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Biomedical subjects

S Krishnan

Publications and source records attributed to S Krishnan.

At least 109 records · Page 6Linked to original sources
PubMed1992

Testing for creatine kinase and creatine kinase-2 in Ontario: reference ranges and assay types.

In 1991, 246 and 136 Ontario laboratories performed total creatine kinase (CK; EC 2.7.3.2) and creatine kinase-2 (CK-2) assays, respectively. A questionnaire mailed to these laboratories requested information about the types of assay used, the origin of their reference ranges, and the source of their instruments and reagents. All laboratories used current test formulations for CK, although seven laboratories did not assay at 37 degrees C. For CK, 69% of all laboratories reported different upper reference limits for men and women (5th-95th percentiles: 160-250 and 115-215 U/L, respectively); 31% reported similar ranges for both sexes. Fifty-six percent derived their own ranges; the remainder used either kit inserts or literature references, and nearly 60% of this latter group claimed to have validated these suggested ranges before use. For 6% of all laboratories, their pediatric ranges were similar to their adult ranges. For CK-2, only 32% used their own reference range; the remainder used kit inserts or literature references, but only 49% of this group validated these ranges before use. Reference limits (5th-95th percentiles) for CK-2 were as follows: activity 6-24 U/L; fraction of CK, 0.022-0.06; and, for mass assays, 5-10 micrograms/L and relative index 0.015-0.04.

Creatine Kinase↗
PubMed1991

Structural and functional characterization of Factor VIII-delta II, a new recombinant Factor VIII lacking most of the B-domain.

A recombinant Factor VIII (Factor VIII-delta II) consists of a unique polypeptide chain of 165 kDa deleted from the major part of the B-domain and from the cleavage site at Arg-1648-Glu-1649 found in plasma-derived Factor VIII. It was expressed in mammalian cells in serum-free medium containing von Willebrand factor and purified by a one-step immunopurification. The recombinant Factor VIII was characterized as a single active peak when subjected to f.p.l.c., in contrast with the plasma-derived molecule. Its coagulant activity was decreased in the presence of EDTA, suggesting that a bivalent ion is required, as for plasma-derived Factor VIII. The activation by thrombin and the inactivation by activated protein C were studied and the resulting molecular forms were analysed by f.p.l.c. and SDS/PAGE. The results clearly demonstrate that, despite the structural differences between plasma-derived and recombinant Factor VIII, activation and inactivation of Factor VIII-delta II generate proteolysed complexes similar to that described for plasma-derived Factor VIII. Thus this deleted recombinant Factor VIII, which is processed similarly to plasma-derived Factor VIII, should be normally integrated in the regulation system of Factor X activation in the blood-coagulation cascade.

Amino Acid Sequence↗
PubMed1991

Thrombin cleavage analysis of a novel antihaemophilic factor variant, factor VIII delta II.

Factor VIII delta II is a genetically engineered deletion variant of factor VIII expressed by recombinant Chinese hamster ovary cells, in which a major portion of the central (B) domain and a part of the light chain (Pro771-Asp1666) are missing. After immunoaffinity purification, the kinetics of thrombin cleavage of the novel molecule was analysed by SDS/PAGE, Western blotting and N-terminal amino acid sequencing. Thrombin first cleaves factor VIII delta II at Arg740-Ser741 to generate the 90-kDa heavy chain and an 80-kDa fusion polypeptide consisting of the remaining portion of the B domain and the 73-kDa light chain. The 90-kDa fragment is further cleaved, giving rise to 50-kDa and 40-kDa fragments while the 80-kDa fragment generates a 71/73-kDa doublet. The 71/73-kDa doublet, 50-kDa and 40-kDa fragments were further analysed by N-terminal amino acid sequencing and found to correspond to the predicted amino acid sequences. Our study shows that, in spite of the 900 amino acid deletion present in factor VIII delta II, the essential structural elements required for thrombin activation are conserved.

Amino Acid Sequence↗
PubMed1991

The effect of Hoe-427 (an ACTH4-9 analog) on free-choice ethanol consumption in male and female rats.

