The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif.

The ROX1 gene encodes a repressor of the hypoxic functions of the yeast Saccharomyces cerevisiae. The DNA sequence of the gene was determined and found to encode a protein of 368 amino acids. The amino-terminal third of the protein contains a high-mobility-group motif characteristic of DNA-binding proteins. To determine whether the Rox1 repressor bound DNA, the gene was expressed in Escherichia coli cells as a fusion to the maltose-binding protein and this fusion was partially purified by amylose affinity chromatography. By using a gel retardation assay, both the fusion protein and Rox1 itself were found to bind specifically to a synthetic 32-bp DNA containing the hypoxic consensus sequence. We assessed the role of the general repressor Ssn6 in ANB1 repression. An ANB1-lacZ fusion was expressed constitutively in an ssn6 deletion strain, and deletion of the Rox1 binding sites in the ANB1 upstream region did not increase the level of derepression, suggesting that Ssn6 exerts its effect through Rox1. Finally, ROX1 was mapped to yeast chromosome XVI, near the ARO7-OSM2 locus.     

Calcium-dependent Modulation of the Agonist Affinity of the Mammalian Olfactory Cyclic Nucleotide-gated Channel by Calmodulin and a Novel Endogenous Factor

The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca²⁺ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC₅₀) was reversibly increased from 3 μm to about 30 μm. This Ca²⁺-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure to 3 mm Mg²⁺+ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca²⁺ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca²⁺-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the affinity of the channel for CAM. The affinity shift induced by Ca²⁺-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR, ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated by an as yet unidentified factor distinct from CAM. Received: 26 December 1995/Revised: 14 March 1996     

Effect of radiotherapy and chemoradiotherapy on circulating antioxidant system of human uterine cervical carcinoma

Rats bearing the Yoshida AH-130 ascites hepatoma showed important changes in lipid metabolism. The presence of this rapidly growing tumour induced a significant reduction in the intestinal absorption of an oral [^l4C]triolein load but without changes in whole body oxidation of the tracer to CO₃. Both white (WAT) and brown (BAT) adipose tissue lipoprotein lipase (LPL) activities were increased at day 4 of tumour growth, changes that seem to be related with those observed in [¹⁴C] lipid accumulation; however, heart LPL activity was increased at day 7 but there was no change at day 4. In addition, there was a marked hyperlipemia in the tumour-bearing animals, whereas the blood ketone body concentrations were lower in these animals in comparison with the corresponding pair-fed group. The in vivo lipogenic rate was increased in liver of the tumour-bearing animals (day 4); conversely, it was decreased in WAT and skeletal muscle (day 4) and IBAT (day 7) of the AH-130-bearing rats. It may be suggested that the increased liver lipogenic rate associated with tumour burden is the main factor contributing to the hyperlipidaemia present in the Yoshida AH-130 bearing rats.     

Corticosterone induces steroidogenic lesion in cultured adult rat Leydig cells by reducing the expression of star protein and steroidogenic enzymes

The present study was designed to investigate the dose-dependent direct effect of corticosterone on adult rat Leydig cell steroidogenesis in vitro. Leydig cells were isolated from the testis of normal adult male albino rats, purified on discontinuous Percoll gradient and plated in culture plates/flasks overnight at 34 degrees C in a CO(2) incubator under 95% air and 5% CO(2) using DME/F12 medium containing 1% fetal bovine serum. After the attachment of cells, serum-containing medium was removed and cells were exposed to different doses (0, 50, 100, 200, 400, and 800 nM) of corticosterone using serum-free fresh medium for 24 h at 34 degrees C. At the end of exposure period, cells were utilized for assessment of the activities and mRNA expression of steroidogenic enzymes (cytochrome P(450) side chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and cytochrome P(450) aromatase) and steroidogenic acute regulatory protein gene expression. Testosterone and estradiol production were also quantified. Activities of cytochrome P(450) side chain cleavage enzyme, 3beta- and 17beta-hydroxysteroid dehydrogenases were declined significantly in a dose-dependent manner after corticosterone exposure, while their mRNA expression were significantly reduced at higher doses of corticosterone exposure. The activity and mRNA expression of cytochrome P(450) aromatase registered a significant increase at 100 nM dose of corticosterone whereas at 200-800 nM doses both the activity as well as the mRNA levels was significantly reduced below the basal level. StAR protein gene expression was significantly inhibited by higher doses of corticosterone employed. At all doses employed, corticosterone significantly reduced the production of testosterone by Leydig cells, while estradiol level registered a significant increase at 50 and 100 nM doses but at higher doses, it registered a significant decrease when compared to basal level. It is concluded from the present in vitro study that the molecular mechanism by which corticosterone reduces the production of Leydig cell testosterone is by reducing the activities and mRNA expression of steroidogenic enzymes and steroidogenic acute regulatory protein.     

