Nitrogen fixation and nitrogen metabolism in heliobacteria

Three species of anoxygenic phototrophic heliobacteria, Heliobacterium chlorum, Heliobacterium gestii, and Heliobacillus mobilis, were studied for comparative nitrogen-fixing abilities and regulation of nitrogenase. Significant nitrogenase activity (acetylene reduction) was detected in all species grown photoheterotrophically on N₂, although cells of H. mobilis consistently had higher nitrogenase activity than did cells of either H. chlorum or H. gestii. Nitrogen-fixing cultures of all three species of heliobacteria were subject to switch-off of nitrogenase activity by ammonia; glutamine also served to switch-off nitrogenase activity but only in cells of H. mobilis and H. gestii. Placing photosynthetically grown heliobacterial cultures in darkness also served to switch-off nitrogenase activity. Dark-mediated switch-off was complete in lactate-grown heliobacteria but in pyruvate-grown cells substantial rates of nitrogenase activity continued in darkness. In all heliobacteria examined ammonia was assimilated primarily through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway although significant levels of alanine dehydrogenase were present in extracts of cells of H. gestii, but not in the other species. The results suggest that heliobacteria, like phototrophic purple bacteria, are active N₂-fixing bacteria and that despite their gram-positive phylogenetic roots, heliobacteria retain the capacity to control nitrogenase activity by a switch-off type of mechanism. Because of their ability to fix N₂ both photosynthetically and in darkness, it is possible that heliobacteria are significant contributors of fixed nitrogen in their paddy soil habitat.     

Optimal growth of Streptococcus cremoris HP in milk is related to β- and ϰ-casein degradation

Summary Initiation of growth and the growth rate of Streptococcus cremoris HP in a complete synthetic medium supplemented with an enzymatic digest of casein appeared to be inhibited by (di)hydrogen phosphate. The inhibitory effect of the inorganic phosphate fraction was counteracted by -glycerophosphate.Growth experiments involving different caseins or combinations of caseins as the only source of nitrogen (and essential amino acids) were performed in an adapted medium in which optimal growth was expected to depend only on the type of nitrogen source. Maximal growth occurred on a combination of -casein and a relatively low concentration of . It approximated the growth on milk added to the medium. These results and those showing the capacity of the organism to grow on milk-derived fractions suggest that in milk it is mainly the soluble (-) casein fraction together with the easily accessible hydrophilic part of micellar , which maintain optimal growth.     

Sertoli cells of adult rats “in vitro”

Summary A cell line obtained from isolated seminiferous tubules of adult rat testis has been studied in vitro over a period of 35 days.Light and electron microscopic studies performed from hour 2 to the end of culture have shown the presence of a monomorphic cell population. After 5–6 days of culture the cells formed a monolayer. The cytoplasm of the cells contained numerous lipid bodies and produced numerous projections. The nucleus showed several indentations and one or more nucleoli. From the 9th to the 15th day of culture the cells developed a large amount of endoplasmic reticulum, Golgi apparatus and aggregates of electron dense granules. From the 20th to 40th day the cell cultures progressively degenerated.Immunochemical analysis of the culture medium revealed the presence of estradiol-17, which reached its maximum production rate from the 8th day to the 18th day of culture. Corresponding to cell involution estradiol concentration underwent a rapid decrease.On the basis of morphological and biochemical data the cells could be considered Sertoli cells.This work was supported by Grants n. 74.00155.04 and n. 75.01224.04 from the Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy, and by Istituto di Ricerca F. Angelini, Rome, ItalyPart of this work was presented at the 10th Italian Congress of Electron Microscopy. Ostuni 1–4 October 1975The excellent technical assistance of Miss Laura Vassallo, Daniela Venturini and Mr. Massimo Rosati and Mario Termine is deeply appreciated     

Phosphorylation of pollen proteins in relation to self-incompatibility in rye (Secale cereale L.)

The gametophytic two-locus self-incompatibility (SI) system in rye was investigated in view of a possible involvement of protein phosphorylation and Ca²⁺ as constituents of a signal transduction mechanism. Phosphorylation kinetics in pollen grains was found to be significantly different after in vitro treatment of pollen with either cross or self stigma proteins, with a pronounced phosphorylation activity in self-treated pollen grains. Loss of SI in self-compatible (SC) mutants was associated with a significantly decreased basic phosphorylation activity in untreated pollen grains as compared to SI genotypes. Separation of phosphorylated pollen proteins by SDS-PAGE reveals four major proteins in the MW range of 43–82 kDa which were differently phosphorylated in SI vs SC genotypes as well as in cross vs self-treated pollen grains. Application of different protein kinase inhibitors and the Ca²⁺ antagonists verapamil and La³⁺ to isolated stigmas resulted in an inhibition of the SI response in in vitro self-pollination. The role of protein kinases and Ca²⁺ as constituents of a putative SI-specific signal transduction mechanism is discussed.     

Conformation of the lecithin molecule with attached water molecules

CNDO/2 studies on the conformation of the chain of lecithin indicated a strong preference for a gauche-gauche arrangement about the phosphodiester group. Folding the chain about and 4 was energetically very favorable. Hydration of the same segment revealed three levels of water-binding energies. The ion-dipole interactions of water and the choline moiety were energetically non-substantial. In contrast, binding of water to the unesterified phosphate oxygens produced the highest enthalpies. Attachment of water to the esterified phosphate oxygens or the ester oxygens of the chain resulted in intermediate binding strengths. By investigating complete incorporation of nine water molecules into a chosen lipid structure, a plausible lecithin-water geometry was deduced for a liquid crystalline system.     

Penicillinase activity in three strains ofClostridium butyricum

Three strains ofClostridium butyricum exhibited elevated minimal inhibitory concentrations (MICs) to penicillin (64–1,024 g/ml), ampicillin (32–256 g/ml), carbenicillin (128–1,024 g/ml), and oxacillin (32–64 g/ml). Cephalosporin/cephamycin agents were more active than penicillin drugs. All isolates were found to possess a -lactamase. The -lactamases were primarily cell associated during the logarithmic phase of growth. Stationary-phase cells released most of the enzyme into the culture medium. Cephalothin supplementation of broth cultures with concentrations equivalent to one-eighth of the MIC significantly increased the quantity of -lactamase synthesized. The -lactamases produced by these three isolates exhibited greatest activity with penicillin followed by ampicillin>cephaloridine>carbenicillin and oxacillin. No enzymatic activity was observed using cephalothin, cephalexin, cefazolin, cefoxitin, or cephamandole substrates. The -lactamases were inhibited by clavulanic acid and para-chloromurcuribenzoate and not inhibited by cloxacillin. Each enzyme exhibited an isoelectric point of 4.2.     

The chromosomal localization of human β-galactosidase revisited: a locus for β-galactosidase on human chromosome 3 and for its protective protein on human chromosome 22

Summary A series of man-Chinese hamster and man-mouse somatic cell hybrids was investigated to study the localization of the genes coding for the human lysosomal enzyme -galactosidase (EC 3.2.1.23) and for its protective protein. Using a monoclonal antibody, raised against human placental -galactosidase, it was observed that the structural locus for the -galactosidase polypeptide is located on chromosome 3. The nature of the involvement of chromosome 22 in the expression of human -galactosidase was elucidated by metabolic labelling of the hybrids with radioactive amino acids, immunoprecipitation with monoclonal and polyclonal antibodies against -galactosidase, followed by analysis via gel electrophoresis and fluorography.The data show that the presence of chromosome 22 coincides with the presence of a 32 kd protein. This polypeptide, the protective protein was previously shown to be intimately associated with human -galactosidase. In addition, the protective protein was found to be essential for the in vivo stability of -galactosidase by aggregating -galactosidase monomers into high molecular weight multimes. Both chromosome 3 and 22 are therefore necessary to obtain normal levels og -galactosidase activity in human cells.     

Polygonal inequalities as a key to neuronic equations

Neuronic or decision equations, first proposed as a mathematical model of neural activity, have shown, after their exact, compact solution was found, typical behaviours that make them natural tools for General Systems studies. It is shown here that their mathematical investigation is remarkably furthered by generalizing the triangular inequality to polygonal ones. These permit the immediate computation of the tensorial expansion of linearly separable boolean functions, and exhibit clearly the connection between their continuous and discontinuous aspects.     

