Disomic and diploid meiotic products in Saccharomyces cerevisiae. Effect of vincristine, vinblastine, adriamycin, bleomycin, mitomycin C and cyclophosphamide

The effect of some antineoplastic drugs on the induction of disomic and diploid meiotic products in Saccharomyces cerevisiae was evaluated. Vincristine and vinblastine turned out not to be effective on any of the four genetic phenomena studied (sporulation, recombination, disomic induction and diploid induction). Adriamycin showed only slight activity in inducing diploids, particularly during the first meiotic division. Cyclophosphamide was active on both phenomena leading to the formation of disomic and diploid spores. Mitomycin C and bleomycin were effective on the induction of diploids. In all of these inductions, the origin of diploids was due to failure of the second meiotic division. No significant effects were found on recombination frequency. As a general conclusion, one may assume that formation of aneuploid and diploid gametes are two distinct phenomena, not necessarily correlated from the point of view of the mechanism and of the specificity of induction.     

Structural analysis of a cDNA clone from Xenopus laevis containing a repetitive sequence element

Total polysomal RNA from Xenopus laevis stage 40 embryos was probed for the presence of repetitive sequences by Northern blot analysis with a genomic DNA fragment which had previously been shown to contain several repetitive sequence elements (Spohr et al., 1981). The analysis revealed that various presumptive mRNAs contain sequences complementary to the repetitive probe. Consequently, a cDNA library was constructed and screened with the same probe. Forty-eight positive recombinants containing eucaryotic inserts of 300–700 base pairs were isolated and one such clone was characterized in detail. Analysis of its nucleotide sequence revealed the presence of an open reading frame for 118 amino acids. Comparison of nucleotide sequences located 3′ to this presumptive protein coding region with the sequence of the genomic DNA fragment used as a probe clearly identifies and allows one to define the exact location of the repetitive element in the cloned cDNA. This analysis shows furthermore that one portion of the repeated sequence is highly conserved in the two members of this repetitive sequence family, whereas the other part is more divergent. In this area blocks of oligonucleotides are scattered between nonhomologous DNA stretches. The occurrence frequency of the presumptive mRNAs which carry repetitive elements homologous to the used repetitive probe is suggested to be close to that of rare mRNAs.     

Detection of Cystathionine Ketimine and Lanthionine Ketimine in Human Brain

The sulfur containing imino acids cystathionine ketimine (CK) and lanthionine ketimine (LK) have been detected in the human brain by an HPLC procedure. The HPLC procedure takes advantage of the selective absorbance at 380 nm of the phenylisothiocyanate-ketimine adduct. Quantitation of cystathionine ketimine and lanthionine ketimine indicates a mean concentration (mean ± SD, n = 4) of 2.3 ± 0.8 nmol/g for CK and of 1.1 ± 0.3 nmol/g for LK in four human cerebral cortex samples of neurosurgical source. The identification of these cyclic ketimine derivatives of L-cystathionine and L-lanthionine as normal human metabolites in human nervous tissue may have interesting metabolic and physiological implications.     

Translation "in vitro" of globin mRNA from Xenopus laevis

RNA isolated from Xenopus laevis reticulocytes and characterized as globin mRNA (Meza et al., 1978) was tested for its capacity to stimulate "in vitro" a wheat germ translation system, and the ability to synthesize a polypeptide. The latter was identified as globin by its electrophoretic mobility and immunoprecipitation with antiglobin antibody.     

Occurrence of Rhodococcus coprophilus and associated actinomycetes in feces, sewage, and freshwater.

