Protein 1a: A major wheat germ agglutinin binding protein on the surface of human granulocytes associated with the cytoskeleton

Lectin-receptors on leukocyte and endothelial surfaces are becoming more important in the light of increasing evidence which implicates lectin-carbohydrate interactions in diverse physiological phenomena. This study reports the identification of a major 118 kDa granulocyte surface protein, (Protein 1a) which binds the lectin wheat germ agglutinin (WGA), and is distinctly different from reported WGA binding granulocyte membrane proteins. Protein 1a has been isolated from the Triton-soluble and Triton-insoluble lysates of normal individuals and patients with Chronic Myeloid Leukemia (CML) using a combination of differential solubilization, lectin affinity, ion exchange chromatography and HPLC. The protein from the detergent lysates of both normal and CML granulocytes has similar pI values, lectin affinities, and hydrophobicity. However, its solubility in Triton is different in the two cell types. In 71% of CML cases examined, Protein 1a exhibits decreased Triton solubility suggesting its increased association with the cytoskeleton (CSK). Stimulation of normal granulocytes with WGA leads to the translocation of the soluble form of Protein 1a to the Triton-insoluble fraction. This cytoskeletal recruitment of Protein 1a is sustained only under conditions of excess WGA and occupied receptor. The CSK disruptive agent dihydrocytochalasin B (H₂CB) releases the insoluble form of the receptor into the Triton-soluble fraction. Investigation of a CSK-involving process such as ligand internalization revealed that CML granulocytes exhibit slower kinetics of internalization of fluorescent WGA molecules. Since Protein 1a is a major WGA receptor on the granulocyte surface, its decreased Triton solubility in CML granulocytes suggests that this may be one of the factors contributing to the defective receptor-mediated endocytosis of WGA by CML cells, arising as a consequence of altered membrane-CSK interaction — a nodal point in the signal transduction cascade.     

High rate anaerobic digestion of a petrochemical wastewater using biomass support particles

Anaerobic digestion of wastewater from a dimethyl terephthalate plant was studied in continuously stirred tank reactors with plastic net biomass support particles (BSP) at a level of 20% (v/v). The experimental results showed that the BSP system could treat the wastewater at a hydraulic retention time as low as 1.5 d, organic loading as high as 20 kg COD/m3/d and at acidic feed pH as low as 4.5 with 95% COD reduction and biogas production of about 8l/l/d, while the control system without support particles could not treat the wastewater above a 5-d hydraulic retention time, 5 kg COD/m3/d organic loading and a feed pH of 6.0. Thus, augmentation of BSP upgraded the performance of the conventional suspended growth system to an equivalent level to advanced reactors.     

Effect of biotin on alkaloid production during submerged cultivation of Claviceps sp. strain SD-58

Addition of biotin to culture medium NL-406 significantly increased alkaloid yield during submerged cultivation of Claviceps sp. strain SD-58. Alkaloid yield was further enhanced by incorporating leucine in biotin-supplemented culture medium. Increased alkaloid production was associated with an increase in the lipid content of cells and in the number of chlamydospores. Biotin deficiency caused a reduction in alkaloid yield and a parallel decrease in lipid content and chlamydospore numbers.     

Helospectin-like immunoreactivity in the esophagus

Helospectin I and II are two non-amidated, VIP-like peptides, isolated from the salivary gland venom of the lizard Heloderma horridum. The lower esophagus of cat, sheep and man was analyzed for helospectin-like immunoreactivity.

Immunocytochemistry revealed helospectin-immunoreactive nerve fibers in the muscle layers, submucosa and mucosa of all species studied. In myenteric ganglia helospectin-immunoreactive nerve fibers and nerve cell bodies could be seen. Double immunostaining for helospectin and vasoactive intestinal peptide (VIP) revealed their coexistence in nerve fibers and cell bodies throughout the lower esophagus of all species tested. Double immunostaining for helospectin and neuropeptide Y revealed their coexistence in nerve fibres surrounding vascular and non-vascular smooth muscle. In the cat and sheep (but not in man) a subpopulation of the helospectin/VIP-containing fibers stored, in addition, substance P.

The helospectin-immunoreactive material in the esophagus probably constitutes a novel neuropeptide. The distribution of the VIP/helospectin-immunoreactive neurons and fibers indicates their possible involvement in the regulation of motor and secretory activities.     

Evidence for secretion of high molecular weight mucins by canine tracheal epithelial cells in primary culture: Effects of select secretagogues on mucin secretion

