Characterization of Oncorhynchus mykiss 5-hydroxyisourate hydrolase/transthyretin superfamily: Evolutionary and functional analyses

Teleosts have highly diverged genomes that resulted from whole genome duplication, which leads to an extensive diversity of paralogous genes. Transthyretin (TTR), an extracellular thyroid hormone (TH) binding protein, is thought to have evolved from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some stage of chordate evolution. To characterize the functions of proteins that arose from duplicated genes in teleosts, we investigated the phylogenetic relationship of teleost HIUHase and TTR aa sequences, the expression levels of Oncorhynchus mykiss HIUHase and TTR mRNA in various tissues and the biological activities of the O. mykiss re-HIUHase and re-TTR. Phylogenetic analysis of the teleost aa sequences revealed the presence of two HIUHase subfamilies, HIUHase 1 (which has an N-terminal peroxisomal targeting signal-2 [PTS2]) and HIUHase 2 (which does not have an N-terminal PTS2), and one TTR family. The tissue distributions of HIUHase 1 and TTR mRNA were similar in juvenile O. mykiss and the mRNA levels were highest in the liver. The O. mykiss re-HIUHase and re-TTR proteins were both 40–50 kDa homotetramers consisting of 14–15 kDa subunits, with 30% identity. HIUHase had 5-hydroxyisourate (5-HIU) hydrolysis activity with Zn^2 + sensitivity, whereas TTR had ligand binding activity with a preference for THs and several environmental chemicals, such as halogenated phenols. Our results suggest that O. mykiss HIUHase and TTR have diverged from a common ancestral HIHUase with no functional complementation.     

Molecular cloning and catalytic characterization of a recombinant tropine biosynthetic tropinone reductase from Withania coagulans leaf

Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34 kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30% retention of activity at 60 °C) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species.     

The cytoplasmic domain of TGFβR3 through its interaction with the scaffolding protein, GIPC, directs epicardial cell behavior

The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor β Receptor (TGFβR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFβR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFβR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially derived cell invasion in Tgfbr3^−/− hearts. Tgfbr3^−/− epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFβ1 and TGFβ2. Unexpectedly, loss of TGFβR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFβR3 in Tgfbr3^−/− cells rescued deficits in invasion in vitro in response TGFβ1 and TGFβ2 as well as FGF2 and HMW-HA. Expression of TGFβR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3^−/− cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3^+/+ cells decreased invasion in response to TGFβ2, FGF2, and HMW-HA. We conclude that TGFβR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3^−/− mice.     

Development of chiral fluorinated alkyl derivatives of emixustat as drug candidates for the treatment of retinal degenerative diseases

The discovery of how a photon is converted into a chemical signal is one of the most important achievements in the field of vision. A key molecule in this process is the visual chromophore retinal. Several eye diseases are attributed to the abnormal metabolism of retinal in the retina and the retinal pigment epithelium. Also, the accumulation of two toxic retinal derivatives, N-retinylidene-N-retinylethanolamine and the retinal dimer, can damage the retina leading to blindness. RPE65 (Retinal pigment epithelium-specific 65 kDa protein) is one of the central enzymes that regulates the metabolism of retinal and the formation of its toxic metabolites. Its inhibition might decrease the rate of the retina’s degeneration by limiting the amount of retinal and its toxic byproducts. Two RPE65 inhibitors, (R)-emixustat and (R)-MB001, were recently developed for this purpose.     

