Applying malaria parasite's heme detoxification system for screening potential antimalarial drugs.
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Animals↗
Biomedical subjects
R Kannan
Publications and source records attributed to R Kannan.
Bloody diarrhea caused by a needle in the pancreatic head.
Bloody diarrhea in adolescents is not uncommon polyposis, inflammatory bowel disease, and infections are the usual causes. A 13-year-old boy with a metallic foreign body in the head of the pancreas presented with bloody diarrhea. The clinical problems and surgical approach are discussed.
Adolescent↗
Specific and instantaneous one-step chemodetection of histidine-rich proteins by Pauly's stain.
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Animals↗
Heme-artemisinin adducts are crucial mediators of the ability of artemisinin to inhibit heme polymerization.
A lack of molecular understanding of the targets and mechanisms of artemisinin action has impeded the improvisation of more efficient antimalarials based on this class of endoperoxide drugs. We have synthesized a heme-artemisinin adduct designated as "hemart" to discover if it mediates the ability of artemisinin to inhibit heme polymerization. Hemart mimics heme in binding to Plasmodium falciparum histidine-rich protein II (PfHRP II) but cannot self-polymerize. Instead, it inhibits all heme polymerizations, including basal and those triggered by PfHRP II, Monooleoyl glycerol (MOG), or P. yoelii extract. Hemart has an edge over heme in displacing heme from PfHRP II, and either low pH or chloroquine dissociates heme but not hemart from PfHRP II. Our results suggest that hemart, by mimicking heme, stalls all mechanisms of heme polymerization, resulting in the death of the malaria parasite.
Animals↗
Unexpected presentations--four cases of lead poisoning.
OBJECTIVE: To describe the clinical presentation and response to treatment of four patients who presented with abdominal pain and were diagnosed to have lead poisoning. METHODS: After ruling out the more obvious causes of abdominal pain by barium studies, gastrointestinal endoscopies, and biochemical studies, blood lead levels were estimated by atomic absorption spectrophotometry. The patients were treated with oral d-penicillamine. RESULTS: The four patients had blood lead levels from 79 microg/dL to 365 microg/dL. All four of them showed marked improvement in their clinical condition and lowering of blood lead levels on follow up. CONCLUSIONS: Clinicians need to develop a high index of suspicion of lead poisoning as a possible cause of unexplained abdominal pain or altered sensorium, especially against a background of environmental lead contamination in India.
Abdominal Pain↗
Crystal structure of the stromelysin-3 (MMP-11) catalytic domain complexed with a phosphinic inhibitor mimicking the transition-state.
Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP-11) whose proteolytic activity plays an important role in tumorigenicity enhancement. In breast cancer, ST3 is a bad prognosis marker: its expression is associated with a poor clinical outcome. This enzyme therefore represents an attractive therapeutic target. The topology of matrix metalloproteinases (MMPs) is remarkably well conserved, making the design of highly specific inhibitors difficult. The major difference between MMPs lies in the S(1)' subsite, a well-defined hydrophobic pocket of variable depth. The present crystal structure, the first 3D-structure of the ST3 catalytic domain in interaction with a phosphinic inhibitor mimicking a (d, l) peptide, clearly demonstrates that its S(1)' pocket corresponds to a tunnel running through the enzyme. This open channel is filled by the inhibitor P(1)' group which adopts a constrained conformation to fit this pocket, together with two water molecules interacting with the ST3-specific residue Gln215. These observations provide clues for the design of more specific inhibitors and show how ST3 can accommodate a phosphinic inhibitor mimicking a (d, l) peptide. The presence of a water molecule interacting with one oxygen atom of the inhibitor phosphinyl group and the proline residue of the Met-turn suggests how the intermediate formed during proteolysis may be stabilized. Furthermore, the hydrogen bond distance observed between the methyl of the phosphinic group and the carbonyl group of Ala182 mimics the interaction between this carbonyl group and the amide group of the cleaved peptidic bond. Our crystal structure provides a good model to study the MMPs mechanism of proteolysis.
