Prohibition of alcohol in India.
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Alcohol Drinking↗
Biomedical subjects
S Bhattacharya
Publications and source records attributed to S Bhattacharya.
Light-stable rhodopsin. II. An opsin mutant (TRP-265----Phe) and a retinal analog with a nonisomerizable 11-cis configuration form a photostable chromophore.
In order to prepare a completely light-stable rhodopsin, we have synthesized an analog, II, of 11-cis retinal in which isomerization at the C11-C12 cis-double bond is blocked by formation of a cyclohexene ring from the C10 to C13-methyl. We used this analog to generate a rhodopsin-like pigment from opsin expressed in COS-1 cells and opsin from rod outer segments (Bhattacharya, S., Ridge, K.D., Knox, B.E., and Khorana, H. G. (1992) J. Biol. Chem. 267, 6763-6769). The pigment (lambda max, 512 nm) formed from opsin and analog II (rhodospin-II) showed ground state properties very similar to those of rhodopsin, but was not entirely stable to light. In the present work, 12 opsin mutants (Ala-117----Phe, Glu-122----Gln(Ala, Asp), Trp-126----Phe(Leu, Ala), Trp-265----Ala(Tyr, Phe), Tyr-268----Phe, and Ala-292----Asp), where the mutations were presumed to be in the retinal binding pocket, were reconstituted with analog II. While all mutants formed rhodopsin-like pigments with II, blue-shifted (12-30 nm) chromophores were obtained with Ala-117----Phe, Glu-122----Gln(Ala), Trp-126----Leu(Ala), and Trp-265----Ala(Tyr, Phe) opsins. The extent of chromophore formation was markedly reduced in the mutants Ala-117----Phe and Trp-126----Ala. Upon illumination, the reconstituted pigments showed varying degrees of light sensitivity; the mutants Trp-126----Phe(Leu) showed light sensitivity similar to wild-type. Continuous illumination of the mutants Glu-122----Asp, Trp-265----Ala, Tyr-268----Phe, and Ala-292----Asp resulted in hydrolysis of the retinyl Schiff base. Markedly reduced light sensitivity was observed with the mutant Trp-265----Tyr, while the mutant Trp-265----Phe was light-insensitive. Consistent with this result, the mutant Trp-265----Phe showed no detectable light-dependent activation of transducin or phosphorylation by rhodopsin kinase.
Amino Acid Sequence↗
A bacteriorhodopsin analog reconstituted with a nonisomerizable 13-trans retinal derivative displays light insensitivity.
With the aim of preparing a light-insensitive bacteriorhodopsin-like pigment, bacterio-opsin expressed in Escherichia coli was treated in phospholipid-detergent micelles with the retinal analog II, in which the C13-C14 trans-double bond cannot isomerize due to inclusion in a cyclopentene ring. The formation of a complex with a fine structure (lambda max, 439 nm) was first observed. This partially converted over a period of 12 days to a bacteriorhodopsin-like chromophore (ebR-II) with lambda max, 555 nm. An identical behavior has been observed previously upon reconstitution of bleached purple membrane with the analog II. Purification by gel filtration gave pure ebR-II with lambda max, 558 nm, similar to that of light-adapted bacterio-opsin reconstituted with all-trans retinal (ebR-I). Spectrophotometric titration of ebR-II as a function of pH showed that the purple to blue transition of bacteriorhodopsin at acidic pH was altered, and the apparent pKa of Schiff base deprotonation at alkaline pH was lowered by 2.4 units, relative to that of ebR-I. ebR-II showed no light-dark adaptation, no proton pumping, and no intermediates characteristic of the bacteriorhodopsin photocycle. In addition, the rates of reaction with hydroxylamine in the dark and in the light were similar. These results show, as expected, that isomerization of the C13-C14 double bond is required for bacteriorhodopsin function and that prevention of this isomerization confers light insensitivity.
Bacteriorhodopsins↗
Light-stable rhodopsin. I. A rhodopsin analog reconstituted with a nonisomerizable 11-cis retinal derivative.
