Variable Regions 1 and 2 (VR1 and VR2) in JSRV gag Are Not Responsible for the Endogenous JSRV Particle Release Defect

Jaagsiekte sheep retrovirus (JSRV) is a betaretrovirus causing ovine pulmonary adenocarcinoma, a transmissible lung tumor of sheep. A very closely related endogenous retrovirus (enJSRV) occurs as 15 to 20 copies in the genome of all sheep, and is not known to be linked to pathogenesis. We previously localized a particle release defect of the full-length endogenous-derived expression construct pCMV2enJS56A1 to the amino-terminal region of gag that incorporates the two variable regions VR1 and VR2, which harbor the main sequence differences between endogenous and exogenous JSRV in this part of gag. Here, we tested the hypothesis that either or both of these variable regions are responsible for the observed particle release defect in enJS56A1. We found that the PPPPPPPS motif of the exogenous VR1 is neither necessary nor sufficient for particle release. Furthermore, the precise substitution of VR1 and VR2 in the exogenous JSRV expression plasmid pCMV2JS ₂₁, using their enJS56A1-derived counterparts, did not abrogate the ability of the resulting constructs to release particles. The particle release defect of enJS56A1 is therefore not determined exclusively by either VR1 or VR2. These results point to a small number of amino acids lying outside of VR1 and VR2 that may be responsible for the particle defect of enJS56A1 Gag.     

Effects of dopamine agonists on hypothalamic defensive attack in cats

The effects of methamphetamine (MAT) and apomorphine (APO), dopamine agonists, were studied in 16 cats to evaluate their effects on threshold for defensive attack behavior elicited by electrical stimulation of the ventromedial hypothalamic nucleus (VMH). Directed attack and hissing were selected from elementary responses as constituting a defensive attack. Hissing threshold was measured in two situations, one with human provocation and the other without provocation. MAT administered systemically lowered the thresholds for all three types of responses in a dose-related manner (0.5, 1.0, and 3.0 mg/kg). The effects of 1.0 mg/kg of APO were almost identical to those observed with 0.5 or 1.0 mg/kg of MAT. These results suggest that MAT-induced aggressive behavior may be mediated by a dopamine-induced increase in the excitability of the VMH.     

Melanoma cell adhesion molecule (MCAM/CD146) is expressed on human luteinizing granulosa cells: enhancement of its expression by hCG,interleukin-1 and tumour necrosis factor-alpha

Melanoma cell adhesion molecule (MCAM) was originally reported to be involved in the invasion and progression of melanoma. It was also shown to be responsible for the attachment of cells to endothelial cells. In this study, we demonstrated by immunohistochemistry that immunoreactive MCAM was not expressed on granulosa cells in the pre-ovulatory follicle, but it was clearly detected in large luteal cells in corpora lutea from the mid-luteal phase of the menstrual cycle. Northern blotting analysis confirmed the expression of MCAM mRNA in corpus luteum. MCAM was weakly detected by immunocytochemical staining in human luteinizing granulosa cells isolated from patients undergoing IVF treatment. Its expression was found to be increased during time in culture of these cells. Flow cytometry and Northern blot analysis revealed that MCAM expression on luteinizing granulosa cells was enhanced when the cells were cultured for 5 days in the presence of hCG (1 IU/ml) or cytokines such as interleukin-1alpha (10 ng/ml) and tumour necrosis factor-alpha (10 ng/ml). No significant difference of MCAM expression was observed between the cultures under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. These results indicate that luteinizing granulosa cells express MCAM and that MCAM expression is regulated by LH/hCG and cytokines during luteinization. Since MCAM has been reported to mediate cellular interaction with endothelial cells, this molecule may play a role in neovascularization during corpus luteum formation in the human ovary.     

Ultrastructure and synaptic organization of the spinal accessory nucleus of the rat

The accessory nucleus is composed of neurons in the medial column that innervate the sternocleidomastoid muscle, and neurons in the lateral column that innervate the trapezius muscle. We retrogradely labeled these neurons by injection of cholera toxin conjugated horseradish peroxidase into the sternomastoid (SM) or the clavotrapezius (CT) muscles, and investigated fine structure and synaptology of these neurons. Almost all SM and CT motoneurons had the appearance of alpha-motoneurons, i.e., large, oval or polygonal cells containing well-developed organelles, Nissl bodies, and a prominent spherical nucleus. More than 60% of the somatic membrane was covered with terminals. The SM motoneurons (34.4 x 52.2 microm, 1,363.1 microm(2) in a section) were slightly larger than the CT motoneurons (32.8 x 54.2 microm, 1,180.8 microm(2)). The average number of axosomatic terminals in a section was 52.2 for the SM, and 54.2 for the CT motoneurons. More than half of them (58.0%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II) with the SM motoneurons, while 57.9% of them contained round vesicles and made asymmetric synaptic contacts (Gray's type I) with the CT motoneurons. A few C-terminals were present on the SM (3.5) and the CT (3.7) motoneurons. About 60% of the axodendritic terminals were Gray's type I in both the SM and the CT motoneurons. A few labeled small motoneurons were also found among the SM and the CT motoneurons. They were small (19.2 x 26.2 microm, 367.0 microm(2)), round cells containing poorly developed organelles with a few axosomatic terminals (9.3). Only 20% of the somatic membrane was covered with the terminals. Thus, these neurons were presumed to be gamma-motoneurons. These results indicate that the motoneurons in the medial and the lateral column of the accessory nucleus have different ultrastructural characteristics.     

