nanoparticles and this extensive synthesis can elevate the amounts of SNPs in the environment, which, consequently, pose a serious threat to the ecosystem and can bring unwanted environmental effects. As plants are an important part of ecosystem, investigation of toxic effects of SNPs on plants is particularly interesting. This study evaluates the potential risk of SNPs interaction with plants. For this, seeds of Vigna radiata L. were screened in presence of SNPs (20 mgL⁻¹) using the germination, growth, and biochemical parameters as a phototoxicity criterion. The 19.57 nm average-sized SNPs were synthesized via the biosynthesis method. These biosynthesized SNPs were then applied on two varieties of V. radiata (Azri and High cross 404) and found to have variety dependent toxic effects on seed germination, growth, and biochemical parameters. Seed germination, root length, shoot length, fresh weight, chlorophyll, carotenoid, sugar content, and total proteins were reduced by 20, 46, 50, 18, 55, 62, 82, and 67%, respectively, in High cross 404, when compared with control (distilled water). The variety Azri was less sensitive than the variety High cross 404. In conclusion, the results demonstrated that SNPs affect seed germination and seedling growth when internalized and accumulated in plants, revealing that SNPs were responsible for the side effects. More in-depth research is required, in the form of different concentrations of SNPs or different plant species, to draw a logical conclusion and develop legislation about the safe use of biosynthesized SNPs.Supplementary Information The online version contains supplementary material available at 10.1007/s12298-021-01073-4.     

Resistin inhibits glucose uptake in L6 cells independently of changes in insulin signaling and GLUT4 translocation

Elevated levels of resistin have been proposed to cause insulin resistance and therefore may serve as a link between obesity and type 2 diabetes. However, its role in skeletal muscle metabolism is unknown. In this study, we examined the effect of resistin on insulin-stimulated glucose uptake and the upstream insulin-signaling components in L6 rat skeletal muscle cells that were either incubated with recombinant resistin or stably transfected with a vector containing the myc-tagged mouse resistin gene. Transfected clones expressed intracellular resistin, which was released in the medium. Incubation with recombinant resistin resulted in a dose-dependent inhibition of insulin-stimulated 2-deoxyglucose (2-DG) uptake. The inhibitory effect of resistin on insulin-stimulated 2-DG uptake was not the result of impaired GLUT4 translocation to the plasma membrane. Furthermore, resistin did not alter the insulin receptor (IR) content and its phosphorylation, nor did it affect insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation, its association with the p85 subunit of phosphatidylinositol (PI) 3-kinase, or IRS-1-associated PI 3-kinase enzymatic activity. Insulin-stimulated phosphorylation of Akt/protein kinase B-alpha, one of the downstream targets of PI 3-kinase and p38 MAPK phosphorylation, was also not affected by resistin. Expression of resistin also inhibited insulin-stimulated 2-DG uptake when compared with cells expressing the empty vector (L6Neo) without affecting GLUT4 translocation, GLUT1 content, and IRS-1/PI 3-kinase signaling. We conclude that resistin does not alter IR signaling but does affect insulin-stimulated glucose uptake, presumably by decreasing the intrinsic activity of cell surface glucose transporters.     

Solid-state fermentation-supported enhanced production of α-galactosidase by a deoxyglucose-resistant mutant of <Emphasis Type="Italic">Aspergillus niger</Emphasis> and thermostabilization of the production process

The aim of the present investigation was to comparatively evaluate the behaviour of A. niger and its derepressed mutant in production of α-galactosidase in submerged (SmF) and solid state fermentation (SSF) using basal Vogel’s medium or corn steep liquor as nitrogen source and observe the response of latter source under both cultural techniques under different temperature regimes, and determine if SSF can be exploited in a wide range of temperature expected to vary in this fermentation system. All studies were performed in both systems under pre-optimized cultural conditions. Higher melting temperature and negative values of entropy of activation in SSF indicated that the genetic system of both organisms was thermodynamically resistant in the presence of corn steep liquor but sensitive to inactivation in the presence of Vogel’s nitrogen sources in submerged fermentation. This was reflected as the organisms demanded higher magnitudes of energy for product formation in the presence of ammonium salts. Studies on influence of corn steep liquor revealed that it had stabilizing effect too in both fermentation systems but the mutant strain was more stable in both fermentation systems. Because of these properties, the mutant organism may be exploited for bulk production of α-galactosidase in SSF under condition where temperature may fluctuate during fermentation.