PubMed Health⌕ Search

Biomedical subjects

R Kasturi

Publications and source records attributed to R Kasturi.

18 recordsLinked to original sources
PubMed2000

Selection of peptidic mimics of digoxin from phage-displayed peptide libraries by anti-digoxin antibodies.

Since the initial report of the development of methodology to generate high-affinity digitalis-specific (digoxin) antibodies, these antibodies have proven extremely useful tools to monitor digoxin levels in digitalized patients and, as Fab fragments, to reverse toxic digoxin effects in life-threatening digoxin overdoses. These antibodies (both digoxin-specific and ouabain-specific) have been used extensively by investigators for the identification and characterization of putative endogenous digitalis-like factors. In this study, we used two well-characterized mouse anti-digoxin monoclonal antibodies (mAbs), designated 26-10 and 45-20, as binding templates with which to select short bacteriophage-displayed (pIII protein inserted) peptides that are capable of binding to these mAbs and mimicking the conformational structure of digoxin. Selective enrichment from two phage-displayed random peptide libraries enabled us to isolate and identify distinct 15 and 26 amino acid residue peptide inserts that bind with high avidity and idiotypic specificity to the selecting mAbs. Among these displayed inserts a subset was identified whose mAb binding is inhibited by digoxin and whose corresponding synthetic peptides inhibit phage binding. They, therefore, appear to bind at the mAbs digoxin-binding sites. These data provide the first clear evidence that short polypeptides can serve as surrogates for the low molecular mass hapten digoxin.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of human monoclonal antibodies to digoxin.

Fab preparations of sheep polyclonal anti-digoxin Abs have proven useful for reversal of the toxic effects of digoxin overdoses in patients. Unfortunately, the use of foreign species proteins in humans is limited because of the potential for immunological responses that include hypersensitivity reactions and acute anaphylaxis. Immunization of recently developed transgenic mice, whose endogenous micro heavy and kappa light chain Ig genes are inactivated and which carry human Ig gene segments, with a digoxin-protein conjugate has enabled us to generate and isolate eight hybridoma cell lines secreting human sequence anti-digoxin mAbs. Six of the mAbs have been partially characterized and shown to have high specificity and low nanomolar affinities for digoxin. In addition, detailed competition binding studies performed with three of these mAbs have shown them to have distinct differences in their digoxin binding, and that all three structural moieties of the drug, the primary digitoxose sugar, steroid, and five-member unsaturated lactone ring, contribute to Ab recognition.

Animals↗
PubMed1998

Identification of a model cardiac glycoside receptor: comparisons with Na+,K+-ATPase.

The availability of high-affinity anti-digoxin monoclonal antibodies (mAbs) offers the potential for their use as models for the characterization of the relationship between receptor structure and cardiac glycoside binding. We have characterized the binding of anthroylouabain (AO), a fluorescent derivative of the cardiac glycoside ouabain, to mAbs 26-10, 45-20, and 40-50 [Mudgett-Hunter, M., et al. (1995) Mol. Immunol. 22, 477] and lamb kidney Na+, K+-ATPase by monitoring the resultant AO fluorescence emission spectra, anisotropy, lifetime values, and Förster resonance energy transfer (FRET) from protein tryptophan(s) (Trp) to AO. These data suggest that the structural environment in the vicinity of the AO-binding site of Na+,K+-ATPase is similar to that of mAb 26-10 but not mAbs 45-20 and 40-50. A model of AO complexed to the antigen binding fragment (Fab) of mAb 26-10 which was generated using known X-ray crystal structural data [Jeffrey, P. D., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10310] shows a heavy chain Trp residue (Trp-H100) that is close ( approximately 3 A) to the anthroyl moiety. This is consistent with the energy transfer seen upon AO binding to mAb 26-10 and suggests that Trp-H100, which is part of the antibody's cardiac glycoside binding site, is a major determinant of the fluorescence properties of bound AO. In contrast, the generated model of AO complexed to Fab 40-50 [Jeffrey, P. D., et al. (1995) J. Mol. Biol. 248, 344] shows a heavy chain Tyr residue (Tyr-H100) which is part of the cardiac glycoside binding site, located approximately 10 A from the anthroyl moiety. The closest Trp residues (H52 and L35) are located approximately 17 A from the anthroyl moiety, and no FRET is observed despite the fact that these Trp residues are close enough for significant FRET to occur. The energy transfer seen upon AO binding to Na+,K+-ATPase suggests the presence of one completely quenched or two highly quenched enzyme Trp residues approximately 10 and approximately 17 A, respectively, from the anthroyl moiety. These data suggest that the Na+,K+-ATPase Trp residue(s) involved in fluorescence energy transfer to AO is likely to be part of the cardiac glycoside binding site.

