The mRNA regulation of porcine double-stranded RNA-activated protein kinase gene

The double-stranded RNA (dsRNA)-activated protein kinase (PKR), which is one of the products of interferon (IFN)-stimulated genes, participates in the biological actions of IFN such as antiviral effects and immune response. In the present study, we identified the primary structure of porcine PKR proteins by cDNA cloning. Porcine PKR protein consisted of 537 amino acids and had two dsRNA-binding domains similarly existing in PKR proteins of other species. The treatment with IFN-alpha induced the expression of PKR 3.9-fold in a porcine kidney cell line, LLC-PK1. The same results were obtained when the cells were treated with poly(I).poly(C), but treatment with either IFN-gamma or LPS did not induce this gene in LLC-PK1 cells. These results suggest similarity of the regulatory mechanisms in the PKR gene among mammalian species.     

Nitrate content and induction of NADH nitrate reductase in cultured rice cells

The nitrate content of cultured rice cells grown in liquid R-2 medium containing 40 mM nitrate increased rapidly and reached a maximum 6 hr after inoculation. After about 6-hr lag period, NADH nitrate reductase activity increased rapidly to the 24th hr. Promotive effects on the nitrate accumulation and the induction of NADH nitrate reductase were found by the addition of 5 mM ammonium into the medium. Nitrate content increased exponentially as a function of increasing external nitrate concentration between 0 and 80 mM. The NADH nitrate reductase activity also increased from 0 to 40 mM of nitrate media, but leveled off between 40 and 80 mM.

A high level of nitrate content was detected in the cells grown in 10 or 40 mM of nitrate medium without sucrose for 48 hr, suggesting that sucrose in the medium may have no effect on the nitrate accumulation in the early growth period.     

Mechanism-based CYP2D6 inactivation by acridone alkaloids of Indonesian medicinal plant Lunasia amara

Fourteen acridone alkaloids isolated from Lunasia amara Blanco were tested for their mechanism-based inhibition on human liver microsomal dextromethorphan O-demethylation activity, a prototype marker for cytochrome P450 2D6 (CYP2D6). Among the 14 compounds, 5-hydroxygraveroline (1), 8-methoxyifflaiamine (2), lunamarine (3), and lunine (12) increased their inhibitory activity with increasing preincubation time. Then, we further examined the possibility of mechanism-based inhibition on 5-hydroxygraveroline (1) and lunamarine (3), which showed the potent inhibition. Further investigations on 1 and 3 showed that the characteristic time- and concentration-dependent inhibition, which required a catalytic step with NADPH, was not protected by nucleophiles, and was decreased by the presence of a competitive inhibitor. Thus, 1 and 3 were concluded as mechanism-based inactivators of CYP2D6.     

Establishment of a patient-derived xenograft mouse model of pleomorphic leiomyosarcoma

Soft tissue sarcomas are difficult to treat using chemotherapy owing to a current deficiency in candidate drugs for specific targets. Screening candidate compounds and analyzing therapeutic targets in sarcomas is insufficient, given the lack of an appropriate human sarcoma animal model to accurately evaluate their efficacy, as well as the lack of an adequate technical protocol for efficient transplantation and engraftment of sarcoma specimens in patient-derived xenograft (PDX) models. Accordingly, in this study, we sought to identify the optimal type of sarcoma and develop a protocol for generating a PDX model. We characterized a PDX mouse model using histopathological and immunohistochemical analyses to determine whether it would show pathological characteristics similar to those of human sarcomas. We achieved engraftment of one of the 10 transplanted sarcoma specimens, the xenografted tumor of which exhibited massive proliferation. Histologically, the engrafted sarcoma foci resembled a primary tumor of pleomorphic leiomyosarcoma and maintained their histological structure in all passages. Moreover, immunohistochemical analysis revealed the expression of specific markers of differentiation to smooth muscle, which is consistent with the features of leiomyosarcoma. We thus demonstrated that our pleomorphic leiomyosarcoma PDX mouse model mimics at least one aspect of human sarcomas, and we believe that this model will facilitate the development of novel therapies for sarcomas.     

High-resolution linkage mapping of the rat hooded locus

To identify a gene responsible for the hooded phenotype in the rat, high-resolution linkage mapping for the hooded locus was performed using IS (non-hooded) and LEA (hooded) rats. The map revealed that only Kit gene existed in the critical region, suggesting that the Kit is a strong candidate gene. However, mutation was not found in the coding region of the LEA rat Kit gene. Further, the expressions of Kit mRNA were not different in fetal neural tubes and both neonatal and adult skins between IS and LEA rats. Furthermore, Kit-positive cells, possibly melanocytes, were found in the non-pigmented hair follicles of hooded phenotype rats. Several hypotheses are conceivable to account for mechanisms in the appearance of hooded phenotype.     

