Biomedical subjects

T Chikuma

Publications and source records attributed to T Chikuma.

At least 19 recordsLinked to original sources

PubMed2004

Effect of memantine on the levels of glial cells, neuropeptides, and peptide-degrading enzymes in rat brain regions of ibotenic acid-treated alzheimer's disease model.

It has been implicated that glia activation plays a critical role in the progression of Alzheimer's disease (AD). However, the precise mechanism of glia activation is not clearly understood yet. In our present studies, we confirmed our previous results where change the levels of neuropeptides and peptidases in ibotenic acid (IBO) infusion into the rat nucleus basalis magnocellularis, an animal model of AD. Furthermore, we extended our study to investigate a possible protection effect of co-administration on the changes of neuropeptides, and neuronal and glial cells in IBO-infused rat brain by memantine treatment. The levels of substance P and somatostatin were decreased in the striatum and frontal cortex 1 week after IBO infusion, and recovered to the control level by memantine treatment, indicating the involvement of neuropeptides in AD pathology. Furthermore, the immunohistochemical and enzymatic studies of GFAP and CD 11b, and peptidylarginine deiminase, markers of glia, in the striatum and frontal cortex showed the increase in IBO-treated rat brain as compared with controls, while co-administration of memantine and IBO no increase of astrocytes and microglia activation was observed. The present biochemical and immunohistochemical results suggest that glia activation might play an important role to the pathology of AD, and correlate with the changes of neuropeptide levels in AD brain that is recovered by memantine treatment.

Alzheimer Disease↗

PubMed2001

High-performance liquid chromatographic-fluorimetric assay of chymotrypsin-like esterase activity.

A sensitive and reproducible assay for the determination of chymotrypsin-like esterase activity is reported. This method is based on fluorimetric detection of a dansylated amino acid, 5-dimethylaminonaphthalene-1-sulfonyl-L-phenylalanine, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-L-phenylalanine ethyl ester, after separation by high-performance liquid chromatography using a C18 reversed-phase column and isocratic elution. This method is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-L-phenylalanine at concentrations as low as 40 pmol/ml, yields highly reproducible results and requires less than 9.5 min per sample for quantitation. The optimum pH for chymotrypsin-like esterase activity was 7.7-8.3. The Km and Vmax values were, respectively 25 microM and 0.241 pmol/microg protein/h with the use of enzyme extract obtained from mouse kidney. The approximate molecular mass of this enzyme was estimated to be 67000 by gel filtration. Chymotrypsin-like esterase activity was strongly inhibited by N-tosyl-L-phenylalaline chloromethyl ketone. Among the mouse organs examined, the highest specific activity of the enzyme was found in lung. This new method would be useful for clarification of the physiological role of this enzyme.

Chromatography, Gel↗

PubMed2000

A highly sensitive high-performance liquid chromatography-- fluorometric method for the assay of peptidylarginine deiminase activity.

The activity of peptidylarginine deiminase (PAD) has generally been assayed by a colorimetric method using N-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoyl-L-arginine (Bz-L-Arg) as the substrates. The widespread occurrence of citrulline and urea in tissues makes use of this method difficult, especially for small samples. We developed a highly sensitive high-performance liquid chromatography method with N-dansyl-glycyl-L-arginine as the substrate. This method was sensitive enough to determine previously undetectable activity of PAD in HL-60 cells. Two types of PAD (HL-60 cell and brain PAD) could be distinguished by differential competition, using either BAEE or Bz-L-Arg as a preferential substrate in the assay. These data indicate that the present method is applicable to many tissues.

Animals↗

PubMed2000

Anterograde axonal transport of Boc-Arg-Val-Arg-Arg-MCA hydrolyzing enzyme in rat sciatic nerves: cleavage occurs between basic residues.

Axonal transport of Boc-Arg-Val-Arg-Arg-MCA hydrolyzing enzyme activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and peaked 72 h after ligation. The optimum pH for Boc-Arg-Val-Arg-Arg-MCA hydrolyzing enzyme activity was 6.5 to 6.9 and did not require Ca(2+) for the activity. Two molecular forms with enzyme activity were identified by size-exclusion chromatography and the molecular masses of the two enzymes were estimated to be 98 and 52 kDa. Two enzyme activities were strongly inhibited by Hg(2+), Cu(2+) and trypsin inhibitors such as TLCK, antipain and leupeptin. It cleaved the substrate, Boc-Arg-Val-Arg-Arg-MCA, between the dibasic sequence Arg-Arg, and needed a support of aminopeptidase B-like enzyme activity for the liberation of 7-amino-4-methylcoumarin. These results suggest that the enzyme is transported in rat sciatic nerves and involved in the post-translational processing of precursor proteins under the anterograde axonal transport. But there is absolutely no evidence for a role in precursor processing and such a putative role is purely speculative.

Animals↗

PubMed2000

Effects of microiontophoretically-applied opioid peptides on Purkinje cells in the cat cerebellum.

AIM: The purpose of the present study was to examine the effects of microiontophoretically-applied opioid peptides on Purkinje cell of the cerebellum. METHODS: The effects of microiontophoretically-applied morphine, leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), and dynorphin 1-13 (Dyn) on the spontaneous discharge of Purkinje cells in the cerebellum of the anesthetized cat were examined. RESULTS: Microiontophoretic applications of Leu-Enk and morphine produced inhibitory and excitatory responses, respectively in Purkinje cells. Application of both morphine and Leu-Enk induced dose-dependent responses. The excitatory responses were antagonized by naloxone, whereas the inhibitory responses were not. Bicuculline, a GABA-A antagonist, completely abolished both the Leu-Enk- and morphine-induced-inhibitory responses. Iontophoretic application of Met-Enk and dyn produced inhibitory responses only. Met-enk- and dyn-induced inhibition was antagonized by naloxone. CONCLUSION: In Purkinje cell activity, microiontophoretically applied Leu-Enk- and morphine-induced excitation is connected with opiate receptors, whereas inhibition is related to the GABA receptor. However, Met-Enk and dyn produced only inhibitory effects via an opiate receptor in the cerebellum of cats.

Action Potentials↗

PubMed1999

High-performance liquid chromatographic-fluorimetric assay for cathepsin A (lysosomal protective protein) activity.

A rapid and sensitive assay for the determination of cathepsin A activity is reported. This method is based on fluorimetric detection of a dansylated peptide, 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe-L-Leu, after separation by high-performance liquid chromatography using a C18 reversed-phase column and isocratic elution. This method is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe at concentrations as low as 300 fmol, yields highly reproducible results and requires less than 7.0 min per sample for separation and quantitation. The optimum pH for cathepsin A activity was 4.5-5.0. The Km and Vmax values were respectively 14.9 microM and 27.91 pmol/microg/h with the use of enzyme extract obtained from mouse kidney. Cathepsin A activity was strongly inhibited by Ag+, Hg2+, diisopropylfluorophosphate and p-chloromercuriphenylsulphonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in kidney. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.

Animals↗

PubMed1999

The effects of morphine-induced increases in extracellular acetylcholine levels in the rostral ventrolateral medulla of rat.

The present study examined the role of the rostral ventrolateral medulla (RVLM) in the modulation of acetylcholine (ACh) release by morphine. We examined the effect of morphine on the release of ACh in the RVLM of freely moving rats using the in vivo microdialysis method. The basal level of ACh was 303.0 +/- 28.2 fmol/20 microliter/15 min in the presence of neostigmine (10 microM). Morphine at a low dose of 5 mg/kg (i.p.) increased ACh release by the RVLM by 42.4%. A higher morphine dose (10 mg/kg i.p.) significantly increased the release of ACh by 75.4%, with a maximal effect (86.4%) at 75 min. This enhancement following i.p. administration of morphine was reversed by naloxone (1 mg/kg i.p.). Addition of morphine (10(-4) M) to the perfusion medium increased the ACh release by 85.8% of the predrug values. The increased ACh release induced by local application of morphine was reversed by pretreatment with naloxone (1 mg/kg i.p.). The antinociceptive effect of locally applied morphine into the RVLM was assessed using the hot-plate test and tail immersion test in unanesthetized rats. Local application of morphine (10(-4) M) via a microdialysis probe induced an increase in both tail withdrawal and hot-plate response. These findings suggest that morphine seems to exert a direct stimulatory effect on ACh release by the RVLM and that morphine-induced nociception is, in part, activated by the release of ACh in freely moving rats.

Acetylcholine↗

PubMed1999

[Promotion of home medical care and ways to provide medicines to patients at home].

The Medicare Security Law and Fee Schedule for Medical Services have undergone revisions in accordance with the changes in society, and these were introduced on the premise that home medical care be promoted. If home medical care is promoted, it is expected that a considerable proportion of persons who are now hospitalized and depend heavily on medicare will be transferred home, partly because of the shortening of the maximum insured hospitalization time. Thus, it will become necessary to provide medicines including injectables such as TPN to patients at home. Generally speaking, medicines for patients at home are provided by nearby pharmacists based on prescriptions written by the physician in charge of a patient. However, with regard to injectables such as TPN that require aseptic environments for preparation, such requirements can not yet be met satisfactorily owing to a shortage of sufficient provisions at pharmacies and of sufficient technical skills on the part of pharmacists. Currently, there are only 27 pharmacies which are officially recognized as being sufficiently equipped to prepare injectables in aseptic environments, and pharmacists who actually prepare TPN drugs in aseptic environments account for about one third of this number. The current number of pharmacists will obviously not meet the demand for medicines from patients at home, which will undergo a sharp rise in the near future. Accordingly, it is essential to increase the number of pharmacists nationwide who are sufficiently equipped to manage injectables such as TPN in aseptic conditions.

Drug Prescriptions↗

PubMed1998

The effect of methamphetamine on the release of acetylcholine in the rat striatum.

We examined the effect of methamphetamine on the release of acetylcholine in the striatum of freely moving rats, using an in vivo microdialysis method. The basal level of acetylcholine was 3.67+/-0.47 pmol/30 microl per 15 min in the presence of neostigmine (10 microM). Tetrodotoxin (1 microM), a selective blocker of voltage-dependent Na+ channels, markedly inhibited the release of acetylcholine in the striatal perfusates. Apomorphine (1.0 mg/kg, i.p.), a dopamine receptor agonist, also significantly attenuated acetylcholine release. Methamphetamine (0.1 and 0.5 mg/kg, i.p.) did not immediately affect acetylcholine release in the striatum, but a dose of 1.0 mg/kg (i.p.) induced an increase of acetylcholine release in the striatum at 15-60 min. Striatal infusion of methamphetamine (5 and 10 microM) did not influence acetylcholine release. The increase following intraperitoneal administration of methamphetamine was slightly diminished by haloperidol (0.5 mg/kg). After microinjection of the neurotoxin, 6-hydroxydopamine (6 microg/3 microl), in the substantia nigra 7 days before, the increase of acetylcholine induced by the administration of methamphetamine (1.0 mg/kg) was slightly attenuated, whereas the administration of reserpine (2 mg/kg, i.p.) 24 h before, combined with alpha-methyl-p-tyrosine (300 mg/kg, i.p.) 2.5 h before, completely blocked the increase in release of acetylcholine. These findings suggest that methamphetamine exerts an excitatory influence on striatal acetylcholine release in freely moving rats, and that this excitatory effect involves the dopaminergic system and the catecholaminergic system.

Acetylcholine↗

PubMed1998

Effect of pentylenetetrazol treatment on cholecystokinin mRNA and peptide levels in rat hippocampus and cortex.

After treatment with pentylenetetrazol (PTZ), cholecystokinin (CCK) mRNA and CCK-like immunoreactivity (CCK-LI) levels were determined in rat hippocampus and cortex at different time points. In the temporal cortex treatment with 60 mg/kg PTZ, i.p., induced increases of CCK mRNA and CCK-LI levels at 2 days after the injection. In the hippocampus, a similar increase of CCK mRNA level was observed on the second day. By contrast, in the frontal cortex, CCK-LI level was increased at 10 days after the treatment with PTZ. These data show that PTZ increases both CCK mRNA and CCK-LI levels in these rat brain regions at different time.

Animals↗

PubMed1998

Axonal transports of Boc-Gly-Arg-Arg-MCA hydrolysing enzyme in rat sciatic nerves.

Study on neural axon transport is a very useful method to find a neuron-specific protease. In the present study, the enzyme activity (release of 7-amino-4-methyl-coumarin from t-butyloxycarbonyl-glycyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide) was measured in the proximal, middle, and distal segments between 12 and 120 h after double ligations of rat sciatic nerves to find precursor processing enzyme specific for pair of basic amino acid residue. The enzyme activity was significantly increased not only in the proximal but also in the distal segments 12-120 h after the ligation, and the maximal enzyme activity was found in both segments at 72 h. The enzyme activity eluted by anion exchange chromatography of the proximal segment showed at least three peaks, and was slightly higher than the activity of the distal one. The activity in the middle segment was very low in comparison with the activity in the proximal and distal segments. These data indicate that some of the enzymes specific for pair of basic amino acid residue are transported by both anterograde and retrograde axonal flow, and may undergo a neuron-specific processing.

Amino Acid Sequence↗

PubMed1997

High-performance liquid chromatographic-colorimetric assay for glycine carboxypeptidase activity.

A rapid and sensitive assay method for the determination of glycine carboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe, enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a TSK gel ODS-80TM reversed-phase column by isocratic elution. This method is sensitive enough to measure 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 7.5 min per sample for separation and quantitation. The pH optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The Km and Vmax values were respectively 21.1 micromol and 3.73 pmol/microg/h with the use of enzyme extract obtained from bovine pituitary. Glycine carboxypeptidase activity was strongly inhibited by Ag+, Cu2+ and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in testis. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.

Animals↗

PubMed1997

Chemical kindling induced by pentylenetetrazol changes cholecystokinin mRNA and peptide levels in rat frontal cortex.

Kindling model is useful to study the mechanism of learning and memory. Cholecystokinin (CCK) mRNA and CCK-like immunoreactivity (CCK-LI) levels in the hippocampus and frontal cortex of chemically kindled rats were determined at different time points. In the frontal cortex, chronic treatment with pentylenetetrazol (PTZ) (40 mg/kg per day for 8 days) increased CCK mRNA level at 7 days, and decreased CCK-LI level at 2 and 7 days after the last injection. However, neither CCK mRNA nor CCK-LI levels in the hippocampus changed. These results suggest that PTZ-induced kindling increases CCK mRNA expression and CCK-LI release in the frontal cortex.

Animals↗

PubMed1996

A fluorometric assay for measuring deamidase (lysosomal protective protein) using high-performance liquid chromatography.

A rapid and sensitive assay method for the determination of deamidase activity is reported. This method is based on fluorometric detection of a dansylated dipeptide, 5-dimethylaminonaphthalene-1-sulfonyl-D-Tyr-Val (N-Dns-D-Tyr-Val), enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-D-Tyr-Val-NH2 (N-Dns-D-Tyr-Val-NH2), after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This method is sensitive enough to measure N-Dns-D-Tyr-Val at concentrations as low as 100 fmol, yields highly reproducible results and requires less than 8.5 min per sample for separation and quantitation. The optimum pH for deamidase activity was 4.0-4.5. Greater than 5 mM of reduced glutathione was needed for maximal enzyme activity. The Km and Vmax values were respectively 125 microM and 14.12 pmol/micrograms/h with the use of enzyme extract obtained from mouse spleen. Deamidase activity was strongly inhibited by Ag+, Cu2+, diisopropylfluorophosphate, and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in spleen. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this enzyme.

Animals↗

PubMed1996

Distribution and processing of peptidylglycine alpha-amidating monooxygenase activity in rat dorsal root ganglia and sciatic nerves.

Since peptidylglycine alpha-amidating monooxygenase (PAM) and carboxypeptidase H (CPH) are transported by a rapid anterograde axonal flow in rat sciatic nerves, different properties of those enzymes were examined in the cell body (dorsal root ganglia) and axon (sciatic nerves) of rat. The relative enzyme activities of soluble to membrane-associated forms in the sciatic nerves were higher than those in the ganglia. On a gel permeation chromatogram, 3 peaks (100, 70 and 30 kDa) of PAM activity appeared in both tissues. There are main peaks at 70 kDa in the ganglia and at 30 kDa in the sciatic nerves. From these data, we suppose that PAM in rat sciatic nerves is proteolytically processed during the axonal transport of secretion granules.

Acetylcholine↗

PubMed1995

Highly sensitive high-performance liquid chromatography-fluorimetric assay method for carboxypeptidase H activity.

A rapid and sensitive high-performance liquid chromatographic (HPLC)-fluorimetric assay method has been developed for the determination of carboxypeptidase H activity based on the measurement of N-(5-dimethyl-aminonaphthalene-1-sulfonyl)glycine (dansyl-Gly) formed enzymatically from dansyl-Gly-L-Lys or dansyl-Gly-L-Arg. Dansyl-Gly is eluted faster than the substrates with an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer at pH 7.0 containing methanol, but eluted slower with an acidic buffer at pH 4.6. The new HPLC method separates the product and substrate in less than 5 min using an elution buffer at pH 7.0 containing 60% methanol. Using this method carboxypeptidase H activity has been detected in rat sciatic nerves. This HPLC method facilitates the assay of carboxypeptidase H activity in the enzyme samples from various tissues.

Amino Acid Sequence↗

PubMed1994

Characterization of peptidylglycine alpha-amidating monooxygenase in bovine hypothalamus.

In many peptide hormones and neuropeptides, the carboxyl-terminal alpha-amide structure is essential in eliciting their biological activity. In the present study, an enzymatic activity capable of converting 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly(Dabsyl-Gly-Phe -Gly) to 4-dimethylaminoazo-benzene-4'-sulfonyl-Gly-L-Phe-NH2(Dabsyl- Gly-Phe-NH2) was investigated in bovine hypothalamus. The concentrations of copper ion and ascorbic acid required for maximal enzyme activity were 16 microM and 2 mM, respectively. Amidating activity showed a pH profile with two pH optima at acidic pH (around 6.0) and neutral pH (around 7.5). Kinetic studies with the enzyme obtained from bovine hypothalamus demonstrated two distinct Km and Vmax values. The first Km and Vmax values were 142.9 microM and 22.2 pmol/microgram/h and the second Km and Vmax values were 22.7 microM and 4.44 pmol/microgram/h, respectively. Two molecular forms of amidating activity were identified by size-exclusion chromatography and the molecular weight of the two enzymes were estimated to be 49 kDa and 69 kDa.

Amino Acid Sequence↗

PubMed1994

A rapid anterograde axonal transport of carboxypeptidase H in rat sciatic nerves.

Using the highly sensitive HPLC-fluorophotometry technique, anterograde and retrograde axonal transport of carboxypeptidase H (CPH), a putative prohormone processing enzyme that removes a basic amino acid from the C-terminus of a precursor peptide, was measured 12-72 h after double ligations of rat sciatic nerves. CPH-like activity in rat sciatic nerves was 60-fold lower than that in the pituitary gland. CPH-like enzyme activity was rapidly accumulated in the proximal segment and peaked 48 h after ligation. The axonal flow was 100 mm/day, indicating that CPH in rat sciatic nerves is rapidly transported to the nerve terminals as an active form. The properties of the enzyme were similar to those of CPH in the brain: The pH optimum is at 5.5, and the molecular mass is approximately 5 kDa. These results suggest that active CPH in the PNS is transported by a rapid anterograde axonal flow and may play a role in converting proneuropeptides to active neuropeptides under the axonal transport.

Animals↗