<Emphasis Type=Italic>Caenorhabditis elegans</Emphasis>behavioral genetics: where are the knobs?

Thousands of behavioral mutants of Caenorhabditis elegans have been studied. I suggest a set of criteria by which some genes important in the evolution of behavior might be recognized, and identify neuropeptide signaling pathways as candidates.     

Revision of “<Emphasis Type=Italic>Septonema ochraceum</Emphasis>” revealed three new species of <Emphasis Type=Italic>Venturiaceae</Emphasis> and <Emphasis Type=Italic>Herpotrichiellaceae</Emphasis>

Survey of seven strains determined as Septonema ochraceum (Dothideomycetes, inc. sed.) isolated from pine litter or obtained from public collections revealed three new species, Fusicladium cordae, F. sicilianum (Venturiaceae), Cladophialophora matsushimae (Herpotrichiellaceae) and a cryptic species morphologically identical to Devriesia americana (Teratosphaeriaceae), but phylogenetically distinct. Morphological survey and phylogenetic analysis using nucleotide sequence data from the nuclear ribosomal subunit genes indicate a close relationship within three species colonising pine litter needles, F. cordae, F. pini and F. ramoconidii. F. sicilianum is most related to F. rhodense. C. matsushimae represents a species belonging to one of the lineages of the polyphyletic genus Cladophialophora. None of the strains observed can be classified morphologically as S. ochraceum, of which the type material does not exist.     

β-Glycosidase of <Emphasis Type=Italic>Thermus thermophilus</Emphasis> KNOUC202: Gene and biochemical properties of the enzyme expressed in <Emphasis Type=Italic>Escherichia coli</Emphasis>

The β-glycosidase gene of Thermus thermophilus KNOUC202 was cloned, expressed in Escherichia coli JM109(DE3), and the enzyme was purified and characterized. The gene (KNOUC202β-gly) was composed of 1296 bp encoding a β-glycosidase (KNOUC202β-glycosidase) of 431 a.a., belonging to the family 1 of glycosyl hydrolase. The gene was expressed as monomer of 430 a.a. with amino terminal methionine excised in E. col JM109(DE3). The enzyme hydrolyzed β-glycosides whose glycone are galactose, glucose and fucose well, however showed no or very low activity on β-D-glycosides whose glycone are disaccharides and xylose. k _cat of the enzyme for the hydrolysis of p-Nph-β-D-Glcp was lower than those for p-Nph-β-D-Galp and ONPG, however K _m for p-Nph-β-D-Glcp was highly lower than those for p-Nph-β-D-Galp and ONPG resulting in the catalytic efficiency(k _cat/K _m) for the hydrolysis of p-Nph-β-D-Glcp much higher than those for p-Nph-β-D-Galp and ONPG. Optimum pH and optimum temperature of the enzyme were pH 5.4 and 90°C. The enzyme has high thermostability, not losing its activity at 80°C for 2 h in 0.05 M Na-phosphate buffer of pH 6.8 with T _m of 100.0 ± 0.031°C in 0.02 M Tris-HCl buffer of pH 8.2. The b-glycosidase produced a disaccharide composed of galactose as transglycosylation by-product during hydrolysis of lactose.     

Polygenic control for fermentation of β-fructosides in the yeast <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>: New genes <Emphasis Type=Italic>SUC9</Emphasis> and <Emphasis Type=Italic>SUC10</Emphasis>

Using molecular karyotyping and genetic hybridization analysis, two new polymeric β-fructosidase genes, SUC9 and SUC10, were identified in the yeast Saccharomyces cerevisiae, which are located on chromosome XIV and on the chromosome XVI/XIII doublet, respectively. The genes are responsible for fermentation of sucrose and raffinose. The SUC gene genotypes of strains VKM Y-1831 and DBVPG 1340 are SUC2 SUC9 and suc2 ⁰ SUC10, respectively. suc2 ⁰ is a silent sequence. The scientific and applied significance of SUC genes is discussed.     

