Production and Characterization of Monoclonal Antibodies to Wall-Localized Peroxidases from Corn Seedlings

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a M_r 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a M_r 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.     

Reduction of tumor growth following treatment with a glutamine antimetabolite

Assessment of arterial-venous differences across transplanted methylcholanthrene-induced sarcomas in rats revealed significant decreases in plasma concentrations of glutamine, serine and glucose. Treatment with the glutamine antimetabolite, acivicin, significantly reduced tumor weights by 65% at the conclusion of the experiment 34 days after tumor induction. These results suggest that glutamine is an essential metabolic substrate for tumor growth and that blockade of glutamine utilization can inhibit the growth of these transplantable sarcomas.     

Sodium-dependent lysine flux across bullfrog alveolar epithelium

Amino acid transport across the alveolar epithelial barrier was studied by measuring radiolabeled lysine fluxes across bullfrog lungs in an Ussing chamber. In the absence of a transmural electrical gradient, L-[14C]lysine was instilled into the upstream reservoir and the rate of appearance of the radiolabel in the downstream reservoir was determined. Two lungs from the same animal were used simultaneously to determine tracer fluxes both into and out of the alveolar bath. Results showed that the radiolabel flux measured in the alveolar to the pleural direction was greater than that measured in the opposite direction in the presence of sodium in the bathing fluids. The net flux of L-[14C]lysine was saturable with [Na+], with an apparent transport coefficient (Kt) of 28 mM for Na+. Hill analysis of [14C]lysine flux vs. [Na+] indicated a coupling ratio of 1:1 between sodium and radiolabeled L-lysine. Total L-lysine flux as a function of [L-lysine] was also saturable, with Kt of 7.3 mM for L-lysine. Ouabain significantly decreased absorptive (alveolar-to-pleural) radiolabel flux, while slightly increasing the flux observed in the opposite direction. L-leucine completely inhibited absorptive net flux of L-[14C]lysine. alpha-Methylaminoisobutyric acid (MeAIB), on the other hand, only slightly reduced net flux of L-[14C]lysine from the control value. The presence of a net absorptive, Na+-dependent amino acid flux across the alveolar epithelial barrier indicates that the tissue is capable of removing amino acids and sodium from the alveolar fluid by a coupled cotransport mechanism, which may be important for both protein metabolism and fluid balance by alveolar epithelium.     

Effects of T-2 toxin and its congeners on membrane functions of cultured human fibroblasts

Cytotoxicity of T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, and T-2 tetraol was compared between normal human fibroblasts and mutant I-cell human fibroblasts, which only produce 10 to 15% of lysosomal hydrolases present in normal fibroblasts. Both cleavage of 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and cell count by hemocytometer were used for evaluations. For all toxins, dose-related effects on both types of cultures were evident. Cytotoxicity of the above mycotoxins on both cell lines were similar, indicating that lysosomal enzymes were not involved in the toxicity of T-2 toxin and its congeners. An inhibitor of lysosomal cysteine proteases (E-64) did not alter the cytotoxicity of T-2 toxin. The decreasing order of toxicity was T-2 toxin, HT-2 toxin, neosolaniol, acetyl T-2 toxin, and T-2 tetraol in both cell lines. When normal human fibroblasts were loaded with the fluorescent dye Lucifer yellow CH (LY), a subsequent treatment of T-2 toxin did not disrupt lysosomal membranes. The uptake of LY was not affected by T-2 toxin, which indicated that T-2 toxin did not interfere with the endocytic pathway. Results indicate that T-2 toxin and its congeners do not exert their primary toxic effect through lysosomal enzymes, membranes, or via the endocytic pathway.     

Probing the opioid receptor complex with (+)-trans-superfit. I. Evidence that [D-Pen2,D-Pen5]enkephalin interacts with high affinity at the delta cx binding site.

A variety of data support the existence of an opioid receptor complex composed of distinct but interacting mu cx and delta cx binding sites, where "cx" indicates "in the complex." The ability of subantinociceptive doses of [Leu5]enkephalin and [Met5]enkephalin to potentiate and attenuate morphine-induced antinociception, respectively, is thought to be mediated via their binding to the delta cx binding site. [D-Pen2,D-Pen5]Enkephalin also modulates morphine-induced antinociception, but has very low affinity for the delta cx binding site in vitro. In the present study, membranes were depleted of their delta ncx binding sites by pretreatment with the site-directed acylating agent, (3S,4S)-(+)-trans-N-[1-[2-(4-isothiocyanato)phenyl)-ethyl]-3-methy l-4- piperidyl]-N-phenylpropaneamide hydrochloride, which permits selective labeling of the delta cx binding site with [3H][D-Ala2,D-Leu5]enkephalin. The major findings of this study are that with this preparation of rat brain membranes: a) there are striking differences between the delta cx and mu binding sites; and b) both [D-Pen2,D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin exhibit high affinity for the delta cx binding site.     

ACCURACY OF PHYLOGENETIC-ESTIMATION METHODS UNDER UNEQUAL EVOLUTIONARY RATES

A comparative study of the accuracy of three different approaches to phylogenetic estimation was made on simulated data with differing rates of change in different lineages. The three approaches were maximum likelihood, maximum parsimony, and phenetic clustering. The data were generated by simulating genetic drift with different population sizes over a simple four-species tree topology. Although the accuracy of all three approaches was found to be dependent on the number of loci (characters), maximum likelihood was found to perform considerably and consistently better than maximum parsimony or phenetic clustering.     

Affinity purification of human deoxycytidine kinase: avoidance of structural and kinetic artifacts arising from limited proteolysis

Homogeneous deoxycytidine kinase has been isolated from leukemic human T-lymphoblasts by affinity chromatography based on a multisubstrate analog, deoxycytidine 5'-adenosine 5"'-P1,P4-tetraphosphate (dCp4A). Chromatography of extract treated with protease inhibitors yielded a monomeric polypeptide, inasmuch as the Mr of the native protein, 59,300, is comparable to the value of 52,000 from sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric pH was 6.1. But, enzyme isolated without protease inhibitors exhibited two fragments of Mr = 30,000 and 33,000, suggesting that proteolytic cleavage of the parental polypeptide had occurred during affinity chromatography. Both the parental and proteolyzed enzymes phosphorylated deoxyadenosine and deoxyguanosine, as well as deoxycytidine. However, the proteolyzed enzyme had an increased apparent Km for deoxycytidine. In consequence of this, a mixture of the two forms produced bimodal kinetic plots, whereas linear kinetics were displayed by each form alone.     

Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro

This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro. We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase. The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16. These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement. Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16. Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity. These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.