Ethanol consummatory patterns of individual male and female rats and the effects of Hoe-427 (Ebiratide), an ACTH4-9 analog, thereon, were studied in a test system using 24 hour, two-bottle free choice consumption between 0.2% saccharin and 10% ethanol in 0.2% saccharin. Single, daily i.p. doses (0.03mg/rat) of either ACTH4-10 or its analog resulted in a significant reduction of daily ethanol consumption with no effects on saccharin consumption. After 4 days of treatment, male rats consistently exhibited a rebound increase in ethanol consumption; this effect was not seen in females. The daily ethanol consummatory patterns of the female animals seemed to exhibit a 4-6 day cyclic rhythymicity, suggesting an interaction with estrous cycles. These results support a role for ACTH4-10 in the initiation of ethanol consummatory behavior in rats and suggests the existence of sex differences in this phenomenon.

Adrenocorticotropic Hormone↗
PubMed1991

Results of a province-wide quality assurance program assessing the accuracy of cholesterol, triglycerides, and high-density lipoprotein cholesterol measurements and calculated low-density lipoprotein cholesterol in Ontario, using fresh human serum.

To evaluate laboratory performance, eight to 13 samples of fresh human serum from volunteers were sent to 250 laboratories in the Canadian province of Ontario licensed to perform lipid analysis. Fresh human specimens were used because of potential matrix effects with processed materials. We show that on all survey samples, 71% (range, 63% to 82%) of participating laboratories are within +/- 5% of the target cholesterol value and that 93% are within +/- 10%. The goal of the National Cholesterol Education Program for 1992 is total error of no more than +/- 9% for 95% of results. The unblanked triglycerides results show that on all samples 40% (14% to 59%) of participants are within +/- 5% and 68% (range, 31% to 86%) are within +/- 10% of the target value. For triglycerides results from 0.9 to 2.0 mmol/L, 80% or more are within +/- 0.2 mmol/L. Between 2.0 and 3.0 mmol/L, 90% are within +/- 0.3 mmol/L of the target values. For high-density lipoprotein cholesterol, for all samples 35% (range, 24% to 50%) of laboratories are within +/- 5% and 68% (range, 55% to 88%) are within +/- 10%. A range of 80% to 95% of participants are within +/- 0.2 mmol/L of the target values. For calculated low-density lipoprotein cholesterol, 51% and 62% of the laboratories surveyed are within +/- 5%, with 83% and 89% within +/- 10% of the target values. We conclude that the laboratory measurement of lipids is approaching the degree of accuracy and precision required for clinical purposes, and that the use of fresh human serum samples is a viable approach to their proficiency testing.

Blood Chemical Analysis↗
PubMed1990

Interrelationships among major protistan groups based on a parsimony network of 5S rRNA sequences.

To test the validity of the maximum parsimony approach to discern protistan interrelationships, we have derived an optimal network of 16S-like rRNA sequences using our parsimony algorithm and compared it with those reported using the distance matrix method. We have also derived an optimal network topology of 50 5S rRNA sequences through an interactive search using our algorithm. In both these networks, the kinetoplastids and euglenoids form a linkage group with Dictyostelium emerging from its neighbourhood. The cryptophytes, dinoflagellates and chromophytes and green algae emerge as independent lines suggesting that plastids arose more than once during protistan evolution. The large 5S rRNA tree further indicates independent origins of mesozoa and metazoa; kinetoplastids and ciliates; and diphyletic origin of fungi. Comparatively close positions of charales and land plants, chytrids and Zygomycetes, Physarum and amoeba, and red algae and green algae are also seen in this network.

Animals↗
PubMed1990

Proficiency testing for creatine kinase isoenzyme CK-2 (CK-MB) in Ontario.

Three surveys of the measurement and interpretation of creatine kinase (CK; EC 2.7.3.2) isoenzyme 2 (CK-MB) were conducted in Ontario, Canada, in 1989. Of the clinical laboratories participating, 66% used immunological methods and 24% used electrophoretic methods. Although reference ranges and interpretative routines varied widely, 95% of the laboratories reported correct interpretations for 10 of the 15 vials tested. The only major problems occurred with samples with very low total CK activity. Within-survey duplicate results compared well, and 89% of the laboratories had consistent between-survey results, even for specimens with low total CK activity. Errors were proportional to the frequency of use of the different analytical methods. The lyophilized testing material gave higher results with methods for measuring the mass of CK-2, suggesting that the material contained inactive but immunologically intact CK-2. The surveys indicate that laboratories should review their protocols for measuring CK-2 when only a single sample from the patient is available.

