RESUMEN

INTRODUCTION: Repair of obstructive azoospermia caused by childhood herniorrhaphy may be difficult. Therefore, intracytoplasmic sperm injection using testicular sperm is performed. However, vasovasostomy combined with laparoscopic surgery is challenging. CASE PRESENTATION: A 42-year-old man underwent inguinal hernia repair at age 3. He had normal testicular size, azoospermia, normal hormone levels (follicle-stimulating hormone, luteinizing hormone, and testosterone), absence of Y chromosome micro deletion, and karyotype:46XY, t(1:21)(p34.1:q22.3). He was diagnosed with obstructive azoospermia. Repeated intracytoplasmic sperm injections using testicular sperm resulted in miscarriages. Vasovasostomy combined with laparoscopic surgery was subsequently performed. Postoperative semen analysis result was almost normal. After intracytoplasmic sperm injection of ejaculated sperm, his wife got pregnant. CONCLUSION: Even if patients have chromosomal abnormalities, performing microsurgical re-anastomosis first is recommended. To our knowledge, this is the first case of a laparoscopy-assisted vasovasostomy for post-herniorrhaphy vas deferens obstruction in Japan.

RESUMEN

OBJECTIVES: Uterine fibroids are capable of causing infertility, but there are no definite criteria for which laparoscopic uterine myomectomy (LM) is known to be beneficial. To investigate the usefulness of LM, we examined pregnancy rates in patients with infertility with no obvious cause except for the presence of uterine fibroids. MATERIALS AND METHODS: We retrospectively reviewed the clinical records at Suzuki Memorial Hospital between June 2010 and August 2014. We found 60 eligible patients (LM group, 46; non-LM group, 14). The criteria for performing LM were a maximal fibroid diameter of 40 mm or more or the presence of >4 fibroids. RESULTS: The duration of infertility before the first visit was significantly longer in the LM group; although there was no significant difference in the mean patient age and body mass index. Pregnancy was achieved in 45.7% of patients (21/46) in the LM group and 28.6% (4/14) in the non-LM group. There were no pregnancies in patients with >10 fibroids. The postoperative pregnancy rate in the LM group was comparable to previously reported pregnancy rates. CONCLUSIONS: Our criteria for performing LM in patients with no obvious cause for infertility except for uterine fibroids seem appropriate, especially when the fibroids are large and the number of fibroids is between 4 and 9. However, our results suggest that the effectiveness of LM is low in patients with 10 or more uterine fibroids.

RESUMEN

The relationship between the protein composition of rice and nitrogen compounds (amino acids and oligo-peptides) in the produced sake were investigated using endosperm protein mutant rice (LGC-1, LGC-Jun, Kx433, QA28), sake rice (Yamadanishiki) and cooking rice (Nipponbare, Nihonmasari, Koshihikari). The total nitrogen concentration, amino acid concentration and most peptide peak areas determined by RP-HPLC and gel filtration chromatography of the produced sake were lower when sake was made from a low glutelin content rice mutant compared with other rice varieties. The concentration of nitrogen compounds in the sake positively correlated with the glutelin content of the highly milled rice grains used for sake production. Sake produced using a low glutelin content rice mutant is generally evaluated as having a light taste. Our findings suggest that nitrogen compounds (oligo-peptides and amino acids) derived from rice glutelin significantly contribute to the taste of sake.

