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Biomedical subjects

H Nogami

Publications and source records attributed to H Nogami.

At least 73 records · Page 4Linked to original sources

Bioassay of chondrocyte differentiation by bone morphogenetic protein.

For sensitive bioassay of cartilage development in response to bone morphogenetic protein (BMP), the supernatant of interstitial fluid containing crud BMP was obtained from an implanted diffusion chamber, applied to a cellulose acetate film, and stained with Alcian blue. The optical density of the Alcian blue-stained spot was measured by a densitometer for quantitative analysis. Density of the Alcian blue-stained spot was markedly increased when cartilage developed outside the chamber. Occurrence of cartilage-specific proteoglycan-H (PG-H) and Type II collagen was seen in this intensely stained spot by dot-blot analysis. Electron microscopic examination revealed that the residue of interstitial fluid in the implanted diffusion chamber contained many collagen fibrils and proteoglycan granules.

Animals↗

Three distinct molecular species of proteoglycan synthesized by the rat limb bud at the prechondrogenic stage.

To characterize proteoglycans in the prechondrogenic limb bud, proteoglycans were extracted with 4 M guanidine HCl containing a detergent and protease inhibitors from Day 13 fetal rat limb buds which had been labeled with [35S]sulfate for 3 h in vitro. About 90% of 35S-labeled proteoglycans was solubilized under the conditions used. The proteoglycan preparation was separated by DEAE-Sephacel column chromatography into three peaks; peak I eluted at 0.45 M NaCl concentration, peak II at 0.52 M, and peak III at 1.4 M. Peaks I and III were identified as proteoglycans bearing heparan sulfate side chains. The heparan sulfate proteoglycan in peak III was larger in hydrodynamic size than the proteoglycan in peak I. The heparan sulfate side chains of peak III proteoglycan were smaller in the size and more abundant in N-sulfated glucosamine than those of peak I proteoglycan. Peak II contained a chondroitin sulfate proteoglycan with a core protein of a doublet of Mr 550,000 and 500,000. The chondroitin sulfate proteoglycan was easily solubilized with a physiological salt solution and the heparan sulfate proteoglycan in peak I was partially solubilized with the physiological salt solution. The remainder of the proteoglycan in peak I and the heparan sulfate proteoglycan in peak III could be solubilized effectively only with a solution containing a detergent, such as nonanoyl-N-methylglucamide. This observation indicates the difference in the localization among these three proteoglycans in the developing rat limb bud.

Amino Acids↗

Electron-microscopic study on the anterior pituitary gland of spontaneous dwarf rats.

The spontaneous dwarf rat is a novel experimental model animal on the study of pituitary dwarfism. The fine structure of the anterior pituitary cells was studied in the immature and mature dwarf rats. Pituitary glands were removed from 5-, 10-, 20-day-old immature dwarfs, adult (45 days-16 weeks) dwarfs and normal 3-month-old rats and processed for electron-microscopic observation. In the control animals, growth hormone cells were readily identified by their ultrastructural characteristics, such as the presence of numerous electron-dense secretory granules, 300-350 nm in diameter, well developed rough endoplasmic reticulum and a prominent Golgi complex. In contrast, growth hormone cells were not found in the anterior pituitary gland of the spontaneous dwarf rat at any age examined. Other pituitary cell types, i.e., luteinizing hormone/follicle stimulating hormone, thyroid stimulating hormone, adrenocorticotropic hormone and prolactin cells, appeared similar in their fine structure to those found in the control rats. In the pituitary gland of dwarf rats, a number of polygonal cells were observed either with no or relatively few secretory granules. The rough endoplasmic reticulum was arranged in parallel cisternae and the Golgi complex was generally prominent in these cells. In addition, many were found to have abundant lysosomes. A few minute secretory granules were occasionally observed; however, the immunogold technique failed to localize growth hormone or prolactin in the granules. The nature of these cells remained obscure in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Studies on the development of growth hormone and prolactin cells in the rat pituitary gland by in situ hybridization.

