Mechanism of n-butyrate uptake in the human proximal colonic basolateral membranes

Current studies were undertaken to characterize the mechanism of short-chain fatty acid (SCFA) transport in isolated human proximal colonic basolateral membrane vesicles (BLMV) utilizing a rapid-filtration n-[(14)C]butyrate uptake technique. Human colonic tissues were obtained from mucosal scrapings from organ donor proximal colons. Our results, consistent with the existence of a HCO(3)(-)/SCFA exchanger in these membranes, are summarized as follows: 1) n-[(14)C]butyrate influx was significantly stimulated into the vesicles in the presence of an outwardly directed HCO(3)(-) and an inwardly directed pH gradient; 2) n-[(14)C]butyrate uptake was markedly inhibited (approximately 40%) by anion exchange inhibitor niflumic acid (1 mM), but SITS and DIDS (5 mM) had no effect; 3) structural analogs e.g., acetate and propionate, significantly inhibited uptake of HCO(3)(-) and pH-gradient-driven n-[(14)C]butyrate; 4) n-[(14)C]butyrate uptake was saturable with a K(m) for butyrate of 17.5 +/- 4.5 mM and a V(max) of 20.9 +/- 1.2 nmol x mg protein(-1) x 5 s(-1); 5) n-[(14)C]butyrate influx into the vesicles demonstrated a transstimulation phenomenon; and 6) intravesicular or extravesicular Cl(-) did not alter the anion-stimulated n-[(14)C]butyrate uptake. Our results indicate the presence of a carrier-mediated HCO(3)(-)/SCFA exchanger on the human colonic basolateral membrane, which appears to be distinct from the previously described anion exchangers in the membranes of colonic epithelia.     

Remodeling of the left atrium in pacing-induced atrial cardiomyopathy

Rapid atrial pacing produces atrial systolic and diastolic failure characterized by absent atrial booster pump function, increased atrial chamber stiffness, enhanced atrial conduit function, and atrial enlargement. However, the processes underlying these abnormalities are poorly understood. Therefore, we examined left atrial myocardium from dogs with rapid pacing-induced atrial failure (400 bpm for 6 weeks) and from control dogs. Western blotting was used to determine the levels of proteins involved in calcium homeostasis (SERCA 2A, phospholamban, Na⁺-Ca²⁺ exchanger). Matrix metalloproteinase (MMP) activity was measured using gelatin and casein zymography, and levels of tissue inhibitor of metalloproteinase-4 (TIMP-4) and the TIMP-4 complexed with MMPs were measured with Western blot analysis. There were no differences in SERCA 2A or Na⁺-Ca²⁺ exchanger protein levels, but phospholamban level was significantly decreased in atrial samples from rapidly paced dogs (51.2 ± 7.8 vs. 77.0 ± 10.0, p < 0.01). The activity of MMP-9 was selectively and significantly increased by 50%, and the level of complexed TIMP-4 protein was significantly decreased by 50% in samples from dogs with atrial failure. Thus, rapid pacing-induced atrial failure is associated with differential changes in MMP activity, an unchanged number of calcium pumps, and compensatory changes in the level of phospholamban.     

In vitro Plant Regeneration and Genotype Conservation of Eight Wild Species of Curcuma

In vitro protocols for plantlet regeneration and medium-term genotype conservation of eight wild species of Curcuma have been optimized. Both the phenomena were genotype-dependent and influenced significantly by type and concentration of cytokinins used. In general, benzyladenine (BA) was found superior to other cytokinins tested for plantlet regeneration and ,-dimethylallylaminopurine (2iP) for conservation. Number of shoots per culture ranged from 1.3 to 7.2 and conservation period from 264 to 379 d. In 30-d-old cultures, highest frequency of shoot regeneration could be obtained in C. malabarica (7.2 shoots per culture) on MS + 11.4 M zeatin. Curcuma sp. (unidentified wild species) could be conserved for maximum period (379 d) on MS + 24.6 M 2iP followed by C. aromatica (363 d) on MS + 22.8 M zeatin. The tissue culture-raised plantlets were morphologically similar to their parents. The in vitro-conserved plants multiplied rapidly in tissue cultures and produced normal rhizomes upon transfer to soil in net house.     

Extraction of oil from Jatropha curcas L. seed kernels by combination of ultrasonication and aqueous enzymatic oil extraction

Use of ultrasonication as a pretreatment before aqueous oil extraction and aqueous enzymatic oil extraction was found to be useful in the case of extraction of oil from the seeds of Jatropha curcas L. The use of ultrasonication for 10 min at pH 9.0 followed by aqueous oil extraction gave a yield of 67%. However, the maximum yield of 74% was obtained by ultrasonication for 5 min followed by aqueous enzymatic oil extraction using an alkaline protease at pH 9.0. Use of ultrasonication also resulted in reducing the process time from 18 to 6 h.     

