Fibrinogen as a Predictor of Early Neurological Deterioration and Poor Outcome in Acute Ischemic Stroke.

There has been more than a 100 per cent increase in stroke incidence in India from 1970 - 2008. Early Neurological Deterioration (END) is associated with an increased risk of disability and mortality in ischemic stroke patients and approximately 32% of ischemic stroke patients experience END. Although various factors have been identified to predict the occurrence of END in ischemic stroke such as age, gender, diabetes, initial stroke severity, stroke subtype and radiological parameters, similar data for the Indian population is lacking. Fibrinogen is a mediator in the development of coronary artery thrombi and future cardiac events and has been reported to be independently associated with a poor functional outcome. MATERIAL: We enrolled 141 patients with acute ischemic stroke from a single centre. END was defined as a total National Institutes of Health Stroke Scale (NIHSS) score deterioration by 2 or more points within the first week. Patients with a Modified Rankin Scale (MRS) score of 3 or more at discharge, or a stroke recurrence event during hospital stay were said to have a poor outcome. We performed univariate analysis in the total population to develop a logistic regression model to assess potential factors associated with END and poor outcome. OBSERVATION: Fibrinogen levels were higher in the END group than the non - END group (464.57 ± 121.05 vs. 305.0 ± 123.28, p <0.001) and was an independent predictor for END in the logistic regression model (odds ratio 1.011, p <0.001). Increasing age and a higher NIHSS score at admission were other risk factors for developing END. Fibrinogen was also independently associated with poor outcome (odds ratio 1.004, p = 0.038) along with initial NIHSS score and fasting blood sugar level. CONCLUSION: Fibrinogen levels at stroke onset is independently associated with END and a worse hospital outcome in an Indian population subset with ischemic stroke. Routine plasma fibrinogen assays may help clinicians in stratifying patients into a high-risk group, who may require more potent antiplatelet therapy or use of fibrin-depleting agents.

Management of mandibular sub condylar and condylar fractures using retromandibular approach and assessment of associated surgical complications.

PURPOSE: To evaluate surgical complications associated with open reduction and internal fixation of condylar fractures using retromandibular approach in terms of intraoperative and postoperative complications. METHOD: Ten patients with displaced unilateral/bilateral condylar fractures were selected for the study. Intraoperative complications were evaluated in the form of haemorrhage due to damage to retromandibular vein and damage to marginal mandibular branch of facial nerve. Postoperative complications like presence of infection, signs of Frey's syndrome, parotid fistula formation, facial nerve palsy, and discrepancy in occlusion and functions of temporomandibular joint were evaluated at intervals of 24 h, one week, six weeks and three months postoperative. Radiographically, the approximation of fracture fragments, plate fracture and screw loosening on orthopantomograph and Reverse Towne's view were evaluated at intervals of 24 h, six weeks and three months postoperatively. RESULTS: None of the patients suffered from any major complication intra and post operatively. CONCLUSION: Open reduction and internal fixation should be given due consideration in the management of displaced mandibular condylar fractures and is associated with minimal morbidity using retromandibular approach.

Fenitrothion-induced changes in lipids of rats.

The dose-dependent lipid accumulation caused by fenitrothion administration is associated with alteration of the ratio of various components of phospholipids and neutral lipids in various organs of rats up to 48 h. The concentration of triacylglycerol, phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl glycerol increased in liver, kidney and brain in treated rats whereas phosphatidyl serine was greatly reduced. High-performance liquid chromatography and gas-liquid chromatography were used for quantiative analysis of phospholipids and triacylglycerol. The changes in relative composition of various lipids by fenitrothion administration may lead to malfunctioning and alteration of biological properties.

Biodegradation and bioaccumulation of fenitrothion in rat liver.

The biodegradation of fenitrothion O,O-dimethyl-O-(3-methyl-4-nitro phenyl) phosphorothioate was investigated in rat liver after administration of various doses (5 mg/100 g body weight and 20 mg/100 g body weight) in acute treatment and 1 mg/100 g body weight in chronic treatment. High performance liquid chromatography of the pesticide and its metabolites formed in liver in acute treatment showed time-dependent sequential conversion of pesticide into three major metabolites within 24 h. These metabolites were separated and purified to homogenity by HPLC and characterized by IR spectroscopy as O,O-dimethyl-O-(3-methyl-4-amino phenyl) phsophorothioate (metabolite 1), O,O-dimethyl phosphorothioate (metabolite II) and O,O-dimethyl phosphate (metabolite III) in the fi rst dose (5 mg/100 g body weight). Metabolite II was found to be different in the second dose (20 mg/100 g body weight) and identified as O,O-dimethyl O-3-methyl-4-amino phenyl phosphate. The results with the fi rst dose indicated reduction of the nitro group in fenitrothion as step I followed by hydrolytic clevage of the P-O-aryl bond in metabolite I and oxidative desulphurylation of metabolite II. At higher dose (20 mg/100 g body weight) oxidative desulphurylation takes place as step II followed by hydrolysis of metabolite II. The bioaccumulation of fenitrothion within 60 days during chronic treatment showed no metabolite but continuous reduction in fenitrothion concentration, indicating excretion of pesticide and its products in urine and in faeces.

