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Abstract: Sterol glycosyltransferases catalyze the synthesis of diverse glycosterols in plants. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a novel 27β-hydroxy glucosyltransferase was purified to near homogeneity from cytosolic fraction of W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme showed activity with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols/withanolides with β-OH group at C-17, C-21 and C-27 positions. It transferred glucose to the alkanol at C-25 position of the lactone ring, provided an α-OH was present at C-17 in the sterol skeleton. A comparable enzyme has not been reported earlier from plants. The enzyme is distinct from the previously purified W. somnifera 3β-hydroxy specific sterol glucosyltransferase and does not glucosylate the sterols at C-3 position; though it also follows an ordered sequential bisubstrate reaction mechanism, in which UDP-glucose and sterol are the first and second binding substrates. The enzyme activity with withanolides suggests its role in secondary metabolism in W. somnifera. Results on peptide mass fingerprinting showed its resemblance with glycuronosyltransferase like protein. The enzyme activity in the leaves of W. somnifera was enhanced following the application of salicylic acid. In contrast, it decreased rapidly on exposure of the plants to heat shock, suggesting functional role of the enzyme in biotic and abiotic stresses. [Copyright &y& Elsevier]

Abstract: Sterol glycosides are constituents of plant cell membranes. Glucosylations of the sterols are catalyzed by sterol glucosyltransferases (SGTs), which are members of family 1 glycosyltransferases. We have identified the family of SGT genes expressed in the leaves of a medicinal plant Withania somnifera. One member (SGTL1) of this gene family was cloned. The full-length cDNA sequence of SGTL1 represents 2532 bp, comprising untranslated regions (UTRs) of 337 and 89 bp at the 5′ and 3′ ends, respectively. The amino acid sequence deduced from the 2103 bp open reading frame (ORF) showed homology (67–45%) to the reported plant SGTs. The presence of two putative transmembrane domains suggested the association of SGTL1 with membrane. The SGTL1 was expressed in Escherichia coli and recombinant enzyme from the supernatant was partially purified and biochemically characterized. The relative activity and kinetic properties of SGTL1 for different sterols were compared with a recombinant SGT (GenBank Accession No. Z83833) of Arabidopsis thaliana (AtSGT). Both the recombinant enzymes showed activity with 3-β-OH sterols. The distribution of SGTL1 transcript in W. somnifera, as determined by quantitative PCR, showed higher expression in roots and mature leaves. Expression of the SGTL1 transcript in the leaves of W. somnifera was enhanced following the application of salicylic acid. In contrast, it decreased rapidly on exposure of the plants to heat shock, suggesting functional role of the enzyme in biotic and abiotic stresses. [Copyright &y& Elsevier]

Abstract: Sterol glycosyltransferases catalyze the synthesis of diverse glycosteroids in plants, leading to a change in their participation in cellular metabolism. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a cytosolic sterol glucosyltransferase was purified 3406 fold to near homogeneity from W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme was active with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols and phytosterols with 3β-OH group. It showed a low level of activity with flavonoids and isoflavonoids. The enzyme gave maximum K cat/K m value (0.957) for 24-methylenecholesterol that resembles aglycone structure of pharmacologically important sitoindosides VII and VIII from W. somnifera. The enzyme follows ordered sequential bisubstrate mechanism of reaction, in which UDP-glucose and sterol are the first and second binding substrates. This is the first detailed kinetic study on purified plant cytosolic sterol glucosyltransferases. Results on peptide mass fingerprinting and substrate specificity suggested that the enzyme belongs to the family of secondary metabolite glucosylating glucosyltransferases. The enzyme activity exhibited a rapid in vivo response to high temperature and salicylic acid treatment of plants, suggesting its physiological role in abiotic and biotic stress. [Copyright &y& Elsevier]

Background: Increased mitochondrial activities contributing to cancer cell proliferation, invasion, and metastasis have been reported in different cancers; however, studies on the therapeutic targeting of mitochondria in regulating cell proliferation and invasiveness are limited. Because mitochondria are believed to have evolved through bacterial invasion in mammalian cells, antibiotics could provide an alternative approach to target mitochondria, especially in cancers with increased mitochondrial activities. In this study, we investigated the therapeutic potential of bacteriostatic antibiotics in regulating the growth potential of colorectal cancer (CRC) cells, which differ in their metastatic potential and mitochondrial functions. Methods: A combination of viability, cell migration, and spheroid formation assays was used to measure the effect on metastatic potential. The effect on mitochondrial mechanisms was investigated by measuring mitochondrial DNA copy number by qPCR, biogenesis (by qPCR and immunoblotting), and functions by measuring reactive oxygen species, membrane potential, and ATP using standard methods. In addition, the effect on assembly and activities of respiratory chain (RC) complexes was determined using blue native gel electrophoresis and in-gel assays, respectively). Changes in metastatic and cell death signaling were measured by immunoblotting with specific marker proteins and compared between CRC cells. Results: Both tigecycline and tetracycline effectively reduced the viability, migration, and spheroid-forming capacity of highly metastatic CRC cells. This increased sensitivity was attributed to reduced mtDNA content, mitochondrial biogenesis, ATP content, membrane potential, and increased oxidative stress. Specifically, complex I assembly and activity were significantly inhibited by these antibiotics in high-metastatic cells. Significant down-regulation in the expression of mitochondrial-mediated survival pathways, such as phospho-AKT, cMYC, phospho-SRC, and phospho-FAK, and upregulation in cell death (apoptosis and autophagy) were observed, which contributed to the enhanced sensitivity of highly metastatic CRC cells toward these antibiotics. In addition, the combined treatment of the CRC chemotherapeutic agent oxaliplatin with tigecycline/tetracycline at physiological concentrations effectively sensitized these cells at early time points. Conclusion: Altogether, our study reports that bacterial antibiotics, such as tigecycline and tetracycline, target mitochondrial functions specifically mitochondrial complex I architecture and activity and would be useful in combination with cancer chemotherapeutics for high metastatic conditions. [ABSTRACT FROM AUTHOR]

