Molecular cloning of cDNA for rat L-type pyruvate kinase and aldolase B

Two double-stranded cDNA recombinant pBR322 plasmid libraries were constructed starting from high carbohydrate diet rat liver poly(A)+ mRNA, either fractionated by denaturing sucrose gradient centrifugation for the cloning of L-type pyruvate kinase cDNA, or nonfractionated for aldolase B. Both libraries were screened with single-stranded cDNA probes reverse transcribed from fasted or high carbohydrate diet rat liver mRNAs. mRNAs from fasted animals were also fractionated by sucrose gradient centrifugation and mRNAs from the fed animals were, in addition, further purified by high performance liquid gel filtration chromatography. Those clones hybridizing with the "positive" probe (from animals fed the high carbohydrate diet) and not with the "negative" one (from fasted animals) were preselected and their plasmid DNA was purified and analyzed by positive hybridization-selection. Thirty of 4500 bacteria colonies transformed by recombinant plasmids were preselected by differential screening for pyruvate kinase, and 8 of 864 colonies for aldolase B. Twenty-two recombinant plasmids for pyruvate kinase and two for aldolase B were shown to contain specific cDNA inserts by positive hybridization-selection. Plasmids DNAs of some pyruvate kinase and aldolase B clones (whose inserts ranged from 700 to 1050 bases in length) were labeled by nick translation and used as probes for Northern blot hybridization. The pyruvate kinase cDNA probes recognized mainly a 3400-base RNA species which was detected in high carbohydrate diet rat liver, but not in fasted rat liver and in tissues which do not synthesize L-type pyruvate kinase. In addition, some pyruvate kinase probes hybridized with minor RNA species of about 2000 bases in length, only observed after carbohydrate diet. For aldolase B, the recombinant plasmid DNA hybridized with a single RNA species of 1750 bases. This RNA, detected in kidney, small intestine and liver, was induced by a high carbohydrate diet and increased with liver development. The rat probe cross-hybridized with human aldolase B messenger RNA.     

The reactogenicity and immunogenicity of the Urabe Am 9 live mumps vaccine and persistence of vaccine induced antibodies in healthy young children

The immunogenicity and reactogenicity of the Urabe Am 9 mumps virus vaccine strain were studied after the administration of different doses of the vaccine to 197 children ranging in age from seven and a half months to nine years and without a history of mumps. There was no effect of dose on the response in serum neutralizing antibodies in the range of 10(2.9) to 10(4.7) TCID50/dose. In the 90 subjects without detectable serum neutralization antibodies before vaccination seroconversion was obtained in 94.4% after 42 days. Half of a group of 34 seropositive children who were tested also showed a fourfold or greater rise in antibodies. Persistence of vaccine-enhanced haemagluttinin-inhibition (EHI) antibodies was satisfactory as only two of 46 vaccinees followed-up for between 27 and 32 months had undetectable levels of EHI antibodies and the geometric mean titre of vaccine-induced EHI antibodies had only fallen to about one-third by 32 months after vaccination. Although there was serological evidence of a subclinical re-infection in three subjects, to date none of the vaccinees has had clinical mumps indicating that the vaccine confers protection against disease. The vaccine was well tolerated. Furthermore, the majority of the few 'reactions' reported were probably not vaccine-related. It is concluded that the Urabe Am 9 is an acceptable strain for use in live mumps vaccines.     

Comparison of the localization of nucleic acid synthesis during bud formation on leaf fragments and on intact undetached leaves ofBegonia rex Putz.

The budding capacity ofBegonia rex leaf fragments is well known; that of undetached leaves has been shown by us only recently after treating the leaves with 6γγ DMAAP. Benzyladenine is as effective as 6γγ DMAAP in stimulating budding. Lower temperatures (17°, 22–12°, 12°) are also capable of inducing bud formation but only after a small cut has been made in a main vein of the undetached leaf. Root formation can also be provoked on undetached leaves which have a cut in the main leaf vein by higher temperatures (24–22°) or by an IAA treatment. Differences in the first stages of bud formation on leaf fragments and on undetached leaves are observed using histochemical and histoautoradiographic techniques.     

Etude ultrastructurale de l'endoplasme et des vacuoles chez deux types de Dinoflagellés appartenant aux genres Noctiluca (Suriray) et Blastodinium (Chatton)

Résumé L'ultrastructure des éléments cytoplasmiques de deux Dinoflagellés a révélé des différences fondamentales directement liées à leur mode de vie.Chez Noctiluca (Dinoflagellé libre, incolore), les corps de Golgi jouent un rôle important dans la formation des sphères archoplasmiques au moment des divisions et lors de l'élaboration de l'endoglée. Les mitochondries de grande taille, parfois géantes, ont des crêtes nombreuses et une matrice réduite. Elles offrent souvent des relations de contiguïté avec les granulations lipidiques et l'enveloppe nucléaire; elles sont très abondantes dans le tentacule du trophozoïte. Il n'existe pas de plastes. Les inclusions lipidiques abondent ainsi que les trichocystes fibreux et les poches mucifères dont le contenu est polysaccharidique.Chez Blastodinium (Dinoflagellé parasite, coloré sauf une espèce), les corps de Golgi sont très réduits dans le trophocyte, assez rares mais de structure classique dans les sporocytes. Les mitochondries sont de petite taille, pauvres en crêtes et ont une matrice importante chez le trophocyte; elles sont plus grandes dans les sporocytes. Les plastes bien développés, périphériques, possèdent des lamelles stromatiques formées de trois thylakoïdes chez les sporocytes; par contre, chez l'individu végétatif, le plus souvent incolore et relativement protégé de la lumière par plusieurs rangées de sporocytes, les plastes, de grande taille ont un aspect dégénéré: lamelles stromatiques rares, thylakoïdes tassés et stroma important; ce sont des étioplastes. Le cas de Blastodinium contortum hyalinum toujours incolore, est particulier: les plastes existent encore mais sont totalement dégénérés. Il y a très peu de lipides dans le trophocyte, mais ils sont par contre abondants dans les sporocytes. Il existe des trichocystes fibreux et chez l'individu végétatif, des figures myéliniques.Ces importantes variations structurales illustrent parfaitement, dans l'échelle évolutive des Dinoflagellés, l'exemple de deux modes de vie très différents.