With the aim of preparing a light-stable rhodopsin-like pigment, an analog, II, of 11-cis retinal was synthesized in which isomerization of the C11-C12 cis-double bond is blocked by a cyclohexene ring built around the C10 to C13-methyl. The analog II formed a rhodopsin-like pigment (rhodopsin-II) with opsin expressed in COS-1 cells and with opsin from rod outer segments. The rate of rhodopsin-II formation from II and opsin was approximately 10 times slower than that of rhodopsin from 11-cis retinal and opsin. After solubilization in dodecyl maltoside and immunoaffinity purification, rhodopsin-II displayed an absorbance ratio (A280nm/A512nm) of 1.6, virtually identical with that of rhodopsin. Acid denaturation of rhodopsin-II formed a chromophore with lambda max, 452 nm, characteristic of protonated retinyl Schiff base. The ground state properties of rhodopsin-II were similar to those of rhodopsin in extinction coefficient (41,200 M-1 cm-1) and opsin-shift (2600 cm-1). Rhodopsin-II was stable to hydroxylamine in the dark, while light-dependent bleaching by hydroxylamine was slowed by approximately 2 orders of magnitude relative to rhodopsin. Illumination of rhodopsin-II for 10 s caused approximately 3 nm blue-shift and 3% loss of visible absorbance. Prolonged illumination caused a maximal blue-shift up to approximately 20 nm and approximately 40% loss of visible absorbance. An apparent photochemical steady state was reached after 12 min of illumination. Subsequent acid denaturation indicated that the retinyl Schiff base linkage was intact. A red-shift (approximately 12 nm) in lambda max and a 45% recovery of visible absorbance was observed after returning the 12-min illuminated pigment to darkness. Rhodopsin-II showed marginal light-dependent transducin activation and phosphorylation by rhodopsin kinase.
Electrophoresis, Polyacrylamide Gel↗
Quantitative 111In antimyosin antibody imaging to predict the age of myocardial infarction.
To establish whether quantitative 111In antimyosin uptake can be used to predict infarct age, we studied the heart-lung ratio in 107 images from 90 patients at various intervals following a Q-wave infarction. Imaging was performed 24 hours following 111In antimyosin injection. The HLR was measured as the ratio of the maximum counts in the infarcted myocardium to the adjacent lung background. The ratio ranged from 1.26 to 3.87, and declined with increasing infarct age. Infarcts were classified on the basis of age as type I (less than 3 days old), type II (less than 14 days), and type III (less than 90 days). True positive and false positive rates (TPR and FPR), and test-likelihood ratio calculations were performed for HLR thresholds ranging from 1 to 4, for the three infarct types. A FPR of 0% and likelihood ratio of infinity was obtained at a HLR threshold of 2.3 for type I infarcts (TPR 40.8%); at a HLR threshold of 2 for type II infarcts (TPR 50.6%), and a threshold of 1.8 for type III infarcts (TPR 52.6%). The likelihood of each infarct type can be estimated directly from the HLR for values below the above thresholds. These results show that quantitative 111In-antimyosin imaging may be used to predict infarct age.
Antibodies, Monoclonal↗
Entamoeba histolytica extrachromosomal circular ribosomal DNA: analysis of clonal variation in a hypervariable region.
The ribosomal RNA genes of the protozoan parasite Entamoeba histolytica are highly repeated and display restriction fragment length polymorphism. Using a set of four DNA probes spanning the coding region and part of the flanking region of the E. histolytica ribosomal RNA genes, an analysis of the DNA bands generated by EcoRI digestion of Entamoeba DNA is presented. This analysis included five strains of E. histolytica, four strains of E. moshkovskii, and one strain each of E. invadens and E. terrapinae. No common bands were observed between E. histolytica and the other Entamoeba. Within E. histolytica, two bands were conserved in all strains while the others were polymorphic. Detailed analysis of DNA from independently isolated clones of the strain HM-1:IMSS of E. histolytica showed two bands to be highly polymorphic. Of these, the 4.4-kb band of clone 6 was further analyzed. Polymorphism in this band could even be demonstrated in cells of the same clone. Restriction enzyme analysis of this DNA band from two clones of HM-1:IMSS showed that the polymorphism may be due to variable numbers of DraI repeat units present in this DNA stretch.
Animals↗
A second short repeat sequence detected downstream of rRNA genes in the Entamoeba histolytica rDNA episome.
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Animals↗
Recognition of Entamoeba histolytica lipophosphoglycan by a strain-specific monoclonal antibody and human immune sera.
Western blot analysis showed that the monoclonal antibody 2D7.10 recognized lipophosphoglycan (LPG) from Entamoeba histolytica HM-1:IMSS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of [3H]galactose-labeled LPG and Western blot analysis of total lysate of E. histolytica with 2D7.10 revealed patterns similar to that of LPG with 2D7.10. This antibody could also immunoprecipitate purified LPG from the strain HM-1:IMSS after biosynthetically labeling with [3H]galactose and [32P]orthophosphate. However, no immunoprecipitation was observed when 2D7.10 was incubated with [32P]orthophosphate-labeled purified LPG from strain 200:NIH. Sera from patients suffering from invasive amoebiasis also immunoprecipitated 32P-labeled, purified LPG and could immunostain this molecule in Western blots. The human immune sera recognized carbohydrate epitopes but not the associated polypeptides of LPG, as evidenced by sensitivity to periodate digestion, mild acid hydrolysis but not to pronase treatment. It was earlier shown that 2D7.10 binds a carbohydrate epitope in a subset of axenized pathogenic strains of E. histolytica and that this epitope undergoes changes when cultured along with bacteria. These observations suggest that the E. histolytica LPG contains a strain-specific, variable epitope and that LPG is immunogenic in human.
