Decolorization of triphenylmethane, azo, and anthraquinone dyes by a newly isolated Aeromonas hydrophila strain

A broad-spectrum dye-decolorizing bacterium, strain DN322, was isolated from activated sludge of a textile printing wastewater treatment plant. The strain was characterized and identified as a member of Aeromonas hydrophila based on Gram staining, morphology characters, biochemical tests, and nearly complete sequence analysis of 16S rRNA gene and the gyrase subunit beta gene (gyrB). Strain DN322 decolorized a variety of synthetic dyes, including triphenylmethane, azo, and anthraquinone dyes. For color removal, the most suitable pH and temperature were pH 5.0–10.0 and 25–37°C, respectively. Triphenylmethane dye, e.g., Crystal Violet, Basic Fuchsin, Brilliant Green, and Malachite Green (50 mg l⁻¹) were decolorized more than 90% within 10 h under aerobic culture condition and Crystal Violet could be used as sole carbon source and energy source for cell growth. The color removal of triphenylmethane dyes was due to a soluble cytosolic enzyme, and the enzyme was an NADH/NADPH-dependent oxygenase; For azo and anthraquinone dyes, e.g., Acid Amaranth, Great Red GR, Reactive Red KE-3B, and Reactive Brilliant Blue K-GR (50 mg l⁻¹) could be decolorized more than 85% within 36 h under anoxic condition. This strain may be useful for bioremediation applications.     

Chronic PKC-beta2 activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane CFTR targeting

We investigated the effects of chronically applied PKC-stimulating phorbol esters on subcellular CFTR expression and localization in polarized HT-29 Cl.19A monolayers. Modulation of PKC activity with the PKC-beta-specific agonist 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) or nonisoform-selective PMA altered monolayer CFTR immunofluorescence. A decrease in the CFTR signal within the luminal cellular pole was noted with both phorbol esters. Volumetric analysis of the intracellular CFTR signal revealed that both compounds promoted CFTR accumulation into punctate vesicle-like structures found adjacent to the cellular tight junction [labeled with zona occludens (ZO)-1 antibody], extending basally (DOPPA) into the cell. Puncta were more frequent with DOPPA and larger in size with PMA. DOPPA also promoted ZO-1 accumulation at tricellular corners associated with enhanced CFTR puncta number. The observed loss of CFTR immunofluorescence signal induced by low-dose PMA was related to CFTR sequestration into fewer cytoplasmic puncta and correlated with larger increases in PKC substrate phosphorylation. Both phorbol esters downregulated steady-state cellular CFTR mRNA levels by 70%. However, the effects of DOPPA and PMA were largely independent of CFTR biosynthesis: expression levels were 80-85% of control, and the glycosylation status of immunoprecipitated protein remained largely unchanged. Thus changes in cellular CFTR localization correlated with our companion study showing that PMA-induced inhibition of transcellular cAMP-dependent short-circuit current (ISC) was accompanied by cytoplasmic PKC-beta2 accumulation and modest activation of PKC-beta1 and PKC-epsilon. The inhibitory effect of DOPPA on ISC was related solely to increased cytoplasmic PKC-beta2 levels. Thus PKC-beta2 is hypothesized to participate in the regulation of CFTR apical plasma membrane targeting within the constitutive cellular biosynthetic pathway.     

The Induction of Intercellular Adhesion Molecule-1 on Human Umbilical Vein Endothelial Cells by a Heat-Stable Component of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Live Porphyromonas gingivalis enhanced the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of human umbilical vein endothelial cells (HUVECs) in a bacterial dose-dependent manner. Inactivation of P. gingivalis by ultraviolet (UV), heat (56°C, 30 min), or sonication did not alter its stimulatory activity. ICAM-1 expression began to increase at 4 h after stimulation, reached a maximum at 12 h, and remained at the maximum for at least the next 8 h. This time course was similar to that of expression by Escherichia coli LPS. Furthermore, the effect of UV-inactivated P. gingivalis was not inhibited by boiling or polymyxin B treatment. In addition, the effect of P. gingivalis strain W83 on ICAM-1 expression was stronger than that of strain ATCC 33277. Our results suggested that some unidentified, heat-stable proteins, polysaccharides, or lipids may be the stimulatory factor(s), although the participation of LPS could not be completely ruled out. The ability of P. gingivalis to stimulate ICAM-1 expression on endothelial cells may play an important role in the pathogenesis of periodontal disease.     

Expression of Green Fluorescent Protein in <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> Is Affected by Passage Through Host Plants

Xylella fastidiosa, a Gram-negative bacterial plant pathogen, causes Pierces disease of grapevine in North America. In South America the pathogen causes citrus variegated chlorosis, which is widespread in Brazil. We have introduced into Xylella fastidiosa a mini-Tn5 transposon that encodes a green fluorescent protein (GFP) gene optimized for expression in bacteria. The mini-Tn5 derivative was inserted into different sites of the genome in independent transconjugants as determined by Southern blotting. The GFP gene was expressed well and to different levels in different transconjugants. Four independent transconjugants were separately used to inoculate sweet orange and tobacco seedlings. The transconjugants were able to colonize the plants and were subsequently isolated from points distal to the inoculation sites. When the relative fluorescence of the transconjugants that had been passed through either tobacco or sweet orange was compared with that of the same transconjugant maintained continuously in vitro, we observed that passage through either plant host significantly increased the level of expression of the GFP. The increased level of expression of GFP was transient, and was lost upon further culture in vitro. Xylella fastidiosa forms biofilms in planta which are believed to represent a metabolically differentiated state. The increased expression of GFP observed after passage through plants may be accounted for by this phenomenon.     

