Structural characterization of Botryosphaeran: a (1-->3;1-->6)-beta-D-glucan produced by the ascomyceteous fungus,Botryosphaeria sp

The exopolysaccharide, Botryosphaeran, produced by the ligninolytic, ascomyceteous fungus Botryosphaeria sp., was isolated from the extracellular fluid by precipitation with ethanol, and purified by gel permeation chromatography to yield a carbohydrate-rich fraction (96%) composed mainly of glucose (98%). Infra-red and 13C NMR spectroscopy showed that all the glucosidic linkages were in the beta-configuration. Data from methylation analysis and Smith degradation indicated that Botryosphaeran was a (1-->3)-beta-D-glucan with approx 22% side branching at C-6. The products obtained from partial acid hydrolysis demonstrated that the side branches consisted of single (1-->6)-beta-linked glucosyl, and (1-->6)-beta-linked gentiobiosyl residues.     

Peptide inhibition of cytokine-stimulated aromatase activity in breast tissue fibroblasts

The cytokine interleukin-6 (IL-6) and its soluble receptor (IL-6sR) can act synergistically to stimulate aromatase activity in cultured stromal fibroblasts derived from breast tissues. In this study, a 16 amino acid peptide, AROHIB, has been used in an attempt to block the ability of IL-6 plus IL-6sR to stimulate aromatase activity in stromal fibroblasts. Pre-incubation of cells with AROHIB for a 3-h period before the addition of IL-6 and IL-6sR resulted in a marked (67%) reduction in the ability of these factors to stimulate aromatase activity. AROHIB was found to be rapidly degraded when exposed to MCF-7 breast cancer cells or fibroblasts. Analysis by FAB-MS was used to identify the site of peptide cleavage. Subsequently, a series of 10 amino acid peptides, DP1-DP4, were designed, synthesised and tested for their ability to resist proteolytic degradation and to inhibit IL-6 plus IL-6sR-stimulated aromatase activity. Peptide DP2, a modified version of the active fragment of AROHIB, had N-acetyl and C-amino terminal protection and an internal D-amino acid (instead of L form) at the site of proteolytic cleavage. Using cells cultured in the presence of 2% stripped foetal calf serum, peptide DP2 resulted in a 74% reduction in cytokine-stimulated aromatase activity. Under serum-free conditions, peptides DP1-DP3 showed modest inhibitory properties. Results from this study suggest that it may be possible to develop small peptides to inhibit cytokine-stimulated aromatase activity in a tissue-specific manner.     

A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice

A -lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3 region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3 end, with BLG sequences of exon 6/intron 6/exon 7 and the 3 region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3 flanking sequences. The BLG 5 regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.     

Overexpression and purification of Helicobacter pylori flavodoxin and induction of a specific antiserum in rabbits

Flavodoxin from the gastric pathogen Helicobacter pylori has been shown to be the electron acceptor of the essential pyruvate-oxidoreductase enzyme complex and proposed to be involved in the pathogenesis of gastric MALToma. In order to obtain a sufficient amount for biochemical and structural studies, we overexpressed the protein either with a C-terminal His(6) -tag or as a fusion protein upstream of intein- and chitin-binding domains. With both expression systems we succeeded at purifying soluble and functional flavodoxin containing the cofactor FMN. When expressing with a His(6) -tag, we purified approximately 20 mg flavodoxin per liter of bacterial culture, while expression as an intein-CBD fusion protein with autocatalytic removal of the intein-CBD part rendered only approximately 1 mg of purified flavodoxin per liter of bacterial culture. Expressed as an intein-CBD fusion protein, flavodoxin copurified with a C-terminal degradation product, which was not observed for expression with a His(6) -tag. However, we were able to obtain protein crystals suited for X-ray structure determination from flavodoxin expressed as an intein-CBD fusion protein, but not from flavodoxin expressed with a C-terminal His(6) -tag. We further report the induction of a rabbit antiserum specific for H. pylori flavodoxin.     

Chaperone-Assisted Overexpression of an Active -Carbamoylase from Agrobacterium tumefaciens AM 10

The N-carbamoyl- -amino acid amidohydrolase ( -carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 was cloned by polymerase chain reaction in plasmid pET28a and was overexpressed in Escherichia coli JM109 (DE3). However, almost 80% of the enzyme remained trapped in inclusion bodies. To facilitate the expression of the properly folded active enzyme, the chaperones GroEL/ES were coexpressed in plasmid pKY206. This resulted in a 43-fold increase in active enzyme production compared to the wild-type strain. The histidyl-tagged -carbamoylase was purified by a single step nickel-affinity chromatography to a specific activity of 9.5 U/mg protein.     

Down-regulation of particulate protein kinase Cepsilon and up-regulation of nuclear activator protein-1 DNA binding in liver following in vivo exposure of B6C3F1 male mice to heptachlor epoxide

The effects of in vivo administration of the cyclodiene tumor promoter heptachlor epoxide on mouse liver protein kinase C were studied in male B6C3F1 mice by protein kinase C activity assays and Western blotting under conditions known to increase the incidence of hepatocellular carcinoma because protein kinase C is thought to be critical in phorbol ester-induced tumor promotion. Under these test conditions, 20 ppm dietary heptachlor epoxide for 1-20 days increased cytosolic and decreased particulate total protein kinase C activities, while 10 ppm had no effect. Further, total cytosolic and particulate protein kinase C activities were decreased within 1 hour by 10 mg/kg intraperitoneal (i.p.) heptachlor epoxide. Western blotting showed that conventional protein kinase Calpha and beta isoforms were unaffected by heptachlor epoxide. Particulate novel protein kinase Cepsilon, however, was selectively down-regulated by 1, 10, and 20 ppm dietary heptachlor epoxide, whereas the cytosolic isoform was decreased by 1 and 10 ppm heptachlor epoxide for 10 days. The high-dose treatment for 24 hours also decreased particulate novel protein kinase Cepsilon but increased the cytosolic titer. These results demonstrate that this isoform is unique in its sensitivity to heptachlor epoxide. Activator protein-1 DNA binding, a critical factor in tumor promotion, was substantially increased at 3 and 6 hours with 3.7 mg/kg (i.p.) heptachlor epoxide and at 3 and 10 days with 20 ppm dietary heptachlor epoxide. The effects of heptachlor epoxide on protein kinase C and activator protein-1 are similar to those caused by phorbol ester treatments and correlate well to heptachlor levels found to induce tumors in mice. However, heptachlor epoxide did not initially activate protein kinase C with in vivo treatments or with in vitro treatments of a plasma membrane fraction aimed at demonstrating direct activation, as has been shown for phorbol esters. The ability of heptachlor epoxide to down-regulate particulate novel protein kinase Cepsilon correlates to dosages used in in vivo tumor promotion studies. However, this may represent a negative feedback response rather than a causative effect.     

Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP⁺/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, K _m for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg²⁺ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots. Received: 21 June 2000 / Accepted: 28 July 2000     

Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the -glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

One step purification of glucose-6-phosphate dehydrogenase from brain areas by immunoaffinity chromatography

Glucose-6-phosphate dehydrogenase was purified from rabbit brain cortex using a single immunoaffinity chromatographic step and was contaminated only by a 50 kDa protein. The proteins, separated by SDS-PAGE, were sequenced: the glucose-6-phosphate dehydrogenase was blocked at the N-terminal, the co-eluted protein was similar to -tubulin. Our technique can be applied to purification and sequencing of the enzyme from brain areas or to measure its turnover rate in cultured cells.