Disrupted-In-Schizophrenia 1, a candidate gene for schizophrenia, participates in neurite outgrowth.

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11)(q42.1;q14.3) translocation that segregated with schizophrenia in a Scottish family. Predicted DISC1 product has no significant homology to other known proteins. Here, we demonstrated the existence of DISC1 protein and identified fasciculation and elongation protein zeta-1 (FEZ1) as an interacting partner of DISC1 by a yeast two-hybrid study. FEZ1 and its nematode homolog are reported to represent a new protein family involved in axonal outgrowth and fasciculation. In cultured hippocampal neurons, DISC1 and FEZ1 colocalized in growth cones. Interactions of these proteins were associated with F-actin. In the course of neuronal differentiation of PC12 cells, upregulation of DISC1/FEZ1 interaction was observed as along with enhanced extension of neurites by overexpression of DISC1. The present study shows that DISC1 participates in neurite outgrowth through its interaction with FEZ1. Recent studies have provided reliable evidence that schizophrenia is a neurodevelopmental disorder. As there is a high level of DISC1 expression in developing rat brain, dysfunction of DISC1 may confer susceptibility to psychiatric illnesses through abnormal development of the nervous system.

Interchangeable binding of Bcl10 to TRAF2 and cIAPs regulates apoptosis signaling.

Bcl10 was identified as a candidate gene responsible for low grade B cell lymphomas of mucosa-associated lymphoid tissue. Overexpression of Bcl10 in cultured cells was reported to promote apoptosis, however, the mechanism of regulation of apoptosis mediated by Bcl10 has not been demonstrated. In the present study, we analysed the apoptosis signaling pathway mediated by Bcl10, focusing on phosphorylation of Bcl10 and the dynamic interaction with its binding partners during apoptosis. Previously, we have demonstrated that Bcl10 potentially interacts with the other apoptosis regulator, TNF receptor associated factor-2 (TRAF2) and inhibitor of apoptosis proteins (cIAPs). The present results showed that the complex formation of these molecules was regulated by phosphorylation of Bcl10, that is, phosphorylation of Bcl10 resulted in binding of Bcl10 to cIAPs and the dissociation of it from TRAF2. Moreover, hyperphosphorylation of Bcl10 enhanced apoptosis, suggesting that changes in the binding partners of Bcl10 were correlated to the promotion of apoptosis as mediated by Bcl10. Indeed, the mutant which was deleted from the binding site of Bcl10 for cIAPs, could not induce apoptosis. These findings indicate that Bcl10 is a mediator of apoptosis signaling, by switching over binding to cIAPs from TRAF2 through the events of Bcl10 phosphorylation.

Activation of caspase-12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent mechanism in response to the ER stress.

When accumulation of a malfolded protein in the endoplastic reticulum (ER) is induced by various adverse conditions, such as hypoxia, glucose starvation, and perturbation of calcium homeostasis, cells respond to the stress by increasing transcription of genes encoding ER molecular chaperones, a process known as unfolded protein response. The signaling is initiated by IRE1s, ER stress sensors. Alternatively, excessive stress to the ER results in apoptosis. Caspase-12 is known to be essential for this ER stress-induced apoptosis. In this study, we analyzed the detailed regulatory mechanisms of IRE1s during ER stress. We identified c-Jun N-terminal inhibitory kinase (JIK) as a binding partner of IRE1alpha, and JIK was seen to modulate IRE1alpha-TRAF2 (tumor necrosis factor receptor-associated factor 2) complex formation and the resultant alteration to c-Jun N-terminal kinase signaling from IRE1s in response to ER stress. We also demonstrated that TRAF2 interacts with procaspase-12 and promotes the clustering of procaspase-12 and its activation by cleavage in response to ER stress. These results indicate that TRAF2 plays crucial roles not only in the signaling of the c-Jun N-terminal kinase pathway but also in activation of caspase-12 to transduce signals from IRE1s. Thus, we provide a missing link in the ER stress-induced apoptosis-signaling pathway, one which connects the stress sensor molecule IRE1 and the activation of caspase-12.

Short inverted repeats function as hotspots of intermolecular recombination giving rise to oligomers of deleted plastid DNAs (ptDNAs).

We have determined the nucleotide sequences around the junction points of oligomeric-deleted ptDNAs possessing a head-to-head or tail-to-tail configuration from long-term cultured cell lines and albino plants. It was shown that DNA rearrangement occurred by direct fusion of deleted ptDNAs in an inverted orientation, which was linked by an asymmetrical sequence of 254-698 bp derived from either of the ptDNAs joined. It is notable that inverted repeats of 7-14 bp flank the asymmetrical sequences at each of the junction points. These features of the DNA sequence around the junction points are commonly observed in oligomeric ptDNA with a large-scale deletion regardless of the cell lines employed. It is suggested that the short inverted repeats are involved in the intermolecular recombination of ptDNA.

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture.

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.

[Treatment of cancer pain with the percutaneous electrocoagulation of spinal nerves].