Isolation and Characterization of <Emphasis Type="Italic">Citrobacter</Emphasis> Strain HPC255 for Broad-Range Substrate Specificity for Chlorophenols

This study aims at development of an approach for selection of strain, which has capability for oxidation of broad-range of chloro-substitute phenols. A multiplex PCR was optimized targeting loci involved in phenol and chlorophenol degradation, which was used to select activated sludge samples and also to assess the degradative genotype of isolates. The isolated strains were screened on the basis of RAPD analysis. In parallel, physiological experiments were carried out with activated sludge samples and isolated bacteria by respirometric analysis. Based on cluster analysis of RAPD pattern and respirometric data, the isolate G20 was selected and identified by using 16S rDNA sequence analysis as Citrobacter freundii strain HPC255. The strain could oxidize different substituted chlorophenol molecules. Such strains could provide the pool of intermediates, which can further be degraded by the associated population, thus helping in maintaining the synergistic association of catabolic activity in activated sludge.     

Object classification by echolocation in nectar feeding bats: size-independent generalization of shape

The nectar-feeding bat Glossophaga can be trained to discriminate two hollow forms, a hollow hemisphere and a paraboloid with the same diameter and depth, in total darkness. During training a saturation level of about 85-90% correct choices or more can be reached within 50-100 visits. To investigate generalization abilities, the bats were tested with pairs of the same shape but of different size. Although no reward was offered, they preferred the hollow sphere (30 mm and 50 mm diameter, but not 18 mm) over the corresponding paraboloids. Thus, the bats were able to generalize some features of the rewarded form and detect them in forms of the same shape but different size. This transposition is remarkable, since the bats could not use absolute spectral characters, but had to pay attention to size-independent features common to hollow hemispheres. Possible cues are the variation of echoes in dependence of different angles of calling direction (constant in spheres, changing with position in paraboloids) and/or the "timbre" of the echoes, i.e. their spectral pattern independent of their absolute pitch     

Sequence Analysis of DNA Fragments from the Genome of the Primary Endosymbiont of the Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

The whitefly Bemisia tabaci contains a primary prokaryotic endosymbiont housed within specialized cells in the body cavity. Two DNA fragments from the endosymbiont, totaling 33.3 kilobases, were cloned and sequenced. In total, 37 genes were detected and included the ribosomal RNA operon and genes for ribosomal RNA proteins. The guanine plus cytosine of the DNA was 30.2 mol%, different from that of endosymbionts of other plant sap-sucking insects.     

Characterization of the First VanB Vancomycin-Resistant <Emphasis Type="Italic">Enterococcus faecium</Emphasis> Isolated in a Spanish Hospital

We report the detection and characterization of the first vancomycin-resistant VanB-type Enterococcus faecium to be isolated in a Spanish hospital. Sequence analysis of the vanB gene showed that this isolate belonged to subtype vanB2. Moreover, PCR amplification analysis indicated that the vanB gene cluster was linked to a Tn5382-like transposon.     

Body, eye, and chorioallantoic vessel growth are not dependent on cardiac output level in day 3-4 chicken embryos

Normal aerobic metabolic rates persist in the early chicken embryo after elimination of cardiac output, but the dependence of tissue growth and differentiation on blood flow is unknown in these early stages. We partially ligated (25-50% occlusion) the ventricular outflow tract of Hamburger-Hamilton stage (HH) 16-18 embryos, producing a wide range of cardiac output. For the next approximately 48 h (to HH 24), we measured heart rate (HR), stroke volume (SV), and cardiac output (CO), as well as these growth indicators: eye diameter, chorioallantoic vessel density, and body mass. Acutely, HR declined with partial ligation (from 108 to 98 beats/min). Paradoxically, SV and CO decreased sharply in most embryos but increased in others, collectively producing the desired large variation (up to 25-fold) in CO and permitting assessment of tissue growth over a very large range of blood perfusion. Eye diameter doubled (from 0.6 to 1.2 mm) with development from HH 16 to HH 24, but within a developmental cohort there was no significant correlation between eye diameter and CO over a 25-fold range of CO. Similarly, chorioallantoic membrane vessel index was independent of CO over the CO range at all stages. Finally, body mass increase during development was not significantly affected by partial conal truncal ligation. Collectively, these data suggest that normal eye and vessel growth and body mass accumulation occur independent of their rate of blood perfusion, supporting the hypothesis of prosynchronotropy-that the heart begins to beat and generate blood flow in advance of the actual need for convective blood flow to tissues.     

Transformation of verapamil by Cunninghamella blakesleeana

A filamentous fungus, Cunninghamella blakesleeana AS 3.153, was used as a microbial model of mammalian metabolism to transform verapamil, a calcium channel antagonist. The metabolites of verapamil were separated and assayed by the liquid chromatography-ion trap mass spectrometry method. After 96 h of incubation, nearly 93% of the original drug was metabolized to 23 metabolites. Five major metabolites were isolated by semipreparative high-performance liquid chromatography and were identified by proton nuclear magnetic resonance and electrospray mass spectrometry. Other metabolites were characterized according to their chromatographic behavior and mass spectral data. The major metabolic pathways of verapamil transformation by the fungus were N dealkylation, O demethylation, and sulfate conjugation. The phase I metabolites of verapamil (introduction of a functional group) by C. blakesleeana paralleled those in mammals; therefore, C. blakesleeana could be a useful tool for generating the mammalian phase I metabolites of verapamil.