Biomedical subjects

S Tokuyama

Publications and source records attributed to S Tokuyama.

At least 55 records · Page 3Linked to original sources

PubMed1996

Inhibitory effects of diltiazem, an L-type Ca2+ channel blocker, on naloxone-increased glutamate levels in the locus coeruleus of opioid-dependent rats.

To investigate the effects of diltiazem, an L-type Ca2+ channel blocker, on naloxone-precipitated withdrawal and elevations of glutamate levels in the locus coeruleus (LC) of opioid-dependent rats, animals were continuously infused with 26 nmol/microliter/h of morphine or butorphanol intracerebroventricularly (i.c.v.) via osmotic minipumps for 3 days. I.c.v. injection of naloxone (an opioid-receptor antagonist, 48 nmol/5 microliters) precipitated withdrawal signs and increased extracellular fluid levels of glutamate in the LC of morphine- or butorphanol-dependent rats measured by in vivo microdialysis. Meanwhile, concomitant infusion of opioids with diltiazem (10 or 100 nmol/microliter/h) inhibited the withdrawal signs and prevented the elevations of glutamate levels in the LC. These results suggest that an expeditious release of glutamate in the LC region regulated by L-type Ca2+ channels mediated system plays a role in the expression of withdrawal signs from opioids.

Animals↗

PubMed1996

Disialosyl galactosylgloboside as an adhesion molecule expressed on renal cell carcinoma and its relationship to metastatic potential.

Aberrant glycosylation expressed in specific types of human cancer may define stage, direction, and fate of tumor progression. Well-studied examples are expression of sialosyl-Lewis(x) or sialosyl-Lewis(a) in colorectal carcinoma and histo-blood group A and H/Le(y) in lung cancer. In renal cell carcinoma (RCC), expression of sialosyl-Lewis(x) has no correlation with metastatic potential. Clinicopathological studies have revealed that the degree of expression of disialosyl galactosylgloboside (DSGG) and monosialosyl galactosylgloboside is correlated with metastatic potential (to lung and lymph nodes) of RCC and inversely correlated with patient survival. In the present study, we compared the adhesion of RCC lines to sections of various tissues measured by Stamper-Woodruff assay and other similar assays under dynamic flow conditions. Of the eight RCC lines tested, only TOS-1 (which expresses DSGG) bound strongly to lung tissue sections. TOS-1 did not bind to sections of liver, kidney, or lymph nodes. In the same eight RCC lines, we also compared expression of DSGG and monosialosyl galactosylgloboside (reflected by reactivity with RM1 and RM2), overall ganglioside patterns, and correlation with lung tissue-binding ability. Under both static and dynamic flow conditions, the binding of TOS-1 cells to lung alveolar tissue was correlated with their DSGG expression, i.e., the binding was inhibited by RM2 but not by RM1. This binding was also inhibited by sialidase but not by EDTA (i.e., it was CA 2+ independent). The other seven cell lines (TOS-2, TOS-M, SMKT-R1, -R2, -R3, and -R4, and ACHN), which do not express DSGG, showed much weaker adhesion to lung tissue. None of the eight cell lines showed E- or P-selectin-dependent adhesion. These results suggest the existence of a yet-uncharacterized sialoadhesive receptor++ that specifically recognizes DSGG. This receptor could be the binding target in RCC metastasis to lung.

Carbohydrate Conformation↗

PubMed1996

Direct evidence for a role of glutamate in the expression of the opioid withdrawal syndrome.

