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Biomedical subjects

A K Shukla

Publications and source records attributed to A K Shukla.

At least 19 recordsLinked to original sources
PubMed1998

Tracheal aspirate surface tension in babies with hyaline membrane disease: effects of synthetic surfactant replacement.

Our objective was to determine changes in surface tension of tracheal aspirate over the first 4-5 days of life in babies with hyaline membrane disease, with and without synthetic surfactant replacement. Tracheal aspirates were collected prior to and for 96-108 hr after initiation of a randomized double-blind trial of synthetic surfactant (EXOSURF Neonatal) or air-treated control patients. Using the captive bubble technique, we measured minimum surface tension (initial adsorption, first quasi-static compression, dynamic cycling at 30 cpm, second quasi-static compression and 5 min after quasi-static compressions) in 39 surfactant-treated and 44 control babies. We also compared minimum surface tension with the respiratory support provided. Twelve hours after one dose of synthetic surfactant, minimum surface tension on first quasistatic compression decreased significantly from 20.9+/-1.4 to 17.6+/-1.3 mN/m compared to air-treated babies, who did not show any change. Reduction in minimum tracheal aspirate surface tension on first quasi-static compression and during dynamic cycling over 48-60 hr occurred more rapidly in surfactant-treated babies. Ventilator support did not correlate with minimum tracheal aspirate surface tension. We conclude that treatment of babies with synthetic surfactant improved tracheal aspirate minimum surface tension within 12 hr of the first dose and for the next 48-60 hr.

Double-Blind Method↗
PubMed1994

Hydrophilic-interaction chromatography of complex carbohydrates.

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.

Animals↗
PubMed1993

Pattern of childhood trauma. Indian perspective.

Although infectious diseases are still the chief cause of death in children in a developing country like ours, but a definite increase in incident and related mortality due to trauma has been noted in the last decade. The problems relating to pediatric trauma are peculiar to our setup and differ considerably to the severe multiple organ trauma met with in the high velocity vehicular accidents, seen in developed Western countries. The present study identifies patterns of childhood trauma from our region. It comprises 2100 patients admitted over a 3 years period to Pediatric Surgical Unit. Cranial injuries were the most commonly encountered injuries followed by the abdominal and skeletal injuries. Fall from the house roofs is the commonest mode of injury, although road traffic accidents are also recognized to be on the increase. The overall mortality was 7.7%; its chief determinant being the presence of severe head injury. A few important epidemiological factors involved were identified.

Abdominal Injuries↗
PubMed1992

Significance of phototherapy-induced riboflavin deficiency in the full-term neonate.

As a result of impaired fatty acid oxidation, a characteristic urinary dicarboxylic aciduria occurs in the riboflavin deficient animal. We compared the occurrence of riboflavin deficiency induced by phototherapy with changes in urinary organic acid profiles in 8 full-term, breast-fed neonates who received phototherapy for hyperbilirubinemia, and in 10 full-term, breastfed controls. Riboflavin status was assessed by measuring flavin adenine dinucleotide saturation of erythrocyte glutathione reductase. All 8 neonates exposed to phototherapy developed riboflavin deficiency (p less than 0.001). Riboflavin deficiency was progressive with the duration of phototherapy. None of the controls was riboflavin deficient. Urine organic acid profiles indicative of mitochondrial acyl-CoA dehydrogenase activity (fatty acid beta-oxidation, quantitated by gas chromatography mass spectrometry) showed no changes between the study and control groups in mono-, di-, or tricarboxylic acids or other organic acids. The riboflavin deficiency induced by phototherapy in full-term neonates was not of sufficient severity to limit riboflavin-dependent fatty acid oxidation.

Birth Weight↗
PubMed1989

Partial purification and characterization of sialate O-acetylesterase from bovine brain.

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylesterase↗
PubMed1987

Natural occurrence and preparation of O-acylated 2,3-unsaturated sialic acids.

Three O-acylated, unsaturated sialic acids, N-acetyl-9-O-acetyl-, N-acetyl-9-O-lactoyl-, and 2-deoxy-N-glycoloyl-9-O-lactoyl-2,3-didehydroneuraminic acid (5-acetamido-9-O-acetyl-, 5-acetamido-9-O-lactoyl-, and 2,6-anhydro-3,5-dideoxy-5-glycoloylamido-9-O-lactoyl-D-glycero-D-g alacto-non-2- enonic acid) were isolated from urine or submandibular glands of rat, pig, and cow. Mass spectrometric evidence for the existence of 2,3-unsaturated 9-O-acetyl-N-glycoloylneuraminic acid in porcine urine was also obtained. The sialic acids were purified by dialysis, gel- and ion-exchange chromatography, and preparative thin-layer chromatography. They were analyzed by thin-layer chromatography, high-pressure liquid chromatography, and capillary gas-liquid chromatography-mass spectrometry. For comparison, O-acetylated unsaturated sialic acids were synthesized.

