Activity of native vs. synthetic promoters in Brucella

Brucellosis caused by Brucella species is reportedly the most common zoonotic infection worldwide. The bacterial pathogen is also classified by the Centers for Disease Control and Prevention as a category (B) pathogen that has the potential for development as a bioweapon. Although eight genomes of Brucella have been sequenced, little information is available regarding the regulation of gene expression and promoter activity in Brucella spp. We therefore constructed a set of broad-host-range vectors expressing the lacZ reporter gene from various promoters. Four groups of promoters (Brucella native, antibiotic resistant, bacteriophage and synthetic promoters) were tested in vivo and in vitro in Brucella suis. The highest level of heterologous gene expression was achieved with synthetic hybrid trc promoter carrying the adenine-rich upstream element. Furthermore, this demonstrates the usefulness of synthetic promoters for enhanced level of gene expression in Brucella spp.     

Tempol therapy attenuates medial smooth muscle cell apoptosis and neointima formation after balloon catheter injury in carotid artery of diabetic rats

Accumulating data support the hypothesis that reactive oxygen species (ROS) play a critical role in the vascular complications observed in diabetes. However, the mechanisms of ROS-mediated vascular complications in diabetes are not clear. We tested the hypothesis that ROS-mediated increase in proapoptotic factor Bax expression leads to medial smooth muscle cell (SMC) apoptosis that is associated with neointima formation. We used a fructose-rich diet for 4 wk to model Type 2 diabetes in rats. SOD mimetic membrane-permeable 4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempol, 1 mM) was administered in drinking water to scavenge superoxide starting 1 day before surgery and continued during the duration of the experiment. Vascular injury resulted in a significant increase in medial SMC apoptosis that was associated with neointima formation. The number of medial SMC positive for Bax immunostaining significantly increased in injured arteries compared with uninjured arteries. Superoxide scavenging by Tempol treatment inhibited both the Bax-positive index as well as the apoptotic index of medial SMC in response to vascular injury. Tempol treatment inhibited apoptotic loss of medial SMC, thus increasing their density in the injured arteries. These alterations in the media were associated with a marked decrease in neointima formation in injured arteries. We conclude that Bax expression may play an important role in vascular SMC apoptosis and, finally, that this regulatory mechanism is redox sensitive.     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

Leptin downregulates ethanol-induced secretion of proinflammatory cytokines and growth factor

Inflammatory mediators, including cytokines and growth factors are associated with the pathology of chronic liver diseases. The aim of our present work was to study the effect of exogenous leptin and/or ethanol on the secretion of TNF-alpha, IL-6 and TGF-beta1 both in vivo and in vitro. Forty eight hours after the exposure to ethanol (500 mM) significantly elevated the secretion of TNF-alpha, IL-6 and TGF-beta1 in the cell-free culture supernatant (HepG2 and mouse HCC cell lines), which were decreased on leptin (31.2 nM) treatment. Similarly, leptin administration to ethanol (6.32 g kg(-1) body weight) fed mice for 45 days significantly lowered the concentration of these cytokines in the circulation; however, leptin alone (230 microg kg(-1) body weight i.p. administered every alternate day for the last 15 days) had no such significant effect on cytokine secretion both in vivo and in vitro conditions. We conclude that leptin is involved in the protective mechanisms that allow an organism to cope with the potentially autoaggressive effects of its immune system in alcoholic liver disease.     

