Expression pattern of alphavbeta3 and alphavbeta5 integrin mRNA in mouse fetal gonads

The alphavbeta3 and alphavbeta5 integrins are known as transmembrane receptors capable of binding to the RGD amino acid peptide sequence. In mouse early gonadogenesis, some proteins containing the RGD sequence are deposited into extracellular space and participate in morphogenesis. We analyzed the expression patterns of the alphavbeta3 and alphavbeta5 integrins in mouse developing gonads (10.5-13.5 days post coitum) using whole-mount in situ hybridization. The alphav integrin mRNA was homogenously expressed in developing gonadal regions. On the other hand, the beta3 integrin mRNA was found only in large and round cells (presumptive germ cells), whereas beta5 integrin was localized in gonadal somatic cells, with the exception of coelomic epithelial cells. The beta3 integrin-expressed cells were determined to be primordial germ cells because the number of these cells was drastically reduced in busulfan-treated gonads. In this study, we demonstrated that the alphavbeta3 and alphavbeta5 integrins are widely localized in the mouse developing gonads and discussed their presumptive functions on mouse gonadogenesis.     

Development of ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis with truncated 34 kDa proteins

To develop ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the carboxyl termini of the 34 kDa proteins of M. paratuberculosis and Mycobacterium avium subsp. avium (M. avium) were expressed in Escherichia coli expression system. Antibodies specific to M. paratuberculosis were detected with the truncated 34 kDa protein of M. paratuberculosis in ELISA after pre-absorption of serum samples with the truncated 34 kDa protein of M. avium. All the serum samples from cattle confirmed to be infected with M. paratuberculosis were positive and those from healthy cattle were negative in the present ELISA system. These results indicate that the established ELISA detects antibodies specific to M. paratuberculosis with high specificity and sensitivity and is an useful tool for the screening of Johne's disease.     

Analysis of neutral glycosphingolipids from Trypanosoma brucei

Neutral glycosphingolipids (GSLs) were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC), TLC/secondary ion mass spectrometry (TLC/SIMS), and liposome immune lysis assay (LILA). Three species of neutral GSLs, designated as N-1, -2, and -3 were separated on TLC. N-1 GSL migrated very close to glucosylceramide (GlcCer) and N-2 GSL showed the same mobility as lactosylceramide (LacCer). On the other hand, the mobility of N-3 GSL on the TLC plate was slower than globotetraosylceramide (Gb4). In order to characterize the molecular species of neutral GSLs from T. brucei, N-1, -2 and -3 GSLs were analyzed by TLC/SIMS. The TLC/SIMS analysis of N-1 of the parasites revealed a series of (M–H)⁻ ions from m/z 698 to 825 representing the molecular mass range of ceramide monohexoside (CMH) (GlcCer or galactosylceramide). On the other hand, the TLC/SIMS spectra of N-2 GSL revealed a series of (M–H)⁻ ions from m/z 944–987 indicating the molecular mass range of LacCer. In the TLC/SIMS analysis of N-3 GSL, however, the characteristic molecular ions that can elucidate the structure of N-3 GSL were not obtained. In order to confirm the results obtained from TLC/SIMS, N-1, -2, and -3, GSLs were tested by LILA with specific antibodies against GlcCer, LacCer, and Gb4, respectively. N-1 GSL had reactivity to anti-GlcCer antibody and N-2 GSL reacted with the antibody against LacCer. However, N-3 GSL was not recognized by anti-Gb4 antibody. Using anti-GlcCer and anti-LacCer antibodies, furthermore, we studied the expression of GlcCer and LacCer in T. brucei parasites. Both GlcCer and LacCer were detected on the cell surface of T. brucei.     

Availability of Micro-Tom mutant library combined with TILLING in molecular breeding of tomato fruit shelf-life

Novel mutant alleles of an ethylene receptor Solanum lycopersicum ETHYLENE RESPONSE1 (SlETR1) gene, Sletr1-1 and Sletr1-2, were isolated from the Micro-Tom mutant library by TILLING in our previous study. They displayed different levels of impaired fruit ripening phenotype, suggesting that these alleles could be a valuable breeding material for improving shelf life of tomato fruit. To conduct practical use of the Sletr1 alleles in tomato breeding, genetic complementation analysis by transformation of genes carrying each allele is required. In this study, we generated and characterized transgenic lines over-expressing Sletr1-1 and Sletr1-2. All transgenic lines displayed ethylene insensitive phenotype and ripening inhibition, indicating that Sletr1-1 and Sletr1-2 associate with the ethylene insensitive phenotype. The level of ethylene sensitivity in the seedling was different between Sletr1-1 and Sletr1-2 transgenic lines, whereas no apparent difference was observed in fruit ripening phenotype. These results suggested that it is difficult to fine-tune the extent of ripening by transgenic approach even if the weaker allele (Sletr1-2) was used. Our present and previous studies indicate that the Micro-Tom mutant library combined with TILLING could be an efficient tool for exploring genetic variations of important agronomic traits in tomato breeding.     

An H9N2 influenza virus vaccine prepared from a non-pathogenic isolate from a migratory duck confers protective immunity in mice against challenge with an H9N2 virus isolated from a girl in Hong Kong

H9N2 influenza viruses circulate in wild birds and poultry in Eurasian countries, and have been isolated from pigs and humans in China. H9N2 viruses isolated from birds, pigs and humans have been classified into three sublineages based on antigenic and genetic features. Chicken antisera to H9N2 viruses of the Korean sublineage reacted with viruses of different sublineages by the hemagglutination-inhibition test. A test vaccine prepared from a non-pathogenic A/duck/Hokkaido/49/1998 (H9N2) strain of the Korean sublineage, obtained from our influenza virus library, induced immunity in mice to reduce the impact of disease caused by the challenge with A/Hong Kong/1073/1999 (H9N2), which is of a different sublineage. The present results indicate that an inactivated whole virus vaccine prepared from a non-pathogenic influenza virus from the library could be used as an emergency vaccine during the early stage of a pandemic caused by H9N2 infection.     

Effects of coumestrol administration to maternal mice during pregnancy and lactation on renal Ca metabolism in neonatal mice

The present study was conducted to clarify the effects of coumestrol administration to maternal mice during pregnancy and lactation on serum Ca and Ca metabolism in their neonatal mice. From 6.5 to 16.5 days post coitus and from 3 to 10 days after parturition, maternal mice were administered at 200 µg/kg body weight/day of coumestrol. Coumestrol administration did not affect weight gains, serum Ca and the expression of vitamin D receptor (VDR) protein in the kidney of neonatal mice, but weight gains of maternal mice were decreased by coumestrol administration. Coumestrol administration increased the messenger RNA (mRNA) expressions of epithelial Ca channels 1 (ECaC1) and VDR in the kidney of neonatal mice, and also increased the mRNA expressions of ECaC2 in the kidney and small intestine of male neonatal mice. The mRNA expressions of ECaC1, ECaC2, calbindin‐D_9k (CaBP‐9k) and estrogen receptor (ER)α in the kidney and VDR in the small intestine of male neonatal mice were higher than those of female mice. Thus, coumestrol administration to maternal mice during pregnancy and lactation may affect renal Ca metabolism in neonatal mice, especially male neonatal mice via maternal milk.     

Expression and characterization of rainbow trout Oncorhynchus mykiss recombinant myoglobin

Fish Physiology and Biochemistry - Recombinant expression system was established for rainbow trout myoglobin (Mb) considering its unique primary structure of having one unusual deletion and two...     

Cloning,expression, and tissue distribution of bovine interleukin-21

Bovine interleukin-21 (IL-21) cDNA was cloned and sequenced from bovine peripheral blood lymphocytes (PBLs) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin (PHA), and 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h. The open reading frame of the bovine IL-21 cDNA is 459 bp in length and encodes 152 amino acids. The predicted amino acid sequence is 78.2 and 58.5% homologous to the human and murine IL-21 amino acid sequences, respectively. Recombinant bovine IL-21 was expressed by a baculovirus expression system. The bovine IL-21 was processed to the mature form in insect cells and secreted to the supernatant confirmed by N-terminal amino acid sequencing. The recombinant bovine mature IL-21 induced the proliferation of human IL-2-dependent cells, ILT-MAT. The mRNA expression for bovine IL-21 was observed in the spleen, but not in the brain, heart, lung, liver, and kidney. The bovine IL-21 identified in this study may provide new methods for the enhancement of innate immunity in cows.     

Macroscopic study of the functional significance of the forearm muscles in the giant panda

The extensor and flexor group muscles and their related muscles were functional-morphologically observed in the dead body of the giant panda to clarify the action of the forearm and the palm in the manipulation of the species. The Musculus flexor carpi ulnaris had two developed heads, however, we can conclude that the contraction of this muscle slightly changes the angle of the accessory carpal bone to the ulna. The data pointed out that the accessory carpal bone acts as a supporting post, when the giant panda seizes the object. The M. abductor digiti I longus possessed the well-developed origin in both ulna and radius. These findings suggest that this muscle may function as a supinator of the forearm. We also suggest that the well-developed M. pronator quadratus and M. pronator teres, and the proximal part of the M. abductor digiti I longus and the M. supinator may efficiently contribute to the pronator-spinator action of the forearm, when the giant panda brings the food to its mouth using the manipulation system equipped in the palm region.