U-21,963, a New Antibiotic: II. Isolation and Characterization

The isolation and characterization of antibiotic U-21,963 are discussed. This compound is a highly unsaturated monobasic acid with the molecular formula C(9)H(7)NO(3). The molecular weight is 177. It is dextrorotatory, [alpha](D) = +138 degrees , and has a pK(a) of 5.1. The ultraviolet absorption spectrum, which showed a maximum at 223 mmu (epsilon = 15,115), indicates unsaturation alpha-beta to the carboxyl group, and the infrared spectrum suggests the presence of an acetylenic group. Explosive decomposition of U-21,963 at 97 C conforms with the latter. U-21,963 is relatively insoluble in water, but readily soluble in ethyl alcohol, acetone, and halogenated hydrocarbons.     

Pulmonary gas exchange in panting dogs

Pulmonary gas exchange during panting was studied in seven conscious dogs (32 kg mean body wt) provided with a chronic tracheostomy and an exteriorized carotid artery loop. The animals were acutely exposed to moderately elevated ambient temperature (27.5 degrees C, 65% relative humidity) for 2 h. O2 and CO2 in the tracheostomy tube were continuously monitored by mass spectrometry using a special sample-hold phase-locked sampling technique. PO2 and PCO2 were determined in blood samples obtained from the carotid artery. During the exposure to heat, central body temperature remained unchanged (38.6 +/- 0.6 degrees C) while all animals rapidly switched to steady shallow panting at frequencies close to the resonant frequency of the respiratory system. During panting, the following values were measured (means +/- SD): breathing frequency, 313 +/- 19 breaths/min; tidal volume, 167 +/- 21 ml; total ventilation, 52 +/- 9 l/min; effective alveolar ventilation, 5.5 +/- 1.3 l/min; PaO2, 106.2 +/- 5.9 Torr; PaCO2, 27.2 +/- 3.9 Torr; end-tidal-arterial PO2 difference [(PE' - Pa)O2], 26.0 +/- 5.3 Torr; and arterial-end-tidal PCO2 difference, [(Pa - PE')CO2], 14.9 +/- 2.5 Torr. On the basis of the classical ideal alveolar air approach, parallel dead-space ventilation accounted for 54% of alveolar ventilation and 66% of the (PE' - Pa)O2 difference. But the steepness of the CO2 and O2 expirogram plotted against expired volume suggested a contribution of series in homogeneity due to incomplete gas mixing.     

Extraction of melodies in behavioural sequences

A method is proposed that extracts a set of phrases, or “melodies”, from a behavioural sequence, using a technique for extracting and compressing chains based on Information Theory. These melodies are validated by reference to a statistical criterion. An application of this method to the analysis of the behavioural sequences of two groups of mice, the first observed during the day, the second during the night, is described. The advantages and the limitations of the method are discussed.     

Histochemical study on the eccrine glands in the foot pad of the cat

Enzyme and carbohydrate histochemical methods were used to study the secretory activities and secretion properties of the eccrine tubular glands in the foot pad of the cat. The activity spectra of the different oxidative and hydrolytic enzymes investigated indicate high metabolic rates within the secretory epithelium. Additionally the enzyme reactions emphasize a double innervation of the glands by cholinergic and adrenergic nerve fibres. The carbohydrate histochemical differentiation reveals mostly neutral and very few acidic glycoproteins in the secretory cells and the secretion, respectively. Clear (basal) cells contain distinct amounts of glycogen, and dark (superficial) cells show neutral glycoproteins, which reveal after PO-lectin-DAB procedures the following saccharide residues: alpha-D-mannose, alpha-D-galactose, N-acetyl-alpha-D-glucosamine, alpha-L-fucose, beta-D-galactose-D-N-galactosamine, beta-D-galactose, and sialic acid. The results obtained confirm the view that the normal biological functions of the eccrine glands of the feline foot pad are to improve the frictional capacities of the paw and to leave typical scent marks.     

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H(2)-Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H(2)-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 +/- 0.19 mol%), a phospholipid pattern of type II, MK-9(H(4)) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t(d) = 8 h), H(2) plus CO(2) (t(d) = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H(2), nickel was not necessary. The optimum growth temperature was 65 degrees C; no growth was observed below 40 degrees C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70 degrees C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K(m) was 88 mul of CO liter and V(max) was 20.2 mul of CO h mg of protein. The threshold value of S. thermoautotrophicus of 0.2 mul of CO liter was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 mumol min mg of protein. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.     

Mineralocorticoid-induced increase in beta-adrenergic receptors of cultured rat arterial smooth muscle cells

We have investigated the effect of mineralocorticoids on beta-adrenergic receptors in cultured arterial smooth muscle cells. Mineralocorticoid (aldosterone) treatment resulted in a significant increase in beta-adrenergic receptors measured by [3H]dihydroalprenolol (DHA) binding. This effect required at least 20 hours of incubation with aldosterone and was completely blocked by cycloheximide (10 micrograms/ml), indicating protein synthesis was required for this response. Aldosterone at the concentration range of 10(-8)-10(-6) M increased [3H]DHA binding, but was ineffective at 10(-9) M. Scatchard analysis of [3H]DHA binding revealed that the observed significant increase in binding was due to an increased number of binding sites (P less than 0.05), and that the affinity was unchanged. The aldosterone (1 x 10(-8) M) effect was completely blocked by the combination of RU 38486 (10(-6) M) and spironolactone (10(-7) M), but not by the glucocorticoid antagonist RU 38486 alone. While basal c-AMP levels were not changed by aldosterone (10(-6) M) treatment, the isoproterenol (10(-6) M) stimulated level of c-AMP was significantly higher in cells treated with aldosterone (P less than 0.05). We conclude that aldosterone, acting through the mineralocorticoid receptor, has a direct effect on arterial smooth muscle cells mediated through modulation of beta-adrenergic receptors of these cells.     

Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.     

Alveolar slope and dead space of He and SF6 in dogs: comparison of airway and venous loading

Series (Fowler) dead space (VD) and slope of the alveolar plateau of two inert gases (He and SF6) with similar blood-gas partition coefficients (approximately 0.01) but different diffusivities were analyzed in 10 anesthetized paralyzed mechanically ventilated dogs (mean body wt 20 kg). Single-breath constant-flow expirograms were simultaneously recorded in two conditions: 1) after equilibration of lung gas with the inert gases at tracer concentrations [airway loading (AL)] and 2) during steady-state elimination of the inert gases continuously introduced into venous blood by a membrane oxygenator and partial arteriovenous bypass [venous loading (VL)]. VD was consistently larger for SF6 than for He, but there was no difference between AL and VL. The relative alveolar slope, defined as increment of partial pressure per increment of expired volume and normalized to mixed expired-inspired partial pressure difference, was larger by a factor of two in VL than in AL for both He and SF6. The He-to-SF6 ratio of relative alveolar slope was generally smaller than unity in both VL and AL. Whereas unequal ventilation-volume distribution combined with sequential emptying of parallel lung regions appears to be responsible for the sloping alveolar plateau during AL, the steeper slope during VL is attributed to the combined effects of continuing gas exchange and ventilation-perfusion inequality coupled with sequential emptying. The differences between He and SF6 point at the contributing role of diffusion-dependent mechanisms in intrapulmonary gas mixing.     

Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element.

We have cloned three copies of a repeated DNA segment from Borrelia burgdorferi sensu stricto strain B31, present on both circular and linear plasmids of this and other B. burgdorferi sensu lato strains. The DNA sequences are characterized by a highly homologous segment containing two open reading frames (ORFs), ORF-A and ORF-B. Five additional ORFs can be found on the slightly less homologous flanking sequences: ORF-G on the opposite strand upstream of ORF-A, and ORF-C, ORF-D, ORF-E, and ORF-F downstream of ORF-B. The 4.6-kb-long element containing ORF-A through ORF-E is flanked by approximately 180-bp-long imperfect inverted repeats (IRs). The putative gene product of ORF-C displays homology to proteins involved in plasmid maintenance in a number of gram-positive and gram-negative bacteria. ORF-E features several short, highly homologous direct repeats. ORF-A, ORF-B, and ORF-D are homologous to three ORFs on a recently described 8.3-kb circular plasmid of Borrelia afzelii Ip21 that are flanked by similar IRs (J. J. Dunn, S. R. Buchstein, L.-L. Butler, S. Fisenne, D. S. Polin, B. N. Lade, and B. J. Luft, J. Bacteriol. 176:2706-2717,1994). ORF-C and ORF-E, however, are missing from this region on the Ip21 plasmid. Furthermore, the repeated DNA element as defined by the IRs is present in opposite orientations relative to the flanking sequences on the B31 and Ip21 plasmids.