Alkyl-phenyl cleavage of the lignin model compounds guaiacylglycol and glycerol-β-guaiacyl ether by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized guaiacylglycol--guaiacyl ether (I) in high nitrogen, shaking and stationary cultures. 2-(o-Methoxyphenoxy) ethanol (X), 2-(o-methoxyphenoxy) acetic acid (IX) and methoxy-phydroquinone (MHQ) were identified as products of the metabolism of (I). P. chrysosporium also metabolized guaiacylglycerol--guaiacyl ether (IV) in high nitrogen stationary cultures. 2-(o-Methoxyphenoxy)-1,3 propanediol (XII) and 3-hydroxy, 2-(o-methoxy-phenyxy) propionic acid (XIV) were identified as products of the metabolism of (IV). Finally, P. chrysosporium metabolized -deoxyguaiacylglycol--guaiacyl ether (VI) and -deoxyguaiacylglycerol--guaiacyl ether (VII) in limiting nitrogen cultures. 2-(o-Methoxyphenoxy) ethanol (X) and 2-(o-methoxyphenoxy)-1,3 propanediol (XII) were identified as products of the metabolism of VI and VII respectively indicating hydroxylation of those substrates with subsequent alkyl-phenyl bond cleavage. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography - MHQ methoxyhydroquinone     

Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L.

In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5-configurated. The progesterone 5-reductase and the 3-hydroxysteroid-5-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5-reductase was purified 769-fold.Abbreviations DW dry weight - FW fresh weight - PVP polyvinylpyrrolidone     

Stable sulfur isotopic biogeochemistry of the Hubbard Brook Experimental Forest, New Hampshire

In natural ecosystems, differences often exist in the relative abundanceof stable S isotopes (°³⁴S) that can provide clues as tothe source, nature, and cycling of S. Values of °³⁴S inprecipitation, throughfall, soils, soil solution, and stream waters weremeasured at the Hubbard Brook Experimental Forest (HBEF), New Hampshire.Values of °³⁴S in precipitation and throughfall weresimilar to each other but differed seasonally. Precipitation°³⁴S values were higher in the dormant season[°³⁴S = 5.9±0.6 (17)][Mean + SE(N)]than in the growing season [°³⁴S = 5.0±0.6(40)] but throughfall growing-season values were higher[°³⁴S = 5.6±0.6(68)] than for the dormantseason [°³⁴S = 4.9±0.7 (9)]. Different treespecies did not affect throughfall °³⁴S values. In soilsolution, °³⁴S values were higher in the growing season(°³⁴S = 8.9±2.8; 8.8±1.7;and 4.0±0.6 for Oa, Bh, and Bs horizons, respectively) thanin the dormant season (°³⁴S = 5.6±1.5;3.7±2.4; and 3.4±1.2 for Oa, Bh, and Bshorizons, respectively). These seasonal differences in°³⁴S were probably caused by biological isotopicfractionation. The °³⁴S values in streams were generally2 lower and more variable than those in precipitation andthroughfall, suggesting fractionation and/or different isotopic sources inthe soil.     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

DNA variations in regenerated plants of pea (Pisum sativum L.)

Summary The aim of this study was to determine whether DNA variations could be detected in regenerated pea plants. Two different genotypes were analyzed by cytogenetic and molecular techniques: the Dolce Provenza cultivar and the 5075 experimental line. Dolce Provenza regenerated plants showed a reduction in DNA content, particularly at the level of unique sequences and ribosomal genes. Moreover, regeneration was associated with an increase in DNA methylation of both internal and external cytosines of the CCG sequence. On the other hand, the DNA content of the 5075 line remained stable after regeneration. DNA reduction was found only in 5075 plants regenerated from callus cultures maintained for long incubation periods (about a year). The DNA variations observed are discussed both in relation to the genotype source and the role of tissue-culture stress.     

Involvement of Erks activation in cadmium‐induced AP‐1 transactivation in vitro and in vivo

Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category 1 carcinogen. Cadmium-induced upregulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP1 activity by cadmium appears to involve activation of Erks, since the induction of AP1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP1 in AP1luciferase report transgenic mice. Considering the role of AP1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmiuminduced carcinogenesis.     

A neural net capable of competitive and cooperative computation

An algorithm of learning in multilayer threshold nets without feedbacks is proposed. The net is. built of threshold elements with binary inputs. During a learning process each input vector x is accompanied by a teacher's decision ({1,...,M}). The pairs (x[n], [n]) appear in successive steps independently according to some unknown stationary distribution p(x,). The problem of learning of a threshold net has been decomposed to a series of problems of learning of the threshold elements. The proposed learning algorithm of the threshold elements has a perceptron-like form. It was proven that a decision rule of the threshold net stabilizes after a finite number of steps. For definite classes {p(x, )} _* ^K of distributions p(x,), an optimal decision rule stabilizes after a finite number of steps. These classes {p(x, )} _* ^K also contain distributions describing learning processes with perturbations.     

