Search PubMed⌕ Search

Biomedical subjects

S Khurana

Publications and source records attributed to S Khurana.

At least 19 recordsLinked to original sources
PubMed2001

Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton.

We have previously shown that tyrosine phosphorylation of the actin-regulatory protein villin is accompanied by the redistribution of phosphorylated villin and a concomitant decrease in the F-actin content of intestinal epithelial cells. The temporal and spatial correlation of these two events suggested that tyrosine phosphorylation of villin may be involved in the rearrangement of the microvillar cytoskeleton. This hypothesis was investigated by analyzing the effects of tyrosine phosphorylation of villin on the kinetics of actin polymerization by reconstituting in vitro the tyrosine phosphorylation of villin and its association with actin. Full-length recombinant human villin was phosphorylated in vitro by expression in the TKX1-competent cells that carry an inducible tyrosine kinase gene. The actin-binding properties of villin were examined using a co-sedimentation assay. Phosphorylation of villin did not change the stoichiometry (1:2) but decreased the binding affinity (4.4 microm for unphosphorylated versus 0.6 microm for phosphorylated) of villin for actin. Using a pyrene-actin-based fluorescence assay, we demonstrated that tyrosine phosphorylation had a negative effect on actin nucleation by villin. In contrast, tyrosine phosphorylation enhanced actin severing by villin. Electron microscopic analysis showed complementary morphological changes. Phosphorylation inhibited the actin bundling and enhanced the actin severing functions of villin. Taken together our data show that tyrosine phosphorylation of villin decreases the amount of villin bound to actin filaments, inhibits the actin-polymerizing properties of villin, and promotes the actin-depolymerizing functions instead. These observations suggest a role for tyrosine phosphorylation in modulating the microvillar cytoskeleton in vivo by villin in response to specific physiological stimuli.

Actins↗
PubMed2001

The A' and F' pockets of human CD1b are both required for optimal presentation of lipid antigens to T cells.

CD1 proteins are unique in their ability to present lipid Ags to T cells. Human CD1b shares significant amino acid homology with mouse CD1d1, which contains an unusual putative Ag-binding groove formed by two large hydrophobic pockets, A' and F'. We investigated the function of the amino acid residues that line the A' and F' pockets of CD1b by engineering 36 alanine-substitution mutants and analyzing their ability to present mycobacterial glycolipid Ags. Two lipid Ags presented by CD1b were studied, a naturally occurring glucose monomycolate (GMM) isolated from mycobacteria, which contains two long alkyl chains (C54-C62 and C22-C24) and synthetic GMM (sGMM), which includes two short alkyl chains (C18 and C14). We identified eight residues in both the A' and F' pockets that were involved in the presentation of both GMM and sGMM to T cells. Interestingly, four additional residues located in the distal portion of the A' pocket were required for the optimal presentation of GMM, but not sGMM. Conversely, nine residues located between the center of the groove and the F' pocket were necessary for the optimal presentation of sGMM, but not GMM. These data indicate that both the A' and F' pockets of human CD1b are required for the presentation of lipid Ags to T cells.

Alanine↗
PubMed2001

Regulation of phospholipase C-gamma(1) by the actin-regulatory protein villin.

The actin-regulatory protein villin is tyrosine phosphorylated and associates with phospholipase C-gamma(1) (PLC-gamma(1)) in the brush border of intestinal epithelial cells. To study the mechanism of villin-associated PLC-gamma(1) activation, we reconstituted in vitro the tyrosine phosphorylation of villin and its association with PLC-gamma(1). Recombinant villin was phosphorylated in vitro by the nonreceptor tyrosine kinase c-src or by expression in the TKX1 competent cells that carry an inducible tyrosine kinase gene. Using in vitro binding assays, we demonstrated that tyrosine-phosphorylated villin associates with the COOH-terminal Src homology 2 (SH2) domain of PLC-gamma(1). The catalytic activity of PLC-gamma(1) was inhibited by villin in a dose-dependent manner with half-maximal inhibition at a concentration of 12.4 microM. Villin inhibited PLC-gamma(1) activity by sequestering the substrate phosphatidylinositol 4,5-bisphosphate (PIP(2)), since increasing concentrations of PIP(2) reversed the inhibitory effects of villin on PLC activity. The inhibition of PLC-gamma(1) activity by villin was reversed by the tyrosine phosphorylation of villin. Further, we demonstrated that tyrosine phosphorylation of villin abolished villin's ability to associate with PIP(2). In conclusion, tyrosine-phosphorylated villin associates with the COOH-terminal SH2 domain of PLC-gamma(1) and activates PLC-gamma(1) catalytic activity. Villin regulates PLC-gamma(1) activity by modifying its own ability to bind PIP(2). This study provides biochemical proof of the functional relevance of tyrosine phosphorylation of villin and identifies the molecular mechanisms involved in the activation of PLC-gamma(1) by villin.

