Further characterization and immunochemical studies on the carbohydrate specificity of jackfruit (Artocarpus integrifolia) lectin

The lectin from jackfruit (Artocarpus integrifolia) seeds has been purified by Rivanol (6,9-diamino-2-ethoxyacridine lactate) treatment. The specific activity, molecular weights of parent lectin and its subunit, its glycoprotein nature, and hemagglutination-inhibition assays suggest that this preparation is identical to that obtained by affinity chromatography on melibiose-agarose adsorbent (Ahmed, H., and Chatterjee, B. P. (1986) in Lectins, Biology, Biochemistry, Clinical Biochemistry (B?g-Hansen, T. C., and van Driessche, E., eds) Vol. 5, pp. 125-133, Walter de Gruyter, New York). The lectin strongly agglutinates human and several animal erythrocytes. The lectin contains five isolectins of pI values 7.1, 6.85, 5.5, 5.3, and 5.1. It is thermally stable and loses its activity above 75 degrees C. The hemagglutinating activity remains unchanged in the presence of bivalent cations viz., Ca2+, Mg2+, Mn2+, etc. It is a metalloprotein. The lectin retains its activity by dialysis with acetic acid followed by EDTA. It agglutinates Ehrlich ascites cells. Equilibrium dialysis of lectin with melibiose and quenching of fluorescence of 4-methylumbelliferyl-alpha-D-galactopyranoside by the lectin show that homotetrameric jackfruit lectin has two sugar-binding sites. The lectin precipitates well several galactomannans and glycoproteins having terminal D-Gal-alpha-(1----6)- or D-Gal-beta-(1----3)-D-GalNAc residues. It hardly or does not precipitate polysaccharides having terminal D-Gal-alpha-(1----3) residues. Quantitative precipitin-inhibition studies using various haptens suggest that the -OCH2- group at C-1 and -OH groups at C-4 and partially at C-6 in the alpha-glycoside of D-galactose configuration are important for lectin-sugar interaction.     

Effect of polymyxins on the lipopolysaccharide-defective mutants of Proteus mirabilis

The effects of polymyxins (Pmx) B and E on smooth and rough Proteus mirabilis strains were investigated. P. mirabilis mutant R4/028 which completely lacked 4-amino-4-deoxy-L-arabinose was sensitive towards both polymyxins, and the other P. mirabilis strains investigated were resistant. Lipopolysaccharide (LPS) from Pmx-sensitive R4/028 strain, binds 50% more Pmx B than LPS derived from resistant P. mirabilis strains. The presence of iodoacetamide, N-ethylmaleimide and chloramphenicol rendered the Pmx-resistant P. mirabilis strains sensitive towards both polymyxins.     

Prediction of fertilizer phosphate requirement using the Langmuir adsorption maximum

The phosphate adsorption maximum as calculated by the Langmuir equation was used to predict the fertilizer P requirement of wheat (Triticum aestivium, cv. Caldwell) on both virgin and cultivated Decatur clay loam (clayey, kaolinitic, thermic, Rhodic Paleudult) and Hartsells sandy loam soils (fine-loamy, siliceous, thermic, Typic Hapludult). Soils with higher adsorption maximum were found to require more fertilizer P than soils with lower adsorption maximum. For soils 25% saturation of the adsorption maximum gave the optimum dry matter yield. This corresponded to equilibrium P concentration of 0.45 mg L⁻¹ for Decatur cultivated and 0.31 mg L⁻¹ for Decatur and Hartsells virgin soils for optimum dry matter yield. These values are within the range of those reported previously by other investigators working with different soils.     

Polyclonal antibodies against rat liver cytosolic casein kinase II (CK-2) cross-react with CK-2 from other tissues and nuclear form (PK-N2) of the enzyme

Casein kinase II (CK-2) is a ubiquitous serine/threonine protein kinase, and is localized to both the cell nucleus and cytoplasm. Despite extensive biochemical similarities in their properties, there is evidence that the two forms of the enzyme exhibit certain distinctions (1). This prompted us to produce antibodies against CK-2, which could be utilized as a possible tool for investigations of the various forms of this enzyme. Specific polyclonal antibodies against the rat liver cytosolic CK-2 were raised in egg yolk of laying hens; the enzyme had repeatedly failed to elicit an immunogenic response in rabbits. The purified polyclonal antibody (egg yolk immunoglobulin, IgY) recognized all three subunits (42, 38, and 28 kDa) of the enzyme in immunoblots. The antibody when bound to a matrix was capable of removing CK-2 from solution, and the bound enzyme could be recovered from the immunoaffinity matrix with 0.1 M diethylamine. The antibody exhibited a high affinity towards CK-2 prepared from cytosol of liver, ventral prostate, and several other rat tissues, but no immunoreactivity was detected towards a number of other protein kinases tested. The subunits of the nuclear form of CK-2 (PK-N2) migrated differently when electrophoresed in parallel in the same gel. However, the antibody did cross-react with the various subunits of PK-N2 suggesting a significant homology in the immunogenic domains in the various subunits of the two forms of the enzyme.     

