Isolation and characterization of a novel 2-<Emphasis Type=Italic>sec</Emphasis>-butylphenol-degrading bacterium <Emphasis Type=Italic>Pseudomonas</Emphasis> sp. strain MS-1

A novel bacterium capable of utilizing 2-sec-butylphenol as the sole carbon and energy source, Pseudomonas sp. strain MS-1, was isolated from freshwater sediment. Within 30 h, strain MS-1 completely degraded 1.5 mM 2-sec-butylphenol in basal salt medium, with concomitant cell growth. A pathway for the metabolism of 2-sec-butylphenol by strain MS-1 was proposed on the basis of the identification of 3 internal metabolites—3-sec-butylcatechol, 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid, and 2-methylbutyric acid—by gas chromatography-mass spectrometry analysis. Strain MS-1 degraded 2-sec-butylphenol through 3-sec-butylcatechol along a meta-cleavage pathway. Degradation experiments with various alkylphenols showed that the degradability of alkylphenols by strain MS-1 depended strongly on the position (ortho ≫ meta = para) of the alkyl substitute, and that strain MS-1 could degrade 2-alkylphenols with various sized and branched alkyl chain (o-cresol, 2-ethylphenol, 2-n-propylphenol, 2-isopropylphenol, 2-sec-butylphenol, and 2-tert-butylphenol), as well as a dialkylphenol (namely, 6-tert-butyl-m-cresol).     

Clustering by neurocognition for fine mapping of the schizophrenia susceptibility loci on chromosome 6p

Chromosome 6p is one of the most commonly implicated regions in the genome-wide linkage scans of schizophrenia, whereas further association studies for markers in this region were inconsistent likely due to heterogeneity. This study aimed to identify more homogeneous subgroups of families for fine mapping on regions around markers D6S296 and D6S309 (both in 6p24.3) as well as D6S274 (in 6p22.3) by means of similarity in neurocognitive functioning. A total of 160 families of patients with schizophrenia comprising at least two affected siblings who had data for eight neurocognitive test variables of the continuous performance test (CPT) and the Wisconsin card sorting test (WCST) were subjected to cluster analysis with data visualization using the test scores of both affected siblings. Family clusters derived were then used separately in family-based association tests for 64 single nucleotide polymorphisms (SNPs) covering the region of 6p24.3 and 6p22.3. Three clusters were derived from the family-based clustering, with deficit cluster 1 representing deficit on the CPT, deficit cluster 2 representing deficit on both the CPT and the WCST, and a third cluster of nondeficit. After adjustment using false discovery rate for multiple testing, SNP rs13873 and haplotype rs1225934-rs13873 on BMP6-TXNDC5 genes were significantly associated with schizophrenia for the deficit cluster 1 but not for the deficit cluster 2 or nondeficit cluster. Our results provide further evidence that the BMP6-TXNDC5 locus on 6p24.3 may play a role in the selective impairments on sustained attention of schizophrenia.     

Effects of platelet-derived growth factor and fibroblast growth factor on free intracellular calcium and mitogenesis

Although increased free intracellular calcium (Cai) may be one of the main regulators of cell growth and differentiation, studies in cell populations have implied that not all growth factors produce Cai increases. In order to examine in more detail whether Cai increases were related to mitogenesis, we used digital image analysis of intracellular Fura-2 fluorescence to measure Cai in individual BALB/c 3T3 cells stimulated with either platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF). We found that PDGF induced larger and more prolonged Cai increases than FGF did, but that both growth factors induced an initial rapid increase in Cai (less than 2 min) followed by a later sustained increase (greater than 20 min). Only the prolonged Cai increase required extracellular calcium. Following PDGF treatment (1-8 units/ml), the percentage of cells with a large peak Cai increase (greater than twofold) correlated with the percentage of cells made competent (subsequent growth in 1% platelet-poor-plasma). In contrast, purified bovine basic FGF (200-800 pg/ml) and recombinant human acidic FGF (10-300 ng/ml) produced peak Cai increases that were not directly correlated with mitogenesis. In addition, concentrations of intracellular Quin 2 that inhibited Cai transients also inhibited PDGF stimulation but not FGF stimulation of mitogenesis. Thus, Cai increases are necessary for mitogenesis in BALB/c 3T3 cells stimulated by PDGF, but not that stimulated by FGF.     

ON THE ORIGIN OF INCIPIENT REPRODUCTIVE ISOLATION: THE CASE OF DROSOPHILA ALBOMICANS AND D. NASUTA

The nasuta subgroup of Drosophila consists of 12 known species classified within the immigrans group. D. nasuta and D. albomicans are two sibling species widely distributed throughout the Indo-Pacific tropics, which, although morphologically indistinguishable, have different meta-phase-chromosome configurations: chromosomes X and 3 are attached in D. albomicans, so that about 60% of its genes are sex-linked. Our experiments show that, at least in the laboratory, there is no sexual, mechanical, or gametic isolation between the two species. There is, however, hybrid “breakdown” expressed in three ways: 1) reduction in the number of F₂ hybrids produced per culture; 2) reduction in the fertility of F₂ (males) and F₃ (males and females) hybrid progenies; and 3) abnormal sex ratios in the progenies of crosses between strains of certain localities. In experimental populations, the karyotypes of both species are still present in substantial frequencies after 20 generations, although the frequencies of the two karyotypes vary depending on the geographic origin of the strains. Our results support the hypothesis that, in allopatry, the evolution of postzygotic isolation precedes that of prezygotic isolation. The mtDNA is polymorphic in both D. nasuta and D. albomicans and fairly similar between them. Assuming typical rates of mtDNA evolution, the two species would have diverged from each other about 500,000 years ago, whereas the African and Indian populations of D. nasuta (considered to be different subspecies by some authors) might have diverged some 350,000 years ago.     

Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride

Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.     

Visible-Light-Induced Bactericidal Activity of a Nitrogen-Doped Titanium Photocatalyst against Human Pathogens

The antibacterial activity of photocatalytic titanium dioxide (TiO₂) substrates is induced primarily by UV light irradiation. Recently, nitrogen- and carbon-doped TiO₂ substrates were shown to exhibit photocatalytic activities under visible-light illumination. Their antibacterial activity, however, remains to be quantified. In this study, we demonstrated that nitrogen-doped TiO₂ substrates have superior visible-light-induced bactericidal activity against Escherichia coli compared to pure TiO₂ and carbon-doped TiO₂ substrates. We also found that protein- and light-absorbing contaminants partially reduce the bactericidal activity of nitrogen-doped TiO₂ substrates due to their light-shielding effects. In the pathogen-killing experiment, a significantly higher proportion of all tested pathogens, including Shigella flexneri, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Streptococcus pyogenes, and Acinetobacter baumannii, were killed by visible-light-illuminated nitrogen-doped TiO₂ substrates than by pure TiO₂ substrates. These findings suggest that nitrogen-doped TiO₂ has potential application in the development of alternative disinfectants for environmental and medical usages.     

Enhanced expression of Gialpha protein and adenylyl cyclase signaling in aortas from 1 kidney 1 clip hypertensive rats

We have previously shown the augmented levels of Gialpha-2 and Gialpha-3 proteins (isoforms of inhibitory guanine nucleotide regulatory protein (G-protein)), and not of Gsalpha, in the hearts and aortas of spontaneously and experimentally induced hypertensive rats. The increased expression of Gialpha and blood pressure was restored toward WKY levels by captopril treatment, suggesting a role for angiotensin (Ang) II in the enhanced expression of Gialpha protein and blood pressure. This study was undertaken to investigate whether 1 kidney 1 clip (1K-1C) hypertensive rats that exhibit enhanced levels of Ang II also express enhanced levels of Gialpha proteins. Aortas from 1K-1C hypertensive rats were used. The expression of G-proteins was determined at protein levels with immunoblotting techniques, using specific antibodies for different isoforms of G-proteins. The levels of Gialpha-2 and Gialpha-3 proteins were significantly higher in aortas from 1K-1C hypertensive rats than in control rats; Gsalpha levels were unchanged. The inhibitory effect of low concentrations of guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) on forskolin (FSK)-stimulated adenylyl cyclase (AC) activity was significantly enhanced in aortas from 1K-1C hypertensive rats; the inhibitory effect of C-ANP(4-23), which specifically interacts with the atrial natriuretic peptide (ANP)-C receptor, and Ang II on AC was attenuated. GTPgammaS, isoproterenol, glucagon, NaF, and FSK stimulated the AC activity in aortas from control and hypertensive rats to varying degrees; however, the stimulations were significantly lower in hypertensive rats than in control rats. These data suggest that aortas from 1K-1C hypertensive rats exhibit enhanced expression of Gialpha proteins and associated functions.     

Using surface plasmon resonance to directly measure slow binding of low-molecular mass inhibitors to a VanX chip

VanX, a d,d-dipeptidase, is one of five gene products responsible for vancomycin resistance in pathogenic bacteria and is an attractive drug target in circumventing clinical drug resistance. Our previous combinatorial search of VanX substrates in a dipeptide library of d-X(1)-d-X(2) (19(2)=361) forms has led to the discovery of three new compounds (d-Ala-d-Phe, d-Ala-d-Tyr, and d-Ala-d-Trp) having higher k(cat)/K(M) values than those of its natural substrate, d-Ala-d-Ala. Based on structures of newly identified substrates, two representative transition state analogs of substrates, d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) dipeptide phosphonates, used as VanX inhibitor were rationally designed and chemically synthesized. In the synthesis, eight synthetic steps in total were employed for preparing each VanX inhibitor, and their overall isolated yields were 21 and 11% for 6a and 6b, respectively. Binding interactions of d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) with VanX were confirmed unambiguously and measured quantitatively by surface plasmon resonance. The result reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX (for 6a, k(on)=1.18 x 10(3)M(-1)s(-1), k(off)=2.31 x 10(-3) s(-1), K(D)=1.96 microM, chi(2)=0.0737; for 6b, k(on)=1.09 x 10(3)M(-1)s(-1), k(off)=1.80 x 10(-2)s(-1), K(D)=16.5 microM, chi(2)=0.0599). This suggests that only a fraction of the conformers of the inhibitors in solution adopts a conformation best suited for binding interaction with VanX and that the VanX-inhibitor complex may concomitantly undergo a conformational isomerization from an initial but fast weak-binding adduct to slowly convert to a tight-binding complex with a more stable bound geometry. Moreover, in comparison with 6b, an additional aromatic interaction of 6a with the Phe79 residue in the active site of the enzyme, through an energetically favorable face-to-face offset stacked orientation, may account for its higher affinity than 6b to VanX.