Actins↗
PubMed2000

2000 Volvo Award winner in clinical studies: Lumbar high-intensity zone and discography in subjects without low back problems.

STUDY DESIGN: A prospective observational study of patients with low back pain and those without was performed. OBJECTIVE: To investigate the prevalence and significance of a high-intensity zone in a group of patients asymptomatic for low back pain, but who had known risk factors for lumbar disc degeneration. This asymptomatic group was compared with a symptomatic group of patients with respect to the presence of anular high-intensity zone and the pain response with discography. SUMMARY OF BACKGROUND DATA: Some authors have estimated the prevalence of a high-intensity zone in a group of symptomatic patients to be 86%. They have reported a strong correlation between a high-intensity zone and positive discography in patients with low back pain. Other investigators have reported evidence either supporting or discounting these findings. METHODS: Patients with low back pain and those without underwent physical examination, psychometric testing, plain radiograph, magnetic resonance imaging, and discography. The presence of a high-intensity zone, anular disruption, and positive discographic pain then were compared between the two groups. There were strict inclusion criteria for both groups. A total of 109 discs in 42 patients were evaluated in the symptomatic group and compared with 143 discs in 54 patients in the asymptomatic group. The presence of a high-intensity zone was determined by a standardized criteria on T2-weighted magnetic resonance images. Psychometric testing also was administered to each patient before discography. Standard discography was performed on all the patients, and the pain response was recorded using a visual analog scale according to the Walsh et al criteria. RESULTS: The prevalence of a high-intensity zone in the patient populations was 59% in the symptomatic group and 24% in the asymptomatic group. In the symptomatic group, 33 (30.2%) of 109 discs were found to have a high-intensity zone. In the asymptomatic group, 13 of 143 discs were found to have a high-intensity zone. In the symptomatic group, 72.7% of the discs with a high-intensity zone were positive on discography, whereas 38.2% of the discs without a high-intensity zone were positive. In the asymptomatic group, 69.2% of the discs with a high-intensity zone were positive on discography, whereas 10% of the discs without a high-intensity zone were positive. In the patients with normal psychometric testing, 50% of the discs with a high-intensity zone were positive on discography, as compared with 100% positive discography results in patients with abnormal psychometric testing or chronic pain. CONCLUSIONS: The presence of a high-intensity zone does not reliably indicate the presence of symptomatic internal disc disruption. Although higher in symptomatic patients, the prevalence of a high-intensity zone in asymptomatic individuals with degenerative disc disease (25%) is too high for meaningful clinical use. When injected during discography, the same percentage of asymptomatic and symptomatic discs with a high-intensity zone were shown to be painful.

Adult↗
PubMed2000

The rates of false-positive lumbar discography in select patients without low back symptoms.

STUDY DESIGN: Experimental disc injections in subjects with no history of low back symptoms. OBJECTIVE: To determine in an experimental setting the relative pain response and pain-related behavior in selected subjects without a history of low back pain undergoing lumbar discography. This study aimed to select a study population that more closely represented patients undergoing discography in clinical practice. SUMMARY OF BACKGROUND DATA: Previous work has shown that in young, healthy men with little degenerative disc disease and no history of low back problems, discographic injections usually did not cause significant pain. This group differed from the patients who usually undergo discographic evaluation in clinical practice. Most clinical patients are older, have significant degenerative disc disease, have behavioral changes associated with chronic pain, and often have confounding psychosocial troubles. The authors undertook to study discography in subjects without low back pain but with clinical profiles similar to patients undergoing discography in clinical practice. METHODS: Twenty-six individuals, mean age 43 years, with no history of low back pain had lumber discography according to the strict protocol of Walsh et al. Of these, 10 were pain-free; 10 had chronic neck and arm pain, but no low back symptoms; and 6 had primary somatization disorders without low back symptoms.- RESULTS: Significant positive pain response and pain-related behavior with discography were found in 10% of the pain-free group, in 40% of the chronic cervical pain group, and in 83% of the somatization disorder group completing the injections. Twenty-four subjects had negative control discs. Discs with annular disruption were more likely to be painful on injection, particularly in those individuals with ongoing compensation issues, chronic pain, or abnormal psychological testing.- CONCLUSION: If strict criteria are applied, the rate of false-positive discography may be low in subjects with normal psychometric profiles and without chronic pain. Significantly painful injections were very common in subjects with annular disruption and chronic pain or abnormal psychometric testing.

Adult↗
PubMed2000

Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases.

2,5-diketo-D-gluconic acid reductase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent stereo-specific reduction of 2, 5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KLG), a precursor in the industrial production of vitamin C (L-ascorbate). Microorganisms that naturally ferment D-glucose to 2,5-DKG can be genetically modified to express the gene for 2,5-DKGR, and thus directly produce vitamin C from D-glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5-DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics-based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Proteins 2000;39:68-75.

Amino Acid Substitution↗
PubMed2000

Short-term regulation of NHE3 by EGF and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells and fibroblasts.

NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na(+)-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in Vmax of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)i of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number.

Animals↗
PubMed2000

Comparative in vitro efficacy of different platelet glycoprotein IIb/IIIa antagonists on platelet-mediated clot strength induced by tissue factor with use of thromboelastography: differentiation among glycoprotein IIb/IIIa antagonists.

In the present study, the in vitro efficacy of different platelet glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists on platelet-fibrin-mediated clot strength under shear was compared with the antiaggregatory efficacy by using tissue factor (TF) thromboelastography (TEG). The ability of platelets to augment the elastic properties of blood clots under shear conditions was measured by computerized TEG under conditions of maximal platelet activation accelerated by recombinant TF. Under these conditions, platelets significantly enhance clot strength 8-fold (relative to platelet-free fibrin clots). This effect was inhibited to a different extent by various platelet GPIIb/IIIa receptor antagonists; this inhibition appears to be dependent on the transmission of platelet contractile force to fibrin via the GPIIb/IIIa receptors. The GPIIb/IIIa antagonists with high binding affinity for resting and activated platelets and slow platelet dissociation rates (class I) but not those with fast platelet dissociation rates (class II) demonstrated potent and comparable inhibition of platelet aggregation and TF-TEG clot strength. Platelet GPIIb/IIIa antagonists of class I, such as XV459 (free-acid form of roxifiban), DMP802, XV454, and c7E3, demonstrated comparable inhibitory dose responses of TF-TEG clot strength and platelet aggregation, with an IC(50) of 50 to 70 nmol/L. In contrast, platelet GPIIb/IIIa antagonists from class II, with comparable antiaggregatory efficacy, such as DMP728, YZ202 (free-acid form of orbofiban), YZ211 (free-acid form of sibrafiban), YZ751, and other antagonists, have a much lower efficacy in altering the strength of TF-mediated clot formation (IC(50) >1.0 micromol/L). These data suggest differential efficacy among different GPIIb/IIIa antagonists in inhibiting platelet-fibrin clot retraction despite of equivalent antiaggregatory potency.

Abciximab↗
PubMed2000

Exposure of newborn male and female rats to environmental estrogens: delayed and sustained hyperprolactinemia and alterations in estrogen receptor expression.

