Establishment of an optimized set of 406 microsatellite markers covering the whole genome for the Japanese population.

Microsatellites, an essential tool for genetic linkage analyses, are selected in genetic studies on the basis of both informativeness and their positions with respect to one another on the genetic map. In order to establish a microsatellite marker set useful for linkage studies in the Japanese population, we first genotyped 64 unrelated Japanese subjects, using 400 microsatellite markers from a commercially available set (ABI PRISM Linkage Mapping Set-MD10) and then determined the allelic frequencies and heterozygosities for these marker loci in the population. In order to optimize the set, we replaced 41 markers having a heterozygosity lower than 0.6 with as many informative markers in the corresponding loci, and newly added six markers in the set to minimize the several gaps found at intervals of over 20 cM. We finally established a set comprising 406 microsatellites with average intervals of 9cM (maximum, 17 cM) and minimum heterozygosities of over 0.6 (mean, 0.76). All data generated in this study, including the specific polymerase chain reaction (PCR) primer sequences of the newly added markers, are freely available to all researchers at our web site. The genetic tool established here should facilitate genetic linkage studies of various hereditary diseases, especially in the Japanese.

Gender-specific haplotype association of collagen alpha2 (XI) gene in ossification of the posterior longitudinal ligament of the spine.

Among Japanese, ossification of the posterior longitudinal ligament of the spine (OPLL) is a leading cause of myelopathy, showing ectopic bone formation in the paravertebral ligament. We have provided genetic evidence that the collagen alpha2 (XI) (COL11A2) locus of chromosome 6 constitutes susceptibility for OPLL. Five distinct single nucleotide polymorphisms (SNPs), identified in COL11A2, were combined to construct possible haplotypes by the use of a maximum likelihood program. Estimated haplotype frequency was compared in OPLL patients and non-OPLL controls. We report a gender-specific association of the COL11AA2 haplotvpe with OPLL. The frequency of the most commonly observed haplotype was significantly higher in male patients (P = 0.0003) compared with controls, but not in female patients (P = 0.21). OPLL is predominantly observed in males. with a prevalence ratio of 2:1, and our gender-specific associations indicate that genetic factors involving COL11A2 play a specific role in the etiology of OPLL exclusively in males.

A new murine zinc finger gene, Opr.

Here we report a novel murine zinc-finger gene, Opr, belonging to the opa/Zic family. Opr is expressed in the entire embryonic ectoderm before gastrulation, but gradually restricted to the anterior part from the mid to late streak stage. At the beginning of neural induction, Opr is expressed throughout the anterior neural plate, but is soon restricted to the neural ridge. After neural tube closure, its expression is maintained in the dorsal part of the neural tube, except for the roof of the telencephalon. Opr is also expressed in somites and limbs.

[Improving the anti-tumor activity of 5-fluorouracil by methotrexate].

The first clinical application of biochemical modulation (BCM) of 5-fluorouracil (5-FU) was the sequential MTX/5-FU regimen proposed in 1977 by Bertino for the treatment of colorectal cancer. In Japan, sequential MTX/5-FU therapy was mainly used as a new method of treating gastric cancer, and attracted a great deal of attention because it proved effective in many cases of advanced gastric cancer that had been unresponsive to the previous chemotherapy, particularly scirrhous gastric cancer with poor prognosis. Its therapeutic efficacy varied according to histologic type, it was effective in cases of peritoneal dissemination and disseminated intravascular coagulopathy (DIC), it was associated with fewer adverse effects, and it was a multidrug chemotherapy based on a clear rationale. With sequential MTX/5-FU therapy as a starting point, fundamental studies of BCM and its clinical applications have expanded rapidly in Japan. This paper provides an outline of sequential MTX/5-FU therapy from the aspects of its mechanism of action, indications, therapeutic efficacy, relevance to adjuvant therapy, counter-measures to adverse effects, and emergence of resistance to the drugs involved. The high therapeutic efficacy of this therapy in certain histologic types is also discussed, and its combined use with other forms of BCM, as in triple BCM (LV/5-FU + CDDP/5-FU + MTX/5-FU), is introduced.

Effects of lanthanide ions on the binding ability of annexin V to phospholipid vesicle.

The effects of lanthanide ions (Ln(3+)), La(3+), Ce(3+), Gd(3+), and Tb(3+), on the binding ability of annexin V to phospholipid vesicle were studied, and compared with that of Ca(2+). The energy transfer following excitation at 278 nm was accompanied by a small decrease (about 3%) in the fluorescence signal (310-315 nm) of the aromatic amino acid residues, followed by strong re-emission of annexin V-bound Tb(3+) at 547 nm. The results of the titration of Tb(3+)-protein interactions using the rate dialysis method suggest that there are two high affinity metal binding sites on annexin V, to which Tb(3+) binds with an average dissociation constant, Kd, of about 2.11 x 10(-7) M. The Ln(3+) seemed to assist the anticoagulant action of annexin V because it binds more strongly to phospholipid than Ca(2+) does. Annexin V had the same ability to bind phospholipids in 10 mu M Ln(3+) as it did in 1 InM Ca(2+), showing that it binds to anionic phospholipids with Ln(3+) more strongly than it does with Ca(2+).

Anticoagulant action of rare earth metals.

Some of the lanthanides, the rare earth metals, lanthanum (La), cerium (Ce), neodymium (Nd), samarium (Sm), terbium (Tb), dysprosium (Dy), erbium (Er) and ytterbium (Yb) prolonged the clotting time of normal human plasma in a dose-dependent manner when clotting was induced either by thromboplastin or by kaolin in the presence of cephalin and Ca2+. They also prolonged the activated factor X induced clotting time of platelet-rich plasma. The amidolytic activities of activated factor X and thrombin progressively decreased with increasing amount of rare earth metals. These results suggested that the rare earth metals appear to show their anticoagulant effect with mechanisms in part the inhibition of the enzymatic activities of both activated factor X and thrombin.

[Surgical treatment of metastatic lung cancer from colorectal cancers].

Based on conclusions obtained after the observation of 61 colorectal cancer patients with a lung metastasis, the resection of lung metastasis as a therapy was evaluated. Among these 61 patients, only 5 patients had been identified as having a lung metastasis at the time of resection of the primary lesion, whereas the other 56 patients developed the lung metastasis after the curative resection of the colorectal cancer. Only one patient with a synchronous lung metastasis and twelve patients (eleven with a solitary metastatic lesion and one with multiple metastatic lesions) with metachronous lung metastasis underwent removal of the lung metastasis. The three-year survival rate was 65.2% in the metachronous group.

Studies on (n, alpha) recoils of 6Li and 10B in the reactor UTR-Kinki by gas phase electrodeposition.

The energies of recoil particles from (n, alpha) or (n, 2 alpha) reactions of Li and B were investigated by solid state detector and gas phase electrodeposition as previously reported on 228Th series. Nuclear reactor of UTR-Kinki was used as thermal neutron source. Particles from 6Li (n, alpha) 3T and 10B (n, alpha) 7Li were collected on a sheet of triacetate film by gas phase electrodeposition at atmospheric pressure. The film was etched with 6M-NaOH at 50 degrees C. The tracks thus obtained were taken as photographs with a microscope (X600). By counting the number of tracks on the film at various etching times, the energies of alpha-particles were evaluated according to the calibration curve of 227Ac series. Among various peaks of tract density, the two peaks having the evaluated values of 1.85 MeV for 6Li and 1.48 MeV for 10B agreed approximately with the theoretical values of 2.05 MeV and 1.47 MeV of alpha-particle energies for respective reactions.

[Focusing chromatography using a dilute precipitation reagent--separation of 226Ra decayed nuclides by potassium fluoride (author's transl)].

The electrophoresis of 226Ra and it's decayed nuclides was carried out by using hydrochloric acid in the positive electrode cell and aqueous solution of potassium fluoride in the negative electrode cell. Experimental factors, i.e. concentrations of potassium fluoride and hydrochloric acid, intensity of electric field, duration for electrophoresis and pH value of the potassium fluoride solution etc. were examined. The sample solution was of 0.01 M hydrochloric acid with a tracer amount of 226Ra and 210Pb. Each of these nuclides was carrier free and was 4GB1/l(1 muCi/ml) in concentration. For the sake of autoradiography, a photoengraving film (Fujilith Ortho Film, TAC = 135) was made use of examining the locations of the separated radioactive bands obtained on the paper strip. Their beta and gamma radioactivities were detected as the film darkening on developing the film. According to the positions thus detected, the strip filter paper was cut into pieces and the species of radionuclides were determined by the measurements of energies and half-lives on beta and gamma rays. The locations of the separated alpha-radionuclides were decided by etching the surface of it's film with 6 M aqueous solution of sodium hydroxide and the species of radionuclides were determined by the solid state alpha-track detection method. As the result, the optimum conditions for the separation are to use 0.01 M hydrochloric acid in the positive cell and 0.005 M potassium fluoride of pH 6.2 in the negative cell, intensity of the applied electric field being 1000 V/30 cm, and electrophoresis time being 20 minutes. The proposed method gave good results for focusing and separating the radium series nuclides. Bands of 210Bi and 210Po were found on the positive electrode side, while the bands of 226Ra and 210Pb (containing 214Pb) were on the negative electrode side.

[Study on focusing chromatographic simultaneous determinations of 226Ra and its daughter nuclides by means of solid state alpha-tracks detection and beta-autoradiography (author's transl)].

In order to detect to radioactive band on the paper strip developed by focusing chromatography, plate-making-film was used for the autoradiography and beta-spots were photographed. Thereafter the film was etched with sodium hydroxide solution to find the alpha-tracks. Paper strip used for the sample was prepared by the precipitation focusing chromatography of 226Ra and its daughter nuclides using HCl-KF solution as a developer. The film used was not high in its beta-sensitivity, but because of its high resolution good photographic results were obtained according to the intensity of beta-activity when the proper conditions of photographic development were fulfilled. The simple alpha-spectrometry was made possible by counting the numbers of tracks according to the etching depth of the film. The film was hard and thick enough for etching with 6M sodium hydroxide solution at 50 degrees C for more than 50 hrs to measure the depth of tracks.