Small colony variants of <Emphasis Type=Italic>Staphylococcus aureus</Emphasis> — <Emphasis Type=Italic>review</Emphasis>

Bacterial variants of Staphylococcus aureus called small colony variants (SCVs) originate by mutations in metabolic genes, resulting in emergence of auxotrophic bacterial subpopulations. These variants are not particularly virulent but are able to persist viable inside host cells. SCVs show their characteristic auxotrophic growth deficiency and depressed α-cytotoxin activity. Environmental pressure such as antibiotics, select for isogenic SCV cells that are frequently found coexisting with their parent wild-type strains in a mixed bacterial culture. SCV strains often grow on blood agar as non-pigmented or pinpoint pigmented colonies and their key biochemical tests are often non-reactive. Their altered metabolism or auxotrophism can result in long generation time and thus SCV phenotype, more often than not SCV can be overgrown by their wild-type counterparts and other competitive respiratory flora. This could affect laboratory detection. Thus, molecular methods, such as 16S rRNA partial sequencing or amplification of species-specific DNA targets (e.g. coagulase, nuclease) directly from clinical material or isolated bacterial colonies, become the method of choice. Patients at risk of infection by S. aureus SCVs include cystic fibrosis patients (CF), patients with skin and foreign-body related infections and osteomyelitis, as they suffer from chronic staphylococcal infections and are subject to long-term antibiotic therapy. Molecular evidence of SCV development has not been found except for some random mutations of the thymidylate synthase gene (thyA) described in SCV S. aureus strains of CF patients. These variants are able to bypass the antibiotic effect of folic acid antagonists such as sulfonamides and trimethoprim. Resistance to gentamicin and aminoglycosides in the hemin or menadione auxotrophic SCVs was hypothesized as being due to decreased influx of the drugs into cells as a result of decreased ATP production and decreased electrochemical gradient on cell membranes.     

A computational study of the mechanism of the unimolecular elimination of <Emphasis Type=Italic>α</Emphasis>,<Emphasis Type=Italic>β</Emphasis>-unsaturated aldehydes in the gas phase

The mechanism for the decarbonylation of (E)-2-butenal and (E)-2-methyl-3-pheny-2-propenal was studied with different levels of ab initio and DFT methods. Reactants, products and transition structures were optimized for two kinds of reaction channel: a one-step reaction which involves a three-membered cyclic transition state, and a two-step reaction which involves an initial four-membered cyclic transition state. According to our calculations, these two possible mechanisms entail similar energetic costs, and there are only small differences depending on the reactant. The elimination of (E)-2-methyl-3-pheny-2-propenal yields different products depending on the channel followed. Only one of the three possible one-step mechanisms leads directly to (E)-β-methylstyrene (the main product according to experiment). This fact is reasonably well reproduced by our results, since the corresponding transition state gave rise to the lowest activation Gibbs free energy.     

Differences in the timing of cell death,differentiation and function among three different types of ray parenchyma cells in the hardwood <Emphasis Type=Italic>Populus sieboldii</Emphasis> × <Emphasis Type=Italic>P</Emphasis>. <Emphasis Type=Italic>grandidentata</Emphasis>

Differences in the timing of cell death, differentiation and function among three different types of ray parenchyma cells in the hardwood Populus sieboldii × P. grandidentata which form uniseriate and homocellular rays were examined and clarified. Ray parenchyma cells died within 5 years, and the disappearance of nuclei from ray parenchyma cells did not occur successively from the pith side, even within individual radial cell lines of a given ray. Cell death occurred earliest in contact cells, which were connected to adjacent vessel elements through pits, in the fourth annual ring from the cambium. Cell death occurred next in intermediate cells, which were located within the same cell lines as contact cells but were not adjacent to vessel elements, in the fourth annual ring from the cambium. Finally, isolation cells, which were located within the other cell lines of a given ray, died in the fifth annual ring from the cambium. Secondary wall thickenings in contact cells and intermediate cells were initiated before those in isolation cells in the current year’s xylem. Most starch grains were localized in intermediate cells, and there were more lipid droplets in contact cells and intermediate cells than in isolation cells. In addition, the largest quantities of protein were found in contact cells. Our results indicate that the position within a ray and neighboring short-lived vessel elements might affect the timing of cell death and differentiation and, thus, the function of long-lived ray parenchyma cells in Populus sieboldii × P. grandidentata.     

Change in compactness of inclusion bodies of recombinant β-galactosidase expressed in the <Emphasis Type=Italic>araBAD</Emphasis> promoter system of <Emphasis Type=Italic>Escherichia coli</Emphasis>

We investigated the relevance of the relationship between the compactness of β-galactosidase inclusion bodies (β-gal IBs) and their enhanced enzymatic activity with or without the addition of D-fucose (inducer analog) or methyl α-D-glucopyranoside (α-MG, catabolite repressor) after induction in the araBAD promoter system of Escherichia coli. Experiments conducted to evaluate the solubilization of β-gal IBs in guanidine hydrochloride as well as their trypsin degradation and temperature stability revealed that β-gal IBs expressed in response to the addition of D-fucose or α-MG had a looser structure. Additionally, β-gal IBs expressed when D-fucose or α-MG was added were more quickly solubilized in guanidine hydrochloride or degraded by trypsin-treatment than those produced when these compounds were not added. Moreover, the activity of β-gal IBs expressed when D-fucose or α-MG were added was less stable at various temperatures. Consequently, we deduced that the looser structure of β-gal IBs resulted in enhanced enzymatic activity of β-gal IBs upon addition of D-fucose or α-MG after induction.     