Animals↗
PubMed1995

Effects of nine weeks' use of a new stabilized stannous fluoride dentifrice on intrinsic plaque virulence expressed as acidogenicity and regrowth: a modified PGRM study.

A new stabilized stannous fluoride dentifrice, currently marketed as Crest Gum Care has been examined for its effects on intrinsic plaque metabolic and regrowth activity and effects on plaque resistance to SnF2 throughout nine weeks of toothbrushing. Subjects brushed their teeth 1 X, 2 X or 3 X/day with stabilized stannous fluoride dentifrice or placebo dentifrice for nine weeks, presenting in the morning on weeks 3, 6-9 for plaque sampling. Following nine weeks, subjects were crossed-over and repeated the experiment on their alternative assigned product (active SnF2/placebo). Sampled dental plaques were evaluated for standardized glycolysis and regrowth activity using the "Plaque Glycolysis and Regrowth Method" (PGRM). Following the second nine-week treatment period, subjects concluding either placebo or SnF2 toothbrushing participated in a single-treatment PGRM experiment using stabilized stannous fluoride dentifrice. Toothbrushing with stabilized stannous fluoride dentifrice in this experiment produced significant and sustained reductions in both plaque glycolytic and regrowth activity as compared to placebo treated plaques. In the concluding single-brushing PGRM experiment, SnF2 dentifrice was shown to produce equal inhibitory actions in plaque from subjects completing stannous fluoride or placebo treatments. This result confirmed that nine weeks toothbrushing with stabilized stannous fluoride dentifrice produced no development or resistance of plaque to SnF2 inhibition. These results support the strong in vivo antimicrobial actions of the stabilized stannous fluoride dentifrice, Crest Gum Care.

Analysis of Variance↗
PubMed1993

Ca2+, caldesmon, and myosin light chain kinase exchange with calmodulin.

Wheat calmodulin (CaM) was labeled at Cys-27 with the sulfhydryl-specific fluorescent probe 2-(4'-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS), to form MIANS.CaM. In the presence of Ca2+, MIANS.CaM undergoes a large fluorescence increase when it binds myosin light chain kinase (MLCK) and caldesmon (CaD), but little fluorescence change when it binds CaM antagonists or Ca2+. MLCK associates with MIANS.CaM at a rate of 2.8 x 10(7) M-1 s-1 and dissociates at 0.031 s-1 (Kd = 1.1 nM). Protein kinase A phosphorylation of MLCK (P-MLCK) produces a 3.5-fold decrease in its association rate with CaM and a 6-fold increase in its dissociation rate (Kd = 23 nM). CaD associates with MIANS.CaM with a rate of 5.3 x 10(8) M-1 s-1 and dissociates at 57 s-1 (Kd = 108 nM). EGTA disrupts the CaM.MLCK, CaM.P-MLCK, and the CaM.CaD complexes at rates of 3.5 s-1, 6.5 s-1, and 13.5 s-1, respectively. MLCK, therefore, dissociates from CaM more quickly by Ca2+ removal while the lower affinity CaD is dissociated more quickly by competition from higher affinity CaM target proteins than by Ca2+ removal. MLCK binding to CaM slowed Ca2+ dissociation from CaM's C-terminal Ca(2+)-binding sites from 30 s-1 to 6 s-1 while CaD had little effect on Ca2+ dissociation from these sites. During a Ca2+ transient, CaM could exchange with MLCK and CaD rapidly enough for these proteins to be directly involved in the contraction/relaxation cycle of smooth muscle.

