Dynamics of the interaction between the insulin receptor and protein tyrosine-phosphatase 1B in living cells

The dynamics of the interaction of the insulin receptor with a substrate-trapping mutant of protein-tyrosine phosphatase 1B (PTP1B) were monitored in living human embryonic kidney cells using bioluminescence resonance energy transfer (BRET). Insulin dose-dependently stimulates this interaction, which could be followed in real time for more than 30 minutes. The effect of insulin on the BRET signal could be detected at early time-points (30 seconds), suggesting that in intact cells the tyrosine-kinase activity of the insulin receptor is tightly controlled by PTP1B. Interestingly, the basal (insulin-independent) interaction of the insulin receptor with PTP1B was much weaker with a soluble form of the tyrosine-phosphatase than with the endoplasmic reticulum (ER)-targeted form. Inhibition of insulin-receptor processing using tunicamycin suggests that the basal interaction occurs during insulin-receptor biosynthesis in the ER. Therefore, localization of PTP1B in this compartment might be important for the regulation of insulin receptors during their biosynthesis.     

Differential functional properties of Ca2+ stores in pulmonary arterial conduit and resistance myocytes

In smooth muscle cells, oscillations of intracellular Ca2+ concentration ([Ca2+]i) are controlled by inositol 1,4,5-trisphosphate (InsP3) and ryanodine (Ry) receptors on the sarcoplasmic reticulum (SR). Here we show that these Ca2+ oscillations are regulated differentially by InsP3 and Ry receptors in cells dispersed from the main trunk of the pulmonary artery (conduit myocytes) or from tertiary and quaternary arterial branches (resistance myocytes). Ry receptor antagonists inhibit either spontaneous or ATP-induced Ca2+ oscillations in resistance myocytes but they do not affect the oscillations in most conduit myocytes. In contrast, agents that inhibit InsP3 production or activation of InsP3 receptors do not alter the oscillations is resistance myocytes but block them in conduit myocytes. We have also examined the degree of overlap of Ry- and InsP3-sensitive stores in myocytes along the pulmonary arterial tree. In conduit myocytes, depletion of Ry-sensitive stores with repeated application of caffeine in the presence of Ry or in Ca2+ free solutions did not prevent the ATP-induced Ca2+ release from InsP3-dependent stores. However, responsiveness to ATP was completely abolished in resistance myocytes subjected to the same experimental protocol. Thus, InsP3- and Ry-dependent stores appear to be separated in conduit myocytes but joined in resistance myocytes. These data demonstrate for the first time differential properties of intracellular Ca2+ stores and receptors in myocytes distributed along the pulmonary arterial tree and help to explain the distinct functional responses of large and small pulmonary vessels to vasoactive agents.     

Cultural Formulation: Depression and Back Pain in a Young Male Turkish Immigrant in Basel,Switzerland

Culture, Medicine, and Psychiatry -     

Fatty acids as trophic tracers in an experimental estuarine food chain: Tracer transfer

The transfer of fatty acid (FA) biomarkers was assessed by an experimental food chain comprising three trophic levels: leaves of the mangrove Avicennia marina, the grapsid crab Parasesarma erythodactyla, and the blue swimmer crab Portunus pelagicus. FA compositions for each trophic level were investigated through a feeding/starving regime designed to reveal the transfer of FAs along the food chain. Comparison of the FA profiles of the mangrove leaves, the tissues and faecal material of P. erythodactyla suggests that the crab, contrary to previous studies, lacks the necessary enzymes to incorporate some FAs in its diet. Long chain FAs were egested while polyunsaturated acids seemed to be efficiently assimilated. The polyunsaturated FAs 18:2ω6 and 18:3ω3 were identified as useful biomarkers of the mangrove leaves for tracing their transfer to the higher trophic levels. The contribution of these markers to the FA profiles of the crabs was investigated and it was found that both 18:2ω6 and 18:3ω3 could be successfully traced across the first trophic transfer. However, only 18:3ω3 demonstrated a clear second transfer into the tissues of P. pelagicus. Multivariate analysis of the FA profiles of the study organisms was found to be a potentially useful tool for demonstrating differences in diet within a species and also what FAs, and therefore dietary items, are responsible for those differences. MDS analysis of the FA profiles of faecal material from P. erythodactyla showed that this species provides an important ecological link in estuarine systems by providing a substrate for the colonisation of bacteria.     

