Isolation of the ATP-Binding Human Homolog of thearsAComponent of the Bacterial Arsenite Transporter

Arsenite resistance in bacteria is mediated by an efflux pump composed of thearsAandarsBgene products. We have isolated the human homolog of the bacterialarsA(hARSA-I), a member of the ATPase superfamily with no transmembrane domain. Southern and Northern analyses indicated the presence of two cross-hybridizing genes in the human genome and expression ofhARSA-Iin many tissues. A rabbit antiserum raised against a glutathione-S-transferase (GST)/hARSA-I fusion protein identified two cross-reacting proteins of 37 and 42 kDa by Western analysis in two different human cell lines. Overexpression ofhARSA-Iin the embryonal human kidney 293 cell line was accompanied by overproduction of the 37-kDa protein. Biochemical analysis using the GST/hARSA-I fusion protein indicated that hARSA-I is an ATPase analogous to the bacterial ArsA. Thus,hARSA-Iis a new eukaryotic member of a highly conserved ATP-binding superfamily of proteins.     

Guanidine group specific ADP-ribosyltransferase in murine cells

We have identified a guanidine group specific ADP-ribosyltransferase activity, capable of transferring an ADP-ribose group from NAD to a low molecular weight guanidine compound [p-(nitrobenzylidine)amino]guanidine and proteins such as histone and poly-L-arginine, in a variety of murine cell lines. The enzyme activity appears to be associated with an integral membrane protein of apparent molecular weight 30-33 kDa. Incubation of the viable cells in isotonic phosphate buffered saline with [32P]NAD results in the incorporation of label into cellular proteins. Dimethyl sulfoxide treatment of the cells downregulates the transferase activity as well as the ADP-ribosylation of cell proteins with extracellular NAD.     

Protective cellular retroviral immunity requires both CD4+ and CD8+ immune T cells.

We have found previously that postexposure chemoprophylaxis with 3'-azido-3'-deoxythymidine (also known as zidovudine or AZT) in combination with recombinant human alpha A/D interferon fully protected mice exposed to a lethal dose of Rauscher murine leukemia virus (RLV) against viremia and disease. After cessation of therapy, over 90% of these mice were able to resist rechallenge with live RLV, thus demonstrating an acquired immunity. Adoptive cell transfer of 4 x 10(7) cells from immunized mice fully protected naive recipients from viremia and splenomegaly after RLV challenge. However, when these immune T cells were fractionated into CD4+ and CD8+ subpopulations, only partial protection was found when 4 x 10(7) T cells of either subset were given. Full protection against RLV challenge was seen again when the T-cell subsets from immunized mice were recombined and transferred at the same number into naive mice. We conclude that cellular immunity alone is protective and that both CD4+ and CD8+ cell types are required for conferring full protection against live virus challenge.     

MULTIPLE EQUILIBRIA AND MAINTENANCE OF ADDITIVE GENETIC VARIANCE IN A MODEL OF PLEIOTROPY

We describe a multilocus model that incorporates pleiotropic stabilizing selection on a large number of characters. We find many different stable equilibria with different levels of polymorphism and additive genetic variability. The results lend support to Wright's concept of a complex adaptive surface with many peaks of different heights. The model assumes that alleles contribute additively to the characters. We analyze the multilocus model by first considering a two-locus model. The two-locus model depends critically on having loci of different effect and on having the optimum phenotype not be that of a completely heterozygous individual. The effects of different loci need to differ only by less than a factor of two. For the multilocus, multicharacter model, we assume that completely heterozygous individuals do not have the optimum phenotype. By restricting attention to a two-allele model, we also assume that there are no alleles that can affect all characters in all possible combinations of directions.     

Effects of antioxidants and reduced oxygen tension on rat mammary epithelial cells in culture

Summary Free radical damage has the potential to significantly affect the behavior of cells in culture. In this study the effects of antioxidants (superoxide dismutase, catalase, and vitamin E) and lowered oxygen tension (1% oxygen) on primary culture of rat mammary epithelial cells were examined. Rat mammary epithelial cells were dissociated in collagenase with or without the addition of antioxidants and low oxygen tension, then cultured for 10 d in rat-tail collagen gel matrix and fed with Dulbecco’s modified Eagle’sF12 medium supplemented with various hormones and growth factors. Growth potential of the mammary cells was enhanced when antioxidants and low oxygen tension were used, alone or in combination, during the cell dissociation period. Using antioxidants and low oxygen tension during the culture period failed to improve growth potential regardless whether cells were dissociated in standard conditions or with antioxidants and low oxygen tension. The use of antioxidants and low oxygen tension during the cell dissociation period also reduced the degree of keratinization of the cells after 10 d of culture. Using antioxidants and low oxygen tension during the cell culture period did not further reduce keratinization if antioxidants and low oxygen tension were used during the dissociation period, but were effective in reducing keratinization if cells were dissociated in standard condition. In this system, antioxidants and low oxygen tension reduced lipid peroxidation during the cell dissociation period. An iron chelator, desferal, can also reduce lipid peroxidation and enhance growth when used during cell dissociation, suggesting the enhanced growth potential by the addition of antioxidants and low oxygen to be due to the reduction of lipid peroxidation. This study is supported by grants CA05388 and GM11903 to Y. K. H. from the Public Health Service, U.S. Department of Health and Human Services, Washington, DC.     

