Absence of siderophore activity in Legionella species grown in iron-deficient media

Growth of Legionella species in a defined medium deficient in iron did not result in the production of phenolic or hydroxamate siderophores which could be detected by chemical or biological assay methods. Growth of a variety of other gram-negative organisms under the same conditions resulted in the production of both hydroxamate and phenolate siderophores. The iron-deficient medium limited growth of the Legionella species more severely than it did the growth of the other gram-negative organisms. We have concluded that Legionella species do not make the commonly recognized siderophores, probably because they are restricted in their growth to those environments in which inorganic iron is readily available or is supplied in a form bound to an unknown carrier.     

Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal

The difficulties associated with the growth of Legionella species in common laboratory media may be due to the sensitivity of these organisms to low levels of hydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) broth to fluorescent light generated superoxide radicals (3 microM/h) and hydrogen peroxide (16 microM/h). Autoclaved YE medium was more prone to photochemical oxidation than YE medium sterilized by filtration. Activated charcoals and, to a lesser extent, graphite, but not starch, prevented photochemical oxidation of YE medium, decomposed hydrogen peroxide and superoxide radicals, and prevented light-accelerated autooxidation of cysteine. Also, suspensions of charcoal in phosphate buffer and in charcoal yeast extract medium readily decomposed exogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations of bovine superoxide dismutase and catalase also decreased the rate of photooxidation of YE medium. Medium protected from light did not accumulate appreciable levels of hydrogen peroxide, and autoclaved YE medium protected from light supported good growth of Legionella micdadei. Various species of Legionella (10(4) cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide (26.5 microM) in challenge experiments. The level of hydrogen peroxide that accumulated in YE medium over a period of several hours (greater than 50 microM) was in excess of the level tolerated by Legionella pneumophila, which contained no measurable catalase activity. Strains of L. micdadei, Legionella dumoffi, and Legionella bozmanii contained this enzyme, but the presence of catalase did not appear to confer appreciable tolerance to exogenously generated hydrogen peroxide.     

Light-induced Polysome Formation in Etiolated Leaves: Kinetics of Inhibition by Antibiotics

Red light-induced, far red light-reversible increase in etiolated bean (Phaseolus vulgaris, var. Asgrow Valentine) leaf polyribosomes was shown to be sensitive to actinomycin D, cycloheximide, and rifampicin inhibition. Actinomycin prevented response to red light if administered simultaneously with a 10-minute illumination, had no immediate effect if given 2 hours after illumination, but was again rapidly inhibitory at 4 and 6 hours. The effects of actinomycin and far red light were more than additive.     

Andrenocorticotropin and β-lipotropin in the hypothalamus

Adrenocorticotropin and β-lipotropin (β-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-μm sections were immunostained first for either ACTH or β-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and β-LPH demonstrated, immunocytochemically as well as by radioimmunoassay, that the ACTH and β-LPH antisera were directed to the COOH- and NH(2)-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed. Every nerve cell in the arcuate nucleus that contained ACTH also contained β-LPH, but β-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. β-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true. Our observations establish that ACTH and β-LPH are contained in the same nerve cells They stongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell.     

Evolution of alcohol dehydrogenase genes in peonies (Paeonia): phylogenetic relationships of putative nonhybrid species

Alcohol dehydrogenase genes were amplified by PCR, cloned, and sequenced from 11 putative nonhybrid species of the angiosperm genus Paeonia. Sequences of five exons and six intron regions of the Adh gene were used to reconstruct the phylogeny of these species. Two paralogous genes, Adh1A, and Adh2, were found; an additional gene, Adh1B, is also present in section Moutan. Phylogenetic analyses of exon sequences of the Adh genes of Paeonia and a variety of other angiosperms imply that duplication of Adh1 and Adh2 occurred prior to the divergence of Paeonia species and was followed by a duplication resulting in Adh1A and Adh1B. Concerted evolution appears to be absent between these paralogous loci. Phylogenetic analysis of only the Paeonia Adh exon sequences, positioning the root of the tree between the paralogous genes Adh1 and Adh2, suggests that the first evolutionary split within the genus occurred between the shrubby section Moutan and the other two herbaceous sections Oneapia and Paeonia. Restriction of Adh1B genes to section Moutan may have resulted from deletion of Adh1B from the common ancestor of sections Oneapia and Paeonia. A relative-rate test was designed to compare rates of molecular change among lineages based on the divergence of paralogous genes, and the results indicate a slower rate of evolution within the shrubby section Moutan than in section Oneapia. This may be responsible for the relatively long branch length of section Oneapia and the short branch length between section Moutan and the other two sections found on the Adh, ITS (nrDNA), and matK (cpDNA) phylogenies of the genus. Adh1 and Adh2 intron sequences cannot be aligned, and we therefore carried out separate analyses of Adh1A and Adh2 genes using exon and intron sequences together. The Templeton test suggested that there is not significant incongruence among Adh1A, ITS, and matK data sets, but that these three data sets conflict significantly with Adh2 sequence data. A combined analysis of Adh1A, ITS, and matK sequences produced a tree that is better resolved than that of any individual gene, and congruent with morphology and the results of artificial hybridization. It is therefore considered to be the current best estimate of the species phylogeny. Paraphyly of section Paeonia in the Adh2 gene tree may be caused by longer coalescence times and random sorting of ancestral alleles.      

Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.     

Biochemical and serological characteristics of soluble yeast phase antigens of Histoplasma Capsulatum

Soluble antigens of whole yeast-phase cells were extracted with a 0.1 M phosphate buffer containing 0.1 M sodium chloride and 0.02% iodoacetate. After being separated by differential filtration into fractions less than or greater than 50,000 daltons, these antigens were purified by molecular sieve and chromatographic separations on ionic exchange resins. Two high molecular weight fractions obtained from diethylaminoethyl-cellulose (DEAE) at pH 8.0 and 7.0 with tris (hydroxymethyl) aminomethane (Tris) buffer were M antigens; those obtained at pH 4.0 and 4.0 with salt were H antigens. The four fractions had protein to carbohydrate ratios of 7.3, 14.0, 8.4, and 6.5 respectively, and all had essentially the same amino acid composition with no methionine and tyrosine and little histidine, arginine, phenylalanine and lysine. They had high concentrations of glucose, less mannose and traces of galactose. The low molecular weight fractions had the new complex Y antigen, M antigen, and H antigen with protein to carbohydrate ratios of 1.4, 1.4 and 0.3 respectively. The amino acid and sugar composition of Y antigen strongly resembled the composition of the low molecular weight H and M antigens. Unlike the high molecular weight antigens, these low molecular weight antigens had methionine in relatively high concentrations; they had the same sugars as their respective high molecular weight counterparts. The yeast phase antigens differed from their respective mycelial counterparts in the following ways: glucose was the major sugar in the yeast phase with less amounts of mannose and traces of galactose, whereas in the mycelial antigens, mannose was the major sugar, with lesser amounts of galactose, glucose, and hexosamine. The H and M antigens of the yeast phase had high concentrations of glycine and alanine, whereas in the mycelial phase, these antigens had high concentrations of threonine and proline; the H and M antigens of the yeast phase had 5 to 16 times the protein to carbohydrate ratio observed for the same antigens of histoplasmin.