Retrospective study of canine nasal tumor treated with hypofractionated radiotherapy

The object of this study was to evaluate hypofractionated multiportal field and two-portion (rostral and caudal portions divided by the eyelid) radiation therapy for canine nasal tumors. Sixty-three dogs underwent multiportal hypofractionated radiation therapy. The radiation field was divided into rostral and caudal portions by the eyelid. Treatments were performed four times for 57 dogs. The median irradiation dose/fraction was 8 Gy (range, 5-10 Gy); the median total dose was 32 Gy (10-40 Gy). Improvement of clinical symptoms was achieved in 53 (84.1%) of 63 cases. Median survival time was 197 days (range, 2-1,080 days). Median survival times with and without destruction of the cribriform plate before radiotherapy were 163 and 219 days, respectively. There was no significant difference between them. No other factors were related to survival according to a univariate analysis. All radiation side effects, except one, were grade I according to the VRTOG classification. It was not necessary to treat any dogs for skin side effects. One dog (1.6%) developed an oronasal fistula 1 year after completion of radiation therapy. This radiation protocol may be useful in reducing radiation side effects in dogs with cribriform plate destruction.     

Pretreatment of macrophages with the combination of IFN-gamma and IL-12 induces resistance to Leishmania major at the early phase of infection

Interferon (IFN)-gamma is essential but not sufficient to control leishmaniasis. It is known that IFN-gamma is one of the major macrophage-activating cytokines, and the activated macrophages are a principal source of interleukin (IL)-12, which induces autocrine macrophage activation. In this study, the combined effect of IFN-gamma and IL-12 on the susceptibility of macrophages to Leishmania major infection was evaluated. Macrophages pretreated with IFN-gamma and/or IL-12 were infected with the parasites. Four hr post-infection (p.i.), the levels of infection and parasite load in the macrophages treated with the combination of IFN-gamma and IL-12 (IFN-gamma/IL-12) were significantly lower than those in the nontreated cells. However, the macrophages treated with either IFN-gamma or IL-12 did not show resistance to L. major infection. In addition, 72 hr p.i., the IFN-gamma/IL-12-treated and IFN-gamma-treated macrophages showed significantly lower levels of infection and parasite load than the nontreated cells, and higher levels of resistance was observed in the IFN-gamma/IL-12-treated macrophages than in the IFN-gamma-treated macrophages. Although IFN-gamma/IL-12 treatment of macrophages prior to the infection led to the induction of resistance, as described above, this resistance was not induced when these cytokines and the parasites were added simultaneously to the macrophage culture. These results suggest that IFN-gamma/IL-12 treatment prior to the infection restricts the early phase of the infection.     

B cell differentiation in the bursa of Fabricius and spleen of embryos and chicks immediately after hatching

Flow cytometric analysis and immunohistochemical observation were used to qualitatively and quantitatively clarify the nature of B cell differentiation in the bursa of Fabricius of chick embryos and to determine the timing of antibody class switching in chicken spleens based on positivity of IgM and IgG on and in the cells. In the bursa, the sIgM‐positive cell population formed from the 12^th to 15^th day of embryogenesis. The proportion of sIgM‐high expressing (sIgM^high) cells was lower among bursacytes than splenocytes of hatched chicks, suggesting that the sIgM^high bursacytes are to be released to peripheral sites. The proportion of sIgM^high cells was higher at 0 days old than at any other examined stage of development. Colonization of the spleen by B cells occurred between the 18^th day of embryogenesis and 0 days old. Antibody class switching was thought to start in the spleen between 1 and 2 weeks of age, because IgG‐positive cells were present in the spleen of 2‐week‐old chicks, but not 0‐day‐old or 1‐week‐old chicks.     

Bilateral cataract in a cynomolgus monkey

Severe bilateral cataract was found in a 7 year-old naïve female cynomolgus monkey (Macaca fascicularis) 3 months before necropsy. During macroscopic examination, severe opacity and thinning of the lens were observed in both eyes. Histopathology revealed that the lens nuclei and majority of cortex lens fibers had disappeared and become excavated, while the lens fibers in the subcapsular area were swollen and distorted. Other observations included atrophy and vacuolation in the lens epithelial cells and proliferation of spindle cells and collagen fiber beneath the anterior capsule of the right eye. Immunohistochemical staining of these spindle cells revealed the presence of vimentin, cytokeratin and α-smooth muscle actin (α-SMA), which were considered to be derived from lens epithelial cells. This is a rare case of spontaneous, bilateral, hypermature cataract in a cynomolgus monkey.     

Unidentified compound specifically accumulated in soybean nodules formed with H2-uptake negative rhizobium japonicum strains

A large quantity of an unidentified compound was detected by a fluorogenic method in the soybean nodules formed with H₂-uptake negative Rhizobium japonicum strains. The unidentified compound (compound X) reacted with o-phthalaldehyde in the presence of 2-mercaptoethanol giving rise to a fluorescent compound, and was not hydrolyzed in strong acid and strong base. Therefore, the compound X was considered to be one of amines and amino acids. The content of the compound X in the nodules formed with H₂-uptake positive strains was remarkably lower than that in the nodules formed with H₂-uptake negative strains. It was suggested that the compound X may be involved in the process of hydrogen metabolism in soybean nodules. The compound X was not detected in leaves, stems, petioles, and roots of soybean plants, but detected in H₂-uptake negative strains of free-living R. japonicum. In nodules, a great portion of the compound X was distributed into cytosol fraction.