A subclass of cell surface carbohydrates revealed by a CHO mutant with two glycosylation mutations

A novel lectin-resistance phenotype was displayed by a LEC10 Chinese hamster ovary (CHO) cell mutant that was selected for resistance to the erythroagglutinin, E-PHA. Biochemical and genetic analyses revealed that the phenotype results from the expression of two glycosylation mutations, LEC10 and lec8. The LEC10 mutation causes the appearance of N-acetylglucosaminyltransferase III (GlcNAc-TIII) activity and the production of N-linked carbohydrates with a bisecting GlcNAc residue. The lec8 mutation inhibits translocation of UDP-Gal into the Golgi lumen and thereby dramatically reduces galactosylation of all glycoconjugates. This reduction in galactose addition does not, however, cause Lec8 mutants to be very resistant to the galactose-binding lectin, ricin. By contrast, the double mutant LEC10.Lec8 behaved like a LEC10 mutant and was highly resistant to ricin. Based on structural studies of cellular glycopeptides as well as glycopeptides of the G glycoprotein of vesicular stomatitis virus grown in mutant cells, it appears that the ricin resistance of LEC10.Lec8 cells is due to the presence of a small number of Gal residues on branched, N-linked carbohydrates that also carry the bisecting GlcNAc residue. Labelling of N-linked cellular carbohydrates with [3H]galactose was found to occur at a low level for a wide spectrum of cellular glycoproteins in independent Lec8 mutants. Studies of the LEC10.Lec8 mutant have, therefore, led to the identification of a subset of structures that are acceptors for Gal when intra-Golgi UDP-Gal levels are limiting. This mutant also illustrates the potential for regulating cell surface recognition by carbohydrate-binding proteins by altering the expression of a single glycosyltransferase such as GlcNAc-TIII.     

Pyruvate carboxylase from a thermophilic Bacillus. Studies on the specificity of activation by acyl derivatives of coenzyme A and on the properties of catalysis in the absence of activator

1. Oxaloacetate synthesis catalysed by pyruvate carboxylase from a thermophilic Bacillus in the absence of acetyl-CoA required addition of high concentrations of pyruvate, MgATP(2-) and HCO(3) (-), and at 45 degrees C occurred at a maximum rate approx. 20% of that in the presence of a saturating concentration of acetyl-CoA. The apparent K(m) for HCO(3) (-) at pH7.8 was 400mm without acetyl-CoA, and 16mm with a saturating activator concentration. The relationship between reciprocal initial rate and reciprocal MgATP(2-) concentration was non-linear (convex-down) in the absence of acetyl-CoA, but the extent of deviation decreased as the activator concentration was increased. The relationship between reciprocal initial rate and reciprocal pyruvate concentration was non-linear (convex-down) in the presence or absence of acetyl-CoA. 2. The optimum pH for catalysis of oxaloacetate synthesis was similar in the presence or absence of acetyl-CoA. The variation with pH of apparent K(m) for HCO(3) (-) implicated residue(s) with pK(a) 8.6 in catalysis of the activator-independent oxaloacetate synthesis. 3. Linear Arrhenius and van't Hoff plots were observed for the temperature-dependence of oxaloacetate synthesis in the absence of acetyl-CoA over the range 25-55 degrees C. E(a) (activation energy) was 56.3kJ/mol and DeltaH(double dagger) (HCO(3) (-)) (enthalpy of activation) was -38.6kJ/mol. In the presence of acetyl-CoA, biphasic Arrhenius and van't Hoff plots are observed with a change of slope at 30 degrees C in each case. E(a) was 43.7 and 106.3kJ/mol above and below 30 degrees C respectively. 4. Incubation of Bacillus pyruvate carboxylase with trinitrobenzenesulphonate caused specific inactivation of acetyl-CoA-dependent catalytic activity associated with the incorporation of 1.3+/-0.2 trinitrophenyl residues per subunit. Activator-independent catalysis and regulatory inhibition by l-aspartate were unaffected. The rate of inactivation of acetyl-CoA-dependent catalysis by trinitrobenzenesulphonate was specifically decreased by addition of acetyl-CoA and other acetyl-CoA and other acyl-CoA species, but complete protection was not obtained. 5. All alkylacyl derivatives of CoA tested activated Bacillus pyruvate carboxylase; acetyl-CoA was the most effective. The apparent K(a) exhibited a biphasic relationship with acyl-chain length for the straight-chain homologues. Certain long-chain acyl-CoA species showed additional activation at a high concentration. Weak activation occurred on addition of CoA or adenosine 3',5'-bisphosphate, but carboxyacyl-CoA species and derivatives containing a modified phosphoadenosyl group were inhibitory. Thioesters of CoA with non-carboxylic acids, e.g. methanesulphonyl-CoA, serve as activators of the thermophilic Bacillus and Saccharomyces cerevisiae pyruvate carboxylases, but as inhibitors of pyruvate carboxylases obtained from chicken and rat liver. 6. alpha-Oxoglutarate mimics the effect of l-aspartate as a regulatory inhibitor of the pyruvate carboxylases from both the thermophilic Bacillus and Saccharomyces cerevisiae. l-Glutamate was ineffective in both cases.     