Interpretation of metameric architecture in dominant shrubs of the Chilean matorral

Summary The seasonal progression of phenophases in 21 shrub species of the Chilean matorral was analyzed. Five modules or basic units that are responsible for the aboveground architecture of the plants were characterized. These modules appear to be organized in seven different spatial arrangements. In drought-deciduous species a module type with an absolute short shoot with limited apical growth, leafy or spiny, predominated. In evergreen species long shoot and temporal leafy short shoot module types were more frequent. The spatial arrangement of morphologically different modules and the temporal sequence of their formation allow a dynamic interpretation of the modular architecture of the plants.     

Respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus tepidarius (type strain)

Thiobacillus tepidarius (type strain) was grown in microaerophilic conditions, on tetrathionate, thiosulfate or crystalline S^o. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation of the different cultures were measured respirometrically, using exponentially growing cells, with an oxygen electrode. Cells growing on the three different sulfur compounds retain thiosulfate-, tetrathionate, and S^o-oxidizing activities (SOA), but lack respiratory sulfite-oxidizing activity. The SOA for all the cultures was almost totally inhibited by 50 M myxothiazol, an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Tetrathionate- and thiosulfate-oxidizing activities were moderately and weakly inhibited by 50 M totally inhibited (>95%) all respiratory activities. This study suggests that electrons released by S^o oxidation enter the respiratory chain in the quinone-cytochrome b region.Abbreviation SOA sulfur-oxidizing activity     

The effects of collembola grazing on microbial activity in decomposing leaf litter

Summary Ground leaf litter was inoculated with the fungus Coriolus versicolor and incubated in respirometers for 6 days (fresh cultures) or 33 days (senescent cultures) before different number of Folsomia candida were added. Grazing by 5 animals stimulated O₂ consumption in both series of cultures but 10, 15 or 20 animals inhibited microbial respiration. The stimulatory effect was less marked in the senescent cultures. Bacterial and fungal standing crops increased in fresh cultures during the course of the experiment but grazing by collembola increased bacterial and reduced fungal standing, crops in proportion to the grazing intensity. Microbial standing crops were not determined for senescent cultures. Microarthropod feeding activities can therefore exert a strong differential effect on fungal and bacterial populations which has not been previously recognised.     

Genetic heterogeneity of propionic acidemia: Analysis of 15 Japanese patients

Summary Propionic acidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl CoA carboxylase (PCC) activity. We have analyzed the molecular heterogeneity of Japanese propionic acidemia patients using anti-human PCC antiserum and cDNA clones coding for the two protein subunits ( and ) of the enzyme. The steady state levels of both and subunits of PCC from 15 Japanese patients were determined by Western blot. Three patients had neither nor subunits, and the amounts of both and subunits were low in 3 other patients. According to our previous data, we classified these 6 patients as having subunit deficiency. In the remaining 8 patients, subunits were normal, but the subunits were aberrant. Two patients had low levels of normal-sized subunits and 6 had subunits smaller than normal in size and greatly reduced in quantity. These 8 patients were assigned to the subunit deficiency category. One patient had apparently normal and subunits. We could not determine this patient's primary defect. These data reveal the genetic heterogeneity of molecular defects causing propionic acidemia in the Japanese. Southern blot analysis did not reveal any gross alteration in gene structure when DNA was digested withHindIII,EcoRI andTaqI. However, DNA from 3 -subunit-deficient patients, when digested withMspI and probed with PCC cDNA, revealed a unique 2.7-kb band not observed in blots of DNA from any other patient or 15 normal controls. We conclude that this alteredMspI restriction map is the result of a mutation in the subunit gene of these patients.     

Capnocytophaga: New genus of Gram-negative gliding bacteria. II. Morphology and ultrastructure

Gram-negative, anaerobic gliding bacteria were isolated from normal supragingival plaque and from periodontal lesions. Isolates could be divided into two size classes: small 2.4–4.2 m×0.38–0.5 m and large 4.8–5.8 m×0.42–0.6 m cells. The outer membrane was either loose-fitting and wavy, or taut, and of variable thickness. An electron-dense fuzz was discernible on several of the isolates. The periplasmic region was of variable electron-density. The genus Capnocytophaga has been proposed for these organisms based on morphological and cultural characteristics.