Arpp,a new homolog of carp,is preferentially expressed in type 1 skeletal muscle fibers and is markedly induced by denervation

In this study, we isolated and characterized a murine counterpart of the human Arpp (hArpp) gene. Sequence analysis revealed that the murine Arpp (mArpp) gene is almost identical to the Ankrd2 gene, which has recently been isolated as a mouse gene induced in stretched skeletal muscle. The mArpp gene encodes a protein of 332 amino acids that contains four well-conserved ankyrin-repeat domains in the central portion of the protein. The amino acid sequence of mArpp protein (mArpp) is highly homologous to that of mouse cardiac-restricted ankyrin-repeat protein (Carp), which is proposed to be a putative genetic marker for cardiac hypertrophy. Immunohistochemical analysis revealed that mArpp is preferentially expressed in type 1 skeletal muscle fibers, and that mArpp is localized in both the nucleus and the sarcomeric I-band of muscle fibers, suggesting that Arpp may function as a nuclear and sarcomeric protein. Furthermore, mArpp was also expressed in neurons of the cerebellum and cerebrum, the islets of Langerhans in the pancreas, and the esophageal epithelium, suggesting that mArpp may play a functional physiologic role in brain, pancreas, and esophagus as well as in type 1 muscle fibers. Interestingly, although mArpp was localized in both nucleus and cytoplasm in neurons, its localization was restricted to nucleus in pancreas and esophagus, suggesting that intracellular localization of mArpp is regulated in a tissue-specific manner. Furthermore, we found that mArpp- and Carp-expression in skeletal muscle were markedly up-regulated after denervation. Although the elevated expression level of Carp was kept only for two weeks after denervation, that of Arpp was kept at least for 4 weeks, suggesting that mArpp and Carp may play distinct functional roles in denervated skeletal muscle.     

Isolation and radiation hybrid mapping of a dinucleotide repeat polymorphism at the human calcium-sensing receptor (CASR) locus

Calcium-sensing receptor (CASR) in parathyroid gland regulates calcium homeostasis by sensing decreases in extracellular calcium levels and effecting an increase in secretion of parathyroid hormone. A polymorphic dinucleotide (CA) sequence was isolated from a genomic clone containing the human CASR gene and was mapped to 3q13.3–q21. This polymorphism will be useful in the genetic study of disorders affecting calcium metabolism, such as hypercalcemia, hypocalcemia, osteoporosis, hyperparathyroidism, and hypoparathyroidism. Received: June 2, 1998 / Accepted: June 24, 1998     

Caveolar and intercellular channels provide major transport pathways of macromolecules across vascular endothelial cells

Serum macromolecules are transported through the vascular endothelial layer to the interstitium via the caveolae and interendothelial clefts, but the nature of the permeability of these structures is unknown, and the manner of caveola-vesicle transport is controversial. We have developed a method of detecting macromolecular channels using an in situ HRP perfusion into arteries previously perfused with aldehyde and random conventional sectioning for electron microscopy. Using unbiased morphometry, 4.75% of the abluminal caveolae and 15.13% of the intercellular clefts were the tracer-positive in rat aortic endothelium. In rat aortas treated with N-ethylmaleimide, all caveolae and most free vesicles in the cytoplasm except those around the Golgi area were HRP-positive in the endothelial cells; 1.48% of abluminal caveolae were structurally recognized as caveolar channels through the endothelial layer in a plane of single section. The length density of the abluminal caveolae was decreased to about 80% to the physiological control level whereas the larger invaginations were more frequently observed. Moreover 96.17% of the intercellular clefts were HRP-positive. We suggest that a flexible channel-system functions extensively as a macromolecular transport pathway in the arterial endothelium in vivo because the tracer-labeled abluminal caveolae and intercellular clefts should be opened to the luminal surfaces methodologically. We therefore propose that caveolar channels, rather than transcytosis, provide a mechanism of caveola-vesicle transport in the endothelial cells, because free vesicles involved in transcytosis were few in number.     

