An imprinted gene p57KIP2 is mutated in Beckwith-Wiedemann syndrome.

p57KIP2 is a potent tight-binding inhibitor of several G1 cyclin/Cdk complexes, and is a negative regulator of cell proliferation. The gene encoding p57KIP2 is located at 11p15.5 (ref. 2), a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a cancer-predisposing syndrome, making it a tumour-suppressor candidate. Several types of childhood tumours including Wilms' tumour, adrenocortical carcinoma and rhabdomyosarcoma exhibit a specific loss of maternal 11p15 alleles, suggesting that genomic imprinting is involved. Genetic analysis of the Beckwith-Wiedemann syndrome indicated maternal carriers, as well as suggesting a role of genomic imprinting. Previously, we and others demonstrated that p57KIP2 is imprinted and that only the maternal allele is expressed in both mice and humans. Here we describe p57KIP2 mutations in patients with Beckwith-Wiedemann syndrome. Among nine patients we examined, two were heterozygous for different mutations in this gene-a missense mutation in the Cdk inhibitory domain resulting in loss of most of the protein, and a frameshift resulting in disruption of the QT domain. The missense mutation was transmitted from the patient's carrier mother, indicating that the expressed maternal allele was mutant and that the repressed paternal allele was normal. Consequently, little or no active p57KIP2 should exist and this probably causes the overgrowth in this BWS patient.

Diffusion of nodal signaling activity in the absence of the feedback inhibitor Lefty2.

The role of Lefty2 in left-right patterning was investigated by analysis of mutant mice that lack asymmetric expression of lefty2. These animals exhibited various situs defects including left isomerism. The asymmetric expression of nodal was prolonged and the expression of Pitx2 was upregulated in the mutant embryos. The absence of Lefty2 conferred on Nodal the ability to diffuse over a long distance. Thus, Nodal-responsive genes, including Pitx2, that are normally expressed on the left side were expressed bilaterally in the mutant embryos, even though nodal expression was confined to the left side. These results suggest that Nodal is a long-range signaling molecule but that its range of action is normally limited by the feedback inhibitor Lefty2.

GFR alpha3, a component of the artemin receptor, is required for migration and survival of the superior cervical ganglion.

GFR alpha3 is a component of the receptor for the neurotrophic factor artemin. The role of GFR alpha3 in nervous system development was examined by generating mice in which the Gfr alpha3 gene was disrupted. The Gfr alpha3-/- mice exhibited severe defects in the superior cervical ganglion (SCG), whereas other ganglia appeared normal. SCG precursor cells in the mutant embryos failed to migrate to the correct position, and they subsequently failed to innervate the target organs. In wild-type embryos, Gfr alpha3 was expressed in migrating SCG precursors, and artemin was expressed in and near the SCG. After birth, SCG neurons in the mutant mice underwent progressive cell death. These observations suggest that GFR alpha3-mediated signaling is required both for the rostral migration of SCG precursors and for the survival of mature SCG neurons.

Elevation of anti-DNA antibody titer during thyrotoxic phase of silent thyroiditis.

The elevation of anti-DNA antibody titers was observed in two cases of silent thyroiditis; the levels reverted to normal as thyrotoxicosis improved. Biopsy specimens of the thyroid gland revealed chronic lymphocytic thyroiditis. The elevation of anti-DNA antibody titers during the thyrotoxic phase of silent thyroiditis, to our knowledge, has not been reported. In one case, antinuclear antibody testing was also positive during the thyrotoxic phase and became negative in the euthyroid state. These observations provide further support for autoimmunity in silent thyroiditis.

Spaceflight downregulates antioxidant defense systems in rat liver.

Liver antioxidant enzyme activities, mRNA abundance, and glutathione (GSH) status were investigated in male Sprague-Dawley rats placed in an enclosure module aboard Space Shuttle STS-63 for 8 d (F, n = 6). F animals were compared to rats housed in an enclosure module on the ground (G, n = 9), which simulated the vibration and temperature conditions associated with launch and flight, and rats kept under conventional ground vivarium conditions in individual cages (V, n = 6). Spaceflight significantly decreased catalase, GSH reductase, and GSH sulfur-transferase activities in the liver (p < .05). Neither enzyme activity nor enzyme protein content of Cu-Zn and Mn superoxide dismutase (SOD) was affected by flight. The relative abundance of mRNA for Cu-Zn SOD and catalase was significantly decreased comparing F with G rats (p < .05). Spaceflight resulted in a dramatic decrease of liver GSH, glutathione disulfide, and total GSH contents (p < .01), which were accompanied by a lower gamma-glutamyl transpeptidase activity (p < .05). F rats showed a 47% (p < .05) increase in liver malondialdehyde concentration compared to G and V rats. Liver protein content was not affected by flight. These results indicate that spaceflight can downregulate antioxidant defense capacity and elicit an oxidative stress in the liver.

