Purification and characterization of an extracellular β-glucosidase from the anaerobic fungus Piromyces sp. strain E2

An extracellular -glucosidase (EC 3.2.2.21) from the anaerobic fungus Piromyces sp. strain E2 was purified. The enzyme is a monomer with a molecular mass of 45 kDa and a pI of 4.15. The enzyme readily hydrolyzes p-nitrophenyl--d-glycoside, p-nitrophenyl--d-fucoside, cellobiose, cellotriose, cellotetraose and cellopentaose but is not active towards Avicel, carboxymethylcellulose, xylan, p-nitrophenyl--d-galactoside and p-nitrophenyl--d-xyloside. To cleave p-nitrophenyl--d-glucoside the maximum activity is reached at pH 6.0 and 55°C, and the enzyme is stable up to 72 h at 40°C. Activity is inhibited by d-glucurono--lactone, cellobiose, sodium dodecyl sulfate, Hg²⁺ and Cu²⁺ cations. With p-nitrophenyl--d-glycoside, p-nitrophenyl--d-fucoside, and. cellobiose as enzyme substrates, the K _m and V _max balues are 1.5 mM and 25.5 IU·mg^-1, 1.1. mM and 133 IU·mg^-1, and 0.05 mM and 55.6 IU·mg^-1, respectively.     

Flight of the honey bee

Summary Using manometric and gas analytical methods oxygen consumption , carbon dioxide production , respiratory quotientRQ, (Fig. 1A-C) and thorax surface temperature difference T _ts (Fig. 3) were determined in single bees. The animals were either sitting in respiratory chambers or were suspended by the scutum, in which case they were resting, walking (turning a small polystyrene ball) or flying in a closed miniature wind tunnel.During resting (sitting in Warburg vessels) at an ambient temperatureT _a=10°C,RQ was 1.01±0.2 (n=905) with variations due to method (Fig. 1D, E).RQ values during walking were determined in single cases. In no case were they significantly different from 1.00. After the first 10 min of flight meanRQ was 1.00±0.04. It was significantly smaller than 1.00 (RQ=0.97) only during the last 5% of long-time flights (mean flight duration 58.8±28.8 min). With the exception of near-exhaustion conditions no signs of fuels other than carbohydrates were found.Metabolic rateP _m was 19.71±21.38 mW g^–1 during resting at 20°CT _a30°C indicating that many resting bees actively thermoregulate at higherT _a. After excluding bees which were actively thermoregulating, by an approximationP _m was 5.65±2.44 mW g^–1 at 20°CT _a30°C. True resting metabolic rate for sitting bees atT _a=10°C was 1.31±0.53 mW g^–1 (Fig. 2A, B).A significant negative correlation was found between relative (specific) oxygen consumption rel and body massM _b at 85 mgM _b150 mg.At 0°CT _ts16.5°C a significant (-0.01) positive correlation was found between and T _ts in single resting bees: T _Ts+0.099, or betweenP _m and T _ts:P _m=1.343 T _ts+0.581 (Fig. 3D) in ml h^–1,P _m in mW,T in °C).During walking (duration 13.15±5.71 min,n=13) at 12.5°CT _a21°C a stable T _ts of 11.41±3.37°C, corresponding to 167 mW g^–1, was reached for 80 to 90% of the walking time (Fig. 4B).During wind tunnel flights of tethered animals the minimal metabolic power measured in exhaustion experiments was 240 mW g^–1. Calculation of factors of increase inP _m is of limited value in poikilotherms, in which true resting conditions are not exactly defined.     

On the signal transducing mechanisms involved in the synergistic interaction between interleukin-1 and bradykinin on prostaglandin biosynthesis in human gingival fibroblasts

