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Biomedical subjects

R Kluger

Publications and source records attributed to R Kluger.

At least 37 records · Page 2Linked to original sources
PubMed1994

Flow reversal in the descending aorta: a guide to intraoperative assessment of aortic regurgitation with transesophageal echocardiography.

This study assessed the value of biplane transesophageal echocardiographic assessment of diastolic flow reversal in the descending aorta as an alternative to Doppler color flow imaging in determining severity of aortic regurgitation. In 45 patients undergoing cardiac operations, the severity of aortic regurgitation was assessed by semiquantitative grading of the width of the Doppler color flow regurgitant jet relative to the left ventricular outflow tract, and the presence of diastolic flow reversal was assessed with pulsed-wave Doppler measurements at three sites in the descending aorta. In four patients, the diastolic flow reversal method was the only available form of assessment because of inadequate visualization of the left ventricular outflow tract beneath a mitral valve prosthesis. Diastolic flow reversal in the descending aorta was not observed in patients without aortic regurgitation and was always present in patients with severe aortic regurgitation. Aortic valve replacement successfully eliminated descending aortic flow reversal in all 19 patients in whom it was present before valve replacement. Identification of diastolic flow reversal at multiple sites in the descending aorta with biplane transesophageal echocardiography helps to confirm the presence of severe aortic regurgitation and can serve as an alternative method of assessment when visualization of the left ventricular outflow tract is impaired.

Aged↗
PubMed1993

Crystal structure of transketolase in complex with thiamine thiazolone diphosphate, an analogue of the reaction intermediate, at 2.3 A resolution.

The crystal structure of the complex of transketolase and thiamine thiazolone diphosphate has been determined at 2.3 A resolution. The complex has a structure which closely resembles that of this enzyme with the cofactor ThDP. This is consistent with the observation that the binding of the analogue to transketolase involves ground state rather than transition state interactions. Since thiamine thiazolone diphosphate resembles an expected intermediate in the catalytic pathway, the structure of the intermediate was modelled from the crystal structure. Based on this model, enzymic groups responsible for binding of the intermediate and proton transfer during catalysis are suggested.

Binding Sites↗
PubMed1993

Modification of human hemoglobin with methyl acyl phosphates derived from dicarboxylic acids. Systematic relationships between cross-linked structure and oxygen-binding properties.

Human hemoglobin was reacted with five dicarboxylic acid bis(methyl phosphate) reagents under different ligand conditions. The bis(methyl phosphate) reagents tested were derived from fumaric, isophthalic, terephthalic, trans-stilbene-3,3'-dicarboxylic, and trans-stilbene-4,4'-dicarboxylic acids. These acyl phosphate mixed anhydrides are anionic electrophiles and will react with N-terminal amino and lysyl epsilon-amino groups to form amides. The major and many of the minor reaction products that result have been isolated and structurally characterized by globin chain and peptide analysis. Products which are not cross-linked, intrachain linked, and interchain singly and doubly cross-linked occur in proportions which depend upon the reaction conditions and reagent. Modifications of the beta chains were limited to the amino groups of beta 1Val, beta 82Lys, and, to a minor extent, beta 144Lys. In the case of the smaller reagents, the amino groups of alpha 1Val, alpha 99Lys, and, to a minor extent, alpha 139Lys were modified. The oxygen binding affinities of most of the major modified hemoglobins have been measured and are characterized by P50 values from about 1/2 to over 5 times that of unmodified human hemoglobin. Most show strong cooperativity with Hill coefficients (n) of 2.0 or greater. Several of the products that are cross-linked between the beta 1Val of one chain and the beta 82Lys of the other chain have oxygen affinities in a physiologically useful range for oxygen transport and delivery. An inverse linear correlation has been found between the log of P50 and bridging distances for the hemoglobins cross-linked between beta 1Val of one chain and the beta 82Lys of the other chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Three-point cross-linking: potential red cell substitutes from the reaction of trimesoyl tris(methyl phosphate) with hemoglobin.

