Cytosolic Dynamics of Annexin A6 Trigger Feedback Regulation of Hypertrophy via Atrial Natriuretic Peptide in Cardiomyocytes

Malfunctions in regulatory pathways that control cell size are prominent in pathological cardiac hypertrophy. Here, we show annexin A6 (Anxa6) to be a crucial regulator of atrial natriuretic peptide (ANP)-mediated counterhypertrophic responses in cardiomyocytes. Adrenergic stimulation of H9c2 cardiomyocytes by phenylephrine (PE) increased the cell size with enhanced expression of biochemical markers of hypertrophy, concomitant with elevated expression and subcellular redistribution of Anxa6. Stable cell lines with controlled increase in Anxa6 levels were protected against PE-induced adverse changes, whereas Anxa6 knockdown augmented the hypertrophic responses. Strikingly, Anxa6 knockdown also abrogated PE-induced juxtanuclear accumulation of secretory granules (SG) containing ANP propeptides (pro-ANP), a signature of maladaptive hypertrophy having counteractive functions. Mechanistically, PE treatment prompted a dynamic association of Anxa6 with pro-ANP-SG, parallel to their participation in anterograde traffic, in an isoform-specific fashion. Moreover, Anxa6 mutants that failed to associate with pro-ANP hindered ANP-mediated protection against hypertrophy, which was rescued, at least partially, by WT Anxa6. Additionally, elevated intracellular calcium (Ca²⁺) stimulated Anxa6-pro-ANP colocalization and membrane association. It also rescued pro-ANP translocation in cells expressing an Anxa6 mutant (Anxa6^ΔC). Furthermore, stable overexpression of Anxa6^T356D, a mutant with superior flexibility, provided enhanced protection against PE, compared with WT, presumably due to enhanced membrane-binding capacity. Together, the present study delivers a cooperative mechanism where Anxa6 potentiates ANP-dependent counterhypertrophic responses in cardiomyocytes by facilitating regulated traffic of pro-ANP.     

Salmonella engages CDC42 effector protein 1 for intracellular invasion

Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN-7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella-containing vacuoles after long-term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread.     

Understanding the role of starch sheath layer in graviception of Alternanthera philoxeroides: a biophysical and microscopical study

Journal of Plant Research - Plants’ ability to sense and respond to gravity is a unique and fundamental process. When a plant organ is tilted, it adjusts its growth orientation relative to...     

Physico chemical alterations in desugared buffalo milk after fermentation withSaccharomyces fragilis

Summary Buffalo milk was completely desugared by fermentation for 17 h withSaccharomyces fragilis. A thin-layer chromatographic profile indicated the total absence of lactose, galactose, glucose and any oligosaccharides. Desugaring by fermentation did not alter the lipid composition or the protein content of the milk. Gas chromatographic studies indicated the formation of volatile compounds such as acetaldehyde, ethyl acetate, ethyl alcohol, n-propanol, n-butanol and isoamyl alcohol. A pleasant fruity odour observed in the initial stage of fermentation changed to an overripe fruity odour on fermentation. The volatile compounds formed disappeared on spraydrying the milk, leaving only a residual yeasty odour in the desugared milk powder.     

Interferon-inducible gene expression in HL-60 cells: effects of the state of differentiation.

The promyelocytic leukemia line HL-60 can be terminally differentiated in vitro to either monocyte/macrophages or granulocytes. We used this cell line to test whether the state of differentiation of a cell changes its response to interferon (IFN). The characteristics of expression of several IFN-alpha- and IFN-gamma-inducible genes in undifferentiated and differentiated HL-60 cells were examined. p67, an IFN-gamma-inducible protein, was induced similarly in three cell types, whereas another IFN-gamma-inducible protein, p56, was induced strongly only in undifferentiated cells. In contrast, two isozymes of 2,5(A)-synthetase were induced better in differentiated cells in response to either IFN. Several IFN-alpha-inducible mRNAs, e.g., 561, 6-16, 1-8, and 2A, were induced much more strongly in granulocytes than in macrophages or in undifferentiated cells. Electrophoretic mobility shift assays using the IFN-stimulated response element of gene 561 and nuclear extracts of IFN-alpha-treated cells revealed the appearance of one complex and the disappearance of another one, concomitant with differentiation of the cells to granulocytes. These observations suggest that expression of IFN-inducible genes in HL-60 cells is regulated by trans-acting factors whose activity changes with the state of differentiation of the cells. Our study may have implications in the optimal clinical use of IFNs. Inducing cellular differentiation may augment the efficacy of IFNs as antitumor agents.     

Triauxic growth of an oleaginous red yeast <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> on waste ‘extract’ for enhanced and concomitant lipid and β-carotene production

Background

Vegetable ‘mandi’ (road-side vegetable market) waste was converted to a suitable fermentation medium for cultivation of oleaginous yeast Rhodosporidium toruloides by steaming under pressure. This cultivation medium derived from waste was found to be a comparatively better source of nutrients than standard culture media because it provided more than one type of usable carbon source(s) to yeast.