Amino Acid Sequence↗
Glutathione transport in human retinal pigment epithelial (HRPE) cells: apical localization of sodium-dependent gsh transport.
The study was undertaken to identify and localize GSH transport in non-transformed cultured human retinal pigmented epithelial cells (HRPE). In confluent monolayers exhibiting high transepithelial resistance (TER 700-1000 Omega cm(-2)), apical and basolateral GSH uptake were determined after introducing(35)S-GSH (+ 1 m M GSH) to the apical side or basal side in NaCl (Na+ -containing) or choline chloride (Na+ -free) buffers. Cells in growth medium or in incubation buffers were pretreated with acivicin to inhibit gamma-glutamyltranspeptidase (GGT). GSH efflux was measured after labelling the intracellular GSH pool by incubation overnight with 35 S-cysteine and quantitating the release of labelled GSH into the medium. Uptake of GSH was found at both the apical and basolateral membranes of HRPE cells. Inhibition of gamma-glutamyltranspeptidase (GGT) with acivicin did not alter mean GSH uptake (nmol per million cells per 30 min) significantly at the apical (1.63 +/- 0.32 vs 1.45 +/- 0.30; with and without acivicin respectively) or the basolateral (1.17 +/- 0.21 vs 1.44 +/- 0.38) membranes. Transport was verified to be in the form of intact GSH by HPLC. Uptake was unaffected by the removal of Na+ at the basolateral membrane while apical uptake exhibited partial but significant (approximately 40%) Na+ -dependency. Net GSH efflux (nmol per million cells per min) to the apical side of HRPE cells was higher than to the basolateral side in the presence of sodium. Transepithelial flux in the basolateral to apical direction was approximately 17-fold higher than the apical to basolateral direction resulting in a net flux of GSH to the apical side. In conclusion, HRPE cells exhibit GSH transport by Na+ -dependent and Na+ -independent mechanisms. The Na+ -dependent GSH transporter is localized to the apical membrane of HRPE cells.
Biological Transport↗
Vitamin C transport in human lens epithelial cells: evidence for the presence of SVCT2.
Vitamin C [ascorbic acid (AA)] is an important antioxidant present in m M amounts in the aqueous humor. Recently, two specific transporters for vitamin C (SVCT1, SVCT2) have been cloned in the rat and the human. The aim of the present study was to characterize vitamin C transport in an immortalized human lens epithelial cell line (HLE-B3). AA uptake was linear for 120 min in experiments conducted with 14C AA + 40 microM unlabelled AA. Uptake was measured at varying AA concentrations (0.04-1 m M) in Na+-containing and Na+-free buffers for 30 min at 37 degrees C. Effect of potential inhibitors of AA transport was also examined. Presence (or absence) of SVCT1 and SVCT2 was studied by RT-PCR of HLE-B3 poly (A)+ RNA using gene specific primers. Uptake studies revealed that AA uptake was highly Na+-dependent and exhibited saturation. Na+-dependent 14C-AA uptake was strongly inhibited (85-90%) by 10 m M unlabelled AA. Incubation of HLE-B3 cells with cAMP (0.1 m M), cytocholasin B (0.1 m M) and phorbol dibutyrate (1 microM) resulted in partial inhibition (36-51%) of AA uptake. Under similar conditions, D -glucose (10 m M) and staurosporine (0.1 microM) had no effect. RT-PCR showed the presence of SVCT2 while SVCT1 could not be amplified. Exposure to the chemical oxidant tert-butylhydroperoxide (TBH) up-regulated SVCT2 gene expression in HLE-B3 cells. Our data suggest that Na+-dependent transport of AA in normal lens epithelium is most likely mediated by SVCT2 rather than by SVCT1. This transport system may be subject to regulation by oxidant stress and by various second messenger signals.
Antioxidants↗
Human pS2/trefoil factor 1: production and characterization in Pichia pastoris.
The recombinant protein human trefoil factor 1 (hTFF1), formerly called hpS2, has been produced for the first time in a yeast-based expression in Pichia pastoris. hTFF1 was secreted in large amounts in the extracellular medium of P. pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The fermentation broth containing hTFF1 was concentrated by tangential flow filtration prior to purification by anion- and cation-exchange chromatography, followed by preparative high-performance liquid chromatography. The resulting hTFF1 was found to be intact by Western blot analysis. Further analysis revealed mainly the presence of the monomeric form of the hTFF1 peptide. Finally, in vitro, the recombinant hTFF1 was shown to decrease proliferation of the HCT116 cancer cells.
Amino Acid Sequence↗
Regulation of GSH in alphaA-expressing human lens epithelial cell lines and in alphaA knockout mouse lenses.
PURPOSE: To study the mechanism of regulation of GSH in HLE-B3 cells expressing alphaA-crystallin (alphaA) and in alphaA knockout mouse lenses. METHODS: GSH levels and maximal rates of GSH synthesis were measured in immortalized, alphaA-transfected HLE-B3 cells containing varying amounts of alphaA. The mRNA and protein for the rate-limiting enzyme for GSH synthesis, gamma-glutamylcysteine synthetase (GCS), were also determined in alphaA- and mock-transfected cells by Northern blot analysis and Western blot analysis of heavy (GCS-HS) and light (GCS-LS) subunits. The effect of absence of alphaA and alphaB on lens GSH concentrations was evaluated in whole lenses of alphaA knockout and alphaB knockout mice as a function of age. GCS-HS mRNA and protein were determined in young, precataractous and cataractous alphaA knockout lenses. RESULTS: GSH levels were significantly higher in HLE-B3 cells expressing alphaA- compared with mock-transfected cells and were correlated positively with alphaA content. Mean rate of GSH synthesis was also higher in alphaA-expressing cells than in mock controls (0.84 vs. 0.61 nmol. min(-1) per mg protein, respectively). GCS-HS mRNA and GCS-LS mRNA were approximately twofold higher in alphaA-expressing cells, whereas the heavy and light GCS subunit proteins increased by 80% to 100%. In alphaA(-/-) mouse lenses, GSH level was not different from that of wild type up to 2 months from birth, after which it dropped to approximately 50% of controls. On the other hand, GCS-HS and GCS-LS proteins showed a significant decrease before cataract formation as early as 15 days after birth. GSH level in cataract-free alphaB(-/-) lenses was similar to that of wild type for up to 14 months. CONCLUSIONS: Expression of alphaA caused an increase in cellular GSH, in part, because of an increase in mRNA and protein of both GCS subunits. GSH levels decreased with increasing age in cataractous alphaA(-/-) lenses but not in the noncataractous alphaB(-/-) lenses. It is suggested that neonatal precataractous lenses (with normal GSH and decreased GCS) may maintain their GSH level by other compensatory mechanisms such as increased GSH transport.
Animals↗
Neuroblastoma in an adolescent girl.
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Adolescent↗
Angioplasty of membranous obstruction of inferior vena cava.
Membranous obstruction of vena cava (MOVC) is a common cause of hepatic venous outflow obstruction. Surgical procedures utilized to relieve symptoms carry a high morbidity and mortality. A patient who presented with gradual onset ascites and dilated, tortuous veins over the anterior abdominal wall and the back was found to have MOVC by inferior vena cavogram. Balloon dilatation of the obstruction was done successfully through a transvenous approach. Following this, the patient improved remarkably. The procedure is safe, effective and easy to perform. This balloon angioplasty provides alternative method for treatment of membranous obstruction of vena cava.
Adult↗
Streptococcus agalactiae endocarditis.
Streptococcus agalactiae (S. agalactiae) is a rare cause of infective endocarditis, which is associated with a high mortality rate. Endocarditis in adults is generally related to immunocompromised states. We hereby report the case of a 35 year old man who presented with fever and delirium in whom aortic valve endocarditis due to S. agalactiae was detected. Though most patients with S. agalactiae endocarditis need surgical intervention along with antibiotics, our patient improved with medical therapy alone.