Antizyme (AZ) regulates intestinal cell growth independent of polyamines

Since antizyme (AZ) is known to inhibit cell proliferation and to increase apoptosis, the question arises as to whether these effects occur independently of polyamines. Intestinal epithelial cells (IEC-6) were grown in control medium and medium containing 5 mM difluoromethylornithine (DFMO) to inhibit ODC, DFMO + 5 µM spermidine (SPD), DFMO + 5 µM spermine (SPM), or DFMO + 10 µM putrescine (PUT) for 4 days and various parameters of growth were measured along with AZ levels. Cell counts were significantly decreased and mean doubling times were significantly increased by DFMO. Putrescine restored growth in the presence of DFMO. However, both SPD and SPM when added with DFMO caused a much greater inhibition of growth than did DFMO alone, and both of these polyamines caused a dramatic increase in AZ. The addition of SPD or SPM to media containing DFMO + PUT significantly inhibited growth and caused a significant increase in AZ. IEC-6 cells transfected with AZ-siRNA grew more than twice as rapidly as either control cells or those incubated with DFMO, indicating that removal of AZ increases growth in cells in which polyamine synthesis is inhibited as well as in control cells. In a separate experiment, the addition of SPD increased AZ levels and inhibited growth of cells incubated with DFMO by 50 %. The addition of 10 mM asparagine (ASN) prevented the increase in AZ and restored growth to control levels. These results show that cell growth in the presence or absence of ODC activity and in the presence or absence of polyamines depends only on the levels of AZ. Therefore, the effects of AZ on cell growth are independent of polyamines.     

Male accessory gland proteins in Drosophila: a multifaceted field [corrected

Male accessory gland in Drosophila is a secretory tissue of the reproductive system. The proteins synthesized in the accessory gland are tissue specific, stage specific-seen only during the adult stage and sex specific in the sense of male limited expression. These secretions that form a component of the seminal fluid are transferred to the female at the time of copulation and play an important role in reproduction. In conjunction with sperm, these secretory proteins assure reproductive success by reducing the female's receptivity to mating and escalating the rate of egg laying. Some of these proteins are antibacterial in nature with a likely function of protecting the female's genital tract against microbial infection during/after mating. Most of the genes involved in the synthesis of accessory gland proteins are autosomal but a few are still X-linked. Their male specific expression is achieved at the time of sex determination. The level of expression of these genes is dose dependent and they follow Mendelian pattern of segregation. Further, majority of these proteins are rapidly evolving with high rates of non-synonymous substitutions. In this review, by considering the work carried out in different fields, we have tried to generate a comprehensive picture about the male accessory gland and the role of its proteins in the reproduction of Drosophila.     

QTL Mapping of Agronomic Waterlogging Tolerance Using Recombinant Inbred Lines Derived from Tropical Maize (Zea mays L) Germplasm

Waterlogging is an important abiotic stress constraint that causes significant yield losses in maize grown throughout south and south-east Asia due to erratic rainfall patterns. The most economic option to offset the damage caused by waterlogging is to genetically incorporate tolerance in cultivars that are grown widely in the target agro-ecologies. We assessed the genetic variation in a population of recombinant inbred lines (RILs) derived from crossing a waterlogging tolerant line (CAWL-46-3-1) to an elite but sensitive line (CML311-2-1-3) and observed significant range of variation for grain yield (GY) under waterlogging stress along with a number of other secondary traits such as brace roots (BR), chlorophyll content (SPAD), % stem and root lodging (S&RL) among the RILs. Significant positive correlation of GY with BR and SPAD and negative correlation with S&RL indicated the potential use of these secondary traits in selection indices under waterlogged conditions. RILs were genotyped with 331 polymorphic single nucleotide polymorphism (SNP) markers using KASP (Kompetitive Allele Specific PCR) Platform. QTL mapping revealed five QTL on chromosomes 1, 3, 5, 7 and 10, which together explained approximately 30% of phenotypic variance for GY based on evaluation of RIL families under waterlogged conditions, with effects ranging from 520 to 640 kg/ha for individual genomic regions. 13 QTL were identified for various secondary traits associated with waterlogging tolerance, each individually explaining from 3 to 14% of phenotypic variance. Of the 22 candidate genes with known functional domains identified within the physical intervals delimited by the flanking markers of the QTL influencing GY and other secondary traits, six have previously been demonstrated to be associated with anaerobic responses in either maize or other model species. A pair of flanking SNP markers has been identified for each of the QTL and high throughput marker assays were developed to facilitate rapid introgression of waterlogging tolerance in tropical maize breeding programs.