The Constitutive Activity of Epidermal Growth Factor Receptor vIII Leads to Activation and Differential Trafficking of Wild-type Epidermal Growth Factor Receptor and erbB2

A constitutively active epidermal growth factor receptor (EGFR) mutant, EGFR variant III (EGFRvIII), has been detected at high frequencies in certain human cancers. This study evaluated transactivation and trafficking of erbB family members as a result of constitutive EGFR activity in a cancer cell line. Expression of EGFRvIII modulated erbB family members through different mechanisms; the erbB3 mRNA level was reduced, whereas wild-type EGFR (wtEGFR) and erbB2 protein levels were diminished, with no change in their mRNA levels, and there was no change in the erbB4 expression level. Both EGFR and erbB2 were internalized as a result of EGFRvIII''s activity and redistributed to the cell surface upon addition of AG1478, an inhibitor of wtEGFR/EGFRvIII catalytic activity. Acute activation of EGFRvIII by removing AG1478 from cells increased phosphorylation of both wtEGFR and erbB2 and caused differential trafficking of EGFRvIII''s activation partners; wtEGFR was directed primarily to lysosomal compartments and partially to recycling compartments, whereas erbB2 was directed primarily to recycling compartments and partially to lysosomal compartments. Our data demonstrate that the constitutive activity of EGFRvIII is sufficient to trigger endocytosis and trafficking of wtEGFR and erbB2, which may play a role in activating signaling pathways that are triggered during receptor endocytosis. (J Histochem Cytochem 58:529–541, 2010)     

alpha-Thrombin induces rapid and sustained Akt phosphorylation by beta-arrestin1-dependent and -independent mechanisms, and only the sustained Akt phosphorylation is essential for G1 phase progression

In Chinese hamster embryonic fibroblasts (IIC9 cells) alpha-thrombin activates the MAPK(ERK) and phosphatidylinositol 3-OH-kinase (PI 3-kinase)/Akt pathways, and both are essential for progression through the G(1) phase of the cell cycle. We investigated in IIC9 cells, the role of beta-arrestin1 in alpha-thrombin signaling to these pathways. alpha-Thrombin stimulates rapid and sustained PI 3-kinase and Akt activities. Expression of a dominant negative beta-arrestin1 (beta-arrestin1(V53D)) inhibits rapid but not sustained PI 3-kinase and Akt activities. Surprisingly, expression of beta-arrestin1(V53D) does not block activation of the MAPK(ERK) pathway. PI 3-kinase and Akt activities are also inhibited by expression of a beta-arrestin1 mutant, which impairs binding to c-Src (beta-arrestin1(P91G-P121E)), indicating the involvement of c-Src in the rapid stimulation of the PI 3-kinase/Akt pathway. Consistent with these results, PP1, a selective inhibitor of c-Src family kinases, prevents alpha-thrombin-stimulated Akt phosphorylation. Expression of beta- arrestin1(V53D) does not prevent G(1) progression, as its expression has no effect on [(3)H]thymidine incorporation into DNA. In agreement with the ineffectiveness of beta-arrestin1(V53D) to block G(1) progression, cyclin D1 protein amounts and CDK4-cyclin D1 activity is unaffected by expression of beta-arrestin1(V53D). Thus in IIC9 cells, alpha-thrombin activates rapid beta-arrestin1-dependent and sustained beta-arrestin1-independent Akt activity, suggesting that two mechanisms are involved. Furthermore, although blocking the beta-arrestin1-independent PI 3-kinase/Akt pathway prevents G(1) progression, inhibition of the beta-arrestin1-dependent pathway does not, indicating different roles for the rapid and sustained activities.     

Evaluation of low-intensity laser radiation on stimulating the cholesterol degrading activity: Part I. Microorganisms isolated from cholesterol-rich materials

A survey was performed to isolate bacteria and fungi from cholesterol-rich sources including chicken liver, turkey giblets, salmon, lamb, egg yolk, beef brain and shrimps. A total of 34 bacterial and 22 fungal isolates were recovered from the tested sources. The highest count of isolates was recovered from the soil (12 isolates/g), followed by turkey giblets and egg yolk (8 isolates/g, for each). Out of 34 bacterial isolates, five induced the highest level in cholesterol degradation. The most potent bacterial isolate was recovered from turkey giblets and was identified as Streptomyces fradiae. In a trial to increase the cholesterol decomposing potentiality of S. fradiae, low intensity Nd-YAG laser irradiation was evaluated. The exposure of the chlorophyllin – photosensitized bacterium to 210 mW Nd-YAG laser for 8 min induced significant increase in cholesterol degrading activity reaching 73.8% as compared with 54.2% in the case of non-irradiated, non-photosensitized culture. Under the same conditions but using the reaction mixture containing cholesterol as a substrate and extracellular crude enzyme, the percent decomposition reached 53.7% for the irradiated culture as compared to 28.3% in the case of the control. Our data indicate the importance of the photosensitizer in enhancement of laser radiation to stimulate cholesterol decomposition of S. fradiae.     

The comparative gastrointestinal morphology of Jaculus jaculus (Rodentia) and Paraechinus aethiopicus (Erinaceomorpha)

Jaculus jaculus (Lesser Egyptian jerboa) and Paraechinus aethiopicus (Desert hedgehog) are small mammals which thrive in desert conditions and are found, among others, in the Arabian Peninsula. Jaculus jaculus is omnivorous while P. aethiopicus is described as being insectivorous. The study aims to describe the gastrointestinal tract (GIT) morphology of these animals which differ in diet and phylogeny. The GITs of J. jaculus (n = 8) and P. aethiopicus (n = 7) were weighed, photographed, and the length, basal surface areas, and luminal surface areas of each of the anatomically distinct gastrointestinal segments were determined. The internal aspects of each area were examined and photographed while representative histological sections of each area were processed to wax and stained using haematoxylin and eosin. Both species had a simple unilocular stomach which was confirmed as wholly glandular on histology sections. Paraechinus aethiopicus had a relatively simple GIT which lacked a caecum. The caecum of J. jaculus was elongated, terminating in a narrow cecal appendix which contained lymphoid tissue on histological examination. The internal aspect of the proximal colon of J. jaculus revealed distinct V‐shaped folds. Stomach content analysis of J. jaculus revealed mostly plant and seed material and some insects, whereas P. aethiopicus samples showed plant material in addition to insects, indicating omnivorous feeding tendencies in areas where insects may be scarce. J. Morphol. 277:671–679, 2016. © 2016 Wiley Periodicals, Inc.     

Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays

Spleen tyrosine kinase (Syk) is an important non-receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high-throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI-2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze-thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr⁵²⁵/Tyr⁵²⁶ in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high-throughput system.     

Activation of prolyl hydroxylase‐2 for stabilization of mitochondrial stress along with simultaneous downregulation of HIF‐1α/FASN in ER + breast cancer subtype

The present study was undertaken to inquest the chemical activation of prolyl hydroxylase‐2 for the curtailment of hypoxia‐inducible factor‐1α and fatty acid synthase. It was well documented that hypoxia‐inducible factor‐1α and fatty acid synthase were overexpressed in mammary gland carcinomas. After screening a battery of compounds, BBAP‐2 was retrieved as a potential prolyl hydroxylase‐2 activator and validates its activity using ER + MCF‐7 cell line and n‐methyl‐n‐nitrosourea‐induced rat in vivo model, respectively. BBAP‐2 was palpable for the morphological characteristics of apoptosis along with changes in the mitochondrial intergrity as visualized by acridine orange/ethidium bromide and JC‐1 staining against ER + MCF‐7 cells. BBAP‐2 also arrest the cell cycle of ER + MCF‐7 cells at G2/M phase. Afterward, BBAP‐2 has scrutinized against n‐methyl‐n‐nitrosourea‐induced mammary gland carcinoma in albino Wistar rats. BBAP‐2 restored the morphological architecture when screened through carmine staining, haematoxylin and eosin staining, and scanning electron microscopy. BBAP‐2 also delineated the markers of oxidative stress favourably. The immunoblotting and mRNA expression analysis validated that BBAP‐2 has a potentialty activate the prolyl hydroxylase‐2 with sequential downregulating effect on hypoxia‐inducible factor‐1α and its downstream checkpoint. BBAP‐2 also fostered apoptosis through mitochondrial‐mediated death pathway. The present study elaborates the chemical activation of prolyl hydroxylase‐2 by which the increased expression of HIF‐1α and FASN can be reduced in mammary gland carcinoma.     

Non-Carcinogenic Risk Assessment of Heavy Metals and Fluoride in Some Water Wells in the Al-Baha Region,Saudi Arabia

On a global scale, pathogenic contamination of drinking water poses the most significant health risk to humans. However, significant risks to human health may also result from exposure to nonpathogenic, toxic contaminants that are often ubiquitous in waters. The purpose of this study is to determine the levels of heavy metal and fluoride contaminants in water wells used in the Al-Baha region, Saudi Arabia, to evaluate if the levels of metals will have non-carcinogenic effects. Samples were collected from private wells in the area and were analyzed for chemical contamination using approved methods of collection and analysis. Chromium, manganese, zinc, iron, and fluoride were detected in all samples, and were selected for toxicological evaluation. Exposure through ingestion and dermal contact were the scenarios proposed in this study. Chronic daily intakes (CDIs) were estimated for both routes and then compared with health guideline values. The non-cancer risk estimations show that manganese, chromium, and zinc individually have oral Hazard Quotient (HQ) values less than a value of one. Iron and fluoride were found to have oral HQ values greater than 1 in some samples. Also, on considering the additive effect of the contaminants we found that some samples have Hazard Index (HI) values greater than 1, which indicates that there is a concern for chronic non-cancer adverse health effects in case of oral and dermal routes of exposure to water from these wells.     

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.