Tumors are composed of heterogeneous populations of dysregulated cells that grow in specialized niches that support their growth and maintain their properties. Tumor heterogeneity and metastasis are among the major hindrances that exist while treating cancer patients, leading to poor clinical outcomes. Although the factors that determine tumor complexity remain largely unknown, several genotypic and phenotypic changes, including DNA mutations and metabolic reprograming provide cancer cells with a survival advantage over host cells and resistance to therapeutics. Furthermore, the presence of a specific population of cells within the tumor mass, commonly known as cancer stem cells (CSCs), is thought to initiate tumor formation, maintenance, resistance, and recurrence. Therefore, these CSCs have been investigated in detail recently as potential targets to treat cancer and prevent recurrence. Understanding the molecular mechanisms involved in CSC proliferation, self-renewal, and dormancy may provide important clues for developing effective therapeutic strategies. Autophagy, a catabolic process, has long been recognized to regulate various physiological and pathological processes. In addition to regulating cancer cells, recent studies have identified a critical role for autophagy in regulating CSC functions. Autophagy is activated under various adverse conditions and promotes cellular maintenance, survival, and even cell death. Thus, it is intriguing to address whether autophagy promotes or inhibits CSC functions and whether autophagy modulation can be used to regulate CSC functions, either alone or in combination. This review describes the roles of autophagy in the regulation of metabolic functions, proliferation and quiescence of CSCs, and its role during therapeutic stress. The review further highlights the autophagy-associated pathways that could be used to regulate CSCs. Overall, the present review will help to rationalize various translational approaches that involve autophagy-mediated modulation of CSCs in controlling cancer progression, metastasis, and recurrence. [ABSTRACT FROM AUTHOR]

Background: Alterations in mitochondrial signatures such as mitochondrial DNA (mtDNA) content in maternal blood have been linked to pregnancy-related complications. However, changes in maternal mtDNA content, their distribution and associated signaling during normal pregnancies are not clear; which could suggest their physiological role in maternal adaptation to pregnancy related changes and a reference threshold.the Aim Of This Study: to assess the distribution of mtDNA in peripheral blood and their association with circulatory ROS levels across different trimesters of healthy pregnancy.Methods: In this pilot cross sectional study, blood samples of normal pregnant women from each trimester (total = 60) and age-matched non-pregnant (NP) women as control group (n = 20) were analyzed for a) the relative distribution of mtDNA content in cellular and cell free (plasma) fractions using relative quantitative polymerase chain reaction (qPCR) and b) the levels of circulating reactive oxygen species (ROS) by measurement of plasma H2O2. The results were compared between pregnant and NP groups and within trimesters for significant differences, and were also analyzed for their correlation between groups using statistical methods.Results: While, we observed a significant decline in cellular mtDNA; plasma mtDNA was significant increased across all trimesters compared to NP. However, from comparisons within trimesters; only cellular mtDNA content in 3rd trimester was significantly reduced compared to 1st trimester, and plasma mtDNA did not differ significantly among different trimesters. A significantly higher level of plasma H2O2 was also observed during 3rd trimester compared to NP and to 1st trimester. Correlation analysis showed that, while cellular mtDNA content was negatively correlated to plasma mtDNA and to plasma H2O2 levels; plasma mtDNA was positively correlated with plasma H2O2 content.Conclusions: This study suggested that normal pregnancy is associated with an opposing trend of reduced cellular mtDNA with increased circulatory mtDNA and H2O2 levels, which may contribute to maternal adaptation, required during different stages of pregnancy. Estimation of mtDNA distribution and ROS level in maternal blood could show mitochondrial functionality during normal pregnancy, and could be exploited to identify their prognostic/ diagnostic potential in pregnancy complications. [ABSTRACT FROM AUTHOR]

Relative band intensities of C-I on (C) BN-PAGE and (D) in-gel assay were quantitated using ImageJ and normalized with total mitochondrial protein loaded (80 µg). (E) Equal loading of mitochondrial proteins is shown by immunoblotting results of an aliquot of the same samples used for BN-PAGE/in-gel assay, with mitochondrial marker protein VDAC. C-, complex; VDAC, Voltage-dependent anion channel; BN-PAGE, Blue native polyacrylamide gel electrophoresis. [Extracted from the article]