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Biomedical subjects

N Mano

Publications and source records attributed to N Mano.

At least 19 recordsLinked to original sources
PubMed2002

Affinity assembled multilayers for new dehydrogenase biosensors.

We propose a novel approach, which allows the control of the spatial arrangement of redox mediator, coenzyme and enzyme on the electrode at a molecular level, using essentially electrostatic interactions. The first step consists of adsorbing a monolayer of molecules out of a new family of redox mediators, substituted nitrofluorenones. In a second step, a monolayer of calcium cations is immobilized at the interface. It serves as a bridge between the redox mediator and the subsequently adsorbed coenzyme. The weak interaction between a carboxyl group of the redox mediator and the coenzyme's phosphate groups, revealed by QCM measurements, allows the coenzyme to keep its natural activity in the adsorbed state. In the last step, we use the intrinsic affinity of this monolayer of NAD(+) for dehydrogenases to build up a supramolecular sandwich composed of mediator/Ca(2+)/NAD(+)/dehydrogenase. This simple modification procedure, which might constitute a versatile approach for the low cost assembly of well-defined biosensors surfaces, has been successfully applied to the enzymatic detection of glucose, glutamate and alcohol.

Biosensing Techniques↗
PubMed2002

Mediator-modified electrodes for catalytic NADH oxidation: high rate constants at interesting overpotentials.

Carbon paste electrodes were modified with a nitrofluorenone derivative, 2,4,7-trinitro-9-fluorenone, adsorbed on zirconium phosphate (ZP). After electrochemical reduction of the fluorenone derivative, it turns into a very efficient mediator for electrocatalytic NADH oxidation, with a formal potential of about +250 mV vs. Ag/AgCl. The electrochemistry and the electrocatalytic properties of the mediator were investigated with cyclic voltammetry and rotating disk electrode methodology. The second order rate constant with NADH was evaluated and found to be higher than 10(6) M(-1) s(-1), thus approaching true diffusion controlled currents for NADH oxidation.

Carbon↗
PubMed2001

The enantioselective immunoaffinity extraction of an optically active ibuprofen-modified peptide fragment.

Acyl glucuronides are known to produce the covalently bound protein adducts which may be the cause of hypersensitivity and toxic responses to acidic drugs. The structural analysis of the drug-protein adducts is therefore needed. From this point of view, we developed an enantioselective immunoaffinity extraction method, which employs an immobilized antibody to specifically isolate peptide fragments that have been modified with optically active ibuprofen. Rabbits were immunized with (S)-ibuprofen coupled to bovine serum albumin through a beta-alanine group. The elicited antibody strongly recognizes the asymmetric center and the isobutylphenyl moiety of (S)-ibuprofen and its conjugates but has a low affinity for their anti podes. A 0.5-mL aliquot of the immunosorbent (11.5 mg of IgG/mL gel) prepared by immobilization of the antibody was capable of retaining up to 1 microg of (S)-ibuprofen. When a mixture of substance P with (R)- and (S)-ibuprofen-modified substance P was loaded on the immunosorbent, the (S)-ibuprofen-modified substance P was selectively retained. The modified peptide was quantitatively recovered by elution with 10 mM ammonium acetate buffer (pH 5.0)/methanol (5:95, v/v). The proposed method would be useful for the structural characterization of optically active ibuprofen-modified human serum albumin.

Antibodies↗
PubMed2001

Bile acid acyl adenylate: a possible intermediate to produce a protein-bound bile acid.

The non-enzymatic production of a protein-bound adduct by the action of the acyl adenylate of bile acids is described. On incubation of deoxycholyl adenylate with substance P in phosphate buffer, peptides covalently bound with one or two molecules of the bile acid were detected. The modified peptides were structurally characterized by time-of-flight mass spectrometry with matrix-assisted laser desorption/ionization (MALDI-TOFMS) in the post-source decay mode, and by liquid chromatography/electrospray ionization MS/MS. The deoxycholic acid was bound on substance P through the amino group at Arg-1 and/or Lys-3. The adenylate of cholic acid also produced the protein-bound bile acid on incubation with lysozyme, and the binding sites of the cholic acid appeared to be the lysine residues at 1, 33, 97 and 116. The results clearly suggest that bile acid adenylates in vivo may act as active intermediates to produce covalently bound bile acid adducts with peptides and proteins by nucleophilic displacement of the 5'-adenylic acid through the free amino groups.

Adenosine Monophosphate↗
PubMed2001

Electrodes modified with nitrofluorenone derivatives as a basis for new biosensors.

We report about the electrocatalytic properties of electrodes modified by adsorption of nitro-fluorenone derivatives. The stable, adherent monolayer of these catalyst precursors can be transformed electrochemically into the corresponding hydroxylamine compounds (R-NO(2)+4e+4H(+)-->R-NHOH+H(2)O). The completely reversible two electron oxidation of the hydroxylamine leads to the nitroso compounds (R-NHOH-->R-NO+2e+2H(+)) that exhibit high catalytic activity in the electrooxidation of NADH at low overpotentials (-30 mV vs. Ag/AgCl) and therefore constitute a new family of efficient redox mediators for biosensor applications. A significant increase in catalytic activity (up to 500%) is observed after addition of calcium ions to the electrolyte. This is explained by a specific and bridging complexation between the coenzyme's phosphate groups and a carboxyl group present in the catalyst molecule. The interaction favours the contact between NADH and the surface confined catalyst, leading to a higher electron transfer efficiency. This interaction can be used in an approach of molecular level design for controlled monolayer deposition of catalyst, Ca(2+), NAD+ and enzyme. A very simple and inexpensive modification scheme, essentially based on electrostatic attraction, leads to electrodes that can be employed as reagentless biosensors for the electrochemical detection of common and commercially interesting analytes like glucose.

Biosensing Techniques↗
PubMed2001

Sequelae after limb-sparing surgery with major vascular resection for tumor of the lower extremity.

PURPOSE: Limb-sparing procedures have recently replaced amputations as the treatment for tumors invading major vessels of the lower extremity. Major arteries must be reconstructed for limb salvage. The veins are not usually reconstructed. This study was undertaken to investigate the sequelae such as chronic venous disease after venous resection for tumors. METHODS: Ten patients who underwent limb-sparing surgery for a tumor of the lower extremity or retroperitoneum that required major vascular resection were studied. The median follow-up period was 48 months. After combined resection of a major artery and vein, arterial reconstruction was performed. The veins were not reconstructed. The resected veins included the inferior vena cava (n = 2), the external iliac and common femoral veins (n = 3), the superficial femoral vein (n = 3), and the popliteal vein (n = 2). The main outcome measures were clinical classification of chronic venous disease in 10 patients and air plethysmography in seven patients. RESULTS: Clinical classification was C(0A) in 6 patients, C(3A) in 1 patient, C(3S) in 2 patients, and C(4S) in 1 patient. Venous claudication with uncontrollable edema was observed in two patients with C(3S) disease. Pain and itching with inflammatory skin changes were observed in one patient with C(4S) disease. These three patients had undergone resection of the femoral vein, including the deep femoral vein along with proximal adductor muscles. Air plethysmography revealed that the ejection fraction was significantly lower and the residual volume fraction was significantly higher in the three patients with symptoms than in symptom-free patients. CONCLUSIONS: Significant chronic venous disease was observed in the patients who underwent combined resection of the femoral vein, the deep femoral vein, and the adductor muscles for a tumor.

Adult↗
PubMed2001

Simultaneous detection of cholyl adenylate and coenzyme A thioester utilizing liquid chromatography/electrospray ionization mass spectrometry.

It has been proposed that acyl adenylate is first formed during activation of the carboxy group into the acyl CoA thioester, an intermediate in the formation of amino acid conjugates. Acyl CoA synthetases may be responsible for this acyl adenylate formation. Recently, we hypothesized the preferential formation of cholic acid adenylate, a major bile acid, preceding production of the corresponding CoA thioester in incubations with rat liver microsomal fractions. To verify this biosynthetic mechanism, monitoring of the incubation mixture of acyl adenylate together with both substrate and acyl CoA thioester is needed. We have developed a detection method for the simultaneous detection of these cholic acid derivatives utilizing liquid chromatography/electrospray ionization mass spectrometry. The CoA thioester of cholic acid forms a chelation complex with the divalent cations remaining on the silica gel packed into the analytical column. Both the addition of a chelating agent, such as EDTA, to the mobile phase and an adjustment of the mobile phase pH to a weak alkaline effectively removed such chelate formation, producing a sharp CoA thioester peak. For a simultaneous mass spectrometric analysis of cholic acid, the corresponding adenylate and CoA thioester, the combined use of a 300 A particle diameter ODS column and 20 mM ammonium acetate buffer (pH 9.0)/2-propanol/acetonitrile as the mobile phase have been proved to be preferable. To avoid any degradation of the chemically unstable adenylate produced in the incubation, we employed a direct injection of the sample onto a preconcentration column. The obtained results indicated a high sensitivity of this method.

Adenosine Monophosphate↗
PubMed2001

Direct injection method for quantitation of endogenous leukotriene E4 in human urine by liquid chromatography/electrospray ionization tandem mass spectrometry with a column-switching technique.

A new analytical method employing liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a column-switching system was developed for quantitative determination of leukotriene E4 (LTE4) in human urine. A column-switching system using a trapping column, which concentrates the analyte and removes salts and other water-soluble contaminants, allowed direct injection of human urine. Because simultaneously eluted endogenous contaminants suppressed the ionization efficiency of LTE4, good liquid chromatographic separation was very important for establishing this method, notwithstanding the high selectivity of MS/MS. The calibration curve was linear over the range from 10 to 3000 pg/mL, and the method showed good accuracy and precision. This method should therefore be very useful for determination of LTE4 amounts in human urine in studies on leukotriene metabolism and the efficacy of antileukotriene drugs.

Chromatography, Liquid↗
PubMed2000

Infection with Edwardsiella tarda causes hypertrophy of liver cells in the Japanese flounder Paralichthys olivaceus.

To study the direct cause of liver enlargement in the Japanese flounder Paralichthys olivaceus infected with Edwardsiella tarda, the fish were challenged with E. tarda and reared without feeding. The liver of fish exposed to the bacteria was markedly enlarged compared to that of the controls while no severe histopathological change appeared in the organ during the experiments. No notable difference was observed in the crude fat, glycogen, and water content of the liver between challenged and control fish. The size of liver cells and nuclei of the challenged fish was apparently larger than that of the controls. Analysis of crude DNA in the liver suggested that the number of liver cells of starved control fish significantly decreased during the experiment while that of the challenged fish was maintained at a level of the initial control. RNA/DNA ratio of the liver of challenged fish clearly increased while it decreased in the control fish during the experiment. These observations suggest that liver enlargement of flounder infected with E. tarda, at least in the early stage of infection, is not a result of any readily observable histopathological changes and that E. tarda infection causes hypertrophy of the cells, as well as preventing decrease in liver cell number.

Animals↗
PubMed2000

Characterization of Japanese eel immunoglobulin M and its level in serum.

Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790,000; it contained an equimolar heavy chain and light chain with molecular weights of 72,000 and 25,000, respectively. While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains. Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by D-mannose and could be abolished by alpha-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses. The average IgM concentration in the sera of the healthy eels was 3.4 mg ml(-1); it amounted to 10.3% of the total serum protein.

Amino Acid Sequence↗
PubMed1998

Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid CoA thioester.

The substrate specificity of rat liver peroxisomal 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) CoA thioester, was studied. The stable isotope labeled substrates, [3,7,12-18O3]-(25R)- and (25S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on a steroid nucleus of 3,7,12-trioxo-5 beta-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25R)- and 18O-labeled (25S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed delta 24-THCA was determined by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. The delta 24-THCA was derived only from (25S)-THCA CoA thioester, indicating that the 25S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases.

Animals↗
PubMed1998

Structural analysis of N-linked carbohydrate chains of funnel web spider (Agelenopsis aperta) venom peptide isomerase.

The structure of the N-linked carbohydrate chains of peptide isomerase from the venom of the funnel web spider (Agelenopsis aperta) has been analyzed. Carbohydrates were released from peptide isomerase by hydrazinolysis and reductively aminated with 2-aminopyridine. The fluorescent derivatives were purified by phenol/chloroform extraction, followed by size-exclusion HPLC. The structure of the purified pyridylamino (PA-) carbohydrate chains were analyzed by a combination of two-dimensional HPLC mapping, sugar composition analysis, sequential exoglycosidase digestions, and mass spectrometry. The peptide isomerase contains six kinds of N-linked carbohydrate chains of truncated high-mannose type, with a fucose alpha 1-6 linked to the reducing N-acetylglucosamine in approximately 80% of them.

Amino Acid Isomerases↗
PubMed1997

Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ionspray ionization tandem mass spectrometry.

Sphingolipid metabolites ceramide, sphingomyelin, sphingosine, psycosine, sphingosylphosphorylcholine, and dimethylsphingosine were separated and simulataneously quantitated by liquid chromatography/ionspray ionization tandem mass spectrometry (LC/MS/ MS). The use of glassware throughout minimized losses due to adsorption and the pretreatment of this method consisted of simple liquid-liquid extraction procedure with a mixture of chloroform and methanol. After separation on a short C18 silica column eluted in a gradient mode, the metabolites were detected by MS/ MS. This assay allows simultaneously quantification of these metabolites over a range of at least 0.1 to 100 ng/ 10(6) cells. The LC/MS/MS analyses took 10 to 15 min per sample and we could examine up to 50 samples per day. We also detected endogenous sphingosine 1-phosphate in HL-60 cells. The utility of the method was demonstrated by examining changes in metabolites levels in HL-60 cells after treatment with sphingomyelinase. It was found that sphingomyelinase from Bacillus cereus may have selectivity for acyl chain length.

Ceramides↗
PubMed1996

Vitamin B12 affects non-photic entrainment of circadian locomotor activity rhythms in mice.

Administration of vitamin B12 (VB12) has been reported to normalize human sleep-wake rhythm disorders such as non-24-h sleep-wake syndrome (HNS), delayed sleep phase syndrome (DSPS) or insomnia. However, the mechanisms of the action of VB12 on the rhythm disorders are unknown. In the present study, therefore, effects of VB12 on circadian rhythms of locomotor activity were examined in mice. In the first experiment, CBA/J mice were maintained under continuous light condition (LL) or blinded, and after free-running rhythms became stable, the mice were intraperitoneally injected with either VB12 or saline at a fixed time every day. In all the mice with tau > 24 h, saline injections resulted in entrainment of circadian rhythms, whereas not all the mice with tau < 24 h entrained to the injection. In contrast to saline injections, VB12 injections did not always induce entrainment and about half of the mice with tau > 24 h free-ran during the injection. In the second experiment, the amount of phase advances of circadian rhythms induced by a single injection of saline at circadian time (CT) 11 under LL was compared between the mice with and without VB12 silastic tubes. The results showed that the amplitude of phase advances was smaller in the mice with VB12 than those without VB12. In the third experiment, daily injections of saline were given to the mice with VB12 silastic tubes maintained under LL. In this chronic treatment of VB12 as well, attenuating effects of VB12 on saline-induced entrainment were observed. These results suggest that VB12 affects the mechanisms implicated in non-photic entrainment of circadian rhythms in mice.

Animals↗
PubMed1996

Context dependent discharge characteristics of saccade-related Purkinje cells in the cerebellar hemispheres of the monkey.

In the previous paper (Mano et al, 1991), we reported the discharge patterns of saccade-related Purkinje cells during visually guided saccade task, which were recorded from posterior cerebellar hemisphere, the Crus IIa. In the present study, we analysed these P-cell's simple spike activity during the spontaneous saccade in inter-trial intervals (ITI) of visually guided saccade task, comparing with the activity during the visually guided saccade. We found that the modulation of simple spike discharges during spontaneous saccade was weaker than the modulation during the visually triggered saccade. We recorded single unit discharges of Purkinje cells from cerebellar posterior hemisphere (Crus IIa) in awake Japanese monkeys (Macaca fuscata), trained to perform simple reaction time saccade task gazing at a small light rear-projected on to a tangent screen 54 cm in front of the monkey. Horizontal and vertical eye positions were measured by a corneal search coil method. Comparison of simple spike activity associated with spontaneous saccade during ITI to the activity during visually triggered saccade clarified that the discharge patterns of simple spikes are basically the same during both types of the saccades, but the amount of the phasic modulation (increase of decrease of discharge rate) were larger for all directions (up, down, left and right) during visually guided saccade than that during spontaneous saccade in all saccade-related Purkinje cells so far examined in two monkeys. The modulation, however, cannot be assumed to have been induced by the visual stimulus per se. Because, the maximum increase of simple spike discharge rate aligned at saccade onset is larger than that aligned at target jump. And, the half width of the change was wider when aligned at target light jump than when aligned at the onset of saccades, in all the four directions, indicating the changes of the firing rate were more time-locked to the onset of saccadic eye movements than to the triggering visual stimulus. The present findings suggest that the cerebellar hemisphere plays a more important role in the control of externally triggered voluntary eye movements than in the control of self-initiated, self-paced eye movements. We discussed these findings combining with previous findings on limb movement-related P-cells (Mano et al, 1980, 1986, 1989), from view point of the general role of the cerebellar hemisphere in the control of voluntary movements.

Animals↗
PubMed1996

Plasma direct injection high-performance liquid chromatographic method for simultaneously determining E3810 enantiomers and their metabolites by using flavoprotein-conjugated column.

A high-performance liquid chromatographic method using the column-switching technique was developed for the simultaneous determination of E3810 and its metabolites (M1-M4). An avidin column was used for in-line pretreatment to exclude plasma proteins, allowing direct injection of a large volume of plasma. A flavoprotein-conjugated chiral stationary phase in a gradient elution mode then gave baseline separation of E3810 and the four metabolites. The enantiomers of E3810 and M3 were also separated. The method is simple, rapid, accurate, and precise. Since no extraction procedure is employed, which might involve different recoveries of different metabolites, no internal standard is necessary. The method was applied to analyze E3810 and its metabolites in plasma after intravenous injection of the drug in beagle dogs.

2-Pyridinylmethylsulfinylbenzimidazoles↗