O-Glycosylhydrolases of Marine Filamentous Fungi: β-1,3-Glucanases of Trichoderma aureviride

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from the bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl--D-glucosaminidase, -D-glucosidase, -D-galactosidase, -1,3-glucanase, amylase, and pustulanase. -1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that -1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.     

Enzymatic properties of overexpressed human hexokinase fragments

Full-length hexokinase (HK; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), a truncate form of the enzyme lacking the first 11 amino acids (HK-11aa) and the 50 kDa C-terminal half (mini-HK) containing the catalytic domain, were overexpressed and purified to homogeneity to investigate the influence of the N-terminal region of human hexokinase type I (HK) on its regulatory properties. All forms of the enzyme are catalytically active with the HK-11aa being the most active. All the forms of HK showed the same affinity for glucose and MgATP and were also inhibited by glucose 6-phosphate (Glc 6-P) competitively vs. MgATP with similar Kis (28.5-37 M). Glucose 1,6-bisphosphate (Glc 1,6-P2) was also a strong inhibitor of all HKs without significant differences among the different truncate forms of the enzyme (Kis 49.5-59 M). At low concentrations (0-3 mM), Pi was able to reverse the sugar phosphate inhibition of the full-length HK and HK-11aa but not of the mini-HK. In contrast, at high concentrations Pi was an inhibitor of all the hexokinases investigated. These findings confirm that Pi has a low affinity binding site on the C-terminal of HK while counteracts glucose 6-phosphate inhibition by binding to or requiring the N-terminal half of the enzyme. The first 11 N-terminal amino acids influence the specific activity of HK but are unable to affect the kinetic properties investigated.     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

Yeast ribosomal proteins

Summary Homology maps between bacteriophages 81, 80 and were constructed on the basis of electron microscope observation of DNA heteroduplexes. In 81/80 heteroduplex, the left half and the right terminal region of 13% the total molecular length were highly homologous, while the remaining region covering the early gene cluster was entirely nonhomologous. In 81/ heteroduplex, high-degree homologies were detected at the left 14% terminal region covering the head gene cluster, the central 3.8% region covering the att-int-xis region and the 1.3% Q homology region. Low-degree homologies of shorter length were scattered at the tail gene cluster, b2 region, cIII region, PQ region and SR region. Comparing our results with the homology maps of other lambdoid phages reported by Simon et al. (1971) and Fiandt et al. (1971), a phylogenic relation of 81 to other lambdoid phages and the role of recombination in the course of divergence of lambdoid phages are discussed.     

Polymorphism of the HLA DR1 haplotype in the Israeli population investigated at the serological,cellular, and genomic levels

In the present report, we used serological, cellular, and restriction fragment length polymorphism (RFLP) to investigate the DR1 haplotype in the Israeli population. We describe an Israeli homozygous typing cell (HTC), HLA-DwLVA, which defines a new lymphocyte-activating determinant associated with Bw65, DR1 and distinct from Dwl. The parents of this donor, non-Ashkenazi Algerian Jews, are first cousins and share HLA-Cw8, Bw65, BfS, DR1, DQw1, DPw4. No specificity could be assigned to HLA-DwLVA using the 91 Ninth Workshop HTCs. Two families and forty unrelated DR1 individuals were studied with DwLVA and a panel of DR1/Dw1 HTCs. HLA-DwLVA showed segregation as a single determinant within families. This new specificity was present in 24 out of 40 (60%) unrelated DR1 individuals, indicating that in the Israeli population DwLVA is the main lymphocyte-defined determinant associated with the serologically defined DRI specificity, in contrast to non-Jewish Caucasoids where DR1 is significantly associated with Dw1. The vast majority of DwLVA-positive carriers were also Bw65 carriers, indicating that Bw65, DR1, DwLVA may represent a typical allele combination in the Israeli population. The RFLP analysis established the correlation of certain RFLPs with Dw1 and DwLVA. In addition, we describe a cluster of RFLPs that may correspond to a new Dw subtype associated with DR1, for which no serological and cellular reagents have been described so far.     

An improved method,using saturating light pulses,for the determination of photosystem I quantum yield via P700+-absorbance changes at 830 nm

An improved method is introduced for the determination of the quantum yield of photosystem I. The new method employs saturating light pulses with steep rise characteristics to distinguish, in a given physiological state, centers with an open acceptor side from centers with a reduced acceptor side. The latter do not contribute to PSI quantum yield (_I). Oxidation of P700 is measured by a rapid modulation technique using the absorbance change around 830 nm. The quantum yield _I is calculated from the amplitude of the rapid phase of absorbance change (A; 830 nm) upon application of a saturation pulse in a given state, divided by the maximal A (830 nm) which is induced by a saturation pulse with far-red background illumination. Using this technique, _I can be determined even under conditions of acceptor-side limitation, as for example in the course of a dark-light induction period or after elimination of CO₂ from the gas stream. Thus determined _I values display a close-to-linear relationship with those for the quantum yield of PSII (_II) calculated from chlorophyll fluorescence parameters. It is concluded that the proposed method may provide new information on the activity of the PSI acceptor side and thus help to separate the effects of acceptorside limitation from those of cyclic PSI, whenever a non-linear relationship between _II and the P700-reduction level is observed.Abbreviations and Symbols A absorbance change - _I quantum yield of photosystem I - _II quantum yield of photosystem II - PAR photosynthetically active radiation This work was supported by the Deutsche Forschungsgemeinschaft (SFB 176 Molekulare Grundlagen der Signalübertragung und des Stofftransportes in Membranen and SFB 251 Ökologie, Physiologie und Biochemie pflanzlicher Leistung unter Streß).     

Identification and DNA sequence analysis of a γ-type gliadin cDNA plasmid from winter wheat

Summary Recombinant cDNA plasmids possessing the coding sequences for the -type gliadins were isolated from a cDNA library prepared from wheat seed poly (A⁺) RNA. One of these plasmids, pGliB48, specifically hybridizes to poly (A⁺) RNA molecules 1 400–1 500 bases in length that direct the synthesis of polypeptides at 38 Kd and 46 Kd, the latter size characteristic of the -type gliadins. The cDNA sequence of pGliB48 was determined and encompasses the 3 untranslated region as well as 245 amino acids from the C-terminus of the -type gliadin polypeptide. The 5-end of the DNA coding sequence consists of a tandem repeat unit composed of eight amino acids. Localized regions of homology are observed for the /-type and -type gliadin cDNA sequences.     

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

The reaction of methanol dehydrogenase with cytochrome c _L from Methylophaga marina and the reactions of the non-physiological substrates, Wurster's blue and ascorbic acid, with both proteins were studied as a function of temperature (4–32 °C), pressure (1–2000 bar) and ionic strength using the optical high pressure stopped-flow method. The thermodynamic parameters H, S and V were determined for all reactions where electron transfers are involved. These data allowed the determination of the Maxwell relationships which proved the internal thermodynamic consistency of the system under study. A conformational change on the cytochrome c _L level was deduced from both breaks in the Arrhenius plots and the variation of the V with temperature.Abbreviations MOPS 4-morpholinepropanesulfonic acid - CHES 2-(cyclohexylamino)ethanesulfonic acid - MDH methanol dehydrogenase - EDTA ethylenedinitrilotetraacetic acid disodium salt - BTB bromothymol blue (3,3-dibromothymolsulfoneph-thalein) - PQQ 2,7,9-tricarboxy-lH-pyrrolo-[2,3f]quinoline-4,5-dione - cytochrome c _HH mammalian horse heart cytochrome c     

Isolation of a rat immune response gene identical to an alleged mouse A class II β-chain pseudogene

A human HLA-DQ -chain cDNA was used as a probe to identify and isolate a rat major histocompatibility antigen -chain gene from a genomic library constructed in the vector Charon 28 using Wistar rat DNA (RT1 ^u). The isolated exon of the rat gene (RT1.B ₂) encoding a -chain second domain was found to share 93% nucleotide homology with a mouse A ₂ exon. Although the genomic organization of this gene is consistent with the hypothesis that it represents a pseudogene, the remarkable preservation of a specific sequence favors the view that this class II antigen -chain gene has retained its coding function.     

Structure and evolution of the HLA Class II SXβ gene

The class II major histocompatibility complex antigens are cell-surface heterodimers consisting of an a and a chain. Cosmid cloning has shown that the three families of clas II antigens, DR, DQ, and DP, are encoded within the HLA-D region of chromosome 6 as a series of discrete gene clusters. The DP cluster contains two pairs of a and genes, one of which encodes the biochemically-defined DP antigen. In order to assess whether the other two genes, SXa and SX, are also expressed, potential coding regions have been subcloned and sequenced. The SX3 gene is shown to contain region closely homologous to all six exons of DP. A 1 bp deletion in the 2 exon, also observed for the SX4 allele, causes a translation frameshift, suggesting that SX is a pseudogene. However, all the other exons, as well as their splice sites and the putative promoter region, appear to be intact.     

Histochemical and immunochemical studies on mammalian striated muscle fibres

Summary In 15 rabbit and 2 human distinct types of striated muscle, it was shown that all fibres classified as type I (red) by NADH-diaphorase and myofibrillar ATPase reaction exhibit intense fluorescence when treated with a FITC-labelled antibody against human pectoral actomyosin. Type II (white) fibres showing minimal fluorescence, an intermediate fibre type can be differentiated by all three methods. In the rabbit, all three fibre types are found, whereas, in the human muscles studied, only type I and intermediate type fibres were found.Abbreviations ATPase adenosintriphosphatase (EC 3.6.1.3) - anti-PAM anti-human-pectoralis actomyosin - FITC fluoresceine-isothiocyanate     

Nuclear behavior in multinucleate Schizophyllum commune germlings

Summary The ultrastructure of a fir morphological mutant containing multinucleate cells is described in Schizophyllum commune. The germlings of basidiospores which arose from mating fir with wild-type mycelium were studied in culture by phase contrast microscopy to elucidate behavior of multinucleate cells. Nuclear division appeared synchronous from two nuclei yielding four progeny through six nuclei producing twelve products, beyond which loss of synchrony was indicated. Compensatory nuclear migration into an anucleate cell was presumed during synchronous division of nuclear aggregates in the adjacent cell of an individual germling. The migrant nucleus eventually returned to the cell of origin. However, the return route was not via the central pore of the septum but rather occurred at the juncture of the cross-wall with the germling-periphery. Ultrastructure of a partial septum in fir which could accommodate nuclear passage of this sort is described.     

Fluoreszenzmikroskopische Darstellung Aldehydfuchsin-positiver Substanzen mit Crotonaldehyd-Diaminobenzophenon

Zusammenfassung Es wird ein Verfahren zur elektiven Fluorochromierung Aldehydfuchsin-positiver Gewebsstrukturen beschrieben. Bei dem verwendeten Fluorochrom handelt es sich um eine Verbindung, die aus Crotonaldehyd und 4,4-Diamino-benzophenon entsteht.Die nach dieser Methode gefärbten Schnitte können ohne Beeinträchtigung ihrer Fluoreszenz in Eukitt eingeschlossen und aufbewahrt werden. Das fluoreszenzoptische Verhalten der mit Crotonaldehyd-Diaminobenzophenon fluorochromierten Strukturen wird mikrospektrofluorometrisch untersucht.

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Fluorochromation of aldehyde fuchsin-positive substances with crotonaldehyde-diaminobenzophenone

Summary A staining procedure is described for the specific fluorochromation of tissue structures which are aldehyde fuchsin-positive. The fluorochrome is formed by a reaction of crotonaldehyde with 4,4-diamino-benzophenone.The tissue sections stained by this technique can be mounted and preserved in Eukitt without alteration of their fluorescence. Structures fluorochromized with crotonaldehydediaminobenzophenone have been investigated by means of microspectrofluorometry.

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Mit dankenswerter Unterstützung durch das Landesamt für Forschung NRW.     

Effect of deglycosylation on the properties of β-fructofuranosidaseP-1 fromAureobasidium sp. ATCC 20524

Summary Most of the carbohydrate moiety of -fructofuranosidaseP-1 fromAureobasidium sp. ATCC 20524 was removed by endo--N-acetylglucosaminidase F. A subunit of 94000 Da was observed in SDS-PAGE after deglycosylation. TheK _m value for sucrose was not changed by deglycosylation but the stability at pH 4–5 and 50°C was decreased. The deglycosylated enzyme was more sensitive to proteases such as pronase E and subtilisin than the native enzyme. It is considered that the carbohydrate moiety of -fructofuranosidaseP-1 contributes to the stability of the enzyme but is not essential in its catalytic function.     

Effects of G-protein probes on interactions between auxin efflux carriers and phytotropin receptors in zucchini (Cucurbita pepo L.) microsomal membranes

Microsomal vesicles prepared from etiolated hypocotyl tissue of zucchini (Cucurbita pepo L. cv. All Green Bush) exhibited saturable N-1-naphthylphthalamic acid ([³H]NPA) binding, NPA-stimulated association of indol-3yl-acetic acid ([³H]IAA), and saturable binding of guanosine 5-O-[3-thiotriphosphate] (GTP--[³⁵S]). These vesicles were used to test the possibility that NPA receptors might interact with IAA-anion efflux carriers by coupling through a GTP-binding protein (G-protein). Unlabelled GTP--S or guanosine 5-O-[2-thiodiphosphate] (GDP--S) had no effect on saturable NPA binding or on the NPA-stimulated association of IAA with microsomes. NPA did not affect saturable binding of GTP--[³⁵S] to microsomes, either in the presence or absence of saturating concentrations of unlabelled GTP--S or GDP. It is concluded that the occupancy of phytotropin receptors is not transduced to auxin efflux carriers by a GTP-binding protein.     

The influence of externally applied organic substances on phloem translocation in detached maize leaves

Summary Solutions of organic substances show differing influences on the direction of phloem transport of ¹⁴C-labeled assimilates in predarkened maize leaf strips, when externally applied to one end of the strip. One group of substances pushes the assimilates away from the site of application. Examples of this group are 75 mM solutions of sucrose, trehalose, maltose, D-glucose, D-fructose, glucose-6-phosphate, raffinose and galactose. There is strong evidence that pushing substances are taken up from the apoplast and loaded into the phloem. Another group of substances attracts the assimilates, it seems to pull the assimilates in direction to the site of application. Examples of this second group are 75 mM solutions of arabinose, melibiose, myo-inositol, D-mannitol, polyethylene glycol 2000, and Na₂-EDTA (ethylene-diaminetetraacetate). The pulling substances obviously are not taken up into living cells. It is assumed that they accumulate in the apoplast and build up a water stress (water potential), which is counteracted by an increase of solute concentration in the parenchyma, thus creating a sink for assimilates. A third group of substances shows inert behaviour, having no perceptible influence on phloem transport, at least not, when applied as 75 mM solutions. At concentrations of more than 300 mM, inert substances tend to attract assimilates like those of the second group. Inert substances are xylose, sorbose, 2-deoxy-D-glucose, mannose and sorbitol.abbreviation EDTA ethylenediaminetetraacetate Supported by Deutsche Forschungsgemeinschaft     

Zur Brutbiologie des Seggenrohrs?ngers(Acrocephalus paludicola)

Zusammenfassung In der vorliegenden Arbeit werden Beobachtungen ausgewertet, die in den letzten Jahren in einer Seggenrohrsängerpopulation der Mark Brandenburg gesammelt wurden. Brutbiotop ist eine Großseggenwiese. und haben getrennte Reviere, wobei sich -Reviere, -Reviere und auch - und -Reviere zum Teil sehr erheblich überlagern. Keinem konnte ein bestimmtes zugeordnet werden, sie leben also in Keinehe. versuchen, aus der Nestnähe zu vertreiben. Aggressive Handlungen zwischen benachbarten konnten nicht festgestellt werden. Ihre Singflüge unterscheiden sich von denen der Schilfrohrsänger. 28 Eier maßen im Durchschnitt 17,76×13,14 mm. Nur die brüten und Füttern. Die durchschnittliche Fütterungsfrequenz liegt bei 19,7 Fütterungen pro Stunde, die tägliche Aktivitätszeit der ist im Juli etwa 1 Stunde kürzer als im Juni. 13–14 Tage nach dem Schlüpfen verlassen die Jungen das Nest. In der Nestlingsnahrung ist bei Julibruten der hohe Heuschreckenanteil auffällig. Nachdem die Jungen selbständig sind, streunen sie im Brutbiotop umher. Zweitbruten sind sehr wahrscheinlich, konnten aber noch nicht nachgewiesen werden.

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Summary Observations made during the last years on a population of the Aquatic Warbler (Acrocephalus paludicola) in Brandenburg, Germany, gave the following results:In a sedge meadow, withCarex elata the dominant species, males and females ofAcrocephalus paludicola held separate territories, those of males as well of females sometimes considerably overlapping territories held by other individuals of the same or the opposite sex. In no case a certain male could be considered as having formed a firm pair bond with a certain female: on the contrary, the relations between the sexes were very loose, and probably the birds are even promiscuous. Females try to chase away males from the neighbourhood of their nests. Aggressiveness between neighbouring males has not been observed. The display flights of the males are different from those of the Sedge Warbler(A. schoenobaenus). 28 eggs measure on the average 17,76×13,14 mm. Females alone incubate and feed the young, at an average rate of 19,7 times per hour. The young leave the nest 13–14 days after hatching. Young reared in July are fed a great amount of grasshoppers. Second broods probably occur, but could not be proved.

     

Studies on the molecular basis of H+ translocation by cytochromec oxidase

We report here studies which characterize further the interaction ofN,N-dicyclohexylcarbodiimide with cytochromec oxidase leading to inhibition of H⁺ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - NCCD N-(2, 2, 6, 6-tetramethylpiperidyl-1-oxyl)-N-(cyclohexyl)carbodiimide - Hepes 2-(N-2-hydroxyethylpiperazin-N-yl) ethane sulfonate - TMPD N,N,N,N-tetramethylphenylenediamine     

Hydrolysis of oligosaccharides of the β-(1→4)-linked d-xylose series by an endo(1→4)-β-d-xylanase from the anaerobic rumen fungus Neocallimastix frontalis

An endo-(14)--d-xylanase from Neocallimastix frontalis was purified by anion-exchange chromatography. The enzyme had an apparent molecular mass of 30 kDa on SDS-PAGE and exhibited maximum activity at 50°C and at pH values between 6.0 and 6.6. Kinetic studies on the hydrolysis of xylo-oligosaccharides, ranging from xylobiose to xylodecaose, showed that xylohexaose and xyloheptaose were the preferred substrates for the enzyme and that xylobiose, xylotriose and xylotetraose were not hydrolysed. Xylose was not a product of the hydrolysis of any of the xylo-oligosaccharide substrates tested. The enzyme appeared to have a strong preference for the hydrolysis of the internal glycosidic bonds of the oligosaccharides, which is typical of endo-(14)--d-xylanase activity, but it differed from other fungal endo-(14)--d-xylanases in that it had uniform action on the various internal linkages in the xylo-oligosaccharides.V. Garcia-Campayo, S.I. McCrae and T.M. Wood are with The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB, UK     

Comparative immunological and electrophoretic studies on ribosomal proteins of Bacillaceae

Summary The ribosomal proteins from several Bacillus species were compared by two-dimensional gel electrophoresis and immunological methods. The results revealed great heterogeneity among most Bacillus species. Comparison of ribosomal proteins from Bacilli with those of E. coli by two-dimensional gel electrophoresis showed little similarities, while structural homologies could be found by immunological methods. SDS two-dimensional gel electrophoresis revealed that the molecular weight of ribosomal proteins is conserved in all tested bacteria.Paper No. 68 on Ribosomal Proteins. Preceding paper is by Geisser et al., Molec. gen. Genet. 127, 111–128 (1973).