Microphotometric determination of enzymes in brain sections

Summary A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM d-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN₃, 10 mM MgCl₂, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 m. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1, 2-2)     

Mechanism of sugar retention by roots of intact maize and field bean plants

The re-uptake of sugars driven by the proton gradient was studied in sugar net-release and net-uptake experiments using roots of intact maize (Zea mays cv. Blizzard) and field bean (Vicia faba L. cv. Alfred) plants. The net release of sugars into the root medium (0.1 mM CaSO₄) was stimulated by: the protonophore CCCP (10 M); the sulfhydryl reagent NEM (300 M); the specific inhibitor of plasmalemma ATPase vanadate (0.5 mM); and the inhibitor of the glucose carrier phlorizin (2 mM). Net uptake of glucose, fructose and arabinose from 10 M external concentrations was also inhibited by these substances. Surprisingly fusicoccin, a stimulator of net proton release did not effect net sugar uptake. Medium pH values only influenced sugar net uptake if the pH was above 7. It is concluded that a degradation of the proton gradient across the plasmalemma stimulates net sugar release because of disturbed re-uptake of sugars (in particular glucose) via a proton/sugar cotransport system. Thus, the retention of sugars by root cells not only depends on the plasmalemma permeability but also on the electro-chemical proton gradient. If an electro-chemical proton gradient is established by plasmalemma ATPase activity the re-uptake of sugars by proton/sugar cotransport minimizes the release of sugars into the rhizosphere.     

Breeding of a Cyclic Imide-Assimilating Bacterium,Pseudomonas putida s52, for High Efficiency Production of Pyruvate

A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putida s52 as catalyst, 400 mM pyruvate was produced from 500 mM fumarate in a 36-h reaction. Bromopyruvate, a malic enzyme inhibitor, was used for the selection of mutants with higher pyruvate productivity. A bromopyruvate-resistant mutant, P. putida 15160, was found to be an effective catalyst for pyruvate production. Moreover, under batch bioreactor conditions, 767 mM of pyruvate was successfully produced from 1,000 mM fumarate in a 72-h reaction with washed cells from P. putida 15160 as catalyst.     

Theanine Formation by Tea Suspension Cells

The theanine (THE: γ-glutamylethylamide) content and the growth rate of cultured cells of tea (Camellia sinensis L.) were increased greatly to 22.3%, in dry wt. with a medium containing 60 mM nitrate and 25 mM ethylamine as a nitrogen source. The optimum concentrations of nitrate, Mg²⁺, and K⁺ for the growth and formation of THE in suspension cells were 40mM, 3mM, and 104mM, respectively. The yield of THE accumulated in the cultured cells with the medium modified for THE formation was increased greatly due to a great increase of the growth rate.     

Characterization of and Ethanol Hyper-production by Clostridium thermocellum I-1-B

An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological proper­ ties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50%. decrease of growth yield are 27 and 16g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156 h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512mM) ethanol, 8.5 g/liter (94mM) lactate, 2.9 g/liter (48mM) acetate, and 0.9 g/liter (20mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.     

Study of human chromosome V

Summary The induction of fragile sites on human chromosomes has been demonstrated under various conditions that cause thymidylate stress, including exposure to uridine. In this study, we examined common fragile site expression by initially exposing peripheral lymphocytes to uridine, followed by repair of the fragile sites with media containing various concentrations of thymidine. Lymphocytes were cultured in medium 199 with 2 mM uridine. At 0.5, 1, 2, 3, 8, 10, 12, and 18 h before harvest, the uridine medium was removed and replaced by medium containing thymidine at various concentrations. Our results demonstrate that the effect of uridine on chromosome fragility can be reversed by low concentrations of thymidine (2 M up to 200 M) and the rescuing effect of thymidine can be achieved if the cells were treated prior to 2–3 h before harvest. No repair was found if thymidine was added to culture within 2 h prior to harvesting, suggesting that packing of chromosomes is also an important factor in the expression and repair of fragile sites.     

Succinate transport inRhizobium meliloti: Characteristics and impact on symbiosis

The transport of succcinate was studied inRhizobium meliloti M5N1. Succinate accumulation was a saturable function of the succinate concentration, and the apparent Km and Vmax values were respectively 2.9 M and 79 nmoles/min·mg protein. Strong inhibition of succinate transport was observed in the presence of 10 mM DNA and 4 mM azide, whereas arsenate and fluorure had no effect. Fumarate competitively inhibited succinate transport; the Ki value was between 3 and 6 M.A succinate transport mutant ofR. meliloti M5N1 was selected after nitrosoguanidine mutagenesis. It failed to grow on succinate, malate, or fumarate, but grew on arabinose, glutamate, pyruvate, and other carbohydrates. The mutant strain formed white (presumably leghemoglobin deficient) and ineffective nodules, since the acetylene reduction assay showed no nitrogenase activity.     

Fluoride transmembrane exchange in human erythrocytes measured with 19F NMR magnetization transfer

¹⁹F NMR spectra of sodium fluoride in suspensions of human erythrocytes were seen to yield separate resonances for the F^- populations inside and outside the cells. Selective saturation of the magnetization of the intracellular population gave rise to transfer of that saturation to the extracellular population. The extent of magnetization transfer was high and it was blocked by the capnophorin (band 3) anion exchange inhibitor 4,4-dini-trostilbene-2,2-disulfonic acid (DNDS). A series of magnetization-inversion transfer experiments was carried out for the range of intracellular fluoride concentrations of 11 mM to 136 mM and analysed using one-dimensional overdetermined exchange analysis. This yielded an estimate of the equilibrium exchange Michaelis constant and maximal velocity of 27 ± 3 mM and 180 ± 5 × 10^-16 mol cell^-1 s^-1, respectively. There was no alteration of exchange flux of fluoride at an intracellular concentration of 49 mM in the presence of 50 mM glucose; thus suggesting no interaction between glucose and anions in capnophorin-mediated exchange of solutes.     

Activation of pyruvate dehydrogenase in perfused rat heart by dichloroacetate (Short Communication)

The activity of pyruvate dehydrogenase was assayed in extracts of rat hearts perfused in vitro with media containing glucose and insulin±acetate±dichloroacetate. Dichloroacetate (100μm, 1mm or 10mm) increased the activity of pyruvate dehydrogenase in perfusions with glucose or glucose+acetate. Evidence is given that dichloroacetate may facilitate the conversion of pyruvate dehydrogenase from an inactive (phosphorylated) form into an active (dephosphorylated) form.     

Is organic acid required for nutrition of thermophilic fungi?

In contrast to a published report [Wali et al. Arch Microbiol 118:49–53 (1978)], an organic acid is not essential for the growth of thermophilic fungi. The thermophilic fungus, Thermomyces lanuginosus, grows satisfactorily in a synthetic medium containing glucose as carbon source if the pH of the medium is controlled. The control of pH is essential for the concentration of carbon dioxide in the growth medium and the activity of anaplerotic enzyme, pyruvate carboxylase.Abbreviations PEP phosphoenolpyruvate - GDP guanosine 5-diphosphate     

A Highly N-Glycosylated Chitin Deacetylase Derived from a Novel Strain of Mortierella sp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2–4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1–10 mM Co²⁺ and strongly inhibited by 10 mM Cu²⁺. It required at least two GlcNAc residues for catalysis. When (GlcNAc)₆ was used as substrate, K _m and V _max were determined to be 1.1 mM and 54.6 μmol min^?1 respectively.     

Degradation of chlorinated and non-chlorinated aromatic solvents in soil suspensions by pure bacterial cultures

Summary Experiments were performed to test whether or not high concentrations of CaCl₂ (100 mM) are able to arrest and stabilize internal structures and associated functions in Euglena gracilis Z cells stored in darkness at 4° C. Storage of photoheterotrophically grown green cells in high Ca²⁺ media (2–100 mM) retards pheophytinization of the chlorophylls, preserves photosynthetic activities and stabilizes the structural organization of the associated light-harvesting complexes of the photosystem II units. Alterations of photosynthesis and respiration by chlorpromazine or by temperature are strongly reduced in cells stored under such conditions. More precisely, a chlorpromazine inhibition site is evidenced in the mitochondrial electron pathway and its location in the chloroplastic electron pathway is clarified. Adaptation of Euglena cells from 2 mM to 100 mM Ca²⁺ medium is accompanied by an increase both in the externally bound and total internal calcium concentration. A mechanism involving a Ca²⁺ deposit on internal membranes is proposed. Such interpretation is extended to the storage of cells immobilized in Ca²⁺-alginate gel.Nomenclature (Ca²⁺)ex external calcium concentration - Chl chlorophylls - (Cl^–)ex external chloride concentration - CPZ chlorpromazine or 2-chloro-10-(3-dimethylaminopropyl)-phenothiazine - DCMU diuron or (3,4-dichorophenyl)-1,1-dimethylurea - EGTA ethylene glycol-bis(-aminoethylether) N,N,N ,N-tetraacetic acid - Fc initial level of chlorophyll fluorescence with DCMU - Fmax maximal level of chlorophyll fluorescence with DCMU - Fo level of chlorophyll fluorescence after transients - Ft level of chlorophyll fluorescence with DCMU - Pheo pheophytins - PS I and PS II photosystems I and II - SMi storage medium Offprint requests to: C. Tamponnet     

Energy and protein sources for development of pig embryos cultured beyond hatching in vitro

The effects of supplementation of synthetic culture media with different energy and protein sources on in vitro development of pig embryos beyond the 4–8-cell stage have been explored.Minimal Essential Medium (MEM) supplemented with glucose (1 mg/ml) proved superior to Krebs-Ringer Bicarbonate (KRB) supplemented with glucose (1 mg/ml) in its capacity to support embryonic development to the expanded blastocyst stage (P < 0.05). Inclusion of pyruvate (0.25 mM) or lactate (25 mM) in either MEM or KRB based media inhibited embryonic development. As pyruvate and lactate are important and readily utilizable energy sources for development of most other mammalian embryos in vitro, it is suggested that the observed inhibitory effects of these substrates reflect comparatively lower critical ranges of concentrations of pyruvate and lactate for optimum development of pig embryos in vitro.As a supplementary protein source to MEM, heat inactivated (HI) human serum (10% υ/υ) was superior (P < 0.05) to HI-pig serum (10% υ/υ), HI-foetal calf serum (10% υ/υ) or bovine serum albumin (5 mg/ml). The proportion of 4–8-cell pig embryos which developed beyond hatching in MEM supplemented with HI-human serum (> 56%) was higher than any other reported for in vitro culture of pig embryos through the same developmental period and this medium is recommended for future studies on in vitro development of pig embryos from the four-cell through the hatched/expanded blastocyst stages.     

Formation of 2,5-Bis-(d-threo-trihydroxypropyl)pyrazine and Its 6-Isomer by the Oxidative Browning Reaction of d-Xylose with Ammonium Acetate in a Methanolic Medium

A pyruvate kinase-lacking mutant of Brevibacterium flavum produced 22.6 g/liter of l-aspartic acid with glutamic acid as a by-product, when cultured for 48 hr in a medium containing 100 g/liter of glucose. The production clearly depended on the amount of biotin added. This strain, 70, was derived by several steps of mutation from wild strain 2247 producing glutamate, successively via a citrate synthase-defective glutamate auxotroph, strain 214, a prototrophic revertant, strain 15-8, producing 10 g/liter of l-aspartic acid, and an S-(2-aminoethyl)-l-cysteine-resistant mutant, strain 1-231, having low pyruvate kinase and homoserine dehydrogenase and producing lysine. Strain 70, a methionine-insensitive revertant from strain 1-231, had a normal level of homoserine dehydrogenase but no pyruvate kinase. Its citrate synthase activity was about half that of the wild strain at saturated concentrations of the substrates with Michaelis constants for oxalacetate and acetyl-CoA of 110 and 6 times as high as those of the wild-type enzyme, respectively. The mutational step for these alterations in citrate synthase was strain 15-8. Phosphoenolpyruvate carboxylase of strain 70 showed 1.5-fold higher activity in the crude extract at saturated concentrations of phosphoenolpyruvate, a lower Michaelis constant (1.5mM).for the substrate, phosphoenolpyruvate, less sensitivity to the feedback inhibition by aspartate, and higher sensitivities to the activators, acetyl-CoA and fructose-1,6-bisphosphate, than those of the wild strain. The concentrations of aspartate giving 50% inhibition were 6.2- and 4.5-fold higher in the absence and presence of acetyl-CoA, respectively.     

Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.     

Binding sites for horseradish peroxidase on the cell surface

Summary The cytochemical reaction for surface-bound horseradish peroxidase (HRP) on cultured HeLa cells, GH₃ cells, and isolated rat liver cells was suppressed by 30 M monosialoganglioside, by 30 M trisialoganglioside, or by 5 mM CMP-neurminic acid. The reaction was also suppressed by 10 mM chitotriose or by 10 mM UDP-galactose, a galactose acceptor and donor, respectively, for galactosyltransferase. The addition of 2 mM Mn²⁺ to the incubation medium with HRP suppressed the reaction for surfacebound HRP, and the addition of 10–20 mM Ca²⁺ intensified the reaction. The addition of 2 mM Zn²⁺ caused less inhibition than that of 2 mM Mn²⁺, and the addition of 2 mM Co²⁺ caused either a slight inhibition, or no inhibition. These observations support the hypothesis that HRP may be bound to a glycosyltransferase at the cell surface.     

Ammonium photoproduction by free and immobilized cells of Chlamydomonas reinhardtii

Summary Free-living or immobilized Chlamydomonas reinhardtii cells photoproduce ammonium from nitrite in a medium containing 1 mM of l-methionine-d,l-sulphoximine (MSX). Ammonium is accumulated in the medium to 8 mM final concentration, which inhibits nitrite uptake by the MSX-treated cells and consequently the excretion of ammonium is blocked. However, if ammonium was removed from the medium and nitrite and MSX periodically restored, the photoproduction process could be maintained over 96 h, with a final ammonium concentration of about 18 mM for free-living cells and 28 mM for immobilized ones. The MSX-treated cells showed a photoproduction productivity of 1300 mol NH ₄ ⁺ · mg chlorophyll (Chl)^-1, with an average production rate of 14 mol NH ₄ ⁺ · mg Chl^-1 per hour, for calcium alginate-entrapped cells, while the corresponding data for free-living ones was 650 mol NH ₄ ⁺ · mg Chl^-1 and 6.7 mol NH ₄ ⁺ · mg Chl^-1 per hour, respectively. Immobilized cells showed a significant increase in the nitrite uptake rate, probably due to a change in membrane permeability as a consequence of cell-matrix interactions.     

Requirement of chelating compounds for the growth of Corynebacterium glutamicum in synthetic media

Summary The effects of various iron-complexing substances on the growth of Corynebacterium glutamicum in synthetic medium were investigated. The data obtained indicate that C. glutamicum has an absolute requirement for the presence of an iron-complexing compound as a growth factor for rapid and abundabnt growth in synthetic media. This requirement can be met by adding low concentrations (10^–5 M) of certain dihydroxyphenols (catechol, protocatechuate) or relatively high concentratuions (0.1%) of citrate to the medium or by autoclaving a small amount of glucose together with other media components. The addition of catechol or protocatechuate to synthetic media has advantages over media preparation with citrate or autoclaved glucose. In the described synthetic broth supplemented with catechol or protocatechuate growth is largely independent of the iron content of the medium in a range between 0.037 and 0.37 mM.Offprint requests to: W. Liebl     

Antibacterial Activity of l-Lysine α-Oxidase against rec+ and rec− Strains of Bacillus subtilis

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker’s yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker’s yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.