Neurite extension of DRG neurons by gicerin expression is enhanced by nerve growth factor

Gicerin, a cell adhesion molecule, is expressed in dorsal root ganglion (DRG) and sciatic nerves during chick development. This molecule re-appears in these tissues after an injury to the sciatic nerve. In the present study, we investigated the expression of nerve growth factor (NGF) in the regenerating sciatic nerve of chicks and the effects of NGF on the expression and neurite activities of gicerin in DRG. In the sciatic nerve after a crush injury, the expression of NGF and gicerin increased in the Schwann cells and in the nerve fibers, respectively. NGF promoted the neurite projections from in vitro DRG on the gicerin ligands, which were inhibited by anti-NGF antibody. The gicerin mRNA expression increased in the DRG with NGF, which was inhibited by the co-incubation with anti-NGF antibody. These results indicate that NGF might therefore enhance the expression of gicerin in DRG, thereby promoting the gicerin-dependent neurite extension during sciatic nerve regeneration.     

Embryonic development of the pituitary gland in the chick

Pituitary glands of chicken, from stages 20 (70 approximately 72 h of incubation) to 46 (20 days) of Hamburger and Hamilton (1951), were studied by immunocytochemical and histological stainings and India ink injection into blood vessels. Using the distribution pattern of 6 types of immunoreactive adenohypophyseal cells and the location of pituitary stalk as guideposts, we found how specific areas in the epithelium of Rathke's pouch differentiate into specific regions of the adenohypophysis at 20 days. In the sagittal plane, the walls of Rathke's pouch were tentatively divided into the upper part (A(1) + A(2)) and lower part (A(3)) of the anterior wall, and the posterior wall (P(1) + P(2) + P(3)). The cephalic lobe was mainly assembled by the proliferation of parenchymal cells in the areas A(2) + A(3) + P(2) of Rathke's pouch epithelia at 3 days of incubation. The caudal lobe was derived from A(1) + P(1) + P(3). The pars tuberalis was derived from A(1) + A(2). Thus, the avian adenohypophysis is established at 13 days, though the blood supply to the pars distalis is established at 20 days. Therefore, the cephalic lobe and caudal lobe of the pars distalis and the pars tuberalis of the chicken adenohypophysis are derived from specific areas of the cell cords of Rathke's pouch at 3 days of incubation.     

Seasonal changes in the hypothalamo-pituitary-testes axis of the Japanese wood mouse (Apodemus speciosus)

Seasonal changes in the hypothalamo-pituitary-testes axis of the Japanese wood mice (Apodemus speciosus) were studied. The testes, epididymis, pituitary and hypothalamus were compared between mice in the breeding season (July) and non-breeding season (October) using morphological techniques, and the plasma testosterone level was evaluated by enzyme immunoassay. Significant differences in these tissues were observed between the breeding season and the non-breeding season. Specifically, differences in the non-breeding season included 1) a decline in testicular and epididymal weights, arrest of spermatogenesis and decrease of serum testosterone concentration; 2) a decrease in the number of luteinizing hormone (LH)-, follicle stimulating hormone (FSH)-, prolactin (PRL)-, and growth hormone (GH)-immunoreactive cells, and decrease in the size of FSH, PRL, and GH-immunoreactive cells; and 3) an increase in the size of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons. Our findings indicate that the male adult Japanese wood mouse exhibits unique seasonal changes in the hypothalamo-pituitary-testes axis which are not found in laboratory mice.     

Relationship between fatty liver and subsequent development of necrosis, inflammation and fibrosis in experimental alcoholic liver disease

Rats fed a diet varying in the amount of fat, infused with ethanol, were studied to determine the relationship among diet, degree of fatty liver, and development of necrosis, inflammation, and fibrosis. Three groups of experimental animals, male Wistar rats, were fed diets containing 25% fat, 35% fat, and 32% fat with low protein. Morphologic assessment of liver injury was performed monthly by obtaining liver biopsies. The greatest degree of fatty infiltration at 1 month was seen in the high fat-low protein group, the mean fat score (3.8 +/- 0.37) was significantly higher than in the other two groups (P less than 0.05 and P less than 0.01). When the subsequent development of necrosis, inflammation, and fibrosis was related to the degree of fatty infiltration at 1 month, a significant relationship was seen between the number of animals developing these pathologic lesions and the severity of fatty liver. Our results show that the degree of fatty infiltration of the liver, influenced by the dietary intake of both fat and protein, is related to the subsequent development of necrosis, inflammation, and fibrosis in our intragastric feeding model for alcoholic liver disease.     