Suppressors of a Lin-12 Hypomorph Define Genes That Interact with Both Lin-12 and Glp-1 in Caenorhabditis Elegans

The lin-12 gene of Caenorhabditis elegans is thought to encode a receptor which mediates cell-cell interactions required to specify certain cell fates. Reversion of the egg-laying defective phenotype caused by a hypomorphic lin-12 allele identified rare extragenic suppressor mutations in five genes, sel-1, sel-9, sel-10, sel-11 and sel(ar40) (sel = suppressor and/or enhancer of lin-12). Mutations in each of these sel genes suppress defects associated with reduced lin-12 activity, and enhance at least one defect associated with elevated lin-12 activity. None of the sel mutations cause any obvious phenotype in a wild-type background. Gene dosage experiments suggest that sel-1 and sel(ar40) mutations are reduction-of-function mutations, while sel-9 and sel-11 mutations are gain-of-function mutations. sel-1, sel-9, sel-11 and sel(ar40) mutations do not suppress amorphic lin-12 alleles, while sel-10 mutations are able to bypass partially the requirement for lin-12 activity in at least one cell fate decision. sel-1, sel-9, sel-10, sel-11 and sel(ar40) mutations are also able to suppress the maternal-effect lethality caused by a partial loss-of-function allele of glp-1, a gene that is both structurally and functionally related to lin-12. These sel genes may therefore function in both lin-12 and glp-1 mediated cell fate decisions.     

Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp.

Isocitrate lyase was isolated in homogeneous state from a thermophilic Bacillus. The enzyme has a mol.wt. of 180000 and a pI of 4.5 and contains threonine as the N-terminal residue. It resembles in size the cognate enzyme from the mesophilic bacterium Pseudomonas indigofera, but is smaller than the enzyme from the eukaryotic fungus Neurospora crassa. All three lyases are tetramers and similar in amino acid composition, but the thermophile enzyme is distinctive from its mesophilic coutnerparts in possessing a lower catalytic-centre activity, greater resistance to chemical and thermal denaturation and fewer thiol groups and in being strongly activated by salts. Salt activation, by 0.4M-KCl, is about 3-fold at 30 degrees C and pH 6.8 and weakens progressively as the temperature or pH is raised. The activation is probably due to a change in the enzyme conformation caused by the electrolyte modifying the interaction between charged groups or between hydrophobic groups in protein. The possible significance of the salt activation, of the relative paucity of thiol groups and of the greater resistance to chemical denaturants is discussed. Besides its effect on the Vmax., KCl produces large increases in the magnitude of several kinetic parameters. A rise in reaction temperature from 30 to 55 degrees C produces a somewhat similar result. In view of these peculiar features, the patterns of inhibition of enzyme activity by compounds such as succinate and phosphoenolpyruvate were examined at 30 and 55 degrees C in the presence and absence of KCl.     

Ratiometric Ca+2 measurement in human recombinant muscarinic receptor subtypes using the Flexstation scanning fluorometer

In modern drug discovery, numerous assay formats are available to screen and quantitate receptor-ligand interactions. Radioactive assays are “gold standard” because they are fast, easy, and reproducible; however, they are hazardous, produce radioactive waste, require special lab conditions, and are expensive on a large scale. Thus, it provides a lot of importance to the “mix & measure” assays that have an optical readout. Fluorescence techniques are likely to be among the most important detection approaches used for high throughput screening due to their high sensitivity and amenability to automation. The aim of the present study was to determine the functional antagonistic affinities of standard muscarinic antagonists in CHO cells over expressing m1, m3, and m5 receptors and to compare them with the respective binding affinities. This study was further extended to elucidate that Ca⁺² measurement assays can serve as a functional screening tool for GPCRs. For this purpose, standard muscarinic receptor antagonists, namely, tolterodine, oxybutynin, and atropine were used. We determined and compard the IC₅₀ values of these three standard inhibitors in fura 2 AM loaded m1, m3, and m5 overexpressing CHO cells and in radioligand binding assay. Both the assays exhibited comparable rank order potencies of the standard inhibitors. This study suggests that Ca⁺² mobilization assays can be an alternate to radioligand binding assays.     

RNAi‐mediated Ultra‐low gossypol cottonseed trait: performance of transgenic lines under field conditions

Cottonseed remains a low‐value by‐product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra‐low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ‐cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild‐type levels of gossypol and related terpenoids. Although it was a relatively small‐scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild‐type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%–8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi‐based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever‐increasing needs of humanity.     

Dietary Selenium Supplementation Does Not Attenuate Mammary Tumorigenesis-Mediated Bone Loss in Male MMTV-PyMT Mice

Biological Trace Element Research - Bone wasting occurs during the progression of breast cancer and contributes to breast cancer mortality. We evaluated the effect of methylseleninic acid (MSeA),...