Freshwater, sewage, and fecal samples from various sources were examined for Rhodococcus coprophilus, associated actinomycetes, Escherichia coli, and fecal streptococci. Rhodococcus coprophilus was isolated consistently from feces of farm animals, poultry reared in proximity to farm animals, freshwater, and wastewater polluted with animal fecal wastes. It was not isolated from samples of human feces. The ratio of R. coprophilus total actinomycetes was higher in feces from cattle, sheep, ducks, and geese than in specimens from pigs, horses, and fowl. In samples from two freshwater streams polluted by fecal material from farm animals, the ratios of R. copropilus to total actinomycetes were similar to those found in fecal specimens from cattle and sheep. Ratios of fecal coliform to fecal streptococci could not distinguish between fresh human and animal fecal samples and, furthermore, were not reflected in the stream waters polluted by animal fecal material. R. coprophilus has potential in water and dairy bacteriology as a specific indicator organism of fecal pollution due to farm animal wastes.     

Identification of a 3rd Na+ Binding Site of the Glycine Transporter,GlyT2

The Na⁺/Cl^- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na⁺ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na⁺ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na⁺-dependent leucine transporter, LeuT_Aa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na⁺ binding sites in GlyT2, but the physical location of the third Na⁺ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuT_Aa. Although betaine transport by BetP is coupled to the co-transport of two Na⁺ ions, the first Na⁺ site is not conserved between BetP and LeuT_Aa, the so called Na1'' site. We hypothesized that the third Na⁺ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1'' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na⁺ placed at the location consistent with the Na1'' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na⁺ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.     

Inhibition of Bacillus anthracis metallo-β-lactamase by compounds with hydroxamic acid functionality

Abstract

Metallo-β-lactamases (MBLs) that catalyze hydrolysis of β-lactam antibiotics are an emerging threat due to their rapid spread. A strain of the bacterium Bacillus anthracis has its ability to produce and secrete a MBL, referred to Bla2. To address this challenge, novel hydroxamic acid-containing compounds such as 3-(heptyloxy)-N-hydroxybenzamide (compound 4) and N-hydroxy-3-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide (compound 7) were synthesized. Kinetic analysis of microbial inhibition indicated that the both sides of hydroxamic acids containing compound 7 revealed a reversible, competitive inhibition with a K_i value of 0.18?±?0.06?μM. The result has reflected that the both sides of dihydroxamic acids in a molecule play a crucial role in the binding affinity rather than monohydroxamic containing compound 4 which was unable to inhibit Bla2. In addition, in silico analysis suggested that compound 7 was coordinated with a zinc ion in the active site of enzyme. These observations suggest that the dihydroxamic acid-containing compound may be a promising drug candidate, and a further implication for designing new inhibitors of Bla2.     

Distribution of the serine protease HtrA1 in normal human tissues.

The human HtrA family of proteases consists of three members: HtrA1, HtrA2, and HtrA3. In bacteria, the chief role of HtrA is recognition and degradation of misfolded proteins in the periplasm, combining a dual activity of chaperone and protease. In humans, the three HtrA homologues appear to be involved in diverse functions such as cell growth, apoptosis, allergic reactions, fertilization, control of blood pressure, and blood clotting. Previous studies using RNA blot hybridization have shown that the expression of HtrA1 is ubiquitous in normal human tissues. Here we show by immunohistochemistry (IHC) that HtrA1 is widely expressed, although different tissue distributions and/or levels of expression were detected in the different tissues examined. In particular, high to medium HtrA1 expression was detected in mature layers of epidermis, in secretory breast epithelium, in liver, and in kidney tubules of cortex, in concordance with its secretory properties. Furthermore, we show a higher protein expression level in the epithelium of proliferative endometrium, in contrast to epithelium of secretory endometrium, which is almost completely negative for this protein. This suggests a possible role for HtrA1 in the modulation of tissue activity in this organ. The various expression levels in human tissues indicate several possible roles for HtrA1 in different cell types.     

Swarming Behavior of and Hemolysin BL Secretion by Bacillus cereus

An association between swarming and hemolysin BL secretion was observed in a collection of 42 Bacillus cereus isolates (P = 0.029). The highest levels of toxin were detected in swarmers along with swarm cell differentiation (P = 0.021), suggesting that swarming B. cereus strains may have a higher virulence potential than nonswarming strains.