To investigate the role of glutamate in the expression of the withdrawal signs from opioids, rats were intracerebroventricularly (i.c.v.) infused continuously with morphine (a mu-opioid receptor agonist, 26 nmol/microliters per h) or butorphanol (a mixed mu/delta/kappa-opioid receptor agonist, 26 nmol/microliters per h) through osmotic minipumps for 3 days. An i.c.v. injection of glutamate (5 and 50 nmol/5 microliters) dose dependently induced withdrawal signs in morphine- or butorphanol-dependent animals. The withdrawal signs precipitated by the glutamate injection were comparable to those precipitated by an opioid receptor antagonist, naloxone (48 nmol/5 microliters), except for the expression of some specific behaviors and the duration of withdrawal signs. Glutamate or naloxone challenge failed to precipitate any withdrawal signs in saline controlled animals. On the other hand, the expression of the withdrawal signs precipitated by glutamate or naloxone in opioid-dependent animals was completely blocked by pretreatment with MK-801 [a NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptan-5,10-imine], 0.1 mg/kg, i.p. These unique actions of glutamate in continuously opioid-infused rats suggest that a rapid central release of glutamate may be a key factor in the expression of withdrawal signs from opioids. Furthermore, this effect may be mediated by the NMDA subtype of glutamate receptors.

Animals↗

PubMed1996

Overexpression of the gene for N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli and continuous produciton of optically active methionine by a bioreactor.

A DNA sequence encoding N-acylamino acid racemase (AAR) was inserted downstream from the T7 promoter in pET3c. The recombinant plasmid was introduced into Escherichia coli MM 294 lysogenized with a bacteriophage lambda having a T7 RNA polymerase gene. The amount of AAR produced by the E. coli transformant was 1100-fold more than that produced by Amycolatopsis sp. TS-1-60, the DNA donor strain. The AAR was purified to homogeneity from the crude extract of the E. coli transformant by two steps: heat treatment and Butyl-Toyopearl column chromatography. Bioreactors for the production of optically active amino acids were constructed with DEAE-Toyopearl-immobilized AAR and D- or L-aminoacylase. D- or L-methionine was continuously produced with a high yield from N-acetyl-DL-methionine by the bioreactor.

Actinobacteria↗

PubMed1996

Effects of diltiazem, a Ca2+ channel blocker, on naloxone-precipitated changes in dopamine and its metabolites in the brains of opioid-dependent rats.

The effects of diltiazem, an L-type Ca2+ channel blocker, on naloxone (an opioid receptor antagonist)-precipitated withdrawal signs and changes in extracellular levels of dopamine (DA) and its metabolites in various brain regions of morphine (a mu-opioid receptor agonist) or butorphanol (a mu/delta/kappa mixed opioid receptor agonist) dependent rats were investigated using high performance liquid chromatography fitted with an electrochemical detector (HPLC-ED). Rats were rendered opioid-dependent by continuous intracerebroventricular (i.c.v.) infusion with morphine (26 nmol/microliters per h) or butorphanol (26 nmol/microliters per h) for 3 days. The expression of physical dependence produced by these opioids, as evaluated by naloxone (5 mg/kg. i.p.)-precipitated withdrawal signs, was reduced by concomitant infusion of diltiazem (10 and 100 nmol/microliters per h). Under the same condition, naloxone decreased the levels of: DA in the cortex, striatum, and midbrain; 3,4-dihydroxyphenylacetic acid (DOPAC) in the cortex, striatum, limbic areas, and midbrain: and homovanilic acid (HVA) in the striatum, limbic areas, and midbrain regions. In animals rendered dependent on butorphanol, the results obtained were similar to those of morphine-dependent rats except for the changes in DOPAC levels. Furthermore, concomitant infusion of diltiazem and opioids blocked the decreases in levels of DA, DOPAC, and HVA in a dose-dependent manner. These results suggest that the augmentation of intracellular Ca2+ mediated through L-type Ca2+ channels during continuous opioid infusion results in a decrease in extracellular levels of DA and its metabolites in some specific regions, which are intimately involved in the expression of withdrawal syndrome precipitated by naloxone.

Animals↗

PubMed1996

Mu- and delta-opioid receptor antagonists precipitate similar withdrawal phenomena in butorphanol and morphine dependence.

