A novel CLCN1 mutation: P480T in a Japanese family with Thomsen's myotonia congenita.

At least 50 disease-causing mutations in the skeletal muscle voltage-gated chloride channel gene (CLCN1), almost all of which originate from Caucasian families, have been identified. We investigated a Japanese family with Thomsen's myotonia congenita that included 16 affected individuals (8 men and 8 women) through five generations. Polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) screening of 11 members showed an aberrant conformer in exon 13 of CLCN1 complementary DNA (cDNA) in 8 affected and 1 unaffected members. By sequence analysis, we identified a C-to-A transition at nucleotide position 1438, resulting in a substitution of proline for threonine at amino acid position 480 (P480T), the same position of the original mutation (P480L) in Thomsen's disease. The P480T mutation was novel and absent in 100 normal controls. Seven of the 8 affected individuals were heterozygous; another, from affected parents, was homozygous. Clinically, myotonia in the homozygous patient was more severe than that in heterozygous patients, probably due to the gene dosage effect. On a long-train nerve-stimulation test at a rate of 3 Hz, M-wave responses in the homozygous patient showed marked decrement followed by recovery. In contrast, the heterozygous patients showed just a slight decrement or no changes, and none of 2 patients with myotonic muscular dystrophy or 2 normal controls revealed any decrement. Thus, the long-train nerve-stimulation test at a low stimulus frequency may be a useful tool to assess the disease-severity/genotype relationship in myotonia congenita.

Identification of a sequential B-cell epitope on major allergen (Cry j 1) of Japanese cedar (Cryptomeria japonica) pollen in mice.

BACKGROUND: Japanese cedar (Cryptomeria japonica; CJ) pollinosis is one of the most common allergic diseases in Japan. B cell epitopes on Cry j 1, a major allergen of CJ pollen, have been analyzed by the specific monoclonal antibodies to Cry j 1, and most of these epitopes may be conformational, but no previous report has addressed the analysis of sequential epitope mapping with synthetic peptides. The main purpose of the present study is to identify IgE and IgG B cell epitopes on Cry j 1 by using a synthetic peptide approach in mice. METHODS: We synthesized 35 overlapping peptides that cover the entire length of Cry j 1 and examined whether mouse IgE and IgG antibodies produced by immunization with Cry j 1 reacted to the Cry j 1 peptides. RESULTS AND CONCLUSION: We found that mouse IgE and IgG antibodies reacted strongly to Cry j 1 peptide No. 15 ((141)GVEPVHPQDGDALTLRTATN(160)), though those antibodies did not react with other peptides. IgE and IgG antibody binding to peptide No. 15 was completely inhibited by Cry j 1 and the peptide. To determine the minimum epitope in peptide No. 15, we conducted an ELISA inhibition test. IgE and IgG antibody binding to peptide No. 15 was inhibited by smaller peptides of this peptide. We found the core of the epitope to be (145)VHPQDGDA(152).

Oral administration of a dominant T-cell determinant peptide inhibits allergen-specific TH1 and TH2 cell responses in Cry j 2-primed mice.

BACKGROUND: Oral immunotherapy with a peptide for allergic immune responses is theoretically a promising therapy but has not been established yet. OBJECTIVE: To evaluate immune suppressive efficacy of oral administration of an immunodominant peptide, we investigated changes in T-cell proliferation, TH1 - and TH2 -cytokine production, and TH1 - and TH2 -mediated antibody production in mice after oral administration of a peptide. METHODS: Peptide p246-259, containing a dominant T-cell determinant of Cry j 2, which is the major allergen in Japanese cedar pollen, was used in this study. Groups of mice received p246-259 or PBS alone before or after they were primed intranasally with Cry j 2 and cholera toxin. In another experiment mice were primed intraperitoneally with Cry j 2 and alum. Proliferative response and cytokine production by nasal-associated lymph node cells against Cry j 2 were investigated. Amounts of systemic anti-Cry j 2 IgE and IgG antibodies were also measured. RESULTS: Oral administration of the peptide to mice before, or even after, the sensitization induced oral tolerance in T-cell responses against the allergen; the tolerance was associated with decreased production of TH1 (IFN-gamma and IL-2) and TH2 (IL-4) cytokines. Allergen-specific TH1 -mediated (IgG2a and IgG2b) and TH2 -mediated (IgG1 and IgE) antibody responses were also inhibited. CONCLUSIONS: Oral administration of a dominant T-cell determinant peptide induces immunologic tolerance in both TH1 and TH2 cell responses against the whole protein allergen. Our study is the first, to our knowledge, to demonstrate the potential for peptide-based oral immunotherapy in order to treat allergic immune responses.

Erythropoietin prevents place navigation disability and cortical infarction in rats with permanent occlusion of the middle cerebral artery.

Erythropoietin (EPO) prevents the ischemia-induced delayed neuronal death in the hippocampal CA1 field in gerbils. EPO receptor (EPOR) is also expressed in the cerebral cortex but its function is not known. To examine whether EPO has a neuroprotective action in the cortex, EPO was infused into the cerebroventricles of stroke-prone spontaneously hypertensive rats with permanent occlusion of the left middle cerebral artery. Morris water maze test indicated that EPO infusion alleviated the ischemia-induced place navigation disability. The left (ischemic)-to-right (contralateral nonischemic) (L/R) ratio of cerebrocortical area in the EPO-infused ischemic group was larger than that in the vehicle-infused ischemic group. The occlusion caused secondary thalamic degeneration but infusion of EPO prevented the decrease in the L/R ratio of thalamic area and supported neuron survival in the ventroposterior thalamic nucleus. In situ hybridization indicated that EPOR mRNA was upregulated in the periphery (ischemic penumbra) of a cerebrocortical infarct after occlusion of the middle cerebral artery, suggesting that an increased number of EPOR in neurons facilitates the EPO signal transmission, thereby preventing the damaged area from enlarging.

[Complication due to arterial infusion chemotherapy for liver metastasis from colorectal cancer].

OBJECTIVES: Arterial infusion chemotherapy is considered to be an extremely effective treatment for liver metastasis from colorectal cancer in terms of its tumor reduction and preventing recurrence in residual liver after resection. However, there still remain some unclear points as to the influence on hepatic artery and bile duct when this treatment is used over the long term. We report some conclusions obtained by examining cases of hepatic arterial occlusion (stenosis) and biliary complication who received this treatment. MATERIALS AND METHODS: Thirty-six cases who received this treatment over 3 months were the objects of this study, with the aim of direct effect against metastatic focus (21 cases) and prevention of recurrence in residual liver (15 cases). The ages were from 27 to 81; 22 cases were male and 14 were female. Indwelling routes of catheter were gastroduodenal artery (GDA) in 28 cases and femoral artery (FA) in 8 cases. Intermittent high-dose infusion (WHF: 5-FU 1,000 mg/m2/5 hrs qw) was adopted as the method. RESULTS: Hepatic arterial occlusion or stenosis was observed in 12 cases (GDA: 10; FA: 2). There seemed to be no correlation with the total dosage of 5-FU or the number of administrations. Even when hepatic arterial occlusion or stenosis occurred, no change was observed in liver function, and there no death was caused by this. However, CT showed a low-density area followed by atrophy in the right lobe in one case with right hepatic arterial stenosis, despite normal portal blood flow. Of the 6 cases which developed obstructive jaundice, 4 were due to the increase of metastatic focus or lymph nodes, and 1 case without dilatation of bile duct died from suspected sclerosing cholangitis. In this case, ALP had been increasing since 1 month before the onset of jaundice. Another case which developed biloma accompanied by the increase of serum bilirubin improved by discontinuance of chemotherapy. CONCLUSION: Since arterial infusion chemotherapy for liver metastasis from colorectal cancer causes hepatic arterial occlusion (stenosis) at a high rate, early detection of abnormalities by liver function test and imaging diagnosis which leads to early treatment is important.

[Influence of fluid replacement on serum magnesium concentration and proper magnesium supplementation during general anesthesia].

We have studied the influence of fluid replacement on serum magnesium (Mg2+) concentrations, and studied proper Mg2+ supplementation during general anesthesia. Thirty eight patients undergoing elective surgery randomly received: Mg(2+)-free acetated Ringer solution (Group I, n = 15), acetated Ringer solution containing 0.5 mmol.l-1 of Mg2+ (Group II, n = 6), 1.0 mmol.l-1 of Mg2+ (Group III, n = 7), 2.0 mmol.l-1 of Mg2+ (Group IV, n = 6), or 4.0 mmol.l-1 of Mg2+ (Group V, n = 4). Measurements were made on serum and urine Mg2+ concentrations during anesthesia. In Group I, the serum Mg2+ concentrations decreased in correspondence with the water balance. It is suggested that dilution due to the fluid replacement induced the reduction in serum Mg2+ concentrations since the observed urine Mg2+ concentrations were negligible. In Group II-V, the reduction in serum Mg2+ concentrations was inhibited by Mg2+ supplementation, and the serum Mg2+ concentrations remained unchanged in Group IV. We conclude the Mg2+ supplementation is required during anesthesia when a large amount of fluid is infused.

