Characterization of anti‐Müllerian hormone in a case of bovine male pseudohermaphroditism

The current report aimed to characterize plasma anti‐Müllerian hormone (AMH) in bovine male pseudohermaphroditism. The blood AMH concentration in a Japanese Black male pseudohermaphrodite calf was compared with pre‐ and post‐pubertal male and female calves and castrated calves. The concentration in the case was higher than in post‐pubertal males, castrated males, and pre‐ and post‐pubertal female calves (p < .05), but similar to that in pre‐pubertal male calves. After extraction of the testes, the concentration in the case dropped to a certain extent. The extracted testes expressed AMH, as detected by immunohistochemistry. This study is the first to show the characterization of AMH in a male pseudohermaphrodite calf. AMH levels in peripheral blood might be useful to diagnose male pseudohermaphroditism in cattle.     

Changes in plasma concentrations of S100A7 and S100A8 in dairy cows during pregnancy

This study was carried out to examine the changes in plasma concentrations of the Ca‐binding antimicrobial proteins S100A7 and S100A8 during pregnancy in dairy cows. Holstein Friesian cows (n = 19) were inseminated with Holstein Friesian semen. Blood was collected at days 30, 60, 90, 120, 150, 180, 210, 240 and 270 after insemination. Plasma was used for measuring the concentrations of S100A7 and S100A8. Both S100A7 and S100A8 concentrations showed similar patterns during gestation; they increased during the midgestation, between days 90 and 180, and then declined before calving. The findings indicated that plasma concentrations of S100A7 and S100A8 did not change significantly during pregnancy in cows. Further studies are required to determine the roles of S100A7 and S100A8 in physiological function during pregnancy in dairy cows.     

Chemical studies on antioxidant mechanism of curcumin: analysis of oxidative coupling products from curcumin and linoleate

As a part of a research project on the antioxidant mechanism of natural phenolics in food components, curcumin, a turmeric antioxidant, was investigated in the presence of ethyl linoleate as one of the polyunsaturated lipids. During the antioxidation process, curcumin reacted with four types of linoleate peroxyl radicals. Six reaction products were observed in the reaction and subsequently isolated. Their structures were determined by physical techniques, revealing that they have novel tricyclic structures, including a peroxyl linkage. On the basis of the formation pathway for their chemical structures, an antioxidant mechanism of curcumin in polyunsaturated lipids was proposed, which consisted of an oxidative coupling reaction at the 3'-position of the curcumin with the lipid and a subsequent intramolecular Diels--Alder reaction.     

A Study on the Presence of Ferritin-binding Proteins in Fetal Horse Plasma

In mammal circulation, ferritin-binding proteins (FBPs) are thought to be involved in clearance of circulating ferritin after complex formation with it through receptor-mediated uptake. However, there is no report on fetal FBP in fetal circulation. Although iron concentrations of fetal horse plasma were higher than those of adult horse plasma, plasma ferritin concentrations and ferritin-binding activities were found to be significantly lower in fetus than in adult. FBPs were purified from fetal or adult horse plasma on horse spleen ferritin-Sepharose 4B affinity column. Partially affinity-purified fetal horse plasma FBPs were mainly separated into 65 and 41 kDa bands in addition to minor bands with higher molecular masses ranged from 102 to 140 kDa on SDS-PAGE under reducing condition. The adult horse plasma FBPs were separated into 74, 54 and 28 kDa bands, and the 74 and 54 kDa bands reacted with antibodies specific for horse IgM and IgG heavy chains, respectively, by immunoblotting analyses. On the other hand, no antibodies to horse immunoglobulin classes detected any bands in fetal horse plasma FBPs. The affinity-purified adult and fetal horse plasma FBPs did not contain fibrinogen as a plasma specific FBP, probably due to its lower affinity to the ligand ferritin. These results demonstrate the presence of FBPs which are different from adult horse plasma FBPs including anti-ferritin autoantibodies in fetal plasma.     

Isolation and characterization of Campylobacter,Helicobacter, and Anaerobiospirillum strains from a puppy with bloody diarrhea

We carried out a microscopic examination of stools from a 2-month-old female puppy with bloody diarrhea, and this revealed large numbers of different spiral-shaped bacteria. To isolate these organisms, a rectal swab specimen was inoculated onto plates of Skirrow's agar and incubated at 37 degrees C for 6 days in a microaerobic atmosphere. Finally, a total of six different spiral-shaped bacteria (strains G1104, 94105, FR106, B0101, 3J102, and J2103) were isolated. Based on their morphology, biochemical traits, whole-cell protein profiles, and analysis of their 16S rDNA sequences, they were identified as Campylobacter upsaliensis, Helicobacter cinaedi, 'Flexispira rappini', two Anaerobiospirillum spp. with different morphologies, and Helicobacter sp., respectively. Analysis of 16S rRNA gene sequence data for strains 94150 (H. cinaedi) and FR106 (F. rappini) revealed that this approach has limitations when identifying isolates to the species level because of a high degree of sequence homology between these species (>99%) and considerable sequence variation among different isolates within these species. The dog was treated orally with amoxicillin for 3 days, which resolved the diarrhea. However, 1 day after the last dose the bloody diarrhea recurred but regarded to six more days amoxicillin treatment. This suggests a bacterial cause for the diarrhea. The approach to identification to microaerobic spiral-shaped bacteria in diarrheic dogs can be applied further to characterize their role in diarrhea illness.     

Carotenoid Nostoxanthin Production by Sphingomonas sp. SG73 Isolated from Deep Sea Sediment

Carotenoids are used commercially for dietary supplements, cosmetics, and pharmaceuticals because of their antioxidant activity. In this study, colored microorganisms were isolated from deep sea sediment that had been collected from Suruga Bay, Shizuoka, Japan. One strain was found to be a pure yellow carotenoid producer, and the strain was identified as Sphingomonas sp. (Proteobacteria) by 16S rRNA gene sequence analysis; members of this genus are commonly isolated from air, the human body, and marine environments. The carotenoid was identified as nostoxanthin ((2,3,2′,3′)-β,β-carotene-2,3,2′,3′-tetrol) by mass spectrometry (MS), MS/MS, and ultraviolet-visible absorption spectroscopy (UV-Vis). Nostoxanthin is a poly-hydroxy yellow carotenoid isolated from some photosynthetic bacteria, including some species of Cyanobacteria. The strain Sphingomonas sp. SG73 produced highly pure nostoxanthin of approximately 97% (area%) of the total carotenoid production, and the strain was halophilic and tolerant to 1.5-fold higher salt concentration as compared with seawater. When grown in 1.8% artificial sea salt, nostoxanthin production increased by 2.5-fold as compared with production without artificial sea salt. These results indicate that Sphingomonas sp. SG73 is an efficient producer of nostoxanthin, and the strain is ideal for carotenoid production using marine water because of its compatibility with sea salt.     

Measurement of ferritin and anti‐ferritin autoantibodies in serum and colostrum of Holstein and Japanese Black cows

Anti‐ferritin autoantibody is a ferritin‐binding protein commonly found in mammals; it is thought to form an immune complex with ferritin and thereby mediate the rapid clearance of circulating ferritin. The aim of this study is to determine concentrations of ferritin and anti‐ferritin autoantibodies (immunoglobulin (Ig)M, IgG and IgA) in serum and colostrum of Holstein (H) and Japanese Black (JB) cows within 24 h of normal calving. Blood and colostrum samples were collected from cows of various ages (2–11 years) and calving number (1–8 live births). Mean ferritin concentrations were higher in colostrum than in serum for both breeds, and higher colostrum ferritin concentrations were found in H than JB cows. IgA antibodies in serum and colostrum from both breeds had negligible ferritin‐binding activity. For both breeds, IgM and IgG antibodies had higher ferritin‐binding activity in colostrum than in serum. There was a significant correlation between IgM and IgG ferritin‐binding activities in serum and colostrum of H and JB cows. These results suggest that ferritin and IgM and IgG autoantibodies are actively transferred from the blood stream to the colostrum at prepartum or early lactation.     

A Method of Solubilizing and Concentrating Astaxanthin and Other Carotenoids

The valuable marine carotenoid, astaxanthin, is used in supplements, medicines and cosmetics. In this study, crustacyanin, an astaxanthin-binding protein, was used to solubilize and concentrate astaxanthin. The recombinant crustacyanin of European lobster spontaneously formed an inclusion body when it was over-expressed in Escherichia coli. In this study, fusing the NusA-tag to the crustacyanin subunits made it possible to express in a soluble fraction and solubilize astaxanthin in aqueous solution. By cutting off the NusA-tag, the crustacyanin subunits generated the pure insoluble form, and captured and concentrated astaxanthin. Overall, the attaching and releasing NusA-tag method has the potential to supply solubilized carotenoids in aqueous solution and concentrated carotenoids, respectively.     

Seminal plasma lactoferrin but not transferrin reflects gonadal function in dogs

Lactoferrin purified from canine seminal plasma by a three-step chromatography procedure had a molecular mass of 75.2 kDa and cross-reacted with antiserum to equine seminal plasma lactoferrin. Seminal plasma lactoferrin concentrations were determined by a competitive enzyme-linked immunosorbent assay (ELISA) by using rabbit anti-equine lactoferrin antibody and alkaline phosphatase-labeled goat anti-rabbit IgG antibody in 14 normal dogs and found to range from 12 to 197 micro g/ml, with a mean value of 77 +/- 59 micro g/ml (the mean +/- SD). Seminal plasma transferrin concentrations were determined by a sandwich ELISA with goat antibody to canine serum transferrin and alkaline phosphatase-conjugated goat anti-canine transferrin antibody and found to range from 0.32 to 12.6 micro g/m l, with a mean value of 2.44 +/- 3.25 micro g/m l. The lactoferrin concentration significantly correlated with the sperm concentration (r=0.7025, P<0.01), but there was no significant correlation between the seminal plasma transferrin concentration and sperm density. These results indicate that seminal plasma lactoferrin, but not transferrin, reflects gonadal function.