Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.     

Bewegungsstudien an Anatinen

Zusammenfassung Vorliegende Arbeit ist eine Weiterführung derLorenz'schen Bewegungsstudien an Anatinen aus dem Jahre 1941, fortgesetzt an Mischlingen zwischen den dort beschriebenen Arten. Die sich dabei ergebenden Befunde machten eine erneute Untersuchung der Elternarten notwendig. Außerdem wurden einige Arten beobachtet, deren Verhalten noch nicht untersucht worden war. Fragestellung und Begründung werden in der Einleitung gegeben.Im zweiten Abschnitt werden einige der vonLorenz gemachten Beobachtungen berichtigt. So zeigten einige der Kreuzungen mitbahamensis, daß die vonLorenz bei eben dieser Art als Kurzhoch-werden bezeichnete Bewegungsweise dem Ab-auf anderer Schwimmenten homolog ist. Ebenso ist die eine der beiden vonLorenz als Kurzhoch-werden bezeichneten Verhaltensweisen des Krickerpels als Ab-auf zu deuten. Der Gruß desflavirostre-Erpels wurde auch bei weiblichen Tieren gesehen. BeiAnas acuta wurde ein Kinnheben festgestellt, das sich in der Form stark vom Kinnheben beiplatyrhynchos unterscheidet. Als neue Verhaltensweisen wurden u. a. das Haltungannehmen und das Tendieren beimflavirostre-Erpel beschrieben.Im dritten Abschnitt werden einige Verhaltensweisen und ihre Funktion diskutiert und der Versuch gemacht, eine Motivationsanalyse zu geben.(Zeichnungen vonHermann Kacher)     

The neurosecretory system of the adult Calliphora erythrocephala

Summary In continuation of previous light microscopical investigations using darkfield microscopy of the living cells and sections stained with paraldehyde-fuchsin, an electron microscopical study of the medial neurosecretory cells (m.n.c.) of virgin females of Calliphora has been performed. The neurosecretory material consists of elementary granules corresponding in quantity to the amount of secretory material found by the two other methods in flies of the same age and kept on the same diet. The majority of the cells (m.n.c. I) contain granules measuring c. 2000–3000 Å, while fewer cells (m.n.c. II) show a smaller granular diameter (c. 1000–1500 Å). Due to the Tyndall effect the elementary granules are visible when using darkfield microscopy.The granules were seen to be pinched off from the Golgi complexes. These are numerous and well-developed, except in the less active m.n.c. I of the six days old sugar-flies. The reticulum and mitochondria are described. Axoplasmic channels were observed in the m.n.c. I, probably corresponding to structures found by Wigglesworth (1959 and 1960) in other insect neurons with another technique.The fine structure of the giant neurons and the vacuolated cells has been studied, the observations supporting the conclusions of M. Thomsen in a light microscopical study (1965). Lacunae in the ramifying glia are interpreted as belonging to the glial lacunar system described by Wigglesworth (1960).Dedicated to the memory of Ernst Scharrer (1905–1965), pioneer in the study of neurosecretion.We are grateful to the Carlsberg Foundation and the State General Scientific Foundation for financial support.     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

Fine structure of ureteric duct epithelium in the North Atlantic hagfish (Myxine glutinosa L.)

Summary The epithelial cells lining the ureteric duct in the cyclostome, Myxine glutinosa, have a brush border and show specializations of their apical cytoplasm similar to those observed in absorptive proximal tubule cells in higher vertebrate species. These features and the presence of large and numerous cytosomes, presumed to contain lysosomal enzymes, indicate that the ureteric epithelium has taken over some of the functions of the proximal tubule in the atubular kidney of Myxine. Sparsity of basal cytoplasmic processes and mitochondria in the ureteric duct cells appears to correlate with an inability for active, energy-dependent secretory and ion transport functions.This study has been supported by a grant from the Karolinska Institutet Medical School, Stockholm, Sweden (Therese och Johan Anderssons Minne). The author is indebted to Doctor Bertil Swedmark for permission to use the facilities of Kristineberg Marine Biology Station, Fiskebäckskil, Sweden, where hagfish were caught.     

Direct somatic embryogenesis through thin cell layer culture in Panax ginseng

Direct somatic embryos were differentiated on cotyledon transverse Thin Cell Layers (tTCLs) of Panax ginseng after 9 weeks in the Murashige and Skoog basal (MS) medium containing 2,4-d (5M). When MS medium containing 2,4-d (5M) was used for seedling pretreatment and for tTCLs culture, somatic embryos were observed 2 weeks earlier, i.e. after 7 weeks of culture. On the tTCLs from seedlings pretreated with 2,4-d (5M) combined with benzyladenine and zeatin at 0.1 M (BZ), somatic embryos were observed after 6 weeks of culture and the percentage of embryogenesis was higher (62%) than when 2,4-d was used alone for pretreatment (40%). Similar results were also obtained from pretreatment with combinations of 2,4-d (5M) and thidiazuron (TDZ) (0.01, 0.1M). When a combination of 2,4-d (5M) and BZ (0.1M) was used both for seedling pretreatment and for tTCLs culture, both somatic embryos and shoots were observed after only 3 weeks. As the concentration of BZ increased, the percentage of somatic embryogenesis decreased but the percentage of organogenesis increased. Similar responses were obtained with a combination of 2,4-d (5M) and TDZ (0.01M). On the medium containing both NAA (0.3M) and BZ (1M), globular- and heart- stage embryos developed after 4 weeks of culture into cotyledonary-staged embryos which remained dormant after a short elongation of the embryo axis. The importance of seedling pretreatment by growth substances in enhancing somatic embryogenesis is reported.Abbreviations BA 6-benzyladenine - BZ combination of BA and zeatin - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium Murashige and Skoog basal medium - NAA a-naphthaleneacetic acid - TDZ thidiazuron - tTCLs transverse thin cell layers - TCL longitudinal thin cell layer     

Dense-core vesicles in motor nerve terminals

Summary Motor nerve terminals on white and intermediate muscle fibers of the Atlantic hagfish (Myxine glutinosa, L.) contain translucent synpatic vesicles and about 1–2% dense-core vesicles. Terminals on red muscle fibers contain up to 40% dense-core vesicles with diameter 800–1100 Å. Examinations for formaldehyde-induced fluorescence indicate yellow fluorescence (5-HT ?) apparently corresponding with terminal axons on red muscle fibers in craniovelar muscles. Possibly red muscle fibers of Myxine receive monoaminergic innervation.The author is indebted to Dr. Finn Walvig, Biological station, University of Oslo, Drøbak, for supply of hagfishes.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

Techniques for the quantitative study of mutation in plant viruses

Summary Hardly any other virus is chemically and ultramicroscopically as well known as TMV. It is not possible to perform genetic recombinations with this object. The phenomenon of mutation is, however, known and an analysis of the dosis-effect relationship was possible by using the characters chlorotic versus necrotic primary symptoms. Taking into account the phenomenon of interference (mutual exclusion), i.e., comparing the induced mutation frequency with that of a control virus sample diluted to the same level of infectivity, on can perform quantitative analyses. In this way the first chemical mutagensis in the test tube was demonstrated 10 years ago with nitrous acid as mutagenic agent. The criticism raised byBawden to the first publication ofMundry andGierer was already inappropriate at that time. In the meantime it has been demonstrated byWittmann-Liebold andWittmann through analysis of amino acid exchanges in spontaneous mutants and in those isolated after incubation with HNO₂ that the difference between spontaneous and induced mutants demanded byBawden, which cannot be postulated for symptoms in plants, lies, as expected, in amino acid exchanges of the protein coat.

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Zusammenfassung Kaum ein anderes Virus ist chemisch und ultramikroskopisch so gut bekannt wie das TMV. Rekombinations-Genetik ist nicht möglich. Das Phänomen der Mutation ist aber bekannt, und eine Analyse der Dosis-Effekt-Beziehung wurde möglich durch Benutzung der Symptomcharaktere chlorotische versus nekrotische Primärsymptome. Bei Berücksichtigung des Phänomens der Interferenz (mutual exclusion), d. h. wenn man die induzierte Mutationsrate mit der auf gleiche Infektiosität durch Verdünnen der Viruslösung gebrachten als Kontrolle vergleicht, kann eine quantitative Analyse durchgeführt werden. So wurde vor 10 Jahren die erste Chemomutagenese im Reagenzglas mit salpetriger Säure als mutagenes Agens nachgewiesen. Die an der ersten Veröffentlichung vonMundry undGierer vonBawden geäußerte Kritik war schon damals unzutreffend. Inzwischen ist durch die Analyse der Aminosäureaustausche von spontanen und nach Inkubation mit HNO₂ isolierten Mutanten vonWittmann-Liebold undWittmann gezeigt worden, daß die vonBawden geforderte Verschiedenheit spontaner und induzierter Mutanten, die für Symptome an den Pflanzen nicht postuliert werden kann, in den Aminosäureaustauschen des Hüllproteins wie zu erwarten vorhanden ist.

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This paper was a first written for Methods in Virology, Academic Press. The editors and the author did not come to an agreement in the question of citation ofBawden's criticism to the work ofMundry andGierer 1958. It is published here on the occasion of the 10th anniversary of the first chemomutagenesis in the test tube.     

Kinetic,ESR, and trapping evidence for in vivo binding of Mn(II) to glutamine synthetase in brain cells

Mn(II) has been proposed as a potential modulator of various important CNS enzymes, particularly glutamine synthetase, which is compartmentalized in the cytoplasm of glia. Previous studies demonstrated that total glial Mn(II) was 50–57 M, of which 30–40% occurs in the cytoplasm. In the present study, electron spin resonance (ESR) was used to determine that the concentration of free cytoplasmic Mn(II) in cultured chick glial cells is 0.8 (±0.2) M, very near K_d for the GS-Mn(II) complex. No free Mn(II) could be detected in glial mitochondria. Association of Mn(II) with brain glutamine synthetase (GS) was assessed under in vivo conditions in the presence of millimolar Mg(II) by trapping bound⁵⁴Mn(II) ions in the active site with irreversible inhibitors, namely methionine-sulfoximine (MSOX) or specific analogues thereof plus ATP. Ovine brain tissue was lysed directly into buffer containing Mn(II), 3 mM Mg(II), 1 mM MSOX, 1 mM ATP, 200 mM KCl, and 20 mM NaCl. Alternatively, primary cultures of chick glial cells were permeabilized into these inactivation mixtures. -Methyl-d,l-prothionine-S,R-sulfoximine was used to specifically inhibit the mechanistically-related enzyme -glutamyl-cysteine synthetase prior to specific inactivation of GS by -ethyl-d,l-methionine-S,R-sulfoximine. Even inthe presence of 2–3 mM Mg(II), with only 5–10 M Mn(II) present, approximately 20–30% of GS subunits were trapped with bound Mn(II). These results indicate that brain GS exhibits a high degree of specificity for binding Mn(II) over Mg(II) and that Mn(II) binds to GS to a significant extent under in vivo conditions.     

Cellular localization of glycoside hydrolases inBacteroides fragilis

The localizations of six glycosidases produced byBacteroides fragilis—-glucosidase, -glucosidase, -galactosidase, -galactosidase, -N-acetylglucosaminidase, and -l-fucosidase—were studied. Cell fractions and cell extracts were obtained by Triton X-100 release, by disruption by freeze-pressing and sonication, and by osmotic release. Isoelectric focusing of a cytoplasmic and of a Triton X-100 extract of the cell wall fraction was performed and revealed differences in the relative distribution of differently charged forms of -N-acetylglucosaminidase. -Galactosidase and alkaline phosphatase were used as cytoplasmic and periplasmic markers, respectively. It is concluded that inB. fragilis -glucosidase is periplasmic, -l-fucosidase and -galactosidase are cytoplasmic, and -n-acetylglucosaminidase is cell associated and bound to the cell envelope by hydrophobic interactions. -Glucosidase and -galactosidase are localized cytoplasmically and/or located in the cell envelope.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

The in vitro production of an anthocyanin from callus cultures of Oxalis linearis

Callus growth and the production of anthocyanins were sustained on the salts and vitamins of Murashige and Skoog. Callus growth was stimulated at a concentration of 8–32 M -naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d). Benzyladenine (BA) and zeatin at 8 M inhibited callus growth whereas isopentenyladenine (iP) stimulated callus growth. NAA repressed anthocyanin production with an increase in NAA from 8–32 M. Anthocyanin synthesis was promoted by an increase in 2,4-d from 0.5 to 2 M and decreased thereafter up to a concentration 32 M 2,4-d. A concentration of 8 M BA, thidiazuron and zeatin, respectively stimulated pigment production. Sucrose stimulated callus growth at 60 mM and pigment production at 120–360 mM.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - iP isopentenyladenine - TZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-yl-urea - Bu-HCl Butanol-2N HCl - BAW Butanol-acetic acid-water     

The fine structure of the branchial heart appendage of the cephalopod Octopus dofleini martini

Summary The branchial heart appendage of Octopus dofleini martini has been investigated electron microscopically. This organ is dominated by peripherally lobed blood sinuses. It contains free hemocyanin (often aligned in rows), amoebocytes, endothelial cells, and muscle cells which occur mainly in connection with neurons. The neurons are often exposed to the blood. The blood sinuses are enclosed by a basement membrane which contains collagen equivalents and fine fibrillar elements. The sinuses are covered by two different epithelia: 1) the epithelium in the caoity of the appendage consisting of irregularly shaped cells with processes, the so called ( 30 high) podocytes, and 2) the epithelium ( 40 in height) on the surface of the organ, which is composed of two parts: a) a lacuna-forming portion directly adjacent to the basement membrane, which is topped by b) a continuous tissue portion with occasional lacuna-canals. The intercellular spaces of the inner and outer epithelium are connected. The structures of these epithelial cells are discussed in relation to the formation of the pericardial fluid.Our thanks are given to Professor Dr. Georg Kümmel, Freie Universität Berlin, for suggesting the theme and his scientific guidance; to Dr. Kenneth M. Towe, Smithsonian Institution, Washington D.C., for generously allowing the use of his instruments and for his technical assistance; to Mr. Frank Denys, Medical Dental School of Georgetown University, Washington D.C., for sharing with me his technical skills and for making possible the occasional use of a dark room; and finally to Dr. Fred E. Witmer, Office of Saline Water, U.S. Dept. of Interior, Washington D.C., for his help with the translation, and for taking all the side effects of this study as patiently as he did.Supported in part by Grant number GB 17539 of the National Science Foundation. Used as part of a thesis submitted to the Freie Universität Berlin.     

Über fixationsbedingte Unterschiede in der elektronenmikroskopischen Morphologie der Zelltypen im Inselapparat des Frosches Rana ridibunda

Zusammenfassung Langerhanssche Inseln des Frosches Rana ridibunda wurden nach Osmiumtetroxyd-, Glutaraldehyd- und Acroleinfixierung elektronenmikroskopisch untersucht. Nach ausschließlich Aldehydfixierung und Nichtanwendung einer Kontrastierungsmethode fehlt den Sekretgranula der Inselzellen ihr bekannter positiver Kontrast; sie können sogar im negativen Kontrast erscheinen. B-Zellen besitzen runde und nadeiförmige Sekretgranula neben anderen Zelleinschlüssen (Lipidtropfen, lysosomenartige Einschlüsse, Vakuolen, glykogenartiges Material) und zeigen meist eine bipolare Zytoplasmaorganisation. Golgiapparat und Ergastoplasma sind relativ spärlich entwickelt. Während die B-Zellmorphologie keine starke Abhängigkeit von der Art der Fixierung zeigte, erwies sich das Bild der Typ IIundTyp HI-Zellen als stark fixationsabhängig. So scheinen chromophobe Zellen, d.h. Zellen, die leere Bläschen an Stelle der Sekretgranula enthalten, bei Osmiumfixierung aus Typ IIZellen und bei Acroleinfixierung aus Typ III-Zellen zu entstehen. Chromophobe Zellen wurden nach Glutaraldehydfixierung niemals beobachtet. Die bläschenreichen Zellen erscheinen den D-Zellen verschiedener Autoren verwandt zu sein. Das Bild dieser Elemente wird als Fixationsartefakt betrachtet. Ein eigentlicher chromophober Zelltyp scheint demnach in den Langerhansschen Inseln des Frosches nicht zu existieren. Die hellen Zellen der Lichtmikroskopie rekrutieren sich demnach aus verschiedenen Quellen, zu denen granulaarme Formen aller 3 Zelltypen und schlecht erhaltene granulareiche Formen der Typen II und III gehören.

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Summary In a study on the fine structure of the endocrine pancreas in Rana ridibunda, osmium tetroxide, glutaraldehyde, and acrolein have been used as fixatives. After aldehyde fixation alone, without application of any further contrasting procedure, the secretory granules of the islet cells lack their known positive contrast and may show up even in negative contrast. B cells possess round and needle-like secretory granules together with other inclusions (lipid, lysosome-like, vesicular, glycogen-like) and mostly display a bipolar organisation. B cell Golgi apparatus and ergastoplasm are relatively sparse. Whilst B cell structures do not show much variation with the fixative employed, those of type II and type III cells do. Thus, chromophobe cells, i.e. cells containing empty vacuoles instead of secretory granules, seem to arise from type II cells with osmium tetroxide fixation and from type III cells with acrolein fixation. Chromophobe cells were never observed after glutaraldehyde fixation. They are likened to D cells of authors employing osmium fixation and are considered to be artifacts due to fixation. No chromophobe cell proper, therefore, seems to exist in the frog pancreatic islet and the clear cells of light microscopy should stem from several sources including agranular stages of the types B, II, and III and badly preserved granular stages of types II and III.

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Durchgeführt mit dankenswerter Unterstützung durch den Schweizerischen Nationalfonds.

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Zusatz bei der Korrektur. Forssmann, W. G., G. Siegrist, L. Orci, L. Girardier, R. Pictet und Ch. Roullier berichteten [J. Microscopie 6, 279–304 (1967)] über elektronenmikroskopische Beobachtungen an verschiedenen Organen nach Perfusionsfixierung. Die in der vorliegenden Studie nach Immersionsfixierung erhobenen Befunde mit solchen nach Perfusionsfixierung zu erhebenden zu vergleichen, erscheint uns wesentlich.     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

Fragmenta mycologica

Summary Castellani's water culture method for microscopic fungi was re-examined and confirmed in its every detail. It is an ideal method for, at least, the smaller culture collection, in order to avoid continuous, short term subculturing. Benedek's suggestion for establishment of a mycotheca preserved in chlorallactophenol on the analogy of the herbarium of phanerogamists, may fill a long felt gap in mycological laboratories.

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Zusammenfassung Castellani's Wasserkultur-methode für mikroskopische Pilze wurde einer Kontrolluntersuchung unterzogen. Sie konnte in all ihren Einzelheiten bestätigt werden. Sie ist eine ideale Methode, wenigstens für kleine Kultursammlungen, um eine fortgesetzte, kurz-fristige Überimpfung zu vermeiden. Benedek's Vorschlag für die Einrichtung einer Mykotheka in Chlorallactophenol in Analogie mit dem Herbarium für Blütenpflanzen, mag eine lang gefühlte Lücke im mykologischen Laboratorium beseitigen.

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Résumé La méthode deCastellani, la culture en d'eau des champignons microscopiques, a été ré-examinée. Elle pouvait être confirmée en tous les détails. C'est une méthode idéale, au moins, pour la collection de cultures de petite dimension, pour éviter les transferts continus et d'intervalle courte.La suggestion deBenedek pour établir une mycothèque en chlorallactophénol, par analogie avec l' herbier pour les phanérogames peut remplir une lacune, existante pour long temps, aux laboratoires mycologiques.

     

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

Short- and long-term effects of ACTH on the adrenal zona glomerulosa of the rat

Summary Short-term ACTH treatment provoked a decrease in volume of the lipid-droplet compartment in rat zona glomerulosa cells, and a rise in plasma and intracellular concentrations of corticosterone and aldosterone. It enhanced activities of 3-hydroxysteroid dehydrogenase (3HSD), 11-hydroxylase (11OH) and 18-hydroxylase (18OH). Long-term ACTH administration produced a hypertrophy of the zona glomerulosa and its parenchymal cells, a result of the increase in volume of the smooth endoplasmic reticulum and the mitochondrial compartment. The surface area per cell of mitochondrial inner membranes increased; the tubular cristae were transformed into a homogeneous population of vesicles. The plasma and intracellular concentrations of corticosterone further increased, whereas those of aldosterone fell below basal levels (the aldosterone-escape phenomenon). The activities of 3HSD and 11OH were enhanced, that of 180H decreased. Therefore, ACTH stimulates zona glomerulosa growth and transforms parenchymal elements into zona fasciculata celltypes. Cyanoketone nullified acute ACTH effects on plasma and intracellular concentrations of corticosterone and aldosterone, but did not affect the activities of 11OH and 18OH. Chronic ACTH treatment produced similar results, although 18OH activity was not suppressed. The mechanism underlying the aldosterone-escape phenomenon may thus involve a rise in the intracellular concentration of corticosterone, caused by the enhanced synthesis and activation of 3HSD and 11OH.     

A model of associative memory in the brain

A model of associative memory for time varying spatial patterns is proposed and simulated on a digital computer. This is a network composed of many neuron-like elements, and shows an ability for associative memory similar to that of the brain.Suppose a number of sequences of spatial patterns are presented to this network, for example, 12345, ABC, and so on. Then, these patterns are memorized in the network. After that, if any part of one of these sequences, say 23, is presented to the circuit, the rest of the sequence, 45, is recalled following to it. It resembles to such a situation — if we hear a part of a melody which we have memorized in the past, the rest of the melody is recalled even after it is stopped half-way. Although the recalled patterns are not always 100% correct, they are not completely destroyed even if the presented patterns are imperfect.