Novel mutations in TNFRSF1A in patients with typical tumor necrosis factor receptor-associated periodic syndrome and with systemic lupus erythematosus in Japanese

Molecular defects of TNFRSF1A was investigated in members of a family presenting with typical phenotypes of tumor necrosis factor receptor-associated periodic syndrome (TRAPS) and in patients with the autoimmune disorders, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Genomic DNA from the members of a family with typical TRAPS, as well as from 100 patients with SLE, 100 patients with RA and 100 healthy individuals, was studied for mutations in exons 2, 3 and 4 of the TNFRSF1A gene. All individuals were Japanese. Three novel missense mutations were identified in the TNFRSF1A. The C70G mutation was identified in family members with typical TRAPS, which was the second case in eastern Asian population. In addition, the T61I and R104Q mutations were each identified in 2 of the 100 SLE patients. The T61I mutation was identified in one of the 100 healthy individuals. No mutations were identified in the 100 RA patients. Functional analysis revealed that PMA-induced shedding of TNFRSF1A from PBMCs was impaired in a patient carrying T61I. A larger scale of study will clarify whether these two mutations, T61I and R104Q, are associated with chronic inflammatory disorders, such as SLE, or not.     

Dual-viral vector approach induced strong and long-lasting protective immunity against very virulent infectious bursal disease virus

To induce strong protective immunity against very virulent infectious bursal disease virus (vvIBDV) in chickens, two viral vector systems, Marek's disease and Fowlpox viruses expressing the vvIBDV host-protective antigen VP2 (rMDV, rFPV), were used. Most of chickens vaccinated with the rFPV or rMDV alone, or vaccinated simultaneously with both at their hatch (rMDV-rFPV(1d)), were protected against developing clinical signs and mortality; however, only zero to 14% of the chickens were protected against gross lesions. In contrast, gross lesions were protected in 67% of chickens vaccinated primarily with the rMDV followed by boosting with the rFPV 2 weeks later (rMDV-rFPV(14d)). Protection against the severe histopathological lesions of rFPV, rMDV, rMDV-rFPV(1d), and rMDV-rFPV(14d) vaccine groups were 33, 42, 53, and 73%, respectively. Geometric mean antibody titers to VP2 of chickens vaccinated with the rFPV, rMDV, rMDV-rFPV(1d), and rMDV-rFPV(14d) before the challenge were 110, 202, 254, and 611, respectively. Persistent infection of the rMDV in chickens after the booster vaccination with rFPV was suggested by detection of the rMDV genes from peripheral blood lymphocyte DNA at 28 weeks of age. These results indicate that the dual-viral vector approach is useful for quickly and safely inducing strong and long-lasting protective immunity against vvIBDV in chickens.     

Retrograde labeling of mouse spinal descending tracts by a recombinant adenovirus

The present study tested whether a gene-transfer based upon the retrograde axonal transport of the lacZ adenovirus is effective in the spinal descending tracts of the adult mouse. A small volume of a replication-defective recombinant adenovirus encoding E. coli beta-galactosidase was injected into the upper lumbar cord, and, seven days later, the mice were transcardially perfused by a fixative solution. X-gal staining of coronal or sagittal sections of the spinal cord and the brain revealed that many sites of origin for rubrospinal, vestibulospinal, and reticulospinal tracts were retrogradely labeled, whereas few of the corticospinal tract neurons were retrogradely labeled. Ependymal cells surrounding the central canal of the spinal cord, which were located far from the injection site, showed a high expression of beta-galactosidase activity. Motoneurons around the injection site were strongly stained by X-gal staining, and their axons in the ventral root were anterogradely labeled. Afferent fibers in the dorsal root were labeled by the transganglionic transport of beta-galactosidase. To examine the efficacy of the uptake and retrograde transport of HRP and adenovirus, we injected a mixed solution of 10% HRP and recombinant adenovirus. The number of HRP-labeled corticospinal neurons overwhelmed the number of X-gal stained ones, while the numbers of HRP-labeled rubrospinal and subcoeruleus-spinal neurons were smaller in comparison with the numbers of beta-galactosidase-positive counterparts. The present study revealed that the origins for the spinal descending tracts except for corticospinal neurons could be efficiently gene-transferred by the retrograde infection of a recombinant adenovirus. Such a difference in efficacy of retrograde infection among the spinal descending tracts is practically important when an adenovirus-mediated gene transfer is designed to treat certain neurological diseases affecting the spinal descending tracts.