Analysis of the gliadin multigene loci in bread wheat using nullisomic-tetrasomic lines

Summary The polypeptides specified by mRNAs hybridizing to several wheat storage protein cDNAs were determined by one-and two-dimensional gel electrophoresis of hybrid-selected translation products. Some of the polypeptides could be assigned to chromosomes on the basis of results gained from two-dimensional fractionation of the in vitro translation products of poly A⁺ RNA from nullisomic-tetrasomic lines of wheat. cDNA clones belonging to different hybridization groups contained sequences related to different gliadin polypeptide types. In order to determine the chromosomal location and copy number of homologous sequences in the wheat genome, selected cDNA clones were hybridized to restriction endonucleasedigested wheat DNA. The cDNA clones hybridized to sequences derived either from the group 1 chromosomes (-3 gliadin and pTag 544 type) or from the group 6 chromosomes (/, pTag 53 type). The / type sequences are present in 25–35 copies per haploid genome and pTag 544 type sequences in 10–15 copies per haploid wheat genome. Partial sequencing of some of the cDNAs revealed low level homology between the different gliadin cross-hybridization groups, a high-molecular-weight glutenin cDNA sequence and a clone encoding the barley storage protein B-hordein. The significance of these findings is discussed with respect to the probable ancestral relations between wheat endosperm storage protein genes.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Cr(VI) increases tyrosine phosphorylation through reactive oxygen species-mediated reactions

While Cr (VI)containing compounds are well established carcinogens, the mechanisms of their action remain to be investigated. In this study we show that Cr (VI) causes increased tyrosine phosphorylation in human lung epithelial A549 cells in a timedependent manner. Nacetylcysteine (NAC), a general antioxidant, inhibited Cr (VI)induced tyrosine phosphorylation. Catalase, a scavenger of H₂O₂, sodium formate and aspirin, scavengers of hydroxyl radical (OH), also inhibited the increased tyrosine phosphorylation induced by Cr (VI). SOD, an inhibitor of superoxide radical (O₂ ^–), caused less inhibition. ESR study shows that incubation of Cr (VI) with the A549 cells generates OH radical. The generation of radical was decreased by addition of catalase and sodium formate, while SOD did not have any inhibitory effect. Oxygen consumption measurements show that addition of f Cr (VI) to A549 cells resulted in enhanced molecular oxygen consumption. These results indicate that Cr (VI) can induce an increase in tyrosine phosphorylation. H₂O₂ and OH radicals generated during the process are responsible for the increased tyrosine phosphorylation induced by Cr (VI).     

The α2 cDNA sequence of human haptoglobin carries a bacterial promoter functionalin vivo

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete ^2FS precursor protein or only for the subunit have been placed under the control of the P_R promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the P_R promoter, the complete ^2FS constructions constitutively express a smaller polypeptide of 30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (²P_F and ²P_S) located in the duplicated ^2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hp² cDNA sequence, when fused upstream to the cDNA coding for ¹-antitrypsin, constitutively promotesin vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against ¹-antitrypsin or haptoglobin.     

Effects of Lead on Adenylate Cyclase Activity in Rat Cerebral Cortex

Lead decreased in a dose dependent manner the basal AC activity in membranes of rat cerebral cortex (IC₅₀ = 2.5 ± 0.1 M). In membranes preincubated under basal conditions, AC activity was stimulated by approximately two and fourfold by 10 M Gpp(NH)p or forskolin, respectively. Under basal conditions, lead (3 M) inhibited enzyme activity up to 50%, but was not able to inhibit the Gpp(NH)p- or the forskolin-stimulated AC activity. However, in membranes preincubated with Gpp(NH)p (10 M), lead (3 M) had no significant effect on enzyme activity, but it partly blocked the stimulation of AC activity elicited by forskolin (10 M). In membranes preincubated with 10 M lead, the addition of 10 M Gpp(NH)p or forskolin in the incubation medium did not stimulate AC activity. However, when added together in the incubation medium Gpp(NH)p + forskolin produced an increase in enzyme activity. In membranes preincubated with 10 M lead + 10 M Gpp(NH)p, Gpp(NH)p (10 M) or forskolin (10 M) added alone or in combination to the incubation medium did not stimulate AC activity. Moreover, under these latter conditions lead had no further effect on enzyme activity. These results indicate that lead may interact with G-proteins and with the catalytic subunit of cerebral cortical AC to produce inhibition of the enzyme activity.     

Solid phase synthesis of the fibronectin glycopeptide V(Galβ3GalNAcα)THPGY,its β analogue,and the corresponding unglycosylated peptide

The fibronectin fragment VTHPGY and the corresponding glycopeptides V(Gal3GalNAc)THPGY and V(Gal3GalNAc)THPGY were synthesized by the FMOC/solid phase approach. FMOC derivatives of threonine, carrying O-linked, peracetylated Gal3GalNAc chains were used for introduction (HOBt-mediated coupling) of the disaccharide moieties.Abbreviations FMOC 9-fluorenylmethyloxycarbonyl - DMTST dimethyl(thiomethyl)sulfonium triflate - DMF dimethylformamide - BOC butoxycarbonal     

Characterization of the MHC class II region in cattle. The number of DQ genes varies between haplotypes

The organization of the major histocompatibility complex (MHC) class II region in cattle was investigated by Southern blot analysis using human probes corresponding to DO, DP, DQ, and DR genes. Exon-specific probes were also employed to facilitate the assessment of the number of different bovine class II genes. The results indicated the presence of single DO and DR genes, at least three DR genes, while the number of DQ genes was found to vary between MHC haplotypes. Four DQ haplotypes, DQ ^{1 1} to DQ ^{2 4}, possessed a single DQ and a single DQ gene whereas both these genes were duplicated in eight other haplotypes, DQ ^{3 5} to DQ ^{9 12}. No firm evidence for the presence of bovine DP genes was obtained. The same human probes were also used to investigate the genetic polymorphism of bovine class II genes. DQ DQ , DR DR , and DO restriction fragment length polymorphisms (RFLPs) were resolved and in particular the DQ restriction fragment patterns were highly polymorphic. Comparison of the present result with the current knowledge of the class II region in other mammalian species suggested that the DO, DP, DQ, DR, and DZ subdivision of the class II region was established already in the ancestor of mammals. The DP genes appear to be the least conserved class II genes among mammalian species and may have been lost in cattle. The degree of polymorphism of different class II genes, as revealed by RFLP analyses, shows striking similarities between species.     

