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T Uno

Publications and source records attributed to T Uno.

At least 91 records · Page 5Linked to original sources
PubMed1997

Enantioselective high-performance liquid chromatographic determination of nicardipine in human plasma.

A sensitive method for the enantioselective high-performance liquid chromatography (HPLC) determination of nicardipine in human plasma is described. (+)-Nicardipine, (-)-nicardipine and (+)-barnidipine as an internal standard are detected by an ultraviolet detector at 254 nm. Racemic nicardipine in human plasma was extracted by a rapid and simple procedure based on C18 bonded-phase extraction. The extraction samples were purified and concentrated on a pre-column using a C1 stationary phase and the enantiomers of nicardipine are quantitatively separated by HPLC on a Sumichiral OA-4500 column, containing a chemically modified Pirkle-type stationary phase. Determination of (+)- and (-)-nicardipine was possible in a concentration range of 5-100 ng ml(-1) and the limit of detection in plasma was 2.5 ng ml(-1). The recoveries of (+)- and (-)-nicardipine added to plasma were 91.4-98.4% and 93.3-96.7%, respectively, with coefficients of variation of less than 9.0 and 9.4% respectively. The method was applied to low level monitoring of (+)- and (-)-nicardipine in plasma from healthy volunteers.

Calcium Channel Blockers↗
PubMed1997

Coexistence of calcitonin gene-related peptide and NADPH-diaphorase in the canine superior cervical ganglion.

By means of double staining technique of NADPH-diaphorase (NADPH-d) histochemistry and calcitonin gene-related peptide (CGRP) immunohistochemistry, we investigated the coexistence of NADPH-d reactivity and CGRP immunoreactivity in the canine superior cervical ganglion (SCG). Most of NADPH-d reactivity and CGRP immunoreactivity were coexisted in the principal postganglionic neurons. These neurons were distributed throughout the ganglion without specific localization. The present findings suggest the intimate role of CGRP and nitric oxide in postganglionic neurons of the canine SCG.

Animals↗
PubMed1997

Reciprocal size-effect relationship of the key residues in determining regio- and stereospecificities of DHEA hydroxylase activity in P450 2a5.

Collectively, the P450 2a4/2a5 system hyrdoxylates DHEA in at least three positions (7alpha, 7beta, and 2alpha). An individual P450, however, exhibits high specificity to one of these products. Using site-directed mutagenesis of mP450 2a5 from the wild mouse Mus minutoides and bacterial expression, we have associated the function of residues 117, 209, and 481 with the respective specificity observed in each P450. Ala at position 117 determines the 7beta-hydroxylase activity, whereas Val at this position defines the 2alpha-hydroxylase activity. Leu at position 209 is essential for high DHEA 7alpha-hydroxylase activity. The substitutions of residue 481 with various hydrophobic amino acids elicited a profound alteration of the specific hydroxylation rates, but did not influence the regio- and stereospecificities at either of the three positions of DHEA. The alterations caused by residue 481 also depended on the residue identity at position 117 or 209. The results indicate that the sizes of several key residues obey a concerted reciprocal relationship whereby the substrate pocket of the P450s adjusts to accommodate DHEA. A limited molecular modeling study successfully correlates DHEA binding to experimental DHEA hydroxylase activities for a series of mutants at key positions.

Amino Acid Sequence↗
PubMed1997

The pharmacokinetics of water-in-oil-in-water-type multiple emulsion of a new tacrolimus formulation.

We developed a water-in-oil-in-water-type (W/O/W)-type multiple emulsion of a new tacrolimus formulation. A potential approach to avoid the complications of systemic immunosuppression and simultaneously enhance immunosuppressive efficacy is to deliver immunosuppressive agents locally to the site of the target organs. The W/O/W emulsion is dispersed oil drops containing smaller water droplets that allow the delivery of drugs preferentially to the reticuloendothelial systems (RES). Since the liver and the spleen are primary components of the RES, and the brain and the kidney have a poor RES, we hypothesized that a W/O/W emulsion of tacrolimus would possess the pharmacokinetic benefits of local immunosuppression. We evaluated this hypothesis in a rat model. The tacrolimus levels of whole blood, the liver, spleen, brain, and kidney in rats given intravenous emulsions of tacrolimus (W/O/W group) were compared with a group administered tacrolimus alone (T group). There were no significant differences between the pharmacokinetic parameters of W/O/W group and T group based on whole blood data. However, the W/O/W group had significantly decreased tacrolimus levels in the brain and kidney, and significantly increased levels in the liver and spleen compared with the T group. These data suggest that the W/O/W emulsion is applicable as an intravenous drug carrier for local immunosuppression.

Animals↗
PubMed1997

Expression of MAGE genes in ocular melanoma during progression from primary to metastatic disease.

Primary melanomas that form within the eye have a unique pattern of disease progression as compared with melanomas that form within the skin. A high percentage of patients (approximately 50%) develop metastatic tumors that occur predominately in the liver. An unusual characteristic of ocular melanomas is the prolonged disease-free interval that extends for many years between the development of primary and metastatic tumors. It is estimated that the shortest interval between dissemination of tumor cells from the eye and the appearance of clinically detectable metastases is 6 years. A recent report indicated that fresh uveal melanoma tissue and metastatic tumor biopsies failed to express melanoma antigen gene (MAGE)-1, MAGE-2, or MAGE-3. In the present study, we examined the expression of MAGE genes on fresh and cultured tumor cells obtained from an ocular melanoma patient during different stages of progressive disease. MAGE gene expression was determined by reverse transcription-polymerase chain reaction using MAGE-1, MAGE-2 and MAGE-3 specific primers. Our results demonstrate that primary ocular tumor tissue and cultured tumor cells both express significant levels of MAGE-1, 2, and 3 at the time of enucleation. A high percentage of tumor cells within the primary tumor appear to express MAGE as demonstrated by consistent MAGE expression in 16 tumor cell clones. Metastatic liver tumors that developed 3 years after enucleation and 18 years after the initial formation of the primary tumor also expressed high levels of MAGE-1, -2, and -3. MAGE was expressed on fresh tumor tissue from a single biopsy and cultured tumor cells obtained from three of four different metastatic tumor nodules. When the MAGE-negative metastatic tumor cells were treated with the demethylating agent 5-Aza-2-Deoxycytidine (5-Aza-dC), transcription of MAGE-1 was restored, indicating the MAGE genes were not deleted. Our results demonstrate that in some patients, MAGE genes are expressed on primary and metastatic ocular melanomas.

Antigens, Neoplasm↗
PubMed1997

Participation of leukotriene D4 and tumor necrosis factor on lipopolysaccharide-induced airway hyperresponsiveness in guinea pigs.

In guinea pigs, a marked increase in airway responsiveness to acetylcholine (Ach) was observed at 2 h after lipopolysaccharide (LPS) inhalation. To examine the mediators responsible for the airway hyperresponsiveness, the changes of peptide-leukotrienes (LTs), tumor necrosis factor (TNF), interleukin-1 (IL-1), histamine and 5-hydroxytryptamine (5-HT) levels in bronchoalveolar lavage fluid (BALF) were measured. Airway responsiveness to Ach reached a peak 2 h after LPS inhalation. The influx of neutrophil into BALF increased gradually and reached a peak 24 h after LPS inhalation. After the inhalation of LPS, LTD4 and TNF contents in BALF increased within the first 2 h after LPS inhalation. However, other mediators were not detected or increased 6 h after LPS inhalation. Aeroinhalation of LTD4 and murine recombinant TNF-alpha caused airway hyperresponsiveness in guinea pigs. In addition, a LTD4 antagonist, BAYx7195, and an inhibitor of TNF, pentoxifylline, inhibited the LPS-induced airway hyperresponsiveness. These results suggest that LTs and/or TNF play an important role in the onset of airway hyperresponsiveness in guinea pigs.

Acetylcholine↗
PubMed1997

Local control of piriform sinus cancer treated by radiation therapy alone.

Local control rate by radical radiation therapy was analyzed in 33 patients with a piriform sinus cancer. Twenty-five patients (76%) were in stage T3 + T4. Local recurrence-free survival at 3 years was 49% in T1 + T2 and 25% in T3 + T4 (p = 0.01). In T1 + T2 lesions, a biologically effective dose for an acute reaction over 80 Gy and total treatment time less than 70 days appeared to improve local control. In T3 + T4 lesions, good radiation response assessed by the regaining of laryngeal mobility affected local control favorably. An esophageal involvement and destruction of the laryngeal cartilage as well as soft tissue extension precluded the possibility of local control by radiation therapy alone. In addition to the T-stage, other tumor factors should also be considered for predicting local control with radiation therapy.

Aged↗
PubMed1997

Gene transfer of the CD80 costimulatory molecule into ocular melanoma cells using a novel episomal vector.

PURPOSE: The CD80 (B7.1) molecule, which is a necessary costimulatory signal for T-cell activation and proliferation, is a powerful inducer of antitumor immunity. In this study, primary human ocular melanoma cells were transfected with a novel vector (B45-Neo episomal vector) containing the complementary DNA (cDNA) for human CD80 to determine if this vector system is useful for stimulating CD8+ T cells. METHODS: Ocular melanoma cells were transfected with the B45-Neo episomal vector containing the cDNA for human CD80 and were positively selected in medium containing geneticin. The transcription of plasmid cDNA, plasmid copy number, and cell surface expression were determined on transfected tumor cell lines, and cloned tumor cells were obtained by limiting dilution techniques. The stability of CD80 expressed on tumor cells was determined after prolonged culture without geneticin and on irradiated cells. Autologous lymphocytes were restimulated with CD80+ tumor cells in the presence of recombinant interleukin-2 to determine whether CD8+ T cells were stimulated. RESULTS: CD80 was expressed on tumor cells transfected with the B45-Neo vector containing the cDNA for CD80. The level of CD80 expressed on different transfected tumor cell lines was heterogeneous and dependent on the plasmid copy number. High CD80 expression was observed on cloned tumor cells that possessed more than 520 plasmids per cell; intermediate levels were observed on tumor cells with approximately 240 to 520 plasmids. CD80+ ocular melanoma cells maintained a stable CD80 expression even after prolonged culture without geneticin, and on irradiated tumor cells. CD80 expressed on tumor cells was biologically functional and stimulated autologous CD8+ cells. CONCLUSIONS: The B45-Neo episomal vector induces stable expression of the CD80 costimulatory molecule on ocular melanoma cells. Our results indicate that this vector is suitable for experiments designed to genetically engineer ocular melanoma cells to stimulate CD8+ T cells.

B7-1 Antigen↗
PubMed1996

Liquid chromatographic determination of manidipine in serum with electrochemical detection.

A highly sensitive and selective method for determining a dihydropyridine calcium antagonist, manidipine, by liquid chromatography using column switching with electrochemical detection was developed. Manidipine in serum was extracted by a rapid and simple procedure based on C8 bonded-phase extraction and then silica extraction. Manidipine and nilvadipine as an internal standard were separated on a C8 bonded-phase HPLC column and detected by high conversion efficiency amperometric detection at +0.7 V. Manidipine and nilvadipine (I.S.) were separated from an endogenous interference peak in serum and concentrated on a pre-column (C18) by column switching using an isocratic mobile phase, and then the corresponding fractions were introduced to an analytical column with a C8 stationary phase. Determination of manidipine was possible over the concentration range 0.5-10 ng/ml: the limit of detection was 0.3 ng/ml. The recovery of manidipine added to serum was 93.1-98.4% with coefficients of variation of less than 7.1%. The method is applicable to drug level monitoring in the serum of healthy volunteers treated with manidipine and to the analysis of pharmacokinetics.

Calcium Channel Blockers↗
PubMed1996

Relationship of neuropeptides to nitrergic innervation of the canine laryngeal glands.

The participation of vasoactive intestinal polypeptide (VIP) or calcitonin gene-related peptide (CGRP) in the nitrergic innervation of the canine laryngeal glands was investigated using a double-staining technique of NADPH-diaphorase (NADPHd) histochemistry and VIP or CGRP immunohistochemistry. NADPHd-positive nerve fibers with varicosities appeared to terminate in some acinar cells. Double staining revealed that NADPHd reactivity and VIP- or CGRP-like immunoreactivity were colocalized in some nerve fibers distributed around the acini. A cluster of NADPHd-positive cells were occasionally found in the larynx. Many NADPHd-positive cells had VIP-like immunoreactivity and no NADPHd-positive cells were CGRP-like immunoreactive. These findings suggest that nitric oxide participates in the neural control of the laryngeal exocrine secretion in cooperation with intrinsic VIP and/or extrinsic CGRP.

Animals↗
PubMed1996

Tyrosine hydroxylase-immunoreactive cells in the nodose ganglion for the canine larynx.

Several substances have been reported as candidates for the neurotransmitter in the laryngeal afferent system. In the present study we demonstrated that catecholamine is also a candidate neurotransmitter in the canine laryngeal afferent system using tyrosine hydroxylase (TH) immunochemistry in combination with retrograde labelling with cholera toxin B in subunit-conjugated gold (CTBG). A few cells in the nodose ganglion labelled by application of CTBG to the internal branch of the superior laryngeal nerve were also TH-immunoreactive. These cells were also labelled following application of CTBG to the nucleus of the solitary tract. These results indicate that some of the TH-IR cells in the nodose ganglion could be primary afferent neurones for the canine larynx.

Animals↗
PubMed1996

Nitrergic neurons in the canine intrinsic laryngeal muscle.

Nitrergic ganglionic cells located in the canine intrinsic laryngeal muscle were studied by NADPH-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. Cells intensely stained by NADPH-d histochemistry were found between the striated muscle fibers of the intrinsic laryngeal muscle. Most of these cells were bipolar or pseudounipolar in form. Some NADPH-d negative cells were observed to be enveloped in a mesh by varicose NADPH-d positive nerve fibers. The findings obtained by nNOS immunohistochemistry corresponded well with those obtained by NADPH-d histochemistry, indicating that NADPH-d activity in the ganglion in the intrinsic laryngeal muscle is nNOS. The present findings clearly indicate that some of the ganglion cells located in the canine intrinsic laryngeal muscle are nitrergic, and that the ganglionic cells synapse together with the participation of nitric oxide in integrating ganglionic cells.

Animals↗
PubMed1996

Effect of a novel leukotoriene synthesis inhibitor, BAY x1005, on the antigen-and LPS-induced airway hyperresponsiveness in guinea pigs.

Due to the inhibition of 5-lipoxygenase-activating protein (FLAP), BAY x1005 is a new selective inhibitor of leukotriene synthesis. The effects of BAY x1005 on the antigen- and bacterial lipopolysaccharide (LPS)-induced airway hyperresponsiveness in guinea pigs were investigated. Six times provocation of aeroantigen caused biphasic increases in airway resistance which peaked at 1 hr (immediate phase reaction) and 4 hrs (late phase reaction). It also caused airway hyperreactivity to acetylcholine. BAY x1005 at doses of 10 mg/kg and 30 mg/kg significantly inhibited antigen-induced increase in respiratory resistance (Rrs) at 1 and 4 hrs after the last antigen challenge. Simultaneously, BAY x1005 inhibited the antigen-induced airway hyperresponsiveness at doses of 10 and 30 mg/kg and airway eosinophilia (bronchoalveolar lavage study) at a dose of 30 mg/kg. In addition, BAY x1005 at a dose of 30 mg/kg inhibited bacterial LPS-induced airway hyperreactivity to acetylcholine. In this model, BAY x1005 did not affect the increase of the number of leukocytes in bronchoalveolar lavage fluid. These results suggest that BAY x1005 is a potent anti-asthmatic agent with an inhibitory action to airway hyperreactivity.

Acetylcholine↗
PubMed1996

Cysteinyl leukotrienes do not mediate lipopolysaccharide-induced airway hyperresponsiveness in guinea pigs.

Inhalation of bacterial lipopolysaccharide (LPS) by guinea pigs caused bronchial hyperreactivity to acetylcholine with a peak at 2 hr after exposure. Exposure to 0.01% LPS for 30 min resulted in an elevation of cysteinyl leukotrienes (cys-LTs) content in bronchoalveolar lavage fluid (BALF) which was obtained 1 hr after LPS exposure. The cys-LTs antagonist, ONO-1078 (10 mg/kg, p.o.), significantly inhibited LPS-induced bronchial hyperreactivity, but ICI-204,219 (10 mg/kg, p.o.), another cys-LT antagonist, did not. Each dose employed in the present study was sufficient to inhibit LTD4-induced broncho-constriction in guinea pigs. In order to investigate the inhibitory mechanism of ONO-1078, the effect on the LPS-induced production of tumor necrosis factor (TNF) was examined. The amount of TNF in BALF increased significantly 2 hr after exposure to LPS. The inhalation of murine recombinant TNF-alpha (5 x 10(4) u/ml) resulted in bronchial hyperreactivity in guinea pigs. ONO-1078 (10 mg/kg, p.o.) inhibited the increase of LPS-induced TNF in BALF, but ICI-204,219 (10 mg/kg, p.o.) had no effect. These results suggest that TNF plays an important role in the onset of LPS-induced bronchial hyper-reactivity, and that ONO-1078 inhibits the LPS-induced airway hyperreactivity probably due to the inhibition of TNF production.

Acetylcholine↗
PubMed1996

The roles of individual amino acids in altering substrate specificity of the P450 2a4/2a5 enzymes.

A single amino acid substitution is sufficient to alter substrate specificity of P450 enzymes. Mouse P450 2a5, for example, has its substrate specificity converted from coumarin 7- to testosterone 15 alpha-hydroxylase activity by the substitution of Phe at position 209 to Leu. Furthermore, placing Asn at this position confers a novel corticosterone 15 alpha-hydroxylase activity to this P450. Recent site-directed mutational studies show the presence of the topologically common residues, each of which can determine the specificities of various mammalian P450s. For instance, residue 209 (in 2a5) corresponds to a residue at position 206 in rat P4502B1 that regulates its steroid hydroxylase activity. High substrate specificity often observed in an individual P450, therefore, can be determined and altered by the identities of a few critical residues. The structural flexibility of the substrate-heme pocket may also provide P450 enzymes with the ability to display a broad range of substrate specificities. Understanding the underlying principles whereby the flexible pocket determines P450 activities may lead us to the prediction of P450 activities based on the identities of key amino acid residues.

Amino Acids↗