Oxidative d-xylose metabolism of Gluconobacter oxydans

Summary Gluconobacter oxydans subsp. suboxydans ATCC 621 oxidizes d-xylose to xylonic acid very efficiently, although it cannot grow on xylose as sole carbon source. The oxidation of xylose was found to be catalyzed by a membrane-bound xylose dehydrogenase. The xylono--lactone formed in the oxidation reaction is subsequently hydrolyzed to xylonic acid by a -lactonase. The complete oxidation pathway of d-xylose in G. oxydans is evidently located in the periplasmic space.     

Design of superactive and selective integrin receptor antagonists containing the RGD sequence

Summary Integrins play a major role in cell-cell and cell-matrix interactions. The majority of the different types of integrins recognize the tripeptide sequence arginine-glycine-aspartic acid (RGD). To explore the spatial requirements of the pharmacophore for receptor selectivity and high activity, a new procedure, spatial screening, was used. The procedure is based on the experience that the conformation of small cyclic peptides is mainly determined by the chirality of the amino acids (and glycine or proline). For example, cyclic pentapeptides with one d and four l amino acids prefer a II'/ conformation. The sequence RGDFV was shifted around this spatial II'/ template by synthesis of five peptides in which one of the amino acids was used in d-configuration. It turned out that cyclo(-RGDfV-) is a selective inhibitor for the _v3 integrin, which is strongly expressed in cancer cells. Systematic variations with different turn mimetics, retro-inverso structures, modified peptide bonds and sugar amino acids are discussed.     

Displacement of excitatory amino acid receptor ligands by acidic oligopeptides

A number ofD-glutamyl andL-aspartyl dipeptides, glutathione, -D-glutamylglycine and -D-glutamyltaurine, were tested for their efficacy to displace ligands specific for different subtypes of excitatory amino acid receptors from rat brain synaptic membranes. In general, theL enanthiomorphs of -glutamyl peptides were more potent displacers than -D-glutamylglycine and-taurine but the latter were more specific for the quisqualate type of receptors. -L-glutamyl-L-glutamate was the most effective dipeptide in displacing the binding of glutamate, 2-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) and 2-amino-5-phosphonoheptanoate (APH), whereas -L-glutamyl-L-aspartate was the most effective in the binding of kainate. Both oxidized and reduced glutathione were inhibitory, being most potent in the binding of AMPA. -L-Glutamylaminomethylsulphonate was most effective in the binding of APH. The most potent -L-glutamyl peptides (glutathione, -L-glutamyl-L-glutamate,-L-aspartate, and-glycine) may act as endogenous modulators of excitatory aminoacidergic neurotransmission.     

Solubilization and some properties of γ-glutamyltransferase from human brain microvessels

Detergents Triton X-100, sodium deoxycholate, and octyl--D-glucopyranoside, and proteinase papain proved to be excellent agents solubilizing the -glutamyl-transferase (-GT) from human brain cortex microvessels. Ficin also solubilized -GT but to a lesser extent than papain. The relative molecular mass of the detergent-solubilized enzyme form was greater than 200,000 (in the presence of Triton X-100). The relative molecular mass of the proteinase-solubilized form was slightly greater than that of albumine. -GTs of microvessels from five human brain regions and from the choroid plexus were tested for their specificity toward acceptors. The best acceptors were found to be (in decreasing order of activity)l-cystine, glycylglycine,l-glutamine,l-methionine, andl-alanine. The findings suggest that the main features of -GT of the human blood-brain barrier are very similar to those of -GTs from other human tissues.     

Assimilation of γ-butyrobetaine,and d-and l-carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

The metabolic pattern of utilization of [1,2,3,4-¹⁴C, methyl-³H] -butyrobetaine and d-and l-[1-¹⁴C, methyl-³H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on -butyrobetaine, Ps. putida rapidly metabolized -butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or -butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that -butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or -butyrobetaine showed no detectable -butyrobetaine hydroxylase activity.     

Potassium-39 NMR of K+ interaction with the gramicidin channel and NMR-derived conductance ratios for Na+, K+ and Rb+

Summary A potassium-39 NMR study of potassium ion interaction with the gramicidin transmembrane channel in phospholipid bilayers at high ion activity is reported which allows determination of a weak binding constant, K _b ^w 8.3/m, and an off-rate constant for the weak site,k _off ^w 2.6×10⁷/sec. These values are interpreted with the aid of additional NMR data as the binding constant for formation of the doubly occupied channel state and the rate constant for an ion leaving the doubly occupied state. Considering the singly occupied channel state for the potassium ion to be electrically silent at 1 molar ion activity, as with the sodium ion, the single-channel conductance for 100 mV and 30°C calculated to be 29 pS, and using the same approximation with previous NMR results on the sodium and rubidium ions, reasonable conductance ratios were calculated. Further experimental estimates of the other three constants with the experimental location of binding sites and Eyring rate theory to introduce voltage dependence allowed a more complete calculation of the two-site channel. The single-channel conductance for potassium ion is calculated to be 24 pS at 1m activity and 26 pS at 0.6m activity, which compares for diphytanoyl phosphatidylcholine membranes to an experimental most probable single-channel conductance of 25 pS and a mean channel conductance of 20 pS. The calculated conductance ratios using NMR-derived constants were (K)/(Na)=2.0 and (Rb)/(Na)=4.3. These results are close to the experimental values and provide further basis for the use of NMR of quadrupolar ions to provide information on the ionic mechanism of channel transport.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

Synthesis of methyl α- and β-N-dansyl-d-galactosaminides,probes for the combining sites ofN-acetyl-d-galactosamine-specific lectins

The synthesis of the methyl - and -N-dansyl-d-galactosaminides is described using methyl ,-2-azido-2-deoxy-d-galactopyranoside as starting material. This was reduced to the corresponding methyl ,-2-amino-2-deoxy-d-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl ,-N-dansyl-d-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl -N-dansyl-d-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with theN-acetyl-d-galactosamine specific lectin fromErythrina corallodendron.Abbreviations ECorL Erythrina corallodendron lectin - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside Dedicated to Hilde De Boeck (1958–1991).     

Distribution of a dipeptide naphthylamidase in rat tissues and its localisation by using diazo coupling and labeled antibody techniques

Summary Several rat tissues (liver, spleen, kidney, pancreas, skin, heart, lung and brain) were shown to contain a peptidase capable of liberating naphthylamine from glycyl-dl-proline naphthylamide (Gly-Pro-NA). A single DEAE-cellulose chromatography of autodigested homogenates of the above tissues produced a partial separation of the peptidase from the enzymes hydrolysing l-leucine -naphthylamide. The Gly-Pro-NA hydrolysing enzyme was localised in tissue sections by using diazo coupling reaction and indirect immunologic techniques. Antibodies were prepared against the enzyme purified from rat liver and kidney in the rabbit. Rabbit -globulin was localized by using goat anti-rabbit -globulin labeled with fluorescein or with peroxidase.     

Hämatologische beobachtungen an regenbogenforellen (Salmo gairdneriRich.) VII. Färbeindex (FI), absoluter Hämoglobingehalt des Einzelerytrozyten (HbE) and mittlere Hämoglobinkonzentration des Einzelerythrozyten (MCHC)

180 rainbow trouts (Salmo gairdneriRich.), aged from 1 to 3 years, were examined for fluctuations, caused by age and season, by means of colour index (CI), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).CI and MCH behave similarly. Both are increasing until the 2nd year and stay relatively constant thereafter. If the gender is not considered — there are no significant differences in the values of males and females — the CI increases from 1,4 in the first year over 1,6 to 1,7 in the age of 3 years, and the MCH increases from 44,4 over 52,6 , 56,8 , 58,1 to 55,5 .A seasonal periodicity of both indices could not be indicated on not-matured animals (F₂) which were two summers of age. Only, the january values appeared increased — CI: 2, MCH: 68,3 — otherwise the CI varies between 1,8 and 1,7 and the MCH between 53,3 and 59,1 .The MCHC-values of the age groups examined vary between 24,4% and 27,3%. The values of the yearlings form an exception (19,8%). These values certainly are inexact and too low because of the small number of individuals checked (3).

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Mit finanzieller Unterstützung durch die DFG.Institut für Siedlungswasserbau und Wassergütewirtschaft der Universitat Stuttgart Fischtoxikologische Arbeitsgruppe     

Interactions of γ-L-glutamyltaurine with excitatory aminoacidergic neurotransmission

The in vitro effects of -L-glutamyltaurine on different stages of excitatory aminoacidergic neurotransmission were tested with -D-glutamyltaurine as reference. -L-Glutamyltaurine enhanced the K⁺-stimulated release of [³H]glutamate from cerebral cortical slices (25% at 0.1 mM) and slightly inhibited the uptake by crude brain synaptosomal preparations (about 10% at 1 mM). -L-Glutamyltaurine was also a weak displacer of glutamate and its agonists from their binding sites in brain synaptic membrane preparations, being, however, less selective to quisqualate (QA) sites than -D-glutamyltaurine. The basal influx of Ca²⁺ into cultured cerebellar granular cells was not affected by 1 mM -L-glutamyltaurine, but the glutamate- and its agonist-activated influx was significantly inhibited in low-Mg²⁺ (0.1 mM) and Mg²⁺-free media. The glutamate-evoked increase in free intracellular Ca²⁺ and the kainate-activated formation of cGMP in cerebellar slices were both markedly inhibited by 0.1 mM -L-giutamyltaurine. We propose that -L-glutamyltaurine may act as endogenous modulator in excitatory aminoacidergic neurotransmission.     

Evolution of a protein superfamily: Relationships between vertebrate lens crystallins and microorganism dormancy proteins

Summary A search of sequence databases shows that spherulin 3a, an encystment-specific protein ofPhysarum polycephalum, is probably structurally related to the - and -crystallins, vertebrate ocular lens proteins, and to Protein S, a sporulation-specific protein ofMyxococcus xanthus. The - and -crystallins have two similar domains thought to have arisen by two successive gene duplication and fusion events. Molecular modeling confirms that spherulin 3a has all the characteristics required to adopt the tertiary structure of a single -crystallin domain. The structure of spherulin 3a thus illustrates an earlier stage in the evolution of this protein superfamily. The relationship of - and -crystallins to spherulin 3a and Protein S suggests that the lens proteins were derived from an ancestor with a role in stressresponse, perhaps a response to osmotic stress.     

β-d-N-Acetylglucosaminidase,a new cytochemical marker of human lymphocyte subpopulations

Summary -d-N-acetylglucosaminidase staining characteristics of rosetted or non-rosetted normal and malignant lymphoid cells were compared with those observed after nonspecific esterase and acid phosphatase staining. With the three cytochemical techniques a similar staining pattern was observed in T cells (E-rosettes), their subpopulations T and T, B cells and the non-T, non-B cells, as well as in the T cell populations defined with the monoclonal antibodies OKT3,4 and 8. T cells mostly diplayed a dot-like reaction, T and the non-T, non-B cells a fine to heavy granular reaction, while most B cells were negative. OKT4 and OKT8 positive lymphocytes showed for the larger part a dot-like staining pattern, however, the frequency of cells with a granular pattern was distinctly higher in the OKT8, than in the OKT4 positive cells.E(+)mIg(–) and E(–)mIg(–) A.L.L. blasts stained either with a dot-like or granular pattern or failed to react when stained cytochemically for -d-N-acetylglucosaminidase, nonspecific esterase and acid phosphatase activity. Only in a few instances a discrepancy was observed between the types of staining for esterase and acid phosphatase on one hand and those for -d-N-acetylglucosaminidase on the other.     

A sequential response to growth substances in coleoptiles from γ-irradiated wheat

Summary -irradiated wheat seed (500 kr) produces coleoptiles that grow without cell division or DNA synthesis. Apart from an initial 24-hr delay in growth, intact coleoptiles have a pattern of cell elongation similar to normal coleoptiles. The elongation of coleoptiles excised at a size of 2 mm, when the cells are small and just prior to entering a rapid elongation phase, is promoted by kinetin and gibberellic acid (GA₃). Elongation of coleoptiles excised at 8 mm, when the cells are larger and in the rapid elongation phase, is promoted by indoleacetic acid (IAA). This sequential response to growth substances in coleoptiles is remarkably similar to that in normal coleoptiles. The GA₃ response in excised coleoptiles is not inhibited by FUDR, confirming that DNA synthesis is not required for GA₃-induced elongation in coleoptiles.     

Properties of β-glucosidase purified from Aspergillus niger mutants USDB 0827 and USDB 0828

Summary Two extracellular -glucosidases (EC 3.2.1.21) were isolated from Aspergillus niger USDB 0827 and A. niger USDB 0828, and their physical and kinetic properties studied. Both enzymes were very similar in terms of molecular size (230000 Da), pH optimum (pH 4.6), temperature optimum (65° C), stability at high temperatures and substrate preferences. They were capable of hydrolysing -linked disaccharides, phenyl -d-glucoside, p-nitrophenyl -d-glucoside (PNPG), o-nitrophenyl -d-glucoside, salicin and methyl -d-glucoside but lacked activity towards -linked disaccharides, a range of p-nitrophenyl monoglycosides and p-nitrophenyl diglycosides. Both -glucosidases were better at hydrolysing cellobiose than cellotriose, cellotetraose or cellopentaose. For both enzymes, glucose showed competitive inhibition with PNPG as substrate but had no effect with cellobiose. However, the two -glucosidases differed in inhibition by glucono-1,5-lactone and affinity for cellobiose. -Glucosidase from A. niger USDB 0827 also gave lower specific activity, and was more susceptible to metal ions (Ag⁺, Fe²⁺ and Fe³⁺) inhibition than that of A. niger USDB 0828. Correspondence to: Y. K. Hoh     

Synergistic induction of cytotoxicity in macrophages by murine interferon-γ and biological response modifiers derived from microorganisms

Summary The ability of recombinant murine interferon-gamma (rMuIFN-) to activate murine macrophages with or without several biological response modifiers (BRM), including synthetic muramyl dipeptide derivatives (MDPs), was investigated. Mouse peritoneal macrophages were activated by rMuIFN- alone to the cytostatic state, but not the cytolytic state. Other BRM as well as bacterial lipopolysaccharide (LPS), including a lyophilized preparation of an attenuated strain of Streptococcus hemolyticus, a cell wall skeleton of bacillus Calmette-Guerin and synthetic MDPs, were highly active in generating the synergism with rMuIFN-. Macrophages were endowed with the cytolytic activities by combinations of rMuIFN- and MDP-Lys(L18); the combination of 100U/ml of rMuIFN- with 10 ng/ml of MDP-Lys(L18) was sufficient to induce cytolytic activities in macrophages. The synergism was observed when the macrophages primed with rMuIFN- were treated with LPS or MDP-Lys(L18), but not when the sequence of treatment was reversed. The cytotoxicity of macrophages induced by rMuIFN- with MDP-Lys(L18) was suppressed by priming with MDP-Lys(L18). The suppressive effect was also observed by priming with LPS in combinations of rMUIFN- and LPS. The reason for the suppression of macrophage activation by priming with LPS and MDP-Lys(L18) is at present unknown.     

The presence of an endogenous lectin in early embryos ofXenopus laevis

Summary Xenopus laevis embryos were examined for the presence of endogenous carbohydrate binding proteins. Soluble extracts of cleavage, gastrula and neurula embryos are able to agglutinate trypsinized rabbit erythrocytes. Unlike other embryonic lectins this agglutination activity requires the presence of calcium ions but not of sulphydryl reducing agents. It is specifically inhibited by galactose and galactose containing derivatives. Thiodigalactoside is the most potent disaccharide inhibitor followed by lactose and melibiose respectively. Methyl -d-galactopyranoside is a more effective inhibitor than its anomer. N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, methyl -d-mannopyranoside andl-fucose do not inhibit activity at concentrations at or above 25 mM. EDTA and EGTA are also strong inhibitors of this activity. -d-galactoside binding lectins present in the early chick embryo have been implicated in cell to cell and cell to substrate adhesiveness of extraembryonic chick endoderm cells. Since cells of the blastula inXenopus laevis possess surface receptors bearing terminal -d-galactoside groups it is possible that this -d-galactoside binding lectin may play a role in the control of cell surface mediated events during development.     

Malate regulation of Mg2+-ATPase of chloroplast coupling factor 1

The regulatory effects of malate on chloroplast Mg²⁺-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF₁ Mg²⁺-ATPase activity. The subunit of CF₁ may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg²⁺-ATPase was also controlled by the Mg²⁺/ATP ratio in the reaction medium. The enhancing effect of malate on Mg²⁺-ATPase activity depended on the presence of high concentrations of Mg²⁺ in the reaction mixture. Kinetic study showed that malate raised the V_max of catalysis without affecting the K_m for Mg²⁺ ATP. The experiments imply that the stimulation of Mg²⁺-ATPase by malate is probably correlated with the P_i binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF₁ chloroplast coupling factor 1 - CF₁ (-) and CF₁ (-) CF₁ deficient in the and subunit - MF₁ mitochondria coupling factor 1 - NEM N-ethylmaleimide - PMS phenazine methosulfate - OG n-octyl--d-glucopyranoside     

Stereoselective decarboxylation of amino acids in the solid state,with special reference to chiral discrimination in prebiotic evolution

Summary The decarboxylations of sublimated solidd- andl-leucine by nonpolarized -rays give quite different quantum yields, indicating significant selection. The G(CO₂) value for thed-isomer is higher than that for thel-isomer by a factor of 2 within a dose range of 10³–10⁵ rads. The G value for thedl-racemate is close to that of thed-isomer. The effect vanishes if instead of sublimation, crystallization from aqueous solution is the last preparation step. Our results on sublimated leucine agree well with those reported for -induced decarboxylation of solid -phenylalanine prepared similarly by sublimation. The asymmetry increases with longer cooling periods after irradiation. An intrinsic energy difference due to parity nonconservation between enantiomers is discussed as a possible stereoselective mechanism, with special reference to the prebiotic origin of asymmetry in living matter. Other possible sources of the observed effects are also discussed.     

Untersuchungen über die Einwirkung von γ-Strahlen auf die Pflanzenentwicklung und die Blattformen zweier Kartoffelsorten

Zusammenfassung Es wurde über den Einfluß einer 60-Co--Bestrahlung auf die Pflanzenentwicklung und die Blattformen von bestrahlten Bintje- und Sieglinde-Kartoffel berichtet, wobei die Dosen 0, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 und 5000 rad angewandt wurden. Sieglinde weist dabei hinsichtlich Wachstum und Sproßhöhe der Kartoffelstauden eine wesentlich stärkere Empfindlichkeit gegenüber-Bestrahlung auf als Bintje, bei der auch noch bei 5000 rad ein minimales Wachstum der Kartoffelstauden beobachtet werden konnte. Außerdem waren nach Bestrahlung an den Blattfiedern der Kartoffelstauden schon zum Großteil bekannte Erscheinungen wie Strahlensukkulenz und Verwachsung von Endfiederblättern mit einem oder mehreren Nachbarfiederblättern zu erkennen, wohingegen dies bei den mehr gegen den Blattgrund zu befindlichen Fiederblättern nicht auftrat.Die-Bestrahlung wurde am Institut für Biologie und Landwirtschaft im Reaktorzentrum Seibersdorf durchgeführt, wofür wir unseren Dank aussprechen möchten.