Gene families encoding isoforms of two major sesame seed storage proteins, 11S globulin and 2S albumin

Sesame (Sesamum indicum L.) seed has been recognized as a nutritional protein source owing to its richness in methionine. Storage proteins have been implicated in allergenic responses to sesame consumption. Two abundant storage proteins, 11S globulin and 2S albumin, constitute 60-70 and 15-25% of total sesame proteins, respectively. Two gene families separately encoding four 11S globulin and three 2S albumin isoforms were identified in a database search of 3328 expressed sequence tag (EST) sequences from maturing sesame seeds. Full-length cDNA sequences derived from these two gene families were completed by PCR using a maturing sesame cDNA library as the template. The amino acid compositions of these deduced storage proteins revealed that the richness in methionine is attributed mainly to two 2S albumin isoforms and partly to one 11S globulin isoform. The presence of four 11S globulin and three 2S albumin isoforms resolved in SDS-PAGE was confirmed by MALDI-MS analyses. The abundance of these isoforms was in accord with the occurrence frequency of their EST sequences in the database. A comprehensive understanding of these storage proteins at the molecular level may also facilitate the identification of allergens in crude sesame products that have caused severe allergic reactions increasingly reported in the past decade.     

Post-mortem evaluation of expanded polytetrafluoroethylene (ePTFE) used in mitral valve repair in dogs

Mitral valve repair is one of the treatment options for mitral regurgitation. Expanded polytetrafluoroethylene (ePTFE) is a polymer that has been widely used in cardiovascular surgery. In this case series, we report the autopsy and histological findings in 6 dogs that underwent cardiopulmonary bypass for mitral annuloplasty using ePTFE sheets and chordoplasty using ePTFE sutures.From May 2005 to October 2009, 3 female and 3 male dogs with severe mitral regurgitation underwent mitral valve repair. This case series included 3 Cavalier King Charles spaniels, 2 Maltese, and 1 Shih Tzu. The survival period after surgery was 19–72 (35 ± 19) months.In all the cases, autopsy revealed that the ePTFE sheets and sutures were not damaged and well integrated into the surrounding highly differentiated, connective tissues. Low-power microscopy revealed that in all cases, the tissues surrounding the ePTFE sheet in the mitral valve annulus had almost completely been covered by granulation tissue. No inflammatory infiltrate or thrombogenesis was observed around the ePTFE in any of the cases.There was no evidence of reactive changes in the region surrounding the ePTFE. These results suggest that ePTFE has excellent tissue compatibility and durability and can be effectively used for canine mitral valve repair.     

Different thermotolerances in in vitro‐produced embryos derived from different maternal and paternal genetic backgrounds

The present study evaluated the effects of genetic backgrounds on the developmental competence and thermotolerance of bovine in vitro‐produced (IVP) embryos. First, Holstein (Hol) and Japanese Black (JB) oocytes were fertilized with sperm from Hol, JB and a thermotolerant breed (Brahman), and in vitro development was evaluated when the embryos were exposed to heat shock on Day 2 (Day 0 = day of fertilization). Sperm genetic backgrounds affected the developmental competence in controls (P < 0.05). Second, the effect of sperm pre‐incubation for 4 h on subsequent in vitro fertilization was assessed using different sperm genetic backgrounds. The pre‐incubation of sperm did not decrease the embryonic development regardless of the breed of the sperm. A milder heat shock (40.0°C) effect on parthenotes (Hol and JB) and IVP embryos were evaluated. JB parthenotes showed developmental arrest after Day 4, and the rate of development to the blastocyst stage decreased by heat shock, but not in Hol parthenotes. Heat shock decreased developmental competence after cleavage of IVP embryos regardless of genetic background. The thermotolerance of IVP embryos would be controlled by both maternal and paternal factors but genetic involvement was still unclear. Further evaluation is needed to reveal the genetic contribution to thermotolerance.     

Micropores and mesopores in the cell wall of dry wood

To investigate micropores and mesopores in the cell walls of dry wood, CO₂ gas and N₂ gas adsorption onto dry wood were measured at ice-water temperature (273 K) and liquid nitrogen temperature (77 K). CO₂ gas adsorption isotherms obtained were used for determining micropore volumes smaller than 0.6 nm by the HK method (Horvath-Kawazoe method), and N₂ gas adsorption isotherms obtained were used for determining the mesopore volume between 2 nm and 50 nm by the Barrett-Joyner-Halenda (BJH) method. Micropores and mesopores existed in cell walls of dry wood, and the cumulative pore volume was much larger for micropores than for mesopores. Micropores in the cell wall of dry wood decreased with elevating heat treatment temperature, and the decreased micropore was reproducible by wetting and drying. Mesopores did not decrease so much with elevating heat treatment temperature. Micropore volumes for the softwood Hinoki and the hardwood Buna were compared. A larger amount of micropores existed in hardwood Buna than in softwood Hinoki, and this relationship was considered to correspond to the difference in thermal softening properties for lignin in water-swollen Hinoki and Buna. This result probably indicates that micropores in the cell walls of dry wood relate to the structure of lignin.     

Effect of methotrexate exposure at late gestation on development of telencephalon in rat fetal brain

Pregnant rats were treated with 30 mg/kg of methotrexate (MTX) on gestation day (GD) 16, and fetal brainswere examined time-dependently. On GD 20, the appearance of the telencephalon in the MTX group was differentfrom that in the control group, and the major axis of the telencephalon of the MTX group was shortened,compared to that of the control group. In the sagittal section of the telencephalon in the MTX group on GD 20,histopathological findings of deformation and narrowing of the cerebral ventricle, the disturbance of thearrangement of the marginal cell layer of subventricular zone (SVZ) and thickening of telencephalic wall,cortical plate and ventricular zone (VZ)/SVZ were possibly attributable to neuronal migration disorders byMTX. Through all the experimental period, few pyknotic cells or TUNEL-positive cells were observed in theVZ/SVZ of the telencephalic wall and striatum in the control group. On the other hand, in the VZ/SVZ of thetelencephalic wall and striatum in the MTX group, pyknotic cells or TUNEL-positive cells were observed on GD17, and they increased significantly on GD18 and then decreased to the control levels from GD 19 onward. Thephospho-Histone H3-positive rate decreased remarkedly in the VZ/SVZ of the telencephalic wall and striatum ofthe MTX group on GDs 17 and 18, compared to the control group, but they recovered on and after GD 19. Theseresults suggested that there was a high possibility that development of the telencephalon in this periodrequired strong folic acid.     

Meiotic chromosome pairing in intergeneric hybrids of colchicaceous ornamentals revealed by genomic in situ hybridization (GISH)

Diploid and triploid intergeneric hybrids obtained by crosses among Gloriosa superba ‘Lutea’ (2n = 2x = 22), G. ‘Marron Gold’ (2n = 4x = 44), Littonia modesta (2n = 2x = 22), and Sandersonia aurantiaca (2n = 2x = 24) were analyzed for their meiotic chromosome pairing in pollen mother cells by genomic in situ hybridization (GISH) with digoxigenin-labeled total DNA of one parent as probe. Chromosomes from each parent could be clearly distinguished in pollen mother cells of all the five intergeneric hybrids by GISH. For three diploid hybrids, L. modesta × G. superba ‘Lutea’ (2n = 2x = 22), L. modesta × S. aurantiaca (2n = 2x = 23) and S. aurantiaca × G. superba ‘Lutea’ (2n = 2x = 23), 0.04?0.27 autosyndetic bivalents (intragenomic pairing of non-homologous chromosomes) and 0.13?0.36 allosyndetic bivalents (intergenomic chromosome pairing) were observed per pollen mother cell, indicating that there are some homologous chromosomal regions within each genome and among the genomes of Gloriosa, Littonia and Sandersonia. Differences in the average number of allosyndetic bivalents per pollen mother cell among different genome combinations may reflect the evolutionary distances among the three genera, and Gloriosa and Littonia may be closely related to each other, while Sandersonia may have relatively distant relationships with Gloriosa and Littonia. For two triploid hybrids, L. modesta × G. ‘Marron Gold’ (2n = 3x = 33) and S. aurantiaca × G. ‘Marron Gold’ (2n = 3x = 34), no allosyndetic bivalents were observed. Based on the results obtained in the present study, possible utilization of the diploid and triploid intergeneric hybrids for further breeding of colchicaceous ornamentals is discussed.     

Effects of oral exposure of bisphenol A on mRNA expression of nuclear receptors in murine placentae assessed by DNA microarray

Bisphenol A (BPA), a candidate endocrine disruptor (ED), is considered to bind to estrogen receptors and to regulate expressions of estrogen responsive genes. It has also shown evidence of affecting the reproductive, immunological and nervous systems of mammalian embryos. However, the effects of BPA on placentae, a central organ of feto-maternal interlocution, are still unclear. To reveal the mechanisms of BPA effects on placentae in mammals, we compared the mRNA expression of 20 nuclear receptors between placentae of vehicle controls and those of orally BPA exposed pregnant mice by a DNA microarray technique. In murine placentae, mRNAs of 11 nuclear receptors were not detected. However, greater than 1.5 fold changes in mRNA expression of nine nuclear receptors between vehicle control and BPA treated mice were noted. Moreover, remarkable changes in mRNA expression of six non-nuclear receptor proteins were induced by BPA exposure. There were various differences in the effects of BPA on the expression of these mRNAs between the placentae with male embryos and those with female embryos. Such embryo-sex dependent differences are interesting and important pointers to understanding of the endocrine disrupting effect of BPA. The present data indicate that BPA affects the expression of nuclear receptor mRNAs in placentae and may disrupt the physiological functions of placentae.     

Microinsemination with first-wave round spermatids from immature male mice

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.     

An experimental study of apico-aortic valved conduit (AAVC) for surgical treatment of aortic stenosis in dogs

A new valved conduit was developed using a canine aortic valve. The bioprosthetic valve was fixed with glutaraldehyde and epoxy compound (Denacol-EX313/810). A vascular graft composed of ultra-fine polyester fiber (10 mm in diameter, 200 mm in length) was used. Four dogs underwent apico-aortic valved conduit (AAVC) implantation and aortic banding (bypass group, BG), while another 4 dogs underwent aortic banding without AAVC implantation (control group, CG). Cardiac catheterization and angiocardiography were performed for assessment of hemodynamics 2 weeks and 6 months after surgery. Left ventricular systolic pressure, left ventricular end-diastolic pressure and the left ventricular-aortic pressure gradient differed significantly (P<0.01) between the BG and CG dogs. Left ventricular angiocardiography showed patency of the valved conduit in all the BG dogs. Echocardiography was performed before and 2, 4 and 6 months after surgery, and showed that while pressure overload caused concentric myocardial hypertrophy in the CG dogs, the left ventricle dilated eccentrically in the BG dogs. Furthermore, relief of left ventricular pressure overload by AAVC was maintained.