Prenylated chalcones isolated from Crotalaria genus inhibits in vitro growth of the human malaria parasite Plasmodium falciparum

A prenylated chalcone 2 named crotaorixin, has been isolated from the aerial parts of the Crotalaria orixensis. Its structure has been established by extensive 1D and 2D NMR measurements. In vitro antimalarial activity of crotaorixin as well as few prenylated chalcones isolated from C. medicagenia and C. ramosissima were evaluated at three concentrations (50, 10 and 2 microg/ml) against Plasmodium falciparum (Strain NF-54). Compound 3 has exhibited 100% inhibition of schizont maturation at 2 microg/ml concentration.     

High-yield functional expression of human sodium/d-glucose cotransporter1 in Pichia pastoris and characterization of ligand-induced conformational changes as studied by tryptophan fluorescence

Studies on the structure-function relationship of transporters require the availability of sufficient amounts of the protein in a functional state. In this paper, we report the functional expression, purification, and reconstitution of the human sodium/d-glucose cotransporter1 (hSGLT1) in Pichia pastoris and ligand-induced conformational changes of hSGLT1 in solution as studied by intrinsic tryptophan fluorescence. hSGLT1 gene containing FLAG tag at position 574 was cloned into pPICZB plasmid, and the resulting expression vector pPICZB-hSGLT1 was introduced into P. pastoris strain GS115 by electroporation. Purification of recombinant hSGLT1 by nickel-affinity chromatography yields about 3 mg of purified recombinant hSGLT1 per 1-liter of cultured Pichia cells. Purified hSGLT1 migrates on SDS-PAGE with an apparent mass of 55 kDa. Kinetic analysis of hSGLT1 in proteoliposomes revealed sodium-dependent, secondary active, phlorizin-sensitive, and stereospecific alpha-methyl-d-glucopyranoside transport, demonstrating its full catalytic activity. The position of the maximum intrinsic tryptophan fluorescence and titration with hydrophilic collisional quenchers KI, acrylamide, and trichloroethanol suggested that most of Trps in hSGLT1 in solution are in a hydrophobic environment. In the presence of sodium, sugars that have been identified earlier as substrate for the transporter increase intrinsic fluorescence in a saturable manner by a maximum of 15%. alpha-Methyl-d-glucopyranoside had the highest affinity (K(d) = 0.71 mM), followed by d-glucose, d-galactose, d-mannose, and d-allose which showed a much lower affinity. l-Glucose was without effect. d-Glucose also increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary phlorizin, the well-established inhibitor of SGLT1, decreased intrinsic fluorescence by a maximum of 50%, and induced a blue shift of maximum (5 nm). Again, the effects were sodium-dependent and saturable and a high affinity K(d) of 5 muM was observed. In addition the surface of hSGLT1 was labeled with 1-anilinonaphthalene-8-sulfonic acid, a reporter molecule for the surface hydrophobicity. In the presence of sodium, addition of d-glucose decreased ANS fluorescence whereas phlorizin increased ANS fluorescence. Thus three conformational states of SGLT1 could be defined which differ in their packing density and hydrophobicity of their surface. They reflect properties of the empty carrier, the d-glucose loaded carrier facing the outside of membrane and the complex of the outside-orientated carrier with phlorizin.     

Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis

The devR-devS two-component system of Mycobacterium tuberculosis was identified earlier and partially characterized in our laboratory. A devR::kan mutant of M. tuberculosis was constructed by allelic exchange. The devR mutant strain showed reduced cell-to-cell adherence in comparison to the parental strain in laboratory culture media. This phenotype was reversed on complementation with a wild-type copy of devR. The devR mutant and parental strains grew at equivalent rates within human monocytes either in the absence or in the presence of lymphocytic cells. The expression of DevR was not modulated upon entry of M. tuberculosis into human monocytes. However, guinea pigs infected with the mutant strain showed a significant decrease in gross lesions in lung, liver and spleen; only mild pathological changes in liver and lung; and a nearly 3 log lower bacterial burden in spleen compared to guinea pigs infected with the parental strain. Our results suggest that DevR is required for virulence in guinea pigs but is not essential for entry, survival and multiplication of M. tuberculosis within human monocytes in vitro. The attenuation in virulence of the devR mutant in guinea pigs together with DevR-DevS being a bona fide signal transduction system indicates that DevR plays a critical and regulatory role in the adaptation and survival of M. tuberculosis within tissues.