The conformationally constrained N-methanocarba-dT analogue adopts an unexpected C4'-exo sugar pucker in the structure of a DNA hairpin

Incorporation of a bicyclo[3.1.0]hexane scaffold into the nucleoside sugar was devised to lock the embedded cyclopentane ring in conformations that mimic the furanose North and South sugar puckers. To analyze the effects of North-methanocarba-2'-deoxythymidine (N-MCdT) on the B-form DNA, we crystallized d(CGCGAA[mcTmcT]CGCG) with two N-MCdTs. Instead of a duplex, the 12mer forms a tetraloop hairpin, whereby loop N-MCdTs adopt the C4'-exo pucker (NE; P = 50°). Thus, the bicyclic framework does not limit the pucker to the anticipated C2'-exo range (NNW; P = -18°).     

Effects of red bull energy drink on repeated sprint performance in women athletes

Energy drinks are frequently consumed by athletes prior to competition to improve performance. This study examined the effect of Red Bull™ on repeated sprint performance in women athletes. Fifteen collegiate soccer players participated, with mean age, height, and body mass equal to 19.5 ± 1.1 year, 168.4 ± 5.8 cm, and 63.4 ± 6.1 kg, respectively. After performing a familiarization trial, subjects performed three sets of eight bouts of the modified t test after ingestion of 255 mL of placebo or Red Bull 1 h pre-exercise in a randomized, placebo-controlled crossover design. Throughout testing, sprint time, heart rate (HR), and rating of perceived exertion (RPE) were continuously obtained. Repeated measures analysis of variance was used to examine differences in variables between drink conditions. Across athletes, t test time ranged from 10.4 to 12.7 s. Mean sprint time was similar (p > 0.05) between Red Bull (11.31 ± 0.61 s) and placebo (11.35 ± 0.61 s). HR and RPE increased (p < 0.05) during the bouts, but there was no effect (p > 0.05) of Red Bull on either variable versus placebo. Findings indicate that 255 mL of Red Bull containing 1.3 mg/kg of caffeine and 1 g of taurine does not alter repeated sprint performance, RPE, or HR in women athletes versus placebo. One serving of this energy drink provides no ergogenic benefit for women athletes engaging in sprint-based exercise.     

Structure requirements for anaerobe processing of azo compounds: Implications for prodrug design

This Letter generalizes the metabolism of the azo class of compounds by Clostridium perfringens, an anaerobe found in the human colon. A recently reported 5-aminosalicylic acid-based prednisolone prodrug was shown to release the drug when incubated with the bacteria, while the para-aminobenzoic acid (PABA) based analogue did not. Instead, it showed a new HPLC peak with a relatively close retention time to the parent which was identified by LCMS as the partially reduced hydrazine product. This Letter investigates azoreduction across a panel of substrates with varying degrees of electronic and steric similarity to the PABA-based compound. Azo compounds with an electron donating group on the azo-containing aromatic ring showed immediate disproportionation to their parent amines without any detection of hydrazine intermediates by HPLC. Compounds containing only electron withdrawing groups are partially and reversibly reduced to produce a stable detectable hydrazine. They do not disproportionate to their parent amines, but regenerate the parent azo compound. This incomplete reduction is relevant to the design of azo-based prodrugs and the toxicology of azo-based dyes.     

Structure of some East African Glossina fuscipes fuscipes populations

Abstract.  Glossina fuscipes fuscipes Newstead 1910 (Diptera: Glossinidae) is the primary vector of human sleeping sickness in Kenya and Uganda. This is the first report on its population structure. A total of 688 nucleotides of mitochondrial ribosomal 16S2 and cytochrome oxidase I genes were sequenced. Twenty-one variants were scored in 79 flies from three geographically diverse natural populations. Four haplotypes were shared among populations, eight were private and nine were singletons. The mean haplotype and nucleotide diversities were 0.84 and 0.009, respectively. All populations were genetically differentiated and were at demographic equilibrium. In addition, a longstanding laboratory culture originating from the Central African Republic (CAR-lab) in 1986 (or before) was examined. Haplotype and nucleotide diversities in this culture were 0.95 and 0.012, respectively. None of its 27 haplotypes were shared with the East African populations. A first approximation of relative effective population sizes was Uganda > CAR-lab > Kenya. It was concluded that the structure of G. f. fuscipes populations in East Africa is localized.     

Method for tracking nanogel particles in vivo and in vitro

Hydrogels made of N-isopropylacrylamide (NIPA) can be synthesized in the form of highly monodispersed nanoparticles. After synthesis, NIPA hydrogel nanoparticles (nanogels) can be labeled by Alexa Fluor 488 carboxylic acid, 2,3,5,6-tetrafluorophenyl ester through amine-terminated functional groups. This choice of dye is complementary to other biological labeling methods for in vivo studies. When the nanogel/dye nanoparticles are injected into rabbits, they can be imaged via tissue sectioning and confocal microscopy, while nanoparticle concentration can be determined by fluorescent microplate assays. Time-course persistence of nanoparticles in the circulatory system can be readily tracked by direct assay of plasma and urine samples using 485 nm excitation and 538 emission wavelengths to keep background fluorescence to nearly the same level as that found using an empty well. Depending upon how the nanoparticles are injected, circulatory system concentrations can reach high concentrations and diminish to low levels or gradually increase and gradually decrease over time. Injection in the femoral artery results in a rapid spike in circulating nanogel/dye concentration, while injection into the renal artery results in a more gradual increase.     

Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

Background  

Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and insulin-like growth factor binding protein-1 (IGFBP-1), is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE). The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase). However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3) is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase), whose products are required for gluconeogenesis.     

Structure of human nicotinamide/nicotinic acid mononucleotide adenylyltransferase. Basis for the dual substrate specificity and activation of the oncolytic agent tiazofurin.

Nicotinamide/nicotinate mononucleotide (NMN/ NaMN)adenylyltransferase (NMNAT) is an indispensable enzyme in the biosynthesis of NAD(+) and NADP(+). Human NMNAT displays unique dual substrate specificity toward both NMN and NaMN, thus flexible in participating in both de novo and salvage pathways of NAD synthesis. Human NMNAT also catalyzes the rate-limiting step of the metabolic conversion of the anticancer agent tiazofurin to its active form tiazofurin adenine dinucleotide (TAD). The tiazofurin resistance is mainly associated with the low NMNAT activity in the cell. We have solved the crystal structures of human NMNAT in complex with NAD, deamido-NAD, and a non-hydrolyzable TAD analogue beta-CH(2)-TAD. These complex structures delineate the broad substrate specificity of the enzyme toward both NMN and NaMN and reveal the structural mechanism for adenylation of tiazofurin nucleotide. The crystal structure of human NMNAT also shows that it forms a barrel-like hexamer with the predicted nuclear localization signal sequence located on the outside surface of the barrel, supporting its functional role of interacting with the nuclear transporting proteins. The results from the analytical ultracentrifugation studies are consistent with the formation of a hexamer in solution under certain conditions.     

Simultaneous Detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, Three Major Fish Pathogens, by Multiplex PCR

A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.     

Interactions of the high-mobility-group-like Ceratitis capitata C1 proteins with DNA

We have studied the interactions of the high-mobility-group-like proteins (C1a1, C1a2 and C1b) from the fruit fly Ceratitis capitata with DNA. Nitrocellulose filter binding assays, thermal denaturation studies and spectrofluorimetry of the complexes revealed the existence of specific and nonspecific interactions. Thermal denaturation curves showed that the three proteins stabilized the DNA, thus suggesting a preferential binding to double-stranded DNA. The calculation of the thermodynamic parameters of the interactions showed that the nonspecific bindings were characterized by low association constants (Ka) with values ranging from 2.7 X 10(4) M-1 to 2.0 X 10(6) M-1. Also, the cooperativity of these interactions was relatively high (cooperativity factor, w, values ranging over 20-35), and the number of nucleotides involved was low (1-3 base pairs). On the other hand, the existence of specific interactions between C1 proteins and DNA was suggested by two facts: the retention of C. capitata [3H]DNA in nitrocellulose filters was only a low percentage of total input DNA and there was a marked size dependence of the binding (25% retention of a 40-kb DNA and only 3% retention with a DNA of 1 kb). The specific bindings had higher Ka values than the nonspecific ones, and they also were cooperative. Some differences were observed between C1b and the C1a proteins about the way they interact with C. capitata DNA.