Results

HPLC results showed that the extract contained glucose, xylose and glycerol along with other carbon sources, allowing triauxic growth pattern with preferably usage of glucose, xylose and glycerol resulting in enhanced growth, lipid and carotenoid production. Presence of saturated and unsaturated fatty acid methyl esters (FAMEs) (C_14-20) in the lipid profile showed that the lipid may be transesterified for biodiesel production.

Conclusion

Upscaling these experiments to fermenter scale for the production of lipids and biodiesel and other industrially useful products would lead to waste management along with the production of value added commodities. The technique is thus environment friendly and gives good return upon investment.

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Characterization of cysteine thiol modifications based on protein microenvironments and local secondary structures

We have demonstrated earlier that protein microenvironments were conserved around disulfide‐bridged cystine motifs with similar functions, irrespective of diversity in protein sequences. Here, cysteine thiol modifications were characterized based on protein microenvironments, secondary structures and specific protein functions. Protein microenvironment around an amino acid was defined as the summation of hydrophobic contributions from the surrounding protein fragments and the solvent molecules present within its first contact shell. Cysteine functions (modifications) were grouped into enzymatic and non‐enzymatic classes. Modifications studied were—disulfide formation, thio‐ether formation, metal‐binding, nitrosylation, acylation, selenylation, glutathionylation, sulfenylation, and ribosylation. 1079 enzymatic proteins were reported from high‐resolution crystal structures. Protein microenvironments around cysteine thiol, derived from above crystal structures, were clustered into 3 groups—buried‐hydrophobic, intermediate and exposed‐hydrophilic clusters. Characterization of cysteine functions were statistically meaningful for 4 modifications (disulfide formation, thioether formation, sulfenylation, and iron/zinc binding) those have sufficient amount of data in the current dataset. Results showed that protein microenvironment, secondary structure and protein functions were conserved for enzymatic cysteine functions, in contrast to the same function from non‐enzymatic cysteines. Disulfide forming enzymatic cysteines were tightly packed within intermediate protein microenvironment cluster, have alpha‐helical conformation and mostly belonged to CxxC motif of electron transport proteins. Disulfide forming non‐enzymatic cysteines did not belong to conserved motif and have variable secondary structures. Similarly, enzymatic thioether forming cysteines have conserved microenvironment compared to non‐enzymatic cystienes. Based on the compatibility between protein microenvironment and cysteine modifications, more efficient drug molecules could be designed against cysteine‐related diseases.     

Sorption of heavy metals by four basidiomycetous fungi

Biosorptions of Pb²⁺, Cr⁶⁺, Cd²⁺ and Ni²⁺ were investigated, with special emphasis on the first one, using live and dead fungal mycelia. Of the four fungi, namely Polyporus ostreiformis, Volvariella volvacea, Pleurotus sajor-caju and Phanerochaete chrysosporium, the last one was found to be most effective in Pb²⁺ removal. Total biosorption was effected in 6 days up to the Pb²⁺ concentration of 6 mg/l, with a specific uptake of 1.33 mg Pb²⁺/g dry cell mass. The removal of other three metals varied between 28.8–73.3% from a medium containing 4 mg/l of each of the metals.Laboratory facilities created under M.Tech programme in Biotechnology and Engineering (sponsored by Department of Biotechnology, Govt. of India) were partly utilized for this study.     

Effect of cadmium,mercury and copper on partially purified hepatic flavokinase of rat

The effect of cadmium (Cd2+), mercury (Hg2+) and copper (Cu2+) was studied with partially purified flavokinase (ATP:riboflavin 5-phosphotransferase EC 2.7.1.26) from rat liver. All the divalent heavy metal cations inhibited flavokinase activity in a concentration-dependent manner. The inhibitory effect of cadmium on the enzyme was completely reversed by increasing concentration, of Zinc (Zn2+) indicating a competition between Zn2+ and Cd2+ for binding with the enzyme. A competition between riboflavin and Cd2+ is also evident from the present investigation. These observations hint at the possibility that Zn2+ and Cd2+ probably compete for the same site on the enzyme where riboflavin binds. However, inhibition of flavokinase by Hg2+ could not be reversed by Zn2+. Our studies further reveal that hepatic flavokinase appears to contain an essential, accessible and functional thiol group(s) which is evident from a concentration dependent inhibition of activity by sulfhydryl reagent s like parachloromercuribenzoate (PCMB), 5,5-dithiobis (2-nitrobenzoic acid)(DTNB), and N-ethylmaleimide (NEM). Inhibition of flavokinase by sulfhydryl reagents were protected, except in case of NEM inhibition, when the enzyme was incubated with thiol protectors like glutathione (GSH) and dithiothreitol (DTT). Furthermore, the enzyme could also be protected from the inhibitory effect of Cd2+ and Hg2+ by GSH and DTT suggesting that Cd2+ probably interacts with a reactive thiol group at or near the active site of enzyme in bringing about its inhibitory effect. (Mol Cell Biochem 167: 73-80, 1997)