Evaluation of velopharyngeal function using high-speed cine-magnetic resonance imaging based on T2-weighted sequences: a preliminary study.

The objective was to introduce a new technique for visualizing the three-dimensional (3D) movements of velopharyngeal-related muscles using high-speed cine-magnetic resonance imaging (MRI) based on T2-weighted sequences. The evaluation of phonation- and water swallowing-related events was performed in 11 healthy subjects. Specifically, whether cine-MRI could precisely visualize normal velopharyngeal function during these two events was examined. The 3D movements of the soft palate, superior pharyngeal constrictor muscles, and levator veli palatini muscles were visualized in all 11 subjects. A noteworthy finding was that the magnetic resonance signals of the superior constrictor pharyngeal muscles and the levator veli palatini muscles were significantly higher during phonation and during water swallowing than at rest. This initial study suggests that the 3D movements of velopharyngeal-related muscles can be successfully and precisely visualized without side effects. The magnetic resonance signal changes seen in the superior pharyngeal constrictor and levator veli palatini muscles using the technique described here should be useful to develop better methods of evaluation of velopharyngeal function.

Predicting the therapeutic effect of carbamazepine in trigeminal neuralgia by analysis of neurovascular compression utilizing magnetic resonance cisternography.

The aim of this study was to determine whether the evaluation of neurovascular compression (NVC) using new criteria on magnetic resonance (MR) cisternography improves the prediction of the curative effects of carbamazepine (CBZ) in trigeminal neuralgia (TN). In this study, a new analysis of NVC using MR cisternography was performed retrospectively for 280 patients with clinical signs and symptoms suggesting TN. The new analysis examined whether the site of the NVC was less than 3mm from the edge of the exit point and within the first third of the root entry zone on MR cisternography. Prediction of the curative effects of CBZ using the new evaluation of distance to the NVC improved on the predictive ability of the previous method of calculating the NVC volume (Mantel-Haenszel coefficient, P<0.01). In particular, initial treatment with CBZ 100mg/day for 2 weeks appeared more effective for patients with NVC volumes of ≤5mm3 plus a distance to the NVC of ≤3mm, than for those with NVC volumes >5mm3. The evaluation of NVC on MR cisternography using this new approach appears to be more useful than the previous method for predicting the initial treatment response in patients with TN.

Zonal distribution of Purkinje cells in the zebrafish cerebellum: analysis by means of a specific monoclonal antibody.

We have isolated a monoclonal antibody that recognizes a 42-kDa protein from adult zebrafish brain. The antibody stains the typical drop-shaped perikaryon of Purkinje cells and their dendrites. The cerebellum of teleosts has complex features. It is composed of three parts; the valvula cerebelli (Va), the corpus cerebelli (CCe), and the crista cerebellaris (CC). In higher vertebrates, the molecular layer is always found as the most outer layer of the cerebellum, but in teleosts, some of the granular cells are located on the surface of the Va. In higher vertebrates, the boundary between the granular and molecular layers always contains Purkinje cells, but this does not occur in teleosts. The Purkinje cells are found only in a part of the boundary in Va. We have found that the layer containing Purkinje cells forms a continuous zone in the cerebellum in the zebrafish. The complex structure of the cerebellum is more easily understood with the aid of the concept of a "Purkinje zone". The Purkinje zone starts at the caudal end of Val (lateral division of Va), turns at the edge of Va toward Vam (medial division of Va), connects to CCe, and ends at the bottom of CCe. The dendrites are found only on one side of the zone. The dendrites of the Purkinje cells in Vam are planar and are packed regularly, similar to those of higher vertebrates. However, the dendrites in Val and the posterior part of CCe are not planar and are irregularly packed.

Molecular bases of congenital hypopigmentary disorders in humans and oculocutaneous albinism 1 in Japan.

The molecular bases of various types of congenital hypopigmentary disorders have been clarified in the past 10 years. Homozygous gene mutations of enzymes functional in melanogenesis such as tyrosinase, P protein and DHICA oxidase, result in oculocutaneous albinism (OCA) 1, OCA 2, and OCA 3, respectively. The genes responsible for Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS) have also recently been isolated and cloned. The transcription factor paired box 3 (PAX3) works at the promoter region of the microphthalmia-associated transcription factor (MITF) gene, and the MITF transcription factor orders the expression of c-kit, which encodes the receptor for stem-cell factor, which in turn stimulates melanoblast migration from the neural tube to the skin in the embryo. Heterozygous mutations of PAX3, MITF, or c-kit genes induce Waardenburg syndrome (WS) 1/3, WS 2 or Piebaldism, respectively. A defect of endothelin-3 or the endothelin-B receptor produces WS 4. In our examination of 26 OCA 1 patients in Japan, all were found to have homozygous or heterozygous tyrosinase gene mutations at codons 77 or 310. Therefore, mutations at codons 77 and 310 are the major ones in Japanese patients with OCA 1. An autosomal dominant pigmentary disease of dyschromatosis symmetrica hereditaria (DSH) is well known in Japan, and is characterized by a mixture of hypo- and hyper-pigmented macules of various sizes on the backs of the hands and feet. The disease gene and its chromosomal localization have not been identified yet. Our trial of linkage analysis and positional cloning to determine the disease gene is presented.

Exclusion of linkage between dyschromatosis symmetrica hereditaria and chromosome 9.

Dyschromatosis symmetrica hereditaria (DSH) is an autosomal dominant pigmentary disorder, first reported by Toyama in 1910. It is characterized by a mixture of hypopigmented and hyperpigmented macules of various sizes on the backs of the hands and feet. The disease gene of DSH and its chromosomal localization have not yet been identified. A family with DSH and idiopathic torsion dystonia (ITD), a rare neurological disease, was recently reported. Therefore, we speculated that there was a linkage between the DSH gene and the ITD gene, named DYT1 and localized on chromosome 9, and performed linkage analysis between DSH and microsatellite markers on chromosome 9 in three Japanese DSH families (36 patients in total). We obtained a LOD score of < -2 over the whole region of chromosome 9 encompassing DYT1. Thus, we conclude that there is no linkage between DSH and DYT1 as well as any region of chromosome 9.

A splicing mutation of the tyrosinase gene causes yellow oculocutaneous albinism in a Japanese patient with a pigmented phenotype.

BACKGROUND: Yellow oculocutaneous albinism (OCA) that is caused by tyrosinase gene mutations shows two characteristics: extreme hypopigmentation at birth and the eventual development of yellow or blond hair. OBJECTIVE: We studied a Japanese girl who had brown hair, a lighter skin color than her unaffected family and brown eyes at 9 months of age. METHODS: We performed direct sequencing analyses of the tyrosinase gene in her genomic DNA. RESULTS: The patient was a compound heterozygote for the +DeltaC310 mutation (known to result in absent melanogenic activity) and a second t-->a transition at the 3' end of intron 2. CONCLUSION: The t-->a transition has previously been reported as a splicing mutation in other Caucasian patients with a typical yellow OCA phenotype. However, this patient showed much more pigmentation than that reported in Caucasians. Therefore, we estimate that the mild phenotype results from her genetic pigment background.

Sequence-based diagnosis of tyrosinase-related oculocutaneous albinism: successful sequence analysis of the tyrosinase gene from blood spots dried on filter paper.

BACKGROUND: A large number of mutations of the tyrosinase gene result in oculocutaneous albinism (OCA). Therefore, at present, sequence analysis of the tyrosinase gene has become necessary to diagnose OCA patients and their relatives. OBJECTIVE: The aim of this study was to facilitate the sequence-based diagnosis of tyrosinase-related OCA by using small amounts of the patient's blood. METHODS: Blood spots dried on filter papers were used as sources of genomic DNA. The exons and flanking regions of the tyrosinase gene were amplified by polymerase chain reaction (PCR) and were directly sequenced in both directions. RESULTS: We successfully amplified all exons of the tyrosinase gene by PCR and were able to characterize compound heterozygous mutations of R278X and + delta C310 in the patient's gene. CONCLUSION: Recent advances of PCR-related technology allowed us to use fairly limited samples of blood for sequence analysis of the tyrosinase gene.

Oculocutaneous albinism and analysis of tyrosinase gene in Japanese patients.

Oculocutaneous albinism (OCA) is a heterogeneous groups of autosomal-recessive genetic disorders. The molecular pathogenesis of several types of OCA have been clarified in the ten years since our first report in 1989 on a pathologic mutation of the tyrosinase gene. In this article, a new classification of OCA based on genetic evidence is briefly reviewed, and our study on Japanese patients with tyrosinase-negative OCA is summarized.

