Inhibition of prolyl hydroxylase activity by bradykinin analogs containing a prolyl-like residue

A number of substituted bradykinin analogs were prepared in which the proline in position 3 was replaced by analogs of proline. All of the bradykinin analogs, with the exception of l-azetidine-2-carboxyl³-bradykinin showed significant ability to inhibit prolyl hydroxylase activity. Addition of an l-glutamyl residue to the amino terminus of 3,4-dehydro-l-prolyl³-bradykinin and trans-4-hydroxy-l-prolyl³-bradykinin resulted in competitive inhibitors of increased effectiveness with K_i, values approximately 10^?4m. One of the peptides, l-3,4-dehydro-l-prolyl³-bradykinin, appeared to serve as a substrate for prolyl hydroxylase.     

The effect of high temperatures on tissue lactate,pyruvate and venous blood properties in the rat

Brain, muscle and liver pyruvate and L(+) lactate were determined in rats (1) control, T_re=37.2°C; (2) thermal stress T_re=40.8°C; (3) terminal thermal stress T_re=43.8°C, and (4) hypoxic death at reduced oxygen tension. A second experiment was conducted to examine venous blood acid-base parameters in heated rats at imminent death. Group 2 failed to show a significant change in brain lactate concentration, but muscle lactate decreased and liver lactate increased significantly (27 and 48%, respectively). Group 3 showed significant increases of 65 and 125% in the lactate content of brain and liver, respectively, but in this instance no significance was attributed to the 12% decrease in muscle lactate. Hypoxia in Group 4 resulted in the greatest increases in tissue lactate in brain, muscle and liver (130, 50 and 171%, respectively). The pyruvate concentrations of brain and liver in Group 2 exhibited no change, but muscle pyruvate decreased significantly (59%). Group 3 brain and muscle pyruvate decreased significantly by 57 and 74%, while liver pyruvate remained unchanged. Hypoxia (4) produced no significant differences in pyruvate levels from those observed in Group 3. The changes in venous blood properties of rats heated until respiratory movement ceased suggested acute and severe metabolic acidosis while animals exhibiting heat induced spasms and loss of coordination showed only slight decreases in blood pH and bicarbonate levels. Brain water content did not change, but muscle was dehydrated and liver tissue water content increased in rats exposed to lethal temperatures. The results indicate that hypoxia probably occurs in rat tissues at high temperatures, but not to a degree that would result in death.

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Zusammenfassung Gehirn, Muskel und Leberpyruvat and 1-Laktat wurden bestimmt in Ratten (1) Kontrollen, T_re=37,2°C; (2) Hitze T_re=40,8°C; (3) tödliche Belastung T_re=43,8°C und (4) Tod durch Hypoxie bei reduziertem PO₂. In einem zweiten Experiment wurde der Säure Basengehalt des venösen Blutes in erhitzten Tieren vor Eintritt des Todes bestimmt. Gruppe 2 zeigte keine signifikante Änderung in der Gehirnlaktatkonzentration, dagegen fiel das Muskellaktat (–27%) und das Leberlaktat stieg significant(+48%). Gruppe 3 zeigte einen signifikanten Anstieg des Gehirn- (+65%) und Leberlaktats (+125%), während das Muskellaktat fiel (–11%). Hypoxie in Gruppe 4 bewirkte den grössten Anstieg des Laktats im Gehirn (+130%), Muskel (+50%) und Leber (+171%). Die Pyruvatkonzentration in Gruppe 2 zeigte nur im Muskel einen Anstieg auf +59%. In Gruppe 3 fielen das Gehirn- (–57%) und Muskelpyruvat (–74%) signifikant, ohne Änderung in der Leber. Ähnliche Resultate ergaben sich bei Hypoxie in Gruppe 4. Vor dem Eintritt des Hitzetodes war im Blut hochgradige Acidose nachweisbar, während Tiere mit Hitzespasmus und Koordinationsstörungen nur gering niedrigere pH- und Bikarbonatwerte aufwiesen. Der Gehirnwassergehalt blieb unverändert, der Muskel war dehydriert und die Leber zeigte einen höheren Wassergehalt bei letal hohen Temperaturen. Die Ergebnisse zeigen, dass Hypoxia bei Hyperthermie auftritt, doch nicht in dem Ausmasse, um zum Tod zu führen.

