The role of <Emphasis Type="Italic">stj</Emphasis> fimbrial operon in the intestinal persistence of <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium in mice

Salmonella Typhimurium contains 13 operons coding for fimbriae with unique binding specificities to host epithelial surfaces. stj operon is only detected in S. Typhimurium genome suggesting that Stj fimbria may effect serovarspecific virulence characteristics. In this study, the role of stj fimbrial operon in the long-term persistence of S. Typhimurium was identified by competitive infection experiment in genetically resistant mouse (CBA) model system. Knock-out mutation of stjA (major subunit of the Stj fimbria) gene reduced recovery of S. Typhimurium from fecal samples and its colonization to spleen, cecum and mesenteric lymph nodes over a 34-day time period (p < 0.05). This data indicate that stj fimbrial operon has a role in long-term intestinal persistence of S. Typhimurium in CBA mice.     

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Nuclear factor (NF)-κB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-κB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65^−/− murine fibroblasts immortalize at considerably faster rates than RelA/p65^+/+ cells. The ability of RelA/p65^−/− fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65^−/− fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-κB. Altogether, our findings present a fresh perspective on the role of NF-κB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.     

Genotoxic potential of medroxyprogesterone acetate in cultured human peripheral blood lymphocytes

Medroxyprogesterone acetate was studied at three different concentrations (1, 5 and 10 microM), for its genotoxic effects in human peripheral blood lymphocyte culture using chromosomal aberrations and sister chromatid exchanges as parameters. Duplicate peripheral blood cultures were treated with three different concentrations (1, 5 and 10 microM) of medroxyprogesterone acetate. The study was carried out both in the absence as well as in the presence of metabolic activation (S9 mix) with and without NADP. Medroxyprogesterone acetate was found genotoxic at 5 and 10 microM in the presence of S9 mix with NADP. To study the possible mechanism of the genotoxicity of medroxyprogesterone acetate, superoxide dismutase and catalase at different doses were used separately and in combination with 10 microM of medroxyprogesterone at different doses in the presence of S9 mix with NADP. Superoxide dismutase treatment results in an increase of the genotoxic damage but catalase treatment reduce the genotoxic damage of medroxyprogesterone acetate. Catalase treatment in combination with superoxide dismutase also results in the further reduction of the genotoxic damage. The results of the present study reveal that medroxyprogesterone acetate is genotoxic only in the presence of metabolic activation (S9 mix) with NADP. Treatments with superoxide dismutase and catalase suggests the possible generation of reactive oxygen species by redox cycling of various forms of quinones, similar to estrogens, that are the results of aromatic hydroxylation by cytochrome P450s.     

Novel 1-(2-pyrimidin-2-yl)piperazine derivatives as selective monoamine oxidase (MAO)-A inhibitors

In the present study, a new series of 2-[4-(pyrimidin-2-yl)piperazin-1-yl]-2-oxoethyl 4-substituted piperazine-1-carbodithioate derivatives (2a-n) were synthesized and screened for their monoamine oxidase A and B inhibitory activity. The structures of compounds were elucidated using spectroscopic methods and some physicochemical properties of new compounds were predicted using Molinspiration and MolSoft programs. Compounds 2-[4-(pyrimidin-2-yl)piperazin-1-yl]-2-oxoethyl 4-(4-nitrophenyl)piperazine-1-carbodithioate (2j) and 2-[4-(pyrimidin-2-yl)piperazin-1-yl]-2-oxoethyl 4-benzhydrylpiperazine-1-carbodithioate (2m) exhibited selective MAO-A inhibitory activity with IC_50?=_?23.10, 24.14?µM, respectively. Some of the biological results were found in accordance with the obtained in silico data based on Lipinski’s fule of five.     

Role of luteinizing hormone in changes in concentrations of progesterone and luteal blood flow during the hours of a simulated pulse of 13,14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM) in heifers

A bolus treatment (e.g., 25 mg) of prostaglandin F(2alpha) (PGF) in the study of luteolysis in cattle results in dubious interpretations. Therefore, in experiment 1 of the present study, a 13,14-dihydro-15-keto-PGF (PGFM) pulse was simulated by incremental intrauterine (IU) infusion of PGF for 2.7 h on Day 14 postovulation. Concentrations of PGFM during the first hour of infusion and at the maximum were not different between simulated (n = 7) and spontaneous (n = 7) pulses. In experiment 2, four groups (n = 6 per group) were treated at Minute 0 (beginning of infusion) as follows: saline (infused IU), PGF (infused IU), acyline/saline, and acyline/PGF. Two hours before Minute 0, each heifer was given flunixin meglumine to inhibit endogenous PGF secretion, and heifers in the acyline/saline and acyline/PGF groups were given acyline to inhibit luteinizing hormone (LH). Plasma progesterone concentrations were similar among groups during Minutes 0 to 60, with no indication of an initial transient progesterone increase in the two PGF groups. Progesterone began to decrease in the PGF groups at Minute 60 and to rebound at Minute 135 after the PGFM peak at Minute 120. The rebound was complete in association with an increase in LH in the PGF group, but it was not complete when LH was inhibited in the acyline/PGF group. Luteal blood flow increased during PGF infusion in the two PGF groups and remained elevated for approximately 2 h after the PGFM peak in the PGF group but not in the acyline/PGF group. Novel findings were that an initial transient increase in progesterone did not occur with the simulated PGFM pulse and that LH stimulated a progesterone rebound and maintained the elevated luteal blood flow after the PGFM peak.     

Follicle and systemic hormone interrelationships during induction of luteinized unruptured follicles with a prostaglandin inhibitor in mares

The objective was to determine differences in follicle and reproductive hormone characteristics in mares with ovulatory and flunixin meglumine (FM)-induced anovulatory cycles. Estrous mares were given 1500 IU hCG when the follicle was ≥ 32 mm (0 h). In Experiment 1, control mares (n = 7) were not treated further. The remaining mares (n = 11) were given 1.7 mg/kg FM i.v. twice daily, from 0 to 36 h after hCG treatment. Blood samples and ultrasonographic examinations were performed every 12 h. All control mares ovulated normally between 36 and 48 h. In contrast, eight of 11 FM mares did not ovulate, but developed luteinized unruptured follicles (LUFs). Three FM-treated mares did not develop conventional LUFs. Plasma progesterone concentrations were lower (P < 0.05) in LUF mares at 96, 120, and 216 h than in controls, whereas plasma LH concentrations were higher (P < 0.05) between 108 and 120 h in LUF mares than in controls. Plasma concentrations of PGFM and estradiol did not differ significantly between groups. In Experiment 2, the three mares that did not develop LUFs were treated, during the consecutive cycle, with the same dose of FM but with increased frequency at zero, 12, 24, 30, 36, and 48 h after hCG. One mare formed a LUF, whereas the other two did not. These two mares had lower LH concentrations than LUF or control mares in the two consecutive cycles. In conclusion, systemic treatment with FM blocked ovulation in 73% of treated mares. Mares with LUFs had lower progesterone and higher LH concentrations than control mares.     

Effect of Plumbago zeylanica extract and certain curing agents on multidrug resistant bacteria of clinical origin

Alcoholic extract of Plumbago zeylanica (root) was tested against multidrug-resistant clinical isolates of bacteria (Salmonella paratyphi, Staphylococcus aureus, Escherichia coli, Shigella dysenteriae and a R-plasmid-harbouring standard strain, E.coli x⁺). The extract exhibited strong antibacterial activity against all test bacteria irrespective of their antibiotic resistance behaviour. Phytochemical analysis of crude extract revealed the presence of flavonoids, saponins and naphthoquinone. A comparative evaluation of R-plasmid elimination from E. coli x⁺ (pUK 651) by the plant extract, DNA intercalating dyes (acridine orange and ethidium bromide) and a DNA gyrase antagonizing drug (pefloxacin) were made. All these agents could cure R-plasmid effectively at their respective sub-MIC concentrations. Maximum plasmid curing was observed by pefloxacin (88%), followed by ethidium bromide (36%), acridine orange (14%) and alcoholic extract of P. zeylanica (14%). Curing of plasmid pUK651 from E. coli x⁺ was confirmed by determining the loss of resistance markers in the cured derivative culture. This revised version was published online in November 2006 with corrections to the Cover Date.     

The primary structure of alpha-lactalbumin from camel milk

The primary structure of camel alpha-lactalbumin was determined by analysis of the intact protein, and of CNBr fragments and enzymatic peptides from the carboxymethylated protein chain. Results show that camel alpha-lactalbumin has 123 residues and a molecular mass of 14.6 kDa. The amino acid sequence is strictly homologous to alpha-lactalbumins characterized, but also exhibits extensive differences: 39 residues differ in relation to the bovine protein and only 35 residues are conserved among hitherto known alpha-lactalbumins with characterized structures. All residues ascribed critical structural or functional roles are strictly invariant in the camel protein.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.