Glutamate Oxidation by Soybean Cotyledon and Leaf Mitochondria

Mitochondria purified from cotyledons of soybean seedlings fiveto ten days old have the capacity to rapidly oxidize glutamate(measured as glutamate dependent oxygen consumption). This capacitywas greatest at ten days after planting but was very low priorto emergence of cotyledons from the vermiculite and during senescence.Solubilized glutamate dehydrogenase activity, on the other hand,was substantial at two days after planting, peaked at sevendays, then declined and rose again during senescence. It issuggested that mitochondrial glutamate oxidation plays a rolein reserve mobilization and amino acid metabolism during seedlinggrowth. Leaf mitochondria and those from senescing cotyledonscould not sustain rapid rates of glutamate oxidation despiteready oxidation of other substrates and high solubilized glutamatedehydrogenase activity, suggesting an alternative role for theenzyme in these tissues. Possible controlling factors are discussed. ² Present address, Garvan Institute, Darlinghurst, N. S. W.,Australia. ³ Permanent address, Department de Biologia Vegetal, Facultatde Biologia, Universitat de Barcelona, Barcelona, Spain. (Received May 6, 1988; Accepted August 3, 1988)     

Onchocerca volvulus. Monoclonal anti-idiotype antibody as antigen signal for the microfilaricidal cytotoxicity of diethylcarbamazine-treated platelets

Over the past 35 yr, diethylcarbamazine (DEC) has been the most widely used agent for the treatment of filarial diseases, particularly in onchocerciasis. The microfilaricidal action of DEC has been recently shown to be mediated by blood platelets with the additional triggering of a filarial excretory Ag (FEA). This FEA could be detected by using mAb in the serum of infected patients. By using one mAb (IA2(23] directed against Onchocerca volvulus and recognizing circulating Ag (Ab1), we purified by affinity chromatography the target molecule of IA2(23) (an O. volvulus glycoprotein recognized by IA2(23) mAb). This compound had a dose-dependent effect on the cytotoxic action of DEC-treated platelets. We subsequently produced an anti-idiotype mAb to Ab1 (Ab2), and considered the possibility of replacing the O. volvulus glycoprotein recognized by IA2(23) mAb by Ab2. Ab2 was selected according to its ability to inhibit the binding of radioiodinated Ab1 to the filarial target Ag. It induced the production of anti-O. volvulus antibodies (Ab3) in rats. At a constant concentration of DEC platelets, the addition of increasing amounts of Ab2 led to a dose-dependent cytotoxic effect against parasite larvae. Experiments performed with Ab2 on detergent solubilized surface proteins of platelets identified four bands of Mr 18, 26, 43.5, and 100 kDa, supporting the idea of the presence of binding sites on the platelets for a FEA required for the microfilaricidal cytotoxicity of DEC-treated platelets.     

Initial Studies on the Regulation of Toluene Degradation by Pseudomonas Putida F1

Pseudomonas putida Fl oxidizes toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is the substrate for “meta” fission of the aromatic nucleus. Kinetic and induction experiments indicate that the genes encoding enzymes for these reactions are part of an operon, designated the tod operon, that is coordinately induced and regulated. Strains unable to utilize toluene as a growth substrate were isolated at high frequencies by using screening procedures that utilize the redox dye, 2,3,5-triphenyl-2H-tetrazolium chloride. Biochemical characterization of strains with mutations in the structural genes of the tod operon showed that toluene induces the first four enzymes in toluene degradation by P. putida Fl. The isolation and characterization of pleiotropicnegative mutants together with mutants altered in terms of their expression of tod genes suggests that the tod operon may be under the control of a positive regulatory element.     

Anaerobic biotransformations of pollutant chemicals in aquifers

Summary Anaerobic microbial communities sampled from either a methanogenic or sulfate-reducing aquifer site have been tested for their ability to degrade a variety of groundwater pollutants, including halogenated aromatic compounds, simple alkyl phenols and tetrachloroethylene. The haloaromatic chemicals were biodegraded in methanogenic incubations but not under sulfate-reducing conditions. The primary degradative event was typically the reductive removal of the aryl halides. Complete dehalogenation of the aromatic moiety was required before substrate mineralization was observed. The lack of dehalogenation activity in sulfatereducing incubations was due, at least in part, to the high levels of sulfate rather than a lack of metabolic potential. In contrast, the degradation of cresol isomers occurred in both types of incubations but proved faster under sulfate-reducing conditions. The requisite microorganisms were enriched and the degradation pathway forp-cresol under the latter conditions involved the anaerobic oxidation of the aryl methyl group. Tetrachloroethylene was also degraded by reductive dehalogenation but under both incubation conditions. The initial conversion of this substrate to trichloroethylene was generally faster under methanogenic conditions. However, the transformation pathway slowed when dichloroethylene was produced and only trace concentrations of vinyl chloride were detected. These results illustrate that pollutant compounds can be biodegraded under anoxic conditions and a knowledge of the predominant ecological conditions is essential for accurate predictions of the transport and fate of such materials in aquifers.     

