Microbial lipids and stable foam formation in the activated sludge process.

The presence of fats and oils in sewage has been related to the formation of stable foams in activated sludge treatment systems. Foam forming microbes can utilise and, in some cases, store lipid substrates. Since surface lipids would confer the hydrophobicity necessary for flotation on the sludge biomass, the extractable lipids in foaming and non-foaming biomass samples were examined. Both pure mono-cultures and sludge samples were used. The results showed that, whilst there were some differences in the lipid profiles of the mono-cultures, the different sludge types did not show any significant pattern or variation which could be used as a lipid-based explanation for foam formation.

Low-energy collision-induced dissociation of protonated polyamino alcohol derivatives of peptides and N-terminal blocked peptides.

This investigation reports the low-energy collision-induced dissociation of the protonated molecules of polyamino alcohols, formed by chemical reduction of synthetic peptides and N-terminal blocked peptides, in order to evaluate its potential for peptide sequence determination. The --CH2--NH-- cleavage with charge retention on the N-terminus was prominent, and the entire sequence of ions produced in this manner was observed. Some of the sequence ions arising from --CH2--NH-- cleavage accompanied by migration of two hydrogens, and --NH--C alpha H-- cleavage with charge retention on the C-terminus, also occurred prominently. In addition, a great number of internal fragment ions were produced in relatively high abundance; these provided supplementary sequence information and were, in some cases, critical in determining the sequence. The usefulness of this method was exemplified by its application to the sequence determination of bacterial lipopeptides.

Steroid transhydrogenase activity of 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina.

3-Ketosteroid-delta 1-dehydrogenase from Nocardia corallina catalyzes transhydrogenation of 3-keto-4-ene-steroid to 3-keto-1,4-diene-steroid e.g., progesterone to 1,4-androstadiene-3,17-dione. The reaction proceeded linearly at first and then soon slowed down owing to equilibration. The turnover number of this reaction was of the same magnitude as that of the dehydrogenation of 3-keto-4-ene-steroid. The pH optimum was 8.4, which is lower than that of the dehydrogenase reaction. The enzyme has a wide specificity for hydrogen acceptor steroids. The Km' and Kmax' values for these steroids and the values of the corresponding 3-keto-4-ene-steroids were compared. Kinetic studies of the steroid transhydrogenase reaction demonstrated a typical ping-pong mechanism. The enzyme oxidized 1,2-tritiated progesterone and transferred the tritium atoms to the reaction product, 4-androstene-3,17-dione, and water. Transhydrogenation in D2O resulted in the incorporation of a deuterium atom into the C2-position of 4-androstene-3,17-dione. The results indicate that the enzyme catalyzes C1, C2-trans axial abstraction of hydrogen atoms from progesterone, transfer of the 1 alpha-hydrogen to the C1-position of 1,4-androstadiene-3, 17-dione and release of the 2 beta-hydrogen to water. Reaction schemes based on the experimental results are proposed. The enzyme also catalyzes the reduction of 3-keto-1,4-diene-steroids with reduced benzyl viologen.

Parallel antitumor, granuloma-forming and tumor-necrosis-factor-priming activities of mycoloyl glycolipids from Nocardia rubra that differ in carbohydrate moiety: structure-activity relationships.