To our knowledge, this is the first report of high-frequency thermocoagulation applied to the spinal root. We treated 34 patients suffering from cancer pain with this technique. Among these patients, cancer pain occurred due to intrapelvic metastasis in 11 patients, and 13 complained of chest pain due to cancer. Every patient was considered to have good or excellent response when his or her pain score was reduced to 6 points or less from the score before thermocoagulation; 10 points. Based on this criterion, 54.5% and 30.8% of the above-mentioned patients showed good and excellent responses respectively one month after treatment. This technique, therefore, was considered to be effective with less side effects compared with other nerve blocking techniques.

Molecular cloning and characterization of gravity specific cDNA in rice (Oryza sativa L.) suspension callus.

Rice (Oryza sativa L. var. Nipponbare) suspension callus was exposed to gravity stress at 450,000 g for 2 hours, after which poly(A)+RNA was isolated and a cDNA library was constructed. Three different gravity specific cDNAs, namely, GSC 128, GSC 233 and GSC 381 of 0.67, 0.60 and 0.68 kilobase pairs and transcripts of 1.9, 1.6 and 2.0 kb, respectively, were isolated by differential screening and Northern hybridization. The maximum level of transcript was achieved after 4 hours of exposure to gravity at 450,000 g for GSC 128, 2 hours for GSC 233 and 8 hours for GSC 381 followed by a gradual decrease to undetectable levels with the extension of gravitation time. Callus (GSC 128), shoot and callus (GSC 381) and root and callus (GSC 233) specific expression of transcripts was identified. Although the protection of callus by treatment with ABA, kinetin and sucrose extended the period of expression of mRNA in suspension callus after gravity exposure, the expression of gravity-inducible mRNA was exclusively regulated by the degree of callus viability or survival after the stress. In addition, we demonstrated that the level of GSC 381 transcript was markedly increased by exposing the cell to periodical gravity stress, suggesting that this mRNA is expressed and translated into special proteins which are closely related to the survival of the cell against gravity stress. The sequence of GSC 233 and GSC 381, consisting of 417 and 531 base pairs of the longest open reading frames, encode polypeptides with calculated molecular weights of 15.29 and 19.47 kDa, respectively. A sequence homology search against a data bank revealed that GSC 233 and GSC 381 differed from other stress inducible genes in terms of the coding sequence and expression characteristics.

Molecular cloning and characterization of genes related to chilling tolerance in rice.

Polyadenylated RNA was isolated and a cDNA library constructed from seedlings of a chilling-tolerant rice cultivar (Oryza sativa L. subsp. Japonica cv Nipponbare). Four clones were isolated by differential screening. Northern blot hybridization using RNAs from chilling-tolerant (Nipponbare) and -sensitive (IR36) cultivars revealed higher steady-state levels of transcripts for the four genes in Nipponbare than in IR36 maintained at the same low temperatures. The accumulation of transcripts homologous to selected cDNA sequences during chilling were tissue-specific. The nucleotide and deduced amino acid sequences of three clones, pBC121, pBC442, and pBC591, were determined, and no homology was identified by comparison with the latest version of EMBL and LASL gene data bases. The deduced protein sequences from the longest open reading frame of the clones pBC121 and pBC442 are rich in leucine and serine, whereas that of the clone pBC591 contains arginine-rich basic domains.

A successful therapy with plasma exchange for interstitial pneumonia of progressive systemic sclerosis.

A 47-year-old woman who was diagnosed as progressive systemic sclerosis (PSS) had acute and severe interstitial pneumonia. Based on the results of her chest roentgenogram, computed tomography, transbronchial lung biopsy (TBLB) and bronchoalveolar lavage (BAL), her interstitial pneumonia was considered to be atypical of PSS. Although she was treated with corticosteroid, methylprednisolone pulse therapy and immunosuppressive drug, the effect of these drugs was insufficient as treatment for the interstitial pneumonia. Therefore, plasma exchange was attempted. After plasma exchange was carried out for three days, her symptoms improved as well as the laboratory data and chest roentgenogram without any severe side effects. We recommend plasma exchange for interstitial pneumonia of PSS as an effective treatment.

Sequence of three members and expression of a new major subfamily of glutelin genes from rice.

Three members have been isolated of an additional glutelin gene subfamily, named subfamily B, consisting of about five members per haploid rice genome. Restriction fragment length polymorphism analysis showed major differences between Japonica and Indica lines, indicating the divergence of the subfamily since the split between the two varieties. While corresponding exons of the subfamily B showed 80 to 88% nucleotide sequence homology, those exons were only 60-65% homologous to those of the glutelin A subfamily, distinguishing them from the subfamily A. Intron position and derived polypeptide structure, in addition to the nucleotide sequence, confirm the subfamily B members as glutelins. Analysis of RNA from seeds of different stages of development showed that the subfamily B members were expressed at the same time as those of subfamily A, demonstrating coordinated regulation of the two subfamilies.

Binding shift assay of parvalbumin, calmodulin and carbonic anhydrase by high-performance capillary electrophoresis.