Enhanced production and characterization of a β-glucosidase from <Emphasis Type=Italic>Bacillus halodurans</Emphasis> expressed in <Emphasis Type=Italic>Escherichia coli</Emphasis>

A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A ₆₀₀ 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. K _m and k _cat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec⁻¹, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.     

Effect of <Emphasis Type=Italic>Azotobacter chroococcum</Emphasis> on <Emphasis Type=Italic>in vitro</Emphasis> pineapple plants’ growth during acclimatization

Pineapple is one of the most important tropical fruits, but the availability of planting material is insufficient to agricultural demands. Therefore, several pineapple micropropagation protocols have been developed. However, acclimatization of in vitro plants continues to take a prolonged period. Biofertilizers have been found as safe alternatives to improve the agricultural performances of many crops. This study highlights some of the effects of the application of Azotobacter chroococcum (INIFAT5 strain) on in vitro pineapple plants during acclimatization. The bacteria were sprayed immediately after transplanting to the ex vitro environment; the plants were then sprayed every 4 wk. A control group of plants was established. Subsequently, after 5 mo, the evaluated variables included fresh and dry plant weight, plant height (cm), and root length (cm). The anatomy of middle-aged leaves and roots was also studied: transversal thickness and width of cuticle, epidermis, hypodermis, aquiferous parenchyma, and photosynthetic parenchyma. Thickness of root exoderm, external cortex, internal cortex, and stele were also evaluated. In general, the INIFAT5 strain improved the plant development. Results showed that the bacteria significantly provoked changes in the plant fresh weight, the thickness of the leaf abaxial and adaxial cuticles, and the root exoderm width. Contrastingly, A. chroococcum did not affect the thickness of the leaf photosynthetic parenchyma.     

Study of CTX-M Type of Extended Spectrum β-Lactamase among Nosocomial Isolates of <Emphasis Type=Italic>Escherichia coli</Emphasis> and <Emphasis Type=Italic>Klebsiella pneumoniae</Emphasis> in South India

Data on CTX-M type extended-spectrum β-lactamase (ESBL) produced by Gram-negative bacteria by molecular methods are limited from India. This study was conducted to investigate the prevalence of CTX-M type ESBL producing Escherichia coli and Klebsiella pneumoniae from nosocomial isolates in a tertiary care hospital in southern India. A total of 179 clinical isolates of K. pneumoniae (n = 72) and E. coli (n = 107) were obtained in a period of 3 months and assessed for ESBL production phenotypically. Associated resistance to a panel of antibiotics and Minimum Inhibitory Concentration for 3rd generation cephalosporins was determined. Phenotypically ESBL positive isolates were subjected to PCR for bla _CTX-M gene using two sets of primers for the simultaneous detection of all the five major groups of CTX-M types. All the positive isolates were then subjected to a group specific PCR to detect the prevalent group. Out of 179 isolates, 156 (87.1%) were positive for ESBL phenotypically, which includes 39.2% of K. pneumoniae and 60.8% of E. coli. All of them were examined by PCR using two primers for the presence of bla _CTX-M genes. Among the 156 phenotypic positive isolates, 124 (79.4%) were positive for bla _CTX-M genes, of which 45 (36.2%) were K. pneumoniae, 79 (63.7%) were E. coli. When the 124 positive clinical isolates were further tested with CTX-M group-specific primers, all were positive for the CTX-M-1 group. Our findings document evidence of the high prevalence of multidrug resistant CTX-M group 1 type ESBL among nosocomial isolates in this region. High co-resistance to other non-β-lactam antibiotics is a major challenge for management of ESBL infections. This is alarming and calls for the judicious use of carbapenems, especially in developing countries. This has significant implications for patient management, and indicates the need for increased surveillance and for further molecular characterization of these isolates.     

