A Point Mutation in the Two-Component Histidine Kinase BcOS-1 Gene Confers Dicarboximide Resistance in Field Isolates of Botrytis cinerea

ABSTRACT Partial DNA fragments of Botrytis cinerea field isolates encoding the putative osmosensor histidine kinase gene (BcOS1) were cloned by polymerase chain reaction amplification and the predicted amino acid sequences were compared between dicarboximide-sensitive and resistant field isolates. The predicted BcOS1p is highly homologous to osmosensor histidine kinase OS1p from Neurospora crassa including the N-terminal six tandem repeats of approximately 90 amino acids. Four dicarboximide-resistant isolates of B. cinerea (Bc-19, Bc-45, Bc-682, and Bc-RKR) contained a single base pair mutation in their BcOS1 gene that resulted in an amino acid substitution in the predicted protein. In these resistant isolates, codon 86 of the second repeat, which encodes an isoleucine residue in sensitive strains, was converted to a codon for serine. The mutation of Botrytis field resistant isolates was located on the second unit of tandem amino acid repeats of BcOS1p, whereas the point mutations of the fifth repeat of OS1p confer resistance to both dicarboximides and phenylpyrroles and also osmotic sensitivity in Neurospora crassa. These results suggest that an amino acid substitution within the second repeat of BcOS1p is responsible for phenotypes of field resistant isolates (resistant to dicarboximides but sensitive to phenylpyrroles, and normal osmotic sensitivity) in B. cinerea.     

Primary malignant peripheral nerve sheath tumor with eosinophilic cytoplasmic globules arising from the greater omentum in a dog

A 10-year-old Golden Retriever dog had a solitary tumor mass arising from the greater omentum. Histologically, the tumor showed varying cellularity and patterns of cellular arrangement. In dense cellular areas, spindle-shaped cells were arranged in interlacing bundles. The sparse cellular area was characterized by loosely arranged fusiform cells. The neoplastic cells frequently contained PAS-positive eosinophilic globules in the cytoplasm, and mitotic figures were frequently observed. The tumor cells were positive to vimentin, S-100 protein, glial fibrillary acidic protein, myelin basic protein, neuron-specific enolase and myoglobin. The present tumor was diagnosed as a malignant peripheral nerve sheath tumor (MPNST) with eosinophilic cytoplasmic globules arising from the greater omentum. To our knowledge, this may be the first case of primary omental MPNST in domestic animals.     

Trans-endothelial and trans-epithelial transfer of lactoferrin into the brain through BBB and BCSFB in adult rats

The transportation of intravenously administered bovine lactoferrin (bLF) into the cerebrospinal fluid (CSF) was immunohistochemically investigated in adult rats. Administered bLF was detected in the vesicular membranes of endothelial cells in cerebral blood vessels 10 min after the infusion. Numerous immunoreactive small vesicles were also detected at the ependymal cells in the choroid plexus. Moreover, the bLF concentration in the CSF was significantly increased at 1-2 hr after the intravenous infusion of bLF (10 or 30 mg/kg). These findings clearly demonstrate that LF is possibly transported into the brain matter even in adult animals.     

Changes in the population of lymphocytes expressing CD4 and CD8 during the process of atresia of white follicles in hens

The aim of this study was to determine whether the population of lymphocytes expressing CD4 and CD8 molecules changed in the white follicles during atresia in chickens. Frozen sections of healthy, early atretic, advanced atretic and late atretic follicles were immunostained for CD4 and CD8, and the populations of positive cells were analyzed under a light microscope. In the healthy, early atretic and advanced atretic follicles both CD4+ and CD8+ cells were localized in the theca layer, but not in the granulosa layer. However, an influx of CD4+ and CD8+ cells was observed not only in the theca but also in the follicular cavity that was formed by disintegration of the oocyte in late atretic follicles. The frequency of CD4+ T cells in the theca layer did not differ among healthy, early atretic and advanced atretic follicles, but was significantly increased in the late atretic follicles (P < 0.05). The frequency of CD8+ cells showed a pattern of change that resembled that of CD4+ T cells, with a significantly greater population in late atretic follicles than the other follicles (P < 0.05). These results suggest that CD4+ and CD8+ cells are increased in the late atretic follicles, probably to promote the tissue regression.     

A case of spontaneous Zymbal’s gland carcinoma with lung metastasis in an aged Fischer 344 rat

Zymbal’s gland neoplasms are induced in rats through the administration of various carcinogens, but spontaneous neoplasia is rare. This report describes a spontaneous Zymbal’s gland carcinoma with lung metastasis found in an aged male Fischer 344 rat. Macroscopically, the dome-like tumor nodule, approximately 30 mm in diameter with ulceration, was located near the ear canal of the rat. No healthy tissue or structure of Zymbal’s gland was identified on the corresponding side, while the normal salivary glands and a lacrimal gland were observed. Histologically, a large part of the tumor mass was occupied by poorly differentiated neoplastic cells, the shapes of which were oval to polygonal or fusiform. Additionally, clusters of sebaceous-like foamy cells and squamous metaplasia with prominent keratinization were observed. Tumor cells were found to metastasize to the lung; these cells displayed histological similarities, including a sebaceous gland-like pattern, to those in the primary site. The tumor cells were immunohistochemically positive for cytokeratin AE1/AE3 or vimentin but negative for CD68, S100, α-smooth muscle actin, von Willebrand factor, and desmin. Our results indicate that the tumor was a poorly differentiated Zymbal’s gland carcinoma with lung metastasis.     

Analysis of glycosuria in okapi (Okapia johnstoni): Examination of urinary N‐acetyl‐β‐D‐glucosaminidase

We analyzed the urinary excretion of glucose and N‐acetyl‐β‐D‐glucosaminidase (NAG) in six okapis (Okapia johnstoni) in captivity to investigate the cause of their urinary sugar excretion. The urinary glucose‐positive okapi had significantly higher urinary NAG indices than the urinary glucose‐negative okapi. There was also a positive correlation between urinary glucose levels and urinary NAG indices. These results suggest that the proximal tubular function of the glycosuric okapi may have been obstructed, which impaired glucose reabsorption.     

Plant regeneration from protoplasts isolated from primary calli using mature embryos of two Brazilian rice cultivars

A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Abnormal functions of Leydig cells in crossbred cattle–yak showing infertility

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle–yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β-hydroxysteroid dehydrogenase (3βHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle–yak spermatogenesis. Testicular tissues from yaks (1–3 years old) and hybrids (F1–F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle–yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle–yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.     

The effect of cortisol administration on blood plasma immunoglobulin M (IgM) concentrations in masu salmon (Oncorhynchus masou)

Immunoglobulin M (IgM) is known as a main factor in the humoral immune system of teleosts. In the present study, the effect of cortisol on plasma IgM concentrations was investigated using a specific antibody to IgM in masu salmon (Oncorhynchus masou). Cortisol was orally administered each day for 2 weeks at a dose of 1 mg g⁻¹ in the diet, and for the following week the fish were fed a non-treated diet. Blood plasma samples were collected at 0, 1, 2 and 3 weeks after the initiation of treatment. Oral administration of cortisol elevated plasma cortisol concentrations to about 40 ng/ml for 2 weeks after administration and slightly reduced plasma IgM concentration; the suppression was statistically significant one week after the period of hormone administration. However, treatment with cortisol did not affect plasma concentrations of total protein or α1-protein, one of the major serum proteins, during the experimental period. These results indicate that cortisol specifically suppresses plasma IgM concentrations.