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Fine structure of endoplasm and vacuoles in two types of dinoflagellates of the genus Noctiluca and Blastodinium

Summary The fine structure of cytoplasmic elements of two Dinoflagellates reveals fundamental differences directly linked to their way of life.In Noctiluca, free-living, colourless dinoflagellate, the Golgi bodies play an important role in the formation of archoplasmic spheres at the moment of division and when the endoglea is elaborated. The mitochondria of great size, sometimes giant, have very abundant cristae and a reduced matrix. They often show relations of contiguity with the lipid granules and the nuclear envelope; they are very abundant in the tentacle of the trophozoïd. There are no plastids. The lipid inclusions are very abundant and so are fibrous trichocysts and muciferous bags whose content is polysaccharidic.In Blastodinium, parasitic dinoflagellate, coloured except for one species, the Golgi bodies are much reduced in the trophocyte, rather rare but of classical structure in the sporocytes. The mitochondria are of small size, with rare cristae and have an enlarged matrix in the trophocyte; they are bigger in the sporocytes. The plastids are well developed, peripheral, and have stromatic lamellae formed of three thylakoïds in the sporocytes. On the other hand, in the vegetative individual, usually colourless since it is protected from the light by many rows of sporocytes, the large plastids have a degenerated aspect, rare stromatic lamellae, packed thylakoïds and enlarged stroma; they are etioplastids. The case of Blastodinium contortum hyalinum, always colourless, is special: the plastids still exist but are completely degenerated. There are very few lipids in the trophocyte but abundant ones in the sporocytes. Some fibrous trichocysts occur, and in the vegetative individual, some myelinic vacuoles.These important structural variations illustrate very well the example of two very different ways of life, in the evolutionary tree of dinoflagellates.

Cet article fait partie d'un travail d'ensemble constituant une Thèse de Doctorat d'Etat intitulée: La cytologie et la division de deux types de Dinoflagellés: Noctiluca Suriray (genre libre) et Blastodinium Chatton (genre parasite).     

Biosynthesis of methionine in an ethionine-resistant strain ofCandida utilis

The strainCandida utilis T 20 adapted to a high concentration of ethionine, excretes considerable amounts of methionine in a synthetic medium, about 40 times as much as the original non-adapted strain. At the same time, the amount of methionine in yeast cells incrncreased, predominantly in the pool (9 times as much as in the control). This ability to produce greater amounts of methionine in the pool or to excrete it into the medium is not permanent, since after 5 passages on agar with ut ethionine the amount of methionine was practically not increased as compared with the original non-adapted strain. An increase in free methionine and of methionine excreted into the medium was found on cultivating the strain in a molasses-containing medium, too.     

Apolar conjugates of ecdysteroids are not used as a storage form of molting hormone in the argasid tick Ornithodoros moubata

Fifth (last) instar nymphs of th e tick Ornithodoros moubata convert ingested 20-hydroxyecdysone (20E) to apolar conjugates AP2, which are then converted to th e more polar conjugates API. Only small quantities of free hormone were transferred to th e hemolymph and the carcass within t h e first 2 days after the blood meal. The proportion of radiolabel in these two compartments was highest at the time of the endogenous ecdysteroid peak; however, no traces of free [³H]20E were detected. The conversion probably occurs principally in the intestinal cells. Eleven days after ingestion, 84% of the radiolabel is located in the digestive tract, mainly in the form of API conjugates. API obtained in second instar nymphs fed with [3H]ecdysone ([³H]E) remain stable throughout the following nymphal instars. The ecdysteroid moiety of APT remained unchanged. The hydrolysis, although not complete, always yielded a peak comigrating with the reference E but never 20E or any other clearly distinct peaks that may have corresponded to metabolites of 20E. Less label per individual was present in adults, but its nature remained the same, viz., API mainly located in the digestive tract. In females, 2.5% of the label was transferred to the progeny during the first ovipositional cycle. Apolar products (mainly AP2) that accumulated in eggs of females injected with [³H]E or [³H]20E during vitellogenesis remained unchanged during the whole embryonic development. During the molting cycle of larvae, there was only a slight conversion of AP2 to API, but esterase hydrolysis of these products released the same percentages of E and 20E as in the freshly laid eggs. We conclude that in this tick species apolar conjugates of ecdysteroids are inactivation metabolites that are not reutilized during the development of the animal. These metabolites are mainly retained in the tick, probably because of its peculiar blocked midgut. Several studies have shown that in other arthropod species (ticks, spiders, and insects), these apolar metabolites are excreted in the feces.