Mutations induced by ionizing radiation in a plasmid replicated in human cells. I. Similar, nonrandom distribution of mutations in unirradiated and X-irradiated DNA

The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells. Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E. coli and the nature of the mutations was determined by direct sequencing of the tRNA gene. At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%). When control plasmid was replicated directly in E. coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells. The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs. The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences. These mutations at C's were preferentially distributed in the nontranscribed strand. We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation. The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication.     

Malonyl-CoA in skeletal muscle and liver of streptozotocin-diabetic rats.

Malonyl-CoA, the inhibitor of carnitine palmitoyl transferase I, has been examined in this study in the muscle and liver of diabetic rats. Male Sprague-Dawley rats were rendered diabetic with streptozotocin (6 mg/100 g body wt). The gastrocnemius/plantaris muscles and liver samples were frozen at liquid nitrogen temperature. Muscle malonyl-CoA was 1.8 +/- 0.2 pmol/mg in control rats and 1.5 +/- 0.2 pmol/mg in the diabetic rats. This difference was not statistically significant. Liver malonyl-CoA of control rats was 8.6 +/- 0.8 pmol/mg, in comparison to 4.3 +/- 0.6 pmol/mg in diabetic rats. In the liver, high concentrations of malonyl-CoA inhibit fatty acid oxidation and ketogenesis. Failure of malonyl-CoA to decline in muscle in the diabetic may be responsible in part for the diversion of fatty acids to the liver, thereby enhancing hepatic fatty acid oxidation and ketogenesis.     

Parameters of growth in primary cultures and cell lines established from Drosophila imaginal discs

We have further characterised our tissue culture system for the growth in vitro of Drosophila imaginal disc cells, including the culture medium requirements for optimum growth and we have adjusted the protocol recommended for the initiation of cultures. Many imaginal disc fragments become organised into vesicles, and some of these secrete extracellular material into the lumen. Sensory axons differentiate in primary disc cultures, in the absence of bristle formation. The early stages of cell division to form a cell line are recorded.     

Integumental lesions caused by ectoparasites in a wild population of the side-blotched lizard (Uta stansburiana)

Histopathological effects of ectoparasites on integument were examined for a wild population of the side-blotched lizard Uta stansburiana. These included the trombiculid Neotrombicula californica, the pterygosomatid mite Geckobiella texana; the macronyssid mite Ophionyssus natricis (Macronyssidae) and the ixodid tick Ixodes pacificus. A diffuse inflammatory response occurred at the site of chigger and tick attachment which consisted of histiocyte, heterophil, fibroblast and lymphocyte infiltration that often extended into the dermis. Granuloma formation also was noted. The most prevalent parasite was N. californica which frequently occurred in large aggregations above the eyelids. Ectoparasites were most abundant from February through April.     

NH(4)-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH(4). Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH(4). The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O(2)) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH(4)Cl. Both mutants failed to incorporate [C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10 cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a N(2)-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of N inside root and shoot material than the wild type did. The results of our nitrogen balance and N enrichment studies indicated that NH(4)-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

A new enzymatic activity participating in the initiation of glycogen biosynthesis in rat brain

A rat brain extract, able to synthesize from UDP-Glc an alpha-1,4-glucan covalently bound to a protein in the absence of added primer is described. The compound formed is precipitable by dilute trichloroacetic acid (TCA). In the presence of glycogen, added as primer, this molecule is enlarged and is not precipitable by TCA. Unprimed and primed activities differ in several aspects, such as the behavior in the presence of some effectors, and the optimum pH. Umprimed and primed activities presented two pHs optima, both sharing only one. The proteoglucans synthesized under the different pHs gave different patterns after analysis under denaturing PAGE and the oligosaccharides synthesized on the protein backbone differ in the glucosyl length. It is concluded that also in rat brain, the initiation process of glycogen biosynthesis is mediated through the formation of a glycoprotein. Our present results showed that the step of the putative "Glycogen Initiator" proposed by use before, requires two enzymes UDPGlc-transglucosylating activities, Glycogen Initiator 1 and Glycogen Initiator 2, before Glycogen Synthase in the alpha-1,4-glucosidic linkages formation.     

Bioengineering in development of the hybrid artificial pancreas

The hybrid artificial pancreas for treatment of diabetes consists of insulin-secreting pancreatic tissue which is surrounded by a membrane that protects the tissue from rejection by the immune system following implantation. In this paper, we review the alternative therapeutic approaches for diabetes under study and then discuss the technical requirements that must be met by a hybrid device useful to humans. Previous work on intravascular and extravascular immunoisolation devices is reviewed from the standpoint of these requirements, and three critical unresolved issues are discussed: biocompatibility, oxygen supply limitations, and prevention of immune rejection.     

Human N-acetylgalactosamine-4-sulphatase: protein maturation and isolation of genomic clones

N-Acetylgalactosamine-4-sulphatase (EC 3.1.6.1, G4S) is composed of a 57 kDa species in human liver that dissociates into 43 kDa and 8 kDa subunits under reducing conditions and, when deficient, causes the lysosomal storage disorder, mucopolysaccharidosis type VI. We isolated genomic clones containing the G4S first exon, including the leader peptide and the amino terminus of the 43 kDa polypeptide. Amino-terminal amino acid sequences of the 43 kDa and 8 kDa subunits indicated that the 8 kDa component is linked to the 43 kDa polypeptide by a single disulphide bond, does not contain the mannose-6-phosphate lysosomal targeting signal and is at the carboxyl terminus of G4S.