Multiple intravenous injections (30 micrograms, ten times) in ICR mice of trehalose dimycolate and glucose monomycolate from Nocardia rubra, containing C36-48 mycolic acids, showed a prominent antitumor effect on a subcutaneously implanted sarcoma-180, an allogeneic sarcoma of mice with a significant granuloma formation in lungs, spleen and liver. On the other hand, mycoloyl glycolipids other than glucose monomycolate and trehalose dimycolate, such as mannose or fructose mycolate, showed no significant activity for tumor regression or granuloma formation in mice. Trehalose dimycolate and glucose monomycolate from N. rubra, and glucose monomycolate with C56-60 mycolic acids from Rhodococcus terrae also showed a distinctive priming activity for tumor necrosis factor (TNF), when lipopolysaccharide from Escherichia coli was administered as an eliciting agent. The TNF activity in the sera of mice was abrogated almost completely by anti-(murine TNF alpha) antibody with protein-A-agarose. Again in contrast, mannose and fructose mycolate from N. rubra and glucose monomycolate with C30-34 mycolic acids from Rhodococcus equi did not show such activities in mice. Meth-A, a syngeneic fibrosarcoma of BALB/c mice, was less sensitive to administration of glycolipids than sarcoma-180. These results indicated that the existence of a glucose or trehalose molecule was necessary for the expression of immunomodifying activities among various mycoloyl glycolipids differing in carbohydrate structure. However, since the administration of lipopolysaccharide was essentially required as an eliciting agent for the induction of TNF, while no eliciting agent was required for the antitumor activities, TNF does not seem to contribute directly to the antitumor activities of mycoloyl glycolipids in our systems. There was, however, a parallel structure-activity relationship among granuloma-forming, antitumor and TNF-priming activities, indicating that the structures of both the carbohydrate moiety and the mycoloyl residues influenced an initial step, such as macrophage activation, commonly and profoundly.

Mycolyl glycolipids stimulate macrophages to release a chemotactic factor.

We have previously reported that mycolyl glycolipids from Nocardia rubra such as glucose or trehalose mycolates induced granuloma formation in mice. The structure of the carbohydrate moiety of the mycolyl glycolipids influenced the granuloma forming activity profoundly. Here, we have examined the macrophage-chemotactic activity in the culture supernatants stimulated with various glycolipids differing in carbohydrate moiety (trehalose 6,6'-dimycolate, or TDM; glucose monomycolate, or GM; mannose monomycolate, or MM; and fructose monomycolate, or FM). A distinctive chemotactic activity was detected with TDM or GM, but, little or none with MM or FM.

New diterpenoid antibiotics, spirocardins A and B.

New antibiotics spirocardins A and B were isolated from the culture broth of an actinomycete isolated from a soil sample collected near Lake Hibara, Fukushima Prefecture, Japan. The producing strain was classified as Nocardia sp. SANK 64282. The antibiotics were isolated from the culture filtrate by solvent extraction and purified further by silica gel and preparative reverse phase column chromatography. They were primarily active against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus and limited species of Gram-negative bacteria such as Bacteroides fragilis and Klebsiella pneumoniae. They were also moderately active against several species of Mycoplasma. The molecular formulae of spirocardins A and B were C20H30O6 and C20H32O6, respectively. From their physico-chemical characteristics they were revealed to be diterpenoid antibiotics with closely related structures and the former was easily converted to the latter by the reduction with NaBH4.

A possible mechanism of fever induced by Nocardia rubra cell wall skeleton (N-CWS) in experimental animals.

The characteristics of fever elicited by the cell wall skeleton of Nocardia rubra (N-CWS) and by lipopolysaccharide (LPS) were compared in rabbits, and the possible involvement of the antigenicity of N-CWS was investigated in guinea pigs. In rabbits, fever of more than 0.5 degree C developed after an intravenous (i.v.) injection of 10 micrograms/kg or more of N-CWS, and was monophasic with 30-100 micrograms/kg but biphasic with the highest dose of 300 micrograms/kg. LPS elicited fever with similar characteristics at doses of 0.01-0.1 microgram/kg. With both compounds, the fever was inhibited by indomethacin. Tolerance to N-CWS and LPS appeared after dosing with 30 or 0.1 micrograms/kg, respectively for 10 d. In guinea pigs sensitized with N-CWS, challenge with 1 or 10 micrograms/kg of N-CWS 10 d later, which did not induce fever in the nonsensitized animals, caused fever of more than 0.5 degree C, and delayed-type hypersensitivity (DTH) appeared. N-CWS also elicited fever in nonsensitized guinea pigs bearing N-CWS-sensitized lymphocytes or anti-N-CWS antibody; the fever was higher in the guinea pigs sensitized with the lymphocytes than in those with the anti-N-CWS antibody. In brief, single injections of N-CWS and of LPS elicited fever with similar characteristics, although the potency of N-CWS was weaker. With N-CWS, the fever is proposed to be triggered by the antigenicity of the compound itself, because doses as low as 1 or 10 micrograms/kg elicited fever along with immunological response in N-CWS-sensitized animals, but not in nonsensitized ones.