Animals↗
Management of burn contractures of the first web space of the hand.
First web space contractures of the hand affect predominantly the movements at the trapeziometacarpal joint, rendering the thumb ineffective for opposition and thereby destroying the essential elements of grasp and pinch. Adequate release of contractured first web space followed by a local skin flap cover and dynamic abduction splintage for the subsequent 6 weeks gives excellent results. Documenting the first web angle at every follow-up visit can detect early recurrences and the dynamic splintage can be reinstituted to achieve the normal first web space. Secondary surgery in the form of flexor pollicis longus tendon lengthening or opponens-plasty helps to prevent recurrences.
Burns↗
Molecular genetics of inherited retinal degenerations.
There has recently been substantial progress in categorizing the vast range of human retinal degeneration phenotypes. A molecular approach has assigned chromosomal locations for approximately a dozen such diseases and has identified four of the genes involved.
Albinism, Ocular↗
A completed screen for mutations of the rhodopsin gene in a panel of patients with autosomal dominant retinitis pigmentosa.
Recently it has been demonstrated that some families with autosomal dominant retinitis pigmentosa (adRP) have mutations in the rhodopsin gene while others do not. Previously we have identified six such mutations in seven adRP families in this laboratory, one of which was previously described in US patients. We now present a completed screen of the rhodopsin gene in a panel of 39 adRP families, by a rapid screening technique which will be of use for routine diagnosis. Nine different mutations were ultimately found, in a total of twelve of the 39 families. These include the six previously identified mutations, in codons 68-71, 190, 211, 255, 296 and 347, two new ones in codons 53 and 106, and another mutation first identified in a single US patient, in codon 58. Thus approximately 30% of adRP families have 'Rhodopsin RP' while the remainder probably have a defect elsewhere in the genome. Of those families in which rhodopsin mutations have been found, four have been classified D type, three as sectorial RP and the remainder are of uncertain classification. All families excluded from chromosome 3q by linkage have been classified R type. These data suggest a correlation between clinical sub-classification and the underlying rhodopsin/non-rhodopsin heterogeneity.
Amino Acid Sequence↗
Torsade de pointes and long QT syndrome following major blood transfusion.
We report a rare complication following transfusion of a large volume of blood in an adult patient. An electrophysiological disturbance of the cardiac cycle with prolongation of the QT interval developed, which was followed by recurrent episodes of torsade de pointes, a unique form of ventricular tachycardia. The most likely cause of this acquired long QT syndrome was hypomagnesaemia secondary to massive blood transfusion. Treatment with a magnesium infusion restored the QT interval to normal and temporary ventricular pacing prevented further ventricular arrhythmias.
Adult↗
The influence of therapeutic blocking of Gp IIb/IIIa on platelet alpha-granular fibrinogen.
Recent evidence suggests that platelet alpha-granule fibrinogen (fg) is derived from the plasma pool. Since platelets from patients with Type I Glanzmann's thrombasthenia (GT) are deficient in intracellular fibrinogen (fg) it was hypothesized that Gp IIb/IIIa could mediate the uptake of fg. To study the potential role of Gp IIb/IIIa in intracellular fg trafficking, the influence of therapeutic blocking of Gp IIb/IIIa on platelet fg was studied in 12 patients with stable ischaemic heart disease. Patients were either given a single intravenous dose of the monoclonal antibody 7E3 Fab (n = 4) or a combination of bolus and continuous infusion up to 24 (n = 3), 36 (n = 3) or 96 h (n = 2). All patients showed grossly prolonged bleeding times with a significant reduction of ex-vivo ADP induced aggregation. Although, surface Gp IIb/IIIa binding sites were consistently reduced in all patients, there was a variable but delayed decrease in platelet fg relative to vWf:Ag in only six out of the 12 patients studied. The reduction in fg appeared dependent upon both dosage and duration of Gp IIb/IIIa blockade. The study provides further evidence for the novel role of Gp IIb/IIIa in the intracellular trafficking of fg to platelet and megakaryocytic alpha-granules.
Adult↗
Modulation of a surface antigen of Entamoeba histolytica in response to bacteria.
Changes in the cell surface of Entamoeba histolytica, a human intestinal parasite and the causative agent of amebic dysentery, were examined with a monoclonal antibody, 2D7.10, which selectively recognizes carbohydrate epitopes in some axenic amebic strains. While high-level expression of this epitope was observed in axenic amebae, it was either absent or present only in small amounts in xenic amebae. Furthermore, reassociation of the axenic amebae with intestinal flora resulted in loss of the 2D7.10 epitope. Our data suggest that surface antigens of E. histolytica can be modulated in response to bacteria and may provide an explanation for the observed influence of bacteria on amebic virulence.
