pH-dependent modulation of alkaline phosphatase activity in Serratia marcescens

Serratia marcescens is an opportunistic pathogen responsible for causing nosocomial infections, corneal ulcer, necrotizing fasciitis, cellulites, and brain abscess. Alkaline phosphatase (APase) is believed to play an important role in the survival of several intracellular pathogens and their adaptation. We have studied the effect of low phosphate concentration and acid pH on the APase activities of S. marcescens. In a low phosphate medium, some strains of S. marcescens synthesize two different types of APases, a constitutive (CAPase) and an inducible (IAPase). Both the CAPase and IAPase isoenzymes completely lost their enzyme activities at pH 2.3, within 10 min of incubation at 0°C. Acid-treated IAPase isoenzymes I, II, III, and IV solutions when adjusted to pH 7.8 showed recovery of 70%, 52%, 72%, and 60% of the lost activities, respectively. When the pH of the CAPase reaction mixture was raised to pH 7.8, the enzyme activity regained only 5% of its initial activity. Variations in protein concentration also affected the pH-dependent reversible changes of the IAPase activity. The higher the protein concentration, the faster the inactivation of enzyme activity observed at acidic pH at 0°C. Conversely, the lower the protein concentration, the higher the rate of reactivation of enzyme activity observed for IAPase at alkaline pH. Protein interaction studies revealed a lack of similarity between CAPase and IAPase, suggesting separate genetic origin of these potentially virulent genes of S. marcescens. Received: 4 December 2001 / Accepted: 7 January 2002     

A Prospective Analysis of Co-Processed Non-Ionic Surfactants in Enhancing Permeability of a Model Hydrophilic Drug

Paracellular route is a natural pathway for the transport of many hydrophilic drugs and macromolecules. The purpose of this study was to prospectively evaluate the ability of novel co-processed non-ionic surfactants to enhance the paracellular permeability of a model hydrophilic drug metformin using Caco-2 (human colonic adenocarcinoma) cell model. A three-tier screen was undertaken to evaluate the co-processed blends based on cytotoxicity, cellular integrity, and permeability coefficient. The relative contribution of the paracellular and the transcellular route in overall transport of metformin by co-processed blends was determined. Immunocytochemistry was conducted to determine the distribution of tight-junction protein claudin-1 after incubation with the co-processed blends. It was found that novel blends of Labrasol and Transcutol-P enhanced metformin permeability by approximately twofold with transient reduction in the transepithelia electrical resistance (TEER) and minimal cytotoxicity compared with the control, with the paracellular pathway as the major route of metformin transport. Maximum permeability of metformin (～10-fold) was mediated by Tween-20 blends along with >75% reduction in the TEER which was irreversible over 24-h period. A shift in metformin transport from the paracellular to the transcellular route was observed with some Tween-20 blends. Immunocytochemical analysis revealed rearrangement of the cellular borders and fragmentation on treatment with Tween-20 blends. In conclusion, cytotoxicity, cellular integrity, and permeability of the hydrophilic drugs can be greatly influenced by the polyoxyethylene residues and medium chain fatty acids in the non-ionic surfactants at clinically relevant concentrations and therefore should be thoroughly investigated prior to their inclusion in formulations.     

Marine sponge polyketide inhibitors of protein tyrosine kinase.

The marine polyketide natural product, halenaquinone, was shown to be an irreversible inhibitor of pp60v-src, the oncogenic protein tyrosine kinase encoded by the Rous sarcoma virus. This compound had an IC50 of approximately 1.5 microM against pp60v-src and also inhibited the ligand-stimulated kinase activity of the human epidermal growth factor receptor with an IC50 of approximately 19 microM. Halenaquinone blocked the proliferation of a number of cultured cell lines, including several transformed by oncogenic protein tyrosine kinases. Halenaquinol, xestoquinone, halenaquinol sulfate, and several simple synthetic quinone analogs were also shown to inhibit pp60v-src.     

Correlation between Resistin,Tuberculosis and Khat Addiction: A Study from South Western Province of Saudi Arabia

Tuberculosis(TB) is a disease of global significance, which accounts for a death in every 15 seconds. Recent studies shows TB is rising in certain parts of the world, and Saudi Arabia is one of them. Several factor contribute in predisposing the subjects for infection including but not limited to addiction to various compounds which have immune modulation properties, such as amphetamines and Heroin etc. Khat a plant whose leaves are chewed for its euphoric effect in east Africa and Arabian Peninsula including Saudi Arabia, is considered as mildly addictive, and its principle compound, Cathinone shares structural and functional similarity with amphetamine a known immunomodulator. Tuberculosis being a disease of immune modulation has a varied spectrum of complex interplay of proinflammatory molecules, resistin is one of them. In the present study, we try to explore the trinity of khat addiction, serum resistin level and tuberculosis by correlating the serum resistin level in non khat addicted healthy subjects, khat addicted healthy subjects, and in patients, both khat addicted and non khat addicted, with active tuberculosis. We observed significantly higher resistin level among the apparently healthy khat addicted subjects as compared to non addicted healthy controls. Thereafter, when we compare the resistin levels between khat addicted and non khat addicted TB patients we did not found significant difference between the two groups. However bacillary load was observe to be significantly higher among the khat addicted TB patient as compare to non addicted one. Validation of above results in animal model revealed dose dependant increase in bacillary growth in the Wistar rats treated with khat. Taken together these results suggest the role of khat in immune modulation albeit in the limited frame of resistin level.     

Lactic acid stimulates interleukin-23 production by peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide

Lactic acid is the predominant acid present in the vagina. We evaluated the consequences of lactic acid, at physiological levels present in the vagina, on cytokine responses of peripheral blood mononuclear cells (PBMCs) obtained from 10 individuals in the presence or absence of bacterial lipopolysaccharide. Preincubation of PBMCs in 15 mM lactic acid before the addition of lipopolysaccharide resulted in a 246% mean increase in interleukin-23 (IL-23) secretion over that released in the presence of lipopolysaccharide alone (P=0.0068). The lipopolysaccharide-induced production of tumor necrosis factor-α, IL-6, IL-10 and IL-12 was unaffected by lactic acid. IL-23 stimulation was not observed if the lactic acid was neutralized before its addition to the culture medium or if hydrochloric acid was substituted for lactic acid. In the absence of lipopolysaccharide, lactic acid did not stimulate the production of IL-23 or any of the other cytokines. The increase in IL-23 production was proportional to the lactic acid concentration over a 15-60 mM range. We conclude that at body sites characterized by lactic acid accumulation, such as in the human vagina, exposure to gram-negative bacteria results in selective IL-23 production, leading to a subsequent preferential stimulation of the Th17 T lymphocyte pathway.     

Development and characterization of a novel fusion protein composed of a human IgG1 heavy chain constant region and a single-chain fragment variable antibody against Venezuelan equine encephalitis virus

Murine monoclonal antibody 1A4A1 has been shown to recognize a conserved neutralizing epitope of envelope glycoprotein E2 of Venezuelan equine encephalitis virus. It is a potential candidate for development of a second generation antibody for both immunodiagnosis and immunotherapy. In order to minimize the immunogenicity of murine antibodies and to confer human immune effector functions on murine antibodies, a recombinant gene fusion was constructed. It encoded a human IgG1 heavy chain constant region and a single-chain fragment variable antibody of 1A4A1. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The recombinant soluble antibody was demonstrated to retain high antigen-binding affinity to Venezuelan equine encephalitis virus and to possess some human IgG crystallizable fragment domain functions, such as recognition by protein G and human complement C1q binding. On non-reducing and reducing gel electrophoresis analysis of proteolytic fragments of the recombinant antibody, disulfide bond formation was found in the hinge region of the antibody. From these data, it was concluded that the recombinant antibody was capable of antigen recognition, and retained several functional activities. This work forms the basis for characterization of the recombinant antibody as to efficacy in vivo.     

Role of pollination in yield and physicochemical properties of tomatoes (Lycopersicon esculentum)

Very little is known about pollination and its effects on the yield and physicochemical properties of flowering plants in tropical countries. Wind and insect pollinators are among our natural resources because pollination is the most important ecosystem service performed by wind and insects, and is vital to the socio-economic status of human beings. In this experiment, different pollination methods for tomato plants were examined. Self-pollination was encouraged by covering the plants with a plastic sheet. Wind and insects were excluded from these plants, and thus only self-pollination was possible. The experiment occurred during the flowering stage. Wind-pollinated plants were covered with a muslin cloth, which excluded insects, and only wind could pass through the cloth. For insect pollination, plants remained uncovered, allowing free access to insects to pollinate the flowers. At fruit maturity, when fruits were completely red, fruits from each treatment were harvested on the same date and under the same conditions. Results illustrated the substantial importance of insects as pollinators of tomato crops. Open field had greater tomato yield and positive effects on physicochemical properties on fruit than under self and wind pollination.     

In situ end labelling of DNA to detect apoptotic cell death in a variety of human tumours

The present studies illustrate clinical applications of in situ end labelling (ISEL) of DNA to detect apoptosis in a variety of human malignancies including myelodysplastic syndromes (MDS, n=10), non-Hodgkin's lymphoma (NHL, n=10), head and neck cancer (n = 3), breast cancer (n = 1) and cervical cancer (n = 1). These studies also describe a new in situ double labelling technique to detect apoptosis and proliferation (S-phase cells) simultaneously in the same section of plastic embedded tissue. In vivo intravenous infusions of thymidine analogues (i.e. bromodeoxyuridine (BrdU) and/or iododeoxyuridine (IUdR)) followed by their detection with a specific monoclonal antibody in a plastic embedded biopsy, combined with ISEL in the same section, facilitated simultaneous estimations of apoptosis and proliferation. The most salient finding of these studies was excessive apoptosis in MDS including the cells in S-phase as indicated by uniquely double labelled cells in their bone marrow biopsies. On the other hand, a very low degree of apoptosis was observed in NHL and other solid tumours. Moreover, the solid tumours exhibited definite compartments of apoptosis and proliferation. Further experiments are underway to confirm these findings in a larger study in order to design appropriate therapeutic modalities for these disorders.     

Interaction of quercetin with DNA

I n vitro experiments to study interaction of the mutagenic flavonoid quercetin with DNA are described. Calf thymus DNA treated with quercetin for various time periods was subjected to S₁ nuclease hydrolysis. Thermal melting profles of treated DNA were also determined using St nuclease. The rate of DNA hydrolyzed after 1 hr of pre-treatment with quercetin was found to be only about 50% of that in its absence. However, after 10 and 24hrs of treatment with the drug, the rate of S₁ nuclease hydrolysis was observed to be greater than that of native DNA. Thermal melting profiles of DNA, treated with quercetin for 10 and 24 hrs, indicated a slight decrease in melting temperatures. Gel filtration of native DNA, which had been digested with S₁ nuclease after preincubation with quercetin for 24 hrs, indicated the production of various sized degraded molecules. The results suggest that the initial interaction of quercetin with DNA may have a stabilizing effect on its secondary structure, but prolonged treatment leads to an extensive disruption of the double helix.     

Inhibition of repair of O-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells

The pro-mutagenicity of chemically-induced methylation of DNA at the O⁶ position of dexoyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O⁶-methyldeoxyguanosine (O⁶-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O⁶-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O⁶-methylguanine (O⁶-meG). Exposure of cells to 0.6 mM O⁶-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O⁶-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O⁶-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced O[su6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O⁶-medGuo, it is concluded that this lesion is highly pro-mutagenic.