Recombinant human interleukin-1 (IL-1) and bradykinin (BK) synergistically stimulate prostaglandin E₂ (PGE₂) formation in human gingival fibroblasts cultured for 24 h. Neither BK or IL-1 per se, nor their combinations, caused any acute stimulation of cellular cyclic AMP accumulation. BK, but not IL-1, caused a rapid, transient rise of intracellular Ca²⁺ concentration ([Ca²⁺]_i), as assessed by recordings of fura-2 fluorescence in monolayers of prelabelled gingival fibroblasts. IL-1 did not change the effect of BK on [Ca²⁺]_i. Ionomycin and A 23187, two calcium ionophores, synergistically potentiated the stimulatory effect of IL-1 on PGE₂ formation. Three different phorbol esters known to activate protein kinase C also synergistically potentiated the action of IL-1 on PGE₂ formation. Exogenously added arachidonic acid significantly enhanced the basal formation of PGE₂. In IL-1 treated cells, the enhancement of PGE₂ formation seen after addition of arachidonic acid, was synergistically upregulated by IL-1. These data show that i) the synergistic interaction between IL-1 and BK on PGE₂ formation is not due to an effect linked to an upregulation of cyclic AMP or [Ca²⁺]_i; ii) the signal transducing mechanism by which BK interacts with IL-1, however, may be linked to a BK induced stimulation of [Ca²⁺]_i and/or protein kinase C; iii) the mechanism involved in the action of IL-1 may, at least partly, be due to enhancement of the biosynthesis of prostanoids mediated by an upregulation of cyclooxygenase activity.     

Über den Histochemischen Nachweis der β-Glucuronidase, α-Mannosidase und α-Galactosidase mit 1-Naphthylglykosiden

Zusammenfassung Mit simultanen Azokupplungsverfahren und 1-Naphthylglykosiden als Substraten werden Verteilung und Aktivität von -Glucuronidase, -Mannosidase und -Galactosidase bei Ratte, Maus und Meerschweinchen untersucht.Für die -Glucuronidase besteht das Inkubationsmedium aus 5–10 mg 1-Naphthyl-glucuronid (gelöst in 0.4 ml NN-Dimethylformamid) und 0.6 ml 2% Hexazonium-p-rosanilin in 9 ml 0.2 M Acetat-Puffer, pH 5; für die -Mannosidase und -Galactosidase aus der gleichen Menge 1-Naphthyl--mannosid bzw. --galactosid und p-Rosanilin in 9 ml 0.1 M CitratCitronensäure-Phosphatoder Acetat-Puffer, pH 5 bzw. 5.2. Die Spezifität der Nachweisreaktionen sichern qualitative und quantitative Hemmversuche mit verschiedenen 1–4-Lactonen und Galactose ab.Die -Glucuronidase kann bei der Ratte vor allem intralysosomal nachgewiesen werden, z.B. in Niere, Nebenhoden, Uterus, Samenblase, Darm und Respirationstrakt; Mäuseund Meerschweinchengewebe setzen 1-Naphthyl--glucuronid langsamer um. Für die -Mannosidase läßt sich in zahlreichen Organen auch histochemisch die lysosomale Lokalisation des Enzyms beweisen, wobei die Aktivität in Urogenitalsystem, Darm und Speicheldrüsen besonders hoch ist, und in der Mäuseniere geschlechtsspezifische Unterschiede vorkommen. Erstmalig wird die intralysosomale Lokalisation der -Galactosidase gezeigt, die ubiquitär in teilweise hoher Aktivität anzutreffen ist. 6-Br-2-Naphthyl--galactosid eignet sich in Verbindung mit Fast Blue B nicht zur intralysosomalen Lokalisation der -Galaotosidase.Fluorometrische Messungen aller 3 Glykosidasen mit dem entsprechenden 1-Naphthylglykosid ergeben nach Fixation in Formol oder Glutaraldehyd Hemmraten zwischen 90 und 98 %; anschließendes Waschen in Zuckerlösung verdoppelt oder verdreifacht die Restaktivität.

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On the histochemical demonstration of -glucuronidase, -mannosidase and -galactosidase using 1-naphthyl glycosides

