Cloning of estrogen receptor-β cDNA of common Japanese conger Conger myriaster and expression of the gene during oocyte development

ABSTRACT:   Estrogen receptor (ER) cDNA was cloned from the liver of common Japanese conger Conger myriaster . Common Japanese conger ER cDNA contains 2543 nucleotides including an open reading frame that encodes 596 amino acids, with a molecular weight of 66.1 kDa. The deduced amino-acid sequence of the common Japanese conger ER shares 85% identity with that of eel Anguilla japonica ER-β, and 39–46% and 53–59% identity with the deduced sequences of ER-α and ER-β of other species, respectively. In a phylogenetic analysis, the common Japanese conger ER was found to cluster with the ER-β of other species. The common Japanese conger ER-β also has six domains (A–F), which are conserved in the nuclear receptor superfamily. The amino-acid sequences in the C and E domains were highly conserved between common Japanese conger ER-β and other ER. However, the other domains showed low homology. In the female common Japanese conger, ER-β mRNA was highly expressed in the liver and pituitary gland. The expression levels of the ER-β gene increased from the oil droplet stage to the primary yolk globule stage and were maintained until the migratory nucleus stage.     

Preventive effects of amino acids and glutathione on the formaldehyde-induced denaturation of myofibrillar proteins

ABSTRACT:     Formaldehyde (FA)-induced denaturation of myofibrillar proteins and its prevention were investigated by means of measuring the solubility, adenosine triphosphatase (ATPase) activity, and thermal gel formability of myofibrils and surimi proteins in the presence and absence of free amino acids and glutathione, reduced form. The addition of FA decreased the solubility of myofibrils in 0.5 M NaCl at pH 7.0 and 0°C depending on its concentration and incubation time. The solubility decrease was completely inhibited by the presence of equal, twofold, and threefold amounts of cysteine (Cys), glutathione, and histidine (His) to the amount of FA, respectively. Myofibrillar Ca-ATPase was markedly activated at the initial phase and then decreased later by the addition of FA. The K-ATPase was inactivated with an increase in the amount of FA. The FA-induced changes in both ATPase activities were inhibited in the presence of Cys and His. Thermal gel formability of surimi paste increased only in a short period after the addition of a low concentration of FA. Practically, FA inhibited the thermal gelation and setting effect through the inactivation of transglutaminase. In the presence of Cys, His or glutathione, a strong elastic surimi gel was produced because FA-induced detrimental effects were inhibited.     

Indole-3-carbaldehyde: a tyrosinase inhibitor from fungus YL185

Indole-3-carbaldehyde (1) was isolated as a tyrosinase inhibitor from the ethyl acetate-soluble fraction of extracellular fluids of unknown fungus YL185. The partial sequencing data of 18S ribosomal DNA (18S rDNA) indicate that this isolate belongs to the family Polyporaceae or Corticiaceae sensu lato. Indole-3-carbaldehyde inhibited the oxidation of l-3,4-dihydroxyphenylalanine (l-DOPA) by mushroom tyrosinase with a 50% inhibitory concentration (IC₅₀) of 1.3mM and showed inhibitory activity on melanin production in B16 melanoma cells. The aldehyde group of 1 plays an important role in eliciting tyrosinase inhibitory activity.     

Resistance to <Emphasis Type="Italic">Phytophthora infestans</Emphasis>: exploring genes required for disease resistance in Solanaceae plants

The oomycete Phytophthora infestans is the causal agent of potato late blight, one of the most destructive and historically significant pathogens in agricultural production. A virus-induced gene silencing-based screening of the solanaceous model plant N. benthamiana resulted in revealing a wide range of resistance mechanisms of solanaceous plants against this pathogen. In this article, we present an overview of the various pathways involved in the N. benthamiana–P. infestans pathosystem, including some of the follow-up work that was triggered by these findings. The purpose of this review is to assemble these findings and integrate them into our current understanding of plant pathogen defense mechanisms and discuss their potential application for the development of potato resistance to P. infestans.     

Random sequencing of genomic DNA of <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis>

ABSTRACT:   Random sequencing of the genomic DNA of Photobacterium damselae subsp. piscicida was conducted. The sequences were assembled into 930 contigs. The total length of these contigs accounts for 37.5% of the genome of P. damselae subsp. piscicida. The contigs contain 2055 open reading frames (ORFs) that have homology with genes in the GenBank database. Furthermore, some of the ORFs have homology with reported virulence related genes, and are classified as encoding colonization factors, exotoxin, and lipopolysaccharide (LPS). Thirty-seven ORFs have homology with colonization factors. In those colonization factors, 27, three, and four ORFs have homology with polar flagellar-related genes, capsule-related genes, and others (accessory colonization factors, superoxide dismutase (Cu-Zn), MshA biogenesis protein, and heme receptor genes), respectively. Five ORFs have homology with exotoxin-related genes (2 hemolysin, phospholipase, hyaluronidase, lysophospholipase L2 genes). Further, six ORFs have homology with LPS-related genes.     

Anti-Müllerian hormone (AMH) profiles as a novel biomarker to evaluate the existence of a functional cryptorchid testis in Japanese Black calves

Anti-Müllerian hormone (AMH) and testosterone (T) profiles in blood were investigated before and after an hCG stimulation test to assess their sensitivity and specificity for the existence of a functional cryptorchid testis in Japanese Black calves. The hCG (3,000 IU) was administered on Day 0, and peripheral blood was collected on Days 0 (just before hCG injection), 5 and 7 in intact male calves (Intact; n=19), bilateral castrated calves (Castrated; n=17), unilateral cryptorchid calves, which abdominal testis could been extracted (Uni-crypto; n=9). Castration of a descended testis was carried in the Castrated and Uni-Crypto groups on Day -14. The AMH detectability and the optimum cut-off point for T levels using the receiver operating characteristic curve were verified to characterize the cryptorchid testis. AMH values on Day 0 were 21.1 ± 5.1 and 29.0 ± 7.5 ng/ml in the Intact and Uni-crypto groups, respectively (Mean ± SEM). AMH levels were under the detection limit in the Castrated group (i.e., < 0.006 ng/ml). T showed its peak levels on Day 5 in the Intact group (26.8 ± 4.2 ng/ml), while it remained low in the Castrated group (< 0.9 ng/ml) and did not show a significant difference in the Uni-crypto group. The detectable levels for AMH was 0.006 ng/ml, and the optimum cut-off point for T was 0.9 ng/ml; the sensitivity and specificity for evaluation of testicular descent into the scrotum were 1.0 for both the AMH and T levels. The detection rates in the Uni-crypto group using them were 1.0 and 0.57 for AMH on Day 0 and T on Days 5 or 7, respectively. In conclusion, plasma AMH profiles could be used as a novel biomarker to evaluate the existence of a functional cryptorchid testis in Japanese Black calves.     

Effect of roll clearance of mechanical processing of corn silage harvested at the black‐line stage of maturity on carbohydrate and protein utilization in dairy cows

The experiment was conducted to evaluate the effect of roll clearance of mechanical processing of whole plant corn silage (CS) on carbohydrate and protein utilization in dairy cows. Treated CS was harvested at the black‐line stage of maturity and chopped at a theoretical length of cut (TLC) of 9.5 mm without processing or at a TLC of 19 mm with processing at a roll clearance of 1, 3 and 5 mm. Four ruminally and duodenally cannulated dry cows were assigned to a 4 × 4 Latin square design for 14‐day periods. Cows were fed diets containing 77% CS and 23% soybean meal (dry matter basis) to equalize the crude protein supply. Mechanical processing had no significant effect on dry matter intake and neutral detergent fiber digestibility. Ruminal and total tract starch digestibility and total digestible nutrients tended to be higher with processing at a roll clearance of 1 or 3 mm than at 5 mm. Microbial nitrogen efficiency in cows did not differ among all treatment groups. These results suggest that when CS is harvested at the black‐line stage of maturity, roll clearance should be 3 mm or less with a TLC of 19 mm.     

Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1

Neospora caninum is a veterinary medically important pathogen capable of causing abortion in cattle and neuromuscular paralysis in dogs. The surface antigen 1 of N. caninum (NcSAG1) is an important candidate for the development of a diagnostic reagent for neosporosis. In order to establish an effective diagnostic method, the gene encoding truncated NcSAG1 (NcSAG1t) lacking a signal peptide and C-terminal hydrophobic regions was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The purified GST-NcSAG1t was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of N. caninum antibodies in cattle. The ELISA with GST-NcSAG1t clearly differentiated between immunofluorescent antibody test (IFAT)-positive and -negative sera from cattle. In addition, the ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Toxoplasma gondii. Field serum samples collected from cattle in Brazil were examined for the diagnosis of neosporosis by using the ELISA. Of the 197 samples analyzed, 66 (33.5%) samples were positive for antibodies to N. caninum. Of the 66 ELISA-positive samples, 60 (90%) samples were confirmed as positive by Western blot analysis with whole parasite antigens. These results suggest that the recombinant NcSAG1t could be a reliable reagent for use as an antigen in ELISA for the serodiagnosis of N. caninum infection in cattle.     

Genetic detection of Babesia bigemina from Mongolian cattle using apical membrane antigen-1 gene-based PCR assay

We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established SpeI-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90 (33.8%) and 25 (9.4%) of the blood samples showed positive reactions for B. bigemina by the SpeI-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the SpeI-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina.     

Plasmid-mediated florfenicol resistance in Mannheimia haemolytica isolated from cattle

The aim of this study was to analyse a florfenicol-resistant Mannheimia haemolytica isolated from a calf to determine the genetic basis of its florfenicol-resistance. The antimicrobial susceptibility and plasmid content of the isolate were determined. A florfenicol resistant plasmid carrying the floR gene was identified by PCR and transformed into Escherichia coli JM109 and HB101 strains. The plasmid was then mapped and sequenced completely. The isolate was resistant to chloramphenicol, florfenicol, oxytetracycline, kanamycin, dihydrostreptomycin, nalidixic acid, ampicillin, and amoxicillin; it carried a floR plasmid of 7.7kb, designated pMH1405. The mobilisation and replication genes of pMH1405 showed extensive similarity to the 5.1-kb pDN1 plasmid from Dichelobacter nodosus and the 10.8-kb pCCK381 plasmid from Pasteurella multocida. An adjacent 2.4-kb segment was highly homologous to the TnfloR region of the E. coli BN10660 plasmid. A plasmid-mediated floR gene was responsible for florfenicol resistance in the bovine respiratory tract pathogen M. haemolytica. The pMH1405 plasmid is the smallest floR-carrying plasmid reported to date. To the best of our knowledge, this is the first report of a florfenicol-resistant gene in M. haemolytica.