ABSTRACT
Process analytical technology is important for the analysis and control of manufacturing processes. Near-infrared spectroscopy is widely used in various process analytical technologies for the analysis of the chemical componentsof solid dosage forms. Lubrication is an important process carried out before a tablet is produced. In this process, the concentration of lubricant, such as magnesium stearate (StMg), might change for one of many reasons during powder transport, which would be a critical problem such as variation in tablet compressibility and dissolution failure of compressed tablets. Our group investigated the feasibility of the quantitative monitoring of a change in the concentration of StMg in the feeder tube of tableting equipment employing real-time near-infrared spectroscopy.
Subject(s)
Drug Compounding/instrumentation , Drug Compounding/methods , Excipients/analysis , Stearic Acids/analysis , Tablets , Computer Systems , Drug Industry , Least-Squares Analysis , Regression Analysis , Spectroscopy, Near-InfraredABSTRACT
The use of transmission near infrared spectroscopy (TNIRS) is of particular interest in the pharmaceutical industry. This is because TNIRS does not require sample preparation and can analyze several tens of tablet samples in an hour. It has the capability to measure all relevant information from a tablet, while still on the production line. However, TNIRS has a narrow spectrum range and overtone vibrations often overlap. To perform content uniformity testing in tablets by TNIRS, various properties in the tableting process need to be analyzed by a multivariate prediction model, such as a Partial Least Square Regression modeling. One issue is that typical approaches require several hundred reference samples to act as the basis of the method rather than a strategically designed method. This means that many batches are needed to prepare the reference samples; this requires time and is not cost effective. Our group investigated the concentration dependence of the calibration model with a strategic design. Consequently, we developed a more effective approach to the TNIRS calibration model than the existing methodology.
Subject(s)
Spectroscopy, Near-Infrared/methods , Tablets/analysis , Calibration , Chromatography, High Pressure Liquid , Drug Compounding , Drug Industry , Models, Statistical , Multivariate Analysis , Reference Standards , Reproducibility of Results , Spectroscopy, Near-Infrared/statistics & numerical dataABSTRACT
A high-performance liquid chromatographic assay for monitoring the antifungal drug fluconazole in human serum was developed using a C18 reversed-phase column with isocratic elution. The method involved sample clean-up by solid-phase column extraction, and subsequent analysis required only 14 min per sample for separation and quantitation. The assay was precise, with intra- and inter-assay relative standard deviations of < or = 1.5% and < or = 3.1%. The minimum detectable concentration of fluconazole was 0.3 nmol/ml. This assay has the advantage of minimizing the risk of interference from co-administered drugs to critically ill patients undergoing hemodiafiltration.
Subject(s)
Antifungal Agents/blood , Fluconazole/blood , Hemodiafiltration , Aged , Aged, 80 and over , Antifungal Agents/administration & dosage , Antifungal Agents/pharmacokinetics , Chromatography, High Pressure Liquid , Drug Monitoring/methods , Fluconazole/administration & dosage , Fluconazole/pharmacokinetics , Humans , Injections, Intravenous , Limit of Detection , Male , Reproducibility of Results , Solid Phase Extraction , Spectrophotometry, UltravioletABSTRACT
A high-performance liquid chromatographic assay has been developed for the detection and quantification of the conventional postnatal uterotonic drug, methylergometrine, in human breast milk using a C-18 reversed-phase column by isocratic elution. The analytical method consisted of sample clean-up by solid-phase extraction, and the fluorescence detection required only 8.5 min per sample for separation and quantitation. This assay gave intra- and inter-assay coefficients of variation of less than 7.9% and 7.7%, respectively, and the detection limit was approximately 50 pg/ml. This method was applied for drug level monitoring in the breast milk of patients given methylergometrine.
Subject(s)
Methylergonovine/analysis , Milk, Human/chemistry , Oxytocics/analysis , Adult , Chromatography, High Pressure Liquid , Female , Fluorometry/methods , Humans , Methylergonovine/therapeutic use , Oxytocics/therapeutic use , Postpartum Period , Reference Standards , Reproducibility of Results , Solid Phase Extraction/methods , SolutionsABSTRACT
Soluble forms of amyloid-beta (Abeta) have been considered responsible for cognitive dysfunction prior to senile plaque formation in Alzheimer's disease (AD). As its mechanism is not well understood, we examined the effects of repeated i.c.v. infusion of soluble Alphabeta(25-35) on peptidergic system and glial cells in the pathogenesis of AD. The present study aims to investigate the protective effects of memantine on Abeta(25-35)-induced changes in peptidergic and glial systems. Infusion of Alphabeta(25-35) decreased the level of immunoreactive somatostatin (SS) and substance P (SP) in the hippocampus prior to neuronal loss or caspase activation, which is correlated with the loss of spine density and activation of inducible nitric-oxide synthase (iNOS). Biochemical experiment with peptide-degrading enzymes, prolyl oligopeptidase (POP) and endopeptidase 24.15 (EP 24.15) activities demonstrated a concomitant increase with the activation of glial marker proteins, glial fibrillary acidic protein (GFAP) and CD11b in the Abeta-treated hippocampus. Double immunostaining experiments of EP 24.15 and GFAP/CD11b antibodies clearly demonstrated the co-localization of neuro peptidases with astrocytes and microglia. Treatment with memantine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist significantly attenuated Abeta(25-35)-induced changes of neuropeptides, their metabolizing enzymes, glial marker proteins, and activation of iNOS. Taken together, the data implies that memantine exerts its protective effects by modulating the neuropeptide system as a consequence of suppressing the glial cells and oxidative stress in AD model rat brain regions.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Peptides/antagonists & inhibitors , Brain/drug effects , Memantine/pharmacology , Neuroglia/drug effects , Neuropeptides/drug effects , Peptide Fragments/antagonists & inhibitors , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/toxicity , Animals , Biomarkers/metabolism , Brain/metabolism , Brain/physiopathology , CD11 Antigens/drug effects , CD11 Antigens/metabolism , Disease Models, Animal , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Amino Acid Antagonists/therapeutic use , Glial Fibrillary Acidic Protein/drug effects , Glial Fibrillary Acidic Protein/metabolism , Gliosis/drug therapy , Gliosis/physiopathology , Gliosis/prevention & control , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/physiopathology , Male , Memantine/therapeutic use , Nerve Degeneration/chemically induced , Nerve Degeneration/metabolism , Nerve Degeneration/physiopathology , Neuroglia/metabolism , Neuropeptides/metabolism , Nitric Oxide Synthase Type II/drug effects , Nitric Oxide Synthase Type II/metabolism , Oxidative Stress/drug effects , Oxidative Stress/physiology , Peptide Fragments/metabolism , Peptide Fragments/toxicity , Peptide Hydrolases/pharmacology , Rats , Rats, Wistar , Somatostatin/drug effects , Somatostatin/metabolism , Substance P/drug effects , Substance P/metabolismABSTRACT
Prolonged exposure to nitrous oxide (N2O) results in development of acute tolerance to its antinociceptive effect. Cross-tolerance to N2O-induced antinociception is also observed in morphine-tolerant animals. Despite increasing evidence of tolerance development to N2O-induced antinociception, the details of the mechanisms that underlie this tolerance remain unknown. The present study was conducted to investigate the involvement of brain protein kinase C (PKC) isoform in these two types of tolerance to N2O-induced antinociception in mice. Prolonged exposure (41 min in total, including 30 min pre-exposure and 11 min of antinociceptive testing) to 70% N2O produced a reduction in N2O-induced antinociception, indicating development of acute tolerance. The prolonged exposure to 70% N2O caused an activation of PKCgamma isoform in the brain, but not the PKCepsilon isoform. Pretreatment with a PKCgamma-antisense oligonucleotide but not the corresponding mismatch oligonucleotide (i.c.v.) prevented the development of acute tolerance to N2O-induced antinociception. Chronic morphine treatment (10 mg/kg, s.c., b.i.d. for 5 days) resulted in development of tolerance to morphine-induced antinociception and cross-tolerance to N2O-induced antinociception. The development of tolerance to morphine and cross-tolerance to N2O were both inhibited by pretreatment with PKC inhibitor, chelerythrine (1 nmol, i.c.v.). Morphine-tolerant mice showed an activation of PKC within the brain, which was suppressed by pretreatment with chelerythrine (1 nmol, i.c.v.). Thus, activation of brain PKC, in particular, the PKCgamma isoform, appears to play an important role in the development of both acute tolerance and cross-tolerance to N2O-induced antinociception in mice.
Subject(s)
Analgesics, Non-Narcotic/pharmacology , Drug Tolerance/physiology , Nitrous Oxide/pharmacology , Nociceptors/drug effects , Protein Kinase C/physiology , Alkaloids/pharmacology , Analgesics, Opioid/pharmacology , Animals , Behavior, Animal/drug effects , Benzophenanthridines/pharmacology , Drug Interactions , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Male , Mice , Mice, Inbred C57BL , Morphine/pharmacology , Pain Measurement/drug effects , Time FactorsABSTRACT
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.
Subject(s)
Alzheimer Disease/drug therapy , Brain/drug effects , Dopamine Agents/pharmacology , Memantine/pharmacology , Neuropeptides/drug effects , Peptide Hydrolases/drug effects , Alzheimer Disease/chemically induced , Animals , Blotting, Western , Brain/metabolism , Disease Models, Animal , Electrophoresis, Polyacrylamide Gel , Excitatory Amino Acid Agonists/pharmacology , Ibotenic Acid/pharmacology , Immunohistochemistry , Male , Neuroglia/drug effects , Neuroglia/metabolism , Neuropeptides/metabolism , Peptide Hydrolases/metabolism , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Chromatography, High Pressure Liquid/methods , Chymotrypsin/analysis , Esterases/analysis , Spectrometry, Fluorescence/methods , Chromatography, Gel , Hydrogen-Ion Concentration , Reproducibility of ResultsABSTRACT
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.
Subject(s)
Chromatography, High Pressure Liquid/methods , Fluorometry/methods , Hydrolases/analysis , Animals , Arginine/analogs & derivatives , Arginine/analysis , Arginine/metabolism , Brain/enzymology , Citrulline/adverse effects , Dansyl Compounds/analysis , Dansyl Compounds/metabolism , Glycine/analogs & derivatives , Glycine/analysis , Glycine/metabolism , HL-60 Cells/enzymology , Humans , Hydrogen-Ion Concentration , Male , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , Rats , Rats, Wistar , Sensitivity and Specificity , Substrate Specificity , Urea/adverse effectsABSTRACT
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.
Subject(s)
Metalloendopeptidases/metabolism , Oligopeptides/metabolism , Sciatic Nerve/metabolism , Animals , Axonal Transport , Enzyme Activation , Hydrolysis , Male , Metalloendopeptidases/chemistry , Oligopeptides/chemistry , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Cerebellum/physiology , Morphine/pharmacology , Opioid Peptides/pharmacology , Purkinje Cells/physiology , Action Potentials/drug effects , Animals , Cats , Dynorphins/pharmacology , Enkephalin, Leucine/pharmacology , Enkephalin, Methionine/pharmacology , Female , Iontophoresis , Male , Peptide Fragments/pharmacologyABSTRACT
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.
Subject(s)
Carboxypeptidases/metabolism , Chromatography, High Pressure Liquid/methods , Animals , Brain/enzymology , Cathepsin A , Hydrogen-Ion Concentration , Kidney/enzymology , Liver/enzymology , Lung/enzymology , Male , Mice , Mice, Inbred ICR , Spectrometry, Fluorescence , Spleen/enzymology , Testis/enzymologyABSTRACT
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.
Subject(s)
Acetylcholine/metabolism , Medulla Oblongata/physiology , Morphine/pharmacology , Pain/physiopathology , Animals , Dose-Response Relationship, Drug , Extracellular Space , Infusions, Parenteral , Injections, Intraperitoneal , Male , Medulla Oblongata/drug effects , Microdialysis , Morphine/administration & dosage , Naloxone/pharmacology , Neostigmine/pharmacology , Pain/prevention & control , Rats , Rats, Wistar , Tetrodotoxin/pharmacologyABSTRACT
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.
Subject(s)
Home Care Services, Hospital-Based , Pharmacy Service, Hospital , Drug Prescriptions , Humans , Parenteral Nutrition, Home Total , Pharmacy Service, Hospital/supply & distributionABSTRACT
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.
Subject(s)
Acetylcholine/metabolism , Corpus Striatum/drug effects , Dopamine Uptake Inhibitors/pharmacology , Methamphetamine/pharmacology , Adrenergic Agents/pharmacology , Adrenergic Uptake Inhibitors/pharmacology , Animals , Apomorphine/pharmacology , Corpus Striatum/metabolism , Dopamine Agonists/pharmacology , Enzyme Inhibitors/pharmacology , Male , Microdialysis , Oxidopamine/pharmacology , Rats , Rats, Wistar , Reserpine/pharmacology , Tetrodotoxin/pharmacology , alpha-Methyltyrosine/pharmacologyABSTRACT
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.
Subject(s)
Axonal Transport , Coumarins/metabolism , Endopeptidases/metabolism , Multienzyme Complexes , Oligopeptides/metabolism , Sciatic Nerve/enzymology , Amino Acid Sequence , Animals , Chromatography, Gel , Hydrolysis , Kinetics , Male , Mixed Function Oxygenases/metabolism , Protein Precursors/metabolism , Protein Sorting Signals/metabolism , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Cerebral Cortex/drug effects , Cholecystokinin/genetics , Hippocampus/drug effects , Neuropeptides/metabolism , RNA, Messenger/metabolism , Animals , Blotting, Northern , Cerebral Cortex/metabolism , Convulsants , Hippocampus/metabolism , Immunohistochemistry , Male , Pentylenetetrazole , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Cholecystokinin/genetics , Frontal Lobe/drug effects , Kindling, Neurologic/drug effects , Neuropeptides/metabolism , RNA, Messenger/metabolism , Animals , Frontal Lobe/metabolism , Male , Pentylenetetrazole , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Carboxypeptidases/analysis , Chromatography, High Pressure Liquid/methods , Pituitary Gland/enzymology , Viscera/enzymology , Animals , Brain/enzymology , Carboxypeptidases/antagonists & inhibitors , Carboxypeptidases/metabolism , Cattle , Colorimetry , Enzyme Inhibitors/pharmacology , Hydrogen-Ion Concentration , Kidney/enzymology , Liver/enzymology , Lung/enzymology , Male , Metals/pharmacology , Mice , Mice, Inbred ICR , Pituitary Gland/metabolism , Protease Inhibitors/pharmacology , Spleen/enzymology , Testis/enzymologyABSTRACT
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.