A histological evaluation for guided bone regeneration induced by a collagenous membrane

This study was designed to evaluate the histological changes during ossification and cellular events including osteogenic differentiation responding to collagenous bioresorbable membranes utilized for GBR. Standardized artificial bony defects were prepared at rat maxillae, and covered with a collagenous bioresorbable membrane. These animals were sacrificed at 1, 2, 3 and 4 weeks after the GBR-operation. The paraffin sections were subject to tartrate resistant acid phosphatase (TRAP) enzyme histochemistry and immunohistochemistry for alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC). In the first week of the experimental group, woven bone with ALP-positive osteoblasts occupied the lower half of the cavity. The collagenous membrane included numerous ALP-negative cells and OP-immunoreactive extracellular matrices. At 2 weeks, the ALP-, OP- and OC-immunoreactivity came to be recognizable in the region of collagenous membrane. Since ALP-negative soft tissue separated the collagenous membrane and the new bone originating from the cavity bottom, the collagenous membrane appeared to induce osteogenesis in situ. At 3 weeks, numerous collagen fibers of the membrane were embedded in the adjacent bone matrix. At 4 weeks, the membrane-associated and the cavity-derived bones had completely integrated, showing the same height of the periosteal ridge as the surrounding alveolar bones. The collagen fibers of a GBR-membrane appear to participate in osteogenic differentiation.     

Myopathy phenotype in transgenic mice expressing mutated PABPN1 as a model of oculopharyngeal muscular dystrophy

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD)is a late-onset disorder characterized clinically by progressiveptosis, dysphagia and limb weakness, and by unique intranuclearinclusions in the skeletal muscle fibers. The disease is causedby the expansion of a 10-alanine stretch to 12–17 alanineresidues in the poly(A)-binding protein, nuclear 1 (PABPN1;PABP2). While PABPN1 is a major component of the inclusionsin OPMD, the exact cause of the disease is unknown. To elucidatethe molecular mechanism and to construct a useful model fortherapeutic trials, we have generated transgenic mice expressingthe hPABPN1. Transgenic mice lines expressing a normal hPABPN1with 10-alanine stretch did not reveal myopathic changes, whereaslines expressing high levels of expanded hPABPN1 with a 13-alaninestretch showed an apparent myopathy phenotype, especially inold age. Pathological studies in the latter mice disclosed intranuclearinclusions consisting of aggregated mutant hPABPN1 product.Furthermore, some TUNEL positive nuclei were shown around degeneratingfibers and a cluster of it in the lesion in necrotic musclefibers. Interestingly, the degree of myopathic changes was moreprominent in the eyelid and pharyngeal muscles. Further, muscleweakness in the limbs was apparent as shown by the fatigabilitytest. Nuclear inclusions seemed to develop gradually with aging,at least after 1 week of age, in model mouse muscles. We establishedthe first transgenic mouse model of OPMD by expressing mutatedPABPN1, and our model mice appear to have more dramatic alternationsin myofiber viability. ^* To whom correspondence should be addressed. Tel: +81 963736083; Fax: +81 963736599; Email: yamamura{at}gpo.kumamoto-u.ac.jp     

Characterization of very virulent Marek's disease viruses isolated in Japan

Pathogenicity of two isolates of Marek's disease virus (MDV), MS1 and MS2, from chickens was examined in two genetically different strains of chickens, MD-susceptible P-2 chickens and less susceptible PDL-1 chickens. The isolates induced an early mortality syndrome unassociated with lymphoproliferative lesions in P-2 chickens. There were no significant differences in pathogenicity between our isolates and the Md/5 strain of very virulent MDV (vvMDV) in both P-2 and PDL-1 chickens. Protective indices of turkey herpes virus (HVT) vaccine against challenge with MS1 or MS2 in P-2 chickens were 54% and 28%, respectively, whereas HVT gave more than 80% protection in PDL-1 chickens. These results indicate that the two isolates could be classified as vvMDV. In contrast, a bivalent vaccine composed of HVT and serotype 2 MDV, and CVI988 vaccine gave good protection against challenge with the isolates in P-2 chickens; however, the best protection was given by the CBI988 vaccine. This is the first report of isolation of vvMDV in Japan.