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Biomedical subjects

U R Desai

Publications and source records attributed to U R Desai.

At least 19 recordsLinked to original sources
PubMed2001

Choroidal granulomas in systemic sarcoidosis.

PURPOSE: To evaluate the clinical course, including response to therapy, of patients with macular and peripapillary choroidal granulomas secondary to systemic sarcoidosis. METHODS: This is a retrospective case study and literature review. Nine patients with choroidal granulomas were identified. Eight patients had a tissue biopsy confirming sarcoidosis; one was diagnosed from clinical history and typical gallium scan. Ocular examinations included fundus examination, fluorescein angiography, and visual field examination. Eight patients had magnetic resonance imaging (MRI) scans looking for intracranial granulomas. Treatment consisted of oral prednisone in eight patients (one with concomitant subconjunctival triamcinolone); one patient received no treatment because of good vision and granuloma in the nasal retina. Variables studied included visual acuity (VA), response of granulomas to treatment, time to recurrence, and associated anterior segment findings. RESULTS: Eight of nine patients had a solitary lesion whereas one had multifocal involvement. The granulomas ranged in size from one half to four disk diameters. Eight patients had blurry vision; one was asymptomatic. All nine patients had hilar adenopathy and/or pulmonary parenchymal disease. No patient had nonocular neurologic symptoms and in eight patients who underwent MRI examination no intracranial granulomas were detected. Of the eyes that were treated (n = 8) all had decrease in the size of the choroidal mass at an average of 4 months of treatment. Two had complete resolution. Mean follow-up was 29.2 months. At the time of initial diagnosis only one patient had an active anterior uveitis. Five of nine patients had at least one recurrence. Mean time to recurrence was 7.6 months after discontinuing oral prednisone. The VA at presentation ranged from 20/30 to 20/300. Final VA was 20/30 or better in all patients. CONCLUSIONS: Choroidal granulomas related to systemic sarcoidosis respond well to oral corticosteroids. They may recur but good vision can be maintained. They are not typically associated with concomitant iritis and also do not appear to be associated with intracranial granulomas.

Administration, Oral↗
PubMed1998

Mechanism of heparin activation of antithrombin: evidence for an induced-fit model of allosteric activation involving two interaction subsites.

The anticoagulant activation of the serpin antithrombin by heparin pentasaccharide DEFGH was previously shown to involve trisaccharide DEF first binding and inducing activation of the serpin, followed by disaccharide GH binding and stabilizing the activated state [Petitou et al. (1997) Glycobiology 7, 323-327; Desai et al. (1998) J. Biol. Chem. 273, 7478-7487]. In the present study, the role of conformational changes and charged residues of the GH disaccharide in the allosteric activation mechanism was investigated with variant pentasaccharides modified in the GH disaccharide. Perturbation of the conformational equilibrium of iduronate residue G through replacement of the nonessential 3-OH of this residue with -H resulted in parallel decreases in the fraction of residue G in the skew boat conformer (from 64 to 24%) and in the association constant for pentasaccharide binding to antithrombin [(2.6 +/- 0.3)-fold], consistent with selective binding of the skew boat conformer to the serpin. Introduction of an additional sulfate group to the 3-OH of residue H flanking a putative charge cluster in the GH disaccharide greatly enhanced the affinity for the serpin by approximately 35-fold with only a small increase in the fraction of residue G in the skew boat conformation (from 64 to 85%). The salt dependence of binding, together with a recent X-ray structure of the antithrombin-pentasaccharide complex, suggested that the majority of the enhanced affinity of the latter pentasaccharide was due to direct electrostatic and hydrogen-bonding interactions of the H residue 3-O-sulfate with antithrombin. All variant pentasaccharides produced a normal enhancement of antithrombin fluoresence and normal acceleration of factor Xa inhibition by the serpin at saturating levels, indicating that conformational activation of antithrombin was not affected by the pentasaccharide modifications. Rapid kinetic studies were consistent with the altered affinities of the variant pentasaccharides resulting mostly from perturbed interactions of the reducing-end GH disaccharide with the activated antithrombin conformation and minimally to an altered binding of the nonreducing-end DEF trisaccharide to the native serpin conformation. Together, these results support a model in which the conformational flexibility of the G residue facilitates conversion to the skew boat conformer and thereby allows charged groups of the GH disaccharide to bind and stabilize the activated antithrombin conformation that is induced by the DEF trisaccharide.