Possible involvement of a tumor necrosis factor (TNF)-like mediator as an endogenous pyrogen in fever induction by Nocardia rubra cell wall skeleton (N-CWS).

Tumor necrosis factor (TNF), a cytokine produced in macrophages, also acts as an endogenous pyrogen (EP). To investigate whether TNF has a role in the fever induced by Nocardia rubra cell wall skeleton (N-CWS), the relationship between fever and TNF production was studied in guinea pigs. N-CWS injected i.v. to guinea pigs caused biphasic fever and had L-929 cell-killing activity which resembled that of TNF in the sera 30 min before the first phase of fever appeared. In vitro, L-929 cell-killing activity was demonstrated in the culture supernatant of guinea pig peritoneal macrophages pretreated with N-CWS, and the activity increased dependently on N-CWS concentration or culture duration. When the supernatant of the macrophages was fractionated by gel filtration and each fraction was assayed for fever-inducing and L-929 cell-killing activities, the fraction with the cell-killing activity also induced fever with characteristics similar to that by i.v. injection of N-CWS in guinea pigs. These results suggest that TNF acts as an EP on the fever induced by N-CWS in guinea pigs.

Occurrence of trehalose mycolate as the major lipidic component carrying toxic property in clinical nocardia isolates

Cepas Rp-Sn e RP-533 isoladas de dois casos clínicos do Hospital das Clínicas de Ribeiräo Preto e identificadas como nocárdias, produzem micolato de trealose com poder rotatório específico de +38.6- e + 38,0-, respectivamente. Essa fraçäo glicolipidídica possue atividade toxigênica enquanto a de lipídeos neutros mais ácidos graxos livres e a de fosfolipídeos säo desprovidas dessa propriedade

Application of chromatographic methods to the analysis of macrophage factors induced by Nocardia opaca cell walls.

Cell walls from Nocardia opaca induce the production of mitogenic factors by mouse peritoneal macrophages in vitro. These factors stimulate thymocytes from C3H/HeJ mice. Supernatants of peritoneal cell culture exhibiting this activity were fractionated by chromatographic procedures such as gel filtration and metal chelate affinity chromatography and the biological activities assayed. These fractionation studies indicate that several biologically active products occur in the supernatant. Four factors monokines (M) with different apparent molecular masses M1 (100,000), M2 (50,000), M3 (16,000) and M4 (7000) were obtained, one of which (M3) was identical to interleukin 1 (IL1). Several of the biochemical parameters of one of these factors, M2, were analyzed. It was found that this monokine had many properties in common with IL1: stimulation of proliferation of thymocytes from C3H/HeJ mice, similar amino acid composition and mobility during isoelectric focusing.

Effect of B-factor and its analogues on rifamycin biosynthesis in Nocardia sp.

B-Factor, 3'-(1-butylphosphoryl)adenosine, which was isolated from yeast extract, is an inducer of rifamycin production in a rifamycin non-producing Nocardia mutant. Feeding of B-factor to the mutant culture demonstrated that the induction process was triggered during early stationary phase. Rifamycin production in the mutant was also induced by an exogenous supply of 3-amino-5-hydroxybenzoic acid, an intermediate of the antibiotic pathway, suggesting that a step upstream from the intermediate is regulated by B-factor. B-Factor analogues, i.e., alkylesters of 3'-AMP with alkyl side chains of C(2) approximately C(12) and n-butyl esters of 3'-GMP and 2'-AMP all showed the B-factor activity. Among these n-octyl ester of 3'-AMP showed the lowest effective concentration of approximately 3 x 10(-10) M. An intrinsic substance of the Nocardia sp. with potent B-factor activity and a UV absorption maximum at 260 nm was isolated from the cells of the parental strain.

Menaquinone composition in the classification and identification of aerobic actinomycetes.

