Regeneration of plants from callus tissue of the pasture legume Centrosema brasilianum

Plants were regenerated from leaf explants of Centrosema brasilianum cultured in vitro. Callus and buds were produced on Murashige and Skoog medium (MS), 0.8% agar, 0.1 mg/l NAA and 1 mg/l BAP. Regeneration of multiple shoots was achieved by transferring callus onto fresh medium containing 0.01 and 1 mg/l of NAA and BAP, respectively. Shoots formed roots upon transfer to MS with 0.01 mg/l NAA. Plantlets were succesfully transferred to soil. Leaf-derived calli of Centrosema arenarium, C. macrocarpum, C. pascuorum, C. pubescens, and C. virginianum did not produce shoots when cultured in vitro.     

Development of fertilized ovules and their role in the growth of the pea pod

The histological development of fertilized ovules during fruit-set and development in pea ( Pisum sativum L. cv. Alaska) has been investigated. Killing the ovules on day 0 (anthesis) or day 1 prevented fruit-set and resulted in ovary degeneration. When the ovules were destroyed at later stages the ovaries developed, though the rate of growth of the pod was reduced significantly. Pollination in pea occurs normally the day before anthesis, and fertilization of the egg cell 32 to 48 h later. The first divisions of the zygote and endosperm nuclei started simultaneously (ca 48 h after pollination) but the endosperm developed more rapidly than the embryo; the embryo sac cavity was lined with free endosperm nuclei at the time of beginning suspensor elongation. Extracts of endosperm and ovule coats from ovules at day 7 after anthesis showed fruit-set activity in pea, the latter material having about 3 times more activity than the former per ovule basis. These results indicate that fertilization of the ovule is necessary for fruit-set in pea, and that compounds which induce fruit-set are probably synthesized in the ovules following fertilization.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Clinical and molecular heterogeneity of phenylalanine hydroxylase deficiencies in France

RFLPs of 68 normal and 74 mutant alleles at the phenylalanine hydroxylase (PAH) locus were determined in 37 French kindreds. A total of 23 haplotypes, including 18 normal and 16 mutant alleles, were observed. Two-thirds of all mutant alleles were confined within only four haplotypes, while the last third was accounted for by 12 haplotypes, including eight haplotypes absent from Caucasian pedigrees reported thus far. Several mutant haplotypes were present in typical phenylketonuria only, others were present in variants only, and some were present in both. In addition, a particular mutant haplotype (haplotype 2) was found to harbor different mutations in our series, resulting in either typical phenylketonuria or in mild hyperphenylalaninemias. The diploid combination of so many mutant haplotypes in PAH-deficient patients and of compound heterozygosity at the PAH locus in southern Europe might account for the broad spectrum of individual phenotypes observed in France.     

Control of sugar transport in human fibroblasts independent of glucose metabolism or carrier-substrate interaction

Transport regulation by different metabolizable and nonmetabolizable sugars was studied in human fibroblasts. Sugars were classed as glucose-like (D-mannose, 3-0-methyl-D-glucose, thio-D-glucose, and D-allose) and starvation-like (D-galactose, D-fructose, L-glucose, D-xylose, 6-deoxy-D-glucose and 2-deoxy-D-glucose) based on their competence in curbing glucose starvation enhanced transport. No significant correlation existed between the ability of a sugar to curb hexose transport and the KI of that sugar in inhibiting hexose transport. Independence of the transport curb from glucose metabolism was observed since nonmetabolizable analogs of D-glucose when substituted for D-glucose in the culture medium effected glucose [i.e. 3-0-methyl-D-glucose (3-OMG)] and starvation-like (i.e. 6- and 2-deoxy-D-glucose) effects. The KI of inhibition pf 2-deoxy-D-glucose transport for 3-OMG was 8.5 mM, similar to those obtained for 6-deoxyglucose and 2-deoxyglucose on 2-deoxyglycose transport (7.5 and 3.5 mM, respectively) and on 3-0-methylglucose transport (3.5 and 2.5 mM, respectively). An equimolar mixture of D-glucose and 3-OMG (5.55 mM each) was more effective than 11.1 mM D-glucose or 3-OMG alone in curbing hexose transport or reversing hexose starvation induced increases in transport. The effect of 3-OMG may be independent of glucose metabolism but it is possible that 3-OMG structurally mimics a metabolite of glucose that may interact with intracellular regulators of carrier degradation and or expression.     

Characterization of the biosynthesis in vivo of α-aminoadipyl-cysteinyl-valine inPenicillium chrysogenum

Summary The parameters affecting the formation in vivo of -aminoadipyl-cysteinyl-valine (ACV), an intermediate in penicillin biosynthesis, have been established in low- and high-penicillin producing strains ofPenicillium chrysogenum. ACV was found both in cell extracts and in the culture broth filtrates. (¹⁴C)valine, -(¹⁴C)aminoadipic acid and (¹⁴C)cysteine were efficiently incorporated into ACV. Formation of ACV was stimulated by phenylacetic acid when added during the growth of the culture. ACV biosynthesis was enhanced when protein synthesis was blocked with cycloheximide or anisomicin. The ACV-synthesising activity of the culture increased between 24 and 48 h of the culture preceeding penicillin biosynthesis, and remained constant thereafter. A decay of ACV-forming activity was observed when de novo protein synthesis was inhibited with cycloheximide. The apparent half-life of the ACV-synthesising enzyme system was 2.5 h.     

Effect of prolactin and glucocorticoids on P-enolpyruvate carboxykinase activity in liver and mammary gland from diabetic and lactating rats

Summary The administration of 2 bromo--ergocryptine, to reduce serum prolactin decreased the activity of cytosolic P-enolpyruvatc carboxykinase (GTP) (EC4.1.1.32) about 50% in both liver and mammary gland of lactating animals. Adrenalectomy had similar effects to those of bromo-a-ergocryptine. In contrast, there was a 50% increase in enzyme activity in the mammary gland of diabetic, lactating rats and a 10-fold increase in liver as compared with normal rats. P-enolpyruvate carboxykinase activity in mammary gland as liver is coordinately regulated by prolactin, glucocorticoids and insulin.     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine