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Prostaglandin F (PGF) ethanolamide (prostamide F) synthase, which catalyzed the reduction of prostamide H(2) to prostamide F(2alpha), was found in mouse and swine brain. The enzyme was purified from swine brain, and its amino acid sequence was defined. The mouse enzyme consisted of a 603-bp open reading frame coding for a 201-amino acid polypeptide with a molecular weight of 21,669. The amino acid sequence placed the enzyme in the thioredoxin-like superfamily with Cys(44) being the active site. The enzyme expressed in Escherichia coli as well as the native enzyme catalyzed not only the reduction of prostamide H(2) to prostamide F(2alpha) but also that of PGH(2) to PGF(2alpha). The V(max) and K(m) values for prostamide H(2) were about 0.25 micromol/min.mg of protein and 7.6 microm, respectively, and those for PGH(2) were about 0.69 micromol/min.mg of protein and 6.9 microm, respectively. Neither PGE(2) nor PGD(2) served as a substrate for this synthase. Based on these data, we named the enzyme prostamide/PGF synthase. Although the enzyme showed a broad specificity for reductants, reduced thioredoxin preferentially served as a reducing equivalent donor for this enzyme. Moreover, Northern and Western blot analyses in addition to the prostamide F synthase activity showed that the enzyme was mainly distributed in the brain and spinal cord, and the immunohistochemical study in the spinal cord showed that the enzyme was found mainly in the cytosol. These results suggest that prostamide/PGF synthase may play an important functional role in the central nervous system.

Prostaglandin (PG) D(2) ethanolamide (prostamide D(2)) was reduced to 9alpha,11beta-PGF(2) ethanolamide (9alpha,11beta-prostamide F(2)) by PGF synthase, which also catalyzes the reduction of PGH(2) and PGD(2) to PGF(2alpha) and 9alpha,11beta-PGF(2), respectively. These enzyme activities were measured by a new method, the liquid chromatographic-electrospray ionization-mass spectrometry (LC/ESI/MS) technique, which could simultaneously detect the substrate and all products. PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2) were separated on a TSKgel ODS 80Ts column, ionized by electrospray, and detected in the negative mode. Selected ion monitoring (SIM) of m/z 353 ([M-H](-)), 353 ([M-H](-)), 351 ([M-H](-)), 333 ([M-H-H(2)O](-)), 456 ([M+59](-)), and m/z 358 ([M-37](-)) was used for quantifying PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2), respectively. The detection limit for PGF(2alpha) and 9alpha,11beta-PGF(2) was 0.01pmol; that for PGH(2) and PGD(2), 0.1pmol; and that for prostamide D(2) and 9alpha,11beta-prostamide F(2), 0.5 and 0.03pmol, respectively. The LC/ESI/MS technique for measuring PGF synthase activity showed higher sensitivity than other methods. Using this method, we found that Bimatoprost, the ethyl amide analog of 17-phenyl-trinor PGF(2alpha) and an anti-glaucoma agent, inhibited all three reductase activities of PGF synthase when used at a low concentration. These results suggest that Bimatoprost also behaves as a potent PGF synthase inhibitor in addition to having prostamide-like activity.

The amino acid sequence of membrane-associated prostaglandin (PG) E synthase-2 (mPGE synthase-2), which has a broad specificity in its thiol requirement for a catalytic activity, has the consensus region from 104Leu to 120Leu found in glutaredoxin and of thioredoxin. The sequence of Cys-x-x-Cys in the consensus region is the active site for thioredoxin and mPGE synthase-2 also has this amino acid sequence (110Cys-x-x-113Cys). The mutation from 110Cys to Ser or the double mutation from 110Cys and 113Cys to Ser caused loss of PGE synthase activity, whereas the single mutation from 113Cys to Ser did not affect the enzyme activity. These results indicate that 110Cys, but not 113Cys, is the essential amino acid in the active site of mPGE synthase-2. 110Cys is an important amino acid in PGE synthase activity and plays the critical role as Cys at the same position in redoxin. Moreover, we found that the reduced form of lipoic acid (dihydrolipoic acid) serves as one of the natural activators of mPGE synthase-2 in the cells.

The ice-nucleating bacterium Pantoea ananas KUIN-3 accumulated glucose in cells following a shift in temperature (10 degrees C) from the optimum growth temperature (30 degrees C). This accumulation might be caused by the activation of glucose-6-phosphatase. Although this strain after culturing at 30 degrees C was harmed by freezing, the cryotolerance of this strain was reached about 80% after cold acclimation at 10 degrees C.

The ice-nucleating bacterium, Pantoea agglomerans IFO12686, induces the cryoprotective protein (CRP) by cold acclimation at 12 degrees C. The CRP was purified to apparent homogeneity by various chromatographies. We found that the purified CRP was a monomer of approximately 29,000 according to gel filtration chromatography and SDS-PAGE, and was a heat-stable protein. The CRP could protect freeze-labile enzymes, lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH) and isocitrate dehydrogenase (iCDH), against freezing-thawing denaturation. The activity of the CRP was about 3.5 x 10(4) times more effective than bovine serum albumin (BSA) and 2 x 10(6) times than COR26 from the ice-nucleating bacterium Pseudomonas fluorescens KUIN-1. We confirmed that the CRP was a novel protein, as judged by the a different molecule mass from the already-known cryoprotectants, and has an extremely high cryoprotective activity.

We describe a Japanese case of neonatal hyperinsulinism due to a de novo mutation (Gly446Asp) in glutamate dehydrogenase gene (GLUD1). A boy suffered from hypoglycemic coma with relative hyperinsulinemia on day 1 after birth, and received subtotal pancreatectomy. Examination of the resected pancreas revealed a diffuse increase in endocrine cells, consistent with 'nesidioblastosis'. He is now 15 years old and has exhibited mild but persistent hyperammonemia, which is a very unique feature of the disorder caused by GLUD1 activating mutations. He has also been suffering from seizures and mental retardation. Thus, GLUD1 mutations can be a cause of congenital hyperinsulinism in Japanese.

We found that a cold acclimation protein from an ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100 degrees C, cryopreservation at -20 degrees C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12 degrees C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82x10(-10), 4.35x10(-9), 8.98x10(-12), and 3.05x10(-11), respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.

The ice-nucleating bacterium Pantoea agglomerans (Erwinia herbicola) IFO12686 (INA(+)) responds to a decrease in temperature by the induction of proteins. The pattern of protein bands from strain IFO12686 following a shift in temperature from 30 to 12 degrees C could be divided into four major groups: (1) increasing protein bands, (2) decreasing protein bands, (3) increasing--decreasing protein bands, and (4) almost constant protein bands. We identified a cryoprotective function in the increasing protein band found in strain IFO12686. The increasing protein bands that followed a reduction in temperature were considered to have an important role in cold acclimation or adaptation. We showed that these proteins possessed cryoprotective activity when tested against the freeze-labile enzyme lactate dehydrogenase. The strain IFO12686 had greater cryotolerance than Pa. agglomerans IAM1595 (INA(-)), and the degree of cryotolerance was increased by cold acclimation., (Copyright 2000 Academic Press.)

The role of pre- and intraoperative procedures for the localization of insulinomas has been extensively debated. We report a case of successful treatment using preoperative selective intra-arterial calcium injection and intraoperative glucose monitoring. A 12-year-old boy with hypoglycemic attacks had a large insulinoma in the head of the pancreas on computed tomography. Preoperative selective angiography combined with arterial stimulation-venous sampling (ASVS) by intra-arterial injection of calcium revealed no other insulinomas in the body and tail of the pancreas. Elevation of serum glucose on intraoperative monitoring confirmed complete enucleation of the insulinoma. Preoperative ASVS can accurately localize an insulinoma, and may help to increase the success rate of surgery and avoid blind pancreatectomy.

We analyzed dyadic interactions between 12 neutered dogs (6 females and 6 males) and 44 humans (20 women, 14 men, and 10 girls) who were unfamiliar with each other. We also examined the effect of sex differences in dogs and humans as well as age differences in humans on human-dog interactions in a guide-dog training program. Female dogs more actively regulated their distance from humans than male dogs. Dogs made contact with women more frequently than with men, and men made contact with dogs more frequently than women. Girls initiated interactions with dogs more frequently than women; girls formed reciprocal interactions with dogs less frequently than women.

We identified four novel mutant alleles of the insulin receptor gene in three patients with genetic syndromes associated with insulin resistance. Two mutant alleles of the insulin receptor gene were identified in a patient with the Rabson-Mendenhall syndrome who was a compound heterozygote for a mutation at the 3'-splice acceptor site of intron 4 (AG-->GG), the first mutation causing an aberrant splicing at this locus, and a deletion of eight base pairs in exon 12. The second patient with leprechaunism was also a compound heterozygote for a deletion of one base pair in exon 19 and a mutation, Thr910-->Met, which causes impaired receptor processing. Interestingly, the third patient with type A syndrome was a simple heterozygote for the identical one base pair deletion. The fact that the same one base pair deletion links to type A in a simple heterozygote and to leprechaunism in a compound heterozygote appears consistent with the hypothesis that the severity of mutations will determine the phenotype.

Genomic DNA from 19 Japanese patients with congenital lipoid adrenal hyperplasia (lipoid CAH) representing 16 different families was examined to identify the genetic alterations of steroidogenic acute regulatory protein (StAR). Ten of 19 patients had a 46,XX karyotype and nine had a 46,XY karyotype. Six of the 46,XX patients have experienced spontaneous pubertal changes including breast development and irregular menstruation whereas none of the 46,XY subjects displayed pubertal changes. Eight different mutations were identified. Sixteen patients were either homozygotes or compound heterozygotes for the Q258X mutation. The seven other mutations identified were 189delG, 246insG, 564del13bp, 838delA, Q212X, A218V and M225T. The 189delG, 246insG, 546del13bp and Q212X mutants encode truncated proteins. COS-1 cells transfected with expression vectors encoding cDNAs for the mutant StAR proteins which affect the C-terminus, 838delA, A218V and Q258X, exhibited no steroidogenesis enhancing activity. However, the M225T mutant retained some steroidogenic activity. The patient with the M225T mutation had late onset of this disorder and some capacity to secrete testosterone in response to hCG. These findings suggest: (i) that the Q258X mutation can be used as a genetic marker for the screening of Japanese for lipoid CAH, (ii) that the C-terminus of StAR plays an important role in the protein's activity and (iii) that there are differences in the extent of functional impairment of the testis and ovaries in lipoid CAH.

The patient was a male child 6 years and 6 months old. He visited our hospital because of bilateral cryptorchism and true phimosis. Since he was found to have fairly small testis, micropenis, and anosmia, endocrinological examination was performed. The plasma levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were at the lower limit of normal values. LH gave a low reaction to the LH-releasing hormone test, while the reaction of FSH was near the lower limit of normal values. In the human chorionic gonadotropin test, there was no rise in the plasma levels of testosterone. Nor was there any reaction to the venous olfactory test with Alinamine (thiamine propyldisulfide). Accordingly, the patient was diagnosed as having Kallmann's syndrome. A search of the literature reveals few reports that deal with Kallmann's syndrome in infancy. Early diagnosis seems to be of significance, however, to institute early appropriate treatment to prompt the development of proper secondary sexual characteristics and ensure normal future fertility.

The concentration of arginine vasopressin in unextracted random urine was directly measured by radioimmunoassay. The urinary AVP value (Vm) measured by RIA, their AVP value (Vc) revised by urinary creatinine and their AVP value (Vc/op) revised by urinary creatinine and urinary osmotic pressure were applied for discriminate diagnosis of diabetes insipidus (DI). In children with central DI, Vm was significantly lower then that of normal subjects and the distribution of Vm was different from that of normal subjects. In children with renal DI, the distribution of Vc/op was different from that of normal subjects. In conclusion the children with central DI and renal DI can be discriminated from the normal children in comparison with Vm and Vc/op.

Strychnine at concentrations of 0.1 approximately 1.0 microM induced supersensitivity of rat vas deferens to alpha-agonists but not to other stimulants. Strychnine-treatment resulted in an increase in the number of alpha-adrenoceptors, determined by measuring the binding of [3H]prazosin. Glycine did not have any significant effect on the contracture and did not antagonize the action of strychnine. Thus, specific supersensitivity of alpha-adrenergic system was induced by treatment with low concentrations of strychnine.

T-1982 (cefbuperazone), a new 7 alpha-methoxycephem antibiotic, was fundamentally and clinically studied, and the following results were obtained. The antibacterial activities of T-1982 against clinical isolates of S. aureus, E. coli, K. pneumoniae, S. marcescens, P. mirabilis and P. aeruginosa were determined in comparison with those of CER, CEZ, CMZ and CTT. Against S. aureus, CER and CEZ exhibited excellent activity, whereas T-1982 was less active with the peak MIC of 12.5 micrograms/ml even with the inoculum size of 10(6) cells/ml. The activity of T-1982 was equal to that of CTT and by far superior to that of CER, CEZ and CMZ against E. coli, K. pneumoniae and P. mirabilis, the peak MICs with the inoculum size of 10(6) cells/ml being less than or equal to 0.1-0.2 microgram/ml, less than or equal to 0.1-0.2 microgram/ml and 0.2-0.39 microgram/ml, respectively. Against S. marcescens, T-1982 was superior to CMZ and CTT and 48% of the strains were inhibited by 3.13 micrograms/ml or less, whereas all the strains were resistant to CER and CEZ. The MIC of T-1982 against most strains of P. aeruginosa was more than 100 micrograms/ml. 10 mg/kg or 20 mg/kg of T-1982 was administered by one shot intravenous injection or 1 hour drip infusion to 23 pediatric patients to measure serum levels and urinary recovery. At 30 minutes after one shot injection of 10 mg/kg and 20 mg/kg, the highest serum levels of 22.0-38.8 micrograms/ml and 52.4-80 micrograms/ml were observed, the half-lives being 1.32 hours and 1.76 hours. When given by 1 hour drip infusion, the serum levels attained the peaks of 29.2-42.6 micrograms/ml and 49.0-75.6 micrograms/ml at the end of infusion, the half-lives being 1.24 hours and 1.19 hours. The urinary recovery rates within 6 hours were 74.2-92.5% and 50.2-66.5% by one shot injection and 63.4-84.2% and 53.9-79.0% by drip infusion. T-1982 was administered at a dose of 50 mg/kg by 30 minutes drip infusion to a child with purulent meningitis. The levels of T-1982 in the cerebrospinal fluid at 1 hour after administration were 4.8-6.7 micrograms/ml with the CSF/serum ratios of 4.4-8.4%. A total of 36 pediatric patients (21 cases of respiratory tract infection, 9 cases of urinary tract infection and each 1 case of purulent cervical lymphadenitis, scarlet fever, purulent meningitis, acute colitis, peritonitis and sinusitis) was treated with 40-80 mg/kg/day of T-1982 (252.6 mg/kg/day in purulent meningitis). The response was excellent in 27 patients and good in 7 patients, the efficacy rate being 94.4%. Diarrhea or eruption were observed in each 1 case. No abnormal laboratory findings were noted in any cases.

In this clinical study we evaluated the use of hydroxyapatite ceramic (HAP) implant in conjunction with the surgical treatment of periodontal osseous defects. Fifty-five defects in 23 patients were treated and filled with HAP particles (BDHAP-101). At 6 months and 12 months after the placement of particles, the sites were evaluated by gingival margin heights, periodontal pocket depths, attachment levels, tooth mobility, the gingival index (GI), and radiographic analyses. Oral hygiene status was estimated by the plaque index (PlI). There were significant improvements with regard to probe depth, attachment level, tooth mobility, and GI. The radiographic analysis showed a similar density of the adjacent bone and the particles, suggesting excellent biocompatibility. There were no changes in PlI before or after the placement of implants.

Development, growth, maturation and aging processes of secretory cells of rat salivary glands progress mainly after birth. Nuclear non-histone proteins, phosphorylated actively and reversively, have an important role as regulatory molecules of gene activity and have a possibility to bring about specific changes in these cellular processes. We examined in the present study the age-dependent changes in the phosphorylation of non-histone proteins of rat salivary glands. Nuclei purified from submandibular and parotid glands of 8-week-old rats rapidly incorporated 32P from gamma-32P-ATP into the nuclear phosphoproteins and reached equilibrium within 9 min. A preponderant amount of the 32P was present in non-histone proteins. The levels of phosphorylation of non-histone proteins in salivary gland nuclei increased rapidly after birth, reaching a maximum in both gland nuclei of 4-week-old rats and then decreasing to the levels observed in submandibular and parotid gland nuclei from 20 and 16-week-old rats, respectively. These levels were still maintained in nuclei from aged rats. Moreover, age-dependent changes in the protein kinase activity of submandibular and parotid gland nuclei were linked up with the changes in the phosphorylation of non-histone proteins. However, changes were not observed in the phosphorylation of histone proteins after birth. These results suggest that protein kinase activity in salivary gland nuclei may have an important role on age-dependent changes in cell function, mediated through the control of the phosphorylation of non-histone proteins.

The stimulatory effect of forskolin on amylase secretion was studied by pretreating rat parotid tissue with forskolin for 10 min, incubating it in medium without forskolin for 10 min, and then treating it with forskolin again. Pretreatment with 10 microM forskolin for 10 min resulted in increased amylase secretion and enhanced accumulation of cyclic AMP in the tissue during the second incubation with forskolin. In the presence of colchicine or vinblastine, the enhancement in cyclic AMP accumulation during the second incubation with forskolin was prevented, but the increased amylase secretion remained unchanged. The increased amylase secretion was counteracted only in the presence of concanavalin A. On the other hand, increased amylase secretion induced by isoproterenol (IPR) pretreatment was counteracted by colchicine, vinblastine, concanavalin A or strychnine. These data suggest that the total amount of cyclic AMP in the tissue does not have any essential role in the supersensitivity of the amylase secretory response, and that the supersensitivity induced by forskolin differs from that induced by IPR.

Rat vas deferens has been shown to become supersensitive to alpha-adrenergic agonists on brief treatment with epinephrine (Epi-treatment). Epi-treatment increased the contractile response and the number of alpha-adrenoceptors in the tissue. The application of colchicine (2.5 mM) during Epi-treatment prevented the supersensitivity-like phenomena and increase in number of alpha-adrenoceptors. Vinblastine (10 microM) also counteracted the effect of Epi-treatment but cytochalasin B (100 microM) and strychnine (2.5 mM) did not. It is suggested that the supersensitivity-like phenomena induced in rat vas deferens by Epi-treatment were due to a change in microtubular components of the membrane.

In rat parotid tissue, amylase secretion and accumulation of cyclic AMP were not selective responses to the different beta-subtypes, beta 1 and beta 2. However, the supersensitivity of the amylase secretory response induced by brief pretreatment with beta-agonist was due specifically to a beta 2-adrenergic response.

Urinalysis of alkaptonuria using NMR spectroscopy revealed the abnormal amount of homogentisic acid. Qualitative and quantitative determination was performed simultaneously with untreated urine sample and within 15 min.

Late infantile metachromatic leukodystrophy (MLD) was successfully diagnosed in utero by demonstrating the absence of arylsulfatase-A in amniotic fluid using diethylaminoethyl-Sepharose column chromatography. Diagnosis by amniotic fluid using an ion-exchange column is more rapid and reproducible as compared with those reported previously. The diagnosis was confirmed by the absence of arylsulfatase-A in fetal brain, liver, and kidney tissues as well as by the marked accumulation of sulfatide in kidney. The kidney is the most appropriate organ for the demonstration of sulfatide accumulation in fetal tissues in MLD.