Summary By means of simultaneous azo coupling using 1-naphthyl glycosides as substrates the distribution and activity of -glucuronidase, -mannosidase and -galactosidase have been investigated in rats, mice and guinea-pigs.For -glucuronidase the incubation medium consists of 5–10 mg 1-naphthyl--glucuronide (dissolved in 0.4 ml NN-dimethyl formamide) and 0.6 ml 2% hexazonium-p-rosaniline in 9 ml 0.2 M acetate buffer, pH 5; for -mannosidase and -galactosidase of the same quantities of 1-naphthyl--mannoside and -galactoside respectively and p-rosaniline in 9 ml 0.1 M citrate, citric acid-phosphate or acetate buffer, pH 5. Qualitative and quantitative inhibition tests using various 1–4 lactones and galactose prove the reaction specifity of the methods presented here. -Glucuronidase can be detected especially in lysosomes of rat organs, e.g. kidney, epididymis, uterus, vesicular gland, intestine and respiratory tract; tissues from mice and guineapigs exhibit a slower splitting rate for 1-naphthyl glucuronide. As to -mannosidase its lysosomal localization becomes apparent in many organs also by means of histochemistry. The urogenital system, intestine and the salivary glands belong to the structures with the highest amount of -mannosidase, and in the mouse kidney sex differences occur. For the first time -galactosidase can be demonstrated unequivocally in the lysosomes of rat, mouse and guineapig tissues in which this enzyme displays a high overall activity. 6-Br-2-Naphthyl--galactoside and Fast Blue B for postcoupling are not able to detect the lysosomal localization of -galactosidase.Fluorometric measurements of these 3 glycosidases by means of the corresponding 1-naphthyl glycoside reveal inhibition rates between 90 and 98% following fixation in formol or glutaraldehyde. Washing in sugar solution raises enzyme activity two or three times.

     

The effects of aflatoxin B1 on immature germinating maize (Zea mays) embryos

Immature maize (Zea mays L.) embryos were treated with aflatoxin B₁ concentrations, ranging from 0.1 g ml^–1 to 25 g ml^–1. Below 5 g ml^–1 aflatoxin B₁, root and shoot elongation was not significantly inhibited. Ultrastructurally, root tip cells showed little deterioration, except a possible diffused clearing in mitochondria and plastids. As the toxin concentration was increased above 5 gml^–1, shoot, and particularly root elongation, was progressively inhibited. Associated with this, there was an apparent decrease in the ribosome population. Furthermore, membranes, particularly the vacuolar membrane, became abnormal and vacuolar distension occurred. At 20 and 25 g ml^–1, these effects were exacerbated, and mitochondria and plastid structure was disrupted. At these concentrations, there was evidence of a disruption in lipid metabolism. The results are discussed in the context of known aflatoxin effects on cellular control mechanisms and ultrastructure in animal systems.     

The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L.

The metabolism and growth-promoting activity of gibberellin A₂₀ (GA₂₀) were compared in the internode-length genotypes of pea, na le and na Le. Gibberellin A₂₉ and GA₂₉-catabolite were the major metabolites of GA₂₀ in the genotype na le. However, low levels of GA₁, GA₈ and GA₈-catabolite were also identified as metabolites in this genotype, confirming that the le allele is a leaky mutation. Gibberellin A₂₀ was approximately 20 to 30 times as active in promoting internode growth of genotype na Le as of genotype na le. However, the levels of the 3-hydroxylated metabolite of GA₂₀, GA₈ (2-hydroxy GA₁), were similar for a given growth response in both genotypes. In each case a close linear relationship was observed between internode growth and the logarithm of GA₈ levels. A similar relationship was found on comparing GA₂₀ metabolism in the three genotypes le ^d, le and Le. The former mutation results in a more severe dwarf phenotype than the le allele (which has previously been shown to reduce the 3-hydroxylation of GA₂₀ to GA₁). These results indicate that GA₂₀ has negligible intrinsic activity and support the contention that GA₁ is the only GA active per se in promoting stem growth in pea.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Control of yeast fed-batch process through regulation of extracellular ethanol concentration

At high growth rates, the biomass yield of bakers yeast (Saccharomyces cerevisiae) decreases due to the production of ethanol. For this reason, it is standard industrial practice to use a fed-batch process whereby the specific growth rate, , is fixed at a level below the point of ethanol production, i.e., _crit. Optimally, growth should be maintained at _crit, but in practice, this is difficult because _crit is dependent upon strain and culture conditions. In this work, growth was maintained at a point just above _crit by regulating ethanol concentration in the bioreactor. The models used for control design are shown, as are the experimental results obtained when this strategy was implemented. This technique should be applicable to all microorganisms that exhibit an overflow type metabolism.     

Normal meiosis in two 47,XYY men

Summary Details of testicular histology and meiosis are given for two 47,XYY men, one an oligospermic childless individual, the other a fertile man with near-normal spermatogenic activity in his testes. Examination of the chromosomes at meiosis, with Q and C staining, gave no evidence for the occurrence of the second Y chromosome in the germ line of either individual.     

