Post-proline cleaving enzyme. Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man.

Synthesis and application of the first fluorogenic substrate, N-carbobenzoxyglycylprolyl-4-methylcoumarinyl amide (Z-Gly-Pro-MeCouNH) for the determination of the post-proline cleaving enzyme (EC 3.4.21.-) were reported. Maximal activity of the enzyme purified from lamb kidney for the new substrate was observed at pH 7.0. This substrate showed a higher affinity (Km = 0.02 mM) for the enzyme than the proline containing substrates studied previously and allowed the detection of 10-50 ng post-proline cleaving enzyme activity per ml sample after a 1 min incubation period. Distribution of post-proline cleaving enzyme and other proline specific peptidases in rat tissues was studied using Z-Gly-Pro-MeCouNH and other proline-containing substrates. High post-proline cleaving enzyme activity was observed in testis, liver and skeletal muscle. Inhibition experiments indicated that post-proline cleaving enzyme activity was completely inactivated by 0.1 mM diisopropylphosphofluoridate and Z-Gly-Pro-chloromethylketone, as had been found in the case of the enzyme isolated from lamb kidney. Activity in human body fluids was also tested for levels of post-proline cleaving enzyme activity using Z-Gly-Pro-MeCouNH and semen was found to show the highest cleaving activity.

Enzyme immunoassays of kanamycin group antibiotics with high sensitivities using anti-kanamycin as a common antiserum: reasoning and selection of a heterologous enzyme label.

An antiserum against kanamycin (anti-KM) was elicited in rabbits immunized with a kanamycin immunogen prepared by a three-step procedure using N-(m-maleimidobenzoyloxy)succinimide as a cross-linker. KM and tobramycin (TOB) were labeled with beta-D-galactosidase utilizing another cross-linker, N-(gamma-maleimidobutyryloxy)succinimide. The labeled KM showed very strong affinity to anti-KM antiserum and that of TOB had an adequate affinity to anti-KM. Increases in the assay sensitivities at the B/B0 value of 50% of KM and dibekacin were 183- and 191,000-times, respectively, on changing the enzyme label from KM to TOB. The optimal conditions for highly sensitive enzyme immunoassay (EIA) of KM using anti-KM and the enzyme labeled with TOB with satisfactory accuracy and precision were determined. Highly sensitive EIAs of four KM analogs with measurement ranges of 1 to 100 ng/tube were also developed using the labeled TOB and anti-KM as common reagents. Various commonly used drugs were found to have little reactivity in this immunoassay, indicating that the EIA is specific to KM and its analogs. The reasoning and the selection of TOB as the label are also discussed.

Characterization of [(3)H]glutamate binding sites on frozen sections from rat adrenal.

Some biochemical characteristics of [(3)H]glutamate (Glu) binding sites on frozen sections from the rat adrenal glands were studied. Adrenal frozen sections exhibited stereo-selective, saturable and temperature-dependent binding of [(3)H]Glu. An agonist for one of the subclasses of central Glu receptors, quisqualic acid (QA), elicited a significant inhibition of the binding, whereas neither N-methyl-d-aspartic acid nor kainic acid, agonists for other subclasses of the receptors, had such a significant effect on the binding at the concentration range similar to QA. In vitro addition of sodium acetate (100 mM) resulted in a significant inhibition of [(3)H]Glu binding to frozen sections of the rat adrenal glands. It thus appears that there exist QA-sensitive binding sites of [(3)H]Glu in the rat adrenal glands which exhibit pharmacological characteristics distinctly different from those in the brain.

Possible interaction of [(3)H]glutamate binding sites with anion channels in rat neural tissues.

The effect of various ions on [(3)H]l-glutamic acid (Glu) binding was examined using crude synaptic membrane preparations from the rat brain. In vitro addition of sodium acetate (1-100 mM) exhibited a significant enhancement of the binding in a concentration dependent manner. Ammonium chloride (20 mM) prevented the potentiation by sodium acetate at 2 degrees C, whereas sodium acetate exerted an inhibitory action on the ammonium chloride-induced augmentation of the binding at 30 degrees C. Ammonium chloride (1-100 mM) itself elicited a temperature dependent stimulation of the binding, which was invariably attenuated by an antagonist for the anion channels such as picrotoxinin (10(?3) M) as well as by inhibitors of anion transport including ethacrynic acid (10(?3) M) and 4,4?-diisothiocyanatostilbene-2,2?-disulfonic acid (10(?4)?10(?3) M), respectively. The later two inhibitors also caused a significant additional raise of the sodium acetate-induced enhancement of the binding. A significant augmentation of the binding resulted from the addition (20 mM) of various anions known to penetrate the anion channels such as bromide, iodide, nitrate, bicarbonate and thiocyanate in a permeability related manner, while that of non-permeable anions including fluoride, sulfate, acetate, formate, phosphate, oxalate, lactate, succinate and tartarate had no such a profound effect on the binding. Addition of d-aspartic acid resulted in the complete abolition of the Na(+)-dependent binding while sparing the Cl(?)-dependent binding. Scatchard analysis revealed that Cl(?) ions induced a two-fold increase in the number of the binding sites without affecting their affinity, whereas Na(+) ions reduced the affinity with a concomitant increase of the number of the binding sites. Addition of quisqualic acid (10(?5)?10(?3) M) inhibited the Cl(?)-dependent binding of [(3)H]Glu to a significantly greater extent than the inhibition on Na(+)-dependent binding. N- Methyl- d -aspartic acid and kainic acid exerted no preventive action on the basal, Cl(?)-dependent and Na(+)-dependent binding. respectively. The highest basal binding activity was found in the retina among various central structures examined. A significant basal binding activity of [(3)H]Glu was also detected in the pituitary and adrenal but not in the kidney. Chloride ions exhibited a significant facilitation of [(3)H]Glu binding to central regions without altering that to peripheral tissues such as pituitary and adrenal. In contrast, Na(+) ions induced significant attenuation of the binding to the pituitary, adrenal and retina despite the occurrence of augmentation of the binding to other central structures. These results suggest the Glu binding sites may be linked to the anion channels in the rat central nervous system and that this linkage may be absent from the pituitary, adrenal and retina.

Effects of the synthesized growth hormone releasing peptide, KP-102, on growth hormone release in sodium glutamate monohydrate-treated low growth rats.

KP-102 (D-Ala-D-beta-Nal-Ala-Trp-D-Phe-Lys-NH2), a new second generation hexapeptide, has a potent growth hormone (GH)-releasing action in vivo and in vitro. Here, we evaluated the GH-releasing action of KP-102 under pentobarbital (PB) anesthesia in neonatally sodium-glutamate-monohydrate-treated low growth (NMSG-LG) rats. The plasma GH level in NMSG-LG rats after i.v. administration of KP-102 at 100 micrograms/kg was 1/6.7 (95% C.L. 1/14.7 - 1/3.0) of that in normal rats given the same dose (p < 0.01). However, the increase was significant compared with that in normal rats after saline administration (p < 0.01). The plasma GH releasing action of KP-102 at 100 micrograms/kg i.v. in rats with lesions in the bilateral hypothalamic arcuate nuclei (ARC), was about 1/6.3 (95% C.L. 1/12.4 - 1/3.2) of that in normal rats under PB anesthesia (p < 0.01). When KP-102 was injected into the ARC at doses of 0.0002, 0.02 and 2 micrograms/rat, GH release was dose-related (p < 0.01) under PB anesthesia. KP-102 at 2 micrograms i.c.v. also increased the plasma GH levels (p < 0.01) to about 1/8.3 (95% C.L. 1/22.7 - 1/3.1) of that by systematic administration, at the same potency as the ARC injection (1/13.7 and 95% C.L. 1/37.2 - 1/5.0). These findings suggest that KP-102 potently stimulates the GH release by a direct or indirect antagonism of somatostatin (SRIF) and growth hormone releasing hormone (GHRH) release in the hypothalamus and by a direct action on the pituitary. Furthermore, the GH-releasing action of KP-102 was similar and additive upon both regions in vivo at the maximum effective dose. Moreover, since the GH-release in response to KP-102 administration differed between NMSG-LG and normal rats, and since KP-102 increased the GH release even in NMSG-LG rats, it should be evaluated in the hypophysial GH secretion tests, and may be used to treat the hypophysial GH secretion insufficiency.

Glutathione-induced inhibition of Na+-independent and -dependent bindings of L-[3H]glutamate in rat brain.

Reduced glutathione (GSH, 10(-7)-10(-3) M) was found to exert a profound suppressive action on the Na+-independent and -dependent bindings of L-[3H]glutamic acid (Glu) in a temperature-independent manner. Similarly significant reduction of the bindings resulted from the addition of oxidized glutathione (GSSG). Scatchard analysis revealed that GSH as well as GSSG invariably decreased the affinity of the binding sites for [3H]Glu without significantly affecting the number of the binding sites. These results suggest that GSH (GSSG) may in part participate in the synaptic transmission at central Glu neurons through interaction with the receptors and/or the uptake sites for Glu.

[Reproductive and developmental toxicity study of a new antineoplastic agent, S-1 (IV)--Perinatal and postnatal study in rats by oral administration].

S-1 is a newly developed antineoplastic agent consisting in a molar ratio of 1:0.4:1 mixture of tegafur (FT), 5-chloro-2, 4, dihydroxypyridine (CDHP) and potassium oxonate (Oxo) was administered orally to SD rats at doses of 0, 1, 4 and 7 mg/kg/day (as a dose of FT) during the perinatal and postnatal periods to examine its effect on dams and postnatal growth of the offspring. A group as the control was treated only with medium (0.5% hydroxypropyl methylcellulose) solution. The administration of 7 mg/kg/day to dams caused suppression in body weight gains and in food consumption during the treatment period. No adverse effects of S-1 on the length of gestation, gestation index, delivery and nursing ability were found. The administration of 4 and 7 mg/kg/day caused suppression in body weight gains in offspring of both sexes. Significant decrease in kidney weights were observed in females of the 4 mg/kg/day group and in both sexes of the 7 mg/kg/day group. No adverse effects of S-1 were found in number of live offspring at birth, sex ratio of live offspring, number of dead offspring at birth, birth index, viability index, weaning index, incidence of external anomalies, general conditions, postnatal development, reflex responses, motor coordination, emotional behavior, learning ability, skeletons, necropsy findings or reproductive functions. No adverse effects of S-1 on F2 offspring were found in any treatment groups. Under the conditions of the present study, the non-observed effect dose levels of S-1 was 4 mg/kg/day for general toxicology of dams, 7 mg/kg/day for reproductive ability of dams, 1 mg/kg/day for postnatal growth in F1 offspring and 7 mg/kg/day for postnatal growth in F2 offspring.

[Peri- and postnatal study of cefodizime sodium in mice--intravenous administration from late gestation through period of lactation].

Peri- and postnatal study of cefodizime sodium (THR-221), a new developed cephem-type antibiotics, was carried out with ICR mice. THR-221 at dose levels of 0, 300, 1000 and 3000 mg/kg/day were administered intravenously to pregnant and delivered dams from day 15 of gestation through day 21 of lactation. No changes in body weights of dams in all treated groups but slight decrease in food consumptions of 3000 mg/kg/day group were observed. Treated sites, tails of a few dams in this group, were affected with inflammatory lesions because of repeated dosing. Neonates from dams treated with 3000 mg/kg/day were slightly decreased in body weight at birth. At term sacrifice of F 1 of 10 weeks age, absolute and relative spleen weights were decreased in male 3000 mg/kg/day group and in female 1000 and 3000 mg/kg/day group. No effects on other physical, behavioral or reproductive ability examinations of F 1 offspring were showed. It is suggested that no effect dose level of THR-221 is 1000 mg/kg/day and that of F 1 offspring is 300 mg/kg/day.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 26). Detection of 1,3-dinitrobenzene-induced histopathological changes in testes and epididymides of rats with 2-week daily repeated dosing.

As part of a collaborative work, male rats were administered 1,3-dinitrobenzene (1,3-DNB) daily at 0, 25 and 50 mg/kg/day from the age of 6 weeks for 4 weeks (4-week exp.), or at 25, 50 and 75 mg/kg/day from the age of 8 weeks for 2 weeks (2-week exp.). After the end of each administration period, all survivors were sacrificed, and their testes and epididymides were removed, weighed and examined histopathologically. The following results were obtained. In the 4-week exp.: At 50 mg/kg/day, the weights of testes and epididymides showed decrease with macroscopic atrophy. The testicular spermatogenic epithelium showed decrease in the number of sperm-spermatocytes, degeneration/necrosis, giant cell formation and vacuolation, reduction in sperm counts also being evident in the ducts of the epididymides. In the 2-week exp.: At 50 and 75 mg/kg/day, the weights of testes and/or epididymides showed decrease with macroscopic atrophy. Several histopathological changes in the testes and epididymides were essentially the same changes as in the group given 50 mg/kg/day in the 4-week exp., with a clear relation. These results indicate that a 2-week administration period is sufficient to detect testicular and epididymal histopathological changes induced by 1,3-dinitrobenzene in male rats.

[Reproductive and developmental toxicity study of mofezolac (N-22) (1)--Study by oral administration of N-22 prior to and early stages of pregnancy in rats].

Mofezolac (N-22), a newly developed analgesic and anti-inflammatory agent, at dose levels of 0, 10, 30 and 90 mg/kg/day were administered orally to males from pre-mating to mating period and to females from pre-mating to early gestation period. Effects on reproductive performance of both sexes, especially reproductive capability, and development of offspring were examined. 1. In male parents, no changes in body weight and food consumption were found in all male groups, but the increase in gastric mucosal lesions such as ulcers were observed in 30 and 90 mg/kg/day groups. 2. In female parents, the decrease in body weight and food consumption of 90 mg/kg/day group during early gestation period were found, but at necropsy no changes were shown in all female groups. 3. Reproductive capability, mating and pregnancy performance were not affected. 4. No effects of N-22 on fetuses were observed. 5. The suggestions were as follows: No effect dose levels (NOELs) for male and female general-toxicologically were 10 and 30 mg/kg/day, respectively. NOELs for reproductive capability and for fetal development were 90 mg/kg/day.

[Reproductive and developmental toxicity study of mofezolac (N-22) (4)--Study by oral administration of N-22 during the perinatal and lactation periods in rats].

peri- and postnatal study of mofezolac (N-22), a new developed analgesic and anti-inflammatory agent, was carried out with Wistar rats. N-22 at dose levels of 0, 25, 50 and 100 mg/kg/day were administered orally to pregnant and subsequent delivered dams from day 17 of gestation through day 21 of lactation. Body weight gains of dams treated with 50 and 100 mg/kg/day were depressed during perinatal period. Prolongation of pregnancy period, low performance of pup care with decrease in body weights and food consumptions were observed in 100 mg/kg/day group. Decrease in number and birth index, increase in number of stillborns and tendency to decrease in viability index on day 4 were found in 100 mg/kg/day group. Other parameters of development, behavior or reproductive capability of F1 animals showed no changes related to administration of N-22. It was suggested that no effect dose levels of N-22 were 25 mg/kg/day for dams viewpoint of general toxicity and that was 50 mg/kg/day for dams on reproductive performance and for offspring on development.

[Reproductive and developmental toxicity study of suplatast tosilate (IPD-1151T) (2)--Teratological study in rats by oral administration].

A teratological study of suplatast tosilate (IPD-1151T), a new anti-allergic agent which has a suppressive action on IgE antibody formation, was conducted with pregnant Wistar rats. Dosage levels of IPD-1151T 0, 300, 900 and 2700 mg/kg/day were administered to dams orally by gavage on days 7 through 17 of gestation. Two-thirds of dams per group was caesarean-sectioned on day 20 of gestation and their fetuses were removed for examination of external, visceral and skeletal anomalies. The remaining one-third was allowed to deliver naturally. F1 neonates were examined developmental, functional and behavioral parameters and reproductive abilities. The results were as follows: 1. Toxicities on F0 dams in the 2700 mg/kg/day group were salivation, piloerection, and decreases in body weight and food consumption. Seven animals (19.4%) showed severe toxicity and were dead. Toxicity in the 900 mg/kg/day group was a slight decrease in food consumption. The dosage level of 300 mg/kg/day was non-toxic. 2. Toxicities on F1 fetuses in the 2700 mg/kg/day group were a decrease in body weight and an increase in visceral anomalies (main one was ventricular septal defect that might be related to developmental retardation). No toxicities were seen in the 300 and 900 mg/kg/day. 3. In F1 neonates, suppressions of body weight were observed clearly in the male and female 2700 mg/kg/day and slightly in the male 900 mg/kg/day groups. But no changes in parameters of development, function, behavior or reproductive ability were seen in any dosed groups. It was suggested that no effective dose levels of IPD-1151T were 300 mg/kg/day for F0 dams and F1 neonates, and 900 mg/kg/day for F1 fetuses.

[Reproductive and developmental toxicity study of suplatast tosilate (IPD-1151T) (3)--Teratological study in rabbits by oral administration].

A teratological study of suplatast tosilate (IPD-1151T), a newly developed anti-allergic agent, was carried out in pregnant NZW rabbits to assess the effects on dams and fetuses. IPD-1151T was administered to dams orally at dose levels of 0, 100, 300, 450 and 700 mg/kg/day from day 6 through day 18 of gestation, and their fetuses were removed for teratological evaluation. The results were as follows: 1. In dams, marked increase in the incidence of abortion, and decrease in body weight gain, food consumption and feces mass were shown in the 700 mg/kg/day group. Slight decrease in body weight gain and food consumption were seen in the 450 mg/kg/day group, but no toxicities were observed in the 300 mg/kg/day or less groups. 2. In fetuses, marked increase in embryo-fetal deaths and decrease in alive fetal body weights and placental weights, but no teratogenicity were shown in the 700 mg/kg/day group. There were no fetal toxicity or teratogenicity in the 450 mg/kg/day or less groups. 3. No effective dose levels were 300 mg/kg/day for maternal general toxicity and 450 mg/kg/day for maternal reproductive toxicity and for fetuses.

[Reproduction study of 1,1,3-trimethyl-5-phenylbiuret (ST-281): teratological study in rabbits by oral administration].

Teratogenicity of 1,1,3-trimethyl-5-phenylbiuret (ST-281), a new anti-rheumatic agent, was evaluated in rabbits. ST-281 at doses of 0, 50, 100, 200 and 400 mg/kg/day were administered orally to pregnant NZW rabbits from day 6 to day 18 of pregnancy. Body weight and food consumption at the administration and the subsequent periods were significantly decreased in 400 mg/kg/day group, and 5 dams (41.7%) affected severely were dead. No remarkable changes were investigated in findings at near-term caesarean section in any dosed group including 400 mg/kg/day. In visceral and skeletal examinations, no significant increase in incidence of abnormal fetuses were observed. This report suggests that ST-281 has no embryotoxicity or teratogenicity in rabbits.

[Local irritation studies of cefodizime sodium].

Four local irritation studies of cefodizime sodium (THR-221), a new developed cephem-type antibiotics, were carried out with NZW rabbits. 1. In eye irritation test, 25% THR-221 water solution had no irritancy on eye mucosa in rabbits. 2. In single injective intramuscular irritation study, regardless of solvents (water or 0.5% lidocaine), 25% THR-221 solution had irritancy equal to 0.75% acetic acid. But recovery process of the muscle injured with THR-221 was faster and better than with 0.75% acetic acid. 3. In five days injective muscular irritation study, the irritancy of 25% THR-221 water solution on the muscle was milder than that of CTT or CET. Histopathological damage with THR-221, necrosis/degeneration of muscle fibers and edema/hemorrhage in interstitium etc., were well recovered. 4. In vessel irritation study, 10% or more THR-221 water solution had irritancy on ear vessel. THR-221, as same as CTT, caused organized thrombi and inflammation at the surrounding area. The degree of irritation of 20% THR-221 solution was slightly stronger than that of 20% CET, but weaker than that of 20% CTT. 5. In a clinical phase, it is to be desired that THR-221 like as CTT or CET shall be avoided repeated intramuscular or intravenous injections at the same site.

Comparison of sperm motility test methods (except computer-assisted sperm analysis) in rats under the condition of alfa-chlorohydrin treatment--collaborative investigation.

A comparison among rat sperm motility test methods including percent of motile sperm (% Motile), scoring method (Scoring), Ishii's method, Progressive Motility Test (PMT) and Sperm Quality Analyzer (SQA), was conducted using data gathered from eleven laboratories. As a unified study design, mature male rats were orally treated daily for approximately 1 week with alpha-chlorohydrin (ACH), which is known to affect the sperm motility at the epididymis, at dose levels of 2.5, 5 and 10 mg/kg, and then subjected to more than two test methods for sperm motility in each laboratory. Scoring (4 or 5 grades), Ishii's method, PMT and SQA showed high sensitivity for the detection of the effects of ACH, which were not considered to be inferior to a computer-assisted sperm analyzer (CASA). Longer incubation time before testing was considered to contribute to detecting the effects of ACH. In particular, we realized that Scoring was a favorable method even if the demerit of poor objectivity was allowed for. Percent Motile showed lower sensitivity than other test methods. The differences in sensitivity between % Motile and other methods were considered to be based on whether the defects of progressive motion could be detected. Although % Motile cannot clearly judge whether immotile sperm are dead or alive, the value is a great help for the interpretation of the result from other methods. Based on the characters for detectability, objectivity and efficiency, the most suitable method of sperm motility should be selected according to the purpose of the toxicity study.

Lipophilic statins can be osteogenic by promoting osteoblastic calcification in a Cbfa1- and BMP-2-independent manner.

Mevastatin (3-10 microM) and fluvastatin (0.1-10 microM), but not pravastatin, were found to promote calcification of MC3T3-E1 cells and their subclone MC4, in either the presence or absence of 3 mM inorganic phosphate stimulus. The mechanism of action was examined. Gel retardation assay and immunocytochemical analysis of core binding factor (Cbfa1) revealed that mevastatin and fluvastatin completed the nuclear export of Cbfa1, possibly thereby reducing the induction of the stably transfected p6OSE2-luc gene, and then promoted Cbfa1-independent calcification, which invariably occurred in both wild type and dominant negative Cbfa1-expressing cells. The induction of the bone morphogenetic protein-2 (BMP-2) gene promoter failed to respond to the statins. All the effects of the cell-permeable statins were negated by mevalonate pathway metabolites (geranylgeranylpyrophosphate > farnesylpyrophosphate > mevalonate) and reproduced by toxin B (a Rho-specific inhibitor), but not totally by Y27632 (a ROCK-specific inhibitor). The results suggest that lipophilic statins can be osteogenic by promoting Cbfa1- and BMP-2-independent calcification processes.