Floral sexuality and breeding system in gum karaya tree, Sterculia urens

Comprehensive studies were carried out on phenology, floral sexuality, pollination biology, pollen-pistil interaction, breeding system and fruit and seed set on three populations of gum karaya tree (Sterculia urens). The species is andromonoecious and produces a large number of male and a limited number of bisexual (functionally female) flowers. The numbers of male and bisexual flowers varies not only between trees but also during the flowering period within a tree. Each male flower produces about 5000 fertile pollen grains. Neither in morphology nor in number, is there any difference between pollen grains in the bisexual and male flowers. However, pollen grains of bisexual flowers are completely sterile and incapable of siring any seeds. Their anthers, however, serve to attract pollinators; the emasculated bisexual flowers fail to do so. Thus S. urens is apparently andromonoecious but exhibits cryptic monoecy. That the species is self-incompatible was confirmed by controlled pollinations. The self-incompatibility is of the late-acting type and manifests after the entry of the pollen tube into the ovule. Apis indica is the only pollinator recorded by us and wind plays no role in pollination. The efficacy of pollination is low as only 56% of flowers were estimated to be pollinated. The pollen load on one-third the number of pollinated stigmas was lower than the number of ovules present. Fruit set under open pollination is poor and is highly variable from tree to tree (0.7–3.2%). Apart from pollination constraint, limited resource availability may also contribute to low fruit set.     

Comparative residual toxicities of pesticides to the predator Agistemus industani (Acari: Stigmaeidae) on citrus in Florida

Residual toxicities of registered and selected experimental pesticides used on citrus against Agistemus industani Gonzalez (Acari: Stigmaeidae) were compared. Pesticides considered highly toxic to A. industani were: abamectin 0.15 EC at 731ml/ha+FC 435-66 petroleum oil at 46.8l/ha, pyridaben 75WP at 469g/ha, ethion 4EC at 7.01l/ha+FC 435-66 petroleum oil at 46.8l/ha, propargite 6.55 EC at 3.51l/ha, chlorfenapyr 2SC at 1.46l/ha applied alone or in combination with FC 435-66 petroleum oil at 46.8l/ha, sulphur 80DF at 16.81kg/ha, dicofol 4EC at 7.01l/ha, fenbutatin oxide 50WP at 2.24kg/ha, benomyl 50WP at 2.24kg/ha, benomyl 50WP at 1.68kg/ha+ferbam 76 GF at 5.60kg/ha, ferbam 76GF at 11.21kg/ha, neem oil 90EC at 46.8l/ha, and copper hydroxide DF (40% metallic copper) at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha. Pesticides that were moderately to slightly toxic included: copper sulphate 98% at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha, fenbuconazole 2F at 280ml/ha+FC 435-66 petroleum oil at 46.8l/ha, FC 435-66 petroleum oil applied alone at 46.8l/ha or 23.4l/ha, and diflubenzuron 25WP at 1.40kg/ha. Pesticides that were non-toxic included: fenbuconazole 2F at 585ml/ha, malathion 57EC at 5.85l/ha, FC 435-66 petroleum oil at 46.8l/ha, carbaryl 80S at 3.36kg/ha, chlorpyrifos 4EC at 4.68l/ha, and formetanate 92SP at 1.12kg/ha. Understanding the toxic effects of field weathered pesticides against key predacious mite species is important for effective IPM. The results of this study provide a comparison of direct and indirect toxic effects of various pesticides to A. industani under field conditions.     

The toxicity of azidothymidine (AZT) on human and animal cells in culture at concentrations used for antiviral therapy

AZT, a chain terminator of DNA synthesis originally developed for chemotherapy, is now prescribed as an antihuman immunodeficiency virus (HIV) drug at 500 to 1500 mg/person/day, which corresponds to 20 to 60 M AZT. The human dosage is based on a study by the manufacturer of the drug and their collaborators, which reported in 1986 that the inhibitory dose for HIV replication was 0.05 to 0.5 M AZT and that for human T-cells was 2000 to 20,000 times higher, i.e. 1000 M AZT. This suggested that HIV could be safely inhibited in humans at 20 to 60 M AZT. However, after the licensing of AZT as an anti-HIV drug, several independent studies reported 20-to 1000-fold lower inhibitory doses of AZT for human and animal cells than did the manufacturer's study, ranging from 1 to 50 M. In accord with this, life threatening toxic effects were reported in humans treated with AZT at 20 to 60 M. therefore, we have re-examined the growth inhibitory doses of AZT for the human CEM T-cell line and several other human and animal cells. It was found that at 10 M and 25 M AZT, all cells are inhibited at least 50% after 6 to 12 days, and between 20 and 100% after 38 to 48 days. Unexpectedly, variants of all cell types emerged over time that were partially resistant to AZT. It is concluded that AZT, at the dosage prescribed as an anti-HIV drug, is highly toxic to human cells.