The symmetrical trifunctional cross-linking reagent trimesoyl tris(methyl phosphate) (3), reacts selectively with amino groups (beta 1Val and beta 82Lys) in the diphosphoglycerate binding site of human hemoglobin A, producing cross-linked tetrameric species in good yield. A major species is triply linked, alpha alpha beta 1(82) greater than B beta 82, where B symbolizes benzene-1,3,5-tricarbonyl. Both this triply linked species and the doubly linked species, alpha alpha beta 1B beta 82, produced from deoxyhemoglobin have a considerably lower oxygen affinity than does native hemoglobin while maintaining a high degree of cooperativity (n50 = 2.4), making them potentially useful as red cell substitutes, in principle delivering twice as much oxygen as whole blood between pO2 = 100 and = 40 Torr. The yield of products indicates that triply and doubly linked species form in parallel so that there are independent routes to each. It is proposed that differences in routes are due to stereoisomerism about the amide bonds which form from reaction of the reagent with the protein.

Amino Acid Sequence↗
PubMed1990

Pre-bypass myocardial ischaemia in patients undergoing coronary artery surgery.

One hundred patients undergoing elective coronary artery surgery were studied to determine the incidence of pre-bypass myocardial ischaemia. Leads II, aVF and V5 of the electrocardiogram (ECG) were recorded at five-minute intervals from arrival in the anaesthetic room until onset of cardiopulmonary bypass. Thirteen patients developed sixteen episodes of significant ST depression on the ECG during the study period. Three patients were diagnosed as having postoperative myocardial infarction by ECG criteria and creatine phosphokinase-MB rise above 80 units. None of these patients had pre-bypass ST depression. Comparisons of these results with similar studies are presented.

Adult↗
PubMed1989

Double-blind trial comparing epidural lignocaine with epidural fentanyl for anaesthesia during extracorporeal shock wave lithotripsy.

A randomised double-blind trial was undertaken to compare epidural lignocaine 1.5% with adrenaline to epidural fentanyl (100 micrograms in saline 10 ml) in forty unpremedicated patients undergoing extracorporeal shock wave lithotripsy without ancillary procedures. Midazolam only was used for sedation. There was no significant difference in intraoperative pain scores with several patients in each group complaining of pain. Decreases in blood pressure occurred in both groups but were more pronounced in the lignocaine groups (36% vs 25% maximum decrease from control, P = 0.004). Heart rate was increased in the lignocaine group compared with control and to the fentanyl group, but there was no difference in respiratory rates within or between groups. Pruritus was more common in the fentanyl group and leg heaviness in the lignocaine group, but neither was troublesome. There was no difference in urological outcome at one month. Both techniques were satisfactory for the majority of patients, but the fentanyl group had a shorter preparation time and less cardiovascular changes.

Adult↗
PubMed1988

Epidural opioids as anaesthesia for extracorporeal shock wave lithotripsy in two patients with cardiac disease.

Two patients with cardiac disease underwent Extracorporeal Shock Wave Lithotripsy (ESWL) in the Dornier HM3 machine. In order to minimize cardiovascular changes, epidural opioids (fentanyl 100 micrograms in 10 ml saline was used in one patient and meperidine 50 mg in 10 ml saline in the other patient) were used as an alternative to either general anaesthesia or epidural local anaesthesia. Both patients displayed haemodynamic stability, remained pain-free and experienced no complications. Epidural opioids proved a suitable form of anaesthesia for ESWL in these patients.

Analgesics, Opioid↗
PubMed1988

Anaesthesia for extracorporeal shockwave lithotripsy at the Victorian Lithotripsy Service--the first 300 patients.

A prospective survey was undertaken of the anaesthesia for the first 300 patients at the Victorian Lithotripsy Service. The majority (71.7%) were not hospitalised on site, including four quadriplegics and two ASA grade IV patients. Two hundred and eighty-three (94.3%) patients received continuous lumbar epidural anaesthesia, sixteen (5.3%) received general anaesthesia and one received a spinal anaesthetic. Eighty-two patients (27.3%) underwent ancillary procedures at the time of extracorporeal shockwave lithotripsy. The most common intraoperative complications were hypotension (72 patients, 25.4%) and shivering (39 patients, 13.8%). Arrhythmias occurred in nine (3%) patients. There was a 90% patient follow-up rate and the most common postoperative complication was backache (101 patients, 37.4%). The problems of anaesthesia for extracorporeal shockwave lithotripsy are discussed. Epidural anaesthesia offers a number of advantages for this procedure and proved very suitable for the majority of patients.

