Intrinsic tyrosine kinase activity is required for vascular endothelial growth factor receptor 2 ubiquitination, sorting and degradation in endothelial cells

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis co-ordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.     

Role of the Cldn6 cytoplasmic tail domain in membrane targeting and epidermal differentiation in vivo

It is widely recognized that the claudin (Cldn) family of four tetraspan transmembrane proteins is crucial for tight junction assembly and permeability barrier function; however, the precise role of the tail and loop domains in Cldn function is not understood. We hypothesized that the cytoplasmic tail domain of Cldn6 is crucial for membrane targeting and hence epidermal permeability barrier (EPB) formation. To test this hypothesis via a structure-function approach, we generated a tail deletion of Cldn6 (CDelta187) and evaluated its role in epidermal differentiation and EPB formation through its forced expression via the involucrin (Inv) promoter in the suprabasal compartment of the transgenic mouse epidermis. Even though a functional barrier formed, Inv-CDelta187 mice displayed histological and biochemical abnormalities in the epidermal differentiation program and stimulation of epidermal cell proliferation in both the basal and suprabasal compartments of the interfolliclar epidermis, leading to a thickening of the epidermis after 1 week of age that persisted throughout life. Although some membrane localization was evident, our studies also revealed a significant amount of not only Cldn6 but also Cldn10, Cldn11, and Cldn18 in the cytoplasm of transgenic epidermal cells as well as the activation of a protein-unfolding pathway. These findings demonstrate that the overexpression of a tail truncation mutant of Cldn6 mislocalizes Cldn6 and other Cldn proteins to the cytoplasm and triggers a postnatal increase in proliferation and aberrant differentiation of the epidermis, emphasizing the importance of the Cldn tail domain in membrane targeting and function in vivo.     

Restoration of morphine-induced alterations in rat submandibular gland function by N-methyl-D-aspartate agonist

The effects of morphine, 1-aminocyclobutane-cis-1,3-dicarboxylic (ACBD; NMDA agonist) and 3-((R)2-carboxypiperazin-4-yl)-propyl-l-phosphoric acid (CPP; NMDA antagonist) and their concurrent therapy on rat submandibular secretory function were studied. Pure submandibular saliva was collected intraorally by micro polyethylene cannula from anaesthetized rats using pilocarpine as secretagogue. Intraperitoneal injection of morphine (6 mg/kg) induced significant inhibition of salivary flow rate, total protein, calcium, and TGF-beta1 concentrations. Administration of ACBD (10 mg/kg) and CPP (10 mg/kg) alone did not influence secretion of submandibular glands. In combination therapy, coadministration of CPP with morphine did not influence morphine-induced changes in salivary function while ABCD could restore all morphine-induced changes. In combination treatment, ACBD prevented morphine-induced reduction of flow rate, total protein, calcium, and TGF-beta1 and reached control levels. It is concluded that morphine-induced alterations in submandibular gland function are mediated through NMDA receptors.     

Dietary Supplementation with tBHQ,an Nrf2 Stabilizer Molecule,Confers Neuroprotection Against Apoptosis in Amyloid β-Injected Rat

Nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates the up-regulation of cytoprotective genes via the antioxidant response element (ARE). There is significant evidence that oxidative stress is a critical event in the pathogenesis of AD. Considering the protective role of Nrf2 against oxidative injury, we studied to determine whether in vivo toxicity of amyloid β (Aβ) can be attenuated by tBHQ, an Nrf2 stabilizer, Using an Aβ injection model. We demonstrated that pre-activation of endogenous Nrf2 by tBHQ attenuated Aβ-induced caspase-3 expression. tBHQ enhanced GSH, decreased MDA level, and inhibited NF-κB. This investigation provides the first documentation of tBHQ’s neuroprotective effect through decrease of Aβ accumulation in rat brain. Our results show the involvement of Hsp-70 in this protective effect. In summary tBHQ treatment for 1 week prior to Aβ injection protected against the oxidative damage, apoptosis and Aβ accumulation in rats.     

Detection of homologous recombination events in SARS-CoV-2

Biotechnology Letters - The COVID-19 disease with acute respiratory symptoms emerged in 2019. The causal agent of the disease, the SARS-CoV-2 virus, is classified into the Betacoronaviruses family....     

Response Surface Methodology for Optimizing the Bovine Serum Albumin Fibrillation

Amyloid fibrils are considered as nanostructures that could be formed by ordered self-assembly of the partially-folded states of many different peptides or proteins. In this study, bovine serum albumin was used as a model protein whose ordered aggregation (fibrillation) was optimized. Response surface methodology (RSM) was used in a design that contained a total of 30 experimental trials. The first 24 were organized in a factorial design and from 25 to 30 involved the replications of the central points. Data obtained from RSM were subjected to the analysis of variance (ANOVA) and analyzed using a second order polynomial equation. Subsequent testing of the suggested experimental parameters was done in vitro with Congo red spectrophotometric assay. Protein concentration, pH, temperature and time of incubation were the variables used in this study. Responses were assessed by measuring absorbance in 540?nm (characteristic of amyloid formation) and maximal wavelength. Concomitant effects of variables were assessed in surface plots that each considered two of the variables. Interestingly, the pattern obtained by monitoring absorbance at 540?nm and absorbance in maximal wavelength were identical in most cases. We are reporting the optimum concentration of protein, pH, temperature and time at 5?mg?ml^?1, 3.02 and 72?°C and 48?h, respectively. Our findings suggest that use of Congo red spectrophotometric test, as a simple and affordable assay could be suggested as a first test for assessing fibril formation of proteins. Absorbance in maximal wavelength is recommended as a significant indication of fibril formation.     

Lactic acid production by loofah-immobilized Rhizopus oryzae through one-step fermentation process using starch substrate

Rhizopus oryzae PTCC 5263 capacity in synthesis of lactic acid (LA) from 10 g/l of soluble potato starch was determined using one-step fermentation process. Pellets were the favorable growing form of the free cells. The extent of the natural ability of the test fungus on biofilm formation on loofah sponge was examined by immobilizing R. oryzae (LIRO). The maximum LA concentration for the free cells and LIRO within 96 h was 3 and 4 g/l, respectively. In terms of specific starch utilization rate (\(q_{\text{s}}\)) and specific LA formation (\(q_{\text{p}}\)), LIRO performed more favorably compared to the free cells (\(q_{{{\text{s}}_{\text{F}} }} > q_{{{\text{s}}_{\text{LIRO}} }}\) and \(q_{{{\text{p}}_{\text{F}} }} < q_{{{\text{p}}_{\text{LIRO}} }}\)). Cell immobilization strategy was undertaken for the column reactor studies based on the statistically optimized levels of the inoculum size and temperature. Maximum production of the LA by the LIRO using an airlift reactor with net draft tube was 5 g/l obtainable within 48 h.

     

iPSC modeling of stage-specific leukemogenesis reveals BAALC as a key oncogene in severe congenital neutropenia

1. Download : Download high-res image (88KB)
2. Download : Download full-size image