Environmental estrogens (xenoestrogens) are synthetic compounds that are abundant in the environment and mimic natural estrogens. The estrogenicity of two such compounds, bisphenol A (BPA) and octylphenol (OP), during development of the neuroendocrine system was investigated. The objective was to compare the effects of neonatal exposure to BPA, OP, and diethylstilbestrol (DES), a potent synthetic estrogen, on prepubertal serum PRL levels and estrogen receptor (ER) expression in the anterior pituitary and medial basal hypothalamus. Receptor expression in the uterus and prostate, two peripheral estrogen-responsive tissues, was also examined. Newborn male and female Fischer 344 rats were s.c. injected on days 1-5 after birth with corn oil (control), BPA and OP (100 or 500 microg/day), or DES (5 microg/day). Rats were bled on days 15, 20, and 25 and on the day of death (day 30), and serum PRL was analyzed by RIA. Relative expressions of ERalpha and ERbeta were determined by RT-PCR. BPA and OP induced delayed, but progressive, increases in serum PRL levels, up to 3-fold above control levels, in both males and females. The low dose of either compound was equally or more effective as the high dose in eliciting and sustaining elevated serum PRL levels, namely hyperprolactinemia. In contrast, the DES treatment resulted in a transient rise in serum PRL levels. BPA, OP, and, to a lesser extent, DES increased the expression of both ERalpha and ERbeta in the anterior pituitary of males, but not females, whereas the hypothalamic ERs were less responsive to these compounds. DES treatment caused down-regulation of ERalpha expression in the uterus and up-regulation of ERbeta in the prostate, whereas BPA or OP was without effect. In conclusion, exposure of newborn rats of either sex to environmental estrogens results in delayed and sustained hyperprolactinemia and differential alterations in ER expression in the hypothalamus and pituitary. DES appears to target the lower reproductive tract more effectively than the neuroendocrine system.

Animals↗
PubMed1999

The epithelial Na(+)/H(+) exchanger, NHE3, is internalized through a clathrin-mediated pathway.

Trafficking of the Na(+)/H(+) exchanger isoform 3 (NHE3) between sub-apical vesicles and apical membrane of epithelial cells is a suggested mechanism of regulation of NHE3 activity. When epitope-tagged NHE3 was stably expressed in NHE-deficient Chinese hamster ovary cells, a sizable fraction was found in recycling endosomes. This system was used to analyze the mechanism of endocytosis of NHE3. Immunofluorescence and radiolabeling experiments showed that inhibition of clathrin-mediated endocytosis using hypertonicity, acid treatment, or K(+) depletion inhibited internalization of NHE3. Moreover, transient transfection of an inhibitory mutant of dynamin (DynS45N) blocked the clathrin-mediated uptake of transferrin, as well as the endocytosis of NHE3. In ileal villus cells, endogenous NHE3 was also found to co-purify with isolated clathrin-coated vesicles, thereby confirming their association in native tissues. The role of COP-I subunits in the intracellular traffic of NHE3 was evaluated using ldlF cells, which bear a temperature-sensitive mutation in the epsilon-COP subunit. At the permissive temperature, NHE3 distributed normally, whereas at the restrictive temperature, which induces rapid degradation of epsilon-COP, NHE3 was still internalized, but its subcellular distribution was altered. These results indicate that endocytosis of NHE3 occurs primarily via clathrin-coated pits and vesicles and that normal intracellular trafficking of NHE3 involves an epsilon-COP-dependent step.

Amino Acid Sequence↗
PubMed1999

Prophylactic oral amiodarone compared with placebo for prevention of atrial fibrillation after coronary artery bypass surgery.

BACKGROUND: Postoperative atrial fibrillation occurs in 20% to 40% of patients undergoing coronary artery bypass grafting (CABG) and contributes to delayed recovery, increased length of stay, and increased hospital cost. Measures at preventing postoperative atrial fibrillation have had mixed results. We report a double-blind trial comparing oral amiodarone with placebo for the prevention of atrial fibrillation after CABG. METHODS AND RESULTS: All patients undergoing CABG were considered eligible. Exclusion criteria included bradycardia (<50 beats/min), prior Atrial fibrillation, concurrent therapy with antiarrhythmic drugs, or concomitant valve surgery. Patients were given 2 g of amiodarone (73 patients) or placebo (70 patients) in divided doses 1 to 4 days before surgery and 400 mg daily for 7 days postoperatively. Atrial fibrillation occurred in 24.7% (18 of 43) of patients receiving amiodarone and 32. 8% (23 of 70) of patients receiving placebo (P =.30). Heart rate at onset of atrial fibrillation was 133.4 +/- 26.6 beats/min for amiodarone compared with 152.9 +/- 31.6 beats/min for placebo (P =. 04). Duration of atrial fibrillation was 10.2 +/- 8.1 hours for amiodarone compared with 16.2 +/- 27.5 hours for placebo (P =.67). Patients receiving both beta-blockade and amiodarone had a 16.7% incidence of atrial fibrillation compared with 31.9% in the remaining patients (P =.10). Atrial fibrillation was associated with an increased cost of $7011 compared with those who remained in sinus rhythm ($23,869 +/- $20,894 vs $16,857 +/- $5401 in sinus rhythm). Hospital cost of those taking amiodarone was $18,895 +/- $13,267 compared with $18,839 +/- $11,537.18 for placebo (P =.42). CONCLUSION: Postoperative CABG atrial fibrillation is associated with prolonged hospital stay and increased cost. Prophylactic oral amiodarone did not statistically alter the incidence or duration of atrial fibrillation after CABG, although favorable trends were noted. Hospital cost was not affected by therapy with amiodarone.

