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We have identified several interspecific pairs of S haplotypes having highly similar SRK and SP11/SCR sequences between Brassica oleracea and Brassica rapa. The recognition specificities of S haplotypes in these pairs were examined with three different methods. Stigmas of interspecific hybrids between an S-32 homozygote in B. oleracea and an S-60 homozygote in B. rapa, which were produced to avoid the interspecific incompatibility between the two species, showed incompatibility to the pollen of an S-8 homozygote in B. rapa and to the pollen of an S-15 homozygote in B. oleracea, while it showed compatibility to the pollen of other S haplotypes, suggesting B. oleracea S-32 and B. rapa S-60 have the same recognition specificity as B. rapa S-8 and B. oleracea S-15. Pollen grains of transgenic S-60 homozygous plants in B. rapa carrying a transgene of SP11-24 from B. oleracea were incompatible to B. rapa S-36 stigma, indicating that B. oleracea S-24 and B. rapa S-36 have the same recognition specificity. Application of the SP11 protein of B. rapa S-41 and S-47 onto the surface of B. oleracea S-64 stigmas and S-12 stigmas, respectively, resulted in the incompatibility reaction to pollen grains of another S haplotype, but application onto the stigmas of other S haplotypes did not, suggesting that B. oleracea S-64 stigmas and S-12 stigmas recognized the B. rapa SP11-41 and SP11-47 proteins as self SP11 proteins, respectively. Besides having evolutionary implications, finding of many interspecific pairs of S haplotypes can provide insight into the molecular mechanism of self-recognition. Comparing deduced amino-acid sequences of SP11 proteins and SRK proteins in the pairs, regions of SP11 and SRK important for self-recognition are discussed.

Objectives/Hypothesis: We report the first case of laryngeal stenosis with granuloma in a patient with severe congenital neutropenia (SCN). Study Design: Case report and retrospective review. Methods: Review of medical records. Results: A 6-year-old female who was diagnosed with SCN presented with a cough and wheezing. An endoscopic study revealed laryngeal stenosis with granuloma. Tracheotomy and direct laryngoscopy were performed under general anesthesia, and administration of granulocyte colony-stimulating factor was started. The laryngeal granuloma disappeared 3 weeks later, and the tracheal stoma was closed. Conclusions: Presence of a laryngeal lesion should be considered in SCN patients with persistent airway symptoms.

Nucleotide sequences of orthologs of the S-locus related gene, SLR1, in 20 species of Brassicaceae were determined and compared with the previously reported SLR1 sequences of six species. Identities of deduced amino-acid sequences with Brassica oleracea SLR1 ranged from 66.0% to 97.6%, and those with B. oleracea SRK and SLR2 were less than 62% and 55%, respectively. In multiple alignment of deduced amino-acid sequences, the 180-190th amino-acid residues from the initial methionine were highly variable, this variable region corresponding to hypervariable region I of SLG and SRK. A phylogenetic tree based on the deduced amino-acid sequences showed a close relationship of SLR1 orthologs of species in the Brassicinae and Raphaninae. Brassica nigra SLR1 was found to belong to the same clade as Sinapis arvensis and Diplotaxis siifolia, while the sequences of the other Brassica species belonged to another clade together with B. oleracea and Brassica rapa. The phylogenetic tree was similar to previously reported trees constructed using the data of electrophoretic band patterns of chloroplast DNA, though minor differences were found. Based on synonymous substitution rates in SLR1, the diversification time of SLR1 orthologs between species in the Brassicinae was estimated. The evolution and function of SLR1 and the phylogenetic relationship of Brassiceae plants are discussed.

PCR (polymerase chain reaction)-RF(restriction fragment)-SSCP (single-strand conformation polymorphism) – designated here as PRS – is a combined method of SSCP and PCR-RFLP (restriction fragment length polymorphism) – designated as CAPS (cleaved amplified polymorphic sequence) – and was efficient in detecting intraspecific variation of the SLR1 gene in Brassica oleracea. One to six nucleotide changes in restriction fragments of the SLR1 gene were detected as different bands in PRS. In an analysis of randomly chosen DNA fragments in cabbage, PRS detected DNA polymorphism between different cultivars with more than 60% of the primer pairs used except for a combination of two cultivars having highly similar characteristics. In rice, no DNA polymorphism was found between two Japonica cultivars, while more than 80% of the primer pairs showed DNA polymorphism between Japonica cultivars and Indica cultivars. PRS had a 1.5- to twofold greater ability to detect DNA polymorphism in these cabbage and rice cultivars than CAPS. The present study indicated that PRS is potentially useful for the identification of crop cultivars and genetic mapping of DNA fragments including genes of interest.

Brassica self-incompatibility (SI) is controlled by SLG and SRK expressed in the stigma and by SP11/SCR expressed in the anther. We determined the sequences of the S domains of 36 SRK alleles, 13 SLG alleles, and 14 SP11 alleles from Brassica oleracea and B. rapa. We found three S haplotypes lacking SLG genes in B. rapa, confirming that SLG is not essential for the SI recognition system. Together with reported sequences, the nucleotide diversities per synonymous and nonsynonymous site (πS and πN) at the SRK, SLG, and SP11 loci within B. oleracea were computed. The ratios of πN:πS for SP11 and the hypervariable region of SRK were significantly >1, suggesting operation of diversifying selection to maintain the diversity of these regions. In the phylogenetic trees of 12 SP11 sequences and their linked SRK alleles, the tree topology was not significantly different between SP11 and SRK, suggesting a tight linkage of male and female SI determinants during the evolutionary course of these haplotypes. Genetic exchanges between SLG and SRK seem to be frequent; three such recent exchanges were detected. The evolution of S haplotypes and the effect of gene conversion on self-incompatibility are discussed.