Airborne <Emphasis Type=Italic>Aspergillus</Emphasis> and <Emphasis Type=Italic>Penicillium</Emphasis> in the atmosphere of Szczecin, (Poland) (2004–2009)

The investigation into airborne fungal spore concentrations was conducted in Szczecin (Poland) between 2004 and 2009. The objective of the studies was to determine a seasonal variation in concentrations of amerospores on the basis of meteorological parameters. The presence of spores in Szczecin was recorded using a volumetric method. Fungal spores were present in the air in high numbers in late summer and early autumn. The highest concentrations were noted in September, October and November. The peak period was recorded in August, September, October and November. The highest annual number of spores occurred in 2005 and 2007 and the lowest in 2006. High values of daily concentration of amerospores occurred during the afternoon and late at night. In 2005 and 2007 the late-night maximum was overdue about 1 or 2 h. For daily values of dew point temperature and relative humidity, the coefficients were positive, significant for p = 0.001 and ranged from 0.342 to 0.258. The average wind speed was positively correlated for p = 0.01 and the coefficient was 0.291. The similar relations were noted for hourly values of spore concentrations for p = 0.05, p = 0.01 and p = 0.001. For these spore types, the dew point temperature and relative humidity appeared to be the most influential factor.     

β<Subscript>2</Subscript> Adrenoreceptor-Mediated Noradrenergic Effect on GABA-ergic Transmission in the <Emphasis Type=Italic>CA1</Emphasis> Zone of the Rat Hippocampus <Emphasis Type=Italic>in vitro</Emphasis>

Using extracellular recording of evoked potentials, we examined the effect of an agonist of β₂ adrenoreceptors, metaproterenol (MPT), on GABA-ergic transmission in the CA1 zone of slices of the rat hippocampus. Isolated application of GABA evoked in these slices rapid reversible suppression of orthodromic population discharges recorded from the pyramidal layer of the above hippocampal zone after electrical stimulation of Schaffer collaterals in the radial layer. In most cases (13 of 19 preparations), combined application of GABA and MPT interfered with the development of the inhibitory GABA effect. In the case of the action of both of the above agents, the amplitude and duration of evoked responses decreased, but these changes were significantly weaker than those observed upon isolated GABA application. Our experiments showed that the noradrenergic system is capable of modulating GABA-ergic inhibition via β₂ adrenoreceptors and, in such a way, is probably involved in regulation of the inhibition level in the hippocampus. Noradrenaline-induced effects on inhibitory neuronal networks in the hippocampus, similarly to those exerted by several other cerebral neurotransmitter systems, underlie the involvement of the noradrenergic cerebral system in a few physiological processes (emotions, attention, and memory) and are related to some pathological states (Alzheimer’s disease, schizophrenia, epilepsy, etc.).     

Comparison of the Antioxidant Properties and the Toxicity of <Emphasis Type=Italic>p</Emphasis>,<Emphasis Type=Italic>p</Emphasis>′-Dichlorodiphenyl Ditelluride with the Parent Compound,Diphenyl Ditelluride

The hypothesis to be tested in this study is whether the introduction of the chloro group into diphenyl ditelluride molecule (p,p′-dichlorodiphenyl ditelluride, compound 1b) alters the antioxidant and scavenging activity of diphenyl ditelluride (compound 1a) in vitro. The results revealed that 1a and 1b had a potent antioxidant activity in vitro. However, the introduction of a functional group, chloro, into diphenyl ditelluride molecule (1b) did not cause great alterations in the antioxidant action of diphenyl ditelluride against lipid peroxidation, protein carbonyl, and scavenging of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) and 2,2′-diphenyl-1-picrylhydrazyl radicals. Based on the in vitro results, different doses (0.25 and 0.75 μmol/kg) of 1a and 1b or vehicle (canola oil, 1 ml/kg) were administered to rats to investigate if the presence of chloro into diphenyl ditelluride molecule reduces its toxicity. The data demonstrate that the chloro group introduced into diphenyl ditelluride molecule did not alter the acute oral toxicity in rats. The administration of compound 1a in rats only altered the urea level, while compound 1b caused alterations in all toxicological parameters analyzed (alanine aminotransferase and aspartate aminotransferase activities, urea and creatinine levels) in plasma of rats. The results of the present investigation support similar antioxidant and scavenging activities of 1a and 1b in rat liver homogenate in vitro. Furthermore, the presence of chloro into diphenyl ditelluride molecule did not alter the mortality index but increased toxicity of diphenyl ditelluride in rats.     

High-level expression,purification and characterization of a recombinant medium-temperature <Emphasis Type=Italic>α</Emphasis>-amylase from <Emphasis Type=Italic>Bacillus subtilis</Emphasis>

Alpha-amylases are important industrial enzymes with a wide range of applications. Although medium-temperature alpha amylase (AmyE) has some practical advantages, its low yield has limited its applications. When an amyE gene from Bacillus subtilis BF768 was cloned into vector pWB980 and over-expressed in B. subtilis WB600, high activities (723 U ml⁻¹) of secreted AmyE were produced. Recombinant AmyE was purified to a specific activity of 36 U mg⁻¹ having optimal activity at pH 6.0 and 60°C.