Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector

The disamed Ti-binary vector pBIN 6 in Agrobacterium tumefaciens has been used in leaf disc transfomations to produce transgenic apple (Malus pumila Mill.) plants with a nomal phenotype except for a somewhat reduced capacity to root. The presence of the genes for nopaline synthase and neomycin phosphotrans ferase (conferring kanamycin resistance), inserted into the host genome by the vector, was confirmed by Southern blot analysis, the detection of nopaline synthase activity and rooting in the presence of the antibiotic.The nopaline synthase gene continued to be expressed in glasshouse-grown plants several months after removal from in vitro growth conditions.     

Triacylglycerol fatty acid and sterol composition of sediment microorganisms from McMurdo Sound,Antarctica

Summary The triacylglycerol fatty acid and sterol profiles of microorganisms from three McMurdo Sound sediment sites, collected during the austral summer of 1984–1985, were determined using gas chromatography and gas chromatography-mass spectrometry. Comparison of the three sites indicated that Cape Evans contained the greatest concentration of triacylglycerol (TG) (220 nmoles/gram dry weight (gdw) of sediment), approximately six to seven times that determined for sediment microorganisms from the Cape Armitage and New Harbor sites. The relative proportion of triacylglycerolderived polyunsaturated fatty acids (PUFA) revealed a somewhat different trend. New Harbor sediment contained the greatest relative proportion of PUFA (22% of triacylglycerol fatty acids), followed by Cape Evans (16%) and Cape Armitage (11%). The proportion of unsaturated fatty acids (poly-and monounsaturated) was relatively constant and ranged from 63% to 71% of the triacylglycerol fatty acids for the three sites. Sterol concentrations varied from 610 pmoles/gdw at Cape Evans, to 370 and 240 pmoles/gdw for Cape Armitage and New Harbor respectively, and was approximately 1% of the total determined lipid. Cholesterol was the major sterol component detected, occurring at similar relative levels (29%) for all three sites. Other sterols present in decreasing order of abundance were 22-dehydrocholesterol, brassicasterol, 24-ethylcholesterol and 24-methylcholesterol. 5-stanols were only minor components of the three sediments, indicating that in situ biohydrogenation of stenols was not a major sterol transformation process in these recent surface oxic sediments.Part 3 in the series: Microbial Ecology in Antarctic Sea-Ice and Benthic Communities     

Knockdown resistance to dichlorodiphenyl-trichloroethane and pyrethroid insecticides in the napts mutant of Drosophila melanogaster is correlated with reduced neuronal sensitivity

Resistance to pyrethroid insecticides and dichlorodiphenyltrichloroethane (DDT) was investigated in the nap^ts (no action potential, temperature sensitive) mutant of Drosophila melanogaster. In surface contact bioassays, the nap^ts strain showed threefold resistance to deltamethrin at the LC₅₀ level when compared to susceptible Canton-S flies. Cross-resistance was also observed to DDT and the pyrethroids NRDC 157 [3-phenoxybenzyl [1R,cis]-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate], fenfluthrin, and MTI-800 [1-(3-phenoxy-4-fluorophenyl)-4-(4-ethoxyphenyl)-4-methylpentane]. The onset of intoxication by pyrethroids in nap^ts flies was markedly delayed, a finding that is consistent with the existence of a resistance mechanism involving reduced neuronal sensitivity. Resistance at the level of the nerve was confirmed by electrophysiological recordings of spontaneous and evoked activity in the dorsolongitudinal flight muscles of poisoned flies. Preparations from nap^ts insects treated with fenfluthrin displayed longer latencies to the appearance of spontaneous activity and also an absence or reduction in burst discharges compared to equivalent preparations from susceptible individuals. These results are discussed in light of competing hypotheses concerning the mechanism underlying knockdown resistance and reduced nerve sensitivity in insects.     

A highly polymorphic locus cloned from the breakpoint of a chromosome 11p13 deletion associated with the WAGR syndrome