Anilino Naphthalenesulfonates↗
PubMed1993

Gene polymorphism of apolipoprotein AI, the major protein of high density lipoprotein in predicting stroke risk among white and black subjects.

BACKGROUND AND PURPOSE: Restriction fragment length polymorphism of the apolipoprotein AI gene, which encodes the most prominent apoproteins in high density lipoprotein (HDL), were investigated using the restriction enzymes Sac I and Pst I in white and black subjects to determine the potential role of genetic variations as stroke risks as determined by carotid stenosis and an atherogenic serum profile, such as elevated total cholesterol and low density lipoprotein (LDL) levels or reduced HDL levels. METHODS: Ninety-eight subjects, including normal control subjects with no carotid stenosis and subjects with carotid stenosis, who were believed to be at stroke risk, were the study subjects and included 70 white and 28 black subjects. RESULTS: Sac I polymorphic S2 allele frequency was higher in stroke-risk groups. Significantly higher levels of serum cholesterol, triglycerides, and LDL (P < .05) and significantly lower levels of HDL (P < .05) were present in the stroke-risk group with carotid stenosis. Our study showed the following: Sac I polymorphism frequency was significantly higher in black than white subjects (chi 2 = 3.92, P < .05). Triglyceride level was significantly higher in white subjects compared with black subjects (P < .05). In white subjects, carotid artery stenosis was associated with significantly elevated total cholesterol and LDL levels (P < .01) but not with Sac I polymorphism. In black subjects, the reverse was seen with the Sac I polymorphic S2 allele associated with carotid bifurcation stenosis but did not reach statistical significance because of the small number of subjects. In addition, Sac I polymorphism did not correlate with any lipid profile. Pst I polymorphism was not associated with an abnormal atherogenic lipid profile or carotid artery stenosis abnormalities. CONCLUSIONS: Our study shows that carotid artery stenosis in white subjects is associated with increased plasma total cholesterol and LDL levels and an atherogenic profile but not with Sac I polymorphism for apoprotein AI. In black subjects, Sac I polymorphism appears to identify individuals with significant carotid stenosis, a necessary precursor to atherothrombotic brain infarction, but not those with elevated total cholesterol or LDL and/or reduced HDL levels. These results suggest that Sac I polymorphism may identify black subjects at increased risk for atherothrombotic brain infarctions.

Aged↗
PubMed1992

Molecular biological studies in atherothrombotic brain infarction.

Strokes due to atherosclerosis are the most prominent neurological disease affecting adults, and efforts to reduce stroke occurrence, in addition to stroke-risk reduction, will require insights into molecular mechanisms. Our studies showing abnormal metabolism of low and high density lipoproteins (LDL and HDL) in vivo and of RFLP in apoprotein AI, the major protein of HDL, in stroke-prone subjects suggest that greater exploration of fundamental mechanisms of atherothrombotic brain infarction (ABI) should yield preventative strategies, the ultimate treatment for strokes.

Apolipoprotein A-I↗
PubMed1992

Restriction fragment length polymorphism of the apoprotein A-I-C-III gene cluster in control and stroke-prone white and black subjects: racial differences.

