Assimilation of ammonia and growth of biotin deficientAspergillus nidulans

Summary Biotin deficiency inAspergillus nidulans has been found to increase the uptake of ammonium ions, associated with a marked increase in the activity of NADP-linked glutamate dehydrogenase, which is found to be the major route of ammonia assimilation in this culture. The results obtained are discussed with respect to the growth ofAspergillus nidulans during biotin deficiency. J. D. Desai gratefully acknowledges the receipt of a research fellowship from the Government of India, Department of Atomic Energy, Bombay.     

Kinetik der hepatischen Farbstoffaufnahme von Indocyaningrün. Einfluss von Bilirubin und Natriumglykocholat

Summary Kinetics of hepatic uptake of indocyanine green, a dye which is used for evaluation of liver function, were studied in the rat. The results indicate that the relationship between ICG-dose and initial hepatic dye uptake obeys Michaelis-Menten kinetics suggesting an interaction of the dye with a carrier or fixed site in the liver cell. Thus it was possible to calculate maximum ICG-uptake (v _max ) and the Michaelis constant (K _m ) of this transport system from several submaximal values.v _max was 7.65 (6-06-9.65)²² mg per 100 g liver/min and K _m 0.56 (0.31–0.81)²². Under the influence of substances which inhibit the elimination of dyes by the liver the parametersv _max and K _m showed changes which allowed characterization of the type of inhibition. While sodium glycocholate had no influence on maximum hepatic ICG-uptake and the Michaelis constant bilirubin caused a significant increase of K _m to 1.29 (0.68–1.90)²² without significantly changingv _max . These data suggest that bilirubin interferes with hepatic uptake of indocyanine green by competitive inhibition and that uptake of bile acids is dependent on a different mechanism.     

Resistance to ethidium bromide inAspergillus nidulans

Summary A mutant ofAspergillus nidulans resistant to ethidium bromide was isolated and the semi-dominant gene responsible for this resistance was allocated on linkage group II at 17.42±3.05 units of recombination from thewA₃ gene. The gene also confers cross-resistance to acriflavin, malachite green and crystal violet. It was also shown that riboflavin is antagonistic to the toxic effect of ethidium bromide, at certain concentrations. The mechanisms which could be responsible for the toxic effect of this drug are discussed and compared with those of acriflavin. The use of theEtb ₁ gene in genetical analysis through the parasexual cycle is suggested.Acknowledgements. The authors are thankful to the National Council for the Development of Science and Technology (CNPq) for financial support through PIG-SIP 04/053 FAPESP.     

Phosphate transport inClaviceps sp. strain SD-58

Summary Phosphate uptake inClaviceps sp. strain SD-58 was found to be linear for 20 min, proportional to cell density in mg/ml, energy dependent, and taking place against a concentration gradient with a K_m value of 45.45×10^–5 M. Osmotic shock treatment to the cell caused a reduction in phosphate uptake associated with the release of binding protein. Partial restoration of uptake was observed on incubation of osmotically shocked cells with shock fluid. The results are discussed with reference to the effect of phosphate on alkaloid synthesis inClaviceps sp. strain SD-58.     

Bicarbonate and chloride transport across rat ileal basolateral membrane

The mechanisms of HCO ₃ ^– and Cl^– transport across basolateral membranes from rat ileum were investigated in isolated vesicles by means of uptake experiments. Neither Cl^–/HCO ₃ ^– exchanger nor Na⁺–(HCO ₃ ^– )_n cotransport seem to be present in ileal basolateral membranes. Moreover Cl^– uptake is unaffected bycis Na⁺ and/or K⁺ gradients, indicating the absence of Na⁺–Cl^–, K⁺–Cl^– and Na⁺–K⁺–2Cl^– symport activity. An electrically conductive pathway seems to be responsible for both HCO ₃ ^– and Cl^– fluxes. Evidence is also given for the presence of a Na⁺/H⁺ exchanger at the basolateral pole of ileal enterocytes.     

Transfer of isolated nuclei into protoplasts ofAspergillus nidulans

Nuclei were isolated from protoplasts of a haploid auxotrophicAspergillus nidulans strain. Transformation of protoplasts prepared from a complementary haploid auxotrophic strain with these purified nuclei resulted in both heterokaryotic and diploid colonies. The nutritionally-complementing colonies appeared at a frequency of 5×10^–7 to 10^–8.     

Adenosine triphosphatase ofAspergillus nidulans: Stimulation by aminoacids

Summary Ca²⁺-dependent ATPase ofAspergillus nidulans was found to be stimulated by aminoacids in vitro. Both histidine and arginine stimulated the enzyme more effectively than the aromatic aminoacids. Supplementation of the growth medium with basic or aromatic aminoacids increased the enzyme activity in vivo 2–6-fold. The enhanced activity observed in these cultures in vivo was not mediated through the synthesis of new isoenzyme, as observed in proteinenriched cultures, but appeared to be through the activation of enzyme activity.     

Purification and properties of glutamine synthetase I fromRhizobium sp. UMKL 20

Summary Glutamine synthetase I was purified fromRhizobium sp. UMKL 20 following polyethylene glycol precipitation. The enzyme had a subunit molecular weight of 58 kd. Apparent K_m values for ammonia and glutamate were 5.6 and 15.2 mM, respectively. Glutamine synthetase I activity was inhibited by several end products of glutamine metabolism. The purified enzyme was highly adenylylated (E _n ^– =8.5).Acknowledgment. I would like to thank Mr J. C. Lai for technical assistance. This work was carried out with the support of Vote F 153/79 from the University of Malaya.     

Interspecific protoplast fusion and complementation inaspergilli

Summary Protoplast fusion and nutritional complementation between auxotrophic mutants ofAspergillus nidulans andAspergillus fumigatus has been achieved. It is concluded that the nutritional complementation may be due to interspecific aneuploidy.The authors thank Mr.L. Manczinger and Mr.Gy. Oravecz for producing and characterizing the required mutants; Dr.D. Goldstein for providing the strainA. nidulans R. 21; Dr.D. Kerridge for the wild-type strainA. fumigatus 5085; and Mr.L. Nagy, MissÉva Sziráki and MissMária Pölös for skilful technical assistance.     

Bactericidal activity againstPseudomonas aeruginosa is acquired by cultured human monocyte-derived macrophages after uptake of myeloperoxidase

Myeloperoxidase (MPO) is an enzyme located within polymorphonuclear neutrophils capable of producting cytotoxic oxidant species that are particularly active against bacteria with polysaccharide capsules.Pseudomonas aeruginosa (10⁶ bacteria per 1 ml) are killed within 1 h in vitro by a MPO/H₂O₂/Cl^– system (48 mU=132 ng of MPO). The question arose as to whether human macrophages would acquire cytotoxic activity when loaded with this enzyme. Monocytes were therefore isolated from human blood and cultured for up to ten days to induce maturation to macrophages. These cells lost endogenous MPO within five days while H₂O₂ production in response to stimulation by phorbol myristate acetate (10^–6M) decreased to 23% within ten days. On the other hand, their capacity to take up exogenous MPO increased fourfold from day three to day ten. Human macrophages cultured from eight days (when both H₂O₂ production and MPO uptake were sufficient) were therefore used to study the effects of MPO uptake on cytocidal activity againstPseudomonas aeruginosa. After a 1 h MPO loading period, macrophages (5×10⁵ cells per ml) were incubated in the presence of bacteria (0.5 to 2×10⁶ bacteria per ml) for 2 h at 37°C. At a bacteria/macrophage ratio of 1, only 34.8±7.0% of bacteria survived (compared to killing by non-loaded macrophages), while 74.4±9.3% survived at a ratio of 4. From these results, we conclude that loading macrophages with exogenous MPO could enhance their microbicidal activity, suggesting a potentially useful therapeutic application.     

Does ammonium uptake influence xylem sap composition inPhaseolus vulgaris L. andGlycine max (L.) Merill?

The question of whether ammonium uptake influences the occurrence of ureides in legumes has been addressed in this study by investigating threeP. vulgaris genotypes as well as one cultivar ofGlycine max. All plants were raised in sand culture during the dry season in northern Thailand and irrigated daily with nitrogen-free nutrient solution, or the same solution containing 12 mol m^–3 nitrogen in the form of (NH₄)₂SO₄ or KNO₃, each treatment consisting of different proportions of either compound. Regression analyses of xylem sap composition relative to ammonium vs. nitrate supply of plants harvested at V4, R1 and R6 indicated close positive correlations of xylem amino nitrogen content and negative correlations with xylem nitrate content and ammonium supply. Statistically significant correlations between relative xylem ureide content and ammonium availability could be established for theP. vulgaris cultivar Brilliant up to stage R1, but not for the other plants investigated. It was concluded that at least for some genotypes of common bean a relationship exists between ureide production and ammonium uptake by the root system. Since the extent to which ureide production is stimulated remains quite small, its relevance to the xylem solute technique for measurement of N₂ fixation may be limited. Nevertheless, due to the possibility of large genotypic differences in the impact of ammonium on ureide production, this factor must be considered in calculations if N₂ fixation is to be determined in soils containing significant amounts of ammonium, e.g. in paddy fields.     

Differential sensitivity of the two phases of ear artery contraction to intimal and adventitial norepinephrine

Summary The biphasic contraction of the rabbit ear artery to norepinephrine (NE) was investigated in the normal (adventitial stimulation) and the everted (intimal stimulation) segment of ear artery. The 2nd phase response showed an intimal ED₅₀ of 8.2×10^–8 M which was significantly (p<0.05) lower than the adventitial ED₅₀ of 42.6×10^–8 M. This difference was abolished by inhibition of neuronal and extraneuronal uptake for NE. The 1st phase response also showed an ED₅₀ for the intimal stimulation (6.9×10^–8 M) which was significantly (p<0.05) lower than adventitial (65.5×10^–8 M). This difference was reduced but not abolished by NE uptake inhibition. This suggsets that some feature of the adrenergic neuroeffector apparatus is asymmetrically arranged to favor fast responses to blood borne NE.Supported by American Heart Association-Greater Los Angeles Affiliate Grant No. 602. We wish to thank Dr John Bevan and Dr Alasdair MacLean for helpful advice.     

Genetic and allied effects of certain esters of inorganic acids inAspergillus nidulans

Resumen Fueron investigados los efectos mutagénicos de cinco esteres de acidos inorgánicos sobre el mutante bi^–1; meth^–1 delAspergillus nidulans. El método empleado fué el teste de retrocruzamiento con algunas variaciones. El dimetil sulfato se ha mostrado como el mas poderoso mutagénico, seguido por el di-etil sulfato. El mecanismo de induccion mutacional fue discutido y se estableció una hipótesis para explicarla.     

Influence de la vagotomie chez la tancheTinea tinca au cours d'une surcharge pluricationique (eau de mer diluée). Variation des cations Na+, K+, Ca++ et de l'intensité de la respiration tissulaire

Summary Respiratory ionic content (Na⁺, K⁺, Ca⁺⁺) of hepatic tissue and ionic excretion measurements were performed in the TenchTinea tinea kept in diluted sea water. After bilateral section of parasympathetic system, the changes of Na/K value are less than in fresh water. The survival and hepatic O₂ uptake were the same. It is suggested than after both vagi section sea water Ca⁺⁺ allowed a best osmoregulation in this species.

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Ce travail a été réalisé avec la collaboration technique de A.Rup.     

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (ΔF508) cystic fibrosis transmembrane conductance regulator

The Ca²⁺ ionophore ionomycin induced cytosolic [Ca²⁺]_i elevation as well as strong activation of Cl⁻ efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl⁻ efflux was abolished by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A₂ had no effect, indicating the involvement of Ca²⁺-dependent Cl^- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A₂. These results indicate that phospholipase A₂ activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999     

Selective antiproliferative effects of thymidine

A substance with antiproliferative bioactivity in an aqueous extract ofCordyline terminalis was purified and identified by mass spectrometry to be the natural nucleoside, thymidine. 10^–5M Thymidine inhibited EL4 cell replication and decreased cell viability after 12–24 h. The effect was highly specific for this nucleoside. Treated cell cultures showed a significant increase in S phase cells and a corresponding decrease in G₁ phase cells. Nitrobenzylthioinosine (which prevented facilitated entry of thymidine) protected cells from the antiproliferative action of thymidine. A human breast cancer cell line (MCF7) was also growth-inhibited by 10^–5M thymidine but a murine lymphoma cell line (K36) was not. Thus, submillimolar thymidine has effects on cell proliferation which are selective both with respect to specificity for the compound and for different tumour cell lines.     

Die α-Glycerophosphat-Oxydation des Heuschreckenbrustmuskels (Locusta migratoria)

Summary The conditions were studied for the glycerophosphate oxidation by homogenate from locust flight muscle, and the O₂-consumption was measured. Maximal oxidation rates were found with 0.087m glycerophosphate, 8 × 10^–6 m cytochromec, 7 × 10^–6 m DPN and pH 7.5. The production of dihydroxyacetone phosphate is followed by further oxidation steps, as could be shown by estimation of the different fractions of acid-soluble phosphate. Comparative studies were made on different insects and vertebrates. The rate of succinate oxidation by insect muscle was found to be ten times higher than that of vertebrate muscle. The relation of glycerophosphate oxidation to succinate oxidation is quite different in insects and vertebrates, but no difference could be detected between the two types of muscle metabolism of insects: the carbohydrate and the fatutilizing type.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

In vitro dopaminergic neuroprotective and in vivo antiparkinsonian-like effects of Δ3,2-hydroxybakuchiol isolated from Psoralea corylifolia (L.)

Cocktail recipes containing Psoralea corylifolia seeds (PCS) are used to empirically treat Parkinson disease. A PCS isolate Δ³,2-hydroxybakuchiol (BU) can inhibit dopamine uptake in dopamine transporter (DAT) transfected Chinese hamster ovary (CHO) cells, and dopamine reuptake blockade may provide an alternative approach for ameliorating parkinsonism. Here, we assessed the potential dopaminergic neuroprotective, and antiparkinsonian-like activity of BU. BU sample size was increased by using a scale-up extraction paradigm. Pharmacologically, BU significantly protected SK-N-SH cells from 1-methyl-4-phenylpyridinium (MPP⁺) insult, produced striking inhibitory actions on dopamine/norepinephrine uptake and WIN35,428 binding in synaptosomes on in vivo administration, and significantly preventing poor performance on rotarod and dopaminergic loss in substantia nigra in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice. BU acts by protecting dopaminergic neurons from MPP⁺ injury and preventing against MPTP-induced behavioral and histological lesions in the Parkinson’s disease (PD) model, possibly by inhibiting monoamine transporters. These findings suggest that BU could be meaningful in PD treatment. Received 14 January 2009; received after revision 22 February 2009; accepted 10 March 2009     

Factors affecting high-frequency fungal protoplast fusion

Summary Factors influencing the fusion frequency of protoplasts have been examined with auxotrophic mutants ofAspergillus nidulans. The optimum conditions were a total of 5 to 15 million protoplasts per ml, and 25% polyethylene glycol (PEG) 4000 or 6000 as fusogenic agent in 10 to 100 mM CaCl₂ solution.