Biosynthesis and localization of glutamylendopeptidase from Bacillus intermedius strain 3-19

The biosynthesis of glutamylendopeptidase from Bacillus intermedius strain 3-19 and localization of the enzyme in the bacterial cells was studied. The synthesis of the enzyme was suppressed by easily metabolizable carbon sources. Inorganic phosphate and NH4+ ions stimulated the production of glutamylendopeptidase. Complicated organic substrates such as casein, gelatine, and haemoglobin did not affect the biosynthesis of the enzyme. The divalent metallic ions Ca2+, Mg2+, Co2+ increased the production of glutamylendopeptidase while Zn2+, Cu2+, and Fe2+ reduced the biosynthesis of proteinase. The rate of synthesis of the enzyme increased when the rate of the bacterial growth decreased. The maximum enzyme activity in the culture fluid was determined at the stationary phase of growth. In the cells glutamylendopeptidase was bound to the cytoplasmic membrane, and the maximal enzyme activity was detected in the stationary growth phase. The results facilitated the development of a medium which yielded the maximum glutamylendopeptidase production by B. intermedius strain 3-19.     

RNA-hydrolyzing activity of human serum albumin and its recombinant analogue

The comparative analysis of RNA-hydrolyzing activity of albumin from human serum and albumin expressed in methylotrophic yeast Pichia pastoris has been carried out. The rate of polyribonucleotide phosphodiester bond cleavage in the presence of recombinant albumin has been found to be similar to that of the reaction mediated by the native protein. According to ³¹P NMR data, RNA hydrolysis follows the mechanism of intermolecular trans-esterification to yield 2′,3′-cyclophosphodiester reaction products that are further slowly hydrolyzed to form nucleoside-3′- and nucleoside-2′-phosphates. Analysis of pH dependence suggests an acid–base mechanism of catalysis. The catalytic activity and substrate specificity of albumin in RNA hydrolysis distinguish it from human ribonucleases.     

Human serum albumin as a catalyst of RNA cleavage: N-homocysteinylation and N-phosphorylation by oligonucleotide affinity reagent alter the reactivity of the protein

Kinetic parameters for the cleavage of UpA site in an oligonucleotide in the presence of human serum albumin (HSA) or one of its clinically relevant modification were measured. The RNA-hydrolyzing activity of HSA was decreased by its nonenzymatic N-homocysteinylation. According to (31)P NMR data, Lys and Tyr residues were the labeling targets when a phosphorylating analog of oligoribonucleotide substrate was employed. The site of tyrosine modification was slowly dephosphorylated. Lys-directed affinity labeling suppressed oligonucleotide cleavage indicating that lysines took part in the reaction.     

Proteolytic cleavage of pertussis toxin S1 subunit is not essential for its activity in mammalian cells

Background  

Pertussis toxin (PT) is an exotoxin virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1) that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2–S5) that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity.     

Length regulation and dynamics of individual telomere tracts in wild-type Arabidopsis

Although length of the telomeric DNA tract varies widely across evolution, a species-specific set point is established and maintained by unknown mechanisms. To investigate how telomere length is controlled in Arabidopsis thaliana, we analyzed bulk telomere length in 14 wild-type accessions. We found that telomere tracts in Arabidopsis are fairly uniformly distributed throughout a size range of 2 to 9 kb. Unexpectedly, telomeres in plants of the Wassilewskija ecotype displayed a bimodal size distribution, with some individuals harboring telomeres of 2 to 5 kb and others telomeres of 4 to 9 kb. F1 and F2 progeny of a cross between long and short telomere parents had intermediate telomeres, implying that telomere length in Arabidopsis is not controlled by a single genetic factor. We provide evidence that although global telomere length is strictly regulated within an ecotype-specific range, telomere tracts on individual chromosome ends do not occupy a predetermined length territory. We also demonstrate that individual telomere tracts on homologous chromosomes are coordinately regulated throughout development and that telomerase acts preferentially on the shortest telomeres. We propose that an optimal size for telomere tracts is established and maintained for each Arabidopsis ecotype.     

Medium for the production of Bacillus intermedius glutamyl endopeptidase by the recombinant Bacillus subtilis

A nutrient medium was elaborated for the efficient production of glutamyl endopeptidase by the recombinant Bacillus subtilis strain AJ73 bearing the Bacillus intermedius 3-19 glutamyl endopeptidase gene within a multicopy plasmid. Optimal concentrations of the main nutrients, peptone and inorganic phosphate, were found using a multifactor approach. To provide for active growth and efficient glutamyl endopeptidase production, the cultivation medium of the recombinant strain should be enriched in phosphorus, organic and inorganic nitrogen sources, and yeast extract. Complex protein substrates, such as casein and gelatin, enhanced the biosynthesis of glutamyl endopeptidase. At the same time, easily metabolizable carbon sources suppressed it. The production of glutamyl endopeptidase was stimulated by the bivalent cations Ca2+, Mg2+, and Co2+.     

Synthetic transformations of higher terpenoids. XXVI. 16-Acetylaminomethyllabdanoids and theirs cytotoxicity

Condensation of methyl 16-aminomethyllambertianate with N-Boc-omega-amino acids leads smoothly to 16-(N-Boc-aminononan)- and 16-(N-Boc-aminoundecan)amidomethyllabdanoids. The amide of bicyclo[2.2.1]heptan-1,2-dicarbocylic acid with a labdanoid substituent was obtained under the reaction of methyl aminomethyllambertianate with bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic anhydride. Intereaction of methyl 16-aminomethyllambertianate with chloroacetyl chloride leads to methyl 16-(chloroacetylaminomethyl)lambertianate; condensation of this compound with amino acid methyl ethers the corresponding amides of methyl lambertianate was obtained. The resulting compounds are more (compared with lambertianic acid) cytotoxicity in the cell lines CEM-13, MT-4 and U-937 with an CCID50 concentration of 3.9-9.9 microM.     

Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

Background  

MicroRNAs (miRNAs) are small regulatory RNAs, some of which are conserved in diverse plant genomes. Therefore, computational identification and further experimental validation of miRNAs from non-model organisms is both feasible and instrumental for addressing miRNA-based gene regulation and evolution. Sugarcane (Saccharum spp.) is an important biofuel crop with publicly available expressed sequence tag and genomic survey sequence databases, but little is known about miRNAs and their targets in this highly polyploid species.