Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.     

Recombinant expression of biologically active rat leptin in Escherichia coli

Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat leptin was recombinantly expressed in Escherichia coli and purified in a bioactive form to provide a further tool for the analysis of leptin functions in rats. Leptin cDNA was cloned by RT-PCR from total RNA of SD rat adipocytes, and overexpression was achieved by subcloning the leptin cDNA into the pET-29a vector, which enabled the recombinant expression of rat leptin as an S-peptide-tagged fusion protein. Since the fusion proteins were expressed in inclusion bodies, after purification of the insoluble fraction, leptin proteins were refolded by sequential dialysis into physiological buffers. The biological activity of this recombinant protein was confirmed in proliferation assays using leptin-sensitive rat insulinoma cells as well as a newly developed leptin-sensitive luciferase assay system. The specific binding of the S-tagged leptin to leptin-receptor-expressing cells was further shown by flow cytometry using fluorescence-conjugated S-proteins.     

Caspase cleavage product lacking amino-terminus of IkappaBalpha sensitizes resistant cells to TNF-alpha and TRAIL-induced apoptosis

In response to a diverse array of signals, IkappaBalpha is targeted for phosphorylation-dependent degradation by the proteasome, thereby activating NF-kappaB. Here we demonstrate a role of the cleavage product of IkappaBalpha in various death signals. During apoptosis of NIH3T3, Jurkat, Rat-1, and L929 cells exposed to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas, serum deprivation, or TNF-alpha, respectively, IkappaBalpha was cleaved in a caspase-dependent manner. In vitro and in vivo cleavage assays and site-directed mutagenesis showed that caspase-3 cleaved IkappaBalpha between Asp31 and Ser32. Expression of the cleavage product lacking amino-terminus (1-31), DeltaIkappaBalpha, sensitized otherwise resistant NIH3T3 fibroblast cells to apoptosis induced by TNF-alpha or TRAIL, and HeLa tumor cells to TNF-alpha. DeltaIkappaBalpha was more pro-apoptotic compared to wild type or cleavage-resistant (D31E)IkappaBalpha mutant and the sensitization elicited by DeltaIkappaBalpha was as effective as that by the dominant negative mutant, (S32,36A)IkappaBalpha, in NIH3T3 cells. DeltaIkappaBalpha suppressed the transactivation of NF-kappaB induced by TNF-alpha or TRAIL, as reflected by luciferase-reporter activity. Conversely, expression of the p65 subunit of NF-kappaB suppressed TNF-alpha-, TRAIL-, and serum deprivation-induced cell death. On the contrary, DeltaIkappaBalpha was less effective at increasing the death rate of HeLa cells that were already sensitive to death signals including TRAIL, etoposide, or taxol. These results suggest that DeltaIkappaBalpha generated by various death signals sensitizes cells to apoptosis by suppressing NF-kappaB activity.     

Estimation of vaginal probiotic lactobacilli growth parameters with the application of the Gompertz model

Lactobacilli are widely described as probiotic microorganisms used to restore the ecological balance of different animal or human tracts. For their use as probiotics, bacteria must show certain characteristics or properties related to the ability of adherence to mucosae or epithelia or show inhibition against pathogenic microorganisms. It is of primary interest to obtain the highest biomass and viability of the selected microorganisms. In this report, the growth of seven vaginal lactobacilli strains in four different growth media and at several inoculum percentages was compared, and the values of growth parameters (lag phase time, maximum growth rate, maximum optical density) were obtained by applying the Gompertz model to the experimental data. The application and estimation of this model is discussed, and the evaluation of the growth parameters is analyzed to compare the growth conditions of lactobacilli. Thus, these results in lab experiments provide a basis for testing different culture conditions to determine the best conditions in which to grow the probiotic lactobacilli for technological applications.     

Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells

Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.     

Neuronal Control of Exocytosis and Calcium Homeostasis

We showed that 5 M acetylcholine (ACh) and 100 M norepinephrine (NE) cause increases in the total Ca²⁺ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 M ACh and 100 M NE increased the free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca²⁺-free external solution caused a [Ca²⁺] _i increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca²⁺ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated.     

Characterization of Cyt2Bc toxin from Bacillus thuringiensis subsp. medellin

We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus.     

Arthrospira maxima prevents the acute fatty liver induced by the administration of simvastatin,ethanol and a hypercholesterolemic diet to mice

An evident fatty liver, corroborated morphologically and chemically, was produced in CD-1 mice after five daily doses of simvastatin 75 mg/Kg body weight, a hypercholesterolemic diet and 20 percent ethanol in the drinking water. After treating the animals, they presented serum triacylglycerols levels five times higher than the control mice, total lipids, cholesterol and triacylglycerols in the liver were 2, 2 and 1.5 times higher, respectively, than in control animals. When Arthrospira maxima was given with diet two weeks prior the onset of fatty liver induction, there was a decrement of liver total lipids (40%), liver triacylglycerols (50%) and serum triacylglycerols (50%) compared to the animals with the same treatment but without Arthrospira maxima. In addition to the mentioned protective effect, the administration of this algae, produced a significant increase (45%) in serum high density lipoproteins. The mechanism for this protective effect was not established in these experiments.