Biosynthesis of lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase.

The formation of N-glycosidic linkages of glycoproteins involves the ordered assembly of the common Glc3Man9GlcNAc2 core-oligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDP-Man as donor, the final reactions from Man5GlcNAc2-PP-Dol to Man9GlcNAc2-PP-Dol on the lumenal side use Dol-P-Man. We have investigated these later stages in vitro using a detergent-solubilized enzyme extract from yeast membranes. Mannosyltransfer from Dol-P-Man to [3H]Man5GlcNAc2-PP-Dol with formation of all intermediates up to Man9GlcNAc2-PP-Dol occured in a rapid, time- and protein-dependent fashion. We find that the initial reaction from Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol is independent of metal ions, but further elongations need Mn2+ that can be partly replaced by Mg2+ or Ca2+. Zn2+ or Cd2+ ions were found to inhibit formation of Man(7-9)GlcNAc2-PP-Dol, but do not affect synthesis of Man6GlcNAc2-PP-Dol. Extension did not occur when the acceptor was added as a free Man5GlcNAc2 oligosaccharide or when GDP-Man was used as mannosyl donor. The alg3 mutant was described to accumulate Man5GlcNAc2-PP-Dol. We expressed a functional active HA-epitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions. This demonstrates that Alg3p represents the mannosyltransferase itself and not an accessory protein involved in the reaction.

Metabolism and bioaccumulation of fenvalerate and its metabolites in rat organs.

Metabolism and bioaccumulation of fenvalerate and its fenvalerate its metabolites in liver, kidney and brain of rat following the oral administration of a sub-lethal dose (15 mg/kg) of the pesticide for 7, 15 and 30 day periods was investigated by high-performance liquid chromatography (HPLC) in terms of the relative mole concentrations in rat tissues. The cleavage of the ester linkage in fenvalerate yielding two metabolites was found to be primary step in the biodegradation of fenvalerate in rat organs. These metabolites were purified to homogeneity by HPLC and characterized by infra-red spectroscopy as 4-chloro-alpha-(1-methylethyl) benzeneacetic acid and 3-phenoxy benzoic acid.

Biodegradation of fenitrothion in soil.

The biodegradation of fenitrothion (an organophosphorus pesticide) in garden soil of western Uttar Pradesh in northern India has been studied. Both natural and autoclaved soil were treated with 10 p.p.m. of fenitrothion at field capacity moisture and incubated for 90 days in glass containers at 25 and 40 degrees C. The soil samples were collected at various intervals between 0 and 90 days and analysed by HPLC for the residual pesticide and metabolites formed. At 0 days the recovery of the pesticide was 98% and the pesticide then degraded linearly with incubation time. The concentration of pesticide in the natural soil decreased with incubation time with concomitant formation of two metabolites at 25 degrees C and three metabolites at 40 degrees C. These metabolites were purified to homogeneity by HPLC and characterized by IR spectroscopy. The results showed oxidative desulphuration of the pesticide at the first step followed by hydrolytic cleavage of P-O-aryl linkage and demethylation at the second and third step, respectively.

Increased H2 production by immobilized microorganisms.

Viable cells of H2-producers (Bacillus licheniformis and a mixed microbial culture) were immobilized on brick dust and in calcium alginate beads. In batch culture, cells of the mixed culture in the free state yielded 8.2 l H2/mol glucose utilized, whereasB. licheniformis evolved 13.1 l H2. Immobilized cells, however, gave 4-fold more H2 than the free bacteria. Highest yields were from the cells immobilized on brick dust. High H2-production rates continued over two rounds of re-use of the immobilized cells.

Metabolic fate of fenitrothion in liver, kidney and brain of rat.

