Identification of three annexin IX isoforms generated by alternative splicing of the carboxyl-terminal exon in silkworm, Bombyx mori.

Annexins (ANXs) are a family of structurally related proteins with Ca(2+)-dependent phospholipid-binding properties. Here we report the cloning of three cDNAs each encoding annexin IX (ANX IX) isoforms from unfertilized eggs of the silkworm, Bombyx mori. The analysis of exon/intron structures showed that the three mRNAs, named ANX IX-A (2300bp), ANX IX-B (1884bp) and ANX IX-C (1409bp), respectively, were generated from a single gene by alternative usage of a 3'-splice site of the last exon. Thus the three isoforms have an identical sequence from amino acid residues 1 to 307 and this region shows approximately 77% identity to Drosophila melanogaster ANX IX. Only amino acid residues 308-324 (A) or 308-323 (B and C), which correspond to the C-terminal tail, are different in the three proteins. A RT-PCR analysis indicated that the three isoforms of silkworm ANX IX were specifically expressed in various larval tissues and development stages. Interestingly, the C-terminal tail in ANXs I, II and V were previously confirmed as a binding region for protein kinase C. Thus generation of the three ANX IX isoforms in the silkworm, that are different from other ANXs, may have a functional significance other than binding to Ca(2+).

Role of interaction between two silkworm RecA homologs in homologous DNA pairing.

Recombinant BmRad51 and BmDmc1, silkworm homologs of the Escherichia coli RecA proteins catalyzing the homologous DNA pairing, were purified from E. coli cells carrying expression vectors. These possessed different enzymatic properties in the joint molecule formation between single-stranded circular DNA and homologous linear double-stranded DNA. The requirement of single-stranded circular DNA for the efficient reaction was twofold higher in BmRad51 than in BmDmc1. Although able to mediate the joint molecule formation independently, a complex of the two enzymes formed prior to single-stranded DNA binding was found to have augmented efficiency of the pairing reaction.

Prevention of lethal acute graft-versus-host disease in mice by oral administration of T helper 1 inhibitor, TAK-603.

Acute graft-versus-host diseases (GVHD) is a major cause of morbidity and mortality in patients undergoing allogeneic bone marrow transplantation (BMT). T helper 1 (Th1)-type cytokines such as interferon-gamma or tumor necrosis factor-alpha have been implicated in the pathogenesis of acute GVHD. TAK-603 is a new quinoline derivative, which is now in clinical trials for use as a disease-modifying antirheumatic drug. In preclinical studies, it inhibited delayed-type hypersensitivity, but not Arthus-type reaction, in mice, and selectively suppressed Th1 cytokine production. Thus, the present study was designed to investigate whether the Th1 inhibitor (TAK-603) ameliorates lethal acute GVHD in a mouse model. Administration of TAK-603 into BALB/c mice given 10 Gy total body irradiation followed by transplantation of bone marrow and spleen cells from C57BL/6 mice markedly reduced the mortality in association with minimal signs of GVHD pathology in the liver, intestine, and skin. TAK-603 reduced not only the production of Th1-type cytokines, but also the proportion of Th1 cells in CD4(+) helper T cells in this GVHD mouse model. These results suggest that TAK-603 could be a potent therapeutic agent for acute lethal GVHD.

Effect of dibutyryl cyclic AMP on axoplasmic transport in the hippocampus.

The effect of dibutyryl cyclic AMP (dbcAMP) on axoplasmic transport of cultured hippocampal neuron cells from postnatal 1-day mice was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Dibutyryl cyclic AMP increased the axoplasmic transport in both anterograde and retrograde directions. The number of particles flowing in the neurites was increased by 0.5 mM dbcAMP. The peak reached about 160% of the initial value. The instantaneous velocity of axoplasmic transport was also increased by 0.5 mM dbcAMP. The average velocity of anterograde and retrograde direction changed respectively from 1.95 +/- 1.01 microm/s (n = 55) to 2.66 +/- 1.26 microm/s (n = 58) and from 1.94 +/- 0.85 (n = 57) to 2.39 +/- 0.93 (n = 57). Rates were 136.1 and 123.1%, respectively. Previously, we have found that acetylcholine suppressed and adrenaline increased the axoplasmic transport in superior cervical ganglion cells. These effects are related to the amount of endogeneous cAMP. The results of the present report suggest that endogeneous cAMP is also related to hippocampal axoplasmic transport.

Cloning and sequencing of cDNA coding for Cry j I, a major allergen of Japanese cedar pollen.

cDNA clones coding for Cry j I, a major allergen of the Japanese cedar (Cryptomeria japonica) pollen, have been isolated. Two of the clones were sequenced and found to code for a putative 21-residue signal peptide and a 353-residue mature protein with a derived molecular weight of 38.5 KDa. Five possible N-linked glycosylation sites were found in the sequence. Comparison of the nucleotide sequences of the two clones revealed sixteen nucleotide differences and these led to five amino acid exchanges in the mature allergen, indicating that an isoform of the Cry j I molecule exists. The deduced amino acid sequence of the Cry j I shows 46-48% identities with those of the Amb a I family and Amb a II, the major allergens of short ragweed. These findings should facilitate study of the structure-function relationship between the allergen and the immunocompetent cells.

[Determinations of silicon and phosphorus in rice planted in a district of high incidence of amyotrophic lateral sclerosis by neutron activation and X-ray fluorescence analyses].

Silicon and phosphorus contents in polished and unpolished rice planted in a district of high incidence of amyotrophic lateral sclerosis (ALS) have been determined by neutron activation and X-ray fluorescence methods, and compared with those from control areas. In the neutron activation analysis, beta-ray spectra of 32P produced by the 31P(n, gamma)32P reaction on polished and unpolished rice were measured with a low background beta-ray spectrometer. In the X-ray fluorescence analysis, characteristic X-rays were analyzed with a wavelength dispersive X-ray fluorescence spectrometer. Silicon contents in polished and unpolished rice from the ALS area are 42 micrograms.g-1 and 370 micrograms.g-1, respectively, and the corresponding phosphorus contents are 1,210 micrograms.g-1, and 3,370 micrograms.g-1, respectively. The data for ALS area are equal to those for the control area within standard deviation.

Deficient cytochrome b5 reductase activity in nontoxic goiter with iodide organification defect.

A 37-yr-old woman with nontoxic goiter is presented. The thyroid 131I uptake at 3 and 24 hr were, respectively, 77.1% and 81.4% dose. Thiocyanate discharged 65.5% of the accumulated 131I in 30 min. In vitro organification of iodine in the thyroid homogenate from the patient was impaired and it was restored to normal by the addition of H2O2, glucose, and glucose oxidase system, FAD, or reduced cytochrome b5. Riboflavin, FMN, oxidized cytochrome b5, oxidized or reduced cytochrome c, NAD(H), and NADP(H) were ineffective in the reaction. The microsomal NADH-cytochrome b5 reductase activity was definitely low in the patient's thyroid. It was augmented to a normal level by incubation of the microsomes with FAD for 30 min or more. The activities of thyroid peroxidase, G6-PD, 6-PGD, catalase, protease, and NADPH-cytochrome c reductase were within normal limits. The major thyroid protein was normal thyroglobulin which could be readily iodinated in the presence of H2O2 and horse radish peroxidase. These findings suggest the correlation of an iodide organification defect with a cytochrome b5 reductase deficiency. Administration of high doses of FAD led to the restoration of thyroidal iodide organification mechanism associated with an increased thyroid hormone production and to a marked decrease of the goiter. Riboflavin was given without effect even at a high dosage level. Consequently, it seems likely that the deficient cytochrome b5 reductase activity in this patient is due to a defect in the biosynthesis of FAD, the coenzyme of the reductase, from riboflavin.