Administration, Oral↗
PubMed1999

Heparin-binding property of human prolactin: a novel aspect of prolactin biology.

Prolactin (PRL) shares several characteristics with growth factors and cytokines, many of which are known to bind to heparan sulfate proteoglycans. In this study we examined the heparin-binding properties of selected members of the PRL/GH family, using heparin affinity columns followed by gel electrophoresis/Western blotting. Purified human PRL and its cleaved 16K fragment, but not human GH or placental lactogen, were retained on the heparin column and were displaced by 0.5 M NaCl. Native PRL in human pituitary extracts and amniotic fluid showed a similar binding affinity to heparin as the purified hormone. None of the other hormones tested, e.g., rat, ovine and bovine PRL, glycosylated ovine PRL or rat GH, bound to heparin. Two consensus heparin-binding sequences are present in human PRL but not in the other hormones included in this study. We postulate that the heparin-binding capability of PRL affects its biological activity as a growth factor and the angiostatic actions of its 16K fragment.

Amniotic Fluid↗
PubMed1999

Proteolysis of human prolactin: resistance to cathepsin D and formation of a nonangiostatic, C-terminal 16K fragment by thrombin.

The N-terminal 16K fragments of rat and human PRLs possess angiostatic activity. 16K PRL has also been detected in vivo in both humans and rats. Based on an in vitro study, cathepsin D, an acid protease, has been implicated in the generation of rat 16K PRL. However, the proteolytic cleavage of human PRL has not been demonstrated. Our objective was to identify an enzyme that is capable of forming an angiostatic human 16K PRL. To confirm the angiostatic action of rat 16K PRL, the fragment was generated by incubating 23K PRL with rat mammary microsomal fraction at pH 3.2. Upon incubation with human umbilical vein endothelial cells (HUVEC), rat 16K PRL, but not 23K PRL, inhibited basal- and basic fibroblast growth factor-stimulated cell proliferation. Intact rat and human PRLs were then incubated with cathepsin D or acidified microsomal pellets of MCF-7 human breast cancer cells. Analysis by SDS-PAGE showed cleavage of rat, but not human, PRL. Next, hormones were incubated with thrombin at pH 7.4. As shown by SDS-PAGE, digestion of both human and rat PRL by thrombin resulted in the formation of 16K fragments. PRL contained within human amniotic fluid was also cleaved by thrombin. Enzyme specificity was supported by prevention of cleavage by the thrombin inhibitor hirudin. When tested with HUVEC, the human 16K PRL was devoid of angiostatic activity. The activity of this fragment in the Nb2 lymphoma bioassay was 10- to 15-fold lower than that of 23K PRL. Mass spectrometry revealed that the fragment has a mass of 16,878.30+/-15.8 Daltons. Subsequent N-terminal sequencing showed that the thrombin cleavage occurred between amino acid residues 53 (Lys) and 54 (Ala), resulting in the formation of a C-terminal, not an N-terminal, 16K fragment. We conclude that, unlike rat PRL, human PRL is resistant to cleavage by cathepsin D. Thrombin at a physiological pH can generate a C-terminal 16K fragment of human PRL that is not angiostatic and retains little mitogenic activity. We suggest that the precise nature of endogenous 16K PRL fragments that are present in human tissues and body fluids should be carefully examined.

