Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma

Stress can alter immunological, neurochemical and endocrinological functions, but its role in cancer progression is not well understood. Here, we show that chronic behavioral stress results in higher levels of tissue catecholamines, greater tumor burden and more invasive growth of ovarian carcinoma cells in an orthotopic mouse model. These effects are mediated primarily through activation of the tumor cell cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway by the beta(2) adrenergic receptor (encoded by ADRB2). Tumors in stressed animals showed markedly increased vascularization and enhanced expression of VEGF, MMP2 and MMP9, and we found that angiogenic processes mediated the effects of stress on tumor growth in vivo. These data identify beta-adrenergic activation of the cAMP-PKA signaling pathway as a major mechanism by which behavioral stress can enhance tumor angiogenesis in vivo and thereby promote malignant cell growth. These data also suggest that blocking ADRB-mediated angiogenesis could have therapeutic implications for the management of ovarian cancer.     

AMPA receptor subunit GluR2 gates injurious signals in ischemic stroke

Ischemic stroke, or a brain attack, is the third leading cause of death in developed countries. A critical feature of the disease is a highly selective pattern of neuronal loss; certain identifiable subsets of neurons--particularly CA1 pyramidal neurons in the hippocampus are severely damaged, whereas others remain intact. A key step in this selective neuronal injury is Ca2+/Zn2+ entry into vulnerable neurons through alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels, a principle subtype of glutamate receptors. AMPA receptor channels are assembled from glutamate receptor (GluR)1, -2, -3, and -4 subunits. Circumstance data have indicated that the GluR2 subunits dictate Ca2+/Zn2+ permeability of AMPA receptor channels and gate injurious Ca2+/Zn2+ signals in vulnerable neurons. Therefore, targeting to the AMPA receptor subunit GluR2 can be considered a practical strategy for stroke therapy.     

Co-refolding of two peptide fragments derived from Agrobacterium tumefaciens beta-glucosidase with catalytic activity

Four sites of the non-homologous region (coding amino acid residues of 347, 421, 466 and 533) of a gene were randomly selected for splitting to investigate the function of β-glucosidase from Agrobacterium tumefaciens in the co-refolding of peptides into the catalytically active enzyme. As a result of gene splitting, four N- and C-terminal domain peptides were obtained as insoluble inclusion bodies. No catalytic activity was observed when these fragments refolded individually. However, a considerable amount of activity was restored when the two fragments derived from N- and C- terminal peptides were co-refolded together. The deletion of amino acid residues in the non-homologous region resulted in a complete loss of enzyme activity, which suggests that truncation of amino acids in this region strongly affects the co-refolding ability of the enzyme to maintain activity.     

Process optimization for microencapsulation of probiotic yeasts

Background

Microencapsulation is a technique which improves the survival and viability of probiotics. We demonstrate encapsulation of five potential probiotic yeasts with alginate and gum as encapsulation matrices to improve their gastrointestinal transit.

Methods

Gum extracted from various cereals viz. rice, oats, barley, finger millet and pearl millet along with alginate have been used to encapsulate five potential probiotic yeasts. Screening was carried out by measuring swelling index, encapsulation efficiency and nutritional value of microcapsules encapsulated with alginate and gum. The concentration of OBG, sodium alginate and inoculum dosage of probiotic yeasts was optimized using response surface methodology (RSM). Efficiency of alginate OBG microcapsules with or without coating materials viz. whey protein and chitosan also tested. The mucoadhesion ability and storage stability of alginate OBG microcapsules with coating materials were tested.

Results

Highest encapsulation efficiency of probiotic yeasts was noted using oats bran gum (OBG) microcapsules along with alginate in all the five probiotic yeasts. Notably whey protein coated microcapsules showed maximum GIT tolerance (95%) and mucoadhesion (90%) for L. starkeyi VIT-MN03. The minimum loss of viability was observed in L. starkeyi VITMN03 microcapsules on 60th day of storage.

Conclusions

This is the first report on optimization and survival of microencapsulated probiotic yeasts under simulated GIT conditions using natural gum and alginate as encapsulation matrices and whey protein as coating material.

     

Health effects in fish from the polluted Orlando Dam and Klipspruit wetland system,Soweto, South Africa

Pollution of aquatic ecosystems often results in adverse environmental disturbances, including physiological and/ or histomorphological changes in fish. The health of Clarias gariepinus sampled from two polluted water bodies, Orlando Dam and a pond in the Klipspruit wetland catchment, Soweto, was investigated in 2015–2016. Limited fish health-related data is available for this highly impacted freshwater ecosystem. Fish were collected between April 2015 and February 2016. A necropsy and a histological assessment were done on various target organs of each fish. Water and sediment samples were analysed for selected organic and inorganic compounds. Macroscopic assessment revealed abnormally shaped urogenital papillae, morphological alterations of the gonads, as well as discoloration of liver tissue. These observations were supported by microscopic evidence of hepatic histological alterations in fish from Orlando Dam, as well as the presence of both female and male gonadal tissue (intersex) in 13.6% and 50% of fish from the wetland pond and the Orlando Dam, respectively. Water analyses showed high levels of faecal coliform bacteria and metal concentrations and sediment analyses showed detectable levels of potential endocrine disrupting chemicals.     

Gain-of-function p53 protein transferred via small extracellular vesicles promotes conversion of fibroblasts to a cancer-associated phenotype

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Fluorescence polarization assay for calmodulin binding to plasma membrane Ca-ATPase: Dependence on enzyme and Ca concentrations

Calmodulin (CaM) is a Ca²⁺ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K_d for the interaction of CaM with the plasma membrane Ca²⁺-ATPase (PMCA), a Ca²⁺ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca²⁺ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K_d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K_d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K_d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca²⁺ dependence follows a simple Hill plot demonstrating cooperative binding of Ca²⁺ to the binding sites in CaM.     

Sodium Phenylbutyrate and Edaravone Abrogate Chronic Restraint Stress-Induced Behavioral Deficits: Implication of Oxido-Nitrosative,Endoplasmic Reticulum Stress Cascade,and Neuroinflammation

Chronic stress exposure can produce deleterious effects on the hippocampus (HC) which eventually leads to cognitive impairment and depression. Endoplasmic reticulum (ER) stress has been reported as one of the major culprits in the development of stress-induced cognitive impairment and depression. We investigated the neuroprotective efficacy of sodium phenylbutyrate (SPB), an ER stress inhibitor, and edaravone, a free radical scavenger, against chronic restraint stress (CRS)-induced cognitive deficits and anxiety- and depressive-like behavior in mice. Adult male Swiss albino mice were restrained for 6 h/day for 28 days and injected (i.p.) with SPB (40 and 120 mg/kg) or edaravone (3 and 10 mg/kg) for the last seven days. After stress cessation, the anxiety- and depressive-like behavior along with spatial learning and memory were examined. Furthermore, oxido-nitrosative stress, proinflammatory cytokines, and gene expression level of ER stress-related genes were assessed in HC and prefrontal cortex (PFC). CRS-exposed mice showed anxiety- and depressive-like behavior, which was significantly improved by SPB and edaravone treatment. In addition, SPB and edaravone treatment significantly alleviated CRS-induced spatial learning and memory impairment. Furthermore, CRS-evoked oxido-nitrosative stress, neuroinflammation, and depletion of Brain-derived neurotrophic factor were significantly ameliorated by SPB and edaravone treatment. We found significant up-regulation of ER stress-related genes in both HC and PFC regions, which were suppressed by SPB and edaravone treatment in CRS mice. Our study provides evidence that SPB and edaravone exerted neuroprotective effects on CRS-induced cognitive deficits and anxiety- and depressive-like behavior, which is possibly coupled with inhibition of oxido-nitrosative stress, neuroinflammation, and ER stress cascade.     

Human granulocyte colony stimulating factor (hG-CSF): cloning,overexpression, purification and characterization

Background  

Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.