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S Tyagi

Publications and source records attributed to S Tyagi.

At least 37 records · Page 2Linked to original sources
PubMed2001

Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles.

Thiamine, a water-soluble vitamin, is essential for normal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety of conditions. To date, however, little is known about the mechanism of thiamine absorption in the native human small intestine. The objective of this study was, therefore, to characterize the mechanism of thiamine transport across the brush-border membrane (BBM) of human small intestine. With the use of purified BBM vesicles (BBMV) isolated from the jejunum of organ donors, thiamine uptake was found to be 1) independent of Na(+) but markedly stimulated by an outwardly directed H(+) gradient (pH 5.5(in)/pH 7.5(out)); 2) competitively inhibited by the cation transport inhibitor amiloride (inhibitor constant of 0.12 mM); 3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structural analogs (amprolium, oxythiamine, and pyrithiamine), but not by unrelated organic cations (tetraethylammonium, N-methylnicotinamide, or choline); 5) not affected by the addition of ATP to the inside and outside of the BBMV; 6) potential insensitive; and 7) saturable as a function of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 +/- 0.08 microM and a maximal velocity of 1.00 +/- 0.47 pmol. mg protein(-1). 10 s(-1). Carrier-mediated thiamine uptake was also found in BBMV of human ileum. These data demonstrate the existence of a Na(+)-independent, pH-dependent, amiloride-sensitive, electroneutral carrier-mediated mechanism for thiamine absorption in native human small intestinal BBMV.

Adenosine Triphosphate↗
PubMed2001

Differential regulation of the expression of Na(+)/H(+) exchanger isoform NHE3 by PKC-alpha in Caco-2 cells.

Na(+)/H(+) exchange (NHE) activity has been shown to be regulated by various external signals and protein kinases in many tissues and cell types. A family of six NHE isoforms has been identified. Three isoforms, NHE1, NHE2, and NHE3, have been shown to be expressed in the human intestine. The present studies were designed to study regulation of these human NHE isoforms by the alpha-isoform of protein kinase C (PKC) in the Caco-2 cell line. The mRNA levels of the NHE isoforms in Caco-2 cells were initially measured by a semiquantitative RT-PCR technique in response to PKC downregulation by long-term exposure to 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h. PKC downregulation resulted in an approximately 60% increase in the mRNA level for NHE3, but not for NHE1 or NHE2. Utilizing dichlorobenzimidazole riboside, an agent to block the synthesis of new mRNA, we demonstrated that the increase in the NHE3 mRNA in response to downregulation of PKC was predominantly due to an increase in the rate of transcription, rather than a decrease in the NHE3 mRNA stability. Consistent with the mRNA results, our data showed that amiloride-sensitive (22)Na(+) uptake was increased after incubation of Caco-2 cells with 1 microM TPA for 24 h. To elucidate the role of PKC-alpha, an isoform downregulated by TPA, the relative abundance of NHE isoform mRNA levels and the apical NHE activity were assessed in Caco-2 cells over- and underexpressing PKC-alpha. Our results demonstrated that NHE3, but not NHE1 or NHE2, mRNA was downregulated by PKC-alpha and that apical NHE activity was higher in cells underexpressing PKC-alpha and lower in cells overexpressing PKC-alpha than in control cells. In conclusion, these data demonstrate a differential regulation of NHE3, but not NHE2 or NHE1, expression by PKC in Caco-2 cells, and this regulation appears to be predominantly due to PKC-alpha.

Blotting, Western↗
PubMed2001

Sulfate and chloride transport in Caco-2 cells: differential regulation by thyroxine and the possible role of DRA gene.