Adult↗
Demonstration in vivo that stromelysin-3 functions through its proteolytic activity.
Stromelysin-3 (ST3), a matrix metalloproteinase (MMP) expressed in aggressive carcinomas, has been shown to promote tumor development in different in vivo experimental models. However, the inability of its mature form to degrade extracellular matrix components casts doubt on whether ST3 functions in vivo as a protease. In this study, we evaluated whether the ST3 tumor-promoting effect could be ascribed to its proteolytic activity and whether this putative protease could be targeted with MMP inhibitors. Catalytically inactive mutant cDNA of human (h) ST3 or mouse (m) ST3 were generated and transfected into MCF7 cells. When injected into nude mice in the presence of matrigel, the mutant-bearing cells did not exhibit the enhanced tumorigenicity elicited by MCF7 cells transfected with wild-type ST3 cDNA. In a second approach, TIMP2 overproduction in MCF7 cells expressing hST3 was induced by retroviral infection. The co-expression of ST3 and TIMP2 failed to enhance the tumorigenicity of MCF7 cells. Notably, matrigel depleted of low-molecular-weight proteins and growth factors failed to promote the tumorigenicity of ST3-expressing MCF7 cells. These findings provide the first in vivo evidence that ST3 is indeed a protease that can modulate cancer progression by remodeling extracellular matrix and probably by inducing it to release the necessary microenvironmental factors. Thus, ST3 represents an interesting target for specific MMP inhibition.
Adenocarcinoma↗
GSH transport in human cerebrovascular endothelial cells and human astrocytes: evidence for luminal localization of Na+-dependent GSH transport in HCEC.
The purpose of the present study was to identify and localize glutathione (GSH) transport in an in vitro tissue culture model of blood-brain barrier (BBB). The localization of Na+-dependent GSH transport in an immortalized cell line of human cerebrovascular endothelial cells (HCEC) and asymmetry of transport in Transwell studies were investigated. Initial studies with cultured HCEC established a significant (45%) Na+-dependency for GSH uptake in cultured HCEC pretreated with acivicin, an inhibitor of gamma-glutamyltranspeptidase (GGT). Transendothelial electrical resistance (TEER) and uptake of [35S]GSH from luminal and abluminal fluids of HCEC were measured in Na+-containing and Na+-free (choline chloride) buffers using cells grown on gelatin-coated membrane filters. TEER of HCEC monolayers in regular medium was 40.1 +/- 8.0 ohms cm2. Human astrocyte-conditioned medium (ACM) caused no change in TEER, but increased GGT activity approximately threefold when measured in cell lysates. Luminal and abluminal GSH uptake increased in a time-dependent fashion and were not affected by inhibition of GGT activity with acivicin. Sodium dependency was only observed for luminal uptake (Na+-containing 2.41 +/- 0.15 vs. Na+-free 0.96 +/- 0.03 pmol/30 min/million cells, p < 0.001) but not for abluminal uptake (1.02 +/- 0.13 vs. 1.11 +/- 09, p > 0.05). Apparent efflux via the luminal membrane was lower in the presence of sodium as compared to that without sodium, further suggesting that a Na+-dependent uptake process for GSH is operative at this membrane. GSH uptake and efflux were also demonstrated in neonatal rat and fetal human astrocytes, both exhibiting partial Na+-dependency of uptake. In conclusion, our results show for the first time, that HCEC and astrocytes take up GSH by both Na+-dependent and -independent mechanisms. The Na+-dependent GSH transport process in HCEC appears to be localized to luminal plasma membranes of HCEC.
Animals↗
Role of the S1' subsite glutamine 215 in activity and specificity of stromelysin-3 by site-directed mutagenesis.
