IMMUNOHISTOCHEMICAL LOCALIZATION OF TYPE I, II, III, AND IV COLLAGENS IN THE LUNG

Type specific rabbit antibodies to bovine type I, 11, 111, and IV (basement membrane) collagens showing no cross-reaction with other types of collagen were prepared by cross-adsorption and diethylamiuoethyl-cellulose romatography. The antibodies to bovine type I and I11 collagens showed a high cross-reaction with the corresponding human collagens, but those to type I1 and IV collagens did moderate and no cross-reactions with human type I1 and IV collagens, respectively. By using these antibodies, tissue distribution of various types of collagen in normal bovine lung was examined by indirect immunofluorescence microscopy. Both type I and I11 collagens were found to distribute widely in the interstitium of bronchial tree, bronchial
lamina propria and of interlobules as well as alveolar nipples and adventitia of pulmonary arteries. Type I1 collagen was located only in bronchial cartilage. The tissues mainly stained for type 11 collagen were the alveolar interstitium (also stained faintly for type I collagen) and the intima and media of the arteries. Type IV collagen was located in a membranous fashion in alveolar septa and bronchial smooth muscles and subepithelial layers as well as capillaries and the intima and media of arteries. ACTA PATHOL. JPN. 31: 601–610, 1981.     

The expression of neuronal nitric oxide synthase and dystrophin in rat regenerating muscles

We investigated the expression of neuronal nitric oxide synthase (nNOS) and dystrophin in the regenerating skeletal muscles of rats after cardiotoxin-induced myonecrosis by immunohistochemical studies and western blot analysis. In normal muscles, nNOS was moderately immunostained on type 2B fibers, but was faintly immunostained on type 2A or type 1 fibers. In immunohistochemical studies of regenerating muscles, nNOS was first observed at the sarcolemma of type 2B fibers on day 10, when the type discrimination between types 2A and 2B was first detected by ATP reactions. Subsequently, the immunostaining of nNOS grew progressively stronger in type 2B fibers, with faint staining in type 2A and type 1 fibers until day 28. Meanwhile, the immunostaining of dystrophin grew stronger equally in all three fibers until day 21. In western blot analysis of regenerating muscles, nNOS regenerated more slowly than dystrophin. The present data suggest that the expression of nNOS is related to the muscle fiber type differentiation, and that the role of nNOS is related to the function of the type 2B fibers of the muscle. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characteristics of histopathological and ultrastructural features of placental villi in pregnant Nepalese women

The placenta is an important functional unit for gas transfer between mother and fetus. The placental membrane, consisting of trophoblast layer interposed between maternal and fetal blood, plays an active role for intensity of respiration, but no morphological evidence has been documented. Until now, it has been reported that fetal growth retardation and increased fetal mortality rate usually could be seen at high altitude. In an attempt to find the cause of high perinatal mortality rate in Nepal, this study was undertaken to examine pathologically about 1000 Himalayan placentas obtained in Nepal and Tibet since 1977, and the results were compared with those of 5500 Japanese placentas at Saitama Medical School since 1990. In this study, characteristics of ultrastructural features of the Nepalese placental villi investigated in recent years are reported. (1) The gross characteristics of placental pathology in the Himalayan group were represented by marked subchorionic fibrin deposits and increased chorionic cysts in contrast to low incidence of intervillous thrombosis compared with those of the Japanese group. (2) As characteristics of histological findings of the placental villi between Himalayan and Japanese groups, the incidence of chorangiosis and chorangioma in the Himalayan group was significantly higher than that in the Japanese group. (3) Accompanying an increase of vasculosyncytial membrane (VSM) in the villi, thickness and separation of basement membrane of the syncytium in addition to increased apoptosis of syncytial cell nuclei were recognized. (4) As characteristic ultrastructural features of chorionic villi of Nepalese placentas, an increase of mitochondria and cystic formation of rough endoplasmic reticulum (rER), in addition to appearance of lamellar bodies similar to alveolar epithelial type II cell in organellae of the syncytium, were observed. These ultrastructural changes of the placental villous capillaries may be ascribed to hypevascularization caused by the chronic hypoxic state. It is, therefore, presumed that trophoblast cells may play an important role for gas transfer mecha-nism under such a hypoxic state at high altitude.     

Anin vitro invasion model for human renal cell carcinoma cell lines mimicking their metastatic abilities

We developed a modifiedin vitro invasion assay system using monolayers of vascular endothelial cells. A type I collagen gel was formed in plastic dishes, and overlaid with type IV collagen. Calf pulmonary arterial endothelial (CPAE) cells were seeded onto these plates, and incubated until they reached confluence. Five human renal cell carcinoma cell lines with various metastatic potentialsin vivo were then seeded on the monolayer CPAE cells, and their colony formation and invasion activities were examined for 9 days. At day 4, the highly metastatic cell lines increased the number of colony foci on monolayer CPAE cells several fold higher than their poorly metastatic counterpart. The horizontal spreading patterns were also different between poorly and highly metastatic cell lines. On day 9, the number of carcinoma foci that penetrated the monolayer of CPAE cells and type IV collagen sheets into type I collagen gels in highly metastatic cell lines greatly increased as compared with that of poorly metastatic cell lines. Ourin vitro invasion assay using monolayer CPAE cells would be useful to evaluate protease activities and colony formation during invasion.     