The current studies were undertaken to establish an in vitro cellular model to study the transport of SO and Cl(-) and hormonal regulation and to define the possible function of the downregulated in adenoma (DRA) gene. Utilizing a postconfluent Caco-2 cell line, we studied the OH(-) gradient-driven (35)SO and (36)Cl(-) uptake. Our findings consistent with the presence of an apical carrier-mediated (35)SO/OH(-) exchange process in Caco-2 cells include: 1) demonstration of saturation kinetics [Michaelis-Menten constant (K(m)) of 0.2 +/- 0.08 mM for SO and maximum velocity of 1.1 +/- 0.2 pmol x mg protein(-1) x 2 min(-1)]; 2) sensitivity to inhibition by DIDS (K(i) = 0.9 +/- 0.3 microM); and 3) competitive inhibition by oxalate and Cl(-) but not by nitrate and short chain fatty acids, with a higher K(i) (5.95 +/- 1 mM) for Cl(-) compared with oxalate (K(i) = 0.2 +/- 0.03 mM). Our results also suggested that the SO/OH(-) and Cl(-)/OH(-) exchange processes in Caco-2 cells are distinct based on the following: 1) the SO/OH(-) exchange was highly sensitive to inhibition by DIDS compared with Cl(-)/OH(-) exchange activity (K(i) for DIDS of 0.3 +/- 0.1 mM); 2) Cl(-) competitively inhibited the SO/OH(-) exchange activity with a high K(i) compared with the K(m) for SO, indicating a lower affinity for Cl(-); 3) DIDS competitively inhibited the Cl(-)/OH(-) exchange process, whereas it inhibited the SO/OH(-) exchange activity in a mixed-type manner; and 4) utilizing the RNase protection assay, our results showed that 24-h incubation with 100 nM of thyroxine significantly decreased the relative abundance of DRA mRNA along with the SO/OH(-) exchange activity but without any change in Cl(-)/OH(-) exchange process. In summary, these studies demonstrated the feasibility of utilizing Caco-2 cell line as a model to study the apical SO/OH(-) and Cl(-)/OH(-) exchange processes in the human intestine and indicated that the two transporters are distinct and that DRA may be predominantly a SO transporter with a capacity to transport Cl(-) as well.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

Mechanism of folate transport across the human colonic basolateral membrane.

Previous studies from our laboratory have demonstrated the existence of a folate transporter in the human colonic apical membranes. The current studies were undertaken to examine the possible presence and function of a folate carrier in the human colonic basolateral membrane vesicles (BLMV). BLMV were purified from mucosal scrapings of colons of organ donors by a Percoll-density gradient centrifugation technique, and uptake studies were performed using a rapid filtration technique. Our results on [(3)H]Pte-Glu uptake are summarized as follows: 1) uptake was sensitive to osmolarity of the incubation medium; 2) Na(+) removal from the incubation medium did not affect folate uptake into BLMV; 3) uptake was significantly increased with decreasing incubation buffer pH from 8 to 4; 4) uptake demonstrated saturation kinetics with an apparent Michaelis constant of 9.6 +/- 0.48 microM and a maximal velocity of 8.10 +/- 0.36 pmol x mg protein(-1) x 10 s(-1); 5) uptake was markedly inhibited by the structural analog methotrexate (inhibitory constant = 8.28 +/- 1.0 microM); 6) uptake into BLMV demonstrated a trans-stimulation phenomenon; 7) anion exchange inhibitors DIDS and SITS significantly inhibited folate uptake; and 8) uptake was potential-insensitive, as voltage clamping of vesicles or making them inside positive with K(+)/valinomycin failed to influence folate uptake. Western blot analysis using purified human colonic basolateral membrane preparations and specific polyclonal antibodies against the human reduced folate carrier (hRFC) has shown expression of the hRFC protein at this membrane domain. These data demonstrate the existence of a pH-dependent, DIDS-sensitive, electroneutral, carrier-mediated mechanism for folate transport across the human colonic basolateral membranes.

Adult↗
PubMed2001

Evidence for the existence of a distinct SO(4)(--)-OH(-) exchange mechanism in the human proximal colonic apical membrane vesicles and its possible role in chloride transport.

Recent studies have demonstrated that mutations in human downregulated in adenoma gene (DRA) result in congenital chloride diarrhea (CLD), and that DRA may be involved in chloride transport across the intestinal apical domains. DRA is highly homologous to sulfate transporters, but not to any member of the anion exchanger gene family (AEs). Our previous studies have characterized the existence of a distinct Cl(-)-OH(-) (HCO(3)(-)) exchanger, with minimal affinity for sulfate in the human colonic apical membrane vesicles (AMV). However, the mechanism(s) of sulfate movement across the colonocyte plasma membranes in the human colon is not well understood. Current studies were undertaken to elucidate sulfate transport pathways in AMVs of human proximal colon. Purified AMV and rapid filtration (35)SO(4)(--) uptake techniques were used. Our results demonstrate the presence of a pH gradient-driven carrier-mediated SO(4)(--)-OH(-) exchange process in the human proximal colonic luminal membranes based on the following: a marked increase in the SO(4)(--) uptake in the presence of an outwardly directed OH(-) gradient; a significant inhibition of SO(4)(--) uptake by the membrane anion transport inhibitor, DIDS; demonstration of saturation kinetics (K(m) for SO(4)(--): 0.80 +/- 0.17 mM and Vmax 649 +/- 74 pmol/mg protein/10 sec); competitive inhibition of SO(4)(--)-OH(-) exchange by oxalate; SO(4)(--) uptake was insensitive to alterations in the membrane potential; and inwardly directed Na(+) gradient under non-pH gradient conditions did not stimulate SO(4)(--) uptake. SO(4)(--) uptake was significantly inhibited by increasing concentrations of chloride (1-10 mM) in the incubation media with a K(i) for Cl(-) of 9.3 +/- 1.4 mM. In contrast, OH(-)/HCO(3)(-) gradient-driven (36)Cl(-) uptake into these vesicles was unaffected by increasing concentrations of sulfate (10-50 mM). The above data indicate that two distinct transporters may be involved in SO(4)(--) and Cl(-) transport in the human intestinal apical membranes: an anion exchanger with high affinity for SO(4)(--) and oxalate but low affinity for Cl(-), and a distinct Cl(-)-OH(-) (HCO(3)(-)) exchanger with low affinity for SO(4)(--).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

