The W‐ and Z‐linked EE0.6 sequences used for molecular sexing of captive Japanese crested ibis on Sado Island

The Japanese crested ibis Nipponia nippon is a critically threatened bird. Accurate sexing is necessary to perform effective management of captive breeding toward a national project for a tentative release of the Japanese crested ibis on Sado Island. A PCR‐based sexing method targeting a 0.6 kb EcoRI fragment (EE0.6) sequence on W chromosome with AWS03 and USP3 primers has been developed for the Japanese crested ibis. However, the primers were selected from the EE0.6 sequences from bird species other than the Japanese crested ibis. In this study, we determined the W‐ and Z‐linked EE0.6 sequences in the Japanese crested ibis, and clarified Japanese crested ibis sequence mismatch in the binding sites of the primers. Further, we found no polymorphism in the primer binding sites among five founder birds for the Sado captive Japanese crested ibis population. These findings validated the PCR‐based sexing method with the AWS03 and USP3 as accurate molecular sexing methods of captive Japanese crested ibis on the Sado Island. Additionally, we designed a primer set for a novel PCR‐based sexing, based on the EE0.6 sequences obtained in this study. This novel sexing method may be useful for future ecological research following the release of Japanese crested ibis on Sado Island. This is the first report to show the EE0.6 sequences in Japanese crested ibis.     

Exophthalmos Caused by Chronic Sinusitis in a Horse: A Case of Exophthalmos Caused by Sinusitis

A 12-year-old Thoroughbred gelding exhibited a unilateral exophthalmos on the left side. There had been no other clinical symptoms, such as rhinorrhea, before the first consultation. Clinical, hematologic, plasma biochemical, cytologic, and computed tomography (CT) examinations and complete necropsy were performed. These examinations did not reveal neoplastic lesions. CT images suggested that maxillary and frontal sinuses may have been filled with fluid. Cytologic and pathologic examinations revealed that the maxillary and orbital bones were pushed up with a large amount of the inflammatory fluid effused by chronic sinusitis. The CT and cytologic examinations were of great use for differential diagnosis in this case. This was an unusual case of exophthalmos induced by deformation of the orbital bones resulting from chronic sinusitis.     

Characterization of canine filaggrin: gene structure and protein expression in dog skin

Background – Filaggrin (FLG) is a key protein for skin barrier formation and hydration of the stratum corneum. In humans, a strong association between FLG gene mutations and atopic dermatitis has been reported. Although similar pathogenesis and clinical manifestation have been argued in canine atopic dermatitis, our understanding of canine FLG is limited. Hypothesis/Objectives – The aim of this study was to determine the structure of the canine FLG gene and to raise anti‐dog FLG antibodies, which will be useful to detect FLG protein in dog skin. Methods – The structure of the canine FLG gene was determined by analysing the publicly available canine genome DNA sequence. Polyclonal anti‐dog FLG antibodies were raised based on the canine FLG sequence analysis and used for defining the FLG expression pattern in dog skin by western blotting and immunohistochemistry. Results – Genomic DNA sequence analysis revealed that canine FLG contained four units of repeated sequences corresponding to FLG monomer protein. Western blots probed with anti‐dog FLG monomer detected two bands at 59 and 54 kDa, which were estimated sizes. The results of immunohistochemistry showed that canine FLG was expressed in the stratum granulosum of the epidermis as a granular staining pattern in the cytoplasmic region. Conclusions and clinical importance – This study revealed the unique gene structure of canine FLG that results in production of FLG monomers larger than those of humans or mice. The anti‐dog FLG antibodies raised in this study identified FLG in dog skin. These antibodies will enable us to screen FLG‐deficient dogs with canine atopic dermatitis or ichthyosis.     

Fecal progestagens to detect and monitor pregnancy in captive female cheetahs (Acinonyx jubatus)

The purposes of the present study were to establish a noninvasive monitoring assay of fecal progestagen measurement to detect pregnancy and to identify the components of fecal progestagens in early, middle and late pregnancy in cheetahs. Feces were collected from 7 female cheetahs and analyzed from 30 days before the last copulation to parturition in 9 pregnancies. Blood was collected from one cheetah. Fecal progestagen and serum progesterone concentrations were determined by enzyme immunoassay (EIA). The profiles of the fecal progestagen concentrations were similar to the serum progesterone profile. Fecal progestagen and serum progesterone concentrations remained at the baseline until copulation. In the mean fecal progestagen profile during pregnancy (92.8 ± 0.4 days; from the last copulation to parturition), the concentrations increased 3-4 days after the last copulation and remained high until parturition. To investigate changes in the components of progestagen metabolites in the tripartite periods of gestation, fecal progestagens were analyzed by HPLC-EIA. Marked immunoreactive peaks consistent with 5α-pregnan-3α/β-ol-20-one and 5α-pregnan-3,20-dione and small peaks consistent with 5β-pregnan-3α/β-ol-20-one were detected. There were no distinct difference in the components of progestagens among the first, second and third trimesters of pregnancy. The hormone assay, as an indicator of fecal 5α-reduced pregnanes, is useful for detecting pregnancy and monitoring pregnant luteal activity in cheetahs.     

