The Microaerophile SPirillum volutans: Cultivation on Complex Liquid and Solid Media

Spirillum volutans grows only under microaerobic conditions in a peptone-succinate-salts broth, but can grow aerobically when the peptone is replaced by vitamin-free acid-hydrolyzed casein broth. The addition of potassium metabisulfite, norepinephrine, catalase or superoxide dismutase (SOD) permitted aerobic growth in peptone-succinate-salts broth. A combination of catalase and SOD had a synergistic effect. S. volutans lacked catalase and had only a low level of peroxidase activity, but did possess SOD activity (12 to 14 U/mg of protein). The organism was found to be extraordinarily sensitive to exogenous hydrogen peroxide. Illumination of peptone-succinate-salts broth generated hydrogen peroxide and rendered the medium inhibitory to growth. A combination of catalase and SOD prevented this inhibition. Growth of S. volutans on solid media, not previously possible, was accomplished by the use of vitamin-free acid-hydrolyzed casein and peptone-succinate-salts agar media; maximum growth responses were dependent on the following combination of factors: addition of bisulfite, catalase, or SOD, protection of the media from illumination, incubation in a highly humid atmosphere, and incubation under atmospheres of 12% oxygen or less. The results indicate that the microaerophilic nature of S. volutans is attributable largely to the high sensitivity of the organism to exogenous hydrogen peroxide and, to a lesser extent, superoxide radicals occurring in the culture medium.     

Two new species of Viola (Violaceae) from the Intermountain West,U.S.A.

Viola lithion andV. franksmithii are rock-dwelling endemics that belong to the North American perennial, caulescent, simple-leaved, blue-, purple-, or white-flowered group of species.Viola lithion, from the White Pine and Pilot mountain ranges of eastern Nevada and adjacent Utah, belongs to sectionChamaemelanium subsectionCanadenses. It most closely resembles the reniform-leaved, Olympic MountainV. flettii, together with which it possibly shares an ancestry with the widespread and allopatricV. canadensis. Viola franksmithii, restricted to the Logan Canyon drainage of the Bear River Range in northern Utah, belongs to sectionRostellatae subsectionRosulantes. It comes closest morphologically toV. howellii of the Coast Range, but likely evolved from the widespreadV. adunca or a common ancestor.     

Dissection of Nodule Development by Supplementation of Rhizobium leguminosarum biovar phaseoli Purine Auxotrophs with 4-Aminoimidazole-5-Carboxamide Riboside

Purine auxotrophs of Rhizobium leguminosarum biovar phaseoli CFN42 elicit uninfected pseudonodules on bean (Phaseolus vulgaris L.). Addition of 4-aminoimidazole-5-carboxamide (AICA) riboside to the root medium during incubation of the plant with these mutants leads to enhanced nodule development, although nitrogenase activity is not detected. Nodules elicited in this manner had infection threads and anatomical features characteristic of normal nodules, such as peripheral vasculature rather than the central vasculature of the pseudonodules that were elicited without AICA riboside supplementation. Although 10⁵ to 10⁶ bacteria could be recovered from these nodules after full development, bacteria were not observed in the interior nodule cells. Instead, large cells with extensive internal membranes were present. Approximately 5% of the normal amount of leghemoglobin and 10% of the normal amount of uricase were detected in these nodules. To promote the development of true nodules rather than pseudonodules, AICA riboside was required no later than the second day through no more than the sixth day following inoculation. After this period, removal of AICA riboside from the root medium did not prevent the formation of true nodules. This observation suggests that there is a critical stage of infection, reached before nodule emergence, at which development becomes committed to forming a true nodule rather than a pseudonodule.     

Survival of toxigenic Pasteurella multocida in aerosols and aqueous liquids.