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Resume On a déterminé les pyruvates et lactates du cerveau, des muscles et du foie de rats présentant 4 particularités différentes: (1) T_re (température rectale) = 37,2°C (contrôle); (2) T_re = 40,8°C (contrainte de chaleur); (3) T_re = 43,8°C (contraite de chaleur limite) et (4) morts par hypoxie par suite de la réduction de la pression partielle de l'oxygène. Dans un second essai, on a déterminé le rapport acides/bases dans le sang veineux de rats sous contrainte de chaleur, juste avant leur trépas. Dans le groupe 2, on n'a pas trouvé de modification significative de la concentration des lactates du cerveau. Par contre, celle des muscles a diminué (–27%) et celle du foie a augmenté (+48%) et cela de façon significative. Dans le groupe 3, on constate une hausse du taux de lactates dans le cerveau (+65%) et dans le foie (+125%), alors que celui des muscles diminue (–11%). L'hypoxie du groupe 4 a provoqué une forte augmentation des lactates aussi bien dans le cerveau (+130%), les muscles (+50%) que le foie (+171%). La concentration des pyruvates ne fut sensiblement modifiée dans le groupe 2 que pour les muscles (+59%). Dans le groupe 3, on en note une diminution significative dans le cerveau (–57%) et dans les muscles (–74%) alors que le taux du foie reste inchangé. On a obtenu des résultats analogues par l'hypoxie dans le groupe 4. On a pu constater une acidose aigue du sang juste avant la mort de chaleur, alors que les animaux atteints de spasmes et de perturbations dans la coordination ne présentaient que peu de variations du pH et du taux de bicarbonate. Dans un état létal par hautes températures, la teneur en eau du cerveau est restée inchangée. Les muscles étaient par contre déshydratés et le foie contenait davantage d'eau. Ces résultats ont montré que, lorsque la température augmente, les animaux souffrent d'hypoxie, mais pas au point d'en mourir.

     

Enzymatic esterifications of functionalized phenols for the synthesis of lipophilic antioxidants

Of seven lipases used to esterify functionalized phenols and fatty acids to synthesize lipophilic antioxidants, that from Candida antarctica lipase B (CAL-B) had the highest catalytic activity. Esterification yields of benzoic acid derivatives catalyzed by CAL-B are below 2% after 7 days. In contrast, relatively high yields were obstained for the esterification of (poly)phenols bearing primary hydroxy groups and aliphatic acids (85% yield within 15 hours). Crude products were purified and used as lipophilic antioxidants in sunflower oil.     

Excision and transposition of two Ds transposons from the bronze mutable 4 derivative 6856 allele of Zea mays L

Summary The regulatory mutation bronze mutable 4 Derivative 6856 (bz-m4 D6856) contains a complex 6.7 kb Dissociation (Ds) element tagged with a duplication of low copy bz 3 flanking sequences (Klein et al. 1988). This creates a unique opportunity to study the transposition of a single member of the repetitive family of Ds elements. Eighteen full purple revertants (Bz alleles) of bz-m4 were characterized enzymatically and by genomic mapping. For 17 of the Bz alleles, reversion to a wild-type phenotype was caused by excision of the 6.7 kb Ds transposon. Nine of these Bz alleles retained the transposon somewhere in their genome. In this study we show that like Ac (Schwartz 1989; Dooner and Belachew 1989), the 6.7 kb Ds element can transpose within a short physical distance, both proximal and distal to its original position. Additional bz sequences have been mapped immediately distal to the mutant locus in bz-m4 D6856; genetic evidence suggests these are flanked by two additional Ds elements. The remaining Bz revertant, Bz :107, arose from excision of a more complex 13 kb Ds element.     

