New furano- and pyrrolo[2,3-<Emphasis Type="Italic">d</Emphasis>]pyrimidine nucleosides and their 5′-<Emphasis Type="Italic">O</Emphasis>-triphosphates: Synthesis and biological properties

Bicyclic furano[2,3-d]pyrimidine ribonucleosides were synthesized by Pd(0)-and CuI-catalyzed coupling of 5-iodouridine with terminal alkynes. The treatment of the resulting nucleosides with ammonia or methylamine solution in aqueous alcohol resulted in pyrrolo-and N ⁷-methylpyrrolo[2,3-d]pyrimidine nucleosides. 5′-O-Triphosphates of bicyclic nucleosides were obtained by the treatment of the nucleosides with POCl₃ in the presence of a “proton sponge.” The 5′-O-triphosphates are not substrates for HCV RNA-dependent RNA polymerase, but are effective substrates for HCV RNA helicase/NTPase and did not inhibit ATP hydrolysis. Only 3-(β-D-ribofuranosyl)-6-decyl-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one showed a moderate anti-HCV activity in the HCV replicon system and efficiently inhibited replication of bovine viral diarrhea virus (BVDV) in KCT-cells, other compounds being inactive. None of the compounds were cytotoxic within the tested range of concentrations.     

Oligo(2′-<Emphasis Type="Italic">O</Emphasis>-methylribonucleotides) and their derivatives: III. 5′-Mono- and 5′-bispyrenyl derivatives of oligo(2′-<Emphasis Type="Italic">O</Emphasis>-methylribonucleotides) and their 3′-modified analogues: Synthesis and properties

5′-Pyrenylmethylphosphamide and 5′-bispyrenylmethylphosphordiamide derivatives of oligo(2′-O-methylribonucleotides) and their analogues with thymidine attached at their 3′-termini by a 3′-3′-phosphodiester internucleotide bond (“inverted” thymidine) were synthesized. The effect of the pyrene residue(s) on the thermal stability of duplexes of the modified oligonucleotides with RNA and DNA was studied. A possibility of detection of hybridization of 5′-mono- and 5′-bispyrenyl derivatives with RNA and DNA targets in solution was demonstrated according to the changes in fluorescence. 5′-Pyrenylphosphamide derivatives of oligo(2′-O-methylribonucleotides) and their inverted analogues were shown to be used as sensitive probes for the detection of single nucleotide polymorphisms in RNA and DNA by the method of thermal duplex denaturation with fluorescence change registration.     

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.     

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.     

Neurotoxic non-proteinogenic amino acid β-<Emphasis Type=Italic>N</Emphasis>-methylamino-L-alanine and its role in biological systems

Secondary metabolites of photoautotrophic organisms have attracted considerable interest in recent years. In particular, molecules of non-proteinogenic amino acids participating in various physiological processes and capable of producing adverse ecological effects have been actively investigated. For example, the non-proteinogenic amino acid β-Nmethylamino-L-alanine (BMAA) is neurotoxic to animals including humans. It is known that BMAA accumulation via the food chain can lead to development of neurodegenerative diseases in humans such as Alzheimer’s and Parkinson’s diseases as well as amyotrophic lateral sclerosis. Moreover, BMAA can be mistakenly incorporated into a protein molecule instead of serine. Natural sources of BMAA and methods for its detection are discussed in this review, as well as the role of BMAA in metabolism of its producers and possible mechanisms of toxicity of this amino acid in different living organisms.     

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.     

<Emphasis Type=Italic>Pseudomonas seleniipraecipitatus</Emphasis> sp. nov.: A Selenite Reducing γ-<Emphasis Type=Italic>Proteobacteria</Emphasis> Isolated from Soil

Microbial fuel cells (MFCs) are a technology that provides electrical energy from the microbial oxidation of organic compounds. Most MFCs use oxygen as the oxidant in the cathode chamber. This study examined the formation in culture of an unidentified bacterial oxidant and investigated the performance of this oxidant in a two-chambered MFC with a proton exchange membrane and an uncoated carbon cathode. DNA, FAME profile and characterization studies identified the microorganism that produced the oxidant as Burkholderia cenocepacia. The oxidant was produced by log phase cells, oxidized the dye 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), had a mass below 1 kD, was heat stable (121°C) and was soluble in ethanol. In a MFC with a 1000 Ω load and ABTS as a mediator, the oxidizer increased cell voltage 11 times higher than atmospheric oxygen and 2.9 times higher than that observed with ferricyanide in the cathode chamber. No increase in cell voltage was observed when no mediator was present. Organisms that produce and release oxidizers into the media may prove useful as bio-cathodes by improving the electrical output of MFCs.     

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.     

Simple synthesis of <Emphasis Type=Italic>P</Emphasis><Superscript>1</Superscript>-(11-phenoxyundecyl)-<Emphasis Type=Italic>P</Emphasis><Superscript>2</Superscript>-(2-acetamido-2-deoxy-α-<Emphasis Type=Italic>D</Emphasis>-galactopyranosyl) diphosphate

A simple method of the synthesis of P ¹-(11-phenoxyundecyl)-P ²-(2-acetamido-2-deoxy-α-D-galactopyranosyl) diphosphate, which is a synthetic lipid acceptor for glycosyl transferases participating in the biosynthesis of O-antigenic polysaccharides of Gram-negative bacteria, is suggested.     

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.     

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.     

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.     

Identification of a <Emphasis Type=Italic>Rice stripe necrosis virus</Emphasis> resistance locus and yield component QTLs using <Emphasis Type=Italic>Oryza sativa</Emphasis> × <Emphasis Type=Italic>O. glaberrima</Emphasis> introgression lines

Background  

Developing new population types based on interspecific introgressions has been suggested by several authors to facilitate the discovery of novel allelic sources for traits of agronomic importance. Chromosome segment substitution lines from interspecific crosses represent a powerful and useful genetic resource for QTL detection and breeding programs.     

4′,6-Dimethoxyisoflavone-7-<Emphasis Type=Italic>O</Emphasis>-β-<Emphasis Type=SmallCaps>d</Emphasis>-glucopyranoside (wistin) is a peroxisome proliferator-activated receptor α (PPARα) agonist in mouse hepatocytes

Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr kinase family, and is associated with different types of cancer and neurodegenerative diseases. Vanillin is a natural compound, a primary component of the extract of the vanilla bean which possesses varieties of pharmacological features including anti-oxidant, anti-inflammatory, anti-bacterial and anti-tumor. Here, we have investigated the binding mechanism and affinity of vanillin to the CAMKIV which is being considered as a potential drug target for cancer and neurodegenerative diseases. We found that vanillin binds strongly to the active site cavity of CAMKIV and stabilized by a large number of non-covalent interactions. We explored the utility of vanillin as anti-cancer agent and found that it inhibits the proliferation of human hepatocyte carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cells in a dose-dependent manner. Furthermore, vanillin treatment resulted into the significant reduction in the mitochondrial membrane depolarization and ROS production that eventually leads to apoptosis in HepG2 and SH-SY5Y cancer cells. These findings may offer a novel therapeutic approach by targeting the CAMKIV using natural product and its derivative with a minimal side effect.     

Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by <Emphasis Type=Italic>Humicola grisea</Emphasis> var. <Emphasis Type=Italic>thermoidea</Emphasis> grown on sugarcane bagasse

The effect of several carbon sources on the production of mycelial-bound β-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated β-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The β-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50°C, respectively. The purified enzyme was thermostable up to 60 min in water at 55°C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60°C. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-Dgalactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-β-Dgalactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-Dfucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude β-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea β-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.