Catalog of Micro-Tom tomato responses to common fungal, bacterial, and viral pathogens

Lycopersicon esculentum cultivar Micro-Tom is a miniature tomato with many advantages for studies of the molecular biology and physiology of plants. To evaluate the suitability of Micro-Tom as a host plant for the study of pathogenesis, Micro-Tom plants were inoculated with 16 well-known fungal, bacterial, and viral pathogens of tomato. Athelia rolfsii, Botryotinia fuckeliana, Oidium sp., Phytophthora infestans, and Sclerotinia sclerotiorum caused typical symptoms and sporulated abundantly on Micro-Tom. Micro-Tom was resistant to Alternaria alternata, Corynespora cassiicola, and Fusarium oxysporum. When Micro-Tom was inoculated with 17 isolates of Ralstonia solanacearum, many isolates induced wilt symptoms. Agrobacterium tumefaciens also was pathogenic, causing crown galls on stem tissue after needle prick inoculation. In Micro-Tom sprayed with Pseudomonas syringae pv. tomato, P. s. pv. tabaci, or P. s. pv. glycinea, bacterial populations did not increase, and yellow lesions appeared only on leaves sprayed with P. s. pv. tomato. Tomato mosaic virus, Tomato aspermy virus, and Cucumber mosaic virus systemically infected Micro-Tom, which developed symptoms characteristic of other cultivars of tomato after infection with the respective virus. These results indicated that Micro-Tom was generally susceptible to most of the important tomato pathogens and developed typical symptoms, whereas certain pathogens were restricted by either hypersensitive resistance or nonhost resistance on Micro-Tom. Therefore, an assortment of Micro-Tom–pathogen systems should provide excellent models for studying the mechanism of susceptible and resistant interactions between plants and pathogens.     

Effects of Swertia japonica extract and its main compound swertiamarin on gastric emptying and gastrointestinal motility in mice

The Swertia japonica is used clinically as a remedy for gastrointestinal symptoms in Japan. We examined the effects of a S. japonica and swertiamarin on gastric emptying and gastrointestinal motility in atropine-, dopamine-, and 5-hydroxytryptamine (5-HT)-treated mice. All three preparations inhibited reductions in gastric emptying and gastrointestinal motility induced by dopamine (1 mg/kg, intraperitoneal injection, ip). Neither the powder, swertiamarin, nor itopride had any effect on the reductions in gastric emptying and gastrointestinal motility caused by 5-HT (4 mg/kg, ip). These findings suggest that the powder and swertiamarin stimulate gastric emptying and gastrointestinal motility by inhibiting the dopamine D₂ receptor.     

Isolation and structural elucidation of some procyanidins from apple by low-temperature nuclear magnetic resonance

Procyanidin fractions from apple were separated according to the degree of polymerization using normal phase chromatography. Evaluation of physiological functionalities of procyanidins requires individual structural determination. However, it is difficult to elucidate the structure of procyanidins, in particular those with (+)-epicatechin (1) or (-)-catechin (2) units, and determine whether the interflavanoid bonds are 4beta-->8 or 4beta-->6 without cleavage and acetylation. Structural determination used LC-MS and low-temperature NMR. Nine procyanidins were separated by preparative HPLC consisting of three well-known procyanidins [procyanidin B1 (3), procyanidin B2 (4), and procyanidin C1 (5)] and six new procyanidins [epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin (6); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-catechin (7); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-epicatechin (8); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-catechin (9); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-epicatechin (10); and epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin (11)]. Compounds 6-11 were detected for the first time as apple constituents.     

Inhibitory effects of apple polyphenols and related compounds on cariogenic factors of mutans streptococci

The inhibitory effects of apple polyphenols (APP) on the synthesis of water-insoluble glucans by glucosyltransferases (GTF) of streptococci of the mutans group and on the sucrose-dependent adherence of the bacterial cells were examined in vitro. APP markedly inhibited the activity of GTF purified from the cariogenic bacterial cells. However, APP showed no significant effect on the growth of the cariogenic bacteria. The strongest GTF inhibitors in APP were apple condensed tannins (ACT), a mixture of procyanidins. The 50% inhibitory doses of ACT against the GTF of S. sobrinus and that of S. mutans were 1.5 microgram/mL and 5 microgram/mL, respectively. The ACT efficacy largely depended upon the degree of polymerization. Interestingly, while the other polyphenols known to inhibit GTF such as tannic acid markedly inhibited salivary alpha-amylase activity, APP and ACT only scarcely inhibited that enzyme activity. This means that APP and ACT might selectively inhibit the bacterial GTF activity under oral conditions.