Quantitation and structures of oligosaccharide chains in human trachea mucin glycoproteins

Human respiratory mucin glycoproteins from patients with cystic fibrosis were purified and oligosaccharide chains were released by treatment with alkaline borohydride. A neutral oligosaccharide alditol fraction was isolated from mucin obtained from a patient with A blood group determinant by chromatography on DEAF-cellulose and individual oligosaccharide chains were then isolated by gel filtration on BioGel P-6 columns and high performance liquid chromatography with gradient and isocratic solvent systems. The structures of the purified oligosaccharides were determined by methylation analysis, sequential glycosidase digestion and H-NMR spectroscopy. The amount of each chain was determined by compositional analysis. A wide array of discrete branched oligosaccharide structures that contain from 3 to 22 sugar residues were found. Many of the oligosaccharides are related and appear to be precursors of larger chains. The predominant branched oligosaccharides which accumulate contain terminal blood group H (Fuc2Ga14) or blood group A (Fuc2(Ga1NAc3) (Ga14) determinants which stop further branching and chain elongation. The elongation of oligosaccharide chains in respiratory mucins occurs on the 3-linked G1cNAc at branch points, whereas the 6-linked GlcNAc residue ultimately forms short side chains with a Fuc2 (Ga1NAc3) Gal4 G1cNAc6 structure in individuals with A blood group determinant.The results obtained in the current studies further suggest that even higher molecular weight oligosaccharide chains with analogous branched structures are present in some human respiratory mucin glycoproteins. Increasing numbers of the repeating sequence shown in the oligosaccharide below is present in the higher molecular weight chains. {ie75-1} This data in conjunction with our earlier observations on the extensive branching of these oligosaccharide chains helps to define and explain the enormous range of oligosaccharide structures found in human and swine respiratory mucin glycoproteins. Comparison of the relative concentrations of each oligosaccharide chain suggest that these oligosaccharides represent variations of a common branched core structure which may be terminated by the addition of a2-linked fucose to the 3/4 linked galactose residue at each branch point. These chains accumulate and are found in the highest concentrations in these respiratory mucins.     

Oxygen-derived free radicals and hemolysis during open heart surgery

Reperfusion injury occurs during open-heart surgery after prolonged cardioplegic arrest. Cardiopulmonary bypass also is known to cause hemolysis. Since reperfusion of ischemic myocardium is associated with the generation of oxygen free radicals, and since free radicals can attack a protein molecule, it seems reasonable to assume that hemolysis might be the consequence of free radical attack on hemoglobin protein. The results of this study demonstrated that reperfusion following ischemic arrest caused an increase in free hemoglobin and free heme concentrations, simultaneously releasing free iron and generating hydroxyl radicals. In vitro studies using pure hemoglobin indicated that superoxide anion generated by the action of xanthine oxidase on xanthine could release iron from the heme ring and cause deoxygenation of oxyhemoglobin into ferrihemoglobin. This study further demonstrated that before the release of iron from the heme nucleus, oxyhemoglobin underwent deoxygenation to ferrihemoglobin. The released iron can catalyze the Fenton reaction, leading to the formation of cytotoxic hydroxyl radical (OH·). In fact, the formation of OH. in conjunction with hemolysis occurs during cardiac surgery, and when viewed in the light of the in vitro results, it seems likely that oxygen-derived free radicals may cause hemolysis during cardiopulmonary bypass and simultaneously release iron from the heme ring, which can catalyze the formation of OH·.     

Persistent measles virus infection enhances major histocompatibility complex class I expression and immunogenicity of murine neuroblastoma cells

The effect of persistent measles virus infection on the expression of major histocompatibility complex (MHC) class I antigens was studied. Mouse neuroblastoma cells C1300, clone NS20Y, were persistently infected with the Edmonston strain of measles virus. The persistently infected cell line, NS20Y/MS, expressed augmented levels of both H-2K^k and H-2D^d MHC class I glycoproteins. Activation of two interferon(IFN)-induced enzymes, known to be part of the IFN system: (2–5)oligoadenylate synthetase and double-stranded-RNA-activated protein kinase, was detected. Measles-virus-infected cells elicited cytotoxic T lymphocytes that recognized and lysed virus-infected and uninfected neuroblastoma cells in an H-2-restricted fashion. Furthermore, immunization of mice with persistently infected cells conferred resistance to tumor growth after challenge with the highly malignant NS20Y cells. The rationale for using measles virus for immunotherapy is that most patients develop lifelong immunity after recovery or vaccination from this infection. Patients developing cancer are likely to have memory cells. A secondary response induced by measles-virus-infected cells may therefore induce an efficient immune response against non-infected tumour cells.     

The “ON”-bipolar agonist,L-2-amino-4-phosphonobutyrate,blocks light-evoked cone contraction in xenopus eye cups

Rhythmic photoreceptor metabolism in relationship to light-dark cycles is now thought be regulated through a retinal feed-back mechanism with dopamine serving as a principal signal initiating light-evoked events. In order to test the hypothesis that depolarizing ON-bipolar neurons participate in the retinal signalling pathway, we determined the effects of L-2-amino-4-phosphonobutyrate (L-APB) on light-evoked cone contraction in eye cups fromXenopus laevis. L-APB blocked the response stereospecifically when applied over a broad concentration range. The high specificity of L-APB in retina suggests that sign-inverting bipolar neurons which depolarize in light are in the signalling pathway. One possibility is that this pathway conveys signals that regulate dopamine release.Special issue dedicated to Dr. Frederick E. Samson.