Isolation of a complementary DNA encoding the bean PR4 chitinase: an acidic enzyme with an amino-terminus cysteine-rich domain

The amino acid sequences of peptides generated by trypsin and chymotrypsin digestions of the acidic PR4 chitinase from bean were determined. Oligonucleotide primers derived from this sequence were used to synthesize a PR4 chitinase-specific probe by PCR-amplification. This probe allowed the isolation of cDNA clones encoding PR4 chitinase that have been sequenced. This acidic and extracellular chitinase shows some homology to the basic isoform from the same plant, and differs from other known acidic chitinases by the presence of an amino-terminal cysteine-rich domain. Southern blot analysis of bean genomic DNA revealed that PR4 chitinase is encoded by a single gene.     

Anaerobic oxidation of 1-n-heptadecene by a marine denitrifying bacterium

Summary A denitrifying bacterium showing typical characteristics of Pseudomonas sp. (Al1) capable of growth on 1-heptadecene as the sole source of carbon and energy has been isolated from a hydrocarbon-polluted marine sediment by using classical enrichment techniques. The generation time for anaerobic growth on 1-heptadecene was 24 h, and the percentage of hydrocarbon degradation under anaerobic conditions ranged from 19 to 23%. The emulsifying capacity was observed and suggested that Al1 cultivated anaerobically on heptadecene produced surface-active agents. Offprint requests to: M. Gilewicz     

Aquabacter spiritensis,gen. nov., sp. nov. an aerobic,gas-vacuolate aquatic bacterium

A gram-negative, rod-shaped, gas-vacuolate, aerobic, heterotrophic bacterium was isolated from Spirit Lake, Washington. It was further characterized by phase-contrast and electron microscopy and phenotypic tests. The isolate does not utilize carbohydrates. Deoxyribonucleic acid-ribosomal ribonucleic acid (rRNA) hybridizations showed that this bacterium is a member of rRNA superfamily IV where it occupies a separate position on the Azorhizobium caulinodans rRNA sub-branch which, is part of an rRNA cluster containing also the Beijerinckia, the Bradyrhizobium japonicum and the Rhodopseudomonas palustris sub-branches. Its relationship to other gas-vacuolated genera within rRNA superfamily IV is discussed. A new genus Aquabacter with one species Aquabacter spiritensis is proposed. The type strain is strain SPL-1 (=ATCC 43981=LMG 8611).     

The generation of transplasmic Drosophila simulans by cytoplasmic injection: effects of segregation and selection on the perpetuation of mitochondrial DNA heteroplasmy

Summary Experimental transplasmic Drosophila simulans were obtained through cytoplasm microinjection between eggs carrying different mitochondrial genomes. These genomes (siII and siIII) show a 1.5% difference in their sequences. They produced a large number of heteroplasmic flies in their F₁ progeny and several flies were still heteroplasmic at the eighth generation. The distribution of frequencies of mitochondrial genotypes in the offspring of heteroplasmic females suggests that the stochastic processes involved in the evolution of experimental heteroplasmy of multiple nucleotide sites are very similar to those previously described for spontaneous length heteroplasmy. In addition, the siII genome has a noticeable advantage over the siIII genome in both directions of injection. This advantage is estimated at 58% per fly generation and 5% per cell generation.     

Preparation of mycelial and yeast-like spheroplasts fromMucor rouxii by a lytic enzyme preparation fromPenicillium islandicum

Spheroplasts ofMucor rouxii were prepared from mycelial and yeast-like cells by use of aPenicillium islandicum enzymatic complex. This enzyme preparation, which presents high chitosanase and chitinase activities, was produced by growingP. islandicum either on mycelial or yeast-like walls ofM. rouxii. The presence of magnesium sulfate as an osmotic stabilizer was critical to obtain high yields of spheroplasts from mycelial forms. In the case of yeast-like cells, pretreatment with β-mercaptoethanol followed by magnesium sulfate was essential for extensive spheroplast production.     

Simultaneous Determination of 3,4-Dihydroxyphenylalanine, 5-Hydroxytryptophan, Dopamine, 4-Hydroxy-3-Methoxyphenylalanine, Norepinephrine, 3,4-Dihydroxyphenylacetic Acid, Homovanillic Acid, Serotonin, and 5-Hydroxyindoleacetic Acid in Rat Cerebrospinal Fluid and Brain by High-Performance Liquid Chromatography with Electrochemical Detection

A method using reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection for the simultaneous determination of tryptophan (TRP), 3,4-dihydroxyphenylalanine (DOPA), and their metabolites in whole brain, small-brain parts, and cerebrospinal fluid of rats has been developed. The sample preparation requires only homogenization in perchloric acid and centrifugation before injection onto the column. With a LiChrosorb RP-18 (10 micrometer) column and a mobile phase consisting of a phosphate (NaH2PO4, 0.1 M)-methanol mixture with octylsulfonate (2.6 x 10(-3) M) at pH 3.35 and 26 degrees C, the separation of DOPA, dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 4-hydroxy-3-methoxyphenylalanine, TRP, 5-hydroxytryptophan (5-HTP), serotonin, and 5-hydroxyindoleacetic acid was achieved. The method has been applied to study the effect of alpha-monofluoromethyldopa alone and in combination with L-DOPA or L-5-HTP, on the catechol and 5-OH indole levels in brain and CSF of the rat.     

In vitro stimulation of mouse NK cell activity by phorbol esters: A pathway different from interferon-induced activation

Tumor promoters of the phorbol diester type were tested in vitro for their effect on mouse natural killer (NK) activity. The 12-O-tetradecanoyl-phorbol-13-acetate (TPA) significantly increased the spontaneous cytotoxicity of spleen cells preincubated with this reagent, with a maximal effect at 10–20 ng/ml. Higher concentrations of the weak tumor promoter phorbol-12, 13-dibenzoate (PDB) were required to display the same NK-enhancing property, while the inactive tumor promoter 4-αphorboldidecanoate was ineffective at any concentration. TPA-responding spleen cells displayed the same characteristics as classical NK cells: they were present in nude mice, did not adhere to nylon wool, bore the Thy-1 antigen, and displayed the same target cell specificity as interferon-activated NK cells. A major difference with the interferon-induced NK activation resides in the fact that the TPA-inducible increase in lytic activity does not require RNA and protein synthesis. Our results suggest two different NK activation pathways for IFN and phorbol esters.     

Postnatal development of the cholecystokinin-gastrin family of peptides in the brain and gut of rat

The average levels of CCK-8- and CCK-like peptides present in aqueous and acidic extracts of the brain of the newborn rat were, respectively, 5.3 and 2.9 pmol/g wet weight. These low levels increased 20- and 10-fold, respectively, so as to attain, at 25–30 days post partum, values comparable to those found in adult brain. There was also a rapid deposition of CCK-gastrin-like peptides in the whole gut between day 20 post coitum and day 10 post partum, when maximal concentrations were attained. Later, less CCK-gastrin peptides than other gut components were accumulated during the progressive weaning of the young rats, so that the average concentrations of CCK-gastrin-like peptides were, at day 30, similar to those observed in adult gut.