3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

Summary A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of¹H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the and backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His¹¹⁹ side chain in the solution and crystal structures has been detected.     

Enzyme polymorphism in a population of Calomys musculinus (Rodentia,Cricetidae)

NAD-linked lactate, malate, glycerophosphate, alcohol and nonspecific dehydrogenases, aspartate aminotransferases, and soluble esterases from extracts of tissues of individuals from a wild population of Calomys musculinus (Rodentia, Cricetidae) have been analyzed by means of starch gel electrophoresis and specific staining. Allelic frequencies and heterozygosity have been determined. Mendelian inheritance of some of the variants detected was confirmed by breeding experiments. Ten out of fifteen (66.6%) of the genetic loci investigated presented polymorphism. Mean heterozygosity per locus was very high (H=0.2014, se 0.046).This work has been supported, in part, by grants from the Secretaria de Ciencia y Tecnología de la Nación (National Program for Endemic Diseases) and from the Fundación Emilio Ocampo. C. N. G. is a Fellow and A. B. a Career Investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina.     

Meiosis readiness in Lilium longiflorum “Croft”

It is proposed that anthers of Lilium longiflorum Croft approaching the end of premeiotic mitosis reach a state described as meiosis readiness after which cells in premeiotic prophase are unable to complete a mitotic division but despiralize to interphase and enter a meiotic division. Many of the laggard premeiotic cells begin despiralization before reaching an extremely contracted state of late prophase. Premeiotic despiralization is not, therefore, attributed to a deficiency in metaphase but to an inability of these cells to complete prophase. Premeiotic despiralization appears to be preceded by a slowing-down of prophase development. There is variation among anthers and anther regions in the onset of prophase retardation and meiosis readiness. It is suggested that meiosis readiness depends upon a gradual accumulation of meiosis-inducing substances in the cytoplasm of the premeiotic cells. It has not been determined whether the cells that undergo premeiotic despiralization give rise to the giant microsporocytes with shattered chromosomes observed at late prophase of meiosis.     

Effect of ethanol on heart rate and blood pressure in nonstressed and stressed rats

The effect of ethanol on the cardiovascular system (ECG, heart rate, blood pressure) was studied in anesthetized, nonstressed or stressed rats. In anesthetized rats, ethanol showed no effect on heart rate or ECG. In nonstressed rats, ethanol sedated the animals but increased heart rate significantly. This ethanol induced tachycardia seemed the result of a direct stimulation of the sympathetic nerves to the heart. Blood pressure was not significantly affected by ethanol in these nonstressed rats. In stressed rats, marked behavioral excitation and significant increases in heart rate and blood pressure were noted. Ethanol pretreatment calmed the animals considerably during restraint. Ethanol did reduce slightly the stress-induced tachycardia but markedly reduced or antagonized stress-induced blood pressure increases. No major changes in the ECG were noted during these studies with the exception of a few individual animals which showed pathologic ECG responses to ethanol. These data show that ethanol affects cardiovascular functions differently in anesthetized, nonstressed or stressed rats, and that ethanol can significantly reduce or antagonize stress-induced behavioral excitation, tachycardia and hypertension.     

Invasive cane toads are unique in shape but overlap in ecological niche compared to Australian native frogs

Invasive species are an important issue worldwide but predicting invasiveness, and the underlying mechanisms that cause it, is difficult. There are several primary hypotheses to explain invasion success. Two main hypothesis based on niche spaces stand out as alternative, although not exclusive. The empty niche hypothesis states that invaders occupy a vacant niche space in the recipient community, and the niche competition hypothesis states that invaders overlap with native species in niche space. Studies on trait similarity/dissimilarity between the invader and native species can provide information on their niche overlap. Here, we use the highly invasive and well‐studied cane toad (Rhinella marina) to test these two hypotheses in Australia, and assess its degree of overlap with native species in several niche dimensions. We compare extensive morphological and environmental data of this successful invader to 235 species (97%) of native Australian frogs. Our study is the first to document the significant morphological differences between the invasive cane toad and a continent‐wide frog radiation: despite significant environmental overlap, cane toads were distinct in body size and shape from most Australian frog species, suggesting that in addition to their previously documented phenotypic plasticity and wide environmental and trophic niche breadth, their unique shape also may have contributed to their success as an invasive species in Australia. Thus, the invasive success of cane toads in Australia may be explained through them successfully colonizing an empty niche among Australian anurans. Our results support that the cane toad's distinct morphology may have played a unique role in the invasiveness of this species in Australia, which coupled with a broad environmental niche breadth, would have boosted their ability to expand their distribution across Australia. We also propose RLLR (Relative limb length ratio) as a potentially useful measure of identifying morphological niche uniqueness and a potential measure of invasiveness potential in anuran amphibians.     

Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

Background  

Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells.     

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting ß-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from

ABSTRACT: BACKGROUND: D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a beta-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. RESULTS: In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52[DEGREE SIGN]C; however, it exhibited over 60% of maximum activity at 30[DEGREE SIGN]C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50[DEGREE SIGN]C. In this study, a recombinant Pichia pastoris yeast strain secreting beta-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting beta-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization of D-glucose and a 30% conversion of D-galactose to D-tagatose. CONCLUSIONS: The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a P. pastoris strain secreting beta-D-galactosidase and recombinant L-arabinose isomerase seems to offer an interesting alternative for the production of D-tagatose from lactose-containing feedstock.