BACKGROUND AND PURPOSE: The presence of known restriction fragment length polymorphisms in the apoprotein A-I-C-III gene cluster, which encodes their respective apoproteins, was investigated using the restriction enzymes Sac I and Pst I to determine the potential role of genetic variations for stroke risk in an American population. METHODS: Ninety-eight subjects (70 white, 28 black subjects), both normal controls with no carotid stenosis and those with carotid stenosis believed at risk for stroke, defined as showing stenosis focally or diffusely at that site, composed the study population. RESULTS: Sac I polymorphic S2 allele frequency was higher in stroke-risk groups, whereas Pst I polymorphic P2 allele frequency was similar in control and stroke-risk groups. Significantly higher levels of serum cholesterol, triglycerides, and low density lipoprotein (p less than 0.05) and significantly lower levels of high density lipoprotein (p less than 0.05) were observed in stroke-risk groups with diffuse stenosis. Results of our study with the two racial groups show the following: the frequency of Sac I polymorphism was significantly higher in American black compared with American white subjects (chi 2 = 3.92, p less than 0.05). Among serum lipids, triglycerides were significantly higher in white compared with black subjects (p less than 0.05). In white subjects, carotid artery stenosis was associated with significantly elevated total cholesterol and low density lipoprotein (p less than 0.01) but not with Sac I polymorphism. In black subjects the converse was observed, namely, the Sac I polymorphic S2 allele seemed to be associated with carotid bifurcation stenosis but did not reach statistical significance because of the small number of subjects. In addition, Sac I polymorphism did not correlate with any lipid profile. Pst I polymorphism was not associated with any lipid profile or carotid artery stenosis abnormalities. CONCLUSIONS: Our results indicate that carotid artery stenosis identifies white subjects with increased plasma total cholesterol and low density lipoprotein, an atherogenic profile, but not with Sac I polymorphism. These findings suggest that carotid bifurcation stenosis in white subjects is associated with an atherogenic lipid profile but not with apoprotein A-I-C-III restriction fragment length polymorphisms. In black subjects, Sac I polymorphism seems to identify those individuals with significant carotid stenosis, a necessary precursor to atherothrombotic brain infarction, but not those with elevated total cholesterol, elevated low density lipoprotein, and/or reduced high density lipoprotein. These results suggest that Sac I polymorphism may identify black subjects at increased risk for atherothrombotic brain infarctions.

Aged↗
PubMed1992

Low and high density lipoprotein metabolism in atherothrombotic brain infarction.

BACKGROUND AND PURPOSE: Elevated low density lipoprotein and reduced high density lipoprotein cholesterol may increase the risk of atherothrombotic brain infarction, but the metabolic mechanisms accounting for this relation are poorly understood. METHODS: The kinetic parameters of low density and high density lipoprotein were studied in nine subjects with atherothrombotic brain infarction or identifiable (by noninvasive testing) extracranial occlusive disease and in 12 control subjects. Autologous iodine-125-labeled lipoproteins were injected intravenously. Blood samples were drawn 10 minutes after injection and periodically thereafter for 10 days. Kinetic parameters were calculated from the decay curves. RESULTS: The stroke-risk group showed significantly higher triglyceride (p less than 0.05), total cholesterol (p less than 0.02), and low density lipoprotein cholesterol (p less than 0.01). The fractional catabolic rate of low density lipoprotein was significantly lower (p less than 0.001) and the high density lipoprotein rate higher (p less than 0.02) in the stroke-risk group than in the control group. Regression analysis (using all subjects) of serum lipoproteins and their respective fractional catabolic rates correlated significantly (for low density lipoprotein, r = 0.684, p less than 0.001; for high density lipoprotein, r = 0.595, p less than 0.002). Mean percent stenosis showed a significant relation with triglyceride level (r = 0.678, p less than 0.01) and low density lipoprotein cholesterol (r = 0.535, p less than 0.02) but not with high density lipoprotein cholesterol. Mean percent stenosis also showed correlation with both fractional catabolic rate of low density lipoprotein (r = 0.667, p less than 0.002) and with serum high density lipoprotein levels (r = 0.504, p less than 0.02). CONCLUSIONS: Our study provides insights into the role of altered low and high density lipoprotein metabolism in the pathogenesis of carotid stenosis. The statistically significant association of serum lipoprotein metabolic rates with carotid stenosis, rather than their respective serum concentrations, implies that metabolic parameters may be more important in predicting stroke risk.

Carotid Stenosis↗
PubMed1990

Molecular biology of atherothrombotic brain infarction.

Because reduced high density lipoproteins may contribute to atherothrombotic brain infarction, we performed molecular biologic and metabolic studies to characterize high density lipoprotein metabolism with respect to its role in reverse cholesterol transport, to clone the high density lipoprotein receptor, and to determine gene polymorphism for apolipoprotein A-I, the major protein of high density lipoprotein, because altered structure may impair reverse cholesterol transport. For high density lipoprotein metabolism measurements, high density lipoprotein 3 was isolated, purified, and labeled with iodine-125. The radiolabeled high density lipoprotein 3 was reinjected, and daily blood samples were taken for 10 days. Synthesis rates and fractional catabolic rates were determined from the specific activities and daily decrements. Preliminary data indicate that stroke-prone individuals' fractional catabolic rates for high density lipoprotein 3 are twice those of normal individuals. Also, the conversion of high density lipoprotein 3 to high density lipoprotein 2 is reduced in these individuals, suggesting that high density lipoprotein may be abnormally processed in individuals prone to atherothrombic brain infarctions. We surveyed more than 100 patients with carotid stenosis using a 2.2-kb probe for the apolipoprotein A-I gene. A subset of these patients displays polymorphism with restriction enzymes SacI or PstI. These preliminary findings suggest that gene polymorphism for apolipoprotein A-I may provide a molecular clue of atherothrombic brain infarction.

Arteriosclerosis↗
PubMed1989

A novel cDNA extension procedure. Isolation of chicken fatty acid synthase cDNA clones.

We have developed a simple and versatile cDNA extension method using lambda-exonuclease-generated single-stranded DNA as a primer. This plasmid-based cDNA extension method can be used to synthesize unidirectional extensions of the existing cDNA clones or subcloned fragments of the untranslated and exon regions of genomic DNA clones. The method is simple to use and involves no addition of linkers or tailing. We have successfully used this method to isolate 4.6 kilobase pairs of chicken fatty acid synthase cDNA clones, starting from the fragment of a genomic clone coding for the untranslated region of the fatty acid synthase mRNA. About 2.8 kilobase pairs of the cDNA coding for the chicken fatty acid synthase has been sequenced. The sequence has an open reading frame coding for 945 amino acids of the fatty acid synthase. In the sequence, we have identified the enoyl reductase, NADPH binding region, a putative beta-ketoacyl reductase region, and the entire sequences of acyl carrier protein and the thioesterase domains. The arrangement of these partial activities in this sequence confirms the arrangement of these activities as determined through partial proteolytic mapping studies. The amino acid sequence of chicken fatty acid synthase deduced from cDNA sequences shows a high degree of homology with the rat fatty acid synthase sequence, suggesting that these multifunctional proteins are conserved evolutionarily.

Amino Acid Sequence↗
PubMed1988

Characterization of a genomic and cDNA clone coding for the thioesterase domain and 3' noncoding region of the chicken liver fatty acid synthase gene.

The fatty acid synthase (FAS) of animal tissue is a dimer of two identical subunits, each with a Mr of 260,000. The subunit is a single multifunctional protein having seven catalytic activities and a site for binding of the prosthetic group 4'-phosphopantetheine. The mRNA coding for the subunit has an estimated size of 10-16 kb, which is about twice the number of nucleotides needed to code for the estimated 2300 amino acids. We have isolated a positive clone, lambda CFAS, containing FAS gene sequences by screening a chicken genomic library with a segment of a 3' untranslated region of goose fatty acid synthase cDNA clone, pGFAS3, as a hybridization probe. The DNA insert in lambda CFAS hybridizes with synthetic oligonucleotide probes prepared according to the known amino acid sequence of the thioesterase component of the chicken liver fatty acid synthase [Yang, C.-Y., Huang, W.-Y., Chirala, S., & Wakil, S.J. (1988) Biochemistry (preceding paper in this issue)]. Further characterization of the DNA insert shows that the lambda CFAS clone contains about a 4.7-kbp segment from the 3' end of the chicken FAS gene that codes for a portion of the thioesterase domain. Complete sequence analyses of this segment including S1 nuclease mapping, showed that the lambda CFAS clone contains the entire 3' untranslated region of the chicken FAS gene and three exons that code for 162 amino acids of the thioesterase domain from the COOH-terminal end of the fatty acid synthase. Using the exon region of the genomic clone, we were able to isolate a cDNA clone that codes for the entire thioesterase domain of chicken liver fatty acid synthase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Colchicine inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase in cultured avian liver explants.

Chicken embryo liver explants cultured in chemically defined medium in the absence of serum provide a unique system to probe into the mechanism of insulin induction of lipogenic enzymes. Colchicine at concentrations of 0.2 and 1 microM in the culture medium caused inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase by 50 and 90%, respectively. As measured by immunochemical techniques, the inhibition of the induction of these two enzyme systems resulted from the decreased content of the delta 9-terminal desaturase component of the stearoyl-CoA desaturase and the fatty acid synthetase. Colchicine, however, had no effect on the general protein synthesis, nor did it affect the malic enzyme, which is induced by triiodothyronine but not by insulin. Also, colchicine had no influence on the binding of 125I-insulin to isolated plasma membrane. Pretreatment of liver explants with insulin for 0.5-1 h and subsequent incubation in insulin-free media for 48 h resulted in induction of the desaturase and fatty acid synthetase. However, inclusion of colchicine in the media for 3 h subsequent to the treatment with insulin completely abolished the inductive effect of insulin, suggesting that colchicine affects events occurring subsequent to insulin binding to the cell surface membranes. Since lumicolchicine, an inactive isomer of colchicine, had no effect on insulin action, it is suggested that the inhibition of insulin induction of the desaturase and synthetase is related to the depolymerizing action of colchicine. Therefore, in eliciting long-term responses to insulin, microtubular integrity of the cell may be required for the transfer of a putative from cell surface insulin receptor to intracellular sites.

Animals↗
PubMed1983

Increased synthesis and accumulation of phospholipids during differentiation of 3T3-L1 cells into adipocytes.

Conversion of 3T3-L1 preadipocytes to fully developed adipocytes in culture under the influence of dexamethasone, 1-methyl-3-isobutyl xanthine, and insulin offers a unique system to investigate differentiation-related changes in lipid metabolism. Depending on the type of isotopic precursors ([3H2]O, 32Pi, and [1-14C]acetate) used, a 10-170-fold increase in the rate of incorporation into lipid was observed in 3T3-L1 adipocytes which contained 5-10-fold higher amounts of cellular protein/culture than preadipocytes. In preadipocytes and adipocytes, the major lipids synthesized were phospholipids, triglycerides, and fatty acids. The rate of 32Pi incorporation into total lipids was 21-fold higher in adipocytes than that in preadipocytes, and 90% of the total radioactivity in both preadipocytes and adipocytes was contributed by phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). The content of phospholipids was 7-8-fold higher/culture in adipocytes than that in preadipocytes, and 65 and 80% of the total phospholipids of preadipocytes and adipocytes, respectively, were composed of PC and PE. Based on DNA measurements, there was a 3-fold increase in phospholipids and an approximately 3-fold increase in protein/cell. During 3T3-L1 adipose conversion, a detectable increase in cellular protein, triglycerides, and phospholipids was observed on day 3 after induction and, thereafter, these constituents increased continuously up to day 11. Among the different phospholipids of both preadipocytes and differentiating 3T3-L1 cells, PC, PE, and PI exhibited significant changes in their rate of 32Pi incorporation during adipogenesis. While there was a continuous increase in the incorporation of 32Pi into PI, there was preferential incorporation of 32Pi into PC and PE, the significance of which is not clear. The increase in phospholipids and protein content during adipogenesis suggests that phospholipids are required for membrane biosynthesis.

Acetates↗