The present study aimed to analyse how anatomical regeneration contributes to functional recovery after experimental spinal cord repair. Thoracic spinal cord of neonatal rats was completely transected to make a gap and repaired by grafting a section of embryonic spinal cord. Six weeks after surgery, outcome of locomotor performance was assessed using an open field locomotor scale (BBB scale). Axonal regeneration across the repaired site was quantitatively assessed in the raphe, vestibular, and red nuclei and the sensorimotor cortex by a retrograde tracing method. The rats that had no labelled neurons in any of the supraspinal nuclei showed no hind-forelimb coordination. The rats that had labelled neurons in the brainstem nuclei but not in the sensorimotor cortex showed hind-forelimb coordination of varying grades depending on the amount of regeneration. The rats that had labelled neurons in all of the examined nuclei showed almost normal locomotion. In addition to a relationship between distribution of the labelled neurons and functional recovery, a positive correlation was observed between number of the labelled neurons in each of the supraspinal nuclei and locomotor performance of the rat. Thus the grade of restored function appeared to be regulated by distribution and number of fibres regenerated across the repaired site and into the target region. These results suggest that accurate reconstruction of neural connections is essential for significant functional recovery after spinal cord repair.

For the extirpation of hypopharyngeal carcinoma, in most cases laryngopharyngectomy is indicated because many case of that demonstrate advanced stages. However, in case of localized tumor, partial laryngopharyngectomy can be performed to preserve the laryngeal function. The authors have experienced a case of second-staged reconstruction using hinge flap after failure of forearm free flap reconstruction. The swallowing and phonatory function has preservated with this method. A 51-year-old male was diagnosed to have hypopharyngeal carcinoma of the right pyrif orm sinus (T1N2bM0) and underwent partial laryngopharyngectomy, bilateral neck dissection, and reconstruction of hypopharynx using a free forearm flap. However, necrosis of free flap occurred, we had to perform extirpation of total necrotic flap. To control infections and avoid further complications, we chose the second-staged reconstruction, and at first we performed pharyngeal-cutaneous f istulaplasty. After that, we reconstructed using hinge flap and deltopectoral flap. After all, his laryngeal function (swallowing and phonation) has preserved.

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS 46 (S 46 in B. rapa) and BoS 7 (S 7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS 47 and BoS 12 , and BrS 8 and BoS 32 , having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS 46 and BoS 15 showed incompatibility with a BoS 7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS 46 and BoS 7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S-locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS 46 and BoS 7 . These results suggest that the DNA sequences of the regions intervening between the S-locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes.

There is accumulating evidence that fibroblasts are target cells for steroids such as sex hormones and corticoids. The characteristics of fibroblasts vary among tissues and organs. Our aim in this study is to examine differences in responses to steroid hormones among fibroblasts from different cervicothoracic regions. We compared the actions of steroid hormones on cultured fibroblasts from the vocal folds, which are considered to be the primary target of steroid hormones, and the trachea and esophagus in adult male rats. Expression of steroid hormone receptors (androgen receptor, estrogen receptor α, and glucocorticoid receptor) was identified by immunofluorescence histochemistry. Androgen receptor was much more frequently expressed in fibroblasts from the vocal fold than in those from the trachea and esophagus. Cell proliferation analysis showed that administration of testosterone, estradiol, or corticosterone suppressed growth of all 3 types of fibroblasts. However, mRNA expression for extracellular matrix–associated genes, including procollagen I and III and elastin, and hyaluronic acid synthase I was elevated only by addition of testosterone to fibroblasts from the vocal fold. These results indicate that each steroid hormone exerts region-specific effects on cervicothoracic fibroblasts with different properties through binding to specific receptors.

In higher plants, the self-incompatibility mechanism is important for inhibition of self-fertilization and facilitation of out-crossing. In Brassicaceae, the self-incompatibility response is mediated by allele-specific interaction of the stigma-localized S-locus receptor kinase (SRK) with the pollen coat-localized ligand (SCR/SP11). All self-incompatible Brassicaceae plants analyzed have been found to have the SRK and SCR/SP11 genes in the S-locus region. Although Arabidopsis thaliana is self-compatible, transformation with functional SRK-SCR genes from self-incompatible Arabidopsis species confers the self-incompatibility phenotype to A. thaliana. The allele-specific interaction between SRK and SCR activates the downstream signaling cascade of self-incompatibility. Yeast two-hybrid analysis with a kinase domain of SRK as bait and genetic analysis suggested several candidate components of self-incompatibility signaling in Brassica. Recently, A. thaliana genes orthologous to the identified genes for Brassica self-incompatibility signaling were evaluated by using a self-incompatible transgenic A. thaliana plant and these orthologous genes were found not to be involved in self-incompatibility signaling in the transgenic A. thaliana. In this review, we describe common and different aspects of S-locus genomic regions and self-incompatibility signaling between Brassica and Arabidopsis.

The Graduate School of Agricultural Science, Tohoku University, launched an Agri-Reconstruction Project in 2011 immediately after the March 11 Great East Japan Earthquake disaster, and this continues to date. The project’s objective is to support the agricultural, forestry and fisheries reconstruction process in the tsunami disaster area. The activities have been implemented through more than 40 research projects along the Tohoku region including the Rapeseed Project for Restoring Tsunami-Salt-Damaged Farmland.