Three-dimensional ultrasonography in the first trimester of human pregnancy

Our purpose was to visualize normal embryonal and fetal surface anatomical structures in the first trimester of human pregnancy by use of three-dimensional ultrasonography with a specially developed abdominal three-dimensional transducer. Four embryos and 31 fetuses of 8-13 weeks gestation were studied with a specially-developed abdominal three-dimensional transducer (3.5 MHz). This imaging system can provide conventional two-dimensional ultrasonography images and can also generate, within seconds, high-quality three-dimensional images in the surface and transparent mode with no need for an external workstation. The percentage of surface anatomical structures visualized at each gestational age interval using two-dimensional and three-dimensional ultrasonography is presented. Head and trunk were depicted in all cases. The number and the clarity of visualization of face, upper and lower extremities, hand, and foot increased with advancing gestation. The free loop of the umbilical cord was depicted in most cases. The number of depictions of abdominal cord insertion, midgut herniation, and yolk sac decreased with the increase of gestation. Genitals could not be identified in the first trimester. The ability to view some surface anatomical structures (face, hand, and foot) was better with three-dimensional ultrasonography than with two-dimensional ultrasonography. Three-dimensional ultrasonography provides a novel means for visualization of surface anatomical structures of the embryo and early fetus. These results suggest that three-dimensional ultrasonography can become an important modality in future embryological and early fetal research and in detection of embryonic and fetal developmental disorders in the first trimester of pregnancy.      

Expression of estrogen receptor alpha (ER alpha) in the rat temporomandibular joint

Numerous epidemiological studies have pointed out a higher frequency of temporomandibular disorder (TMD) in women than in men, which indicates the involvement of a sex hormone, such as estrogen, in the pathogenesis of TMD. Although estrogen is known to play pivotal roles in osteoarthrosis or rheumatoid arthritis in systemic joints, there have been few reports about the role of estrogen in the temporomandibular joint (TMJ). The effect of estrogen is generally mediated by the estrogen receptors (ERs) ER alpha (the predominant type) and ER beta. In this study we examined the expression of ER alpha protein and mRNA in the TMJ of adult male rats by immunocytochemistry and in situ hybridization histochemistry. Intense ER alpha immunoreactivity was localized in the synovial lining cells, stromal cells in the articular disc, and chondrocytes in the TMJ. These ER alpha-immunopositive synovial lining cells are characteristic of cytoplasmic processes identified with confocal and immunoelectron microscopy, which indicates that they are synovial type B cells. In situ hybridization histochemistry confirmed intense signals for ER alpha in the synovial lining cells and the sublining fibroblasts at mRNA levels. The nuclei of chondrocytes showed an intense immunoreaction for ER alpha in the maturative and hypertrophic layers of the articular cartilage. In addition to the nuclear localization of ER alpha, a weak immunoreaction appeared in the cytoplasm of some ER alpha-positive cells. These findings support the hypothesis that TMJ tissue-at least in the male rat-has the potential to be an estrogen target tissue.     

Autosomal linkage analysis of a Japanese single multiplex schizophrenia pedigree reveals two candidate loci on chromosomes 4q and 3q.

We analyzed a large multiplex schizophrenia pedigree collected in mid-eastern Japan using 322 microsatellite markers distributed throughout the whole autosome. Under an autosomal-dominant inheritance model, the highest pairwise LOD score (LOD = 1.69) was found at 4q (D4S2431: theta = 0.0), and LOD scores at two other loci 3q (ATA34G06) and 8q (D8S1128) were 1.62 and 1.46, respectively. In multipoint analysis, LOD scores of the regions on 4q and 3q remained at a similar level; however, the LOD score of the region on 8q apparently decreased. Additional dense map analysis revealed haplotypes on 4q and 3q regions shared by affected individuals. On chromosome 4q, the haplotype spanning about 8 centiMorgans (cM) was shared by four of six genotyped individuals with schizophrenia and one affected individual whose haplotype was estimated. On 3q, the haplotype spanning about 20 cM was shared by five genotyped individuals with schizophrenia. We obtained two candidate regions of major susceptibility loci for schizophrenia on chromosomes 3q and 4q.     

Adenosine inhibits the synaptic potentials in rat septal nucleus neurons mediated through pre- and postsynaptic A1-adenosine receptors.

Intracellular and voltage-clamp recordings were made from neurons in rat brain slices containing dorsolateral septal nucleus (DLSN), in vitro. Bath application of adenosine (100 microM) produced a hyperpolarization (2-15 mV) in 46% of DLSN neurons (AH-neurons); in the remaining 54% neurons (non-AH-neurons), no hyperpolarization to adenosine was observed. Adenosine (1-300 microM) depressed not only the excitatory postsynaptic potential (EPSP) but also the inhibitory postsynaptic potential (IPSP) and the late hyperpolarizing potential (LHP) evoked by stimulation of the hippocampal CA3 area or the fimbria/fornix pathway in both AH- and non-AH-neurons. In non-AH-neurons, adenosine did not block current responses resulting from glutamate, muscimol or baclofen applied directly to DLSN neurons. In AH-neurons, adenosine partially depressed the baclofen-induced outward current. Adenosine did not block the directly-evoked IPSP (monosynaptic IPSP) as well as the glutamate-induced (hyperpolarizing) postsynaptic potential (PSP) that is mediated by GABA released from interneurons. These results suggest that adenosine does not directly inhibit the release of GABA. The effects of adenosine was mimicked by selective A1-receptor agonists and was blocked by selective A1-receptor antagonists. Pertussis toxin (PTX) blocked the hyperpolarization induced by adenosine or baclofen applied exogenously. Adenosine consistently produced presynaptic inhibition of the EPSP even in DLSN neurons treated with PTX. We conclude that adenosine inhibits neurotransmission between the hippocampus and septum through activation of pre- and postsynaptic A1-receptors which couple with G-proteins of different PTX-sensitivity or with distinct transduction processes at pre- vs. postsynaptic sites.