The first exon of the rat aldolase C gene is essential for restoring the chromatin structure in transgenic mice.

A 13-kb fragment of the rat aldolase C gene contains sufficient information for gene expression. Transgenic mice carrying the 13-kb fragment showed restoration of chromatin structure and tissue-specific, copy number-dependent expression. To localize the regulatory elements responsible for restoring chromatin structure, several mutated constructs were used to produce transgenic mice. Three activities were examined: recreation of DNase hypersensitive sites, restoration of methylation status, and copy number-dependent expression. Deletions of the 3'-flanking region did not affect those activities. Deletion of seven introns affected the mRNA levels but not the restoration of the chromatin structure. The insertion of the LacZ gene into the first exon of the transgene interfered with both the restoration of the chromatin structure and the copy number-dependent expression in transgenic mice. DNase I footprinting assays revealed that brain-specific factors bind to the sequence disrupted by the LacZ insertion. These results suggest that the sequence in the first exon is essential for restoring the chromatin structure of the rat aldolase C gene.

Determination of the reducing terminal sugars of glycosaminoglycans using 2-aminopyridine.

The fluorescence labeling method (Takemoto, H. et al. (1985) Anal. Biochem. 145, 245-250) has been shown to have high sensitivity for measuring the sugar composition of glycoproteins. In the present study, its applicability for analysis of the reducing terminal sugars of glycosaminoglycans was investigated. The procedure involved coupling of glycosaminoglycans with 2-aminopyridine, followed by hydrolysis and N-acetylation, and then analysis by high-performance liquid chromatography on a reverse-phase column. The method was found to be useful for simultaneous determination of acidic, neutral and amino sugars at the reducing termini of glycosaminoglycan moieties.

A case of scleroderma with Sjögren's syndrome developed after mammoplasty.

A case of scleroderma and Sjögren's syndrome, which occurred 9 years after mammoplasty, was reported. The association between mammoplasty and connective tissue disorders was considered to be more than coincidental. The prolonged hypersensitization by injected material may be responsible for the development of connective tissue disorders, that is, a human adjuvant disease.

Enzyme immunoassay of bradykinin.

An enzyme immunoassay of bradykinin was developed by using beta-D-galactosidase as a labeling enzyme. Bradykinin was conjugated to beta-D-galactosidase with a new coupling agent of a hetero bis-functional type, N-(m-maleimidobenzoyloxy)-succinimide (MBS). Antisera were obtained from rabbits immunized with bradykinin linked to albumins (ovalbumin or bovine serum albumin) with toluene-2,4-diisocyanate. Double antibody method was employed to separate the antibody-bound antigen from free. The enzyme activity in the precipitate was measured with a fluorogenic substrate, 4-methyl-umbelliferyl-beta-D-galactoside. This assay is based on heterogeneous competitive binding between unlabeled and labeled antigens, so that unlabeled bradykinin reduces binding of bradykinin-enzyme conjugates to the antibody. A standard inhibition curve was linear between 3 and 300 ng bradykinin/assay tube.

Assay methods for prekallikrein and kininogens and their applications.

Prekallikrein activity in plasma was assayed using a synthetic peptidyl fluorogenic substrate (carbobenzoxy-L-phenylalanyl-L-arginine 4-methylcoumarinyl-7-amide), after activation of prekallikrein by acetone and kaolin. For total kininogen assay, the pretreatment of plasma at pH 2.0 was the best to eliminate bradykinin potentiators and kininase activity, before addition of trypsin to convert kininogen to bradykinin. Assay method of high molecular weight (HMW) kininogen was established by conversion of HMW-kininogen to bradykinin through activation of Hageman factor by glass powder and that of low molecular weight (LMW) kininogen was also by treatment of HMW-kininogen-depleted plasma in the same way as that for total kininogen. The marked reduction of prekallikrein and HMW-kininogen, not of LMW-kininogen, was found in pleural fluid of rat carrageenin pleurisy, and in plasma after i.v. injection of bromelain in rats. Members of the pedigree of hereditary angioneurotic edema patients also show low levels of prekallikrein and kininogens in plasma.

Comparison of the inpatients admitted during the recent 5 years (January 1994-December 1998) with those admitted during the previous 5 years (January 1989-December 1993): a clinico-statistical study.

As society ages, the composition of the diseases that occur within it changes accordingly. With that in mind, we examined the characteristics and trends in the recent inpatients and compared these recent inpatients with those of a previous report to identify the changes that accompany the aging of society. Subjects were 1,534 cases (men 56.9%, female 43.1%, average age 47.1 years) who were hospitalized at Kurume University Hospital for treatment during the 5-year period from January 1st, 1994 through December 31st, 1998. The ratio of inpatients over 65 years old was about 1.8 times higher than in the previous study, showing a clear trend toward an increased overall age of inpatients. As for the types of disease observed, the most common malignancies were epithelial tumor, followed by other benign tumors, as well as 76 cases which included diseases resembling tumor (epulis and exostosis etc.). A majority of the patients (55.6%) were directed to the Hospital by their dentist, a finding similar to that of the previous report. As for geographical distribution, 93.3% of the inpatients lived within 40 km of the center of Kurume City where our oral surgery is located, an increase of about 10% from the last report. In other words, our results showed a reduction in the sphere of treatment distribution.

[Evaluation of time course and inter-relationship of inflammatory mediators in experimental inflammatory reaction].

Inflammatory signs, such as heat, redness, swelling and pain, have been described from the Greek era. In these phenomena various endogenous active substances, i.e., inflammatory mediators, could cause and manifest vascular dilatation, a vascular permeability increase and sensitization of pain receptors, etc. In order to evaluate the roles of inflammatory mediators, we have studied the time courses of inflammatory reaction along with detection of various active substances directly or indirectly in the experimental animal model of pleurisy, such as rat carrageenin-induced, and zymosan-induced pleurisy. These pleurisies showed almost similar time courses of pleural exudate accumulation and neutrophil migration. However, mediators detected in the exudates of such pleurisies were different; in carrageenin-induced pleurisy bradykinin and prostacyclin (PGI2) caused exudate formation, while zymosan-induced pleurisy showed early degradation of mast cells and activation of complements, followed by an increase in platelet activating factor (PAF). In both pleurisies TNF alpha, IL-1, IL-6 and CINC (cytokine-induced neutrophil chemoattractant) appeared similarly in the exudates to cause chemoattractant for neutrophils. TNF alpha and IL-1 could stimulate to produce IL-6 and IL-8. While prostaglandins may regulate cytokine production via a cellular cAMP-dependent mechanism. Thus one should consider the time for application of anti-inflammatory drugs, such as cyclooxygenase inhibitor, indomethacin, since it causes increases in TNF alpha and IL-1 production by reducing PGI2 and prostaglandin E2 (PGE2) levels. In conclusion, inflammatory reaction has its own automatic regulation mechanism through complex cross talks between inflammatory mediators.

[Convulsive seizure after craniotomy--comparison of isoflurane anesthesia and sevoflurane anesthesia].

The incidence of postoperative convulsive seizure was compared retrospectively during either isoflurane or sevoflurane anesthesia in patients after craniotomy. Overall, 5 of a total of 125 patients suffered convulsive seizures after craniotomy: 2 (4%) of 45 patients with isoflurane anesthesia and 3 (4%) of 80 patients with sevoflurane anesthesia. No significant differences were observed between these two groups. The duration of anesthesia and duration of the MAC.h were similar between these two groups. The occurrence of postoperative convulsive seizure was not related with the inhalational anesthetic agents, the underlying disorders and patient characteristics. It is suggested that either isoflurane or sevoflurane anesthesia in neurosurgical patients may have no specific relationship with the postoperative convulsive seizures.

[Intravenous infusion of prostagrandin E1 during and after gastrectomy on postoperative hepato-renal functions in senile patients].

Effect of intravenous prostagrandin E1 (PGE1) during and after surgery on postoperative hepatorenal functions in senile patients was evaluated in 36 elective surgical patients ranged in age from 60 to 85 years. The patients with carcinoma of the stomach underwent total or subtotal gastrectomy under isoflurane anesthesia. These patients were devided into two groups. Eighteen patients received intravenous PGE1 at a rate of 0.03-0.13 micrograms.kg-1.min-1 during surgery and 0.03 microgram.kg.min-1 after surgery until 9:00 am on the first operative day. The remaining 18 patients did not receive PGE1 and served as the control group. Serum GOT and GPT levels in both groups increased significantly at emergence from anesthesia compared with the preanesthetic levels and then declined to the preanesthetic levels on the 3rd postoperative day. Thereafter they increased significantly again on the 7th postoperative day. Serum GOT levels in the PGE1 administered group were significantly lower than those in the control group at the emergence from anesthesia. Serum GPT levels in the PGE1 group tended to be lower than those in the control group on awakening from anesthesia and on the first postoperative day. Serum gamma-GTP levels were stable postoperatively but they increased significantly on the 7th postoperative day in both groups. Serum bilirubin levels were within normal limits in both groups. Postoperative serum levels of urea nitrogen and creatinine were at the preanesthetic levels in both groups. Our findings suggest that continuous intravenous administration of PGE1 during and after surgery is beneficial in attenuating hepatic injury in senile patients for gastrectomy. However, protective effect of PGE1 on postoperative renal function was found to be vague in this study.