Creatine Kinase↗
PubMed1987

Permanent changes in muscle and motoneurones induced by nerve injury during a critical period of development of the rat.

The sciatic nerve was crushed in rats at different times during the first two weeks after birth. Following reinnervation the recovery of the fast and slow muscles and their motoneurones was compared. The main factor affecting recovery of muscle weight and tension was the age at which the nerve was crushed; the earlier the injury the greater the impairment. However, recovery also depended upon muscle type. The fast muscles, tibialis anterior and extensor digitorum longus, always recovered less well than the slow soleus muscle. The greatest difference in recovery was seen when the nerve was crushed between 3 and 6 days of age. The fatigue resistance of fast muscles was markedly increased after nerve injury at any time during the first two postnatal weeks and was greatest when the nerve crush was done soon after birth. However, this change was not just related to muscle weakness as the increase in fatigue resistance after nerve crush at 5 and 12 days was similar regardless of the difference in recovery of the muscles. Retrograde labelling of motoneurones with HRP demonstrated that about 60-70% of motoneurones innervating fast or slow muscles were lost following sciatic nerve crush at birth. It is concluded that motoneurone loss probably accounts for most of the impairment of soleus after postnatal nerve crush but only partly explains the poor recovery of fast muscles.

Animals↗
PubMed1986

Purification and some properties of three serine carboxypeptidases from Aspergillus niger.

Three enzymes exhibiting peptidyl-L-amino acid hydrolase and esterase activities have been purified by immobilized metal-ion affinity chromatography and ion-exchange chromatography. The three enzymes were entirely free of the acid protease activity that normally exists along with them in the crude culture filtrates of Aspergillus niger. Although all three exo-peptidases possessed nearly identical molecular weights (ca. 140,000), isoelectric points (ca. 5.0) and other properties, their affinities for the two substrates tested, carbobenzoxy-L-Glu-L-Tyr and benzoyl L-arginine ethyl ester, differed. All three peptidases were inhibited by phenylmethanesulphonyl fluoride, indicating that they are serine carboxypeptidases. They were also inhibited by tosyl phenylalanine chloromethyl ketone, suggesting the presence of a histidyl residue in their active sites. The differences in the number of accessible histidyl residues on the enzyme surfaces could explain the differences in their retentions on Cu2+-iminodiacetate-Sepharose 6B.

Aspergillus niger↗
PubMed1986

New support for the large-scale purification of proteins.

We propose a new affinity sorbent, matrix-linked histidine, for the large-scale purification of proteins. A variety of proteins and certain peptides, each distinct from the other, were purified. Immunoglobulin G from human placenta was chosen for a detailed study concerning the effects of coupling, spacer-arm and other parameters. Multiple interactions such as charge-transfer and other ionic reactions have been suggested to be responsible for the interactions between proteins and ligand.

Animals↗
PubMed1986

Plasma metabolism of apolipoprotein A-IV in humans.