Mivazerol, a Novel α2-Agonist and Potential Anti-Ischemic Drug, Inhibits KCl-Stimulated Neurotransmitter Release in Rat Nervous Tissue Preparations

Abstract: In this study, we have investigated the effect of mivazerol, [3-(1H-imidazol-4-yl)methyl-1]-2-hydroxy-benzamide hydrochloride, a new α₂-agonist lacking hypotensive properties and a potential anti-ischemic drug, on the evoked release of norepinephrine, aspartate, and glutamate in tissue preparations from hippocampus, spinal cord T1–T5 section, rostrolateral ventricular medulla, and nucleus tractus solitarii of the brainstem of rat. A simple and efficient in vitro procedure to study pharmacologically the release of norepinephrine and glutamate is described. Tissues were chopped into (0.3 × 0.2 × 0.2 mm³) sections and the resulting minces were used for this study. Exposure to KCl (10–75 mM) for 5 min served as a stimulus for the release response. One, S (for aspartate and for glutamate release), or two such stimuli, S₁ and S₂ (for norepinephrine release) were conducted. The release of norepinephrine (+150% above baseline) was inhibited in a dose-dependent manner by mivazerol in hippocampus (IC₅₀ = 1.5 × 10^?8M), spinal cord (IC₅₀ = 5 × 10^?8M), rostrolateral ventricular medulla (IC₅₀ = 10^?7M), and nucleus tractus solitarii (IC₅₀ = 7.5 × 10^?8M), and by clonidine in hippocampus (IC₅₀ = 5 × 10^?8M), spinal cord (IC₅₀ = 4.5 × 10^?8M), rostrolateral ventricular medulla (IC₅₀ = 2.5 × 10^?7M), and nucleus tractus solitarii (IC₅₀ = 10^?7M). This effect was counteracted by the selective α₂-antagonists yohimbine and rauwolscine. A significant glutamate and aspartate release response was also induced by KCl (35 mmol/L) in hippocampus (+250 and +135%, respectively) and spinal cord (+120 and +55%, respectively), in vitro. However, neither mivazerol nor clonidine, at doses up to 10 µM, had any significant effect on KCl-induced glutamate release in spinal cord, whereas mivazerol blocked completely the release of both amino acids in hippocampus and only the release of aspartate in spinal cord. On the other hand, clonidine (1 µM) was only effective in reducing by 40% the release of aspartate in hippocampus. These data indicate that (1) inhibition of KCl-induced norepinephrine release by mivazerol is mediated by its action on α₂-adrenergic receptors; (2) at concentrations selective for α₂-adrenergic receptors, only mivazerol was effective in blocking the KCl-induced glutamate release in hippocampal tissue; and (3) at the same concentrations, both mivazerol and clonidine were unable to inhibit glutamate release in the spinal cord. These data suggest that prevention of hyperadrenergic activity by mivazerol in perioperative patients may be mediated through its effect on the release of norepinephrine and/or the release of glutamate and aspartate in regions of the CNS that are involved in the control of cardiovascular homeostasis.     

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.]

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - BAP 6-benzylaminopurine - KN Kinetin     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Neurological disorder and excessive accumulation of calcium in brain of clinically vitamin A-deficient rats

Three groups of rats were fed two types of synthetic diets for 52 d. The—A group was allowed free access to a vitamin A-deficient diet and showed classical signs of vitamin A deficiency. The brain was the only organ in our experiment where no significant weight difference was present among the three groups. In the brain, calcium concentration was significantly higher in the—A group when compared with the PF (Pair-fed; allowed restricted amount of control diet) and +A groups (allowed free access to control diet). In the tibia, calcium and magnesium concentrations were significantly lower in the—A group when compared with other two groups. Excessive accumulation of calcium in brain and apparently similar unbalance in bone, mineral concentration were observed in central nervous system (CNS) degenerative diseases. Our results suggest that abnormal metabolism of calcium and magnesium in some tissues and excessive accumulation of calcium in brain may be responsible for the development of neurological disorders in vitamin A-deficient rats.