The relative involvement of mu- and delta-opioid receptors in the mediation of butorphanol-, as compared to morphine-, dependence was examined with the use of highly selective antagonists at mu- and delta-opioid receptors. Extracellular fluid levels of glutamate (Glu) and aspartate (Asp) were measured within the pontine locus coeruleus following precipitation of withdrawal from dependence on either butorphanol or morphine in conscious Sprague-Dawley rats. Dependence was induced by intracerebroventricular (i.c.v.) infusion of butorphanol (26 nmol/mu l/h), morphine (26 nmol/mu l/h) or saline vehicle (1 mu l/h) for 3 days by means of an osmotic minipump. Microdialysis probes (2 mm tip) were inserted into the locus coeruleus 24 h before precipitation of withdrawal by i.c.v. injection of either the mu-opioid receptor antagonist, D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; 48 nmol/5 mu l or 48 nmol/5 mu l), or the delta-opioid receptor antagonist, naltrindole (17-cyclopropy;methyl-6,7-dehydro-4,5-epoxy-3, 14-dihydroxy-6,7,2'3'-indolmorphinan hydrochloride; 48 nmol/5 mu l or 100 nmol/5 mu l). Baseline levels of Glu ranged from 9.59 + or - 1.27 to 12.84 + or - 3.01 mu M in the various treatment groups. Levels of Asp were similar. Precipitation of withdrawal by CTOP elicited significant increases of Glu and Asp in both morphine- and butorphanol-dependent rats. Maximal increases in Glu of 425% and 258% above baseline levels were elicited in the first 15 min microdialysis sample following i.c.v. injection of CTOP in morphine- and butorphanol-dependent rats, respectively. Behavioral signs of withdrawal were greater in morphine than butorphanol-dependent groups. The i.c.v. treatment with naltrindole elicited increases in Glu and Asp that were similar, although less marked, than those precipitated by CTOP treatment. Administration of naltrindole produced equivalent signs of withdrawal in both morphine- and butorphanol-dependent rats. Withdrawal from dependence on both morphine and butorphanol is characterized by elevations in coerulear levels of excitatory amino acids. Responses elicited following the use of selective mu- and delta-opioid receptor antagonists to precipitate withdrawal suggest that the role played by these receptors in mediation of the signs and symptoms of withdrawal do not differ greatly between butorphanol- and morphine-dependent rats.

Animals↗

PubMed1996

Naloxone-precipitated changes in biogenic amines and their metabolites in various brain regions of butorphanol-dependent rats.

Influence of a naloxone (an opioid receptor antagonist) challenge (5 mg/kg, IP) on levels of biogenic amines and their metabolites in various brain regions of rats infused continuously with butorphanol (a mu/delta/kappa mixed opioid receptor agonist; 26 nmol/microliter/h) or morphine (a mu-opioid receptor agonist; 26 nmol/microliter/h) was investigated using high-performance liquid chromatography with electrochemical detection (HPLC-ED). Naloxone precipitated a withdrawal syndrome and decreased the levels of: dopamine (DA) in the cortex and striatum, 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum, homovanilic acid (HVA) in the striatum, limbic, midbrain, and pons/medulla regions in butorphanol-dependent rats. However, the levels of norepinephrine (NE), serotonin (5-hydroxytryptamine; 5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in the regions studied were not affected by naloxone-precipitated withdrawal. In addition, naloxone increased the HVA/DA ratio in the cortex, while this ratio was reduced in the limbic, midbrain, and pons/medulla. The reduction of 5-HIAA/5-HT ratio was also detected in the limbic area. In the animals rendered dependent on morphine, the results obtained were similar to those of butorphanol-dependent rats except for changes of 5-HIAA levels in some brain regions. These results suggest that an alteration of dopaminergic neuron activity following a reduction of DA and its metabolites in specific brain regions (e.g., striatum, limbic, midbrain, and pons/medulla) play an important role in the expression of the opioid withdrawal syndrome.

Animals↗

PubMed1996

The effect of ginseng extract on locomotor sensitization and conditioned place preference induced by methamphetamine and cocaine in mice.

Repeated i.p. injections of 2 mg/kg methamphetamine (MA) or 20 mg/kg cocaine at 48-h intervals induced reverse tolerance to their ambulation-enhancing effects (behavioral sensitization). Furthermore, the reappearance of the sensitized state was observed at the time of readministration of MA or cocaine even after a 30-day discontinuation of drug administration. A concomitant injection of ginseng extract (GE), 200 mg/kg, i.p., suppressed the development of reverse tolerance and the reappearance of sensitization to MA and cocaine. Conditioned place preference to MA (1, 2, and 4 mg/kg, i.p.) and cocaine (1, 4, 10, and 20 mg/kg, i.p.), was completely blocked by GE, 200 mg/kg, i.p. combined treatment with MA of cocaine. Meanwhile, spontaneous motor activity and place preference were not affected by GE alone. These results provide evidence that GE may be useful clinically for the prevention of adverse actions of MA and cocaine.

Animals↗

PubMed1996

Inhibition of endothelium-dependent relaxation by hemoglobin in rabbit aortic strips: comparison between acellular hemoglobin derivatives and cellular hemoglobins.

Hemoglobin (Hb)-based artificial oxygen carriers are supposed to induce vasoconstriction through the inactivation of endothelium-derived relaxing factor (EDRF). We examined the vasoconstrictive activity of acellular Hb and cellular Hb solutions in rabbit aortic strips. Unmodified Hb, pyridoxalated Hb, bovine unmodified Hb, haptoglobin-Hb complex (Hp-Hb), and polyoxyethylene glycol-conjugated Hb (PEG-Hb) were used as acellular Hbs having different molecular masses. Cellular Hbs included liposome-encapsulated Hb and red blood cells (RBC). In the first experiment, Hb (10 ng/ml to 1 mg/ml) was cumulatively added to the tissues in which steady-state relaxation was evoked by acetylcholine (ACh) after precontraction induced by phenylephrine. Although all Hb solutions induced a dose-dependent reversal of ACh-induced relaxation, the most potent vasoconstrictive effect was noted with acellular Hbs, and their contractile activities were almost the same independent of molecular mass. On the other hand, liposome-Hb and RBC showed reduced potencies in this order. These results indicate the importance of cellularity as the major factor determining Hb-related EDRF inactivation. In another experiment, the tissues were exposed to Hb at 0.01, 0.1, or 1 mg/ml for 30 min and ACh-induced relaxation was recorded after the complete removal of Hb in an organ bath chamber. Exposure to unmodified Hb at > 0.1-mg/ml concentrations significantly reduced the ACh-induced relaxation, whereas the relaxation was not affected by PEG-Hb, Hp-Hb, liposome-Hb, or RBC. These results suggest that unmodified Hb might be persistently associated with tissues and thereby inhibit ACh-induced relaxation. From these findings, we propose two attributes of Hb-related inhibition of endothelium-dependent relaxation: Acellular Hbs inhibit EDRF more efficiently in the luminal space than cellular Hbs, and unmodified Hb can also inhibit it adluminally and/or adventitially.

Acetylcholine↗

PubMed1996

Antinociceptive effect of dihydroetorphine following various routes of administration: a comparative study with morphine.

Using various routes of administration, the antinociceptive effects of dihydroetorphine (DHE) and morphine were measured in mice. Regardless of the route of systemic and local administration, DHE (1-20 micrograms/kg, i.p.; 1-10 micrograms/kg, s.c.; 1-10 micrograms/kg, i.v.; 10-1000 micrograms/kg, p.o.; 10-100 ng/mouse, intracerebroventricularly (i.c.v.); and 10-100 ng/mouse, intrathecally (i.t.)) and morphine (1-20 mg/kg, i.p.; 1-10 mg/kg, s.c.; 1-10 mg/kg, i.v.; 10-100 mg/kg, p.o.; 1-10 micrograms/mouse, i.c.v.; and 0.5-3 micrograms/mouse, i.t.) produced an antinociceptive effect in a dose-dependent manner, as evaluated by the tail pinch method. However, the duration of the antinociceptive effect of DHE was shorter than that of morphine. The efficacy ratio of the antinociceptive effect between DHE and morphine was approximately 1000 to 1500: 1 by parenteral administration (i.p., s.c., or i.v.) and about 100: 1 by the oral route. Meanwhile, using direct application into the central nervous system (CNS) (i.c.v. or i.t.), the effect of DHE was only 10 to 20 times that of morphine. These data suggest that DHE has an ideal quality as an analgesic by systemic administration which is a more convenient application than local injection, since only a minimum dose of DHE is needed to induce suitable potency of antinociception, and the duration of the effect is short. Further, these unique characteristics of DHE might lead to the prevention of the development of dependence by avoiding accumulation of the drug in the CNS.

Analgesics, Opioid↗

PubMed1996

A potent mu-opioid receptor agonist, dihydroetorphine, fails to produce the conditioned place preference in mice.

Reinforcing effects of dihydroetorphine (DHE) and morphine were evaluated by the conditioned place preference paradigm. Both DHE and morphine produced an antinociceptive effect in a dose-dependent manner. On the other hand, DHE (0.1, 1, 3 and 10 micrograms/kg, i.p.) failed to induce the conditioned preference, while morphine (0.1, 1, 3 and 10 mg/kg, i.p.) caused a dose-dependent preference for the drug-paired place. Thus, these characteristic properties of DHE make it attractive for development as a novel potent analgesic compound that has less dependence liability.

Analgesics, Opioid↗

PubMed1996

[QOL and nursing care of a ventilator-assisted child at home].

QOL is the important issue for chronically ill children. We visited a girl, 8 years old and 24 hours dependent on a ventilator. We talked to her, and observed her daily life, mainly on the following points: (1) what she is doing; and (2) how often, and what kind of care she needs at school and at home. Then we discussed the problems, QOL and nursing care. She enjoyed her school life with her many friends, and received respiratory care, sanitary care, and other care for 11 hours and 19 minutes in average per day. 93.1% of the care was taken by her parents and volunteer care givers, so that they wanted to have nurses care for her. Many kinds of nursing care service were necessary as soon as possible.

Child↗

PubMed1995

Possible involvement of protein kinases in physical dependence on opioids: studies using protein kinase inhibitors, H-7 and H-8.

Effects of a cAMP-dependent protein kinase and protein kinase C inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) and a cAMP- and cGMP-dependent protein kinase inhibitor, H-8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide), on the behavioral signs of naloxone (an opioid receptor antagonist)-precipitated withdrawal syndrome and effects of H-7 on the change of protein kinase C activity in the pons/medulla region induced by morphine (a mu-opioid receptor agonist) or butorphanol (a mu/delta/kappa mixed opioid receptor agonist) were investigated in this study. Rats were intracerebroventricularly (i.c.v.) infused with morphine (26 nmol/microliters/h) or butorphanol (26 nmol/microliters/h) through osmotic minipumps for 3 days. In some groups, either saline or drug-treated groups were concomitantly infused with H-7 (1 and 10 nmol/microliters/h) or H-8 (10 nmol/microliters/h). The expression of physical dependence produced by morphine or butorphanol, as evaluated by naloxone (5 mg/kg i.p.)-precipitated withdrawal signs, was reduced by concomitant infusion of H-7 or H-8. In the same condition, morphine and butorphanol chronic treatment enhanced (28.1% and 26.3% enhancement over the saline-treated group, respectively) cytosolic protein kinase C activity in the pons/medulla, but not in the membrane fraction. Furthermore, concomitant infusion of H-7 inhibited the enhancement of protein kinase C activity. These results indicate that various types of protein kinases may play an important role in the development and/or expression of physical dependence on opioids. Among them, the enhancement of cytosolic protein kinase C activity in the pons/medulla region seems to be one of the major underlying mechanisms in opioid physical dependence.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Ca2+ channel blocker, diltiazem, prevents physical dependence and the enhancement of protein kinase C activity by opioid infusion in rats.

The influence of an L-type Ca2+ channel blocker, diltiazem [(2S-cis)-3-(acetyloxy)-5-[2-(dimethylamino)-ethyl]-2,3-dihydro-2- (4- methoxyphenyl)-1,5-benzothiazepin-4(5H)-one], on the behavioral signs of naloxone (opioid receptor antagonist)-precipitated withdrawal syndrome and the enhancement of protein kinase C activity in the pons/medulla regions of rats rendered dependent on morphine (mu-opioid receptor agonist) or butorphanol (mu/delta/kappa mixed opioid receptor agonist) was investigated. The expression of physical dependence produced by continuous intracerebroventricular (i.c.v.) infusion of morphine (26 nmol/microliters per h) or butorphanol (26 nmol/microliters per h) for 3 days, as evaluated by naloxone (5 mg/kg, i.p.)-precipitated withdrawal signs, was dose dependently attenuated by concomitant infusion of diltiazem (10 and 100 nmol/microliters per h). Furthermore, diltiazem (100 nmol/microliters per h) completely inhibited the enhancement of cytosolic protein kinase C activity in the pons/medulla regions in rats rendered dependent by continuous infusion with morphine or butorphanol. These results suggest that the augmentation of intracellular Ca2+ concentration mediated through L-type Ca2+ channels during continuous opioid infusion leads to the enhancement of cytosolic protein kinase C activity in the pons/medulla region which is intimately involved in the development and/or expression of physical dependence on opioids.

Animals↗

PubMed1995

Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60.

Thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40 degrees C, was purified to homogeneity and characterized. The relative molecular mass (M(r)) of the native enzyme and the subunit was estimated to be 300,000 and 40,000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50 degrees C and 7.5 respectively. The enzyme was stable at 55 degrees C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-L- or D-methionine, N-acetyl-L-valine, N-acetyl-L-tyrosine and N-chloroacetyl-L-valine. In addition, the enzyme also catalyzed the racemization of the dipeptide L-alanyl-L-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and ethyl ester derivatives of N-acetyl-D- and L-methionine were not racemized. The apparent Km values for N-acetyl-L-methionine and N-acetyl-D-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co2+, Mn2+ and Fe2+ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH2-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835-840] was above 80%.

Actinobacteria↗

PubMed1995

Simultaneous measurement of biogenic amines and their metabolites in rat brain regions after acute administration of and abrupt withdrawal from butorphanol or morphine.

Changes in levels of biogenic amines and metabolites were measured using high performance liquid chromatography fitted with an electrochemical detection in various rat brain regions after acute administration of and abrupt withdrawal from continuous intracerebroventricular infusion of butorphanol (a mu/delta/kappa mixed opioid receptor agonist) or morphine (a mu-opioid receptor agonist). A single dose of butorphanol (26 nmol/5 microliters) or morphine (26 nmol/5 microliters) increased levels of 3,4-dihydroxyphenylacetic acid in the striatum and limbic region and of homovanilic acid in the cortex, striatum, and limbic region. In animals which had been infused with butorphanol (26 nmol/microliters/hr) or morphine (26 nmol/microliters/hr) for 3 days, an increase in dopamine turnover was observed. The levels of 3,4-dihydroxyphenylacetic acid was decreased and that of homovanilic acid was increased in the striatum, limbic region, and midbrain immediately after termination of opioid infusion. Both dopamine metabolites (in these areas) were decreased at 2 and 6 hr after butorphanol or morphine withdrawal. Changes in norepinephrine, serotonin, and 5-hydroxyindoleacetic acid levels in some brain regions were observed in the morphine-, but not in butorphanol-dependent rats. These data suggest that the increase and the decrease in dopaminergic activity, but not noradrenergic and serotonergic neurons, in the some brain regions are closely associated with the production of antinociception of and the expression of withdrawal syndrome from butorphanol and morphine, respectively.

Animals↗

PubMed1995

Cloning, DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli.

The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage lambda-4 from the DNA library of Amycolatopsis sp. TS-1-60, a rare actinomycete, using antiserum against the enzyme. The cloned gene was subcloned and transformed in Escherichia coli JM105 using pUC118 as a vector. The AAR gene consists of an open-reading frame of 1104 nucleotides, which specifies a 368-amino-acid protein with a molecular mass of 39411Da. The molecular mass deduced from the AAR gene is in good agreement with the subunit molecular mass (40kDa) of AAR from Amycolatopsis sp. TS-1-60. The guanosine plus cytosine content of the AAR gene was about 70%. Although the AAR gene uses the unusual initiation codon GTG, the gene was expressed in Escherichia coli using the lac promoter of pUC118. The amount of the enzyme produced by the transformant was 16 times that produced by Amycolatopsis sp. TS-1-60. When the unusual initiation codon GTG was changed to ATG, the enzyme productivity of the transformant increased to more than 37 times that of Amycolatopsis sp. TS-1-60. In the comparison of the DNA sequence and the deduced amino acid sequence of AAR with those of known racemases and epimerases in data bases, no significant sequence homology was found. However, AAR resembles mandelate racemase in that requires metal ions for enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacteria↗

PubMed1995

[Clinical significance of proliferating cell nuclear antigen (PCNA) and argyrophilic nucleolar organizer region (AgNOR) in testicular tumors].

BACKGROUND: Proliferative potential of testicular tumor was assessed by immunohistochemistry using anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody and silver staining of argyrophilic nucleolar organizer region (AgNOR), and the results were compared with clinical course. METHODS: Fourty-five patients with testicular tumor and 10 normal testicles were investigated. All specimens were fixed with 10% buffered formaliun within 24 hours. The interval between patient's recognition of testicular swelling for the first time and the date for operation was designated as M (months), and the specimen weight was designated as g (gram). Growth rate of the primary tumor was estimated roughly by g/M. RESULTS: PCNA positive rates in seminomas, non-seminomas and normal testes were 70.5 +/- 19.1% (mean +/- S.D.), 80.4 +/- 10.5% and 17.7 +/- 7.8%, respectively. PCNA positive rate in stage I seminoma (64.4 +/- 19.9%) was significantly lower than stage II and III seminoma (83.6 +/- 7.3%) (p < 0.05), but not significant between stage I non-seminoma and stage II, III non-seminoma. The mean numbers of AgNOR per nucleus in seminomas, non-seminomas and normal testes were 8.09 +/- 1.35 (mean +/- S.D.), 6.89 +/- 1.43, 4.18 +/- 1.60, respectively and significantly higher in testicular tumors than normal testes. There were, however, no significant difference between their clinical stages. Logarithmic significant correlation was observed between PCNA positive rate and the growth rate in primary lesion, but no correlation was found between mean number of AgNOR per nuclei and the growth rate. Of the 10 patients with stage I non-seminomas received surveillance policy, four relapsed. Of the 10 patients, 4 of the 6 patients with higher than 75% PCNA positive rate relapsed, whereas, none of the 4 patients with lower than 75% PCNA positive rate relapsed. There were no significant correlation between PCNA positive rate and number of AgNOR. CONCLUSION: PCNA is more accurate indicator of growth potential in testicular tumor than AgNOR and may predict recurrence in stage I non-seminomatous testicular tumor patients followed by surveillance policy.

Carcinoma, Embryonal↗