The survival of toxigenic Pasteurella multocida in air and liquids was studied to identify possible risk factors in the etiology of atrophic rhinitis. In aerosols, at low relative humidity (28%), the viability of toxigenic P. multocida 5 min after aerosolization was at least 22% of its initial value. Viability at low relative humidity declined to 8% after 45 min. Viability at high relative humidity (79%) was 69% after 5 min and declined to 2% after 45 min. Survival of toxigenic P. multocida in liquids depended on storage and constituents in the liquid. Toxigenic P. multocida became nonculturable 1 to 14 days after inoculation in water and artificial seawater, depending on the storage temperature. Toxigenic P. multocida stored at 37 degrees C could be detected for up to 6 days in pig slurry and more than 36 days in Bacto Tryptose broth and nasal lavages. However, in Bacto Tryptose broth and nasal lavages stored at 4 degrees C, P. multocida was detected for up to 14 days whereas at 15 and 37 degrees C it was detected for more than 49 days. These results suggest that aerosols and fomites can play a role in the transmission of atrophic rhinitis.     

Examination of phenylalanine microenvironments in proteins by second-derivative absorption spectroscopy

We have employed near ultraviolet derivative absorption spectroscopy to study the microenvironments of phenylalanine residues in proteins. The use of second-derivative uv spectra in the 250- to 270-nm range effectively suppresses spectral contributions from tryptophan and tyrosine residues. Fitting a polynomial to the numerically calculated second-derivative spectrum allows precise determination of the position of the negative derivative peak near 258 nm. This position is shown to be correlated with the polarity of the microenvironments of phenylalanine residues. This approach allows monitoring of changes in the state of phenylalanine side chains during folding/unfolding of the proteins. In addition, this method permits perturbation of protein samples with ethylene glycol to be used to establish the relative degree of solvent exposure of protein phenylalanine.     

Demonstration of a heterogeneous transcription pattern of thyroglobulin mRNA in human thyroid tissues.

Previous reports on human thyroglobulin (hTg) modifications in thyroid carcinomas prompted us to study hTg mRNA in thyroid adenomas and carcinomas. The quantification of hTg mRNA showed a decrease in its levels of expression in both pathological conditions which differed by a factor of 2 between adenomas and carcinomas. Furthermore, PCR was used to analyse the characteristics of hTg mRNA by amplifying 4 regions of the hTg mRNA. When applied to 2 normal, 17 benign and 13 malignant pathological tissue specimens, PCR showed no modification in the size of Tg mRNA. However, abnormal sized cDNAs appeared in all tissues with no distinction between the pathologies; the Restriction Fragment Length Polymorphism study of these cDNAs suggests the existence of alternate splicing patterns in thyroglobulin mRNAs.     

Feeding rats a diet enriched with saturated fatty acids prevents the inhibitory effects of acute and chronic ethanol exposure on the in vitro uptake of hexoses and lipids

Chow-fed rats were given 15% ethanol in their drinking water for 4 weeks, and then for the next 2 weeks of ethanol exposure they were fed isocaloric semisynthetic diets enriched in either saturated (S) or polyunsaturated (P, linoleic acid) fats. Food intake was lower in ethanol-fed (ETH) than in control (C) rats, but the average body weight gain was similar in ETH and C fed S or P. Intestinal dry weight and the percentage of the intestinal wall comprised of mucosa were more than 2-fold higher in ETH than C fed P, whereas these values were 50% lower in ETH than C fed S. The in vitro jejunal uptake of glucose and galactose was higher in ETH than C fed S, whereas the converse was true when feeding P. These effects were due to differences in the values of the maximal transport rate (Vmax), the Michaelis constant (Km), and the contribution of passive permeation. The relative permeability of the intestine to lipids was unchanged by giving ethanol or by feeding S or P, but the individual rates of uptake of most medium- and long-chain fatty acids and cholesterol were lower in ETH fed P as compared with S. In a second series of studies the acute effect of ethanol exposure was examined: animals were fed S or P for 2 weeks and the intestine was then removed: when 5% ethanol was added directly to the test solutions, there was lower in vitro jejunal and ileal uptake of glucose and higher jejunal uptake of 18:2 when rats were previously fed P, but not in those fed S. In summary; (1) feeding an isocaloric polyunsaturated fatty acid diet has a trophic effect on the intestinal mucosa of animals chronically drinking ethanol; and (2) feeding rats a diet enriched with saturated fatty acids prevents the inhibitory effects of acute and chronic ethanol exposure on the in vitro jejunal uptake of glucose, galactose and lipids observed in animals fed a polyunsaturated diet. Thus, the effect of chronic consumption of ethanol on the active and passive jejunal uptake of nutrients is influenced by the type of lipids in the animal's diet.     

Investigation of the structural requirements for peptide precursor processing in AtT-20 cells using site-directed mutagenesis of proadrenocorticotropin/endorphin.

Mouse pro-ACTH/endorphin (or POMC) contains in its sequence each of the four possible pairs of basic amino acids recognized as potential cleavage sites in the production of bioactive peptides from higher mol wt precursors: KR (lysine-arginine), RR, RK, and KK. To examine the structural requirements for processing and routing in one region of pro-ACTH/endorphin, a reporter mutation was introduced into the mouse pro-ACTH/endorphin cDNA; a methionine residue was mutated to an isoleucine residue to allow biosynthetic double labeling with [3H]Ile and [35S]Met. Analysis of stable cell lines expressing the reporter cDNA indicated that this mutation did not affect processing or secretion. Therefore, additional mutations were introduced on the reporter background to investigate important structural features of the precursor. First, the tripeptide signal for N-linked glycosylation in the N-terminal glycopeptide (Asn65,Ser66,Ser67) was disrupted by the conservative substitution of asparagine65 with a glutamine residue. Secondly, O-glycosylation was prevented by substitution of threonine45 with an alanine residue. Finally, lysine50 was mutated to an arginine residue, transforming the RK doublet preceding the gamma 3MSH sequence into an RR doublet. The results show that the enzymatic machinery of AtT-20 cells fails to cleave efficiently at the Arg-Lys (RK) site even after elimination of any possible structural hindrance by carbohydrate side-chains. Elimination of O-linked oligosaccharides to the N-terminal side of gamma 3MSH did not allow cleavage at the RK site, and elimination of N-linked oligosaccharides did not alter the processing and routing of pro-ACTH/endorphin in AtT-20 cells. However, mutation of the RK sequence to RR allowed extensive cleavage regardless of the occurrence of O- or N-glycosylation.     

The ordered secretion of bioactive peptides: oldest or newest first?

The order of secretion of newly synthesized and older bioactive peptides was investigated using primary rat intermediate pituitary melanotropes, which synthesize, store, and secrete peptides derived from pro-ACTH/endorphin (PAE; also POMC). PAE-derived peptides produced by the cells were biosynthetically labeled by incubating the cells with radioactive amino acids at various times preceding the period during which secretion was examined; secreted and cellular peptides were characterized and quantitated by immunoprecipitation, using affinity-purified antibodies to selected regions of PAE, followed by polyacrylamide gel electrophoretic analysis. Release in the absence of secretagogues (basal or constitutive release) was compared to release in the presence of maximally effective levels of 8-bromo-cAMP and BaCl2 (stimulated or regulated release). Both cell types showed short-lived preferential basal release of newly synthesized and not fully mature peptides (less than 2-3 h old). Conversely, the cells showed preferential stimulated secretion of older peptides. A process of maturation occurred, taking 2-4 h, after which the secretion of newly synthesized and older peptides in response to secretagogues was nearly indistinguishable for the smallest product peptides. The data support a model of gradual processing of peptides from precursors into smaller products and maturation from molecules only available for basal release into peptides available for stimulated secretion as well as for basal release. Basal secretion was found to include mature peptides as well as intermediates and precursor molecules. The data do not support the existence of any preferential regulated secretion of newly synthesized peptides.