Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA₃ inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA₃, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [9R-5P]DHZ 9--d-ribofuranosyl-dihydrozeatin-monophosphate - [9R-5P]iP 6-isopentenyl-9--d-ribofuranosyladenine-monophosphate - [9R-5P]Z 9--d-ribofuranosyl-zeatin-monophosphate - [9G]BA 6-benzyl-9--d-glucopyranosyladenine - [9G]DHZ 9--d-glucopyranosyl-dihydrozeatin - [9G]iP 6-isopentenyl-9--d-glucopyranosyladenine - [9G]Z 9--d-glucopyranosyl-zeatin - [9R]BA 6-benzyl-9--d-ribofuranosyladenine - [9R]DHZ 9--d-ribofuranosyl-dihydrozeatin - [9R]iP 6-isopentenyl-9--d-ribofuranosyladenine - [9R]Z 9--d-ribofuranosyl-zeatin - BA 6-benzyladenine - DHZ dihydrozeatin - ES⁺ LC-MS/MS HPLC coupled Electrospray Tandem Mass Spectrometry - f.m. fresh mass - mT 6-(3-hydroxybenzyl)adenine - IMA imazalil - iP isopentenyladenine - NAA 1-naphthalene acetic acid - NFT Nutrient Film Technique - (OG)[9R]DHZ O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin - (OG)[9R]Z O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin - (OG)DHZ O--d-glucopyranosyl-dihydrozeatin - (OG)Z O--d-glucopyranosyl-zeatin - PAR Photosynthetic Active Radiation - PBZ paclobutrazol - PRO prochloraz - TDZ thidiazuron - TRI triflurnizole - Z zeatin     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

Syntheses ofγ-fluoro-α-amino acids

Summary Methods for the synthesis of racemic and optically active title compounds are presented. Key step of these four-step procedures is the alkylation with 1-bromo-2-fluoroalkanes of glycine-ester-derived imines in anhydrous medium using lithium diisopropylamide as a base at low temperature or phase transfer catalyzed alkylation with 50% NaOH and triethylbenzylammoniumchloride as the phase transfer catalyst, respectively. Subsequent three-step deprotection gave the free acids in 13–33% overall yield. Deracemization of-fluoro--aminobutyric acid methyl and ethyl esters with-chymotrypsin was shown to give the (–)-enantiomers of the esters and (+)--fluoro--aminobutyric acid in >98% ee, while from thetert-butylester the opposite stereochemical result was observed giving the (–)-acid with 88% ee. Optically active-fluoro--amino acids were synthesized alternatively by phase transfer catalysis with N-benzyl-cinchonium chloride or using an auxiliary-directed asymmetric alkylation of the imine derived from (R)-(+)-camphor or (R)-(+)-2-hydroxypinan-3-one. These processes gave different enantiomers of-fluoro--aminobutyric acid via a monomeric lithium enolate in the first or a dimeric lithium enolate in the second case, respectively. The enantiomeric excess can be improved by lithium/magnesium exchange.     

Glutathione synthesis in maize genotypes with different sensitivities to chilling

The effect of chilling on enzymes, substrates and products of sulfate reduction, gultathione synthesis and metabolism was studied in shoots and roots of maize (Zea mays L.) genotypes with different chilling sensitivity. At full expansion of the second leaf, chilling at 12 °C inhibited dry weight increase in shoots and roots compared to controls at 25 °C and induced an increase in adenosine 5-phosphosulfate sulfotransferase and -glutamylcysteine synthetase (EC 6.3.2.2) activity in the second leaf of all genotypes tested. Glutathione synthetase (EC 6.3.2.3) activity was about one order of magnitude higher than -glutamylcysteine synthetase activity, but remained unchanged during chilling except for one genotype. During chilling, cysteine and glutathione content of second leaves increased to significantly higher levels in the two most chilling-tolerant genotypes. Comparing the most tolerant and most sensitive genotype showed that chilling induced a greater incorporation of³⁵S from [³⁵S]sulfate into cysteine and glutathione in the chilling-tolerant than in the sensitive genotype. Chilling decreased the amount of³⁵S-label incorporated into proteins in shoots of both genotypes, but had no effect on this incorporation in the roots. Glutathione reductase (EC 1.6.4.2) and nitrate reductase (EC 1.6.6.1) activity were constitutively higher in the chilling-tolerant genotypes, but showed no changes in most examined genotypes during 3 d at 12 °C. Our results indicate that in maize glutathione is involved in protection against chilling damage.Abbreviations APSSTase adenosine 5-phosphosulfate sulfotransferase - EC -glutamylcysteine - GR glutathione reductase - OSH glutathione - NR nitrate reductase We thank M. Suter for preparing [³⁵S]adenosine 5-phosphosulfate, Dr. A. Fleming (both our Institute) for correcting the English and M. Soldati (Eschlikon, Switzerland) for his help with the plant material. This work was supported by COST 814 Crop development for the wet and cool regions of Europe.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Synthesis of symmetric disulfides as potential alternative substrates for trypanothione reductase and glutathione reductase: Part 1

Summary The synthesis of a series of symmetrical disulfides as potential substrates of trypanothione reductase and glutathione reductase was described. The key intermediate in the synthetic approach was the choice of S-^tbutylmercapto-L-cysteine (1). The spermidine ring in the native substrate, trypanothione disulfide (TSST), was replaced with 3-dimethyl-aminopropylamine (DMAPA), while the-Glu moiety was replaced by phenylalanyl or tryptophanyl residues. The same modifications in the-Glu moiety of glutathione disulfide (GSSG) were applied.     

β-D-Glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1→3),(1→4)β-D-glucan synthase

Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (14)-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four `U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called `hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species,Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a `class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (14)-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (14)-glycan synthases in plants. For example, the mixed-linkage (13)(14)-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of other -linked cross-linking glycans. In this respect, the enzymatic properties of the mixed-linkage -glucan synthases not only provide special insight into the mechanisms of (14)-glycan synthesis but may also uncover the genes that encode the synthases themselves.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

High irradiance response promotion of a subsequent light induction response in Sinapis alba L.

Relative quantum responsivity curves for inhibition of hypocotyl elongation in Sinapis alba L. seedlings previously grown in white light confirm that a marked end of day inhibition response can be induced by a monochromatic light treatment (30 min) at the end of the light period. In dark grown seedlings, however, no growth inhibition can be induced by a 30 min monochromatic light treatment. A prerequisite for an induction response appears to be a pretreatment with continuous light. Far red light is most effective with blue and red light showing a lesser effectiveness. The light pretreatment also shows a marked fluence rate dependency with respect to its ability to allow an induction response to manifest itself. The pretreatment required shows all the characteristics of a classical HIR response. The appearance of the effect in plants treated with the herbicide SAN 9789 seems to exclude chlorophyll as being the photoreceptor.Abbreviations SAN 9789 4-chloro-5-(methylamino)-2-(, , -trifluoro-m-tolyl)-3(2H)-pyridazinone - RG9 light long wavelength far red light (Schott RG9 colour glass) - FR far red light - WL white light - BL blue light - RL red light - D darkness - P_tot total phytochrome - P_fr far red absorbing form of phytochrome - HSR high irradiance response     

A neural net capable of competitive and cooperative computation

An algorithm of learning in multilayer threshold nets without feedbacks is proposed. The net is. built of threshold elements with binary inputs. During a learning process each input vector x is accompanied by a teacher's decision ({1,...,M}). The pairs (x[n], [n]) appear in successive steps independently according to some unknown stationary distribution p(x,). The problem of learning of a threshold net has been decomposed to a series of problems of learning of the threshold elements. The proposed learning algorithm of the threshold elements has a perceptron-like form. It was proven that a decision rule of the threshold net stabilizes after a finite number of steps. For definite classes {p(x, )} _* ^K of distributions p(x,), an optimal decision rule stabilizes after a finite number of steps. These classes {p(x, )} _* ^K also contain distributions describing learning processes with perturbations.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

A mathematical theory of the functional dynamics of cortical and thalamic nervous tissue

It is proposed that distinct anatomical regions of cerebral cortex and of thalamic nuclei are functionally two-dimensional. On this view, the third (radial) dimension of cortical and thalamic structures is associated with a redundancy of circuits and functions so that reliable signal processing obtains in the presence of noisy or ambiguous stimuli.A mathematical model of simple cortical and thalamic nervous tissue is consequently developed, comprising two types of neurons (excitatory and inhibitory), homogeneously distributed in planar sheets, and interacting by way of recurrent lateral connexions. Following a discussion of certain anatomical and physiological restrictions on such interactions, numerical solutions of the relevant non-linear integro-differential equations are obtained. The results fall conveniently into three categories, each of which is postulated to correspond to a distinct type of tissue: sensory neo-cortex, archior prefrontal cortex, and thalamus.The different categories of solution are referred to as dynamical modes. The mode appropriate to thalamus involves a variety of non-linear oscillatory phenomena. That appropriate to archior prefrontal cortex is defined by the existence of spatially inhomogeneous stable steady states which retain contour information about prior stimuli. Finally, the mode appropriate to sensory neo-cortex involves active transient responses. It is shown that this particular mode reproduces some of the phenomenology of visual psychophysics, including spatial modulation transfer function determinations, certain metacontrast effects, and the spatial hysteresis phenomenon found in stereopsis.List of Symbols (t) Post-synaptic membrane potential (psp) - Maximum amplitude of psp - t Time - The neuronal membrane time constant - Threshold value of membrane potential - r Absolute refractory period - Synaptic operating delay - v Velocity of propagation of action potentil - x Cartesian coordinate - _jj (x) The probability that cells of class j are connected with cells of class j a distance x away - b _jj The mean synaptic weight of synapses of the jj-th class at x - _jj The space constant for connectivity - _e Surface density of excitatory neurons in a one-dimensional homogeneous and isotropic tissue - _i Surface density of inhibitory neurons in a one-dimensional homogeneous and isotropic tissue - E(x, t) Excitatory Activity, proportion of excitatory cells becoming active per unit time at the instant t, at the point x - I(x, t) Inhibitory Activity, proportion of inhibitory cells becoming active per unit time at the instant t, at the point x - x A small segment of tissue - t A small interval of time - P(x, t) Afferent excitation or inhibition to excitatory neurons - Q(x, t) Afferent excitation or inhibition to inhibitory neurons - N _e (x, t) Mean integrated excitation generated within excitatory neurons at x - N _i (x, t) Mean integrated excitation generated within inhibitory neurons at x - _e [N _e ] Expected proportion of excitatory neurons receiving at least threshold excitation per unit time, as a function of N _e - _i [N _i ] Expected proportion of inhibitory neurons receiving at least threshold excitation per unit time, as a function of N _i - G( _e ) Distribution function of excitatory neuronal thresholds - G( ₁ ) Distribution function of inhibitory neuronal thresholds - ₁ A fixed value of neuronal threshold - h(N _e ; ₁) Proportion per unit time of excitatory neurons at x reaching ₁ with a mean excitation N _e - 1[ ] Heaviside's step-function - R _e (x, t) Number of excitatory neurons which are sensitive at the instant t - R _i (x, t) Number of inhibitory neurons which are sensitive at the instant t - R _e Refractory period of excitatory neurons - r _i Refractory period of inhibitory neurons - E(x, t) Time coarse-grained excitatory activity - I(x, t) Time coarse-grained inhibitory activity - Spatial convolution - Threshold of a neuronal aggregate - v Sensitivity coefficient of response of a neuronal aggregate - E(t) Time coarse-grained spatially localised excitatory activity - I(t)> Time coarse-grained spatially localised inhibitory activity - L ₁,L ₂,L,Q See § 2.2.1, § 2.2.7, § 3.1 - Velocity with which retinal images are moved apart - Stimulus width - E _o, I _o Spatially homogeneous steady states of neuronal activity - k _e ,k _ij S _e S _ij See § 5.1     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Intrinsic fluorescence studies on saccharide binding toArtocarpus integrifolia lectin

The combining region ofArtocarpus integrifolia lectin has been studied by using the ligand-induced changes in the fluorescence of the lectin. The saccharide binding properties of the lectin show that C-l, C-2, C-4, and C-6 hydroxyl groups of D-galactose are important loci for sugar binding. The -anorner of galactose binds more strongly than its -counterpart. Inversion in the configuration at C-4 as in glucose results in a loss of binding to the lectin. The C-6 hydroxyl group is also presumably involved in binding as D-fucose does not bind to the lectin.The lectin binds to the Thomsen-Friedenreich antigen (Gal(13)GalNAc) more strongly than the other disaccharides studied, viz. Gal/ (14) Gal and Gal (13) GlcNAc, which are topographically similar to T-antigen. This observation suggests that the combining region ofArtocarpus lectin is complementary to that of T-antigen.Solvent accessibility of the protein fluorophores have been probed by the quenching of protein fluorescence by Iodide ion in the absence and presence of sugar. In the presence of sugar a slight inaccessibility of the fluorophores to the solvent has been observed.Abbreviations MeGal 1-O-methyl--glucopyranoside - MeGal 1-O-methyl--glucopyranoside - GalNAc 2-acetamido-2-deoxy-galactose - Gal Galactose     

Primary structure of the marmoset (Saguinus fuscicollis) hemoglobin. I. Use of tryptic maleylated peptides in the solubilization and sequence elucidation of the α- and β-chains

The primary structure of adult marmoset hemoglobin has been determined. The - and -chains of HbA were separated on a CM23 column in 8 M urea using a sodium phosphate gradient. Tryptic digests of the - and -chains were fractionated on a Dowex 50W-X2 column using a pH and pyridine acetate gradient. Large peptide fragments were obtained by the cyanogen bromide cleavage of the - and -chains, as well as by tryptic digestion of the maleylated - and -chains. The sequence was derived from the amino acid compositions and sequences of the individual tryptic peptide, automated sequence determination of intact - and -chains, as well as automated sequence determination of cyanogen bromide fragments and tryptic maleylated peptides derived from the - and -chains. The complete structure of marmoset adult hemoglobin is closely homologous to that of other primate hemoglobins. The sequence of the marmoset -chain differs from the -chain of human HbA at positions 8, 19, 23, 68, and 116. The -chain from marmoset HbA differs from the -chain of human HbA at positions 5, 13, 21, 50, 87, and 125.This work was supported in part by funds from a Physicians' Medical Education and Research Foundation Grant of the University of Tennessee Memorial Research Hospital and by NIH General Research Support Grant FR-5541 to the institution.     

Convergent synthesis of neoglycopeptides by coupling of 2-bromoethyl glycosides to cysteine and homocysteine residues in T cell stimulating peptides

The 2-bromoethyl -glycosides of the disaccharide galabiose [Gal(1-4)Gal] and the trisaccharides globotriose [Gal(1-4)Gal(1-4)Glc] and 3-sialyllactose [Neu5Ac(2-3)Gal(1-4)Glc] have been prepared by improved routes. The 2-bromoethyl glycosides were then used in cesium carbonate promoted alkylations of the sulfhydryl groups of cysteine and homocysteine residues in T cell stimulating peptides. This convergent and general approach was used to prepare 16 neoglycopeptides which were obtained in 52–95% yields after purification by HPLC. 1H NMR spectroscopy revealed that -elimination and epimerization of neoglycopeptide stereocentres did not occur during the synthesis.     

Location by fluorescence microscopy of glycosidases and a xylanase in the anaerobic gut fungi Caecomyces communis,Neocallimastix frontalis,and Piromyces rhizinflata

-d-Glucosidase, -d-fucosidase -d-xylosidase, and -cellobiopyranosidase activities in Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata, located with fluorescent conjugates, occur throughout the whole thallus as from zoospore germination and disappear before sporulation. -d-Galactosidase and -l-arabinopyranosidase activities are low or nonexistent. A xylanase, detected by indirect immunofluorescence, was observed at the surface of the vegetative cells, vesicles, or rhizoids. Cross-reactions prove the existence of analogies in structure among the enzymes of these anaerobic gut fungi.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following -keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): -ketoisovalerate, -keto-n-valerate, -ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. -Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of -ketoisovalerate or other -keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n _H -values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 M) inactivated the enzyme irreversibly. The 3-phosphate of coenzyme A and the free carboxyl group of -ketoisovalerate were involved in optimal binding of these substrates, but 3-dephospho-acetyl-coenzyme A and the methylester of -ketoisovalerate were also converted by this enzyme. A CH₃–CH₂-grouping of the -keto acids seemed to be necessary for binding this substrate.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

Die Bedeutung der “feedback”-Hemmung der Threonin-Desaminase für die Synthese von Valin und Leucin

Zusammenfassung In Arthrobacter Stamm 23 führte die durch Mutation verursachte allosterische Unempfindlichkeit der Threonin-Desaminase zur dereprimierten Bildung der Enzyme im Isoleucin-Valin-Leucin-Biosyntheseweg. Derepression erfolgte auch, wenn Wildtypzellen in Gegenwart von -Ketobuttersäure inkubiert wurden. In beiden Fällen wurde Isoleucin überproduziert und ins Kulturmedium ausgeschieden. Wie aus Wachstumsexperimenten hervorging, verursachte der Überschuß an -Ketobuttersäure im Medium primär einen Valin- und Leucin-Mangel, der zu einer vorübergehenden Wachstumshemmung führte. Durch die dereprimierte Bildung der Enzyme im Isoleucin-Valin-Biosyntheseweg konnte die Wachstumshemmung überwunden werden.Der vorübergehende Hemmeffekt der -Ketobuttersäure ließ sich auf eine Konkurrenz der Substrate am ersten gemeinsamen Enzym im Isoleucin-Valin-Biosyntheseweg, der Acetohydroxysäure-Synthase, zurückführen. Wegen des niedrigen K _m-Wertes für -Ketobuttersäure wird dieses Substrat vom Enzym bevorzugt umgesetzt. Durch gaschromatographische Bestimmungen der Acetoin- und Acetyläthylcarbinol-Bildung in Enzymtests mit variierten Substrat-Konzentrationen konnte nachgewiesen werden, daß relativ geringe Konzentrationen an -Ketobuttersäure genügen, um die -Acetolacetat-Bildung vollständig zu unterdrücken. Diese Ergebnisse erklären die durch -Ketobuttersäure verursachte vorübergehende Wachstumshemmung bei Bakterien.

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The effect of the feedback inhibition of threonine deaminase on valine-leucine biosynthesis

In Arthrobacter strain 23 the allosteric insensitivity of threonin deaminase caused by mutation resulted in derepressed formation of the enzymes of the isoleucine-valine-leucine pathway. Derepression was also observed, when wild type cells were incubated in the presence of -oxobutyrate. In both cases isoleucine was overproduced and excreted. As growth experiments indicated the excess of -oxobutyrate in the medium caused endogenous valine and leucine deficiency and a transient inhibition of growth. Derepressed formation of the isoleucinevaline biosynthetic enzymes resulted in relief of growth inhibition.The transient inhibitory effect of -oxobutyrate has been traced back to substrate competition at the first enzyme common to the isoleucine and valine pathway, acetohydroxy acid synthase. Due to the low K _m of the enzyme for -oxobutyrate this substrate is preferentially converted. As proven by gaschromatographical measurements of acetoin and acetylethyl carbinol produced in enzyme (acetohydroxy acid synthase) assays with varied substrate concentrations, relatively low concentrations of -oxobutyrate are able to suppress the formation of -acetolactate completely. These results explain the transient inhibitory effect of -oxobutyrate on the growth of bacteria.

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Abkürzungen -KBS -Ketobuttersäure - FAD Flavin-adenin-dinucleotid - AHS Acetohydroxysäure - IPM Isopropylmalat - TPP Thiaminpyrophosphat