Isolation and characterization of acetonitrile utilizing bacteria

Summary Bacteria utilizing high concentrations of acetonitrile as the sole carbon source were isolated and identified asChromobacterium sp. andPseudomonas aeruginosa. Maximum growth was attained after 96 h of incubation andP. aeruginosa grew slightly faster thanChromobacterium sp. The strains were able to grow and oxidize acetonitrile at concentrations as high as 600 mM. However, higher concentrations inhibited growth and oxygen uptake. Degradation studies with (¹⁴C)acetonitrile indicated 57% of acetonitrile was degraded byPseudomonas aeruginosa as compared to 43% byChromobacterium. The isolates utilized different nitrile compounds as carbon substrates.     

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Gene response upon illumination in forming mRNA encoding peroxisomal glycollate oxidase

Glycollate oxidase is a constituent of leaf peroxisomes. Its biosynthesis is, like the biosynthesis of many chloroplastic proteins, controlled by light, via phytochrome. The level of mRNA coding for glycollate oxidase was determined at different stages of greening of etiolated plant cells. The appearance of glycollate oxidase mRNA in the cytoplasm was measured by hybridization with cDNA containing part of the coding sequence for glycollate oxidase. cDNA was prepared from enriched mRNA, inserted into the Pst I site of pBR 322, and cloned in Escherichia coli DH-1. By differential colony hybridization and hybrid selection, a clone containing a 670 bp sequence complementary to mRNA encoding glycollate oxidase was selected and identified. Northern blot hybridization was used to investigate mRNA levels induced by light. It was found that continuous light affected the formation of glycollate oxidase mRNA. When a large population of microbodies was present in the cells being induced, the immediate mRNA increase was very pronounced, and was detectable as little as 20 min after the beginning of the light treatment. In contrast, a lag period in the mRNA increase was observed when the induction was performed with etiolated leaves which are characterized by the occurrence of a rather small population of microbodies. For comparison, we measured the time-course of formation of mRNA coding for a light-induced chloroplastic protein, i.e., a protein of the light-harvesting complex. The time-courses of levels of the two mRNAs indicate that the program of gene expression differs between the two particular proteins destined either for chloroplasts or for peroxisomes. The formation of glycollate oxidase mRNA could also be stimulated by a short pulse of light, a treatment of 15 s being a sufficient trigger.     

Irregular meiosis in a somatic hybrid between S. bulbocastanum and S. tuberosum detected by species-specific PCR markers and cytological analysis

A system of randomly amplified polymorphic DNA (RAPD) markers was developed to facilitate the transfer of S. bulbocastanum (blb) genes into the S. tuberosum (tbr) genome by hybridization and backcrossing. DNA from tbr, blb and the hexaploid hybrid was used as a template for polymerase chain reaction (PCR) amplification. Polymorphic RAPD products, originating from 10-mer primers, specific for blb were cloned and sequenced at their ends to allow the synthesis of 18-mer primers. The 18-mer primers allowed a more reproducible assay than the corresponding RAPDs. Of eight 18-mer primer pairs, four amplified the expected products specific for blb. However, the stringency of the primer annealing conditions needed to be carefully optimized to avoid amplification of the homeologous tbr product, suggesting that the original RAPD polymorphisms were due to single base-pair changes rather than deletions or insertions. Two primers used for amplification of backcross 2 progeny segregated in a 11 (presence:absence) ratio; the other two were unexpectedly absent. The most likely explanation for the loss of these markers is irregular meiosis in the original hexaploid hybrid and subsequent elimination of chromosomes. Cytological analysis of the meiosis in the hybrid demonstrated widespread irregular pairing and the presence of lagging univalents. In addition, the first backcross individual used as the parent for the second backcross had 54 chromosomes instead of the predicted 60. In conclusion, our results demonstrate that PCR technology can be used for the efficient isolation of taxon-specific markers in Solanum. Furthermore, by the use of these markers we detected the loss of chromosomes that was subsequently shown by cytological analysis to be caused by irregular meiosis of the somatic hybrid.