Self-incompatibility (SI) in Brassica is sporophytically controlled by the multi-allelic S locus. SI phenotypes of the stigma and pollen in an S heterozygote are determined by the two S haplotypes it carries; the two haplotypes may be co-dominant or exhibit a dominant/recessive relationship. Because the S receptor kinase (SRK) gene of the S locus was recently shown to determine the S haplotype specificity of the stigma, we wished to investigate whether SRK also plays a role in the dominance relationships between S haplotypes. We crossed plants carrying an SRK 28 transgene with plants homozygous for one of five S haplotypes that are either co-dominant with, or recessive to, S 28 haplotype in the stigma, and analyzed the SI phenotypes of the progeny. In all cases, the SI phenotype of the stigma of plants carrying the SRK 28 transgene could be predicted by the known dominance relationships between the S haplotype(s) and the S 28 haplotype. Moreover, in the S 43 homozygote carrying the SRK 28 transgene where the S 43 phenotype in the stigma was masked by the presence of the SRK 28 , the transcript level of SRK 28 was found to be much lower than that of SRK 43 . All these results suggest that the dominance relationships between S haplotypes in the stigma are determined by SRK, but not by virtue of its relative expression level.

The MIP-MOD (for MOD-locus associated Major Intrinsic Protein) gene encodes an aquaporin-like product, and has been reported to be a candidate for the MOD gene which is required for the self-incompatibility response in Brassica rapa. In an antisense suppression experiment designed to investigate the role of MIP-MOD, we found that levels of MIP-MOD mRNA in the stigmas of fourteen antisense transgenics, as well as in the self-incompatible cultivar Osome (Osm), were much lower than in the stigmas of the self-incompatible S8 homozygous (S8) strain. Therefore, we analyzed the molecular structure of the MIP-MOD gene in three B. rapa strains: S8, Osm, and the self-compatible var. Yellow Sarson (YS). Nucleotide sequence analysis of the MIP-MOD genes isolated from the three strains revealed that all three encode the same amino acid sequence and that YS and Osm contain the same MIP-MOD allele, designated MIP-MOD(YS). Analysis of other self-incompatible B. rapa strains that are homozygous for the MIP-MOD(YS) allele indicated that high levels of MIP-MOD transcripts are not essential for the self-incompatibility response. Furthermore, a MOD mutant generated by gamma-irradiation was found to contain a wild-type MIP-MOD gene that is expressed at normal levels. These data suggest that MIP-MOD is not MOD itself. We suggest that this gene should be renamed MLM (for MIP gene linked to MOD).

Self-incompatibility in Brassicaceae plants is sporophytically controlled by a single multi-allelic locus (S locus), which contains at least three highly polymorphic genes expressed in the stigma (SLG and SRK) and in the pollen (SCR/SPII), Using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis with SLG-specific primer pairs, the S haplotypes of F 1 hybrid and open-pollinated commercial cultivars of Brassica rapa were identified. The number of S haplotypes detected in the F 1 hybrid cultivars of Chinese cabbage, kamatsuna, pak-choi, turnip, open-pollinated cultivars of Chinese cabbage and turnip were 9. 9, 4, 11, 13 and 12, respectively, Nine of them had different PCR-RFLP profiles from those of the S-tester lines that determined the SLG sequences. Four SLG sequences in the F 1 hybrid cultivars were determined and named S 53 , S 54 , S 55 and S 56 , respectively. It is demonstrated that the PCR RFLP analysis using specific primer pairs of SLG and SRK is useful for identification of the S haplotypes, in both, S homozygous and S heterozygous plants of B. rapa. The possibility of using this method routinely in breeding programmes, and in the evaluation of F 1 hybrid seed purity, is discussed.

We describe a 64-year-old man with ‘citrullinemia type II’ whose serum citrulline levels fluctuated between normal and abnormally high during episodic manifesting periods. Elevations of the serum threonine/serine ratio and pancreatic secretory trypsin inhibitor level are very useful diagnostic markers. Our patient’s cerebrospinal fluid citrulline level was also elevated, and T1-weighted magnetic resonance images revealed high-intensity signals at the bilateral internal capsule and the cerebral peduncles. Single-photon emission computed tomography of his brain showed reduced bilateral temporal lobar blood flow. Even if the serum citrulline level is within the normal range, citrullinemia should be considered in adult patients without primary liver dysfunction who show episodic consciousness disturbance, psychotic symptoms or both.

Self-incompatibility (SI) in Brassica is regulated by a single multi-allelic locus, S, which contains highly polymorphic stigma-expressed genes, SLG and SRK. While SRK is shown to be the determinant of female SI specificity, SLG is thought to assist the function of SRK. Here we report that the SLG genes of self-incompatible S18 and S60 homozygotes of Brassica oleracea have an in-frame stop codon and a 23 bp deletion resulting in a frame-shift, respectively. The finding that these SLG genes do not encode functional SLG proteins suggests that SLG is not essential for SI. The possible role of SLG in SI was discussed.