Variation amongEucalyptus species in resistance to eucalyptus longhorned borer in Southern California

Eucalyptus L'Héritier (Myrtaceae: Leptospermoideae) species are native to the Austro-Malaysian region, but have been widely planted in temperate and subtropical regions around the world. In most regions whereEucalyptus have been imported, the Eucalyptus Longhorned Borer (Phoracantha semipunctata F.) (Coleoptera: Cerambycidae) has been accidently introduced. Larvae of the beetle bore through the bark and mine along the cambium of stressed trees, usually killing their host. We report here the relative susceptibilities of 12Eucalyptus species in two mixed-species plantations in California, USA. These trees were stressed by water deficit resulting from a prolonged drought.Eucalyptus species that appeared resistant to the borer includedE. camaldulensis Dehnhardt,E. cladocalyx F. Muller,e. sideroxylon A. Cunn. ex Woolls, andE. trabutii (anE. camaldulensis hybrid). Species that were more susceptible to attack wereE. diversicolor F. Mueller,E. globulus LaBillardière,E. grandis Hill ex Maiden,E. nitens (Deane & Maiden),E. saligna Sm., andE. viminalis LaBillardière. Survival of trees was influenced by fine-scale moisture variation resulting from slope and irrigation effects. Resistance characteristics of theseEucalyptus species did not correlate with taxonomic relatedness or bark characteristics, but did correspond to drought tolerance traits in their native habitat.Eucalyptus species that were resistant to attack byP. semipunctata were those that are most tolerant of drought in Australia.     

On the radiation chemistry of some pyrimidine-electron adducts: reaction of pyrimidine negative ions with t-butanol radicals.

The rates of decay of the electron adducts of thymine, thymidylic acid and of uracil at pH approximately 8-5 and at wave-lengths of 340 or 350 nm in the presence of t-butanol as scavenger for OH radicals are not influenced significantly by the addition of 0-1 M NaClO4. It follows that the decay reactions do not take place between negatively-charged species. The best explanation seems to be an efficient reaction of the electron adducts with the t-butanol radicals formed by reaction of OH radicals with the t-butanol.     

Cloning of the putative aldehyde dehydrogenase,aldA, gene fromStreptomyces aureofaciens

A Streptomyces aureofaciens gene, gap, encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was previously identified. Hybridization studies suggested the presence of a second gap gene in S. aureofaciens. To clone the gene, S. aureofaciens subgenomic library was screened with an oligonucleotide probe encoding a peptide motif conserved in all GAPDH. 3352 bp positive BamHI fragment was identified, the length of which correlated with the hybridization signal. The nucleotide sequence of the fragment was determined, and analysis of the sequence revealed the presence of three open reading frames (ORF). However, none of the genes coded for GAPDH. All three genes formed an operon, consisting of gene orf251, with a high homology to a conserved gene present only in archaeabacteria, and the aldA and adhA genes homologous to various eukaryotic and prokaryotic aldehyde- and alcohol-dehydrogenases, with maximum homology to the phenylacetaldehyde dehydrogenases and arylalcohol dehydrogenases, respectively.     

Comparative changes in the blood-brain barrier and cerebral infarction of SHR and WKY rats

Hypertension is involved in the exacerbation of stroke. It is unclear how blood-brain barrier (BBB) tight-junction (TJ) and ion transporter proteins critical for maintaining brain homeostasis contribute to cerebral infarction during hypertension development. In the present study, we investigated cerebral infarct volume following permanent 4-h middle cerebral artery occlusion (MCAO) and characterized the expression of BBB TJ and ion transporter proteins in brain microvessels of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats at 5 wk (prehypertension), 10 wk (early-stage hypertension), and 15 wk (later-stage hypertension) of age. Hypertensive SHR show increased infarct volume following MCAO compared with WKY control rats. BBB TJ and ion transporter proteins, known to contribute to edema and fluid volume changes in the brain, show differential protein expression patterns during hypertension development. Western blot analysis of TJ protein zonula occludens-2 (ZO-2) showed decreased expression, while ion transporter, Na(+)/H(+) exchanger 1 (NHE-1), was markedly increased in hypertensive SHR. Expression of TJ proteins ZO-1, occludin, actin, claudin-5, and Na(+)-K(+)-2Cl(-) cotransporter remain unaffected in SHR compared with control. Selective inhibition of NHE-1 using dimethylamiloride significantly attenuated ischemia-induced infarct volume in hypertensive SHR following MCAO, suggesting a novel role for NHE-1 in the brain in the regulation of ischemia-induced infarct volume in SHR.