Asunto(s)

Bebidas Alcohólicas/análisis , Oryza/química , Proteínas de Plantas/química , Aminoácidos/análisis , Culinaria , Glútenes/análisis , Mutación , Nitrógeno/análisis , Compuestos de Nitrógeno/análisis , Oryza/clasificación , Oryza/genética , Péptidos/análisis , Gusto

RESUMEN

Glutelin, the major storage protein of rice seed, consists of microheterogenous subunits and partially exists in a macromolecular form that is polymerized by intersubunit disulfide bonds. In order to analyze the glutelin subunits using high-throughput CE, we first identified a sample preparation procedure suitable for CE. The polymerized glutelin treated with a reductant could not dissociate into its constituent monomer subunits when it was dissolved in an acidic solution. However, the glutelin dissociated into its subunits and component α and ß polypeptides when it was denatured and reduced by an appropriate amount of urea and 2-mercaptoethanol at a specific incubation time and temperature. The molecular species of the completely dissociated α and ß polypeptides were identified and quantitatively analyzed by CE using glutelin mutants. The CE analysis also demonstrated that the actual subunit variation in terms of the charge and/or size of the ß polypeptides is much smaller than predicted when compared with that of α polypeptides, even under denaturing and reducing condition. Thus, the combined analytical system described here will be useful for basic and applied research, such as the kinetic characterization of higher-order structure and the quantitative evaluation of glutelin in a large number of diverse rice varieties.

Asunto(s)

Electroforesis Capilar/métodos , Glútenes/química , Oryza/química , Subunidades de Proteína/química , Electroforesis en Gel de Poliacrilamida , Glútenes/análisis , Glútenes/metabolismo , Modelos Lineales , Mercaptoetanol/química , Desnaturalización Proteica , Temperatura , Urea/química

RESUMEN

In order to analyze mutations induced by gamma irradiation in higher plants, we irradiated rice with gamma rays and screened for mutations expressing phenotypes of glutinous endosperm (wx), chlorophyll b deficiency, endosperm protein deficiency, gibberellin-related dwarfism, and shortened plastochron-in order to clarify types of mutations. Nucleotide sequence analysis showed that the most frequent mutation induced by gamma rays was deletion, particularly small deletion. Of the 24 mutations, 15 were small deletions (1-16 bp), four were large deletions (9.4-129.7 kbp), three were single-base substitutions, and two were inversions. Deletions 100 bp-8 kbp in length were not found, suggesting that gamma irradiation is unlikely to induce deletions of 100 bp to 8 kbp but is more likely to induce deletions between 1 and several ten bp or those of around 10 kbp or more. Based on the results, reverse genetics applications may be possible for gamma irradiation-induced deletions in rice by mismatch cleavage analysis used in Targeting Induced Local Lesions IN Genomes (TILLING) to detect small deletions and base substitutions or by using array comparative genomic hybridization (aCGH) to detect large deletions.

Asunto(s)

ADN de Plantas/genética , Rayos gamma , Mutación/genética , Oryza/genética , Oryza/efectos de la radiación , Proteínas de Plantas/genética , Clorofila/deficiencia , Cartilla de ADN/química , Cartilla de ADN/genética , Endospermo , Eliminación de Gen , Giberelinas/metabolismo , Glútenes/genética , Meristema , Plantas Modificadas Genéticamente , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN

RESUMEN

To obtain fundamental information for nutritional improvement of rice (Oryza sativa) seed proteins, the alpha polypeptides of the major storage protein glutelin varied over the genus Oryza were qualitatively and quantitatively characterized with unique methods. The polypeptides were maximally separated by two-dimensional electrophoresis (2D-PAGE) composed of nonequilibrium pH gradient gel electrophoresis (NEPHGE) and higher temperature SDS-PAGE. Then the subunit for each polypeptide spot was identified with the sequential immunodetection called a step-by-step detection method, making use of highly subunit-specific antibodies. The comparative analysis showed considerable variation in the accumulation level of A-type and B-type glutelin subunits and found unknown glutelin subunits that were unable to be identified with the antibodies used. Wild species accumulating a high amount of lysine-rich B-type glutelin subunits and unknown unique subunits were identified as they might play a crucial role in nutritional quality improvement of the cultivated rice.

Asunto(s)

Electroforesis en Gel Bidimensional/métodos , Glútenes/química , Inmunoensayo/métodos , Oryza/química , Péptidos/química , Electroforesis en Gel de Poliacrilamida , Genoma de Planta , Glútenes/metabolismo , Oryza/genética , Oryza/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Semillas/química , Semillas/metabolismo , Temperatura

RESUMEN

In efforts to find genetic resources with high nutritional value of rice seed, we assessed the diversity of the major storage protein glutelin in 13 wild and 2 cultivated rice species by a unique SDS-PAGE method and subunit-specific antibodies. Maximum separation of microheterogeneous glutelin alpha-polypeptides, which is a prerequisite for the diversity evaluation, could be attained by SDS-PAGE performed at higher temperature (45 degrees C) than the generally employed temperatures (4-25 degrees C). Seven antipeptide antibodies were raised against subunit-specific epitope sequences designed at five sites from four variable regions spanning the glutelin alpha-polypeptides. High specificity of each antibody was confirmed using rice glutelin mutants, and demonstrated considerable variation in amino acid sequence and accumulation level of glutelin subunit in wild species, in combination with the higher-temperature SDS-PAGE. The degree of the variation was, however, changed according to the site of variable regions and the type of subunit. Some wild species accumulated nutritious GluB subunits more than cultivated rice. The wild species Oryza longiglumis and O. brachyantha had glutelin with low reactivity against most antibodies examined in this study, reflecting the significant divergence. Such wild species may hopefully serve as important genetic resources for nutritional improvement of cultivated rice.

Asunto(s)

Electroforesis en Gel de Poliacrilamida/métodos , Glútenes/química , Oryza/química , Secuencia de Aminoácidos , Glútenes/inmunología , Datos de Secuencia Molecular , Oryza/genética , Subunidades de Proteína/inmunología , Alineación de Secuencia

RESUMEN

In the course of evolution, a gene is often duplicated in tandem, resulting in a functional redundancy. The analysis of function of these genes by raising double mutant might be difficult because they are very tightly linked. We described here a mutant of such a tandem duplicated gene. glu1 is a gamma-ray-induced rice mutant, which lacks an acidic subunit of glutelin, a major seed storage protein. We found that glu1 harbors a 129.7-kb deletion involving two highly similar and tandem repeated glutelin genes, GluB5 and GluB4. The deletion eliminated the entire GluB5 and GluB4 gene except half of the first exon of GluB5. GluB5 and GluB4 have the same amino acid sequence in the acidic subunit, suggesting that only the mutation involving both GluB5 and GluB4 results in the lack of the glutelin acidic subunit deleted in glu1. Our finding suggests that gamma-ray can be an effective mutagen to analyze tandem repeated and functionally redundant genes.

Asunto(s)

Rayos gamma , Eliminación de Gen , Genes de Plantas/efectos de la radiación , Glútenes/metabolismo , Oryza/genética , Oryza/efectos de la radiación , Secuencias Repetidas en Tándem/efectos de la radiación , ADN de Plantas/química , ADN de Plantas/genética , ADN de Plantas/efectos de la radiación , Regulación hacia Abajo/genética , Regulación hacia Abajo/efectos de la radiación , Evolución Molecular , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes de Plantas/genética , Glútenes/química , Glútenes/genética , Familia de Multigenes/genética , Familia de Multigenes/efectos de la radiación , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Secuencias Repetidas en Tándem/genética

RESUMEN

Chlorophyll degradation is an aspect of leaf senescence, which is an active process to salvage nutrients from old tissues. non-yellow coloring1 (nyc1) is a rice (Oryza sativa) stay-green mutant in which chlorophyll degradation during senescence is impaired. Pigment analysis revealed that degradation of not only chlorophylls but also light-harvesting complex II (LHCII)-bound carotenoids was repressed in nyc1, in which most LHCII isoforms were selectively retained during senescence. Ultrastructural analysis of nyc1 chloroplasts revealed that large and thick grana were present even in the late stage of senescence, suggesting that degradation of LHCII is required for the proper degeneration of thylakoid membranes. Map-based cloning of NYC1 revealed that it encodes a chloroplast-localized short-chain dehydrogenase/reductase (SDR) with three transmembrane domains. The predicted structure of the NYC1 protein and the phenotype of the nyc1 mutant suggest the possibility that NYC1 is a chlorophyll b reductase. Although we were unable to detect the chlorophyll b reductase activity of NYC1, NOL (for NYC1-like), a protein closely related to NYC1 in rice, showed chlorophyll b reductase activity in vitro. We suggest that NYC1 and NOL encode chlorophyll b reductases with divergent functions. Our data collectively suggest that the identified SDR protein NYC1 plays essential roles in the regulation of LHCII and thylakoid membrane degradation during senescence.

Asunto(s)

Complejos de Proteína Captadores de Luz/metabolismo , Oryza/fisiología , Hojas de la Planta/crecimiento & desarrollo , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Clorofila/metabolismo , Oscuridad , Genes Reporteros , Cinética , Complejos de Proteína Captadores de Luz/genética , Datos de Secuencia Molecular , Oryza/clasificación , Oryza/crecimiento & desarrollo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN de Planta/genética , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

Rice glutelin, which accounts for 70-80% of the total proteins of the seeds, consists of two nutritionally different subfamilies (A and B types). Although the similarity in primary sequences between the two subfamilies is as high as 60%, we established conditions to discriminate the two subfamilies when low amounts of antigen are analyzed by immunoblot methods. The glutelin alpha polypeptides can be resolved into six bands labeled alpha1 to alpha6 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gel filtration analysis showed that glutelin exists as a polymerized and a smaller molecular weight form. Immunoblot analysis of SDS-PAGE resolved polypeptides showed that alpha2, alpha3, and alpha4 are an A type and that these A types as well as alpha1, a B type, are polymerized. The polymerization tendency clearly differed between the two subfamilies except for alpha1, which may be derived from GluB-4 as suggested by analysis using Escherichia coli expression systems of glutelin cDNA regions corresponding to alpha polypeptides. GluB-4 and all the A type subunits have an extra Cys residue in the hypervariable regions, corresponding to the C-terminal region of alpha polypeptide. Accordingly, the extra Cys residue is hypothesized to be responsible for the polymerization of glutelin.

Asunto(s)

Glútenes/química , Oryza/química , Secuencia de Aminoácidos , Cromatografía en Gel , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Glútenes/genética , Oryza/genética , Estructura Secundaria de Proteína , Semillas/química , Semillas/genética

RESUMEN

Low glutelin content1 (Lgc1) is a dominant mutation that reduces glutelin content in rice grains. Glutelin is a major seed storage protein encoded by a multigene family. RNA gel blot and reverse transcriptase-mediated PCR analyses revealed that Lgc1 acts at the mRNA level in a similarity-dependent manner. In Lgc1 homozygotes, there is a 3.5-kb deletion between two highly similar glutelin genes that forms a tail-to-tail inverted repeat, which might produce a double-stranded RNA molecule, a potent inducer of RNA silencing. The hypothesis that Lgc1 suppresses glutelin expression via RNA silencing is supported by transgenic analysis using this Lgc1 candidate region, by reporter gene analysis, and by the detection of small interfering RNAs. In this context, Lgc1 provides an interesting example of RNA silencing occurring among genes that exhibit various levels of similarity to an RNA-silencing-inducing gene. Possible mechanisms for gene silencing of the glutelin multigene family by Lgc1 are discussed.

Asunto(s)

Glútenes/genética , Oryza/genética , Interferencia de ARN/fisiología , Secuencia de Aminoácidos , Clonación Molecular , Metilación de ADN , ADN de Plantas/química , ADN de Plantas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glútenes/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes/genética , Mutación , Oryza/crecimiento & desarrollo , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Semillas/genética , Semillas/crecimiento & desarrollo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido