Comparison between D-[3-3H]- and D-[5-3H]glucose and fructose utilization in pancreatic islets from control and hereditarily diabetic rats

The metabolism of D-glucose and/or D-fructose was investigated in pancreatic islets from control rats and hereditarily diabetic GK rats. In the case of both D-glucose and D-fructose metabolism, a preferential alteration of oxidative events was observed in islets from GK rats. The generation of 3HOH from D-[5-3H]glucose (or D-[5-3H]fructose) exceeded that from D-[3-3H]glucose (or D-[3-3H]fructose) in both control and GK rats. This difference, which is possibly attributable to a partial escape from glycolysis of tritiated dihydroxyacetone phosphate, was accentuated whenever the rate of glycolysis was decreased, e.g., in the absence of extracellular Ca(2+) or presence of exogenous D-glyceraldehyde. D-Mannoheptulose, which inhibited D-glucose metabolism, exerted only limited effects upon D-fructose metabolism. In the presence of both hexoses, the paired ratio between D-[U-14C]fructose oxidation and D-[3-3H]fructose or D-[5-3H]fructose utilization was considerably increased, this being probably attributable, in part at least, to a preferential stimulation by the aldohexose of mitochondrial oxidative events. Moreover, this coincided with the fact that D-mannoheptulose now severely inhibited the catabolism of D-[5-3H]fructose and D-[U-14C]fructose. The latter situation is consistent with both the knowledge that D-glucose augments D-fructose phosphorylation by glucokinase and the findings that D-mannoheptulose, which fails to affect D-fructose phosphorylation by fructokinase, inhibits the phosphorylation of D-fructose by glucokinase.     

Preparation, single-crystal X-ray diffraction and high-resolution NMR spectroscopic analyses of N-[(1,4-anhydro-5-deoxy-2,3-O-isopropylidene-D,L-ribitol)-5-yl]trimethylammonium iodide

The synthesis and isolation of 1,4-anhydro-5-deoxy-5-iodo-2,3-O-isopropylidene-D,L-ribitol and N-[(1,4-anhydro-5-deoxy-2,3-O-isopropylidene-D,L-ribitol)-5-yl]trimethylammonium iodide are described. The products were examined by (1)H, (13)C NMR spectroscopy, and N-[(1,4-anhydro-5-deoxy-2,3-O-isopropylidene-D,L-ribitol)-5-yl]trimethylammonium iodide was additionally analyzed by X-ray crystallography.     

Identification of nuclear type II [(3)H]estradiol binding sites as histone H4

[3H]Luteolin binds covalently to uterine nuclear type II sites [B. Markaverich, K. Shoulars, M.A. Alejandro, T. Brown, Steroids 66 (2001) 707] and was used to identify this protein(s). SDS-PAGE analyses of [3H]luteolin-labeled type II site preparations revealed specific binding to 11- and 35-kDa proteins. The 11-kDa protein was identified as histone H4 by amino acid sequencing. Western blotting confirmed that the 11- and 35-kDa proteins were acetylated forms of histone H4. Anti-histone H4 antibodies (but not H2A, H2B, or H3 antibodies) quantitatively immunoadsorbed type II binding sites from nuclear extracts. Binding analyses by [3H]estradiol exchange, using luteolin as a competitor, detected specific type II binding activity to histone H4 (but not histones H2A, H2B, or H3) generated in a rabbit reticulocyte lysate translation system and confirmed that histone H4 is the type II site.     

Synthesis of (2R,5S)-dihydroxymethyl-(3R,4R)-dihydroxypyrrolidine (DGDP) via stereoselective amination using chlorosulfonyl isocyanate

A stereoselective approach for synthesizing (2R,5S)-dihydroxymethyl-(3R,4R)-dihydroxypyrrolidine 1 (2,5-dideoxy-2,5-imino-d-glucitol, DGDP) was achieved using a seven-step approach starting from 2,3,4,6-tetra-O-benzyl-d-mannose (7). Key steps for the preparation of the title compound 1 involved the regioselective and diastereoselective amination of the cinnamyl anti-1,2-polybenzyl ethers 5 and 6 using chlorosulfonyl isocyanate (CSI) and ring cyclization to form the pyrrolidine ring. The reaction between anti-1,2-polybenzyl ether 5 and CSI in toluene at 0 degrees C afforded the corresponding anti-1,2-amino alcohol 4 as a major product with a diastereoselectivity of 16:1 in 76% yield. The mechanism underlying these reactions may be explained by the neighboring-group effect leading to the retention of stereochemistry.     

(3)H](2S,4R)-4-Methylglutamate: a novel ligand for the characterization of glutamate transporters

[(3)H](2S,4R)-4-Methylglutamate ([(3)H]4MG), used previously as a ligand for low-affinity kainate receptors, was employed to establish a binding assay for glutamate transporters (GluTs), as 4MG has also been shown to have affinity for the glial GluTs, GLT1 and GLAST. In rat brain membrane homogenates in the presence of Na(+) ions at 4 degrees C, specific binding of [(3)H]4MG was rapid and saturable (t(1/2) approximately 15 min), representing > 90% of total binding. Dissociation of [(3)H]4MG occurred in a biphasic manner, however, saturation studies and Scatchard analysis indicated a single site of binding (n(H) = 0.85) and a K(d) of 6.2 +/- 0.8 microM with a B(max) of 111.8 +/- 23.8 pmol/mg protein. Specific binding of [(3)H]4MG was Na(+)-dependent and inhibited by K(+) and HCO(3-). Pharmacological inhibition with compounds acting at GluTs revealed that Glu, D- and L-aspartate, L-serine-O-sulfate and Ltrans-pyrrolidine-2,4-dicarboxylate fully displaced specific binding. Drugs having preferential affinity for GLT1, kainate, dihydrokainate and Lthreo-3-methylglutamate, all inhibited approximately 40% of specific binding. The inhibition pattern of L-serine-O-sulfate in the presence of a saturating concentration of dihydrokainate was suggestive of [(3)H]4MG also labelling GLAST. 6-Cyano-7-nitroquinoxaline, a kainate receptor antagonist, and a range of Glu receptor agonists and antagonists failed to significantly inhibit [(3)H]4MG binding. The pharmacological profile of binding of [(3)H]4MG resembled that found for [(3)H]D-aspartate, a ligand specific for GluTs, reinforcing the hypothesis that [(3)H]4MG was labelling GluTs in this assay. Together, these data illustrate the development of an efficient, economic binding assay that is suitable for the characterization of different subtypes of GLuTs.     

Proposal of protocols using D-glutamine to optimize the 2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay for indirect estimation of microbial loads in biofilms of medical importance

Due to technical problems, biofilm biomasses are difficult to be precisely determined. One reliable strategy is based on the colorimetry of formazan compounds derived from tetrazolium salt reduction. XTT presents some desirable properties that make the biofilm measurements easier. However, cells entrapped within the extracellular matrixes normally do not metabolize the tetrazolium equally, leading to underestimation of cell contents. This study evaluated the effectiveness of d-glutamine, a plerotic substrate of tricarboxilic acid cycle (TAC), as inducer of XTT reduction. The metabolic activities of aerobic and anaerobic 48 h-old monospecific biofilms of Pseudomonas aeruginosa ATCC®27853™, Klebsiella pneumoniae ATCC®13883™, Staphylococcus epidermidis ATCC®12228™, Streptococcus mutans ATCC®25175™, and Candida albicans SC5314 were evaluated. Results showed that d-glutamine 50 mM (for P. aeruginosa, K. pneumoniae, and S. epidermidis) and 25 mM (for S. mutans and C. albicans) may enhance the detection of soluble formazan in a significant manner, what becomes the XTT reduction assay more robust.     

Structures and Properties of a Diastereoisomeric Molecular Compound of (2S,3S)- and (2R,3S)-N-Acetyl-2-amino-3-methylpentanoic Acids

An X-ray crystal structural analysis revealed that (2S,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-L-isoleucine; Ac-L-Ile) and (2R,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-D-alloisoleucine; Ac-D-aIle) formed a molecular compound containing one Ac-L-Ile molecule and one Ac-D-aIle molecule as an unsymmetrical unit. This molecular compound is packed with strong hydrogen bonds forming homogeneous chains consisting of Ac-L-Ile molecules or Ac-D-aIle molecules and weak hydrogen bonds connecting these homogeneous chains in a fashion similar to that observed for Ac-L-Ile and Ac-D-aIle. Recrystallization of an approximately 1:1 mixture of Ac-L-Ile and Ac-D-aIle from water gave an equimolar molecular compound due to its lower solubility than that of Ac-D-aIle or especially Ac-L-Ile. The results suggest that the equimolar mixture of Ac-L-Ile and Ac-D-aIle could be obtained from an Ac-L-Ile-excess mixture by recystallization from water.     

Construction and co-expression of polycistronic plasmid encoding d-hydantoinase and d-carbamoylase for the production of d-amino acids

d-Hydantoinase and d-carbamoylase genes from Agrobacterium radiobacter TH572 were cloned by polymerase chain reaction (PCR). The plasmid pUCCH3 with a polycistronic structure that is controlled by the native hydantoinase promoter was constructed to co-express the two genes and transformed into Escherichia coli strain JM105. To obtain the highest level of expression of the d-carbamoylase and avoid intermediate accumulation, the d-carbamoylase gene was cloned closer to the promoter and the RBS region in the upstream of it was optimized. This resulted in high active expression of soluble d-hydantoinase and d-carbamoylase that is obtained without any inducer. Thus, by the constitutive recombinant JM105/pUCCH3, d-p-hydroxyphenylglycine (d-HPG) was obtained directly with 95.2% production yield and 96.3% conversion yield.     

Chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone from D-fructose 1,6-diphosphate

The selective chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDF) from D-fructose 1,6-diphosphate in the presence of reduced nicotinamide-adenine-dinucleotides (NAD(P)H) was investigated by means of HPLC-DAD and HPLC-UV-MS/MS. The temperature optimum for HDF formation was 30 degrees C, whereas the pH value (pH 3-10) and chemical nature of the buffer had no significant influence. A linear correlation of reaction time and D-fructose 1,6-diphosphate concentration with the obtained HDF yield was observed. Proteins appeared to have a stabilizing effect. The NAD(P)H were mandatory, even in the presence of protein, implying a non-enzymatic hydride-transfer to an unknown intermediate which finally leads to the selective formation of HDF. The hydride-transfer was confirmed by the application of selectively pro-4R or pro-4S deuterium labeled NADH resulting in each case in the formation of HDF exhibiting a deuterium labeling of approx 30% and employment of [4R,S-(2)H(2)]-NADH led to a deuterium labeling of approx 66%. The incubation of [1-(13)C]-D-fructose 1,6-diphosphate with [4R,S-(2)H(2)]-NADH revealed that the hydride is transferred to C-5 or C-6 of the D-fructose 1,6-diphosphate skeleton. Thus, a chemical HDF formation from D-fructose 1,6-diphosphate under physiological reaction conditions was shown and for the first time to our knowledge a non-enzymatic hydride-transfer from NADH to a carbohydrate structure was demonstrated.     

Characterization of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands to thermolysin

In this study, glycyl-D-phenylalanine (Gly-D-Phe), glycyl-L-leucine (Gly-L-Leu), and D-phenylalanine (D-Phe) were characterized for their abilities as affinity ligands to thermolysin. Each of the ligands was immobilized to the resin. The optimum pH for adsorption of thermolysin is 5.0-6.0 for each of the ligands. By the affinity column chromatography in which 2mg thermolysin was applied onto 4 ml volume of the resins at pH 5.5, the adsorption ratios based on casein hydrolysis activity were 100% for each of the ligands. However, the adsorption ratios of the resins containing Gly-L-Leu and D-Phe, unlike that of Gly-D-Phe, were progressively decreased with increasing the amounts of thermolysin applied to the column. Measurement of adsorption isotherms showed that the association constant to thermolysin at pH 5.5 of the resins containing Gly-D-Phe was (3.3+/-0.8)x10(5)M(-1), while those of Gly-L-Leu and D-Phe were approximately ten times less. This result is coincident with the observations of performances in affinity column chromatography. On the other hand, maximum thermolysin binding capacities were almost the same among the resins examined. These results indicate that Gly-D-Phe is more suitable than Gly-L-Leu and D-Phe as an affinity ligand for purification of thermolysin.     

A facile synthesis of (1S,5R,6S)-5-azido-6-benzyloxycyclohex-2-en-1-ol

A facile and short synthesis of (1S,5R,6S)-5-azido-6-benzyloxycyclohex-2-en-1-ol (1) has been achieved in high yield starting from 4,5-epoxycyclohex-1-ene by using a catalytic asymmetric allylic oxidation reaction.     

Structure of the O-specific polysaccharide of Proteus vulgaris O15 containing a novel regioisomer of N-acetylmuramic acid, 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O15 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, and H-detected 1H,(13)C HMQC experiments. The polysaccharide was found to contain an ether of GlcNAc with lactic acid, and the following structure of the repeating unit was established:-->3)-alpha-D-GlcpNAc4(R-Lac)6Ac-(1-->2)-beta-D-GlcpA-(1-->3)-alpha-L-6dTalp2Ac-(1-->3)-beta-D-GlcpNAc-(1-->where L-6dTal and D-GlcNAc4(R-Lac) are 6-deoxy-L-talose and 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose, respectively. The latter sugar, which to our knowledge has not been hitherto found in nature, was isolated from the polysaccharide by solvolysis with anhydrous triflic acid and identified by comparison with the authentic synthetic compound. Serological studies with the Smith-degraded polysaccharide showed an importance of 2-substituted GlcA for manifesting of the immunospecificity of P. vulgaris O15.     

Total synthesis and identification of two diastereoisomers of 1-methyl-2-[N-(p-tolylsulfonyl)-2-pyrrolidinyl]ethyl beta-L-fucopyranoside

The reaction of a racemic mixture of (2R,2'S)- and (2S,2'R)-N-(p-tolylsulfonyl)-2-pyrrolidinyl-2-propanol, prepared from (S)-proline, with 2,3,4-tri-O-acetyl-alpha-L-fucopyranosyl trichloroacetimidate led to both diastereoisomers of the title compound after O-deacetylation.     

The identification of (3R,4S)-5-fluoro-5-deoxy-D-ribulose-1-phosphate as an intermediate in fluorometabolite biosynthesis in Streptomyces cattleya

(3R,4S)-5-Fluoro-5-deoxy-D-ribulose-1-phosphate (5-FDRulP) has been identified as the third fluorinated intermediate on the biosynthetic pathway to fluoroacetate and 4-fluorothreonine in Streptomyces cattleya. 5-FDRulP is generated after formation of 5'-fluoro-5'-deoxyadenosine (5'-FDA) and then phosphorolysis of 5'-FDA to 5-fluoro-5-deoxy-D-ribose-1-phosphate (5-FDRP) by the action of a purine nucleoside phosphorylase. An isomerase mediates the conversion of 5-FDRP to 5-FDRulP. The identity of the (3R,4S) diastereoisomer of 5-FDRulP was established by comparative (19)F{(1)H} NMR studies whereby 5-FDRulP that accumulated in a cell free extract of S. cattleya, was treated with a phytase to generate the non-phosphorylated sugar, 5-fluoro-5-deoxy-D-ribulose (5-FDRul). This S. cattleya product was compared to the product of an in-vitro biotransformation where separately 5-fluoro-5-deoxy-D-ribose and 5-fluoro-5-deoxy-D-xylose were converted to 5-fluoro-5-deoxy-D-ribulose and 5-fluoro-5-deoxy-D-xylulose respectively by the action of glucose isomerase. It was demonstrated that 5-fluoro-5-deoxy-D-ribose gave the identical diastereoisomer to that observed from 5-FDRulP.     

Synergism between hydrogen sulfide (H(2)S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom

Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10-320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-N(G)-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and beta-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-gamma-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H(2)S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H(2)S and NO act synergistically in effecting SNTX-induced vasorelaxation.     

Synthesis and evaluation of 6-[1-(2-[(18)F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline for positron emission tomography imaging of the metabotropic glutamate receptor type 1 in brain

The purpose of this study was to synthesize 6-[1-(2-[¹⁸F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline ([¹⁸F]FPTQ, [¹⁸F]7a) and to evaluate its potential as a positron emission tomography ligand for imaging metabotropic glutamate receptor type 1 (mGluR1) in the rat brain. Compound [¹⁸F]7a was synthesized by [¹⁸F]fluorination of 6-[1-(2-bromo-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline (7b) with potassium [¹⁸F]fluoride. At the end of synthesis, 1280-1830 MBq (n = 8) of [¹⁸F]7a was obtained with >98% radiochemical purity and 118-237 GBq/??mol specific activity using 3300-4000 MBq of [¹⁸F]F⁻. In vitro autoradiography showed that [¹⁸F]7a had high specific binding with mGluR1 in the rat brain. Biodistribution study using a dissection method and small-animal PET showed that [¹⁸F]7a had high uptake in the rat brain. The uptake of radioactivity in the cerebellum was reduced by unlabeled 7a and mGluR1-selective ligand JNJ-16259685 (2), indicating that [¹⁸F]7a had in vivo specific binding with mGluR1. Because of a low amount of radiolabeled metabolite present in the brain, [¹⁸F]7a may have a limiting potential for the in vivo imaging of mGluR1 by PET.     

Baclofen-induced,calcium-dependent stimulation of in vivo release ofd-[3H]aspartate from rat hippocampus monitored by intracerebral microdialysis

The release ofd-[³H]aspartate (used as a tracer for endogenous glutamate and aspartate) was studied at high K⁺ (100 mM) and under ischemia in rats implanted with 0.3 mm diameter dialysis tubing through the hippocampus. The effect on thed-[³H]aspartate release of the two -aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) and (±)--(p-chlorophenyl)GABA (baclofen), which specifically activate GABA_A and GABA_B receptors, respectively, was studied. Initial experiments employing HPLC analysis showed a coincident increase in the amounts of glutamate, aspartate and the amount of radioactivity following introduction of K⁺ (100 mM) or a period of ischemia suggesting that thed-[³H]aspartate labels the transmitter pools of the two amino acids under the present experimental conditions. The presence of 10 mM baclofen or 10 mM THIP in the perfusion medium did not inhibit ischemia inducedd-[³H]aspartate release. On the contrary, 10 mM baclofen alone (but not 0.1 or 1 mM) in the perfusion medium induced release ofd-[³H]aspartate in a calcium dependent manner, whereas 10 mM THIP had no significant releasing effect.Special issue dedicated to Dr. Elling Kvamme     

Enzymatic synthesis of D-glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) and NMR analysis of its isomeric forms

D-Glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) was prepared from D-glucosone (D-arabino-hexos-2-ulose) by enzymatic conversion with hexokinase. The isomeric composition of D-glucosone 6-phosphate in aqueous solution was quantitatively determined by NMR spectroscopy and compared to D-glucosone. The main isomers are the alpha-anomer (58%) and the beta-anomer (28%) of the hydrated pyranose form, and the beta-D-fructofuranose form (14%).     

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.     

Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase

The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1-->3)](n)-beta-D-Glcp-MeUmb, where n=1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1-->3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2).