Allosteric Regulation↗
PubMed1998

Mechanism of heparin activation of antithrombin. Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin.

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of approximately 15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.

Antithrombin III↗
PubMed1997

Intraocular pressure elevation after simple pars plana vitrectomy.

PURPOSE: The purpose of the study is to determine the incidence, timing, and severity of variability in the intraocular pressures (IOPs) from baseline after simple pars plana vitrectomy. METHODS: A prospective study was performed in 25 consecutive patients undergoing simple pars plana vitrectomy. Intraocular pressures were measured before surgery, immediately after surgery, and then at 2, 4, 6, 12, and 24 hours after surgery. RESULTS: The mean IOP was elevated significantly 2 hours after surgery when compared with the mean immediate postoperative IOP (30.3 mmHg +/- 11.0 mmHg vs. 17.4 mmHg +/- 7.0 mmHg, P < 0.001). A steady decline was seen at all succeeding timepoints. The 24-hour mean (17.3 mmHg +/- 4.3 mmHg, P = 0.923) was similar to baseline. Ninety-two percent of eyes had a 2-hour postoperative IOP that was higher than the IOP at the completion of surgery. Forty percent of patients required medical management for IOP greater than or equal to 30 mmHg. CONCLUSIONS: Significant IOP elevation can occur after simple pars plana vitrectomy. The optimal time for detecting the pressure rise during the first 24 hours is 2 hours after surgery.

Adult↗
PubMed1997

Combined pars plana vitrectomy and scleral buckling for pseudophakic and aphakic retinal detachments in which a break is not seen preoperatively.

BACKGROUND AND OBJECTIVE: This study presents a group of patients with a pseudophakic or aphakic retinal detachment (RD) and an unseen retinal break who were treated with a pars plana vitrectomy (PPV), fluid-air exchange, internal drainage, and endolaser in addition to a scleral buckle (SB) procedure. PATIENTS AND METHODS: The charts of 10 consecutive patients with a pseudophakic or aphakic RD and an unseen retinal break who were treated with a PPV, fluid-air exchange, internal drainage, endolaser, and an SB were reviewed for preoperative and postoperative visual acuity, postoperative status of the retina, and surgical complications. RESULTS: All of the patients with a postoperative follow-up of at least 6 months continue to maintain an attached retina after one operation. Visual acuity has improved by at least 2 lines on the Snellen chart in 7 patients, remained the same in 2 patients, and decreased in 1 patient. CONCLUSIONS: This pilot study shows good anatomic results when performing a PPV, fluid-air exchange, internal drainage, and endolaser together with an SB for pseudophakic or aphakic patients with an RD and an unseen break.

Aged↗
PubMed1996

Differential behavior of (25R)-5,6-epoxyspirostan-22 alpha-O-3 beta-ol and (25R)-5,6-epoxyspirostan-22 alpha-O-3 beta, 4 beta-diol toward Dowex.

The acid-catalyzed hydrolytic cleavage of the 5,6-epoxyspirostane derivatives by the cation exchange resin Dowex 50W X8 has been exploited with the goal of developing synthetic protocols toward 3,4,5,6-polyhydroxyspirostane analogs that can serve as intermediates to potential biologically active compounds. Whereas the diastereomers (25R)-5 alpha, 6 alpha-epoxyspirostan-22 alpha-O-3 beta-ol and (25R)-5 beta, 6 beta-epoxyspirostan-22 alpha-O-3 beta-ol yield two products, (25R)-6 beta-methoxyspirostan-22 alpha-O-3 beta, 5 alpha-diol and (25R)-spirostan-22 alpha-O-3 beta, 5 alpha, 6 beta-triol on Dowex treatment in water-methanol, the alpha- and beta-diastereomers of the 5,6-epoxy derivative of 3 beta, 4 beta-diol provide a single product, (25R)-3 beta, 6 beta-dihydroxy-5 alpha-spirostan-4-one, in good yields. The structures of these products have been confirmed using 1H NMR, 13C NMR, and 1H-1H J-correlated spectroscopies. Multifunctional product formation suggests tremendous utility of Dowex in steroid synthesis. The product formation has been rationalized on the basis of differential conformational constraints of the A/B rings of the different epoxides in directing the reaction course. The reaction shows an interesting example of stereoelectronic effect of a single hydroxy group in discriminating solvent participation.

Anion Exchange Resins↗
PubMed1996

Vitrectomy and juvenile epiretinal membrane.

The cases of two children who had idiopathic epiretinal membranes are reported. Causes for juvenile epiretinal membranes, including trauma, pars planitis, toxocariasis, Coats' disease, or combined hamartomas, were not present. Both patients previously had documented normal vision in the affected eye. Observation revealed deterioration of vision, and a pars plana vitrectomy and a membranectomy were performed. The 5-year -old girl's vision improved from counting fingers at 5 feet to 20/80. The 12-year-old boy's vision improved from 20/200 to 20/80. Selected cases of juvenile epiretinal membranes may benefit from surgical excision.

Child↗
PubMed1995

Molecular weight of heparin using 13C nuclear magnetic resonance spectroscopy.

Heparin is a polydisperse, heterogeneous polysaccharide that has been used as an anticoagulant for the past 50 years. The molecular weight determination of this important drug has traditionally relied on gel permeation chromatography, which requires the use of well-defined molecular weight standards that are not easily obtained. We have investigated the use of 13C-NMR spectroscopy for measuring the number average molecular weight of heparin. The signal intensities of the reducing end and internal anomeric carbons, having distinctive chemical shifts in the 13C-NMR spectrum, were used to determine the molecular weight. Distortionless enhancement polarization transfer was found to provide a better quantitation of signal intensities of anomeric carbons than broad band decoupling or selective decoupling of anomeric protons. Signal averaging over 300,000 transients, requiring approximately 48 h on a 360 MHz NMR spectrometer, resulted in the measurement of the number average molecular weight (approximately 10,000 Da) of heparin. 13C-NMR spectroscopy does not require the use of difficult to obtain molecular weight standards and thus is particularly well-suited for workers in the pharmaceutical industry.

Animals↗
PubMed1995

Strategy for the sequence analysis of heparin.

The versatile biological activities of proteoglycans are mainly mediated by their glycosaminoglycan (GAG) components. Unlike proteins and nucleic acids, no satisfactory method for sequencing GAGs has been developed. This paper describes a strategy to sequence the GAG chains of heparin. Heparin, prepared from animal tissue, and processed by proteinases and endoglucuronidases, is 90% GAG heparin and 10% peptidoglycan heparin (containing small remnants of core protein). Raw porcine mucosal heparin was labelled on the amino termini of these core protein remnants with a hydrophobic, fluorescent tag [N-4-(6-dimethylamino-2-benzofuranyl) phenyl (NDBP)-isothiocyanate]. Enrichment of the NDBP-heparin using phenyl-Sepharose chromatography, followed by treatment with a mixture of heparin lyase I and III, resulted in a single NDBP-linkage region tetrasaccharide, which was characterized as deltaUAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp -(1-->O-Ser-NDBP (deltaUAp is 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid). Several NDBP-octasaccharides were isolated when NDBP-heparin was treated with only heparin lyase I. The structure of one of these NDBP-octasaccharides, deltaUAp2S(1-->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp(1-->3)-beta-D- Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp-(1-->O-Ser NDBP (S is sulphate, Ac is acetate), was determined by 1H-NMR and enzymatic methods. Enriched NDBP-heparin was treated with lithium hydroxide to release heparin, and the GAG chain was then labelled at xylose with 7-amino-1,3-naphthalene disulphonic acid (AGA). The resulting AGA-Xyl-heparin was sequenced on gradient PAGE using heparin lyase I and heparin lyase III. A predominant sequence in heparin at the protein core attachment site was deduced to be -D-GlcNp2S6S(or 6OH)(1-->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S (or60H) (1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNp2S6S( or 6OH)(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpAc (1- ->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-++ +GlcAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xyl-AGA.

Animals↗
PubMed1994

Interaction of heparin with synthetic antithrombin III peptide analogues.

Heparin-binding proteins may contain specific patterns of basic amino acids, called consensus sequences, that interact with heparin. Small peptides were synthesized that contained consensus sequences (i.e. FAKLNCRLYRKANKSSK) or disrupted consensus sequences (i.e. K136-->A) based on the known sequence of antithrombin III (amino acid residues 123-139). These peptides were then examined in both competitive and non-competitive binding experiments using bioassays, fluorescence spectroscopy, affinity chromatography and n.m.r. spectroscopy. Both the consensus and disrupted-consensus peptide bound to heparin. Peptides with consensus sequences bound specifically to the pentasaccharide antithrombin III-binding site within heparin. In contrast, peptides with disrupted consensus sequences showed no specificity, binding to any sequence within heparin. Proton nuclear Overhauser enhancement spectroscopy demonstrated the proximity of leucine and tyrosine (within the consensus sequence) to the N-acetyl moiety found primarily within the pentasaccharide antithrombin III-binding site of heparin. This experiment confirmed the findings of the other techniques and helped to localize the binding sites in both peptides and heparin. A model is proposed for both specific and non-specific heparin interaction with consensus and disrupted-consensus peptides.

Amino Acid Sequence↗
PubMed1994

Low molecular weight dermatan sulfate as an antithrombotic agent. Structure-activity relationship studies.

A structure-activity relationship of low molecular weight dermatan sulfate was undertaken to understand better this new non-heparin, glycosaminoglycan-based antithrombotic agent. A dermatan sulfate prepared from bovine intestinal mucosa [average molecular weight (MWavg) 25,000], and currently in clinical trials as an antithrombotic agent, was used in this study. Dermatan sulfate was partially depolymerized using hydrogen peroxide and copper(II) as catalyst to MWavg 5600 to obtain a low molecular weight dermatan sulfate. This low molecular weight dermatan sulfate was then fractionated by gel permeation chromatography to obtain four subfractions having MWavg 7800, 5500, 4200 and 1950. The dermatan sulfate, low molecular weight dermatan sulfate and its subfractions showed substantially different optical rotations. The 1H-NMR spectroscopic analysis of dermatan sulfate samples showed some differences including increased content of GalpNAc4S6S residues and improved resolution in ring resonances for low molecular weight dermatan sulfate fractions, primarily the result of reduced molecular weight and lowered heterogeneity. Saccharide compositional analysis relied on chondroitin ABC lyase treatment followed by capillary electrophoresis. Polyacrylamide gel-based oligosaccharide mapping was also performed by treating dermatan sulfate samples with chondroitin B, AC and ABC lysases. These analyses showed increased amounts of sulfation as the MWavg decreased. In vitro bioassay showed maximum anti-Xa activity in the 4.2 kDa fraction and maximum heparin cofactor II-mediated anti-IIa activity in the 5.5 kDa fraction. The in vivo antithrombotic activity of these fractions was measured using a modified Wessler stasis thrombosis model. The 4.2 kDa fraction showed greater antithrombotic activity than the other low molecular weight dermatan sulfate fractions, dermatan sulfate, and low molecular weight dermatan sulfate. This enhanced activity may result from several structural features of the 4.2 kDa fraction including: a high content of 4,6- and 2,4-disulfated disaccharide sequences; the requirement of specific chain length; a change in the ratio of iduronic to glucuronic acid; and the presence of chondroitin ABC lyase resistant material.

Animals↗
PubMed1994

Molecular weight of low molecular weight heparins by 13C nuclear magnetic resonance spectroscopy.

Heparin and low molecular weight heparins are polydisperse polysaccharides with a degree of polymerization ranging from 4 to approximately 40. The determination of their average molecular weights has traditionally relied on size exclusion chromatography involving the use of oligosaccharides of known size and molecular weight as standards. 13C NMR spectroscopy is applied for the first time to obtain the molecular weights of low molecular weight heparins. The signal intensities of the reducing end and internal anomeric carbons, having distinctive chemical shifts in the 13C NMR spectrum, are measured to determine the molecular weight. Compared to techniques utilizing broad band decoupling or selective decoupling of anomeric protons, distortionless enhancement polarization transfer pulse sequence gave better quantitation of signal intensities of anomeric carbons. Molecular weight was calculated from the calibrated ratio of signal intensities of the anomeric carbons of reducing end groups and internal residues, and the disaccharide compositional analysis. The calibrated signal intensity ratio is determined using the T1 relaxation rates of anomeric carbons of model oligosaccharides. The disaccharide composition of low molecular weight-heparins is obtained using capillary electrophoresis. Signal averaging over 40,000-90,000 transients, requiring a total of 12-18 h on a 360-MHz NMR spectrometer was adequate to measure molecular weights in the range of 3000-7000. The measured molecular weights of twelve low molecular weight heparins, analyzed by this 13C NMR spectroscopic technique, correlated well with the number average molecular weights obtained using high performance-gel permeation chromatography and gradient polyacrylamide gel electrophoresis. In addition to establishing the number average molecular weight, the 13C NMR spectra helped distinguish the structural properties of different commercially prepared low molecular weight heparins.

Carbohydrate Sequence↗
PubMed1994

New approaches for the preparation of hydrophobic heparin derivatives.

A heparin derivative sufficiently lipophilic to be bound to plastics, forming blood-compatible supports, or to be used as an anticoagulant by transdermal or oral routes would be of great pharmaceutical interest. For such applications, the functional groups within heparin's antithrombin III binding site, responsible for its anticoagulant activity, cannot be modified. Chemistry is described in which lipophilic substituents were attached to the reducing termini of heparin chains. Substituents introduced at this position had a minimal effect on the antithrombin III binding sites found in heparin's interior. These derivatives, with enhanced hydrophobicities, were prepared using two distinctly different approaches. First, octyl isocyanate and octadecyl isocyanate were coupled to the core peptide of peptidoglycan heparin to form octyl- and octadecyl-peptidoglycan heparin. These octyl- and octadecyl-peptidoglycan heparins were then purified by hydrophobic interaction chromatography on phenyl-Sepharose CL-4B, demonstrating their enhanced hydrophobicities. Second, the lipophilic acyl hydrazides of various long chain fatty acids were coupled to heparin's reducing end. Caprylic (C8), capric (C10), lauric (C12), and stearic (C18) hydrazide derivatives of heparin were prepared using this approach. Only the stearyl hydrazide derivative of heparin showed a measurable increase in lipophilicity. This result demonstrated that a single small linear C8, C10, or C12 aliphatic chain was ineffective in enhancing the hydrophobicity of the highly negative, polyanionic heparin molecule. Two lipophilic chains, lauryl (C12) and stearyl (C18), were then coupled to a single heparin chain, resulting in a heparin derivative having enhanced hydrophobicity. All the heparin derivatives prepared in this study maintained some of their anticoagulant activity.

Antithrombin III↗
PubMed1993

Substrate specificity of the heparin lyases from Flavobacterium heparinum.

A detailed knowledge about the substrate specificities of the heparin lyases is necessary when using these enzymes as tools for elucidating the sequence of heparin and heparan sulfate. The substrate specificity of heparin lyases I, II, and III have been profiled with structurally defined, heparin-derived oligosaccharides. The primary substrate specificities of heparin lyases I and III require the presence of 2-O-sulfated alpha-L-idopyranosyluronic acid and beta-D-glucopyranosyluronic acid residues, respectively, at the linkages being cleaved. Heparin lyase II demonstrates an intriguingly broad primary specificity for oligosaccharides, acting at linkages containing alpha-L-idopyranosyluronic and beta-D-glucopyranosyluronic acid as well as at linkages containing alpha-L-galactopyranosyluronic acid residues. In addition to their primary specificities, each lyase also demonstrates secondary specificities under forcing conditions. Differences in the sulfation pattern within uronic acid residues and sulfation of adjacent residues has profound impact on the ease of lyase cleavage of a glycosidic linkage. Specifically, heparin lyases I and III exhibit secondary specificity for oligosaccharides containing an unsulfated alpha-L-idopyranosyluronic acid residue. The lack of sulfation on residues adjacent to the linkage undergoing cleavage increases the action of heparin lyase III on a glycosidic linkage. In contrast, reduced sulfation on adjacent residues make glycosidic linkage resistant to heparin lyase I. The primary and secondary specificity can be rationalized on the basis of most favorable solution conformation of the uronic acid residues.

Carbohydrate Conformation↗