Effects of X-ray irradiation on genomic DNA methylation levels in mouse tissues.

Effects of ionizing radiation on the level of genomic DNA methylation in liver, brain and spleen of mouse as well as in two kinds of cultured cells were examined by high-performance liquid chromatography. Ten Gy of whole body X-radiation reduced the 5-methyldeoxycytidine contents by about 40% within 8 hours after irradiation in liver. Similar effects were observed at 4 or 7 Gy of X-ray irradiation. However, no such change was detected in brain, spleen and cultured cells. The data indicate that radiation-induced alteration in genomic DNA methylation is not ubiquitous among different tissues and cells.

Sequence analysis of the human tyrosinase promoter from a patient with tyrosinase-negative oculocutaneous albinism.

We examined the tyrosinase gene from a patient with tyrosinase-negative oculocutaneous albinism (OCA). First we studied the protein coding region, exon/intron junctions, and the proximal promoter region (positions -300 to +1) of her tyrosinase gene by direct sequencing. Although the results showed that she was heterozygote for the R77Q mutation, we could find no other mutation. To find a second mutation and compare the sequence in the 5'-flanking region of her tyrosinase gene between two OCA alleles, we amplified a 2422-bp stretch (positions -2065 to +357, including R77Q mutation site) by PCR, and cloned it into a plasmid vector. As a result, we discovered a difference between two OCA alleles in the GA repeat region. Therefore, we expect that the polymorphism in the GA repeat region of the tyrosinase gene will be used as a flanking marker of the OCA allele.

Intake ratio of water-insoluble dietary fiber to the water-soluble one in Japanese: an estimation on dietary records and the secular consideration on the national nutrition survey.

Thirty-four foods were analyzed in order to determine the content of water-soluble dietary fiber (SDF) and insoluble dietary fiber (IDF). Using the results with the standard table for 227 foods, the intake ratio of IDF/SDF of an average Japanese was calculated for the period 1946-1990. The ratio was 3.22 in 1990 as calculated on the food intakes shown in the national nutrition survey, and the secular change was not detected since 1946 when the ratio was 3.30. The ratio was also shown to be well preserved between types of households including the age of the head. Using dietary records of 60 healthy city workers (average 42.8 years) for 4 weeks, however, the weekly average ratio for an individual was found to vary in the range of 2.25-5.13 although the total average for 60 individuals was 3.33. Thus, the well preserved IDF/SDF intake ratio for an average Japanese showed, on the contrary, a wide variation of food selection between each person.

Spontaneous mutant frequency of lacZ gene in spleen of transgenic mouse increases with age.

Spontaneous mutant frequency of lacZ gene in spleen of transgenic MutaTM mouse was examined at different ages. It was (3.2 +/- 1.3 (SD)) x 10(-5) at newborn and increased almost linearly with age up to (8.3 +/- 1.8) x 10(-5) at one year. Since the mutation of the gene is not likely to be subject to selection in vivo, the data support the idea that spontaneous mutation takes place throughout aging process and accumulates with age if not selected out by cell death.

Effects of energy restriction on age-associated changes of DNA methylation in mouse liver.

DNA methylation is known to change with age in several mammalian species. Here we have examined the effect of dietary energy restriction on this age-associated change in liver DNA of C3H/SHN mice. The total 5-methyldeoxycytidine level in the genome decreased slightly soon after energy restriction started. The effect, however, diminished with time and no appreciable difference was detected at middle and old ages. The degree of methylation at the c-myc gene, on the other hand, was not affected by energy restriction at early periods, but the age-dependent alterations at later ages were repressed. This is a new finding to show that DNA methylation is one of the molecular indices of aging affected by energy restriction. It suggests an importance of DNA methylation in the aging process.

Comparison of the age- and tumor-associated changes in the c-myc gene methylation in mouse liver.

Study of DNA methylation of the c-myc gene in liver tumors of BCF1 mice treated with various agents revealed frequent alteration from that in normal liver. The alteration, however, was complex and showed either an increase or a decrease of methylation to various degrees. On the other hand, the tumors obtained from middle-aged C3H/He mice, which develop liver tumors spontaneously at a high incidence rate starting in middle age, showed predominantly increases of methylation in the c-myc gene, while the large tumors found in older mice revealed decreases. The normal part of the liver showed a slight gradual age-dependent increase. These suggest that hypermethylation of the c-myc gene is common to aging and early tumor development in liver. Thus the alteration of DNA methylation seems to be a good clue to investigate why the tumor incidence rate increases rapidly as individuals grow older.

