Legionella sainthelensi: a new species of Legionella isolated from water near Mt. St. Helens

Six strains of a new species, Legionella sainthelensi, were isolated from freshwater in areas affected by the volcanic eruptions of Mt. St. Helens in the state of Washington. Strains of L. sainthelensi are culturally and biochemically similar to other legionellae. They grow on buffered charcoal yeast agar but not on media that lack cysteine. They are gram-negative, nonsporeforming, motile rods that are positive in reactions for catalase, oxidase, gelatin liquefaction, and beta-lactamase. They are negative in reactions for urease, hydrolysis of hippurate, reduction of nitrates, fermentation of glucose, and blue-white autofluorescence. Their cell wall fatty acid composition is qualitatively similar to those of other legionellae, with 50 to 62% branched-chain fatty acids. They contain the isobranched-chain 14- and 16-carbon acids and anteisobranched-chain 15- and 17-carbon acids and relatively large amounts of straight-chain 16-carbon acid. All strains of L. sainthelensi contain approximately equal amounts of ubiquinones Q9, Q10, Q11, and Q12, a pattern similar to those of Legionella bozemanii, Legionella dumoffi, and Legionella longbeachae. Serological cross-reactions were observed between L. sainthelensi, both serogroups of L. longbeachae, and Legionella oakridgensis. Three strains of L. sainthelensi were greater than 90% related by DNA hybridization. The type strain of L. sainthelensi, Mt. St. Helens 4, was 36% related to the type strain of L. longbeachae and 3 to 14% related to the other nine described Legionella species.     

Synthesis and bioassay of isoprenoid 3-alkylthio-1,1,1-trifluoro-2-propanones: Potent,selective inhibitors of juvenile hormone esterase

Four 3-alkylthio-1,1,1-trifluoro-2-propanones with juvenile hormone-like side chains were prepared from citronellol and homogeraniol. These substrates were designed as possible transition-state analogs for the juvenile hormone (JH)-specific esterases present in insects. These four isoprenoid trifluoromethyl ketones were assayed in vitro with JH esterase and general esterases from larvae of the cabbage looper, Trichoplusia ni (Lepidoptera, Noctuidae), and with eel acetylcholinesterase and bovine chymotrypsin. JH esterase inhibition I₅₀ values were in the nanomolar range for all four compounds, while the other esterases had I₅₀'S which were 10³ to 10⁵ higher. The high selectivity of these inhibitors is believed to be due to their similarity in size and functionality to natural JH III. Treatment of T. ni larvae in vivo with solutions of the most active analog, 3-[(E)-4,8-dimethyl-3,7-nonadienylthio]-1,1,1-trifluoro-2-propanone (DNTFP) causes a dose-dependent delay in pupation and a concurrent selective inhibition of JH esterase. These data support the hypothesis that the reduction in in vivo JH titer in larval T. ni is due, in part, to hydrolysis of the hormone by selective esterases. DNTFP appears to be competing with JH for the active site of JH esterase.     

CALCIUM ION DEPENDENCE OF THE 2-SITE IMMUNORADIOMETRIC ASSAY OF MOORE''S S-100 PROTEIN

Abstract— The two-site immunoradiometric assay (two-site IRMA) for a specific protein of the nervous system, S-100, is carried out by reaction of the S-100 protein solution with a solid-phase anti(S-100) followed by a second reaction in which the insoluble product is incubated with purified, radioactive anti(S-100). Unreacted labeled antibodies remain in solution and are washed away. As the amount of S-100 increases, the radioactivity in the solid-phase increases. The most significant assay variable is the effect of calcium on the assay dose-response. 0.1 mM-EDTA causes a total inhibition of the dose-response curve which is reversed by increasing the concentration of calcium ions. The dose-response reaches a maximum at 1.0mM-Ca²⁺. then becomes progressively inhibited as the Ca²⁺ concentration is increased further. Previous immunochemical studies of S-100 which did not allow for this effect must now be interpreted with caution.     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by regulation of hyphal development

The morphological effects of biotin and L-arginine on fruiting body formation of the ascomycete Sordaria macrospora are investigated by scanning electron and light microscopy. Biotin is recognized as an elongation factor and arginine as a branching factor in vegetative and reproductive hyphae. In the absence of exogenous biotin, development is blocked after the ascogonium-core hypha stage of protoperithecial morphogenesis, whereas linear growth of the myceliar front is maintained. The addition of exogenous arginine to a biotin deficient culture induces the formation of numerous side branches even in the older mycelium. Fruiting body formation, however, remains blocked at the protoperithecial stage as before, because of the inability of the side branches to elongate. When biotin and arginine are administered simultaneously, a most vigorous branching and growth are induced in the older mycelium, accompanied by a rapid and maximal formation of fruiting bodies. The results are summarized in a model of the exogenous control of hyphal morphogenesis. The model is designed to explain the relationship between fruiting and hyphal density as well as the edge effect on fruiting body formation.     