Children with constitutional deletions of chromosome 11p13 suffer from aniridia, genitourinary malformations, and mental retardation and are predisposed to develop bilateral Wilms tumor (the WAGR syndrome). The critical region for these defects has been narrowed to a segment of band 11p13 between the catalase and the beta-follicle-stimulating hormone genes. In this report, we have cloned the endpoints from a WAGR patient whose large cytogenetic deletion, del(11)(p14.3::p13), does not include the catalase gene. The deletion was characterized using DNA polymorphisms and found to originate in the paternally derived chromosome 11. The distal endpoint was identified as a rearrangement of locus D11S21 in conventional Southern blots of the patient's genomic DNA, but was not detected in leukocyte DNA from either parent or in sperm DNA from the father. The proximal endpoint was isolated by cloning the junction fragment and was mapped in relation to other markers and breakpoints. It defines a new locus in 11p13-delta J, which is close to the Wilms tumor gene and the breakpoint cluster region (TCL2) of the frequent t(11;14)(p13;q11) translocation in acute T-cell leukemia. An unusual concentration of base pair substitutions was discovered at delta J, in which 9 of 44 restriction sites tested (greater than 20%) vary in the population. This property makes delta J one of the most polymorphic loci on chromosome 11 and may reflect an underlying instability that contributed to the original mutation. The breakpoint extends the genetic map of this region and provides a useful marker for linkage studies and the analysis of allelic segregation in tumor cells.     

Influence of wetland hydroperiod on diversity and abundance of metamorphosing juvenile amphibians

Numbers of successfully metamorphosing juvenile amphibians were tabulated at three wetlands in South Carolina, U.S.A. using terrestrial drift fences with pitfall traps. A relatively undisturbed Carolina bay was studied for eight years, a partially drained Carolina bay for four years, and a man-made borrow pit for three years. Annual production of juveniles at the undisturbed Carolina bay ranged from zero to 75,644 individuals of 15 species. Fewer individuals of fewer species typically metamorphosed at the borrow pit than at the undisturbed bay, with the least numbers at the partially drained Carolina bay. Both total number and species diversity of metamorphosing juveniles at each site each year showed a strong positive correlation with hydroperiod, i.e., the number of days a site contained standing water that year. Data for one common anuran species and the most common salamander species were analyzed separately by multiple regression, in addition to the community analyses. For the mole salamander, Ambystoma talpoideum, hydroperiod was a significant predictor of the number of metamorphosing juveniles, but the number of breeding females was not. For the ornate chorus frog, Pseudacris ornata, the number of breeding females was a significant predictor of the number of metamorphosing juveniles, but hydroperiod was not. Variation in the dates of wetland filling and drying interacts with other factors to determine amphibian community structure and diversity. Either increasing or decreasing the number of days a wetland holds water could increase or decrease the number and species diversity of amphibians in and around a wetland.     

Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.     

Discrepancy in divergence of the mitochondrial and nuclear genomes ofDrosophila teissieri andDrosophila yakuba

Summary Restriction sites were compared in the mitochondrial DNA (mtDNA) molecules from representatives of two closely related species of fruit flies: nine strains ofDrosophila teissieri and eight strains ofDrosophila yakuba. Nucleotide diversities amongD. teissieri strains and amongD. yakuba strains were 0.07% and 0.03%, respectively, and the nucleotide distance between the species was 0.22%. Also determined was the nucleotide sequence of a 2305-nucleotide pari (ntp) segment of the mtDNA molecule ofD. teissieri that contains the noncoding adenine+thymine (A+T)-rich region (1091 ntp) as well as the genes for the mitochondrial small-subunit rRNA, tRNA^f-met, tRNA^gln, and tRNA^ile, and portions of the ND2 and tRNA^val genes. This sequence differs from the corresponding segment of theD. yakuba mtDNA by base substitutions at 0.1% and 0.8% of the positions in the coding and noncoding regions, respectively. The higher divergence due to base substitutions in the A+T-rich region is accompanied by a greater number of insertions/deletions than in the coding regions. From alignment of theD. teissieri A+T-rich sequence with those ofD. yakuba andDrosophila virilis, it appears that the 40% of this sequence that lies adjacent to the tRNA^ile gene has been highly conserved. Divergence between the entireD. teissieri andD. yakuba mtDNA molecules, estimated from the sequences, was 0.3%; this value is close to the value (0.22%) obtained from the restriction analysis, but 10 times lower than the value estimated from published DNA hybridization results. From consideration of the relationships of mitochondrial nucleotide distance and allozyme genetic distance found among seven species of theDrosophila melanogaster subgroup, the mitochondrial nucleotide distance observed forD. teissieri andD. yakuba is anomalously low in relation to the nuclear genetic distance.     

Glutathione Turnover in Cultured Astrocytes: Studies with [15N]Glutamate

The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20-40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady-state incubation medium with 0.05 mM L-cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.     

