Histochemical demonstration of monoamine oxidase in the hypothalamo-hypophysial system of the tree sparrow and the rat

Summary Localization of monoamine oxidase (MAO) was investigated essentially according to the method of Glenner et al. (1957) in the hypothalamo-hypophysial system of the tree sparrow and the rat. The hypothalamic neurosecretory cells of both species showed relatively weak MAO activity. A similar localization of MAO activity was observed in the median eminence of both species: (1) slight or no MAO activity was observed in the ependymal layer, (2) relatively strong activity was revealed in the tissue just beneath the ependymal layer, (3) strong activity was revealed in the outer layer, particularly in the tissues surrounding capillary loops of the primary plexus. It is suggested that an adrenergic mechanism functions in the median eminence. In the pars nervosa, strong reaction was observed in the rat, while a weak reaction occurred in the tree sparrow. However, the color and the size of formazan crystals deposited in the rat pars nervosa differed from those in the hypothalamus. As a whole, the distribution of the neurosecretory material differed from the localization of MAO activity in the hypothalamo-hypophysial system. It is discussed that the neurosecretory neuron is not adrenergic but cholinergic.Aided by Grant A-3678 from the United States Public Health Service. The authors are indebted to Dr. S. Kambara, Zoological Institute, and Dr. H. Hirano, Department of Anatomy, University of Tokyo, for their valuable advice, and also to Assoc. Prof. S. Yamamoto, Department of Hygiene, University of Tokyo, for making available some facilities. They also wish to thank Dr. L. M. Barbato, University of Illinois, and Mr. K. Asami, National Institute of Radiological Sciences, Chiba, and Mr. Suzuki, Research Laboratory, Chugai Pharmaceutical Company Ltd., Tokyo, for the kind supply of MAO inhibitors.     

Cascade transduction and production of the light-nitric oxide signal from shoots to roots in maize seedlings exposed to enhanced UV-B radiation

The biomasses, rate of apparent nitric oxide (NO)-release, nitric oxide synthase (NOS) activity as well as β-d-endo and exo-glucanase activity of the cell wall were analyzed and determined in the roots of maize seedlings. It was found that rhizospheric treatments of 2-phenyl-4,4,5,5-tetramethlimida-zoline-l-oxyl-3-oxide (PTIO), a NO scavenger, and radiation of enhanced ultraviolet-B (UV-B) to aerial parts of the seedling markedly inhibited the rate of NO release in roots, raised the activity of β-d-endo and exo-glucanase, and increased the biomasses of roots. The patent inhibitor, N-nitro-l-arginine (LNNA), of NOS was unable to inhibit NOS activity and NO generation. Inversely, reactive oxygen species (ROS) eliminator, N-acetyl-cysteine (NAC), stimulated the rate of NO release. There is no relationship between NOS activity and the rate of NO release. The latter showed a positive correlation with nitrate reductase (NR) activity, whereas it showed a negative correlation with the bio-masses and the activity of β-d-endo and exo-glucanase. All results implicated that NO was a by-product generated by NR catalysis, whereas NR activity was sensitively repressed by the systemic signal network (involved in ROS) induced by enhanced UV-B. It indicated that the downstream signal molecule of enhanced UV-B light is probably ROS which decreased NO generation through inhibiting NR activity. The endogenous NO generated by NR catalysis is perhaps such a messenger for restraining β-d-endo and exo-glucanase activity that the root growth was retarded.     