The influence of Gln215 in stromelysin-3 (MMP-11), a residue located in the S1' subsite, was determined by producing three single mutants of this position. As compared to wild-type stromelysin-3, the kinetic parameters K(M) and k(cat) for the degradation of the fluorogenic substrate Dns-Pro-Leu-Ala-Leu-Trp-Ala-Arg-NH(2) (Dns-Leu) by these mutants indicated that the Gln/Leu substitution led to a 4-fold decrease in catalytic efficiency, whereas the mutations Gln/Tyr and Gln/Arg increased this parameter by a factor 10. The cleavage of alpha1-protease inhibitor (alpha1-PI), a natural substrate of stromelysin-3, by these mutants was also determined. Their relative activities for the degradation of alpha1-PI correspond to those observed with the synthetic substrate Dns-Leu. The catalytic efficiency of wild-type stromelysin-3 and its mutants to cleave the P1' analogue of Dns-Leu, containing the unusual amino acid Cys(OMeBn) (Dns-Cys(OMeBn)), was also determined. The values of the specificity factor, calculated as the ratio (k(cat)/K(M))Dns-Cys(OMeBn))/(k(cat)/K(M))Dns-Leu, were observed to vary from 26 for the wild-type stromelysin-3 to 120 for the Gln/Leu mutant and 25 for the Gln/Arg mutant. The Gln/Tyr mutant did not cleave the substrate when its P1' position is substituted by the unusual amino acid Cys(OMeBn). Altogether these observations established that both the catalytic activity and the specificity of stromelysin-3 are dependent on the nature of the residue in position 215. Finally, the cleavage efficiency of the Dns substrates by three representative matrixins, namely, MMP-14 (215 = Leu), MMP-1 (215 = Arg), and MMP-7 (215 = Tyr), was determined. Interestingly, the trends observed for these enzymes were similar to those established for the three mutants of stromelysin-3, pointing out the influence of position 215 toward the selectivity in this family of enzymes.
Animals↗
Phosphinic pseudo-tripeptides as potent inhibitors of matrix metalloproteinases: a structure-activity study.
Several phosphinic pseudo-tripeptides of general formula R-XaaPsi(PO(2)-CH(2))Xaa'-Yaa'-NH(2) were synthesized and evaluated for their in vitro activities to inhibit stromelysin-3, gelatinases A and B, membrane type-1 matrix metalloproteinase, collagenases 1 and 2, and matrilysin. With the exception of collagenase-1 and matrilysin, phosphinic pseudo-tripeptides behave as highly potent inhibitors of matrix metalloproteinases, provided they contain in P(1)' position an unusual long aryl-alkyl substituent. Study of structure-activity relationships regarding the influence of the R and Xaa' substituents in this series may contribute to the design of inhibitors able to block only a few members of the matrix metalloproteinase family.
Kinetics↗
Protection from oxidant injury by sodium-dependent GSH uptake in retinal Müller cells.
Glutathione (GSH) is known to play an important role in regulating oxidative damage to cells. The present study was initiated to examine the effect of exogenous GSH on oxidative injury in a retinal Müller cell line and to characterize GSH transport in these cells. Rat Müller cells (rMC-1) were incubated with varying concentrations of t-butylhydroperoxide (t-BHP) to induce oxidative stress, and cell viability was measured after addition of GSH. In other studies, kinetics of GSH uptake and Na+-dependency were examined by incubating cells with35S-GSH in Na+-containing and Na+-free buffers. GSH uptake was studied with GSH at concentrations varying from 0. 05-10 m m in NaCl buffer. In the presence of sodium, extracellular GSH provided protection against t-BHP-induced oxidant injury to rMC-1 cells; in contrast, the amino acid precursors of GSH did not have any effect on cell viability. GSH was taken up by rMC-1 cells in a concentration- and sodium-dependent manner. Kinetic studies revealed both a high affinity (Km approximately 0.31 m m) and low affinity Km( approximately 4.2 m m) component. Furthermore, GSH depletion had no significant effect on the rate of GSH uptake. The results show that physiological concentrations of GSH can protect Müller cells from oxidative injury. Both Na+-dependent and Na+-independent transport systems for GSH exist in Müller cells, and the Na+-dependent GSH transporter may be involved in the protective role of GSH.
Animals↗