Detection of Cl--HCO3- and Na+-H+ exchangers in human airways epithelium.

Molecular species of the Na(+)-H(+) exchanger (NHE) and anion exchanger (AE) gene families and their relative abundance in the human airway regions were assessed utilizing RT-PCR and the RNase protection assay, respectively. Organ donor lung epithelia from various bronchial regions (small, medium, and large bronchi and trachea) were harvested for RNA extraction. Gene-specific primers for the human NHE and AE isoforms were utilized for RT-PCR. Our results demonstrated that NHE1, AE2, and brain AE3 isoforms were expressed in all regions of the human airway, whereas NHE2, NHE3, AE1, and cardiac AE3 were not detected. RNase protection studies for NHE1 and AE2, utilizing glyceraldehyde-3-phosphate dehydrogenase as an internal standard, demonstrated that there were regional differences in the NHE1 mRNA levels in human airways. In contrast, the levels of AE2 mRNA remained unchanged. Differential regional expression of NHE1 isoform may be related to a higher acid load in the tracheal epithelial cells than in epithelia of distal airways. Fluctuations in PCO(2) during inspiration and expiration are probably larger in the tracheal lumen than in the lumen of distal airways with associated larger swings in intracellular pH with each respiratory cycle. Immunohistochemical staining for AE2 protein demonstrated localization to the epithelial cells of human bronchial mucosa.

Bronchi↗
PubMed2001

Bacterial adherence to polymethylmethacrylate posterior chamber intra ocular lenses.

PURPOSE: Bacterial adherence to intraocular lenses (IOLs) has been incriminated in the pathogenesis of postoperative endophthalmitis. Staphylococcus epidermidis is the most common organism isolated. We studied the in-vitro adhesion of Staphylococcus epidermidis to Polymethylmethacrylate (PMMA) IOLs and the effect of duration of exposure to adherence. METHODS: Two groups of 10 IOLs each were incubated in Staphylococcus epidermidis suspension for 2 minutes and 20 minutes respectively. Adhesion of bacterial cells was determined by counting the number of viable bacteria attached to IOLs. RESULTS: The mean bacterial adherence with 2 minutes incubation was 12,889 +/- 7,150 bacteria/ IOL and with 20 minutes incubation was 84,226 +/- 35,024 bacteria/IOL (P< 0.01). CONCLUSION: Our results show that Staphylococcus epidermidis adheres to PMMA IOLs in vitro and the degree of adherence is less for shorter duration of exposure. We conclude that viable bacteria irreversibly adherent to IOLs may play a role in the pathogenesis of postoperative endophthalmitis. Shorter duration of operative manipulation and exposure to contaminating sources may decrease the chances of postoperative endophthalmitis.

Bacterial Adhesion↗
PubMed2000

Combined transformation and genetic technique verification of protein-protein interactions in the yeast two-hybrid system.

The yeast two-hybrid system is frequently used to identify protein-protein interactions. The assay is based on the functional reconstitution of a transcriptional activator. Since an indirect phenotype of the positive clones is the basis for selection of positive interacting clones, the two-hybrid screens are vulnerable to false positives. Here we report a screening protocol based on the sequential use of the cotransformation approach followed by the genetic method for verifying true two-hybrid interactions. Using this procedure, we have screened a cDNA library and have been able to isolate true positives from the yeast two-hybrid screen.

Gene Library↗
PubMed2000

A DNA damage signal is required for p53 to activate gadd45.

We provide direct evidence that overexpression of p53 is not sufficient for robust p53-dependent activation of the endogenous gadd45 gene. When p53 was induced in TR9-7 cells in the absence of DNA damage, waf1/p21 and mdm2 mRNA levels were increased, but a change in gadd45 mRNA was barely detectable. Activation of the gadd45 gene was observed when camptothecin was added to cells containing p53 in the absence of a further increase in the p53 level. Phosphorylation of p53 at serine 15 and acetylation at lysine 382 were detected after drug treatment. It has been suggested that p53 posttranslational modification is critical during activation. However, inhibition of these modifications by wortmannin was not sufficient to block the transactivation of gadd45. Interestingly, after camptothecin treatment, increased DNase I sensitivity was detected at the gadd45 promoter, suggesting that an undetermined DNA damage signal is involved in inducing chromatin remodeling at the gadd45 promoter while cooperating with p53 to activate gadd45 transcription.

Acetylation↗
PubMed2000

Intercellular adhesion molecule 1 and beta2 integrins in C1q-stimulated superoxide production by human neutrophils: an example of a general regulatory mechanism governing acute inflammation.

OBJECTIVE: To investigate the role of intercellular adhesion molecule 1 (ICAM-1) and beta2 integrins in the production of superoxide (O2-) by C1q-stimulated human polymorphonuclear leukocytes (PMN). METHODS: PMN were pretreated with F(ab')2 fragments of monoclonal antibodies (mAb) that blocked or did not block beta2 integrin-mediated adhesion. The cells were added to wells coated with C1q, and the production of O2- was monitored kinetically as a color change due to reduction of cytochrome c. In some experiments, C1q was co-immobilized with purified ICAM-1. RESULTS: Blocking mAb to the shared beta2 integrin subunit, CD18, completely inhibited the O2- response triggered by immobilized C1q, while blocking mAb to the alpha subunits of the beta2 integrins each partially blocked the O2- response. PMN treated with C1q were found to activate the beta2 integrins lymphocyte function-associated antigen 1 and CR3 for binding to ICAM-1. Co-immobilization of ICAM-1 with C1q cooperatively triggered O2- production by PMN. CONCLUSION: beta2 integrin binding to an ICAM provided an essential costimulatory signal for O2-production triggered by C1q in PMN. Our findings suggest a model for PMN activation in which 2 stimuli are required for O2- production: a first signal that also activates PMN beta2 integrins, followed by a second, beta2 integrin-mediated signal, which occurs physiologically upon PMN binding to ICAM-1. The requirement for this dual signal for PMN generation of O2- would serve as a regulatory mechanism to limit the production of O2- to a tissue environment where C1q, or some other stimulus, is colocalized with stromal cells bearing up-regulated ICAM-1. This mechanism may explain why all tissues can express ICAM-1 and may explain in part why inhibitors of tumor necrosis factor alpha, a major physiologic stimulus of ICAM-1 up-regulation, are potent antiinflammatory agents.

Antibodies, Blocking↗
PubMed2000

Evidence for a Na+-H+ exchange across human colonic basolateral plasma membranes purified from organ donor colons.

The mechanism(s) of electrolyte transport across the human colonic contraluminal domain is not well understood. Current studies were undertaken to develop a technique for the isolation and purification of the human colonic basolateral membrane vesicles (BLMV) and to examine the presence of a Na+-H+ exchange process in these membranes. BLMV were purified from mucosal scrapings of organ donor proximal colons utilizing a Percoll density gradient centrifugation technique, and Na+ transport was examined utilizing a rapid filtration, technique. Our data demonstrate that purified basolateral membranes were enriched 10- to 11-fold in Na+, K+-ATPase activity compared to crude homogenate. Results consistent with the Na+-H+ exchange in BLMV are as follows: (1) an outwardly directed H+ gradient stimulated 22Na uptake; (2) 22Na uptake was markedly inhibited by EIPA and amiloride; (3) H+-gradient-stimulated 22Na uptake was not inhibited by bumetanide, SITS, DIDS, acetazolamide, phenamil and benzamil; (4) 22Na uptake was voltage insensitive; (5) 22Na uptake demonstrated saturation kinetics; (6) 22 Na uptake was markedly inhibited by Na+ and Li+ but was unaffected by N-methyl glucamine+, choline+, and NH4+. Immunoblotting studies demonstrated this Na+-H+ exchanger isoform to be represented by NHE1. In conclusion, a technique has been established for the purification of functional human proximal colonic BLMV, and an electroneutral Na+-H+ exchange process has been demonstrated in these membranes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Wavelength-shifting molecular beacons.

We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light are assigned to two separate fluorophores in the same probe. These probes contain a harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic light source, an emitter fluorophore of the desired emission color, and a nonfluorescent quencher. In the absence of complementary nucleic acid targets, the probes are dark, whereas in the presence of targets, they fluoresce-not in the emission range of the harvester fluorophore that absorbs the light, but rather in the emission range of the emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed energy from the harvester fluorophore to the emitter fluorophore by fluorescence resonance energy transfer, and it only takes place in probes that are bound to targets. Wavelength-shifting molecular beacons are substantially brighter than conventional molecular beacons that contain a fluorophore that cannot efficiently absorb energy from the available monochromatic light source. We describe the spectral characteristics of wavelength-shifting molecular beacons, and we demonstrate how their use improves and simplifies multiplex genetic analyses.

Alleles↗
PubMed2000

Rapid identification of Candida dubliniensis using a species-specific molecular beacon.

Candida dubliniensis is an opportunistic fungal pathogen that has been linked to oral candidiasis in AIDS patients, although it has recently been isolated from other body sites. DNA sequence analysis of the internal transcribed spacer 2 (ITS2) region of rRNA genes from reference Candida strains was used to develop molecular beacon probes for rapid, high-fidelity identification of C. dubliniensis as well as C. albicans. Molecular beacons are small nucleic acid hairpin probes that brightly fluoresce when they are bound to their targets and have a significant advantage over conventional nucleic acid probes because they exhibit a higher degree of specificity with better signal-to-noise ratios. When applied to an unknown collection of 23 strains that largely contained C. albicans and a smaller amount of C. dubliniensis, the species-specific probes were 100% accurate in identifying both species following PCR amplification of the ITS2 region. The results obtained with the molecular beacons were independently verified by random amplified polymorphic DNA analysis-based genotyping and by restriction enzyme analysis with enzymes BsmAI and NspBII, which cleave recognition sequences within the ITS2 regions of C. dubliniensis and C. albicans, respectively. Molecular beacons are promising new probes for the rapid detection of Candida species.

Candida↗
PubMed2000

C5a-stimulated human neutrophils use a subset of beta2 integrins to support the adhesion-dependent phase of superoxide production.

Isolated human polymorphonuclear neutrophils (PMN) responded to human C5a with an immediate, transient release of superoxide lasting from 0.5 to 5 min. This was followed by a second release of superoxide, which began at 10 min after addition of C5a, was sustained for more than 30 min, and required ICAM-1 immobilized in the wells. F(ab')2 monoclonal antibody (mAb) preparations were used to dissect the role of individual beta2 integrins and to avoid the confounding effects of ligating Fc receptors. Anti-CD18 mAb treatment of the PMN had no effect on the immediate first phase but completely inhibited the second, adhesion-dependent phase of superoxide production. Anti-CR3 mAb only inhibited the adhesion phase of superoxide production partially, implying that other beta2 integrins were involved. A mixture of anti-CD11a, anti-CD11b, and anti-CD11c was not able to block superoxide production completely, suggesting a role for alphad/beta2. Surprisingly, blocking anti-LFA-1 mAb had no effect on superoxide production. Consistent with this observation, immobilized, purified ICAM-2, a specific counter-receptor for LFA-1, did not support the adhesion-dependent phase of-superoxide production. Thus, PMN treated with C5a used signals via CR3, P150/95, and alphad/beta2, but not LFA-1, to support superoxide production. LFA-1 has been shown by others to mediate most of the adhesion necessary for transendothelial migration in vivo. The inability of LFA-1 ligation to stimulate superoxide production may be an important means of preventing blood-vessel damage when PMN migrate across the endothelium.

Antibodies, Monoclonal↗
PubMed1999

Race-specific HIV-1 disease-modifying effects associated with CCR5 haplotypes.

Genetic variation in CC chemokine receptor 5 (CCR5), the major HIV-1 coreceptor, has been shown to influence HIV-1 transmission and disease progression. However, it is generally assumed that the same CCR5 genotype (or haplotype) has similar phenotypic effects in different populations. To test this assumption, we used an evolutionary-based classification of CCR5 haplotypes to determine their associated HIV-1 disease-modifying effects in a large well-characterized racially mixed cohort of HIV-1-seropositive individuals. We demonstrate that the spectrum of CCR5 haplotypes associated with disease acceleration or retardation differs between African Americans and Caucasians. Also, we show that there is a strong interactive effect between CCR5 haplotypes with different evolutionary histories. The striking population-specific phenotypic effects associated with CCR5 haplotypes emphasize the importance of understanding the evolutionary context in which disease susceptibility genes are expressed.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Thermodynamic basis of the enhanced specificity of structured DNA probes.

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

Base Sequence↗
PubMed1999

Multiplex detection of four pathogenic retroviruses using molecular beacons.

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100, 000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.

Base Sequence↗