Differences in extracellular enzymatic activity between Candida dubliniensis and Candida albicans isolates

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.     

Heterostyly: speciation within a species

Almost all organisms in nature show nonrandom mating to different degrees. Two extreme results of nonrandom mating are speciation and sexual differentiation. Heterostyly is a form of sexual differentiation considered to have evolved to resolve conflicts between male and female functions of hermaphrodite flowers. Our study examines necessary and sufficient conditions for establishment of heterostyly using a configuration individual-based model. Previous models assume invasion of a mutant phenotype into a population with monomorphic wild phenotype. In contrast, our model demonstrates that heterostyly can be established from a population with continuous phenotypic variation, a proposition that requires simpler assumptions than the previous hypotheses. Results of our simulation show that genetic linkage between stigma and anther heights is essential for establishment of heterostyly. Dominance effects on the genes for stamen or stigma heights are not necessary, but they promote evolution of heterostyly. Probability of evolution of heterostyly also depends on the functional relationship between stigma–anther distance and strength of sexual interference, and the distance and probability of pollen deposition success. Parallelity and difference between speciation and sexual differentiation are also discussed.     

Highly potent PDE4 inhibitors with therapeutic potential

The hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system prompted us to design and synthesize a hydrophilic piperidine analog to improve the side effect profile of Ariflo 1, which is an orally active second-generation PDE4 inhibitor. During evaluation of various water-soluble piperidine analogs, 2a-b, 11b-14b, and 17a showed therapeutic potential in cross-species comparison studies. The following three findings were obtained: (1) The hydroxamic acid group, a well known metal chelator, caused a marked increase of inhibitory activity. (2) Water-soluble piperidine analogs lacked the configurational isomerism of Ariflo 1 without loss of inhibitory activity. (3) Replacement of the 4-methoxy residue with a difluoromethoxy residue led to an increase of in vivo potency. Structure-activity relationships are presented. Single-dose rat pharmacokinetic data for 11b, 12b, and 17a are also presented.     

Continuous subcutaneous injection reduces polymorphonuclear leukocyte activation by granulocyte colony-stimulating factor

The use of granulocyte colony stimulating factor (G-CSF) for recovery from neutropenia has been established; however, acute lung injury due to G-CSF-induced polymorphonuclear leukocyte (PMN) activation is a serious complication. This study was designed to compare the activation of PMN with single bolus administration and continuous administration of G-CSF. Healthy volunteers (age 33.8 +/- 1.4 yr; n = 6) received a single bolus injection of 50 microm/m2 of G-CSF (SI; n = 6) or continuous subcutaneous injection of 50 microm/m2 of G-CSF for 24 h (CI; n = 6) and were followed for 48 h. Circulating leukocyte counts, markers of activation on PMN, and circulating levels of G-CSF, IL-6, and PMN elastase were measured. SI rapidly increased serum G-CSF levels, which peaked at 4 h, whereas CI gradually increased G-CSF levels, which remained at a steady level from 8 to 24 h. SI caused a rapid decrease in PMN counts at 0.5 h followed by sustained increase to peak at 12 h. CI gradually increased PMN counts, which peaked at 24 h, but the peak values were not significantly different between the groups. SI-induced activation of PMN, which was characterized by increased expression of CD11b, decreased expression of L-selectin, and increased F-actin content, led to increases in serum IL-6 and PMN elastase level. Such changes were all attenuated with CI (P < 0.05). We conclude that continuous subcutaneous injection of G-CSF resulted in a marrow response similar to that to a single injection but yielded reduced PMN activation.     

Composition and structure of the centromeric region of rice chromosome 8

Understanding the organization of eukaryotic centromeres has both fundamental and applied importance because of their roles in chromosome segregation, karyotypic stability, and artificial chromosome-based cloning and expression vectors. Using clone-by-clone sequencing methodology, we obtained the complete genomic sequence of the centromeric region of rice (Oryza sativa) chromosome 8. Analysis of 1.97 Mb of contiguous nucleotide sequence revealed three large clusters of CentO satellite repeats (68.5 kb of 155-bp repeats) and >220 transposable element (TE)-related sequences; together, these account for approximately 60% of this centromeric region. The 155-bp repeats were tandemly arrayed head to tail within the clusters, which had different orientations and were interrupted by TE-related sequences. The individual 155-bp CentO satellite repeats showed frequent transitions and transversions at eight nucleotide positions. The 40 TE elements with highly conserved sequences were mostly gypsy-type retrotransposons. Furthermore, 48 genes, showing high BLAST homology to known proteins or to rice full-length cDNAs, were predicted within the region; some were close to the CentO clusters. We then performed a genome-wide survey of the sequences and organization of CentO and RIRE7 families. Our study provides the complete sequence of a centromeric region from either plants or animals and likely will provide insight into the evolutionary and functional analysis of plant centromeres.     

Structure-activity study of L-amino acid-based N-type calcium channel blockers

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.     

Repeated cocaine administration changes the function and subcellular distribution of adenosine A1 receptor in the rat nucleus accumbens

Adenosine A1 receptor (A1) protein and mRNA is increased in the nucleus accumbens following repeated cocaine treatment. In spite of this protein up-regulation, A1 agonist-stimulated [35S]GTPgammaS binding was attenuated in accumbens homogenates of rats withdrawn for 3 weeks from 1 week of daily cocaine injections. Cellular subfractionation revealed that the discrepancy between total A1 protein and G protein coupling resulted from a smaller proportion of receptors in the plasma membrane. The decrease in functional receptor in the plasma membrane was further indicated by diminished formation of heteromeric receptor complex consisting of A1 and dopamine D1A receptors. To explore the functional significance of the altered distribution of A1 receptors, at 3 weeks after discontinuing repeated cocaine or saline, animals were injected with cocaine and 45 min later the subcellular distribution of A1 receptors quantified. Whereas a cocaine challenge in repeated saline-treated animals induced a marked increase in membrane localization of the A1 receptor, the relative distribution of receptors in repeated cocaine rats was not affected by acute cocaine. These data suggest that the sorting and recycling of A1 receptors is dysregulated in the nucleus accumbens as the consequence of repeated cocaine administration.     

DNA damage by ethylbenzenehydroperoxide formed from carcinogenic ethylbenzene by sunlight irradiation

Ethylbenzene, widely used in human life, is a non-mutagenic carcinogen. Sunlight-irradiated ethylbenzene caused DNA damage in the presence of Cu2+, but unirradiated ethylbenzene did not. A Cu+ -specific chelator bathocuproine inhibited DNA damage and catalase showed a little inhibitory effect. The scopoletin assay revealed that peroxides and H(2)O(2) were formed in ethylbenzene exposed to sunlight. These results suggest that Cu+ and alkoxyl radical mainly participate in DNA damage, and H(2)O(2) partially does. When catalase was added, DNA damage at thymine and cytosine was inhibited. Ethylbenzenehydroperoxide, identified by GC/MS analysis, induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and caused DNA damage at consecutive guanines, as observed with cumenehydroperoxide. Equimolar concentrations of H(2)O(2) and acetophenone were produced by the sunlight-irradiation of 1-phenylethanol, a further degraded product of ethylbenzene. These results indicate a novel pathway that oxidative DNA damage induced by the peroxide and H(2)O(2) derived from sunlight-irradiated ethylbenzene may lead to expression of the carcinogenicity.     

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid beta-oxidation. The impairment was reversed to the wild-type levels by treatment with 17beta-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid beta-oxidation and in maintaining lipid homeostasis.