Clinical characteristics of chronic cutaneous graft-versus-host disease in Japanese leukemia patients after bone marrow transplantation: low incidence and mild manifestations of skin lesions.

We investigated the incidence of chronic graft-versus-host disease (GVHD) and skin and oral lesions in chronic GVHD in 90 Japanese leukemia patients surviving over 100 days after bone marrow transplantation (BMT) from HLA-compatible siblings. The clinical characteristics of chronic cutaneous GVHD occurring in eight of these patients are summarized. There were some differences from previous reports in the USA and Europe. The incidence (8/29: 28%) of skin lesions in chronic GVHD was low in contrast to the previously reported high incidence (79-90%). Cutaneous manifestations in six out of eight patients with chronic GVHD tended to be mild, and lichen-planus-like eruptions in the skin and scleroderma-like lesions, which are well known as representative cutaneous manifestations of chronic GVHD, were rare. Furthermore, our statistical analysis indicated that there was no relationship between pretransplant total body irradiation and GVHD prophylaxis or acute GVHD and the onset of chronic cutaneous GVHD. The present study suggests that the differences from previous reports in the USA and Europe concerning chronic cutaneous GVHD may be attributable to differing genetic backgrounds in Japanese and western populations.

[Assessment of self monitoring of blood glucose (SMBG) and continuous subcutaneous insulin infusion (CSII) in diabetic pregnant women].

We instructed pregnant women with insulin dependent diabetes mellitus (IDDM) or noninsulin dependent diabetes mellitus (NIDDM) how to monitor their own blood glucose concentrations and evaluated the efficiency and feasibility of continuous subcutaneous insulin infusion (CSII) therapy. Self-monitored glucose concentrations with a reflectance meter correlated with those of hospital laboratory measurements (hexokinase method) with a coefficient of more than 0.9. Glycosylated hemoglobin (HbA1) levels of the patients were normalized with insulin treatment based on the self-monitored glucose concentrations. In pregnant women with NIDDM who monitored their blood glucose concentrations before breakfast, the fasting glucose concentrations could be lowered by insulin administration and the duration of hospitalization could be shortened compared to non-monitored patients. Although diurnal variations were prominent in pregnant women with IDDM and precise control of their blood glucose concentrations was difficult with conventional insulin administration, even if the patients had monitored their glucose concentrations 7 times a day, the mean glucose concentrations and M values could be kept in the optimum range in patients treated with CSII. These methods have contributed to the improvement in maternal and infant complications.

[Effect of pregnancy on hemoglobin A1 in normal and diabetic women (author's transl)].

Hemoglobin A1, a normal minor variant of adult HbA in which the N-terminal valine of the beta chain is glycosylated by a Schiff base. The increased levels of HbA1 in diabetic subjects have been correlated with various indicators of diabetic control during preceding weeks, suggesting that HbA1 measurement is an index of long term blood-glucose control. Considerable attention is now paid to diabetic control during pregnancy because maternal hyperglycemia is believed to be partly responsible for increased prevalence of fetal morbidity and mortality. HbA1 was assayed during pregnancy with the Helena glycosylated hemoglobin quik column kit using an ion exchange resin. 1. HbA1 correlated with HbA1c during pregnancy, r = 0.834, p less than 0.001. 2. HbA1 did not alter during pregnancy as compared to the non-pregnant group, 7.26 +/- 0.85% vs 7.09 +/- 0.60%. 3. There was no relationship between HbA1 and hemoglobin. 4. A significant direct relationship was not found for HbA1 and the infant weight. 5. The diabetic group had elevated levels of HbA1 to the normal group but gradually decreased to normal levels after a rigid control was achieved. 6. None of pregnant diabetics whose HbA1 was normal on presentation gave large baby for gestational age. It is speculated that these fluctuation in HbA1 are most likely due to changes in long-term blood glucose control. HbA should decline to reach a constant, low level as soon as possible in diabetic pregnancy. This tool should add in management of diabetic pregnancy to improve the outcome of pregnancy.