The mechanism of inactivation of glucose oxidase from Penicillium amagasakiense under ambient storage conditions

Glucose oxidase (GOx) from Penicillium amagasakiense has a higher specific activity than the more commonly studied Aspergillus niger enzyme, and may therefore be preferred in many medical and industrial applications. The enzyme rapidly inactivates on storage at pH 7.0-7.6 at temperatures between 30 and 40 °C. Results of fluorimetry and circular dichroism spectroscopy indicate that GOx inactivation under these conditions is associated with release of the cofactor FAD and molten globule formation, indicated by major loss of tertiary structure but almost complete retention of secondary structure. Inactivation of GOx at pH < 7 leads to precipitation, but at pH ≥ 7 it leads to non-specific formation of small soluble aggregates detectable by PAGE and size-exclusion chromatography (SEC). Inactivation of P. amagasakiense GOx differs from that of A. niger GOx in displaying complete rather than partial retention of secondary structure and in being promoted rather than prevented by NaCl. The contrasting salt effects may reflect differences in the nature of the interface between subunits in the native dimers and/or the quantity of secondary structure loss upon inactivation.     

Spotting the right location— imaging approaches to resolve the intracellular localization of invasive pathogens

Background

A common strategy of microbial pathogens is to invade host cells during infection. The invading microbes explore different intracellular compartments to find their preferred niche.

Scope of Review

Imaging has been instrumental to unravel paradigms of pathogen entry, to identify their exact intracellular location, and to understand the underlying mechanisms for the formation of pathogen-containing niches. Here, we provide an overview of imaging techniques that have been applied to monitor the intracellular lifestyle of pathogens, focusing mainly on bacteria that either remain in vacuolar-bound compartments or rupture the endocytic vacuole to escape into the host's cellular cytoplasm.

Major Conclusions

We will depict common molecular and cellular paradigms that are preferentially exploited by pathogens. A combination of electron microscopy, fluorescence microscopy, and time-lapse microscopy has been the driving force to reveal underlying cell biological processes. Furthermore, the development of highly sensitive and specific fluorescent sensor molecules has allowed for the identification of functional aspects of niche formation by intracellular pathogens.

General Significance

Currently, we are beginning to understand the sophistication of the invasion strategies used by bacterial pathogens during the infection process- innovative imaging has been a key ingredient for this.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.     

Phytoglycoprotein (38 kDa) induces cell cycle (G₀/G₁) arrest and apoptosis in HepG2 cells

Styrax japonica Siebold et al Zuccarini (SJSZ) has been used to heal inflammation and bronchitis as folk medicine in Korea. Firstly, glycoprotein isolated from SJSZ (SJSZ glycoprotein) has a molecular weight with 38 kDa and consists of carbohydrate (57.64%) and protein (42.35%). In the composition of SJSZ glycoprotein, carbohydrate mostly consists of glucose (28.17%), galactose (21.85%), and mannose (2.62%) out of 52.64%, respectively. The protein consists of Trp (W, 7.01%), Pro (P, 6.72%), and Ile (I, 5.42%) out of 42.35% as three major amino acids, while total amount of other amino acids is 23.20%. The purpose of this study is to know whether the SJSZ glycoprotein (38 kDa) induces the cell cycle arrest and apoptosis in HepG2 cells. Cytotoxicity was evaluated using MTT and lactate dehydrogenase assay and amount of intracellular reactive oxygen species (iROS) and nitric oxide (NO) was measured using fluorescence microplate reader. Activities of cell cycle-related proteins [p53, p21, p27, Cyclin D1, and cyclin-dependent kinase (CDK)4] and apoptosis-related factors [iNOS, Bid, Bcl-2/bax, cytochrome c, caspase-9, caspase-3, and poly-(ADP-ribose) polymerase (PARP)] were assessed by Western blot and fluorescence-activated cell sorter (FACS) analysis. In the cell cycle-related proteins, SJSZ glycoprotein (50 μg/ml) significantly enhances the expression of p53, p21, and p27, whereas it suppressed the activity of cyclin D1/CDK4. In the apoptosis-related factors, SJSZ glycoprotein (50 μg/ml) stimulates to increase iROS, and NO, to activate iNOS, Bid, Bcl-2/bax, cytochrome c, caspase-9, caspase-3, and PARP. SJSZ glycoprotein (50 μg/ml) has potent effect to arrest cell cycle from G(0) /G(1) to S and to induce apoptosis in HepG2 cells.