Summary The purpose of this investigation was to provide evidence for the secretion of high molecular weight mucins, CTM-A and CTM-B, in primary culture of canine tracheal epithelial (CTE) cells. The cells were isolated from tracheas of mongrel dogs by pronase treatment. Primary cultures of the epithelial cells were established using ICN cellagen inserts in Dulbecco’s modified Eagle’s/F12 medium supplemented with growth factors and could be maintained for up to 23 days. The evidence for the mucin secretion in culture medium and their localization in the cells was established by a) positive immunocytochemical staining using specific antibodies developed against purified native as well as deglycosylated CTM-A and CTM-B; b) incorporation of labeled amino acids, followed by electrophoresis and autoradiography detection of glycoconjugates purified from the culture medium; c) comparison of the amino acid compositions of mucin purified from canine tracheal pouch secretions and that purified from the culture medium; and d) Western blot analyses using specific polyclonal antibodies directed against deglycosylated CTM-A and CTM-B. Immunoaffinity purified secreted labeled glycoconjugates were resistant to hyaluronidase treatment. The effects of cyclic AMP (1 × 10⁻⁵ M), dibutyryl cyclic AMP (1 × 10⁻⁵ M), 8-bromocyclic AMP (1 × 10⁻⁵ M), and prostaglandin E₁ (1 × 10⁻⁶ M) on mucin secretion by CTE cells were also investigated. Secretion of mucins by CTE cells in culture was considerably more enhanced by 8-bromocyclic AMP than that observed for other secretagogues used in this study.     

Cyclic AMP metabolism and lipolysis in cultured human adipocyte precursors

Triacylglycerol breakdown (lipolysis) results from a series of reactions culminated by activation of "hormone-stimulated" triacylglycerol lipase, an enzyme unique to adipose tissue. We have studied various components of the lipolytic process in human omental adipocyte precursors differentiating in culture. The levels of cyclic AMP, the "second messenger" of lipolytic hormones, were about sixfold higher in fat cell precursors than those in abdominal skin fibroblasts. L-Isoproterenol resulted in significant elevation of cyclic AMP levels in both cell types. Preincubation of intact adipocyte precursors with insulin resulted in significant enhancement of "low Km" cyclic AMP phosphodiesterase activity; in contrast, this hormone had no effect on fibroblast phosphodiesterase activity, a distinctive biochemical difference despite the morphological similarities between the two cell types during the early stages of adipocyte precursor maturation. Incubation of adipocyte precursors with isoproterenol resulted in the release of fatty acids into the medium, findings indicative of "hormone-stimulated" lipase activity and, hence, the operation of the entire "lipolytic cascade"; isoproterenol-stimulated lipolysis was inhibited by insulin. Release of fatty acids from fibroblasts was not observed. Thus, "hormone-stimulated" lipolysis and insulin stimulation of cyclic AMP phosphodiesterase activity are expressed during early stages of human adipocyte precursor differentiation.     

Purification of the glycoprotein lectin from the broad bean (Vicia faba) and a comparison of its properties with lectins of similar specificity.

1. The lectin from the broad bean (Vicia faba) was purified by affinity chromatography by using 3-O-methylglucosamine covalently attached through the amino group to CH-Sepharose (an omega-hexanoic acid derivative of agarose). Its composition and the nature of its subunits were compared with concanavalin A and the lectins from pea and lentil. 2. Unlike the other three lectins, broad-bean lectin is a glycoprotein; a glycopeptide containing glucosamine and mannose was isolated from a proteolytic digest. 3. The mol.wt. is about 47500; the glycoprotein consists of two apprently identical subunits, held together by non-covalent forces. Fragments of the subunits, similar to those found in concanavalin A and soya-bean agglutinin, were found in active preparations. 4. Broad-bean lectin was compared with concanavalin A and the lectins from pea and lentil in an investigation of the inhibition of their action by a number of monosaccharides, methyl ethers of monosaccharides, disaccharides and glycopeptides. The most striking differences concern 3-O-substituted monosaccharides, which are strong inhibitors of the action of broad-bean, pea and lentil lectins but not of the action of concanavalin A. There is, however, no strong inhibition of the action of these lectins by 3-Olinked disaccharides.     

Growth,behaviour, and educational achievement of Jamaican children with sickle-cell trait.

A longitudinal study of the mental and physical development of 200 children with normal haemoglobin and 21 with the sickle-cell trait was carried out in a small rural community in Jamaica. At about 2 and 10 years of age heights and weights showed no significant differences. At about 10 years of age classroom behaviour, sociability, and educational achievement were similar. The results suggest that the sickle-cell trait does not affect growth and mental development.     

PEGylation of lysine residues improves the proteolytic stability of fibronectin while retaining biological activity

Excessive proteolysis of fibronectin (FN) impairs tissue repair in chronic wounds. Since FN is essential in wound healing, our goal is to improve its proteolytic stability and at the same time preserve its biological activity. We have previously shown that reduced FN conjugated with polyethylene glycol (PEG) at cysteine residues is more proteolytically stable than native FN. Cysteine‐PEGylated FN supported cell adhesion and migration to the same extent as native FN. However, unlike native FN, cysteine‐PEGylated FN was not assembled into an extracellular matrix (ECM) when immobilized. Here, we present an alternative approach in which FN is preferentially PEGylated at lysine residues using different molecular weight PEGs. We show that lysine PEGylation does not perturb FN secondary structure. PEG molecular weight, from 2 to 10 kDa, positively correlates with FN–PEG proteolytic stability. Cell adhesion, cell spreading, and gelatin binding decrease with increasing molecular weight of PEG. The 2‐kDa FN–PEG conjugate shows comparable cell adhesion to native FN and binds gelatin. Moreover, immobilized FN–PEG is assembled into ECM fibrils. In summary, lysine PEGylation of FN can be used to stabilize FN against proteolytic degradation with minimal perturbation to FN structure and retained biological activity.