Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetyl-CoA acetyltransferases (3-ketothiolases A and B) were purified from Alcaligenes eutrophus . Enzyme A was active with only acetoacetyl-CoA and 3-ketopentanoyl-CoA, whereas enzyme B was active with all the 3-ketoacyl-CoAs (C₄−C₁₀) tested. Enzyme A appeared to be a tetramer ( M _r 70 000) with identical subunits ( M _r 44 000) and enzyme B had a similar M _r of 168 000 (containing M _r 46 000 subunits). Enzymes A and B had isoelectric points of 5.0 and 6.4, respectively. The stoichiometry of the reactions catalysed by each enzyme was confirmed. K _m values of 44 μM and 394 μM for acetoacetyl-CoA, and 16 μM and 93 μM for CoA, were determined with enzymes A and B, respectively. Enzymes A and B gave K _m values of 1.1 mM and 230 μM, respectively, for acetyl-CoA. The condensation reaction was potently inhibited by CoA in both cases.     

A Simple Procedure for Crystallization of the Schiff Reagent

Formation of crystals in Schiff reagents prepared from SO₂ gas previously has been reported either soon after preparation, using high dye concentrations and heating, or after long periods of storage at room temperature. With the first type of procedure only a low yield of crystals accompanied by dye precipitation was obtained. Crystallization without dye precipitation took place if the reagent, prepared with pararosaniline base or chloride in a saturated SO₂ solution, was stored for a sufficient time at room temperature in partly filled flasks. These crystals remained colorless if washed with acid alcohol after being separated by filtration. Schiff reagents layered with paraffin oil or supplemented with 0.1 M hydroquinone took much longer to crystallize, suggesting that crystallizaticn is promoted by the partial oxidation of sulfurous acid to sulfuric acid. A high yield of crystals can be obtained at room temperature after as little as 24 hr by adding 0.04 M of H₂SO₄ to a Schiff reagent prepared with 2% pararosaniline chloride in a saturated SO₂ solution. A Schiff reagent prepared with only 0.2% of these crystals gives an intense staining in the Feulgen and in the Periodic acid-Schiff reactions.     

Metabolism of cell wall polysaccharides from persimmon fruit. Pectin solubilization during fruit ripening occurs in apparent absence of polygalacturonase activity

Pectins from persimmon ( Diospyros kaki L.) fruit pericarp were sequentially extracted with 0. 05 M trans -1,2-diaminocyclohexane-N,N, N', N'-tetraacetic acid (CDTA), 0. 05 M Na₂CO₃ (1°C) and Na₂CO₃ (20°C) and the carbohydrate composition and metabolism during development determined. Young persimmon fruits contained a large proportion of pectins, 46% by dry weight, that decreased to 20% with ripening. This decrease occurred in the CDTA and Na₂CO₃ (1°C) fractions, mainly composed of uronic acids, and represents a net loss of uronic acids, arabinose and galactose. The amount of non-cellulosic neutral sugars was especially high in the Na₂CO₃ (20°C) fraction. The loss of pectins was also accompanied by a depolymerisation of the polysaccharides extracted in the three pectic fractions. However, none of these changes can be attributed to the action of polygalacturonase activity. Proteins were extracted from the pericarp tissue, but endopolygalacturonase (EC 3. 2. 1. 15) activity, determined as a decrease in viscosity of polygalacturonic acid, was not observed in the extract. Determination of exopolygalacturonase (EC 3. 2. 1. 67) activity by measuring the release of reducing groups from polygalacturonic acid was also negative. The results presented indicate that polygalacturonase is not responsible for the metabolism of pectins during persimmon fruit ripening.     

Method for Staining Bacterial Flagella

The following method of staining bacterial flagella is ecommended for use on smears made from suspensions of 10 to 16-tour agar slant cultures, incubated 30 minutes at 37°C before spreadng on thoroly cleaned and named slides:

1. Cover with fixative (100 cc. of 1/4 sat. aqu. solution picric acid, with 5 g. tannic acid and 7.5 g. ferrous sulfate).
2. Wash with tap water, dry and cover with Fontana spirochaete stain; heat to steaming and allow to act for 1 to 2 minutes. Wash in ap water. The stain is prepared as follows: To 25 cc. 2% AgNO₃ add dilute ammonia till the precipitate which forms redissolves; then add more AgNO₃ till a faint turbidity results. A clear solution is useess.

     

GENERAL PATTERN, 35SO4-INCORPORATION AND INTRACELLULAR LOCALIZATION OF GLYCANS IN DEVELOPING SEA URCHIN (ANTHOCIDARIS) EMBRYOS

Glycosaminoglycan (GAG) prepared from sea urchin embryos ( Anthocidaris crassispina ) at various stages with or without pulse ³⁵SO₄-labelling was separated into various fractions by chromatography on DEAE-cellulose with a linear NaCl concentration gradient: fraction "P" (nonacidic) and fractions "A" through "F" (of increasing acidities). The ³⁵SO₄-radioactivity was negligible in "P" and "A", largest in "B" and "C", and decreased in the other fractions three alphabetical order. During development (hatched blastulae to gastrulae) the glycans in fractions "P" and "A" decreased in amount, whereas those in "E" and "F" increased. "E" contained heparin-like (AMPS-1) and dermatanpolysulfate-like (AMPS-2) GAG in addition to a sulfated fucogalactan-like (E₁) glycan. Another sulfated fucogalactan-like (F₁) glycan was found in "F". A sulfated polysialic acid-like (S₁) glycan was found in "C". An EDTA-extract of gastrulae gave AMPS-2, E₁ and F₁. The mitochondria-rich fraction gave AMPS-1, whereas the yolk granule-rich fraction gave S₁. Most of the other still unidentified components in "B", "C", and "D" appeared to be derived from glycoproteins and were mainly located in the crude yolk-mitochondrial and cytosol fractions.     

Presence of glycerol teichoic acid in the cell wall of Lactobacillus kefiranofaciens

The accessory polymer of the capsular polysaccharide of a 'kefiran'-producing Lactobacillus kefiranofaciens was studied. The teichoic acid of L. kefiranofaciens K₁ was extracted from the cell with 5% trichloroacetic acid (w v) at 5°C for 24 h and was purified by ion-exchange chromatography on DEAE-Toyopearl 650 M with a linear gradient of (NH₄)₂CO₃; the yield was only 2%. The teichoic acid has a molecular weight of 22000 and is composed of D-glucose, 2-acetamide-2-deoxy-D-glucose, glycerol and phosphorus in a molar ratio of 1.0 : 2.0 : 2.3 : 1.1. The low yield of teichoic acid suggested that kefiran is the main accessory polymer in the cell-wall of L. kefiranofaciens.     

Comparison of Various Oxidants for Alum Hematoxylin

Oxidation of 100 mg of hematoxylm in 40 ml of a 3% solution of EAI(SO₄),₂. 12H₂O with 20 mg iodine, 4.5 mg of NaIO₃. 3.5 mg of H₅IO₆, 3.5 mg of NaIO₄ or 9 mg KMnO₄ gives equivalent results as shown by both staining and absorbance measurements Placing the prepared solutions at 55 C for 18 hours increased absorbance by 1/4 to 1/3 over those kept at room temperature. Two months after preparation, solutions initially placed at 55 C for 18 hours, with no air space in the bottle, had not increased in absorbancy. In simiiar preparations kept at room temperature the absorbance increased from initial values to that of those that had been heated. Solutions in bottles with air space also showed this increase and also had a scum on the surface and sufficient precipitate to require filtering before use.     

Calmodulin antagonists inhibit germination of Bacillus cereus T spores

M. RACINE, J. DUMONT, C.P. CHAMPAGNE AND A. MORIN. 1991. The effects of lactose, ammonium and phosphate on the production of extracellular polysaccharide from Propionibacterium acidi-propionici VM-25 were studied in whey-based media. The polysaccharide was composed of a water-soluble fraction (15% w/w), a water-insoluble fraction (27% w/w) and ca 65% (w/w) of ash. Up to 15 g/l of polysaccharide was produced during growth on partially deproteinated whey, supplemented with lactose, NH₄ Cl and KH₂ PO₄, after incubation at pH 7.0 and 25.C for 90 h. The final viscosity of the medium remained under 20 centipoises at the end of the fermentation (100–140 h). The fermentation of whey enabled a reduction of the lactose content up to 50%. The polysaccharide-containing fractions were composed of glucose, galactose, mannose, rhamnose and fucose and had M, < 5800. The polysaccharide may have applications as a low viscosity stabilizing agent.     

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P₂) and slices of rat cerebral cortex were investigated. The P₂ fraction had a Na⁺-dependent high-affinity uptake system for cysteine sulfinic acid (K_m, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [¹⁴C]cysteine sulfinic acid from preloaded cortical slices or P₂ fractions was examined by a superfusion method, which avoided reuptake of released [¹⁴C]cysteine sulfinic acid. High K⁺ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P₂ fraction in a partly Ca²⁺-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P₂ fraction.     

MDL 72145, an Enzyme-Activated Irreversible Inhibitor with Selectivity for Monoamine Oxidase Type B

Abstract: MDL 72145, ( E )-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine hydrochloride, was designed and synthesised as a potential enzyme-activated irreversible inhibitor of monoamine oxidase (MAO). In vitro , the compound displayed time-dependent pseudo-first-order irreversible inhibitory characteristics with high selectivity for the B form of rat brain mitochondrial MAO At 10°C the K_t and T₅₀ values for the B enzyme were 40 μ M and 1.7 min, respectively, while these same kinetic constants for the A enzyme were 131 μ M and 14.5 mm, respectively. Selective protection against inactivation of the two forms of MAO by MDL 72145 was obtained by preincu-bating the enzyme with suitable concentrations of the selective A and B substrates, 5-hydroxytryptamine and benzylamine.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Novel cytotoxic diterpenes from the stem of dysoxylum kuskusense

Three novel diterpenes, dysokusones A (1), B (2), and C (3), were isolated from the stem of Dysoxylum kuskusense as cytotoxic substances. The structures were established by spectroscopic examinations. Compounds 1, 2, and 3 were cytotoxic toward HL-60(TB) cells with EC₅₀ values of 2.25, 6.35, and 2.37 μM, respectively. Compound 1 also displayed cytotoxicity against K-562 and NCI-H522 cells with EC₅₀ values of 5.04 and 4.80 μM, respectively.     

Formation of 2-hydroxy-4-methylpentanoic acid from l-leucine by Clostridium butyricum

Abstract Five Clostridium butyricum strains of different origin were grown in trypticase-yeast extract-hemin medium with or without d-glucose (TGYH or TYH medium, respectively) and in a synthetic basal medium with d-glucose (BMG medium). 2-Hydroxy-4-methylpentanoic acid was detected by gas chromatography-mass spectrometry (GC-MS) for the five strains whether grown in TGYH or TYH medium (270 or 170 μM, respectively). In BMG medium supplemented with l-leucine (10 mM), the concentration of this metabolite was strongly increased (2.8 mM versus 10 μM in the control). After culture in TGYH or TYH medium supplemented with l-( methyl -²H₃)leucine, 2-hydroxy-4-([²H₃]methyl)pentanoic acid was detected by GC-MS. This observation demonstrates that C. butyricum is able to convert l-leucine into the corresponding 2-hydroxy acid and opens a new aspect in the study of C. butyricum metabolism.     

Fractional and structural characterization of hemicelluloses isolated by alkali and alkaline peroxide from barley straw

Eight hemicellulosic fractions were obtained by sequential treatment of dewaxed barley straw with 0.1 M NaOH at 45 °C for 3 h, 0.25, 0.5, 1.0, 1.5, 2.0, and 3.0% H₂O₂ at 45 °C for 3 h at pH 11.5, and 10% KOH–1% Na₂B₄O₇·10H₂O at 28 °C for 15 h under continuous agitation. The yields of the fractions were 8.0, 3.1, 3.3, 3.3, 2.2, 2.0, 2.0, and 9.9%, respectively, of the initial amount of barley straw, corresponding to the dissolution of 21.6, 8.4, 8.9, 8.9, 5.9, 5.4, 5.4, and 26.7% of the original hemicelluloses. Meanwhile, the successive treatment also solubilized 29.1, 15.8, 14.6, 10.8, 4.5, 3.2, 2.7, and 3.7% of the original lignin, respectively. This sequential extraction together resulted in dissolution of 91.1% of the original hemicelluloses and 84.8% of the original lignin. The 0.1 M NaOH-soluble hemicellulosic fraction contained mainly xylose, glucose, and arabinose, 44.2, 15.7, and 15.2%, respectively, while the 10% KOH–1% Na₂B₄O₇·10H₂O-soluble fraction predominated in xylose, 75.0%. The six alkaline peroxide-soluble fractions were composed of 50.3–54.4% xylose, 14.7–16.9% arabinose, 6.8–10.7% glucose, 6.8–8.5% glucuronic acid or 4-O-methyl- -glucuronic acid, 0.4–1.5% mannose, and 0.3–1.2% rhamnose. All the hemicellulosic fractions contained substantial amounts of glucuronoarabinoxylans and noticeable quantities of β-glucans. In comparison, the six hemicellulosic fractions, isolated with alkaline peroxide, had much higher molecular weights (56,890–63,810 g mol⁻¹) than those of the two hemicellulosic preparations (28,000–29,080 g mol⁻¹), isolated with alkali in the absence of hydrogen peroxide. The thermal stability of the hemicelluloses increased with an increment of their molar mass.     

Ascorbic acid as negative effector of the peroxidase-catalyzed degradation of indole-3-acetic acid

Ascorbic acid is a strong inhibitor of indole-3-acetic oxidation catalyzed by commercial horse-radish peroxidase. In the presence of excess ascorbic acid, the indole-acetic acid oxidation catalysis is apparently blocked. The activity of peroxidase for indoleacetic acid at pH 3.7 and 33°C, in the presence of 2,4-dichlorophenol and MnCl₂ as promotors was measured by polarographic technique. The K_m was 0.27 m M and the maximum velocity was 1.02 mmol O₂ (mg protein)⁻¹ min⁻¹. Dixon plots lead to an apparent K_i of 1.25 (μ M for ascorbic acid and the inhibition was apparently competitive. Ascorbic acid, besides appearing to be a strong inhibitor of the IAA oxidase activity of peroxidase, seemed to protect IAA from total degradation. Addition of more than 5 μ M ascorbic acid produced both an exponential increase in the lag time before the onset of reaction and, at the end, an oxidation protection of 26 μ M IAA when 111 μ M IAA was present at the stawrt. The possibility of ascorbic acid-IAA auxin from endogenous oxidation in plants, is proposed.     

Synthesis of low molecular weight alginic acid nanoparticles through persulfate treatment as effective drug delivery system to manage drug resistant bacteria

The purpose of this study was to prepare low molecular weight alginic acid (LMWA) nanoparticles by cation-induced, controlled gelification of depolymerized alginic acid for effective drug delivery to drug resistant bacteria. The depolymerization reaction was performed using potassium persulfate oxidation at an optimized condition. The optimized conditions for depolymerization were anticipated to be 37°C, pH 4, 2 days reaction time, and a 0.075 M concentration of potassium persulphate containing 0.001 M silver nitrate in the final reaction mixture. Gel permeation chromatography showed depolymerized alginic acid had an average molecular weight of 20.95 ± 0.49 kDa. Depolymerized alginic acid was also characterized for its structural integrity by X-ray diffraction, nuclear magnetic resonance, and Fourier transform spectroscopy. Depolymerized alginic acid was used to prepare low molecular weight nanoparticles with a particle size of 54 ± 0.41 nm, and a zetapotential of −32.2 ± 3.91 mV. The nanoparicles were then subjected to tetracycline loading. In vitro drug loading and drug release efficiencies after 100 h were determined to be 66.56 ± 1.88 and 61.8 ± 0.141%, respectively. Finally, the minimal inhibitory concentration and a putative mode of action for the tetracycline nanoparticles were determined using tetracycline resistant bacteria, Escherichia coli XL-1.     

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells.

This study examines the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)₂D₃ or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.     

Preferential In Vivo Incorporation of [3H]Arachidonic Acid from Blood into Rat Brain Synaptosomal Fractions Before and After Cholinergic Stimulation

Abstract: Awake adult male rats were infused intravenously with [³H]arachidonic acid for 5 min, with or without prior administration of an M1 cholinergic agonist, arecoline (15 mg/kg i.p.). Methylatropine was also administered (4 mg/kg s.c.) to control and arecoline-treated animals. At 15 min postinfusion, the animals were killed, brains were removed and frozen, and subcellular fractions were obtained from homogenates of whole brain. Total radioactivity and radioactivity in various lipid classes were determined for each fraction following normalization for exposure by use of a unidirectional incorporation coefficient, k ^⋆_brain. In control animals, incorporation was greatest in synaptosomal and microsomal fractions, accounting for 50 and 30% of total label incorporated into membrane lipids, respectively. Arecoline increased incorporation in these two fractions by up to 400% but did not increase incorporation into the myelin, mitochondrial, or cytosolic fractions. Of the incorporated radioactivity, 50–80% was in phospholipid in microsomal and synaptosomal fractions, indicating that phospholipid is the major lipid affected by cholinergic stimulation. These results demonstrate that plasma [³H]arachidonic acid is preferentially incorporated into phospholipids of synaptosomal and microsomal fractions of rat brain. Cholinergic stimulation increases incorporation into these fractions, likely by activation of phospholipase A₂ and/or C in association with acyltransferase activity. Thus, intravenously infused radiolabeled arachidonic acid can be used to examine synapse-mediated changes in brain phospholipid metabolism in vivo.     

Modification of corn cob meal with quarternary ammonium groups

Corn cob meal was modified with quarternary ammonium groups and subsequently extracted with 80% ethanol, water, and 5% NaOH. The fractions obtained had lower polydispersities, values, and yields than unmodified material. The yields are lower than those obtained on bagasse under the same conditions. The modification caused the drastic degradation of the ethanol-lignin (EL) fraction. The one-step extraction with NaOH/H₂O₂ gave 28·8% yield of material (calculated on the starting material) which contained 12·0% Klason lignin, and had the highest polydispersity (4·3, ). The water-soluble fractions consisted of arabinoglucuronoxylan and alkali-soluble fractions of xylan without other sugar moieties. The water-soluble fraction from NaOH/H₂O₂ extraction contained arabinoglucuronoxylan with modified arabinose and acid units. By this method higher yields could be obtained than on bagasse treated by the sequential extraction.