Adolescent↗
PubMed1986

Methyl acetyl phosphate: a novel acetylating agent. Its site-specific modification of human hemoglobin A.

A novel acetylating agent, methyl acetyl phosphate (MAP), has been designed to react with a nucleophile near an anion binding site of proteins. We examined the effect of MAP on hemoglobin (Hb), which has a well defined binding site for 2,3-diphosphoglycerate (DPG), to determine whether this reagent recognizes the DPG binding site. The progress of the reaction was monitored by ion-exchange high-performance liquid chromatography (HPLC) on a TSK CM-SW column. Modified Hb was initially chromatographed on CM-52 and then separated into its component chains. The alpha- and beta-chains from modified and unmodified Hb were digested by TPCK-trypsin. The peptide mixtures were chromatographed on Whatman ODS-3 reversed-phase HPLC columns and the peptide maps of modified and unmodified chains were compared. The peaks formed by the modification with MAP were further purified on YMC ODS-S5 columns and then subjected to amino acid analysis on a Dionex D-500 instrument after acid hydrolysis. We found that the newly formed peptides are beta T1 and beta T14 + 15 and that the loss of a peptide corresponding to beta T9 and beta T 10 + 11 is significant. No change in the alpha-chains was observed. The results suggest that MAP is indeed specific for the DPG binding site, as the above peptides contain the amino acid residues involved in the binding of DPG. We have assigned the acetylation sites as Val-1(beta), Lys-82(beta) and Lys-144(beta).

2,3-Diphosphoglycerate↗
PubMed1986

Site-specific modification of hemoglobin by methyl acetyl phosphate.

Methyl acetyl phosphate, which was originally synthesized as a site-specific reagent for hydroxybutyrate dehydrogenase [R. Kluger and W.-C. Tsui (1980) J. Org. Chem. 45, 2723], also has an affinity for the binding site for 2,3-diphosphoglycerate in hemoglobin. Three residues in or near this cleft between the beta-chains are acetylated by this reagent, i.e., Val-1, Lys-82, and Lys-144. There is no detectable acetylation of any of the amino groups of the alpha-chain. These results indicate the specificity of methyl acetyl phosphate in its reaction with hemoglobin.

Acetylation↗
PubMed1986

Reaction of the anionic acetylation agent methyl acetyl phosphate with D-3-hydroxybutyrate dehydrogenase.

Methyl acetyl phosphate is a competitive inhibitor of the reduction of acetoacetate by D-3-hydroxybutyrate dehydrogenase. The material also irreversibly inactivates the enzyme. The kinetics of the inactivation are consistent with methyl acetyl phosphate acetylating the conjugate base of a hydrogen bond donor. Protection offered by a substrate analogue (methyl acetonylphosphonate) in the presence of coenzyme implicates reaction at the cationic active site. Reversible protection by the amino group reagent 2,3-dimethylmaleic anhydride suggests that methyl acetyl phosphate reacts with an amino group. Sulfhydryl reagents and acetyl phosphate, a poorer acetylating agent, do not inactivate the enzyme. The pH dependence of the inactivation suggests that the acetylation occurs at a site that has a pKa of 8.2. The utility of methyl acetyl phosphate and other acyl phosphate monoesters in reacting with lysines adjacent to cationic sites of enzymes, hemoglobin, and histones is noted.

Hydroxybutyrate Dehydrogenase↗
PubMed1984

Interaction of thiamin diphosphate and thiamin thiazolone diphosphate with wheat germ pyruvate decarboxylase.

The interactions of the apoenzyme of wheat germ pyruvate decarboxylase with thiamin diphosphate and with thiamin thiazolone diphosphate have been investigated. The results test hypotheses concerning the structure of the transition state for decarboxylation of the enzyme-bound adduct of pyruvate and thiamin diphosphate. Thiamin thiazolone diphosphate, a possible transition state analogue, binds to the apoenzyme by a two-step process. The first is slow and reversible (k = 200 M-1 s-1; K = 5 X 10(-7) M). The second step is irreversible (k = 1 X 10(-6) s-1). The rate constant for activation by thiamin diphosphate is 160 M-1 s-1. Thiamin diphosphate is released very slowly from the holoenzyme (k = 2 X 10(-5) s-1). Thiamin thiazolone diphosphate competitively inhibits activation of the apoenzyme by thiamin diphosphate, Ki = 2 X 10(-6) M. Km for thiamin diphosphate is only 3 times larger. Thiamin thiazolone is solvated preferentially to thiamin in 2-butanol, a medium whose polarity should resemble that of the binding site. It is concluded that the observed high affinity of thiamin thiazolone diphosphate for the apoenzyme is the result of a combination of effects which do not require the assumption that it is an analogue of the transition state for the decarboxylation of enzyme-bound 2-(2-lactyl) thiamin diphosphate.