Truncated glucagon-like peptide-1 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Identification of a mammalian analogue of the reptilian peptide exendin-4.

To find mammalian analogues of exendin-4, a peptide from Helodermatidae venoms that interacts with newly discovered exendin receptors on dispersed acini from guinea pig pancreas, we examined the actions of recent additions to the vasoactive intestinal peptide/secretin/glucagon family of regulatory peptides. In every respect tested, the truncated form of glucagon-like peptide-1, GLP-1(7-36)NH2, mimicked the actions of exendin-4. Like exendin-4, GLP-1(7-36)NH2 caused an increase in acinar cAMP without stimulating amylase release. GLP-1(7-36)NH2-induced increases in cAMP were inhibited progressively by increasing concentrations of the specific exendin-receptor antagonist, exendin(9-39)NH2. In dispersed acini from guinea pig and rat pancreas, concentrations of GLP-1(7-36)NH2 that stimulated increases in cAMP caused potentiation of cholecystokinin-induced amylase release. Binding of 125I-[Y39]exendin-4 or 125I-GLP-1(7-36)NH2 to dispersed acini from guinea pig pancreas was inhibited by adding increasing concentrations of unlabeled exendin-4 or GLP-1(7-36)NH2. We conclude that the mammalian peptide GLP-1(7-36)NH2 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Exendin(9-39)NH2, a competitive antagonist of the actions of GLP-1(7-36)NH2 in pancreatic acini, may be a useful tool for examining the physiological actions of this peptide.     

Exendin-4, a new peptide from Heloderma suspectum venom, potentiates cholecystokinin-induced amylase release from rat pancreatic acini.

We examined the actions of exendin-4, a new peptide isolated from Heloderma suspectum venom, on dispersed acini from rat pancreas. Exendin-4 caused a 3-fold increase in cAMP but did not alter cellular calcium concentration. Exendin-4-induced increases in cAMP were inhibited by an exendin-receptor antagonist, exendin (9-39)NH2, but not by VIP-receptor antagonists. Whereas up to 1 microM exendin-4 alone did not alter amylase release, potentiation of enzyme release was observed when the peptide (greater than 30 pM) was combined with cholecystokinin. Potentiation of amylase release was also observed when exendin-4 was combined with carbamylcholine, bombesin or a calcium ionophore, A23187. These results indicate that stimulation of exendin receptors on rat pancreatic acini causes an increase in cellular cAMP. Although this increase in cAMP alone does not result in amylase release, combination of exendin-4 with agents that increase cell calcium results in potentiation of amylase release.     

Mapping of multiple subunits of the neuronal nicotinic acetylcholine receptor to chromosome 15 in man and chromosome 9 in mouse

The alpha 3, alpha 5, and beta 4 genes (human gene symbols CHRNA3, CHRNA5, and CHRNB4 respectively; mouse gene symbols Acra-3, Acra-5, and Acrb-4, respectively) are members of the nicotinic acetylcholine receptor gene family and are clustered within a 68-kb segment of the rat genome (Boulter et al., 1990, J. Biol. Chem. 265:4472). By somatic cell hybrid analysis, three cDNAs corresponding to these genes were used to map the homologous loci to human chromosome 15 and to mouse chromosome 9. Linkage analysis using CEPH pedigrees showed that the CHRNA5 gene was closely linked to the following chromosome 15 loci: D15S46, D15S52, D15S28, D15S34, and D15S35. Using interspecies crosses in mice, the Acra-5 gene was found closely linked to the Mpi-1 locus. The mapping of these members of a neurotransmitter receptor gene family may facilitate the identification of relationships between the neurotransmitter receptors and murine or human phenotypes.     

Purification and structure of exendin-3, a new pancreatic secretagogue isolated from Heloderma horridum venom.

An amino-terminal histidyl structure (His1) is characteristic of most peptides in the glucagon superfamily. An assay for His1 peptides performed by amino-terminal amino acid sequencing was used to screen venom from the Gila monster lizard, Heloderma horridum. Two His1 peptides were identified: helospectin and a new His1 peptide that has been named exendin-3 to indicate that it is the third peptide to be found in an exocrine secretion of Heloderma lizards which has endocrine activity, the first two being helospectin (exendin-1) and helodermin (exendin-2). In the lot of H. horridum venom tested, exendin-3 was 5-10-fold more abundant in molar concentration than helospectin. The structure of exendin-3 was analyzed by amino acid sequencing and mass spectrometry. Exendin-3 is a 39-amino acid peptide with a mass of 4200. It contains a carboxyl-terminal amide and has a strong homology with secretin at its amino-terminal 12 amino acids. The complete structure of exendin-3 is His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala- Val-Arg - Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser- amide. It is 32 and 26% homologous with helospectin and helodermin, respectively. It has greatest homology with glucagon (48%) and human glucagon-like peptide-1 (50%). Exendin-3 (3 microM) stimulated increases in cellular cAMP and amylase release from dispersed guinea pig pancreatic acini.