Acylation↗
PubMed1987

Migration of O-acetyl groups in N,O-acetylneuraminic acids.

Highly purified N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2), N-acetyl-7-O-acetylneuraminic acid (Neu5,7Ac2) and N-acetyl-7,9-di-O-acetylneuraminic acid (Neu5,7,9Ac3) were used to study spontaneous migrations of acetyl groups between hydroxyl groups. The techniques applied involved thin-layer chromatography, gas-liquid chromatography/mass spectrometry, high-performance liquid chromatography and 360-MHz 1H-NMR spectroscopy. It was found that at pH values at which no significant de-O-acetylation is observed: (a) Neu5,7Ac2 can easily be transformed into Neu5,9Ac2, (b) Neu5,7,9Ac3 yields an equilibrium of Neu5,7,9Ac3 and Neu5,8,9Ac3 in a molar ratio of approximately 1:1, and (c) Neu4,5Ac2 does not give rise to O-acetyl migrations. The importance of these findings is discussed in terms of the biosynthesis of O-acetylated sialic acids.

Chemical Phenomena↗
PubMed1986

Reconstitution of the masking effect of sialic acid groups on sialidase-treated erythrocytes by the action of sialyltransferases.

Glutardialdehyde-fixed or native rat erythrocytes were partially desialylated by the action of Vibrio cholerae sialidase, resulting in the binding of these cells to homologous peritoneal macrophages. Resialylation of these erythrocytes by purified alpha-(2----3)- or alpha-(2----6)-sialyltransferases with CMP-N-acetylneuraminic acid led to the incorporation of 60-80% of the enzymically released sialic acid. Binding of the resialylated erythrocytes to peritoneal macrophages was reduced when compared with corresponding, partially desialylated erythrocytes. Thus, the amount of transferred sialic acid was sufficient to demonstrate reconstitution of the masking effect of sialic acids.

Animals↗
PubMed1986

Analysis of sialidase and N-acetylneuraminate pyruvate-lyase substrate specificity by high-performance liquid chromatography.

A rapid and sensitive assay by high-performance liquid chromatography for determination of the activity and substrate specificity of sialidase (EC 3.2.1.18) and N-acetylneuraminate lyase (EC 4.1.3.3) is described. Sialic acids were separated on a strong anion-exchange resin using 0.75 mM sodium sulfate as elution medium. This method allows the determination of a minimum amount of 200 pg (0.6 pmol) of sialic acid. Usually the enzyme mixtures were directly applied to the column without prior purification of substrates and products. The action of sialidase was studied either by the decrease of sialyllactose concentration or by the amount of sialic acid liberated. The relative hydrolysis rates of N-acetylneuraminyl-alpha(2-3)-lactose, N-glycolylneuraminyl-alpha(2-3)-lactose, N-acetylneuraminyl-alpha(2-6)-lactose, N-acetyl-9-O-acetylneuraminyl-alpha(2-3)-lactose, and N-acetyl-4-O-acetylneuraminyl-alpha(2-3)-lactose by Vibrio cholerae sialidase were 100, 88, 25, 12, and 0, respectively. The activity of N-acetylneuraminate lyase from Clostridium perfringens was determined by measuring the rate of disappearance of sialic acids or the formation of acylmannosamines, which is possible in the same chromatogram. Relative cleavage rates of N-acetylneuraminic acid, N-glycolylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, and N-acetyl-4-O-acetylneuraminic acid were found to be 100, 67, 24, 3, and 0, respectively. Comparison of the substrate specificities shows that substituents on the neuraminic acid molecule influence the reactions of both enzymes in a similar way.

Chromatography, High Pressure Liquid↗
PubMed1985

Structural parameters and natural occurrence of 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid.

2-Deoxy-2,3-didehydro-N-glycoloylneuraminic acid has been found to occur in porcine, bovine and equine submandibular glands as well as in the urine of pig, horse and rat. This novel, unsaturated sialic acid was isolated by gel filtration and ion-exchange chromatography. Final purification was achieved by column chromatography or by preparative thin-layer chromatography on cellulose. The structural analysis was performed by combined capillary gas-liquid chromatography/mass spectrometry. The various data were compared with those from synthetic 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid. Besides of the unsaturated N-glycoloylated sialic acid, also the corresponding N-acetylated derivative was present in the materials analyzed. The inhibitory effect of 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid on Vibrio cholerae sialidase using N-acetylneuraminyl-(alpha 2----3)-lactose as substrate is slightly higher (50% inhibition at 10 microM) when compared with 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (50% inhibition at 15 microM).

Animals↗
PubMed1985

Determination of 3-deoxy-D-manno-octulosonic acid (KDO), N-acetylneuraminic acid, and their derivatives by ion-exchange liquid chromatography.