[A method of interval estimation for number of patients in the nationwide epidemiological survey on intractable diseases].

A method for estimating the intervals for the number of patients with intractable diseases from nationwide epidemiological surveys was developed under the assumption that response is independent of frequency of patients. This method is based on data utilizing the number of response hospitals classified by their reported number of patients. The approximate 95% confidence intervals of numbers of several intractable diseases patients were estimated using this method.

Amino acid sequence of guinea pig liver transglutaminase from its cDNA sequence.

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma-carboxamide groups of protein-bound glutaminyl residues. These enzymes are involved in many biological phenomena. In this paper, the complete amino acid sequence of guinea pig liver transglutaminase, a typical tissue-type nonzymogenic transglutaminase, was predicted by the cloning and sequence analysis of DNA complementary to its mRNA. The cDNA clones carrying the sequences for the 5'- and 3'-end regions of mRNA were obtained by use of the sequence of the partial-length cDNA of guinea pig liver transglutaminase [Ikura, K., Nasu, T., Yokota, H., Sasaki, R., & Chiba, H. (1987) Agric. Biol. Chem. 51, 957-961]. A total of 3695 bases were identified from sequence data of four overlapping cDNA clones. Northern blot analysis of guinea pig liver poly(A+) RNA showed a single species of mRNA with 3.7-3.8 kilobases, indicating that almost all of the mRNA sequence was analyzed. The composite cDNA sequence contained 68 bases of a 5'-untranslated region, 2073 bases of an open reading frame that encoded 691 amino acids, a stop codon (TAA), 1544 bases of a 3'-noncoding region, and a part of a poly(A) tail (7 bases). The molecular weight of guinea pig liver transglutaminase was calculated to be 76,620 from the amino acid sequence deduced, excluding the initiator Met. This enzyme contained no carbohydrate [Folk, J. E., & Chung, S. I. (1973) Adv. Enzymol. Relat. Areas Mol. Biol. 38, 109-191], but six potential Asn-linked glycosylation sites were found in the sequence deduced.(ABSTRACT TRUNCATED AT 250 WORDS)

[Dietary habits among smokers].

Differences in dietary habits between smokers and non-smokers were examined, using the data of well-designed case-control study of colon cancer. Smokers took much more coffee, beef and sake than non-smokers, and smokers tended to take meals irregularly. These dietary habits showed dose-response relationship in the amount of smoking. Odds ratio of proportion of smokers with the above four factors to those without any factors was 10.5, and those with two or three factors were 2-6. These results suggest that the interaction of smoking and dietary habits should be analysed in epidemiological study on cancer.

Effects of ceruloplasmin and the haptoglobin-hemoglobin complex on the oxygen-dependent reduction in growth of human diploid fibroblasts in serum-free, albumin containing medium.

We examined that growth-promoting activity of two different human albumin (HSA) preparations for human diploid fibroblasts in serum-free RITC 80-7 medium. The activity of one preparation (sample A) was affected markedly by environmental oxygen, whereas the other (sample B) was little affected. Sample B contained ceruloplasmin (Cp) and haptoglobin (Hp) as impurities. To detect the generation of superoxide anion in the media the amount of reduction of cytochrome c that is inhibited by superoxide dismutase (SOD) was determined. In an aerobic environment it was relatively large in comparison with reduction inhibited in a hypoxic environment. Reduction in the sample A with HSA-supplemented medium was relatively large in comparison with that in sample B with HSA-supplemented medium. The reduction of cytochrome c also was inhibited by Cp (25 mg/l) and catalase (4000 units/ml). Moreover, SOD, Cp, catalase and Hp.Hb (but not Hp) partially prevented oxygen-dependent reduction in growth in an aerobic environment when added to sample A HSA-supplemented medium. These results suggest that Cp and Hp.Hb act as an antioxidants in culture.

Quality deterioration of winged bean during storage.

Seeds of winged bean (Psophocarpus tetragonolobus) were stored under four different sets of conditions; 65% relative humidity (RH) at 5 degrees C, 35% RH at 37 degrees C, 65% RH at 37 degrees C, and 85% RH at 37 degrees C. After storage for three and five months, samples were taken out to explore quality changes during storage. The germination capacity decreased during storage at high temperatures. Although little change was found in content in extractable oil, a decrease in the iodine value and an increase in the acid value and in the TBA value were significant during storage at high temperature and high moisture, indicating deterioration of oil. Aldehyde in defatted bean extract increased under all conditions for storage. Protein extractability was greatly reduced at 36 degrees C and 85% RH, while no reduction was observed at other conditions.