β-Alanine transport into plasma membrane vesicles derived from rat brain synaptosomes

Transport of -alanine has been demonstrated in membrane vesicles isolated from rat brain, using artificially imposed ion gradients as the sole energy source. The uptake of -alanine is strictly dependent on the presence of Na⁺ and Cl^– in the medium, and the process can be driven either by an Na⁺ gradient (out > in) or by a Cl^– gradient (out > in) when the other essential ion is present. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of ionophore valinomycin and anions with different permeabilities. -Alanine uptake is inhibited by the presence of GABA.     

Responses of the teleost Hoplias malabaricus to hypoxia

Synopsis Oxygen uptake (VO₂) during graded hypoxia, rate of hypoxia acclimation, breathing frequency (f_R), breath volume (V_{S, R}) and gill ventilation (V_G) were measured in Hoplias malabaricus. Normoxia and hypoxia acclimated fish had similar and constant VO₂ and V_G in a range of water PO₂ from 150 to 25 mmHg. Hypoxia acclimated fish showed significantly higher VO₂ in severe hypoxia (PO₂ <15 mmHg). Normoxia acclimated fish showed symptoms similar to hypoxic coma after 1 h of exposure to water PO₂ of 10 mmHg whereas the same symptoms were observed only at PO₂ of 5 mmHg for fish acclimated to hypoxia. Fish required 14 days to achieve full acclimation to hypoxia (PO₂ ≥25 mmHg). Lowering of water PO₂ from 150 to 25 mmHg resulted in normoxic fish showing a 3–2 fold increase in V_G. The increase was the result of an elevation in V_{S, R} rather than f_R. Among normoxia acclimated specimens, small fish showed a higher V_G per unit weight than the large ones in both normoxia (PO₂ =150 mmHg) and hypoxia (PO₂ = 15 mmHg). A decrease in the ventilatory requirement (V_G/VO₂) with increased body weight was recorded in hypoxia (PO₂ = 15 mmHg).     

Genetic studies of the Macushi and Wapishana Indians

Summary Blood samples from 509 Macushi and 623 Wapishana Amerindians of Northern Brazil and Southern Guyana have been analyzed with reference to the occurrence of rare variants and genetic polymorphisms of the following 25 systems: (i) Erythrocyte enzymes: acid phosphatase-1, adenosine deaminase, adenylate kinase-k, carbonic anhydrase-1, carbonic anhydrase-2, esterase A_1,2,3, esterase D, galactose-1-phosphate uridyltransferase, isocitrate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, nucleoside phosphorylase, peptidase A, peptidase B, phosphoglucomutase 1, phosphoglucomutase 2, phosphogluconate dehydrogenase, phosphohexoseisomerase, triosephosphate isomerase and (ii) Serum proteins: albumin, ceruloplasmin, haptoglobin, hemoglobin A, hemoglobin A₂ and transferrin. Fifteen different rare variants were detected, involving 11 of these systems. In addition, a previously undescribed variant of ESA_1,2,3 which achieves polymorphic proportions in both these tribes is described. Excluding this variant, the frequency of rare variants is 1.1/1000 in 12510 determinations in the Macushi and 4.7/1000 in 15 396 determinations in the Wapishana. The ESA_1,2,3, polymorphism was not observed in 382 Makiritare, 232 Yanomama, 146 Piaroa, 404 Cayapo, 190 Kraho and 112 Moro. Irregularities in the intratribal distribution of this polymorphism in the Macushi and Wapishana render a decision as to the tribe of origin impossible at present. Gene frequencies are also given for previosly described polymorphisms of 5 systems: haptoglobin, phosphoglucomutase 1, erythrocyte acid phosphatase, esterase D, and galactose-1-phosphate-uridyl-transferase.Research supported by the National Science Foundation and the Energy Research and Development Administration.     

Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH—luc fusion to be observed by a dark-adapted eye and photographed.     

Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis

Summary Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 by fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1–5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS : a heterodimeric -ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a -ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORFl-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM 160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.     

Comparative value of four arcelin variants in the development of dry bean lines resistant to the Mexican bean weevil

Wild Phaseolus vulgaris L. accessions containing arcelin codominant alleles 1 through 5 were reconfirmed and characterized for resistance to the Mexican bean weevil, Zabrotes subfasciatus (Boheman) (Coleoptera: Bruchidae). Accession G 02771 (arcelin 5) had the highest level of antibiosis resistance, followed by G 12952 (arcelin 4), G 12882 (arcelin 1) and G 12866 (arcelin 2). Arcelin 3 accessions conferred the lowest levels of resistance. As the presence of arcelin is inherited as a single dominant gene, a backcross breeding program has been used to transfer resistance to the Mexican bean weevil from wild beans to bean cultivars using serological techniques to detect the presence of arcelin and replicated insect feeding tests to measure resistance levels. Progeny containing arcelin 1 showed resistance equal or superior to that of the resistant check. Arcelin 2-deerived lines had intermediate levels of resistance while no resistant progenies were obtained from crosses with arcelin 3 and 4 sources. Results are discussed in relation to the deployment of arcelin alleles in bean cultivars.

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Valeurs comparées de 5 types d'arcéline dans l'obtention de lignées de Phaseolus vulgaris résistantes à Zabrotes subfasciatus

Résumé La résistance à Zabrotes subfasciatus est associée à la présence d'arcéline, une nouvelle protéine des graines, découverte chez quelques populations de Phaseolus vulgaris. 5 types d'arcéline, hérités comme allèles codominants ont été décrits dans la littérature. Nous avons reprécisé les différentes populations contenant différents types d'arcéline et caractérisé leurs résistances à Z. subfasciatus. La population G 02771, correspondant à l'arcéline 5, présente la résistance la plus élevée par antibiose, suivie de G 12952 (arcéline 4), G 12882 (arcéline 1) et G 12866 (arcéline 2). Les populations contenant l'arcéline 3 présentent le moins de résistance à Z. subfasciatus.Un programme de croisements en retour associé à des tests sérologiques pour déceler la présence d'arcéline chez les descendants jeunes et des expériences répétées d'alimentation par les insectes vec BC2F₃ a été réalisé pour transférer la résistance de populations naturelles à des cultivars de haricots. Les lignées, provenant de croisements avec des populations sauvages avec de l'arcéline 1, ont été fortement résistantes à Z. subfasciatus. Les lignées contenant de l'arcéline 2 ont été considérées comme ayant une résistance intermédiaire. Les lignées avec arcélines 3 et 4 étaient sensibles. Les raisons de l'échec du transfert de la résistance élevée des parents contenant de l'arcéline 4, sont inconnues. On a constaté que la concentration de l'arcéline dans les lignées contenant cet allèle était très faible, tandis que la concentration en arcéline 1 restait remarquablement élevée. Les recherches sont poursuivies pour déterminer les raisons de l'absence de transfert de l'arcéline 4 chez les descendants contenant cet allèle. Quoi qu'il en soit, les caractéristiques agronomiques et les qualités des lignées résistantes (codées RAZ) ont été évaluées en vue d'une diffusion pour les programmes nationaux de recherche des pays de basses altitudes intertropicaux ou Zabrotes subfasciatus fait des dégâts importants.

     

Chromosome Interactions in DROSOPHILA MELANOGASTER. II. Total Fitness

In a large experiment, using nearly 200 population cages, we have measured the fitness of Drosophila melanogaster homozygous (1) for the second chromosome, (2) for the third chromosome, and (3) for both chromosomes. Twentyfour second chromosomes and 24 third chromosomes sampled from a natural population were tested. The mean fitness of the homozygous flies is 0.081 ± 0.014 for the second chromosome, 0.080 ± 0.017 for the third chromosome, and 0.079 ± 0.024 for both chromosomes simultaneously. Assuming that fitnesses are multiplicative (the additive fitness model makes no sense in the present case because of the large selection coefficients involved), the expected mean fitness of the homozygotes for both chromosomes is 0.0066; their observed fitness is more than ten times greater. Thus, it appears that synergistic interactions between loci are considerable; and that, consequently, the fitness function substantially departs from linearity. Two models are tentatively suggested for the fitness function: a "threshold" model and a "synergistic" model.—The experiments reported here confirm previous results showing that the concealed genetic load present in natural populations of Drosophila is sufficient to account for the selective maintenance of numerous polymorphisms (of the order of 1000).     

Volatile constituents of Cupressus dupreziana and the sesquiterpenes of Cupressus sempervirens

Analysis of wood essential oil of Cupressus dupreziana revealed 26 components: 13 monoterpenes and 13 sesquiterpenes. The main components were carv     

Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites.

A liquid medium was developed for the continuous cultivation of Trypanosoma cruzi. Among the several highly purified macromolecules tested only bovine liver catalase, horseradish peroxidase, lactoperoxidase, and bovine hemoglobin supported the continuous growth, at high yield, of mice-virulent Trypanosoma cruzi; other hemoproteins were inactive. Bovine liver catalase showed optimal Trypanosoma cruzi growth-promoting activity, parasites reaching 20 × 10⁶ parasites/ml (95% epimastigotes) at about 10 days in most of the 45 subpassages to date. Furthermore, this protein in the incubation medium provided all the amino acid requirements of actively growing parasites, thus eliminating the need for exogeneous free amino acids. Additional experiments revealed that the hemoprotein's growth-promoting activity was independent of any enzymatic activity and that reconstituting the exact protein composition by means of exogeneous amino acids did not support parasite multiplication, suggesting the importance of the primary structure of the active proteins for growth-promoting activity. These active macromolecules supported the multiplication of five different strains of Trypanosoma cruzi, but did not support Leishmania brasiliensis or Leishmania mexicana proliferation, suggesting species specificity.