Animals↗
Segmental vascular responses to voltage-gated calcium channel potentiation in rat lung.
We determined lung vascular responses to voltage-gated Ca2+ channel potentiation with BAY K 8644 (BAY). We anesthetized 46 rats (Sprague-Dawley; halothane and pentobarbital) and then excised and perfused their lungs at constant blood flow of 25 +/- 2 (SE) ml.kg-1.min-1 at constant airway and left atrial pressures of 5 and 6 cmH2O, respectively. Pulmonary arterial pressure (Ppa) increased from 13.3 +/- 0.3 cmH2O at baseline to 17.3 +/- 1.3 cmH2O after BAY (2.8 x 10(-6) M; n = 5; P less than 0.01). As determined by micropuncture, arteriolar and venular (Pven) pressures did not change. Increase of perfusate Ca2+ (16 x 10(-3) M; n = 8) similarly increased Ppa. NG-mono-methyl-L-arginine (2 x 10(-4) M), an inhibitor of endothelium-derived relaxing factor, augmented the pressor effect of BAY when given after (n = 4) but not before (n = 4) BAY (P less than 0.01). Prior cyclooxygenase blockade with indomethacin (5 mg/kg; n = 5) attenuated the Ppa response to BAY (P less than 0.01). None of these agents changed Pven. To confirm vasoactivity in veins, we induced smooth muscle depolarization with KCl (20 x 10(-3) M; n = 6) and receptor-mediated responses with histamine (3 x 10(-4) M; n = 7). Both of these agents increased Pven markedly (P less than 0.01). We interpret that, in rat lung, BAY causes arterial but not venous constriction, because the venous segment differs from the arterial with regard to Ca2+ channel potentiation.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Binding of radiolabelled luteinizing hormone to intact and ovariectomised rat uterus.
Binding of ovine LH to uterine tissue preparation from intact and ovariectomised rat clearly indicates that uterus possesses specific binding sites for LH. Binding characteristics of LH to uterine tissue preparation from intact rat showed saturability with high affinity and low capacity. Scatchard plot analysis showed dissociation constant of the specific binding site to be 0.12 x 10(-9) mol/l and the number of binding sites was 2.31 +/- 0.05 f mol/mg protein. Ovariectomy did not change the binding affinity but effected a decrease in the number of binding sites (1.7 +/- 0.08 f mol/mg protein). LH treatment of ovariectomized (ovx) rat had no effect on binding affinity but significantly increased the number of binding sites (3.23 +/- 0.1 f mol/mg protein). Reduction of uterine weight due to ovariectomy and marked increase of ovx rat uterine weight by LH administration indicate a source of estrogen in ovx rat. An in vitro uterine tissue slice (from intact and ovx rat) incubation showed depletion of 17 beta-estradiol (E2) content in ovx rat which significantly elevated on LH addition. Data suggest that LH binding to rat uterine tissue has biological relevance.
Animals↗
Purification of C-reactive protein from Channa punctatus (Bloch).
C-reactive protein (CRP) is found to be a normal component of serum of freshwater air-breathing murrel Channa punctatus. Based on the property of binding with C-polysaccharide (CPS) of pneumococcus bacteria in presence of Ca2+, CRP was purified by phosphorylcholine-Sepharose affinity column chromatography. Molecular weight of the intact protein molecule was estimated to be approximately 141,000 by gel filtration. In non-reduced and reduced conditions the molecule showed molecular weight approximately 28,000 and 14,000 respectively in SDS-PAGE. Monospecific antisera was raised against the affinity purified CRP and used as a tool to detect CRP in the hepatic cytosol and egg extract. The level of CRP in the normal serum was estimated to be 220 micrograms/ml.
Animals↗
Responsiveness to gonadotropin releasing hormone of human term trophoblast cells in vitro: induction by estradiol.
Human trophoblast cells isolated from term placentae by enzymatic treatment and maintained in culture, spontaneously secrete hCG after 72hrs but fail to increase their gonadotropin secretion in response to exogenous GnRH. Treatment with estradiol for 24 hrs prior to challenge with GnRH induces the responsiveness to GnRH stimulation. This action of estradiol on trophoblast cells in independent of their stage of differentiation. The competitive displacement analysis of [125I] GnRH binding to estradiol-primed trophoblast cells indicated a single class of low affinity sites with Ka of 1.6 x 10(6) M-1, similar to that observed previously in placental membranes. To our knowledge this is the first demonstration of induction of GnRH receptors by estradiol in human trophoblast cells.
Binding, Competitive↗