The metabolic fate of fenitrothion, O,O-dimethyl-O-(3-methyl-4-nitro phenyl) phosphorothioate, was investigated in rat tissues during the first 24 hours following the intramuscular administration of the pesticide in the animal. High performance liquid chromatography (HPLC) of the pesticide and its metabolites formed in liver, kidney and brain showed the time-dependent sequential conversion of the pesticide into three major metabolites. These metabolites were separated and purified to homogeneity by HPLC and characterized by IR spectroscopy as O,O-dimethyl-O-)3-methyl-4-aminophenyl) phosphorothioate (metabolite I), O,O-dimethyl phosphorothioate (metabolite II), and O,O-dimethylphosphate (metabolite III). These results indicated reduction of the nitro group in fenitrothion aas the first step, followed by the hydrolytic cleavage of the P-O-aryl bond in metabolite I and the oxidative desulphurylation of metabolite II. Fenitrothion was found to induce ultrastructural changes in liver cells especially after 12 h exposure nuclear membrane was completely distorted, nuclear intactness was totally lost and smooth endoplasmic reticulum and Golgi apparatus were abnormally enlarged. In 24 h, however, the regeneration of the nuclear material was observed.

Biodegradation of a carbamate pesticide, Propoxur, in rat tissues.

Propoxur (Baygon, 2-isopropoxyphenyl N-methylcarbamate) is a carbamate pesticide commonly used against house insects. When the insecticide was administered intramuscularly in rats it was converted to a new metabolite which was found to be present in the serum, liver, kidney and brain 6 h after the administration of the pesticide. The metabolite was purified by high performance liquid to chromatography and comparison of the infrared spectra of Propoxur and the metabolite showed that a deamination reaction was responsible for the formation of the metabolite from the parent pesticide. The pesticide also induced haematological changes such as an increased level of total bilubrin, amylase and glutamic-oxalacetic transaminase and decrease of cholinesterase activity, indicating damage of the liver and nervous system in rats.

Purification to homogeneity and characterization of a 1,3-beta-glucan (callose) synthase from germinating Arachis hypogaea cotyledons.

A 1,3-beta-D-glucan (callose) synthase (CS) from a plasma membrane fraction of germinating peanut (Arachis hypogaea L.) cotyledons has been purified to apparent homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino-terminal analysis, and the Western blots pattern. The purification protocol involved preparation of a high specific activity plasma membrane fraction, selective solubilization of the enzyme from the membrane with 0.5% digitonin at a protein-to-detergent ratio of 1:6, sucrose gradient centrifugation, and chromatography on hydroxylapatite and DEAE-Sephadex A-50. The purified CS shows a molecular mass of approximately 48,000 by SDS-PAGE, pH optimum of 7.4, leucine as the amino-terminal residue, Km for UDP-glucose of 0.67 mM, and Vmax of 6.25 mumol/min/mg protein. The enzyme is specific for UDP-glucose as the glucosyl donor and required Ca2+, at an optimum concentration of 2-5 mM, for activity. The enzyme activity was inhibited by nucleotides (ATP, GTP, CTP, UTP, UDP, and UMP). The enzyme activity was also inhibited by the addition of EDTA or EGTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The reaction product formed during incubation of UDP-[14C]glucose and cellobiose with purified enzymes was susceptible to digestion by exo-(1,3)-beta-glucanase, but was resistant to alpha- and beta-amylases and to periodate oxidation, indicating that the polymer formed was 1,3-beta-glucan, and beta-1,4 and beta-1,6 linkages were absent.

Biosynthesis of lipid-linked oligosaccharides. I. Preparation of lipid-linked oligosaccharide substrates.

In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.

Partial purification of a mannosyltransferase involved in the O-mannosylation of glycoproteins from Saccharomyces cerevisiae.

The mannosyltransferase that catalyses the transfer of mannose from dolichyl-phosphate-mannose (Dol-P-Man) to the hydroxyl group of serine/threonine residues in the acceptor peptide (Tyr-Asn-Pro-Thr-Ser-Val) was partially purified approximately 150-fold from the microsomal membrane fraction of Saccharomyces cerevisiae. The membrane-bound enzyme was solubilized with 0.5% Triton X-100 at a protein:detergent ratio of 2:1, and was then purified by ion-exchange chromatography on DEAE-cellulose, followed by hydroxyapatite column chromatography. The partially purified enzyme had a pH optimum of 7.2 and required Mg2+ at an optimum concentration of 10 mM for activity. The apparent mol. wt of the enzyme, as estimated by gel filtration on Sephacryl S-300, was approximately 125 kDa. The activity of the partially purified enzyme was greatly stimulated by phosphatidylcholine (PC), while other naturally occurring phosphoglycerides had no significant effect. The extent of activation of mannosyltransferase activity was greatly affected by the number of carbons and the degree of saturation/unsaturation of the fatty acid substituents, as well as by their position on the glycerol moiety of the PC molecule. Maximum stimulation of the mannosyltransferase activity was induced by a PC derivative in which both sn-1 and sn-2 positions on the glycerol moiety were occupied by C12:0 fatty acids. In general, mannosyltransferase was found to exhibit greater specificity for the L-alpha-PC derivatives in which the sn-2 position of the glycerol contained a saturated fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of dolichyl-P-mannose:Man5(GlcNAc)2-PP-dolichol mannosyltransferase.

