<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.     

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.     

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.     

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.     

Decolourisation and dephenolisation potential of selected <Emphasis Type=Italic>Aspergillus</Emphasis> section <Emphasis Type=Italic>Nigri</Emphasis> strains – <Emphasis Type=Italic>Aspergillus tubingensis</Emphasis> in olive mill wastewater

Aspergillus section Nigri strains Aspergillus aculeatus Ege-K 258, A. foeditus var. pallidus Ege-K156, A. niger Ege-K 4 and A. tubingensis Ege-K 265 were used to treat olive mill wastewater (OMW) in an investigation aimed at exploring their dephenolisation and decolourisation ability and, consequently, the economic feasibility of using any or all of these strains in a pre-treatment step in the processing of OMW. Of these strains A. tubingensis Ege-K 265 resulted in an 80% decolourisation of twofold-diluted OMW and a 30% decolourisation of undiluted OMW; in addition, it was able to remove approximately 30% of all phenolic compounds in both twofold-diluted and undiluted OMW. We conclude that A. tubingensis Ege-K 265 could be effectively used in the pre-treatment step of a combined aerobic-anaerobic process to solve the environmental problems caused by OMW in Mediterranean countries.     

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.     

Peroxidase,acid phosphatase,RNase and DNase activity and isoform patterns during <Emphasis Type=Italic>in vitro</Emphasis> rooting of <Emphasis Type=Italic>Petunia</Emphasis> × <Emphasis Type=Italic>hybrida</Emphasis> microshoots

Specific activities and isoform patterns of peroxidases, acid phosphatases, DNases and RNases were studied in relation to in vitro rooting of Petunia × hybrida microshoots in the presence of 4 μM indole-3-butyric acid (IBA). Specific activities of the above enzymes increased in the course of rooting. Rhizogenesis could be related with an increased specific activity of peroxidases during the initiation phase, in parallel with increased lignin content. Twelve peroxidases, six anionic (A1–A6) and six cationic (C1–C6), seven acid phosphatases (ACP1–ACP7), seven RNases (R1–R7) and four DNases (D1–D4) isoforms were detected following native PAGE. Variation in the number of the above isoforms and their quantity was observed during different stages of rooting. Particularly, A2, A3, C3, C4, C5, ACP2, R1, R2, R3, and D4 isoforms appeared after the induction phase and could be related to emergence of root primordia. Additionally, R3 and D4 could be associated with cell division and differentiation, since these are only expressed in rooted microshoots. Moreover, the higher number of roots in IBA-treated microshoots could be related to the higher expression of RNase and DNase isoforms during initiation and expression phases.     

Antimutagenic effect of lectin and <Emphasis Type=Italic>N</Emphasis>-methyl-<Emphasis Type=Italic>N’</Emphasis>-nitro-<Emphasis Type=Italic>N</Emphasis>-nitrosoguanidine on induced mutagenesis in mammalian cells in vitro

Factors and ways in which macromolecules influence the mutation process are considered. An antimutagenic effect is demonstrated in a study of the combined influence of lectins and the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) on mutagenesis in Chinese hamster cells. Thus, in different schemes of treatment of cells with albumin and MNNG, the experimental frequency of mutations induced by the two agents was statistically reliably lower than the theoretically expected level for their independent combined action. The possibility that there exist common targets and mechanisms through which different types of mutagenic agents act on the cell DNA is discussed.     

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.     

Synthesis and use of <Emphasis Type=Italic>N</Emphasis>,<Emphasis Type=Italic>N</Emphasis>-di-Boc-glutamate γ-semialdehydes and related aldehydes

Summary.  This review article focuses on the synthesis and reactions of N,N-di-Boc glutamate and aspartate semialdehydes as well as related aldehydes. These building blocks are prepared according to various strategies from glutamic and aspartic acids and find interesting synthetic applications. In the first part, the methods for the synthesis of N,N-di-Boc-amino aldehydes are summarized. The applications of these chiral synthons for the synthesis of unnatural amino acids and other bioactive compounds are discussed in the second section. Received April 24, 2002 Accepted August 13, 2002 Published online January 30, 2003 Authors' address: Prof. Violetta Constantinou-Kokotou, Chemical Laboratories, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece, E-mail: vikon@aua.gr Abbreviations: AcNH-TEMPO, 4-acetamido-2,2,6,6-tetramethyl-1-piperidinyloxy free radical; AIBN, 2,2′-azobis(2-methylpropionitrile); Aliquat, methyltrioctylammonium chloride; Bn, benzyl; Boc, tert-butoxycarbonyl; Bu^t, tert-butyl; m-CPBA, 3-chloroperoxybenzoic acid; DAST, diethylaminosulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; (R,R)-(+)-DET, (R,R)-(+)-diethyltartrate; DIBALH, diisobutyl aluminium hydride; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; Et₃N, triethylamine; KHMDS, potassium bis(trimethylsilyl)amide; (S)-LLB, lanthanium-lithium-bis-metallic binaphthol catalyst; MsCl, methanesulfonyl chloride; NEM, N-ethylmorpholine; NMO, 4-methylmorpholine N-oxide; PPA, propylphosphonic acid anhydride; TBHP, tert-butyl hydroperoxide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSI, 1-(trimethylsilyl)imidazole; Trt, trityl.     

Proteolysis of <Emphasis Type=Italic>α</Emphasis>-amylase from <Emphasis Type=Italic>Saccharomycopsis fibuligera</Emphasis>: characterization of digestion products

α-Amylase from Saccharomycopsis fibuligera R-64 was successfully purified by butyl Toyopearl hydrophobic interaction chromatography, followed by Sephadex G-25 size exclusion and DEAE Toyopearl anion exchange chromatography. The enzyme has a molecular mass of 54 kDa, as judged by SDS PAGE analysis. Upon tryptic digestion, two major fragments with relative molecular masses of 39 kDa and 10 kDa, which resemble the A/B and C-terminal domains in the homologous Taka-amylase, were obtained and were successfully separated with the Sephadex G-50 size exclusion column. The 39-kDa fragment demonstrated a similar amylolytic activity to that of the undigested enzyme. However, it was found that the K _m value of the 39-kDa fragment was about two-times higher than that of the undigested enzyme. Moreover, thermostability studies showed a lower half-life time for the 39-kDa fragment. These findings suggest that the 39-kDa fragment is the catalytic domain, while the 10-kDa fragment is the C-terminal one, which plays a role in thermostability and starch binding. Although the undigested enzyme is able to act on raw starches at room temperature, with maize starches as the best substrate, neither the undigested enzyme nor the fragments adsorb the tested raw starches. These results propose Saccharomycopsis fibuligera α-amylase as a raw starch-digesting but not adsorbing amylase, with a similar domain organization to that of Taka-amylase A.     

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.     

Mapping species differences for adventitious rooting in a <Emphasis Type=Italic>Corymbia torelliana</Emphasis> × <Emphasis Type=Italic>Corymbia citriodora</Emphasis> subspecies <Emphasis Type=Italic>variegata</Emphasis> hybrid

Quantitative trait loci (QTL) detection was carried out for adventitious rooting and associated propagation traits in a second-generation outbred Corymbia torelliana × Corymbia citriodora subspecies variegata hybrid family (n = 186). The parental species of this cross are divergent in their capacity to develop roots adventitiously on stem cuttings and their propensity to form lignotubers. For the ten traits studied, there was one or two QTL detected, with some QTL explaining large amounts of phenotypic variation (e.g. 66% for one QTL for percentage rooting), suggesting that major effects influence rooting in this cross. Collocation of QTL for many strongly genetically correlated rooting traits to a single region on linkage group 12 suggested pleiotropy. A three locus model was most parsimonious for linkage group 12, however, as differences in QTL position and lower genetic correlations suggested separate loci for each of the traits of shoot production and root initiation. Species differences were thought to be the major source of phenotypic variation for some rooting rate and root quality traits because of the major QTL effects and up to 59-fold larger homospecific deviations (attributed to species differences) relative to heterospecific deviations (attributed to standing variation within species) evident at some QTL for these traits. A large homospecific/heterospecific ratio at major QTL suggested that the gene action evident in one cross may be indicative of gene action more broadly in hybrids between these species for some traits.     

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.