Animals↗
PubMed1999

Simplified gene-fragment phage display system for epitope mapping.

We describe a simple and efficient system for epitope mapping by cloning random gene fragments into a specially designed gIIIp-based phage display vector. DNA encoding the antigen of interest is PCR-amplified and partially digested with DNaseI to generate 50-150-bp-long fragments, which are polished with T4 DNA polymerase and dephosphorylated. These fragments are cloned at the 5' end of the gIII after linearizing the vector with SmaI/SrfI, and the ligation is carried out in the presence of restriction enzyme SrfI. The restriction enzyme in the ligation reaction recuts the self-ligated vector but not the recombinants, since ligation with foreign fragments destroys the enzyme recognition site. Dephosphorylation of inserts prevents their chimerization and ensures ligation of single insert per vector molecule. Thus, using the above strategy, which prevents self-ligation of both the insert and the vector, the overall cloning efficiency and, thereby the library size, is improved more than 10-fold compared to the standard blunt-end, ligation-based methods for making similar libraries. The library is further enriched by a single-step infection of E. coli by phages obtained from primary transformants. This step eliminates all the phages that carry insert that are not in-frame with gIIIp and therefore do not display gIIIp. We have shown the utility of the above system in constructing a glutathione-S-transferase (GST) gene-fragment library in phages and identifying the epitope recognized by a monoclonal antibody against GST.

Amino Acid Sequence↗
PubMed1999

Cysticercus immunoblot assay in patients with single, small enhancing lesions and multilesional neurocysticercosis.

A single, small (< 20 mm), ring or disc shaped contrast enhancing lesion located at the cortical-subcortical junction with minimal or no surrounding edema on computed tomography is the commonest mode of presentation of neurocysticercosis in the Indian subcontinent. Serum samples of 37 patients with these single, small enhancing lesions (SSEL's) and five patients with typical multilesional parenchymal neurocysticercosis were tested by the electro-immunoblot transfer (EITB) assay and the enzyme linked immunosorbent assay (ELISA). EITB was positive in 18 patients (48.64%) and ELISA was positive in 21 patients (56.76%) with SSEL's. On the other hand EITB was positive in all five patients (100%) and ELISA was positive in four patients (80%) with multilesional neurocysticercosis. The low sensitivity of the EITB in the SSEL's is probably linked to an insufficient immune stimulation provided by a single cysticercus cyst.

Humans↗
PubMed1998

Crystal structure of 2,5-diketo-D-gluconic acid reductase A complexed with NADPH at 2.1-A resolution.

The three-dimensional structure of Corynebacterium 2, 5-diketo-D-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-A resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate. Thus the three-dimensional structure has now been solved for a prokaryotic example of the aldo-keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of L-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-L-gulonate from D-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest.

Bacterial Proteins↗
PubMed1998

Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger.

Na+/H+ exchanger isoform 3 (NHE3), an epithelial brush border isoform of the Na+/H+ exchanger gene family, plays an important role in reabsorption of Na+ in the small intestine, the colon, and the kidney. In several cell types, phorbol 12-myristate 13-acetate (PMA) acutely inhibits NHE3 activity by changes in Vmax, but the mechanism of this inhibition is unknown. We investigated the role of subcellular redistribution of NHE3 in the PMA-induced inhibition of endogenous brush border NHE3 in a model human colon adenocarcinoma cell line, Caco-2. Subcellular localization of NHE3 was examined by confocal morphometric analysis complemented with cell surface biotinylation and compared with NHE3 activity evaluated by fluorometric measurement of intracellular pH. PMA inhibited NHE3 activity by 28% (p < 0.01), which was associated with a decrease of the ratio of the brush border/subapical cytoplasmic compartment of NHE3 from approximately 4.3 to approximately 2.4. This translocation resulted in 10-15% of the total cell NHE3 being shifted from the brush border pool to the cytoplasmic pool. These effects were mediated by protein kinase C, since they were blocked by the protein kinase C inhibitor H7. We conclude that inhibition of NHE3 by protein kinase C in Caco-2 cells involves redistribution of the exchanger from brush border into a subapical cytoplasmic compartment, and that this mechanism contributes approximately 50% to the overall protein kinase C-induced inhibition of the exchanger.

Caco-2 Cells↗