Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H₂O₂ exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H₂O₂ exposure. The present work shows that this tick cell line could tolerate high H₂O₂ concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control.     

Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT)

Hypoxia occurs during the development of the placenta in the first trimester and correlates with both trophoblast differentiation and the induction of telomerase activity through hTERT expression. We sought to determine the mechanism of regulation of hTERT expression during hypoxia. We show that hypoxia-inducible factor 1alpha (HIF-1alpha) and hTERT expression in the human placenta decrease with gestational age and that these are overexpressed in preeclamptic placenta, a major complication of pregnancy. Hypoxia not only transactivates the hTERT promoter activity but also enhances endogenous hTERT expression. The hTERT promoter region between -165 and +51 contains two HIF-1 consensus motifs, and in vitro reporter assays show that these are essential for hTERT transactivation by HIF-1. Introduction of an antisense oligonucleotide for HIF-1 diminishes hTERT expression during hypoxia, indicating that upregulation of hTERT by hypoxia is directly mediated through HIF-1. Our results provide persuasive evidence that the regulation of hTERT promoter activity by HIF-1 represents a mechanism for trophoblast growth during hypoxia and suggests that this may be a generalized response to hypoxia in various human disorders including resistance to cancer therapeutics by upregulating telomerase.     

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was <1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application.     

In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the alpha subunits OASA1 and OASA2

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, alpha and beta, and converts chorismate to anthranilate. Two or more AS alpha-subunit genes have been identified and characterized in several land plants. Although alpha subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS alpha-subunit genes (OASA1 and OASA2) in rice (Oryza sativa ). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS beta subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two beta subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a beta subunit to achieve maximal enzymatic activity even with NH(4)(+) as the amino donor. The V (max) and K (i) for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.     

Significance of low-frequency local fluctuation motions in the transmembrane B and C α-helices of bacteriorhodopsin,to facilitate efficient proton uptake from the cytoplasmic surface,as revealed by site-directed solid-state <Superscript>13</Superscript>C NMR

¹³C NMR spectra of [1-¹³C]Val- or -Pro-labeled bacteriorhodopsin (bR) and its single or double mutants, including D85N, were recorded at various pH values to reveal conformation and dynamics changes in the transmembrane -helices, in relation to proton release and uptake between bR and the M-like state caused by modified charged states at Asp85 and the Schiff base (SB). It was found that the D85N mutant acquired local fluctuation motion with a frequency of 10⁴ Hz in the transmembrane B -helix, concomitant with deprotonation of SB in the M-like state at pH 10, as manifested from a suppressed ¹³C NMR signal of the [1-¹³C]-labeled Val49 residue. Nevertheless, local dynamics at Pro50 neighboring with Val49 turned out to be unchanged, irrespective of the charged state of SB as viewed from the ¹³C NMR of [1-¹³C]-labeled Pro50. This means that the transmembrane B -helix is able to acquire the fluctuation motion with a frequency of 10⁴ Hz beyond the kink at Pro50 in the cytoplasmic side. Concomitantly, fluctuation motion at the C helix with frequency in the order of 10⁴ Hz was found to be prominent, due to deprotonation of SB at pH 10, as viewed from the ¹³C NMR signal of Pro91. Accordingly, we have proposed here a novel mechanism as to proton uptake and transport based on a dynamic aspect that a transient environmental change from a hydrophobic to hydrophilic nature at Asp96 and SB is responsible for the reduced pK_a value which makes proton uptake efficient, as a result of acquisition of the fluctuation motion at the cytoplasmic side of the transmembrane B and C -helices in the M-like state. Further, it is demonstrated that the presence of a van der Waals contact of Val49 with Lys216 at the SB is essential to trigger this sort of dynamic change, as revealed from the ¹³C NMR data of the D85N/V49A mutant.     

New analogue of arenastatin A, a potent cytotoxic spongean depsipeptide, with anti-tumor activity

Two analogues possessing steric hindered substituents on C-15 of arenastatin A (1), a potent cytotoxic spongean depsipeptide, were synthesized and shown to enhance stability in mouse serum. Notably, 15-tert-butylanalogue (6) with higher cytotoxicity exhibited in vivo anti-tumor activity through iv administration different from 1. Additionally, conformation analysis among the two analogues and arenastatin A (1) indicated that the torsion angle from C-14 to C-20 is a conclusive factor for the potent cytotoxicity of 1.