Response and interaction of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and arbuscular mycorrhizal fungi in the soybean rhizosphere

Regulatory response and interaction of Bradyrhizobium and arbuscular mycorrhizal fungi (AMF) play a vital role in rhizospheric soil processes and productivity of soybean (Glycine max L.). Nitrogen (N) and phosphorus (P) are essential nutrients for plant growth and productivity, the synergistic interaction(s) of AMF and Bradyrhizobium along with rhizospheric beneficial microorganisms stimulate soybean growth and development through enhanced mineral nutrient acquisition (N and P) and improved rhizosphere environment. Such interactions are crucial, especially under low-input eco-friendly agricultural cropping systems, which rely on biological processes rather than agrochemicals to maintain soil quality, sustainability, and productivity. Furthermore, enhancement of N-fixation by root nodules along with AMF-mediated synergism improves plant P nutrition and uptake, and proliferation of phosphate-solubilizing fungi. However, the genetic and/or allelic diversity among native strains, their genes/enzymes and many environmental factors (e.g., soil organic matter, fertilizers, light, temperature, soil moisture, and biotic interactors) affect the interactions between AMF and Bradyrhizobium. New information is available regarding the genetic composition of elite soybean inoculant strains in maximizing symbiotic performance, N-fixing capabilities and depending on N and P status the host-mediated regulation of root architecture. Overall, for sustainable soybean production systems, a deeper understanding of the interaction effects of Bradyrhizobium and AMF co-inoculation are expected in the future, so that optimized combinations of microorganisms can be applied as effective soil inoculants for plant growth promotion and fitness. The objective of this review is to offer insights into the mechanistic interactions of AMF and Bradyrhizobium and rhizopheric soil health, and elucidate the role of environmental factors in regulating growth, development and sustainable soybean productivity.     

The apoptotic actions of platelets in acute ischemic stroke

Stroke is a disease that affects the blood vessels that supply blood to the brain. Although platelets are implicated in the pathophysiology of stroke the mechanism is still not clear and there antiplatelet agents available for the prevention and treatment of stroke. We herein examined the relationship between the potential cytokine, TNF-α platelet activation and apoptosis in acute ischemic stroke patients. We selected 60 patients (mean age 57.9 ± 10.2 years) who had not taken any antiplatelet drugs for 14 days. A group of 45 participants (mean age 51.05 ± 9.07 years) were selected as the control group. For both the patients and for the control group, P-selectin (CD62p) and Annexin-V binding, cytochrome-c levels, caspase-3 gene expression and caspase-3 releasing and plasma TNF-α levels were measured in platelets. The results showed significant increase in plasma TNF-α and platelet Annexin-V, CD62p, cytochrome-c and caspase-3 gene expression in stroke patients compared to the control group. The data of this work suggests that inflammation may have a role in platelet apoptosis in stroke which may suggest a new aspect of the role of inflammation in the development of acute ischemic stroke.     

Transient receptor potential ankyrin-1 (TRPA1) modulates store-operated Ca2 + entry by regulation of STIM1-Orai1 association

TRPA1 is a non-selective Ca^2 + permeable channel located in the plasma membrane that functions as a cellular sensor detecting mechanical, chemical and thermal stimuli, being a component of neuronal, epithelial, blood and smooth muscle tissues. TRPA1 has been shown to influence a broad range of physiological processes that involve Ca^2 +-dependent signaling pathways. Here we report that TRPA1 is expressed in MEG01 but not in platelets at the protein level. MEG01 cells maturation induced by PMA results in attenuation of TRPA1 protein expression and enhances thapsigargin-evoked Ca^2 + entry without altering the release of Ca^2 + from intracellular stores. Inhibition of TRPA1 by HC-030031 results in enhancement of both thrombin- and thapsigargin-stimulated Ca^2 + entry. Co-immunoprecipitation experiments revealed that TRPA1 associates with STIM1, as well as Orai1, TRPC1 and TRPC6. Downregulation of TRPA1 expression by MEG01 maturation, as well as pharmacological inhibition of TRPA1 by HC-030031, results in enhancement of the association between STIM1 and Orai1. Altogether, these findings provide evidence for a new and interesting function of TRPA1 in cellular function associated to the regulation of agonist-induced Ca^2 + entry by the modulation of STIM1/Orai1 interaction.     

O-GlcNAcylation-Inducing Treatments Inhibit Estrogen Receptor α Expression and Confer Resistance to 4-OH-Tamoxifen in Human Breast Cancer-Derived MCF-7 Cells

O-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the β-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP₃ production. We observed that these treatments stimulated PIP₃ production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy.     

RABL6A,a Novel RAB-Like Protein,Controls Centrosome Amplification and Chromosome Instability in Primary Fibroblasts

RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53−/− MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis.