A novel cryoprotective protein (CRP) with high activity from the ice-nucleating bacterium, Pantoea agglomerans IFO12686.

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.

Development of play behavior between potential guide dogs for the blind and human raisers.

This study shows longitudinal development of human-dog interactions in a guide dog raising program. Social play interactions between potential guide dogs for the blind (puppies) and their adult female raisers (PWs) were videotaped at home during the period from two months to 11 or 12 months of the puppies' age. The puppies and PWs established close proximity relationships by two to three months of age. The older the puppies became, the shorter time the puppies and PWs spent in intensive fighting and chasing play. The older the puppies became, the longer the puppies and PWs spent in waiting, seeking and possessing together play which needed the puppies' self-control, concentration and ability to cooperate with the PWs. Whether or not these behavioral tendencies which the puppies showed were appropriate for guide dog candidates is also discussed.

Inappropriate behavior of potential guide dogs for the blind and coping behavior of human raisers.

Inappropriate behaviors of potential guide dogs (puppies) for the blind and coping behaviors of their adult female raisers (puppy walkers: PWs) were videotaped in their play situation at home from when the puppies were 2-11 or 12 months of age. The frequency of inappropriate behavior decreased with an increase in the puppies' age, suggesting that human-dog relationships became friendlier. The PWs tended to use moderate coping behaviors to stop the inappropriate behaviors of the puppies. Rejecting interaction with the puppies was effective for stopping the puppies from biting the PWs. Forcible stopping was effective for stopping the puppies from damaging objects. Not responding to the puppies was effective for stopping the puppies from biting the PWs, barking/growling and damaging objects.

A Japanese case of congenital hyperinsulinism with hyperammonemia due to a mutation in glutamate dehydrogenase (GLUD1) gene.

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.

Severe hypercalcaemia and respiratory insufficiency associated with infantile hypophosphatasia caused by two novel mutations of the tissue-nonspecific alkaline phosphatase gene.

UNLABELLED: We report the case of a male patient with infantile hypophosphatasia associated with severe hypercalcaemia and mild respiratory insufficiency. At the age of 2 months, severe hypercalcaemia, low levels of serum alkaline phosphatase activity, and elevated urinary excretion of calcium and phosphoethanolamine were noted. Radiological findings showed generalized osteopenia and disturbed and irregular ossification of the metaphyses. Their involvement had spontaneously improved at the age of 6 months. A genetic study revealed that the tissue-nonspecific alkaline phosphatase gene of the patient had two novel mutations, K207E and G409C, derived from the mother and father, respectively. A reconsitution experiment revealed that both mutant gene products had low but significant enzymatic activity. CONCLUSION: The detection of tissue-nonspecific alkaline phosphatase gene mutations and expression studies to determine the enzymatic activity of mutant gene products was useful for assessing the clinical course of this patient with hypophosphatasia.

Accurate localization of an insulinoma by preoperative selective intra-arterial calcium injection and intraoperative glucose monitoring.

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.

Characterization and properties of intracellular proteins after cold acclimation of the ice-nucleating bacterium Pantoea agglomerans (Erwinia herbicola) IFO12686.

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.

A cold acclimation protein with refolding activity on frozen denatured enzymes.

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.

Human-dog interactions in a guide-dog training program.

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.

Four mutant alleles of the insulin receptor gene associated with genetic syndromes of extreme insulin resistance.

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.

Analysis of the steroidogenic acute regulatory protein (StAR) gene in Japanese patients with congenital lipoid adrenal hyperplasia.

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.

Association of idiopathic hypopituitarism and syringomyelia.

The magnetic resonance in an 8-year-old boy with hypopituitarism and cervical syringomyelia are presented. Magnetic resonance imaging of the brain clearly demonstrated syringomyelia in the cervical spinal cord and transection of the pituitary stalk, findings identical to those reported in the literature. Both disorders have been seen in association with a difficult labor, so we suggest that this is not a chance finding.

Age-dependent changes in the phosphorylation of nuclear proteins of submandibular glands in isoproterenol-treated rats.

Age-dependent changes in the regulation of the phosphorylation of nuclear proteins were investigated in relation to DNA synthesis in rat submandibular glands after injection of isoproterenol (IPR). The level of phosphorylation of nuclear proteins in the tissue increased rapidly after birth, reaching a maximum at 4 weeks, and then decreased to the level of 52-week-old rats. The level of protein kinase activity in nuclei varied in parallel with that of phosphorylation of nuclear non-histone proteins after birth. The time after the injection of IPR required to initiate the phosphorylation of nuclear non-histone proteins of rat submandibular glands, which occurs prior to the onset of RNA synthesis that precedes the replication of DNA, increased with age. These results suggest that this delayed onset of phosphorylation plays a regulatory role in cell proliferation and cell function during aging.

Structure of the human glucokinase gene and identification of a missense mutation in a Japanese patient with early-onset non-insulin-dependent diabetes mellitus.

Glucokinase is thought to play a glucose-sensor role in the pancreas, and abnormalities in its structure, function, and regulation can induce diabetes. We isolated the human glucokinase gene, and determined its genomic structure including exon-intron boundaries. Structure of the glucokinase gene in human was very similar to that in rat. Then, by screening Japanese diabetic patients using polymerase chain reaction--single strand conformation polymorphism (PCR-SSCP) and direct-sequencing strategies, we identified a missense mutation substituting arginine (AGG) for glycine (GGG) at position 261 in exon 7 of the glucokinase gene in a patient with early-onset non-insulin-dependent diabetes (NIDDM).

[Kallmann's syndrome in a child--a case report].

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 association of hypopituitarism with small pituitary, invisible pituitary stalk, type 1 Arnold-Chiari malformation, and syringomyelia in seven patients born in breech position: a further proof of birth injury theory on the pathogenesis of "idiopathic hypopituitarism".

We report seven cases of hypopituitarism all having a history of breech delivery, asphyxia at birth, and syringomyelia. A small pituitary gland was found on MRI or CT in six cases, invisible pituitary stalk on MRI in five cases, and type 1 Arnold-Chiari malformation in six cases. A constellation of these abnormalities are best explained by traction of brain and spinal cord of the subjects exerted during breech delivery and further support the primary role of birth trauma in the genesis of "idiopathic hypopituitarism".

[Radioimmunoassay of arginine vasopressin in unextracted random urine: clinical application for discrimination of diabetes insipidus].

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.