Consequences of harvesting for genetic diversity in American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.): a simulation study

American ginseng, Panax quinquefolius L., is one of the most heavily traded medicinal plants in North America. The effect of harvest on genetic diversity in ginseng was measured with a single generation culling simulation program. Culling scenarios included random harvest at varying levels, legal limit random harvest and legal limit mature plant harvest. The legal limit was determined by the proportion of legally harvestable plants per population (% mature plants per population). Random harvest at varying levels resulted in significant loss of genetic diversity, especially allelic richness. Relative to initial levels, average within-population genetic diversity (H _e) was significantly lower when plants were culled randomly at the legal limit (Mann–Whitney U=430, p<0.001) or when only mature plants were culled (Mann–Whitney U=394, p<0.01). Within-population genetic diversity was significantly higher with legal limit mature plant harvest (H _e=0.068) than when plants were culled randomly at the legal limit (H _e=0.064; U=202, p<0.01). Based on these simulations of harvest over one generation, we recommend that harvesting fewer than the proportion of mature plants could reduce the negative genetic effects of harvest on ginseng populations.     

Influence of Ca2+ channel modulators on [3H]purine release from rat cultured glial cells

[³H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca²⁺-free medium +0.5 mM ethylene glycol-bis(-aminoethylether)N,N,N,N-tetracetic acid (EGTA) reduced the electrically evoked [³H]purine release. Nimodipine only at the concentration of 10 M modified [³H]purine outflow whereas 0.1 M -conotoxin and 0.03–0.1 M nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 M -conotoxin +0.1 M nitrendipine antagonized the evoked [³H]purine release similarly to each drug given alone. Neither nitrendipine nor -conotoxin influenced the uptake of⁴⁵Ca²⁺ by the cultures. The treatment of cells with the Ca²⁺ agonist Bay K 8644 did not affect [³H]purine release or the⁴⁵Ca²⁺ uptake. The drug did not either alter [Ca²⁺]_i, evaluated by loading the cells with 3 M Fura-2/AM. 10–30 M 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca²⁺ discharge, significantly reduced the evoked [³H]purine release. On the other hand, 2 M thapsigargin, an inhibitor of the ion store Ca²⁺ ATPase, was able to increase either the culture [³H]purine release or the [Ca²⁺]_i. Together, the findings indicate that voltage-sensitive calcium channels (VSCC_s) of the neuronal N and L-types are not involved in the modulation of [³H]purine release from rat cultured astrocytes whereas Ca²⁺ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCC_s seem to be able to affect [³H]purine outflow with mechanisms other than VSCC gating.     

Toxicity of Mercaptoethanol to Mutant Strains of the Yeast Pichia methanolica Growing on Different Carbon Sources

Addition of -mercaptoethanol at a concentration of 2–3 mM to media containing methanol, glucose, or yeast extract caused a 50% inhibition of the growth of wild-type yeastPichia methanolica; mercaptoethanol at a concentration of 0.7 to 25 mM inhibited the growth of the mutant strain ecr1. The mutation mth1 of P. methanolica repressed its ability to consume methanol and was accompanied by the loss of alcohol oxidase (EC 1.1.3.13) activity. -Mercaptoethanol restored the ability of mth1 mutant cells to grow on methanol and stimulated their growth under derepression conditions. The growth effect of -mercaptoethanol during derepression was accompanied by partial restoration of alcohol oxidase activity.     

A manganese requirement for patulin biosynthesis by cultures of Penicillium urticae

Antibiotic production by submerged cultures of Penicillium urticae required manganese supplementation of the media. Thus, manganese supplementation (152 M) allowed accumulation of patulin to high concentrations (2 mol/mL), whereas manganese deficiency (1.53 M) resulted in the accumulation to similar levels of the first committed pathway intermediate, methyl-salicylic acid, without significant patulin accumulation. Preliminary studies suggest that a similar manganese effect may occur in other fungal species.     

Ultrastructure of a cave-wall cyanophyte-Gloeocapsa NS4

Gloeocapsa strain NS4, a cyanophyte (cyanobacterium) which grows in low light levels inside cave entrances, was studied in the electron microscope by thin sectioning and freeze-etching. The cells are surrounded by a microfibrillar sheath divided by dense lamellae, which are probably an acidic mucopolysaccharide. Inside this is a typical Gramnegative cell wall. Double-replica freeze-fracture showed that the outer envelope of the wall fractures to give two faces each consisting of densely-packed particles; the particles of the outer leaflet seem to consist of subunits arranged in a hollow cylinder. A structural model of the outer envelope is proposed. The plasma membrane fractures to give a PF face with 3000 9 nm particles m^-1 and an EF face with 150–700 11–12 nm particles m^-1. The thylakoids are arranged in a pattern not previously found in a unicellular cyanophyte, parallel arrays which intersect, and may fuse with, the plasma membrane. The thylakoid membranes have 2,850 particles m^-1, mean size 10.9 nm, on the PF face and 560 particles m^-1, mean size 12.3 nm, on the EF face. Phycobilisomes are difficult to see, but may be unusually large. These ultrastructural features may be adaptations to a very low light habitat.     

Genetic transformation of the pathogenic fungus Wangiella dermatitidis

Genetic transformation of Wangiella dermatitidis was studied using three plasmid vectors (pAN7-1, pWU44, and pKK5) and both electroporation and polyethyleneglycol-mediated methods. pAN7-1 contains the E. coli hygromycin B (HmB) phosphotransferase (hph) gene. Expression of the hph gene confers resistance to antibiotic HmB. Selection for resistance, indicative of transformation, resulted in 10–203 HmB-resistant colonies/g pAN7-1 on medium containing 100 g HmB/ml. Strains of W. dermatitidis used in this study have innate sensitivity to HmB at a critical inhibitory concentration of 20–40 g/ml. Vectors pWU44 and pKK5 contain a URA5 gene from Podospora anserina. A ura5 auxotroph of W. dermatitidis was transformed to prototrophy with pWU44 or pKK5 by complementation. Transformation frequencies for these two plasmids were between 17–50 transformants/g vector DNA. Southern blotting analysis and polymerase chain reaction detection of DNA from putative transformants confirmed transformation.     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

Der Bau des Wurzelscheitels vonCasuarina suberosa Otto etDietr

Zusammenfassung Der Wurzelvegetationspunkt vonCasuarina ist nach dem Dikotylenschema (offener Typus) gestaltet. Die Dermatogenaufspaltungen, die zur Ausbildung der seitlichen Haubenteile führen, können einwandfrei nachgewiesen werden.     

Correlation of biochemical blocks and genetic lesions in leucine auxotrophic strains of the imperfect yeast Candida maltosa

Summary The four enzymatic steps in the conversion of -ketoisovaleriate to leucine were examined in the wild type and in 13 leucine auxotrophic strains of Candida maltosa. The genetic lesions in the auxotrophs, involve at least five different loci and are correlated with three enzymatic steps. This was confirmed by gene cloning, protoplast fusion, and enzyme assays. The pathway for leucine biosynthesis in C. maltosa shows general similarity to that of other lower eukaryotes but there are individual differences in the numbers of genes responsible for single enzymatic steps. A disomic state of the chromosomes carrying genes coding for -isopropylmalate synthase and -isopropyl-malate dehydrogenase was elucidated.     

Estimating the time scale of chemical exchange of proteins from measurements of transverse relaxation rates in solution

Chemical (conformational) exchange on the ms-s time scale is reliably identified by the observation of transverse relaxation rates, R_ex, that depend upon the strength of the effective field (_1eff=B_1eff) used in spin lock or CPMG experiments. In order to determine if the exchange correlation time, _ex, is the fast or slow limit, measurements of (i) signal line shape and (ii) temperature dependence of R_ex have been commonly used in studies of stable, small molecules. However, these approaches are often not applicable to proteins, because sample stability and solubility, respectively, limit the temperature range and signal sensitivity of experiments. Herein we use a complex, but general, two-site exchange equation to show when the simple fast exchange equations for R_ex are good approximations, in the case of proteins. We then present a simple empirical equation that approximately predicts R_ex in all exchange regimes, and explains these results in a clear, straightforward manner. Finally we show how one can reliably determine whether _ex is in the fast or slow exchange limit.     

C4 polymorphism in the dog: Molecular heterogeneity of the C4α and C4γ subunit chains

Using an immunoblotting technique and goat antihuman C4, we observed five distinct electrophoretic variants of C4 in a panel of 60 random dogs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated C4 showed that dog C4 is composed of three polypeptide subunit chains (, , and ) and that structural variability occurs within the - and -chain regions. Two distinct molecular weight forms of both the C4- ( _A and _B) and C4-( _A and _B) chain were detected. The variant forms of C4 and C4 were found in association with particular C4 allotypes.