Glutamine and Aspartate Loading of Synaptosomes: A Reevaluation of Effects on Calcium-Dependent Excitatory Amino Acid Release

Guinea-pig cerebral cortical synaptosomes were preincubated for 60 min with 100 microM D-aspartate, L-aspartate, or L-glutamate. The total D- plus L-aspartate content of the synaptosomal fraction increased to 235%, 195%, or 164%, respectively, of the control. Despite this no increase was seen in the very low KCl evoked, Ca2+-dependent release of aspartate. Preincubation with the three amino acids changed the synaptosomal glutamate content to 78% (D-aspartate), 149% (L-aspartate), or 168% (L-glutamate) of control. However there was no statistically significant effect of these preincubations on the extent of Ca2+-dependent glutamate release. Thus the Ca2+-dependent release of aspartate and glutamate is not determined by the total synaptosomal content of these amino acids. The addition of 0.1-0.5 mM glutamine to the incubation caused a massive appearance of glutamate in the extrasynaptosomal medium. Analysis of specific activities showed that glutamine was hydrolysed directly by an extrasynaptosomal glutaminase, and that intrasynaptosomal glutamate was predominantly labelled by uptake of this glutaminase-derived glutamate. No increase was seen in the extent of Ca2+-dependent release of glutamate (by fluorimetry) either after preincubation with glutamine or in the continued presence of glutamine. Thus we are unable to confirm reports that glutamine expands the transmitter pool of glutamate. The extrasynaptosomal glutaminase activity in the synaptosomal preparation was inhibited by Ca2+ and activated by phosphate. Identical kinetics were obtained with "free" brain mitochondria, confirming the origin of the glutamine-derived glutamate.     

Inositol 1,3,4,5-tetrakisphosphate is essential for sustained activation of the Ca2+-dependent K+ current in single internally perfused mouse lacrimal acinar cells

Summary We have examined the effects of various inositol polyphosphates, alone and in combination, on the Ca²⁺-activated K⁺ current in internally perfused, single mouse lacrimal acinar cells. We used the patch-clamp technique for whole-cell current recording with a set-up allowing exchange of the pipette solution during individual experiments so that control and test periods could be directly compared in individual cells. Inositol 1,4,5-trisphosphate (Ins 1,4,5 P₃) (10–100 m) evoked a transient increase in the Ca²⁺-sensitive K⁺ current that was independent of the presence of Ca²⁺ in the external solution. The transient nature of the Ins 1,4,5 P₃ effect was not due to rapid metabolic breakdown, as similar responses were obtained in the presence of 5mm 2,3-diphosphoglyceric acid, that blocks the hydrolysis of Ins 1,4,5 P₃, as well as with the stable analoguedl-inositol 1,4,5-trisphosphorothioate (Ins 1,4,5 P(S)₃) (100 m). Ins 1,3,4 P₃ (50 m) had no effect, whereas 50 m Ins 2,4,5 P₃ evoked responses similar to those obtained by 10 m Ins 1,4,5 P₃. A sustained increase in Ca²⁺-dependent K⁺ current was only observed when inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5 P₄) (10 m) was added to the Ins 1,4,5 P₃ (10 m)-containing solution and this effect could be terminated by removal of external Ca²⁺. The effect of Ins 1,3,4,5 P₄ was specifically dependent on the presence of Ins 1,4,5 P₃ as it was not found when 10 m concentrations of Ins 1,3,4 P₃ or Ins 2,4,5 P₃ were used. Ins 2,4,5 P₃ (but not Ins 1,3,4 P₃) at the higher concentration of 50 m did, however, support the Ins 1,3,4,5 P₄-evoked sustained current activation. Ins 1,3,4 P₃ could not evoke sustained responses in combination with Ins 1,4,5 P₃ excluding the possibility that the action of Ins 1,3,4,5 P₄ could be mediated by its breakdown product Ins 1,3,4 P₃. Ins 1,3,4,5 P₄ also evoked a sustained response when added to an Ins 1,4,5 P(S)₃-containing solution. Ins 1,3,4,5,6 P₅ (50 m) did not evoke any effect when administered on top of Ins 1,4,5 P₃. In the absence of external Ca²⁺, addition of Ins 1,3,4,5 P₄ to an Ins 1,4,5 P₃-containing internal solution evoked a second transient K⁺ current activation. Readmitting external Ca²⁺ in the continued presence internally of Ins 1,4,5 P₃ and Ins 1,3,4,5 P₄ made the response reappear. We conclude that both Ins 1,4,5 P₃ and Ins 1,3,4,5 P₄ play crucial and specific roles in controlling intracellular Ca²⁺ homeostasis.