A liquid chromatography (1.6 MPa) system for the analysis of 3-deoxy-D-manno-2-octulosonic acid (KDO), N-acetylneuraminic acid (Neu5Ac), methyl alpha- and beta-glycosides of Neu5Ac and KDO, alpha-heptosyl-(1----5)-KDO, various sialyllactoses, alpha-KDO-(2----4)-KDO, alpha-KDO-(2----4)-KDO methyl alpha-glycoside, beta-KDO-(2----4)-KDO methyl beta-glycoside, D-glucuronic acid, D-glucurono-3,6-lactone, and D-galacturonic acid has been developed. Separation was achieved within 10 and 30 min by the use of a small column filled with a strongly basic, anion-exchange resin, Aminex A-29, and 0.75 or 10mM sodium sulfate solutions as mobile phases. This method allowed the determination of KDO and sialic acids in amounts of 100 ng (0.5 nmol) and 200 pg (0.6 pmol), respectively.

Chromatography, High Pressure Liquid↗
PubMed1985

Structural analysis of underivatized sialic acids by combined high-performance liquid chromatography-mass spectrometry.

Mass spectra of chemically ionized, positive ions of underivatized N,O-acylated sialic acids, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and sialyl-alpha(2-3)-lactose were obtained by combined high-performance liquid chromatography--mass spectrometry, using a direct liquid inlet system. The mass spectra of the different compounds for which fragmentation schemes are proposed enable the differentiation between sialic acids, although the localization of O-substituents is not possible. However, since the various sialic acids separated well on high-performance liquid chromatography, combined high-performance liquid chromatography-mass spectrometry allowed their unequivocal characterization.

Animals↗
PubMed1985

The receptor-destroying enzyme of influenza C virus is neuraminate-O-acetylesterase.

The nature of the receptor-destroying enzyme (RDE) of influenza C virus has been elucidated by analyzing its effect on the haemagglutination inhibitors rat alpha 1-macroglobulin (RMG) and bovine submandibulary mucin (BSM), respectively. The inhibitory activity of both compounds is abolished by incubation with influenza C virus. After inactivation, RMG and BSM were found to contain reduced amounts of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) and increased amounts of N-acetylneuraminic acid (Neu5Ac). H.p.l.c. analysis revealed that purified Neu5,9Ac2 is converted to Neu5Ac by incubation with influenza C virus. These results demonstrate that RDE of influenza C virus is neuraminate-O-acetylesterase [N-acyl-9(4)-O-acetylneuraminate O-acetylhydrolase (EC 3.1.1.53)]. The data also indicate that haemagglutination-inhibition (HI) by RMG and BSM and most likely virus attachment to cell surfaces involves binding of influenza C virus to Neu5,9Ac2.

Acetylesterase↗
PubMed1984

New techniques for the investigation of structure and metabolism of sialic acids.

Sialic acid analysis in biological material including gangliosides is often confronted with the necessity to determine trace amounts of various N,O-substituted species. Therefore, techniques of high sensitivity and resolution are required, such as capillary gas-liquid chromatography (GLC) and high performance liquid chromatography (HPLC). Both methods in combination with mass spectrometry allow structural analysis of the different neuraminic acid derivatives. Thus, the number of natural sialic acids known so far has increased to more than 30, including non only saturated, but also 2,3-unsaturated and 2,7-anhydro-sialic acids. Furthermore, HPLC has proved to be especially useful for the study of enzyme reactions, as the sialic acids of enzyme assay mixtures in most cases can be analyzed without prior extensive purification or derivatization.

Animals↗
PubMed1984

Identification of a disialoganglioside (GD1a) containing terminal N-acetyl-9-O-acetylneuraminic acid in rat erythrocytes.

Gangliosides containing 350 micrograms of sialic acids were isolated from 2.85 X 10(11) rat erythrocytes and found to be mainly composed of GD1a and an unknown alkali-labile species which was converted to GD1a after treatment with ammonia. Smaller amounts of GM1 and Fuc-GM1 were also present. Identification of the sialic acids of the novel species by thin-layer chromatography, high performance liquid chromatography and gas-liquid chromatography--mass spectrometry revealed the presence of both N-acetylneuraminic acid and N-acetyl-9-O-acetyl-neuraminic acid in about equimolar amounts. Incubation of the isolated ganglioside with Vibrio cholerae sialidase released N-acetyl-9-O-acetyl-neuraminic acid. Non O-acetylated GM1 was identified as the only remaining ganglioside by thin-layer chromatography. Thus this novel ganglioside has the following structure: Neu5,9Ac2 alpha 2-3Gal beta 1-3GalNAc beta 1-4(Neu5Ac alpha-2-3)Gal beta 1-4Glc beta 1-1'Cer.

Animals↗