In Brassica, self-incompatibility genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are located in the S-locus complex region with several other S-linked genes. The S locus is a highly polymorphic region: polymorphism has been observed not only in sequences of SLG and SRK but also in the location of the S-locus genes. In order to compare the physical location of the S-locus genes in various S haplotypes, we used six class-I S haplotypes of B. rapa and seven class-I S haplotypes of B. oleracea in this study. DNA gel blot analysis using pulsed-field gel electrophoresis (PFGE) showed that the physical distances between SLG and SRK in B. rapa are significantly shorter than those in B. oleracea and that the sizes of MluI and BssHII fragments harboring SLG and SRK are less variable within B. rapa than within B. oleracea. We concluded that several large genomic fragments might have been inserted into the S-locus region of B. oleracea after allelic differentiation of S-locus genes.

Several simple methods of DNA preparation from plant tissues were evaluated for PCR-RFLP analyses of SLG and SRK alleles, which can be used for the identification of S haplotypes of breeding lines in broccoli and cabbage (Brassica oleracea L.) and in purity tests of F1 hybrid seeds. On the five methods tested, the NaI method was found to be the most suitable for the amplification of the SLG and SRK alleles. This method enables the use of a single seed as testing material. Using this method, we identified S haplotypes of 31 broccoli and 31 cabbage cultivars. Ninety-four percent of the cultivars of broccoli and 97% of those of cabbage were-single cross F1 hybrids. Nine and 15 S haplotypes were found in broccoli and cabbage, respectively. The small number of S haplotypes in broccoli suggests the importance of incorporating new S haplotypes in the breeding program.

DNA sequences of SLG and SRK , believed to function in self-pollen recognition of self-incompatibility, were analysed and compared between many S haplotypes of Brassica oleracea . Some different S haplotypes have highly similar SLGs (more than 97.5% amino acid sequence identity), while their SRKs are less similar (less than 88% identity). Different lines having the same recognition specificity often show a high degree of sequence variation in SLG (88.5% identity in the S 2 haplotype), but their SRKs are very similar to each other (97.3% identity). S haplotypes, in which SLG protein is undetectable by Western blot analysis, have stop codons in the middle of the coding region of SLG , or lack an SLG gene in the genome, suggesting that these haplotypes do not synthesize the functional SLG protein. The SRK alleles in these haplotypes are normal. The function of SLG and SRK in self-incompatibility and the evolution of these alleles are discussed.

Forty three S tester lines of Brassica oleracea were characterized using DNA and protein gel-blotting analyses. DNA gel-blot analysis of HindIII-digested genomic DNA with class-I and class-II SLG probes revealed that 40 lines could be classified as class-I S haplotypes while three lines could be classified as class-II S haplotypes. The band patterns in the S tester lines were highly polymorphic. Although the S tester lines typically showed two bands corresponding to SLG and SRK in the analysis with the class-I SLG probe, only one band was observed in the S 24 homozygote. This band was identified as SRK, suggesting that this haplotype has no class-I SLG band. In the analysis using the class-II SLG probe, one plant yielded a different band pattern from the known class-II haplotypes, S 2 , S 5 and S 15 . Unexpectedly, this plant was reciprocally cross-incompatible with the S 2 haplotype. Therefore, it was designated as S 2-b . We found an S 13 haplotype having a restriction fragment length polymorphism different from that of the S 13 homozygotes of the S tester line. These findings indicate that S homozygous lines with the same S specificity do not necessarily show the same band pattern in the DNA gel-blot analysis. Soluble stigma proteins of 32 S homozygotes were separated by isoelectric focusing and detected using anti-S 22 SLG antiserum. S haplotype-specific bands were detected in 27 S homozygotes but not in five S homozygotes, including the S 24 homozygote. This is consistent with the observation that the S 24 haplotype had no SLG band.

We found eosinophilic fibrillary neuronal inclusions (EFNI) that were argyrophilic and immunoreactive for anti-ubiquitin in the cerebral cortex of a patient with sporadic amyotrophic lateral sclerosis (ALS) and mild personality changes. Both hematoxylin and eosin and Bodian's preparations revealed the EFNI to be rod-, flame-shaped, or spherical structures existing within the swollen neuronal perinuclear region in the third, fifth, and sixth layers of the fronto-parieto-temporal cortices including the primary motor cortex. On electron microscopy, filamentous profiles aggregated and formed a single bundle or globule in the neuronal perikaryon without any limiting membrane. Most EFNI had a characteristic multiple layer arrangement. The inner core consisted of randomly oriented granule-free tubules with a fuzzy outer contour, measuring 15-20 nm in diameter. The surrounding layer was made up of granule-associated filaments, electrondense free granules, and small vesicular profiles. Large autolysosome-like membrane-bound vesicular profiles were found scattered at the periphery. Neurofilaments were usually mingled with in the surrounding cytoplasm. Many EFNI were also found in dendrites, but only a few in axons. Both granule-free tubules and granule-associated filaments expressed ubiquitin protein epitopes. Aberrant phosphorylation of neurofilament protein and induction of alphaB-crystallin were shown to exist in EFNI-bearing swollen neurons. Despite having a variety of histological appearances, our observations revealed that EFNI all have common immunocytochemical and ultrastructural characteristics, and thus we assume that EFNI represent a series of cytological alterations in the motor and extra-motor cortices of ALS patients.

To clarify the roles of neurotrophins in the human spinal motoneurons, with special reference to amyotrophic lateral sclerosis (ALS), we studied the immunohistochemical localizations of neurotrophins and their receptors in spinal cords of patients with ALS and compared them with controls. In the controls, the majority of motoneurons showed BDNF-, NT3-, trkB- and trkC-like immunoreactivity (-LI) suggesting that the motoneurons receive an autocrine regulation by both BDNF and NT3. In ALS patients, about three-quarters of the motoneurons had degenerated and the remaining motoneurons showed significantly decreased BDNF-LI, increased NGF- and trkA-LI. These findings indicated neurotrophin-switching in the remaining spinal motoneurons of ALS patients from BDNF and NT3 responsive to NGF responsive.

New glasses containing TbF 3 have been prepared in a TbF 3 –BaF 2 –AlF 3 –GeO 2 system. Their respective densities, refractive indexes, and glass transition temperatures are (5.42–6.07) × 10 3 kg m −3 , 1.72–1.73, and 605.1–675.2 °C. Their magnetic susceptibilities are proportional to the contents of TbF 3 in glasses obtained in this study. The values agree with the calculated values. The Curie constant of the 40TbF 3 –20BaF 2 –10AlF 3 –20GeO 2 glass is 5.13 K emu mol −1 ; its effective magnetic susceptibility is 9.96 μ B. The atomic percentage of Tb 3+ in the 40TbF 3 –20BaF 2 –10AlF 3 –20GeO 2 glass is 11%, which is comparable to that of the oxide glass, which is reported as the material for Faraday components.

Amphidiploid species in the Brassicaceae family, such asBrassica napus, are more tolerant to environmental stress than their diploid ancestors. A relatively salt tolerantB. napusline, N119, identified in our previous study, was used. N119 maintained lower Na+content, and Na+/K+and Na+/Ca2+ratios in the leaves than a susceptible line. The transcriptome profiles of both the leaves and the roots 1 h and 12 h after stress were investigated.De novoassembly of individual transcriptome followed by sequence clustering yielded 161,537 nonredundant sequences. A total of 14,719 transcripts were differentially expressed in either organs at either time points. GO and KO enrichment analyses indicated that the same 49 GO terms and seven KO terms were, respectively, overrepresented in upregulated transcripts in both organs at 1 h after stress. Certain overrepresented GO term of genes upregulated at 1 h after stress in the leaves became overrepresented in genes downregulated at 12 h. A total of 582 transcription factors and 438 transporter genes were differentially regulated in both organs in response to salt shock. The transcriptome depicting gene network in the leaves and the roots regulated by salt shock provides valuable information on salt resistance genes for future application to crop improvement.

DNA polymorphism of the S-locus receptor kinase gene (SRK) participating in self-incompatibility in Brassica was analyzed by PCR-RFLP and nucleotide sequencing. In the screening of primers for specific amplification of polymorphic DNA fragments of SRK, the best combination was that of a forward primer (PK1) having the nucleotide sequence of the second exon of S6 SRK and a reverse primer (PK4) having the complementary nucleotide sequence of the fifth exon of S6 SRK. PCR using this primer pair amplified DNA fragments of 0.9–1.0 kb from 36 S haplotypes out of 42 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonuclease(s): 25 types were found in a double digestion with MboI and AfaI. Nucleotide sequencing of the DNA fragments amplified from five S haplotypes showed that the third, fourth, and fifth exons of SRK are highly conserved, and that there are high variations of the second and third introns of SRK, which produced polymorphism of the band pattern in PCR-RFLPs. Another forward primer (PK5) having the nucleotide sequence of the second exon, which is derived from S2 SRK, amplified DNA fragments of almost the same region of SRK from 27 S haplotypes in combination with PK4. Although SRK alleles of the class-II S haplotypes were not amplified, all of the class-I S-haplotypes were amplified with a primer mixture of PK1, PK4 and PK5. The DNA fragments of both SRK alleles in S heterozygotes, or a 1 : 1 mixture of the genomic DNA of different S homozygotes, were amplified without exception, suggesting the usefulness of these primers for the identification of S heterozygotes. The DNA fragment sizes obtained by digestion with restriction endonucleases served as markers for the identification of S haplotypes.

Spinocerebellar ataxia 6 (SCA6) is an autosomal dominant spinocerebellar degeneration caused by the expansion of the polymorphic CAG repeat in the human alpha1A voltage-dependent calcium channel subunit gene (CACNL1A4 gene). We have analyzed 60 SCA6 individuals from 39 independent SCA6 Japanese families and found that the CAG repeat length is inversely correlated with the age of onset (n = 58, r = -0.51, P < 0.0001). SCA6 chromosomes contained 21-30 repeat units, whereas normal chromosomes displayed 6-17 repeats. There was no overlap between the normal and affected CAG repeat number. The anticipation of the disease was observed clinically in all eight parent-child pairs that we examined; the mean age of onset was significantly lower (P = 0.0042) in children than in parents. However, a parent-child analysis showed the increase in the expansion of CAG repeats only in one pair and no diminution in any affected cases. This result suggests that factors other than CAG repeats may produce the clinical anticipation. A homozygotic case could not demonstrate an unequivocal gene dosage effect on the age of onset.

Self-incompatibility in Brassica is controlled by a single multi-allelic locus ( S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene ( SLG ) and an S receptor kinase gene ( SRK ). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris . Sequence comparisons of a large number of SLG alleles and SLG -related genes revealed the following points. ( i ) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG 14 of B. oleracea and SLG 25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. ( ii ) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison ( SLG 8 and SLG 46 of B. campestris ) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. ( iii ) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG / SRK are presented.

Summary Monofactorial gametophytic self-incompatibility in the family Rosaceae is controlled by the single multi-allelic S-locus. In Japanese pear, a naturally occurring self-compatible mutant was found as a sport of a self-incompatible cultivar with an S-genotype of S2,S4. The S,4 allele has been shown to be non-functional in the style of the mutant, but functional in pollen. The present study shows the S2-RNase gene to be transcribed normally in the style of the mutant, but that the S4-RNase gene transcript is not detectable. Furthermore, the S4-RNase gene could not be detected in the mutant by genomic Southern blot, indicating that the mutant lacks the gene. The extent of deletion in the mutant was estimated to be more than 4 kbp, and spans the entire length of the S4-RNase gene. These findings provide evidence that the S-RNase gene is essential for the self-incompatibility reaction in the style, but is not involved in controlling the self-incompatibility behavior of pollen.

Nine mutant lines lacking glutelin subunits were selected from M2 seeds of about 10000 M1 plants mutagenized with gamma rays or EMS and from 1400 mutant lines selected originally for morphological characters. There were three types of mutants, one line lacking the largest subunit among four minor bands of glutelin acidic subunits (Type 1), five lines lacking the second largest subunit band (Type 2), and three lines lacking the third largest subunit band (Type 3). Mutants lacking the smallest subunit band were not found. Type 1 lacked 2 of the 10 spots of glutelin acidic subunits separated by two-dimensional electrophoresis and 1 of the 11 spots of the 57-kDa glutelin precursor. Type 2 lacked 2 spots of acidic subunits and 1 spot of the 57-kDa glutelin precursor, and had low amounts of 1 of the 8 spots of glutelin basic subunits. Type 3 mutants lacked each of 1 spot of the acidic subunits and glutelin precursor and had low amount of 1 spot of the basic subunits. Genetic analysis of the mutated genes showed that these mutant characters were controlled by single recessive genes named glu-1, glu-2, and glu-3, respectively. Mutated genes of different lines of the same type were found to be at the same locus. RFLP analysis of F2 plants between the mutant lines and cv `Kasalath' indicated that glu-1 is on chromosome 2, glu-2 on chromosome 10, and glu-3 on chromosome 1. These mutant genes were combined by crossing, and a line lacking the 3 minor bands of the glutelin acidic subunits was developed. However, the total glutelin content of this line was not remarkably reduced, showing a only 13% decrease.

Water deficit imposed by either drought or salinity brings about severe growth retardation and yield loss of crops. Since Brassica crops are important contributors to total oilseed production, it is urgently needed to develop tolerant cultivars to ensure yields under such adverse conditions. There are various physiochemical mechanisms for dealing with drought and salinity in plants at different developmental stages. Accordingly, different indicators of tolerance to drought or salinity at the germination, seedling, flowering and mature stages have been developed and used for germplasm screening and selection in breeding practices. Classical genetic and modern genomic approaches coupled with precise phenotyping have boosted the unravelling of genes and metabolic pathways conferring drought or salt tolerance in crops. QTL mapping of drought and salt tolerance has provided several dozen target QTLs in Brassica and the closely related Arabidopsis. Many drought- or salt-tolerant genes have also been isolated, some of which have been confirmed to have great potential for genetic improvement of plant tolerance. It has been suggested that molecular breeding approaches, such as marker-assisted selection and gene transformation, that will enhance oil product security under a changing climate be integrated in the development of drought- and salt-tolerant Brassica crops.

From a series of studies on single nucleotide polymorphisms (SNPs) in the bovine growth hormone (GH) gene of Japanese Black cattle, the type-C (127(Val) and 172(Met) ) that is specific for this breed has been intensively focused upon because of the economic importance for carcass traits, such as intramuscular oleic acid contents. In the present study, we intended to analyze the 3-D structure of GH of haplotype C, and developed a novel method to detect the type C gene. Three-D analysis of the type C protein showed that the amino acid residues (127(Val) and 172(Met) ), which are present in the third and fourth helixes, respectively, and are important for binding with GH receptors, are shifted to deeper positions in the molecule compared with that for type A (127(Leu) and 172(Thr) ), implying the alteration of binding interaction with receptors. A novel, efficient and cost-effective method (Dot-blot-SNP technique) for type C genotyping was successfully established, of which the basal method was a reported genotyping of SNPs for a large number of plants, reducing the cost to 10% or less of direct sequencing.

Stylar ribonucleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus x domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.

Hair cell loss induced by aging, ototoxic drugs and noise leads to irreversible hearing loss and balance disorders in mammals due to the failure of hair cells to regenerate. To investigate the possibility of transplantation therapy to repair damaged inner ear, we have examined whether grafted fetal otocyst cells could survive and migrate into injured sensory organs. We obtained otocyst cells from green fluorescein protein (GFP)-transgenic rats on embryonic day 12.5, then transplanted these cells into the inner ears of young rats previously exposed to intense sound. One month after transplantation, the grafted inner ear sensory organs were examined immunohistochemically. Grafted otocyst cells had survived and demonstrated special morphological features in the host organs; cells that migrated into the organ of Corti were similar to supporting cells. These results indicate that injured sensory organs express some kind of scaffolding that plays important roles in the survival and differentiation of the grafted otocyst cells.

SNP markers for QTL analysis of 4-MTB-GSL contents in radish roots were developed by determining nucleotide sequences of bulked PCR products using a next-generation sequencer. DNA fragments were amplified from two radish lines by multiplex PCR with six primer pairs, and those amplified by 2,880 primer pairs were mixed and sequenced. By assembling sequence data, 1,953 SNPs in 750 DNA fragments, 437 of which have been previously mapped in a linkage map, were identified. A linkage map of nine linkage groups was constructed with 188 markers, and five QTLs were detected in two F(2) populations, three of them accounting for more than 50% of the total phenotypic variance being repeatedly detected. In the identified QTL regions, nine SNP markers were newly produced. By synteny analysis of the QTLs regions with Arabidopsis thaliana and Brassica rapa genome sequences, three candidate genes were selected, i.e., RsMAM3 for production of aliphatic glucosinolates linked to GSL-QTL-4, RsIPMDH1 for leucine biosynthesis showing strong co-expression with glucosinolate biosynthesis genes linked to GSL-QTL-2, and RsBCAT4 for branched-chain amino acid aminotransferase linked to GSL-QTL-1. Nucleotide sequences and expression of these genes suggested their possible function in 4MTB-GSL biosynthesis in radish roots.

Rates of nitrogen transformations in the surface aerobic soil of a paddy field (fine-textured strong-gley soils) were estimated by combination of a mathematical model and parallel incubation experiments using 15N as a tracer. The mineralization rate was nearly constant throughout the incubation. However, the ammonium assimilation rate during the initial phase of the incubation was two to three times higher than the mineralization rate, and rapidly decreased with time. This decrease did not follow first-order kinetics for the ammonium content in soil. The nitrification rate increased slightly with time. Reduction of nitrate to ammonium was detectable in all the experiments, but its rate was very low ( The relative magnitudes of the rates of N transformations changed with the incubation temperature. The rates of denitrification plus nitrate assimilation were greatly enhanced at 30°C. The mineralization rate also increased with temperature, but the nitrification rate decreased slightly. The rates of N transformations in the surface layer, especially under aerobic condition, were from 10 to 100 times greater than those in the subsurface layer under anaerobic condition. This indicates that N transformations in the surface layers of paddy soils are quantitatively of great importance.

To elucidate the role and the mechanism of action of nerve growth factor in the adult central nervous system, we investigated the localization of nerve growth factor-like immunoreactivity in adult rat brain, both quantitatively and immunohistochemically, using polyclonal anti-nerve growth factor immunoglobulin G. We raised rabbit polyclonal anti-mouse nerve growth factor antibody with an extremely high titer as 10 −9 determined by an enzyme immunoassay. The affinity-purified anti-nerve growth factor immunoglobulin G specifically recognized nerve growth factor with no cross-reaction to recombinant brain-derived neurotrophic factor and neurotrophin-3 evaluated by an enzyme immunoassay. We quantified nerve growth factor content in each layer of the adult rat cerebral cortex and in each small piece (0.225 mg wet weight tissue) of the diencephalon, brainstem and cerebellum with a highly sensitive two-site enzyme immunoassay. Nerve growth factor content was unevenly distributed in the cerebral cortex (dense in layers II/III and V/VI and sparse in layers I and IV). Moderate to high levels of nerve growth factor were registered in the habenular nuclei, zona incerta, ventral tegmental area, substantia nigra, locus coeruleus, ventral cochlear nucleus, trapezoid body, lateral vestibular nucleus, cerebellar nuclei and paraflocculus. Immunohistochemically, the nerve growth factor-like immunoreactivity was found in the cell bodies, dendrites and axons of adult rat central neurons, not only in the cerebral cortex, hippocampus and basal forebrain, but also in the diencephalon, brainstem and cerebellum. The population of neurons with nerve growth factor-like immunoreactivity was limited, but unexpectedly widespread, and the density of these cells correlated well with the content determined by an enzyme immunoassay in the present and a previous study [Nishio T. et al. (1992) Expl Neurol. 116 , 76–84]. The monoamine neurons, including dopaminergic, noradrenergic and serotonergic neurons, showed intense nerve growth factor-like immunoreactivity, indicating that the central monoaminergic neuronal system may also be involved in the nerve growth factor trophic system. To visualize nerve growth factor transported in the axons and to enhance the immunostaining in the nerve growth factor-producing cells, we injected colchicine, a potent inhibitor of microtubule polymerization and a blocker of axoplasmic transport, into the lateral ventricle of adult Wistar rat brain. Colchicine treatment enhanced the intensities of nerve growth factor-like immunoreactivity in the axons and cell bodies, especially in the axon hillocks and the proximal axons of the nerve growth factor-producing neurons. This observation may suggest the existence of an orthograde axonal transport system for nerve growth factor in the central neurons. These results suggest that nerve growth factor may exist in widespread populations of adult central neurons, including the monoaminergic neurons, and may exert a trophic effect on these neurons through not only a retrograde manner but also an orthograde manner of transport.

We examined the cold stability of microtubules (MT) in biopsied sural nerves. In control nerves, cold treatment reduced the density of MT in unmyelinated axons. The proportion of cold-insoluble MT was approximately 30% by morphological study. By contrast, the extraction study using essentially the same nerves showed that the ratio of cold-insoluble tubulin was 60-75%. These findings suggest the existence of a subpopulation of cold-stable tubulin which may not make up MT. However, in 2 patients with acute dysautonomia the proportion of cold-stable MT or tubulin was decreased compared to that in controls in both the morphological and extraction study. This suggests the altered local process involving MT in primary axonal degeneration.

Brassica napus, an allopolyploid species having the A genome of B. rapa and the C genome of B. oleracea, is self-compatible, although both B. rapa and B. oleracea are self-incompatible. We have previously reported that SP11/SCR alleles are not expressed in anthers, while SRK alleles are functional in the stigma in B. napus cv. 'Westar', which has BnS-1 similar to B. rapa S-47 and BnS-6 similar to B. oleracea S-15. This genotype is the most frequent S genotype in B. napus, and we hypothesized that the loss of the function of SP11 is the primary cause of the self-compatibility of 'Westar'. To verify this hypothesis, we transformed 'Westar' plants with the SP11 allele of B. rapa S-47. All the transgenic plants and their progeny were completely self-incompatible, demonstrating self-compatibility to be due to the S haplotype having the non-functional SP11 allele in the A genome, which suppresses a functional recessive SP11 allele in the C genome. An artificially synthesized B. napus line having two recessive SP11 alleles was developed by interspecific hybridization between B. rapa and B. oleracea. This line was self-incompatible, but F(1) hybrids between this line and 'Westar' were self-compatible. These results suggest that the self-compatibility mechanism of 'Westar' is applicable to F(1) seed production in B. napus.

Among the mutant lines of rice that have been selected for morphological characters, one line, NM67, was found to have a low content of glutelin and a higher content of prolamine in its seed protein than other Japanese cultivars. This mutant is a semi-dwarf and partially sterile line, and its leaves turn yellow before heading. Genetic analysis after backcross to the original cultivar, 'Nihonmasari', revealed the following: (1) the character of low glutelin content was always accompanied by the character of high prolamine content; (2) the low glutelin (and high prolamine) character seemed to be manifested by a single dominant gene; and (3) semi-dwarfness, low fertility and early yellowing leaf of the mutant, which might also be pleiotropy, were controlled by a single recessive gene independent of the gene for protein content. The protein character of NM67 was genetically separated from semi-dwarfness and low fertility, and a new line having low glutelin content and high prolamine content with normal morphological characters comparable to those of the original cultivar was obtained from the progenies of the cross. The possible use of this line as a low protein rice cultivar is discussed.

Rice mutants containing low levels of the 16-kDa allergenic protein, which is the main allergen in the rice grain for patients of atopic dermatitis due to the intake of rice, were screened, and 4 independent mutant lines with small amounts of this protein were found by SDS gel electrophoresis and immunoblot analysis. These mutants were grouped into two types. Two mutant lines, 85KG-4 and 86RG-18, contained low levels of the 16k-Da and 26-kDa polypeptides and a high level of the 57-kDa polypeptide. The 16-kDa polypeptide content of these mutants was about half that of the original cultivars. Homozygous lines were developed, and these showed normal growth and seed set. The other 2 mutant lines, 87KG-970 and 89WPKE-149, showed traces of the 16-kDa and 26 kDa polypeptides and contained a high level of the 13 kDa polypeptide. The homozygous plants of this type were sterile. All of the mutant lines had floury endosperms. Genetic analysis suggested that low 16-kDa polypeptide content is controlled by a single recessive gene. Attempts to separate of the genes for low 16-kDa polypeptide content and floury endosperm by crossing with the original cultivar were unsuccessful, suggesting the tight linkage of these two genes or pleiotropism of a single mutated gene. The relationship between low 16-kDa polypeptide content and the floury character and the possible use of the mutant as a low allergen rice are discussed.

We report on 2 patients with multifocal motor neuropathy with focal conduction block involving motor but not sensory fibers at the site of nerve swelling. A nerve biopsy specimen from adjacent to the enlargement in 1 patient showed a perivascular area containing scattered demyelinated axons surrounded by small onion bulbs. The observed pathological findings are consistent with the conduction block considered characteristic of this neuropathy, although the underlying immunological mechanisms for selectivity and persistence remain undetermined.

A rapid and easy method to discriminate plant cultivars is indispensable to confirm food labeling. We established a fluorogenic ribonuclease protection (FRIP) assay to discriminate Japanese rice (Oryza sativa L.) cultivars based on single nucleotide polymorphisms (SNPs). The FRIP assay uses a hybridization technique between fluorescent probes and the target sequence prepared by run-off transcription, but the requirement of two PCR thermocycles is the problem when preparing template DNA for run-off transcription from rice genomic DNA. In this study, we designed new PCR primers with asymmetric melting temperatures. These primers amplified the target SNP marker containing a T7 RNA polymerase promoter sequence upstream of the target sequence in a single PCR. Moreover, 100 cultivars were discriminated with the patterns of 15 SNPs. The assay can be used as a rapid method of analysis to discriminate Japanese rice cultivars.

DNA methylation is an important epigenetic modification regulating gene expression and transposon silencing. Although epigenetic regulation is involved in some agricultural traits, there has been relatively little research on epigenetic modifications of genes in Brassica rapa, which includes many important vegetables. In B. rapa, orthologs of DDM1, a chromatin remodeling factor required for maintenance of DNA methylation, have been characterized and DNA hypomethylated knock-down plants by RNAi (ddm1-RNAi plants) have been generated. In this study, we investigated differences of DNA methylation status at the genome-wide level between a wild-type (WT) plant and a ddm1-RNAi plant by methylation-sensitive amplification polymorphism (MSAP) analysis. MSAP analysis detected changes of DNA methylation of many repetitive sequences in the ddm1-RNAi plant. Search for body methylated regions in the WT plant revealed no difference in gene body methylation levels between the WT plant and the ddm1-RNAi plant. These results indicate that repetitive sequences are preferentially methylated by DDM1 genes in B. rapa.