Short and Straightforward Synthesis of Triphosphates of Artificial Nucleobase Pairs Displaying Unconventional Pairing Scheme

Concise, facile and efficient synthesis of 5′-O-triphosphates of 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2a]-1,3,5-triazin-4(8H)-one (dP) is reported using a one-pot synthetic procedure.     

Streptomycin affinity depends on 13 amino acids forming a loop in homology modelled ribosomal S12 protein (rpsL gene) of Lysinibacillus sphaericus DSLS5 associated with marine sponge (Tedania anhelans)

Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms.     

Protective Role of Cynodon dactylon in Ameliorating the Aluminium-Induced Neurotoxicity in Rat Brain Regions

Cynodon dactylon (Poaceae) is a creeping grass used as a traditional ayurvedic medicine in India. Aluminium-induced neurotoxicity is well known and different salts of aluminium have been reported to accelerate damage to biomolecules like lipids, proteins and nucleic acids. The objective of the present study was to investigate whether the aqueous extract of C. dactylon (AECD) could potentially prevent aluminium-induced neurotoxicity in the cerebral cortex, hippocampus and cerebellum of the rat brain. Male albino rats were administered with AlCl(3) at a dose of 4.2?mg/kg/day i.p. for 4?weeks. Experimental rats were given C. dactylon extract in two different doses of 300?mg and 750?mg/keg/day orally 1?h prior to the AlCl(3) administration for 4?weeks. At the end of the experiments, antioxidant status and activities of ATPases in cerebral cortex, hippocampus and cerebellum of rat brain were measured. Aluminium administration significantly decreased the level of GSH and the activities of SOD, GPx, GST, Na(+)/K(+) ATPase, and Mg(2+) ATPase and increased the level of lipid peroxidation (LPO) in all the brain regions when compared with control rats. Pre-treatment with AECD at a dose of 750?mg/kg b.w increased the antioxidant status and activities of membrane-bound enzymes (Na(+)/K(+) ATPase and Mg(2+) ATPase) and also decreased the level of LPO significantly, when compared with aluminium-induced rats. The results of this study indicated that AECD has potential to protect the various brain regions from aluminium-induced neurotoxicity.     

Renal Function Trajectories in Patients with Prior Improved eGFR Slopes and Risk of Death

Background

Multiple prior studies demonstrated that patients with early Chronic Kidney Disease (CKD) and positive estimated Glomerular Filtration Rate (eGFR) slopes experience increased risk of death. We sought to characterize patients with positive eGFR slopes, examine the renal function trajectory that follows the time period where positive slope is observed, and examine the association between different trajectories and risk of death.

Methods and Findings

We built a cohort of 204,132 United States veterans with early CKD stage 3; eGFR slopes were defined based on Bayesian mixed-effects models using outpatient eGFR measurements between October 1999 and September 2004; to build renal function trajectories, patients were followed longitudinally thereafter (from October 2004) until September 2013. There were 41,410 (20.29%) patients with positive eGFR slope and they exhibited increased risk of death compared to patients with stable eGFR slope (HR = 1.33, CI:1.31–1.35). There was an inverse graded association between severity of albuminuria and the odds of positive eGFR slope (OR = 0.94, CI:0.90–0.98, and OR = 0.76, CI:0.69–0.84 for microalbuminuria and albuminuria; respectively). Following the time period where positive eGFR slope is observed, we characterized 4 trajectory phenotypes: high eGFR intercept and positive trajectory (HIPT) (12.42%), intermediate intercept and mild negative trajectory (IIMNT) (60.04%), low intercept and fast negative trajectory (LIFNT)(23.33%), and high intercept and fast negative trajectory (HIFNT) (4.20%). Compared to IIMNT (reference group), HIPT is associated with younger age, dementia, HIV, chronic lung disease, peripheral artery disease, weight loss, and inversely associated with albuminuria; LIFNT and HIFNT were associated with diabetes, hypertension, cardiovascular disease, peripheral artery disease, and albuminuria. The risk of death at 9 years was lowest in IIMNT (HR = 1.12, CI:1.09–1.14), highest in HIPT (HR = 1.71, CI:1.63–1.79), and intermediate in LIFNT (HR = 1.36, CI:1.32–1.40) and HIFNT (HR = 1.56, CI:1.45–1.68).

Conclusions

Our results demonstrate that patients with positive eGFR slopes, when followed over longer period of time, follow 4 distinct trajectory phenotypes that have distinct demographic and clinical correlates and are differentially associated with risk of death.