Carboxy-Lyases↗
PubMed1981

Inhibition of bacterial D-3-hydroxybutyrate dehydrogenase by substrates and substrate analogues.

D-3-Hydroxybutyrate dehydrogenase (Pseudomonas lemoignei, EC 1.1.1.30) is subject to substrate inhibition by acetoacetate at concentrations above 5 mM but not by D-3-hydroxybutyrate at concentrations up to 50 mM. NADH causes substrate inhibition at concentrations over 0.1 mM as does NAD. Kinetic analysis suggests that substrate inhibition by acetoacetate is due to its binding to enzyme lacking NADH, a consequence of the ordered bibi mechanism. Substrate inhibition by NADH and NAD arises from binding of these species to a secondary site. This is confirmed by kinetics which indicate that ADP and ATP compete with NAD and NADH at both sites. New analogues of acetoacetate were synthesized to test the specificity requirements of the acetoacetate binding site which has been proposed to contain a hydrogen bond donor and a cation spaced to receive acetoacetate. Both dimethoxyphosphinylacetate and methyl 2-methoxy-phosphinylacetate fulfill the structural requirements and are effective. They thus join methyl acetonylphosphonate as the only known competitive inhibitors for the acetoacetate site, confirming the proposed structure.

Acetoacetates↗
PubMed1980

Phosphonate analogues of pyruvate. Probes of substrate binding to pyruvate oxidase and other thiamin pyrophosphate-dependent decarboxylases.

A number of enzymes catalyze the removal of carbon dioxide from pyruvate through covalent participation of the coenzyme thiamin pyrophosphate. The conversions of the decarboxylated adduct, hydroxyethyl thiamin pyrophosphate, to subsequent products distinguishes the function of these enzymes. Acetaldehyde is produced by pyruvate decarboxylase, acetic acid by pyruvate oxidase and acetyl coenzyme A by pyruvate dehydrogenase. Differences and details of steps prior to decomposition of hydroxyethyl thiamin pyrophosphate can be evaluated through the use of two substrate analogues, methyl acetylphosphonate and acetylphosphonate. Methyl acetylphosphonate and acetylphosphonate are competitive inhibitors toward pyruvate with Escherichia coli pyruvate oxidase and E. coli pyruvate dehydrogenase but the value of the Ki for the oxidase is more than three orders of magnitude higher than for the dehydrogenase. Yeast pyruvate decarboxylase is not inhibited at all under the same conditions. The binding of methyl acetylphosphonate results in ligand-induced changes in the near ultraviolet circular dichorism spectrum of the oxidase. This spectral perturbation is only seen in the presence of the cofactor, thiamin pyrophosphate, strongly suggesting that the inhibitor is binding at the same site as the substrate, pyruvate, on the enzyme. Kinetic data suggest that lipid activators of pyruvate oxidase increase the affinity of the enzyme for pyruvate and its analogues.

Acetaldehyde↗
PubMed1980

Amino group reactions of the sulfhydryl reagent methyl methanesulfonothioate. Inactivation of D-3-hydroxybutyrate dehydrogenase and reaction with amines in water.

Methyl methanesulfonothioate (MMTS) has been used as a sulfhydryl reagent. However, it inactivates D-3-hydroxybutyrate dehydrogenase in a manner that suggests that an amino group may have reacted with the reagent. Nuclear magnetic resonance (NMR) analysis of solutions of glycylglycine, glycine ethyl ester, and imidazole with MMTS indicates that a reaction occurs which produces what is identified by its NMR signal as the methylsulfenamide of the amine. It is suggested that caution should be used when MMTS is employed where reaction with an amine could give an undesired product.

Glycine↗