Menaquinones were the only isoprenoid quinones found in 36 strains representing different species of the genera Nocardia, Mycobacterium, Rhodococcus, Amycolatopsis, Saccharothrix, Streptomyces, Nocardiopsis and Actinomadura. Dihydrogenated menaquinones with nine isoprene units [MK-9(H2)] were the main components isolated from Mycobacterium. Dihydrogenated and tetrahydrogenated menaquinones with eight isoprene units were the predominant compounds identified in typical Rhodococcus and Nocardia strains, respectively. "Nocardia phenotolerans" differed from all of the other Nocardia species included in the study, in that it contained the MK-9(H2) [MK-8(H2)] menaquinone system. Nocardioform bacteria lacking mycolic acids contained tetrahydrogenated menaquinones with nine isoprene units as the main component. The Streptomyces strains studied exhibited complex mixtures of partially saturated menaquinones with nine isoprene units with the hexa- and/or octahydrogenated components predominating. Actinomadurae contained major amounts of hexahydrogenated menaquinones with nine isoprene units. In contrast, the single Nocardiopsis strain examined possessed complex mixtures of menaquinones with ten isoprene units, the dihydrogenated components being main constituents.

A case of lung infection caused by an unusual strain of Nocardia farcinica.

A case of lung infection caused by an unusual strain of Nocardia farcinica is reported. This is the third case of the N. farcinica infection in this country. The strain failed to utilize rhamnose as sole carbon source, but could be identified by a numerical identification method. The mycolic acids contained 1-3 double bonds and the numbers of the carbon atoms of the mycolic acids were 50 to 60, average 56.

High-performance liquid chromatography analysis of mycolic acids as an aid in laboratory identification of Rhodococcus and Nocardia species.

High-performance liquid chromatography analysis of the p-bromophenacyl esters of mycolic acids from whole organisms gave chromatographic patterns that were useful in differentiation of Rhodococcus and Nocardia species. Rhodococcus equi, R. erythropolis, and R. rhodochrous contained more-polar mycolic acids and were easily separated from the less-polar mycolic acid-containing species of R. sputi, R. bronchialis, R. corallinus, R. rubropertinctus, and R. terrae. The less-polar mycolic acid-containing Rhodococcus species showed chromatographic patterns that partially overlapped (in elution times) the patterns of Nocardia asteroides, N. otitidiscaviarum, and N. brasiliensis, but the larger number of peaks in the last species made separation between the genera possible. Distinct chromatographic patterns were found for most species, except for R. equi strains that showed two different patterns. Strains of R. rubropertinctus and R. terrae appeared identical. N. asteroides and N. otitidiscaviarum showed similar mycolic acid patterns.

Isolation of mycolic acid-containing glycolipids in Nocardia rubra and their granuloma forming activity in mice.

Three classes of glycolipids (TMM (trehalose monomycolate), TDM (trehalose dimycolate) and GM (glucose mycolate] containing mycolic acids as hydrophobic components were isolated from a strain of Nocardia rubra (Rhodococcus rubrum) and their structures have been partially characterized using infrared spectrometry, gas-liquid chromatography and gas chromatography-mass spectrometry. Acid or alkaline hydrolysis of isolated glycolipids revealed that trehalose was the sole water soluble component in TMM and TDM, while glucose was the hydrophilic component in GM. On the other hand, saturated, monoenoic and dienoic mycolic acids with carbon atoms ranging from C36 to C50 contained constituents of fatty acid moiety at C44. From the analytical results, TMM, TDM and GM were tentatively identified as trehalose monomycolate, trehalose dimycolate and glucose monomycolate, respectively. The mycolic acid composition differed significantly by the glycolipid classes: the highest amount of saturated mycolic acids were detected in TMM and GM, while a significant amount of dienoic mycolic acids have been found in TDM and the cell wall bound lipid fraction (BL). All these three classes of glycolipids containing mycolic acids showed strong granuloma forming activity in lungs and spleen of ICR mice 1 week after intravenous injection of 100 to 500 micrograms glycolipid in W/O/W micelles containing Freund's incomplete adjuvant. These results indicated that glycolipids containing shorter carbon chain mycolic acids ranging C40-50, corresponding to less acyl numbers or monosaccharides such as glucose, can also produce foreign body-type granuloma in mice without protein antigens.