As assessed by molecular sieve chromatography and quantitation by a specific radioimmunoassay, apoA-IV is associated in plasma with the triglyceride-rich lipoproteins, to a high density lipoprotein (HDL) subfraction of smaller size than HDL3, and to the plasma lipoprotein-free fraction (LFF). In this study, the turnover of apoA-IV associated to the triglyceride-rich lipoproteins, HDL and LFF was investigated in vivo in normal volunteers. Human apoA-IV isolated from the thoracic duct lymph chylomicrons was radioiodinated and incubated with plasma withdrawn from normal volunteers after a fatty meal. Radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, HDL, and LFF were then isolated by chromatography on an AcA 34 column. Shortly after the injection of the radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, most of the radioactivity could be recovered in the HDL and LFF column fractions. On the other hand, when radioiodinated apoA-IV-labeled HDL or LFF were injected, the radioactivity remained with the originally injected fractions at all times. The residence time in plasma of 125I-labeled apoA-IV, when injected in association with HDL or LFF, was 1.61 and 0.55 days, respectively. When 125I-labeled apoA-IV was injected as a free protein, the radioactivity distributed rapidly among the three plasma pools in proportion to their mass. The overall fractional catabolic rate of apoA-IV in plasma was measured in the three normal subjects and averaged 1.56 pools per day. The mean degradation rate of apoA-IV was 8.69 mg/kg X day. The results are consistent with the conclusions that: apoA-IV is present in human plasma in three distinct metabolic pools; apoA-IV associated with the triglyceride-rich lipoproteins is a precursor to the apoA-IV HDL and LFF pools; apoA-IV in LFF is not a free protein and its turnover rate is faster than that of apoA-IV in HDL; since no transfer of apoA-IV from the HDL or the LFF occurs, these pools may represent a terminal pathway for the catabolism of apoA-IV; and the catabolism of apoA-IV in HDL is dissociated from that of apoA-I although both apoproteins may reside on the same lipoprotein particles.

Apolipoprotein A-I↗
PubMed1985

Origin of apolipoprotein A-I polymorphism in plasma.

The origin and the functional significance of apo-A-I polymorphism in man has been investigated. Together with proapo-A-I (identified as A-I1 of the polymorphic series), four other isoforms are found in human plasma, namely A-I2, A-I3, A-I4, and A-I5. A-I3 is the "mature" product of proapo-A-I conversion in plasma. In this study we provide evidence that the other, more acidic, mature apo-A-I isoproteins are derived from A-I3 by a stepwise deamidation process. This conclusion is based on the following observations. 1) Incubation of A-I3 or A-I4, either free or associated with high density lipoprotein, produces a series of more acidic isoproteins corresponding to the sequence found in plasma. The conversion process fits in well with a first order reaction, and A-I3 to A-I4 conversion occurs virtually at the same rate as A-I4 to A-I5 conversion. 2) A-I3 and A-I4 have the same NH2- and C-terminal residues. 3) Formation of apo-A-I acidic isoproteins is accompanied by liberation of ammonia. In order to investigate whether deamidation of apo-A-I results in the production of forms which have different catabolism, a series of turnover studies was carried out in normal volunteers. A-I3 and A-I4 residence times in plasma were, respectively, 3.50 +/- 0.16 and 3.00 +/- 0.10 days (mean +/- S.E.; n = 3). Degradation rate of A-I3 was 8.81 +/- 0.69 mg/kg/day and that of A-I4 was 1.66 +/- 0.15 mg/kg/day (mean +/- S.E.; n = 3). Conversion of A-I3 to A-I4 and A-I4 to A-I5 occurred at the same rate in vivo as that observed in vitro. These results are consistent with the concept that A-I3 is the precursor to the other mature apo-A-I isoforms in plasma. A-I3 is the major isoform through which apo-A-I is eliminated from plasma.

Ammonia↗
PubMed1985

The effect of reducing the peripheral field on motoneurone development in the rat.

The tibialis anterior and extensor digitorum longus muscles of the rat were reduced in size either by crushing the sciatic nerve or by removing part of the muscle tissue during the first postnatal week. Four to 6 weeks later the number and size of the motoneurones supplying these muscles were assessed using retrograde transport of horseradish peroxidase. The pattern of synaptic connections in the muscles supplied by these motoneurones was examined 3-46 weeks after the initial operation using a combined silver cholinesterase stain. The number of labelled motoneurones was not reduced after nerve crush but was reduced to some extent after partial muscle removal. The distribution of motoneurone sizes, however, was altered by both procedures in that the largest motoneurones became smaller. In the muscle both procedures affected synaptic organization. In the case of sciatic nerve crush at 5-6 days the incidence of muscle fibres with more than one endplate and endplates contacted by more than one axon terminal was higher than in normal adult muscles. When part of the muscle was removed, the predominant feature was the persistence of a high incidence of free sprouting nerve fibres. We therefore conclude that reduction of the peripheral field during the postnatal period does affect the development of some motoneurones.

Animals↗