Shifts in mobility caused by binding of Ca2+ to calmodulin and parvalbumin were studied using high-performance capillary electrophoresis in a Tris-glycine buffer, rather than conventional polyacrylamide gel electrophoresis which requires larger amounts of sample and longer assay time. A Zn(2+)-binding protein, carbonic anhydrase, also showed a partial shift in mobility following Zn(2+)-binding.

Genomic DNA sequences of two new genes for new storage protein glutelin in rice.

A new cDNA and two genomic genes encoding the rice storage protein glutelin were isolated and sequenced. The nucleotide sequence of one gene (GluA-3) was completely identical with that of the new cDNA identified here, and the other (GluA-4) was a pseudogene. These glutelin genes were closely related to each other, and belonged to the subfamily A containing the type I (GluA-1) and II (GluA-2) glutelin genes. The Northern blot analysis, using synthetic oligonucleotide specific to the GluA-3 gene as a probe, showed that this gene was expressed earlier than other glutelin genes during seed maturation.

Analysis of the 5' flanking region responsible for the endosperm-specific expression of a rice glutelin chimeric gene in transgenic tobacco.

The 5' upstream region of the rice storage protein type II glutelin gene was examined for its regulatory function in transgenic tobacco. Chimeric genes containing 5' flanking regions of the glutelin gene transcriptionally fused to the beta-glucuronidase (GUS) reporter gene were introduced into the tobacco genome by Agrobacterium tumefaciens-mediated gene transfer. The chimeric genes were expressed specifically in developing seeds, as opposed to leaves and stems, of the transgenic tobacco. Histochemical analysis revealed that the GUS activity was restricted to the endosperm tissue. A deletion series of the 5' flanking region was created from position -1329 to -74 relative to the transcriptional initiation site and similarly examined in transgenic tobacco. Measurement of GUS activity of the seeds from the transgenic plants bearing the chimeric genes indicated that the region between positions -441 and -237 was required for the temporal and endosperm-specific expression of the GUS activity in tobacco. RNA analysis by northern blotting confirmed the importance of the -441 to -237 region. Addition of up to 888 bp to the -441 deletion resulted in little increase in GUS activity, although all constructs expressing the GUS gene showed a similar tissue and temporal regulation pattern.

Interaction of an immature seed-specific trans-acting factor with the 5' upstream region of a rice glutelin gene.

The 5' flanking region of a glutelin gene was analyzed for interactions with nuclear proteins from immature rice seed. The specific region between positions -272 and -99 was shown to interact with nuclear proteins from immature seeds, but not with those of leaves and roots. Methylation interference experiments revealed that one factor interacted with a specific sequence element between positions -130 and -120 relative to the transcriptional start site. The sequence specificity of this DNA-protein interaction was confirmed by competition experiments using synthetic oligonucleotides. By using a synthetic oligonucleotide as a probe it was also shown that the binding activity was closely correlated with the mRNA levels of this gene during seed maturation.

[Studies on stress analysis of laminate veneers].

The stress distributions in the laminate veneers under the two kinds of loadings are analyzed numerically by use of FEM in order to make clear the defects of various enamel preparations and Young's modulus of laminate shell materials in restored teeth. The analytical results are summarized as follows, 1) In both cases of two loading conditions under in this study, the stress distribution in restored teeth varied much with Young's modulus of laminate shell materials. 2) Especially in the case of thin enamel preparation in marginal light-chamber configuration, the higher tensile stress is obtained under the higher Young's modulus in the resin cement layer near the labio-gingival margin. 3) When the high load is applied normal to the incisal edge, considering each material strength, however what kind of laminate shell materials are used, the restored teeth is quite within the bounds of possibility for exploration in facio-gingival region or fracture in vicinity of loading points.

[Stress analysis of porcelain laminate veneers. (1)].

The stress distributions in the porcelain laminates under various kinds loading are analysed numberically in order to make clean the reason of their exfoliation or fracture. The two-dimensional finite element method is used to determine the principal stresses developed in the porcelain laminated and the teeth substance of a restored maxillary central incisor. The thicknesses of enamel preparations are assumed to be 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm and 0.8 mm in the analysis. As a result of this analysis, the following points are made clear: 1) The stress concentration are observed at the vicinity of a loading point and in the cervical regions, independently of the loading conditions on its location and direction. 2) In case of the vertical loading, the location of loading point has no effect on the overall stress magnitude and distribution. 3) In the case when the load is applied in the 45 degrees-direction to the vertical axis, the overall stress level is increased, as the location of loading point is far from the supporting regions. 4) In the case when the load is applied normal to the vertical axis at the incisal edge, the high stress is obtained in comparison with other loading conditions.

[Delayed eruption caused by non-ionic contrast material injection].

An investigation was conducted into the adverse reactions, especially delayed reactions, of non-ionic contrast media. Out of a total number of 3,411 people treated, there were 45 cases (1.32%) in which adverse reactions were observed, and of these 45 cases, 14 cases (0.41% of the total number) showed delayed adverse reactions. Of the aforesaid 14 patients with delayed adverse reactions, all of them developed skin eruptions, but no seriously adverse effects were eventually observed.