Studies on the inactivation of the flavoproteind-amino acid oxidase fromTrigonopsis variabilis

Inactivation ofd-amino acid oxidase occured by different mechanisms. The enzyme showed a rapid loss of activity in the presence of micromolar amounts of Cu²⁺ and Hg²⁺. It was also sensitive to oxidative inactivation by Fe²⁺ and H₂O₂ when both reagents were added in millimolar amounts. When oxidatively inactivatedd-amino acid oxidase and a corresponding non-treated control were modified with the sulfhydryl-modifying, fluorescent reagent monobromobimane and subsequently digested with endoproteinase Glu-C, Cys-298 was identified to be a target for oxidative modification according to differences in the known peptide profile of fluorescence intensity. Another reason for the observed loss of enzyme activity in crude extracts was the specific proteolytic digestion ofd-amino acid oxidase, which was dependent on the growth phase of the cells used. This cleavage was catalyzed by a serine-type proteinase and was the introductory step for the further complete degradation of the enzyme. In addition, a coenriched 50-kDa protein, identified as NADPH-specific glutamate dehydrogenase, significantly decreased the stability of thed-amino acid oxidase activity. Treatment of apo-d-amino acid oxidase fromT. variabilis with monobromobimane resulted in a significantly increased fluorescence of two peptides, neither of which contained any cysteine residue. Thus, an involvement of cysteine residues in binding the FAD coenzyme should be excluded.     

Activity levels of six glycoside hydrolases in apple fruit callus cultures depend on the type and concentration of carbohydrates supplied and the presence of plant growth regulators

Sucrose presence and concentration modulated in different ways and to different extents the activity of six plant glycoside hydrolases (PGHs) extracted from apple callus cultures, both in the water soluble fraction (WS-F) and in the NaCl-released fraction (NaCl-F). β-d-Glucosidase activity increased because of sucrose starvation and the addition of sucrose decreased both WS-F and NaCl-F β-d-glucosidase from calli grown in a Murashige and Skoog’s basal medium with (MSH) or without (MS0) plant growth regulators (PGRs). WS-F and NaCl-F α-l-arabinofuranosidase, NaCl-F β-d-galactosidase and NaCl-F β-d-xylosidase activity reached a maximum when 0.045 M sucrose was added to the MS0 medium with an ensuing decline at higher sucrose concentrations. α-d-Galactosidase and α-d-xylosidase activity reached a maximum when 0.045 M sucrose was supplied and did not decline significantly in 0.09 M sucrose-supplied calli. When the effects of PGR presence or absence were analysed, NaCl-F β-d-glucosidase, α-d-galactosidase, β-d-galactosidase, α-d-xylosidase and β-d-xylosidase activities were found to be higher in MS0 than in MSH. To assess whether sugar effects were sucrose-specific, other sugars (glucose, fructose, galactose, maltose, lactose, raffinose, sorbitol and mannitol) were tested, with or without PGR supplementation. In general, sugar alcohols (mannitol, sorbitol) and some monosaccharides (fructose and glucose in particular) were better inducers of NaCl-F α-l-arabinofuranosidase, β-d-galactosidase and β-d-xylosidase activity than disaccharides (sucrose, maltose, and lactose) or the trisaccharide raffinose. This trend was not widespread to all PGHs assessed since sucrose-supplemented calli displayed higher NaCl-F α-d-galactosidase than those supplemented with glucose, galactose, sorbitol or mannitol. These results show that sugars supplied to callus tissue cultures as a carbon source can also modulate PGH activity. Modulation is different for each PGH, sugar-specific and, at least in the case of sucrose, concentration-dependent. Results also suggest the existence of regulatory interactions between PGRs and sugars as part of an intricate sensing and signalling network. Combination of PGR, sugar type and concentration should be taken into account to maximize each PGH activity for further enzyme studies.     

Characterization of prolidase I and II purified from normal human erythrocytes: comparison with prolidase in erythrocytes from a patient with prolidase deficiency

The effect of various sulfur-containing amino acids on the activities of prolidase isoenzymes I and II isolated from erythrocytes of healthy individuals, and erythrocyte lysates from a patient with prolidase deficiency was investigated. The activity of prolidase I against glycylproline was strongly enhanced by d-methionine. l-Methionine and d,l-methionine slightly enhanced the activity at low concentration, but N-acetyl-l-methionine had no effect. d-Ethionine, l-ethionine, and d,l-ethionine also enhanced the activity of prolidase I. d,l-Homocysteine enhanced the activity at low concentration, but inhibited the activity at 50 mM. The activity of prolidase II against methionylproline was enhanced by d-methionine, d,l-methionine, and l-methionine, but N-acetyl-l-methionine had no effect. d-Ethionine and d,l-ethionine strongly enhanced the activity of prolidase II compared with l-ethionine; d,l-homocysteine weakly enhanced the activity. d,l-Homocysteine-thiolactone inhibited the activities of prolidase I and II in a concentration-dependent manner. The effect of various sulfur-containing amino acids on prolidase activity against methionylproline in erythrocyte lysates from a patient with prolidase deficiency was almost the same as that on prolidase II. The kinetics of the activities of prolidase I, II, and patient prolidase were also studied. Their K _m values were changed by adding sulfur-containing amino acids, but V _max values were unchanged.     

A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland

Summary A technique for the cytochemical demonstration of peroxidase activity in unfixed guinea-pig thyroid tissue is described in this paper. The substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) is oxidized by the peroxidase to form an insoluble reaction product. Optimal results were obtained after 20 min incubation at 37° C in reaction medium containing 1.4mm DAB (in 0.1m Tris-HCl) and 0.15mm hydrogen peroxide at pH 8.0. Peroxidase activity was seen in the thyroid follicle cells as a diffuse brown reaction product (which was more dense and granular in erythrocytes). The enzyme activity was quantified using a scanning-integrating microdensitometer, and the effects of two specific peroxidase inhibitors were evaluated. Both 3-amino-1,2,4-triazole and methimazole inhibited peroxidase activity in the follicle cells (enzyme activity was still seen in the erythrocytes), maximal inhibition occurring at 10mm. Stimulation of peroxidase in the thyroid was observedin vivo (1 I.U. TSH administered every 8 h for two days), with the maximal stimulation occurring after 1 day.     

Occurrence of two l-threonine (l-serine) dehydratases in the thermophile Chloroflexus aurantiacus

The thermophilic phototrophic prokaryote, Chloroflexus aurantiacus was shown to contain high constitutive l-threonine (l-serine) deaminating activity. Separation of cellular proteins by DE 52-cellulose chromatography and by polyacrylamide gel electrophoresis with subsequent activity staining of the gels yielded two bands, one representing an isoleucine-sensitive, the other one an isoleucine-insensitive form of l-threonine dehydratase. Both enzymes had a molecular weight of 120,000 but were distinguished by their different affinities to the two substrates, l-threonine and l-serine.Abbreviations SDH l-serine dehydratase - TDH l-threonine dehydratase     

Occurrence of freed-aspartate and aspartate racemase in the blood shellScapharca broughtonii

Summary Substantial concentrations of D-aspartate were found in several tissues of Scapharca broughtonii together with approximately equal concentrations ofl-aspartate. The foot and mantle extracts also contained an aspartate racemase activity. The formation ofl-aspartate from the Denantiomer by the foot extract was apparently slower than the reverse reaction, and this unbalance seemed to be due to the presence of an enzyme activity which rapidly convertedl-aspartate tol-alanine. The possible role of D-aspartate in the anaerobiosis was discussed.     

The effect of cations on reassociation of the components of the cellulosome cellulase complex synthesized by the bacterium Clostridium thermocellum

The cellulosome multienzyme complex was dissociated into 12–14 components when incubated at 30° C in a reaction mixture that was buffered at pH 5.0 and was 50 mm with respect to sodium dodecyl sulphate and 10 mm with respect to both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT). The dissociated components reassociated into a complex when dialysed against 20 mm TRIS/HCl buffer, pH 7.7, containing 2.5 mm DTT. When incubated in the presence of Ca²⁺ and DTT the reassociated complex had the same activity to hydrogen-bond-ordered cellulose as the undissociated cellulosome. However, when Ca²⁺ ions were incorporated into the TRIS/HCl-DTT dialysis medium the reconstituted complex had very little activity towards cellulose. Other divalent cations such as Mg²⁺ and Ba²⁺ had the same effect, but the monovalent cation Na⁺ resulted in a complex that was very active on crystalline cellulose. The results are interpreted as indicating that the divalent cations bind to one or more of the dissociated polypeptide components and induce changes in conformation that prevent their reassociation into a complex with activity towards crystalline cellulose. Correspondence to: T. M. Wood     

Four glycoside hydrolases are differentially modulated by auxins, cytokinins, abscisic acid and gibberellic acid in apple fruit callus cultures

α-l-Arabinofuranosidase, α- and β-d-xylosidase, and β-d-glucosidase activity was detected in the soluble fraction (S-F) extracted with water and in the NaCl-released fraction (NaCl-F) extracted with a high-salt concentration buffer from apple callus cultures. The activity was found to be differentially modulated by the addition of various plant growth regulators (PGRs) to calluses that had lost their requirement for specific PGRs (“habituation” phenomenon). α-l-Arabinofuranosidase activity was 93%, 130%, 126% and 186% higher in the NaCl-F from IAA-, IBA-, ABA- and GA₃-treated callus than in that extracted from untreated callus while S-F α-l-arabinofuranosidase activity was only 71%, 24%, 55% and 66% higher, respectively. α-d-Xylosidase displayed low activity levels in both S-F and NaCl-F but 2iP-treated callus showed higher α-d-xylosidase activity in both fractions than the control. 2,4-D increased α-d-xylosidase activity by 110% in the NaCl-F but decreased it by 40% in the S-F. β-d-Xylosidase activity increased by 99% in S-F from 2iP-treated callus but slightly decreased in the NaCl-F. In GA₃-treated callus, NaCl-F β-d-xylosidase activity increased by 188%. S-F and NaCl-F from Picloram-treated callus showed undetectable or only slightly noticeable α-l-arabinofuranosidase, α-d-xylosidase and β-d-xylosidase activity. Interestingly, β-d-glucosidase activity rose 28-fold in the S-F extracted from Picloram-treated callus. β-d-glucosidase was the only enzyme assayed that greatly increased its NaCl-F activity after 10 subcultures, and the addition of any PGR to the callus culture –except for Picloram and ABA– decreased its activity, suggesting that this enzyme may be associated with certain stress conditions, such as PGR starvation or Picloram addition. This is the first report on glycoside hydrolases from fruit callus as modulated by different PGRs.     

Regulation of aspartokinase activity in non-fermentative,marine eubacteria

Summary Cell-free extracts of gram-negative, non-fermentative, marine eubacteria were assayed for aspartokinase activity. The organisms tested included polarly flagellated species and groups which had GC contents in their DNAs of 46 to 64 moles % (Alteromonas, Pseudomonas) as well as species which had peritrichous flagellation and moles % GC contents of 53 to 68 (Alcaligenes). The results of these studies suggested that in all the strains tested, aspartokinase activity was catalyzed by a single enzyme. On the basis of the effect ofl-threonine,l-lysine,l-methionine, andl-isoleucine on activity, five different types of aspartokinases (designated I through V) were delineated. In aspartokinase types I through IV,l-threonine andl-lysine inhibited activity by means of a concerted feedback inhibition; in type V, activity was inhibited byl-threonine but unaffected byl-lysine. In types I, III, and IV,l-threonine andl-lysine alone were inhibitory, while in type II these effectors had virtually no effect on activity when tested singly. Three distinct responses were observed in the presence of two other end products of the aspartate pathway,l-methionine andl-isoleucine. In types I and II, these two amino acids usually stimulated activity and overcame the inhibition byl-threonine andl-lysine; in types IV and V,l-methionine andl-isoleucine had no effect; and in type III these amino acids inhibited activity. The results of this study indicate that the aspartokinases of a number of species and groups of marine bacteria have similarities and differences which should be of use in making future taxonomic groupings.     

Characterization of acetyl-CoA: l-lysine N6-acetyltransferase,which catalyses the first step of carbon catabolism from lysine in Saccharomyces cerevisiae

The carbon catabolism of l-lysine starts in Saccharomyces cerevisiae with acetylation by an acetyl-CoA: l-lysine N⁶-acetyltransferase. The enzyme is strongly induced in cells grown on l-lysine as sole carbon source and has been purified about 530-fold. Its activity was specific for acetyl-CoA and, in addition to l-lysine, 5-hydroxylysine and thialysine act as acetyl acceptor. The following apparent Michaelis constants were determined: acetyl-CoA 0.8 mM, l-lysine 5.8 mM, dl-5-hydroxylysine 2.8 mM, l-thialysine 100 mM. The enzyme had a maximum activity at pH 8.5 and 37°C. Its molecular mass, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 52 kDa. Since the native molecular mass, determined by gel filtration, was 48 kDa, the enzyme is a monomer.     

Cholinesterases in the hypothalamo-hypophysial neurosecretory system of the White-crowned Sparrow,Zonotrichia leucophrys gambelii

Summary The distribution of cholinesterases in hypothalamo-hypophysial neurosecretory system of the White-crowned Sparrow has been examined histochemically. The perikarya of the neurosecretory cells of the paraventricular and supraoptic nuclei have a high acetylcholinesterase activity. Acetylcholinesterase activity also occurs in the cells of the infundibular nucleus. The proximal parts of the axons of the cells of the neurosecretory and infundibular nuclei have strong acetylcholinesterase activity and weak non-specific cholinesterase activity. In the median eminence, the activity of acetylcholinesterase is strongest in the palisade layer. In the pars nervosa, there is definite, although weak, acetylcholinesterase activity.This investigation was supported by grants from the National Institutes of Health to Professor Farner (B-1353) and to Dr. Kobayashi (A-3678).     

Induction of aldose reductase activity inCandida guilliermondii by pentose sugars

Summary Cell extracts ofCandida guilliermondii grown ind-xylose,l-arabinose,d-galactose,d-glucose,d-mannose and glycerol as sole carbon sources possessed NADPH-dependent aldose reductase activity, but no NADH-dependent activity was detected.d-xylose andl-arabinose were the best inducers of aldose reductase activity. The highest enzyme activity ind-xylose orl-arabinose-grown cells was observed first withl-arabinose followed byd-xylose as substrates of the enzymatic reaction. However, only low activity was found ind-glucose,d-mannose andd-galactose-grown cells, indicating that these carbon sources cause catabolite repression. Enzyme activities induced ind-xylose-grown cells were twice as high as those obtained from the cells under resting conditions. Furthermore, the level of induction of aldose reductase activity depended on the initial concentration ofd-xylose. The present study shows that aldose reductase activity may be efficiently induced by pentose sugars of hemicellulosic hydrolysates and weakly by hemicellulosic hexoses.     

Ultrastructural localization of some phosphatases in the prothoracic gland of the insect Leucophaea Maderae

Summary The cytochemical localization of acid phosphatase and thiamine pyrophosphatase activity was studied by light and electron microscopy in prothoracic gland cells of the cockroach Leucophaea moderae. Nymphal and young adult animals were used.Prominent sites of acid phosphatase activity included large membrane-bounded dense bodies or lysosomes, and certain cisternae of the Golgi apparatus. The results suggest a possible difference in the enzymatic activity toward glycerophosphate and aromatic phosphates as substrates.Thiamine pyrophosphatase activity was localized in elements of the Golgi apparatus and endoplasmic reticulum, and in lysosome-like dense bodies. This latter activity was abolished by sodium fluoride treatment, whereas the phosphatase activity in the Golgi apparatus and endoplasmic reticulum is unaffected by such inhibition.The cytochemical results confirm through direct evidence the suggestions of Scharrer (1964), that the large dense bodies present in the prothoracic gland cells are lysosomes, and that their activity may be related to stages in the life history of the glands. Furthermore, the lysosomes or their derivative structures may play an essential role in the autolysis of the prothoracic glands toward the end of their active period.The enzymatic activity of the endoplasmic reticulum may indicate the involvement of this organelle in the metabolism of steroid-like precursor materials necessary for the synthesis of ecdysone.This study was supported by U.S.P.H.S. grants 5 T1-MH-6418 and NB-05219, and grant RO 1-AM-3984 to Dr. Berta Scharrer. I would like to express my appreciation to Dr. Scharrer for her encouragement and assistance during this study. I also wish to thank Mrs. Sarah Wurzelmann for her competent technical aid.     

Construction and characterization of a recombinant whole-cell biocatalyst of Escherichia coli expressing styrene monooxygenase under the control of arabinose promoter

A recombinant Escherichia coli (pBAB1) containing styrene monooxygenase (SMO) was developed for the conversion of styrene to enantiopure (S)-styrene oxide that is an important chiral building block in organic synthesis. The styAB genes encoding SMO was cloned into a multicopy plasmid under the tightly regulated promoter of bacterial l-arabinose operon which is inducible by l-arabinose. The recombinant showed that expression level of StyA protein and whole-cell SMO activities were varied depending on the concentration of the inducer l-arabinose. The maximum SMO activity was 108 U/g cdw when the cells were induced with 0.2% l-arabinose. SDS-PAGE and Western blot analyses indicated that whole-cell SMO activity was strongly correlated with the expression level of StyA protein. Organic-aqueous two-phase experiment could yield 50 mM enantiopure (S)-styrene oxide in organic phase in 18 h, but the recombinant SMO activity was unstable during the reaction. The expression of styAB under the control of l-arabinose promoter was significantly repressed in the presence of glucose.     

Assimilation of D-Malate by Rhodobacter capsulatus E1F1

Rhodobacter capsulatus grew by using either L- or D-malate as carbon sources under light/anaerobic conditions. The cellular yields were the same with D- or L-malate. Both L-malate dehydrogenase and L-malic enzyme activities were detected in cell-free extracts from cells grown in both isomers. By contrast, a racemase activity converting D-malate into L-malate was induced only when D-malate was present in the culture medium. This racemase activity was Mn²⁺-dependent and was measured by coupling it either to the malate dehydrogenase or to the fumarase activities. The racemase activity was partially purified by anion-exchange chromatography. Received: 30 November 2000/Accepted: 10 January 2001     

Metabolism of d-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene

Summary The Escherichia coli xylose isomerase gene was transformed into Schizosaccharomyces pombe for direct d-xylose utilization. In order to understand d-xylose metabolism and determine the limiting factors on d-xylose utilization by the transformed yeast, d-xylose transport, xylose isomerization, and xylulose phosphorylation were investigated. The results indicated that low activity of xylose isomerization in the cloned yeast was the limiting step for d-xylose fermentation. An in vitro study showed that yeast proteases decreased xylose isomerase activity. Xylitol, a by-product of d-xylose fermentation, had no effect on the activity of xylose isomerase.     

Preliminary studies on the growth of Pseudomonas sp. BA2 and the production of L-aminoacylase

Summary The bacterial strain Pseudomonas sp. BA2 did not develop l-aminoacylase activity in the absence of N-acetyl-dl-alanine. The maximum growth rate and enzyme production were obtained when the acetylated amino acid was used as the sole carbon and nitrogen source. A maximum biomass of A₆₆₀=1.543, after 24 h of cultivation, was obtained. The l-aminoacylase activity reached the maximum value (5.6 U ml^–1 broth) in the stationary growth phase.