The dolichyl-P-mannose:dolichyl-PP-heptasaccharide alpha-mannosyltransferase (2.4.1.130), which catalyzes the transfer of mannose from dolichyl-P-mannose to the Man5(GlcNAc)2-PP-dolichol acceptor glycolipid, was solubilized from pig aorta microsomes with 0.5% NP-40 and purified 985-fold by a variety of conventional methods. The partially purified enzyme had a pH optimum of 6.5 and required Ca2+, at an optimum concentration of 8-10 mM, for activity. Mn2+ was only 20% as effective as Ca2+, and Mg2+ was inhibitory. The mannosyltransferase activity was also inhibited by the addition of EDTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The enzyme was quite specific for dolichyl-P-mannose as the mannosyl donor and Man5(GlcNAc)2-PP-dolichol as the mannosyl acceptor. The Km values for dolichyl-P-mannose and the acceptor lipid Man5(GlcNAc)2-PP-dolichol were 1.8 and 1.6 microM. On Bio-Gel P-4 columns and by HPLC, the radiolabeled oligosaccharide formed during incubation of dolichyl-P-[14C]mannose and unlabeled Man5(GlcNAc)2-PP-dolichol with the purified enzyme behaved like Man6(GlcNAc)2. This octasaccharide was susceptible to digestion by endoglucosaminidase H, indicating that the newly added mannose was attached to the 6-linked mannose in an alpha 1,3-linkage. This linkage was further confirmed by acetolysis of the oligosaccharide product [i.e., Man6(GlcNAc)2], which gave a labeled disaccharide as the major product (greater than 90%).

Purification, by high-performance liquid chromatography, and characterization, by high-field 1H-n.m.r. spectroscopy, of two fucose-containing pentasaccharides of goat's milk.

Two fucose-containing pentasaccharides were isolated from goat's milk using a Bio-Gel P-4 column, followed by reverse-phase C-18 high-performance liquid chromatography. The structures of the pentasaccharides as characterized by high-field 1H-n.m.r. spectroscopy and enzymatic digestion were found to be [see text] and [see text].

Studies on the determination and degradation of pyrimethamine in mammals.

Treatment of pyrimethamine with blood plasma in vitro yields a metabolite which is also produced when the drug is administered through intravenous injection in the rat. A thin layer liquid chromatographic method for quantitative and qualitative determination of pyrimethamine and its metabolite in plasma and biological tissues is described.

Chemically induced aneuploidy in higher plants.

Although there is extensive literature reporting aneuploidy in plants there has been little consideration of the mechanisms of induction of the observed changes in chromosome number and the role of environmental agents in its induction. This paper collates and analyses the literature on aneuploidy induction in plants with the aim of identifying the mechanisms involved. Many of the major events that occur during cellular segregation, such as respiration, spindle and phragmoplast functions and chromosome doubling, appear to be stressed by agricultural chemicals, drugs, natural and industrial products, leading to aneuploidy in both the mitotic and meiotic cells of approximately 60 plant species. Of the nearly 400 chemicals at present suspected to be aneugenic to plants, approximately 150 are agricultural chemicals, 60 are drugs, 60 are natural substances and 50-60 are industrial products. Nearly half of these agents were reported to affect spindle mechanisms in mitosis and meiosis. About 60 affected phragmoplast function in root meristems. Another 60 chemicals appear to modify chiasma frequencies. Nearly 40 chemicals appear to be clastogenic in such a way that could lead to aneuploidy by a secondary mechanism. About 45 plant species exhibit aneuploidy without any apparent cause or source of stress. This could arise from several metabolic and anthropogenic causes operating alone or together such as: (i) the nutritional status of the soil, (ii) irrigation by polluted water, (iii) the ageing of seeds, (iv) pathogenesis of plants, (v) cryptotoxins in plants, (vi) pesticide applications and (vii) pesticide residues. Environmental factors such as pH and temperature may further compound these stresses. Among the biota, plant systems alone seem to possess all the components to monitor every conceivable type of aneuploidy and respond to all kinds of stress in our environment. There are deficiencies in the work hitherto carried out which need to be rectified. Some aspects have to be reinvestigated with improved protocols. For a perspective understanding of the phenomenon of aneuploidy, so vital to human systems, it is imperative that we devise new approaches, some of which are outlined here.