Cytogenesis of growth hormone and prolactin cells in the rat pituitary gland was studied using in situ cDNA-mRNA hybridization and immunocytochemistry. Frozen or Paraplast sections of fetal and neonatal pituitaries were hybridized with 3H-cDNAs for rat prolactin or growth hormone, and were then processed for autoradiography. A number of growth hormone mRNA-positive cells were encountered throughout the anterior lobe on day 19 of gestation. Individual variation in growth hormone gene expression was observed between fetuses at day 19 of gestation (6 out of 8 fetuses examined were positive for growth hormone mRNA). In contrast, growth hormone mRNA was detected in the all fetuses examined on day 20 or later. The autoradiographic signal (number of reduced silver grains) appeared to increase with later stages of development. Fetal growth hormone mRNA-positive cells were evenly scattered throughout the anterior lobe. Most of them were isolated, however, small clusters of several growth hormone cells were infrequently observed. Prolactin mRNA-positive cells were found first on the 22nd day (the last day of gestation) in 3 of 6 fetuses examined, but were rarely observed on earlier gestational days. By postnatal day 8, prolactin mRNA-positive cells were numerous and the grain density over prolactin cells increased. Both growth hormone and prolactin cells were found as early as 18 days of gestation using immunocytochemistry, although the number of positive cells was very small at this stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Elevated accumulation of hyaluronate in the tubular bones of osteogenesis imperfecta.

The content and composition of glycosaminoglycans in the tubular bones of osteogenesis imperfecta were compared to those in the tubular bones of age-matched controls. Chondroitin sulfate was the major glycosaminoglycan (70-80% of total) both in the normal and pathological bones, and its level, based on the tissue wet weight, was slightly less in the pathological bones. The composition of chondroitin sulfate disaccharide units in the pathological samples was different from those of the control; a lower proportion of chondroitin 4-sulfate unit. Hyaluronate accounted for at most 7% of total glycosaminoglycans from the normal bones. The hyaluronate content of the pathological bones was 1.5- to 3-fold higher than that of the controls. Glycosaminoglycans have been shown to participate in the formation of a functional supramolecular complex in extracellular matrices. Therefore, it may be postulated that the abnormalities in glycosaminoglycan composition in the tubular bones of osteogenesis imperfecta is implicated in some clinical aspects of this connective tissue disorder such as the bony fragility.

Bone and Bones↗

Studies on prolactin and growth hormone gene expression in the pituitary gland of spontaneous dwarf rats.

The spontaneous dwarf rat (SDR) is a newly established experimental model for pituitary dwarfism with an autosomal recessive mode of inheritance. GH and PRL gene expressions were studied in the pituitary gland of this animal. Immunocytochemistry failed to detect any immunoreactive GH cells in SDR pituitary gland, whereas numerous PRL cells were encountered throughout the anterior lobe. Similarly, GH was not detectable in the pituitary cytosol from SDR of 7, 20, and 80 days of age. PRL of the same mol wt as the authentic rat PRL was found in the pituitary glands in all SDRs examined. Since these results were thought to be due to the absence of GH mRNA in the SDR's pituitary gland, we attempted to detect GH mRNA by dot hybridization. Very small amounts of GH mRNA were found in the pituitary glands of both male (2.6% of normal) and female (6.0% of normal) SDRs. Reduced levels of PRL mRNA were also found in the pituitary glands of male (50%) and female (30%) SDRs. Northern blot analyses of total RNA extracts revealed that the GH mRNA of the SDR was similar in size to the mature GH mRNA found in the pituitary glands of control rats. PRL mRNA from the SDR also migrated to the same position as normal PRL mRNA. Nuclear DNA preparations were prepared from normal and SDR livers, and the genomic organizations of the GH and PRL genes were examined using restriction mapping. The DNA fragment length patterns obtained with the cDNA probes for GH or PRL using several restriction enzyme digestions were identical. The results suggest that the GH deficiency in SDR is not due to a gross deletion or rearrangement of the GH gene. It is proposed that SDR may be an excellent experimental model for the study of isolated GH deficiency in the human.

Animals↗

New assessment of airway responsiveness. Effect of pretreatment with procaterol on allergen-induced bronchoconstriction.

We examined airway responsiveness to allergen inhalation using a novel technique by which dynamic compliance (Cdyn) and pulmonary resistance (Rl) are simultaneously calculated by Fourier-series analysis of flow and transpulmonary pressure during tidal breathing. C0 and C0.5 (Cdyn at the frequency of zero and 0.5 Hz, respectively) were computed using the regression line of Cdyn versus frequency measured at the fundamental and first three harmonics in each breathing cycle. First, the validity of this system was tested by comparing Rl, C0 and C0.5 during five consecutive breaths with those obtained by the conventional method. A good correlation was seen in Rl, C0 and C0.5 between the two methods. Second, we studied airway response to allergen inhalation before and after oral administration of a long-acting beta 2-stimulant (procaterol, 50 micrograms or 100 micrograms) or placebo in a double-blind crossover trial in six atopic asthmatic subjects. In control allergen inhalation tests by administration of placebo, Rl increased progressively, and C0.5, expressed as percentage of control compliance at zero frequency (C0.5/COcont), decreased progressively. After 100 micrograms procaterol, Rl response to allergen was almost completely inhibited. However, a decrease in C0.5/C0cont was still observed. These findings suggest that pretreatment of asthmatic patients with procaterol can release allergen-induced bronchoconstriction of the central airways, but cannot release that of the peripheral airways.

Adult↗

Possible site of bronchodilation due to inhaled procaterol aerosol in asthmatic patients.

We studied the effective site of an inhaled aerosol of procaterol, a beta 2-selective adrenergic bronchodilator, in 8 asthmatic patients whose basal lung functions are almost within the normal range in both slow vital capacity (VC) and forced expiratory volume in one second (FEV1.0), and are free from asthmatic attack. In patients who had received procaterol 30 min after inhalation of aerosol, there was no significant change in VC, although FEV1.0, maximal expiratory flow at 50% VC (V50), maximal expiratory flow at 25% VC (V25) and maximal expiratory flow at 30% VC of partial maximal expiratory flow volume curve (V30p) improved significantly. On the other hand, in those who had received placebo, none of the parameters changed. Furthermore, R1 decreased and C0.5 increased significantly during the first 5 min after inhalation of procaterol aerosol. After an interval of 5 min, R1 did not change any further, while C0.5 continued to improve until 30 min after inhalation of procaterol. These results suggest that procaterol may first dilate the large airway and then may gradually dilate the small airway in bronchial asthma.

Adult↗

Defective association between collagen fibrils and proteoglycans in fragile bone of osteogenesis imperfecta.

The relationship between collagen fibrils and proteoglycans in the bone matrices of three osteogenesis imperfecta (OI) patients was observed ultrastructurally to clarify the mechanism responsible for pronounced bony fragility. Collagen fibrils and noncollagenous components were prepared from the bone matrix of patients with osteogenesis imperfecta and a control sample, respectively. Compared with the control, a 95.2% decrease was found in the number of proteoglycan granules periodically associated with the cross-banding of collagen fibrils in one OI sample from a patient with severe bony fragility. Extractability of collagen fibrils and proteoglycans was high in this sample. However, proteoglycans and collagen fibrils could be reattached in vitro to reproduce the condition observed in the controls. There was no decrease in the number of proteoglycan granules attached to prepared collagen fibrils in a sample from a patient with predominantly bowing deformity of bones. Hypothetically, the separation of proteoglycans from collagen fibrils in OI may be associated with increased bony fragility. In osteogenesis imperfecta patients, the mechanism inducing bowing deformity appears to be different from that inducing fragility.

Bone and Bones↗

Uptake and retention of 3H-estradiol by gonadotrophs and lactotrophs in the pituitary glands of the guinea pig, hamster and gerbil.

Nuclear uptake and retention of 3H-estradiol by luteinizing hormone (LH) and prolactin (PRL) cells was examined in three species of rodents (guinea pigs, hamsters and gerbils) using the combined techniques of immunocytochemistry and autoradiography. Castrated animals were injected with 3H-estradiol and decapitated 1.5 h later. The pituitary glands were processed for thaw-mount autoradiography followed by conventional immunocytochemical staining for LH and PRL. 3H-estradiol accumulated in more than 80% of the anterior pituitary cells in the gerbils, while only 33 and 22% of the cells accumulated 3H-estradiol in the hamsters and guinea pigs, respectively. A varying percentage of immunoreactive LH and PRL cells in all three species were found also to contain binding sites for estradiol. Some LH and PRL cells in hamsters and guinea pigs and only some in PRL cells of gerbils were found to be devoid of grains. Quantitative analysis revealed that the number of grains per nucleus differed considerably from cell to cell. LH cells of guinea pigs accumulated much larger amounts of 3H-estradiol than did the PRL cells, while the LH cells in the hamsters and gerbils accumulated only slightly more 3H-estradiol than the PRL cells. These results confirm the previous observations in rats and baboons that demonstrated tremendous species differences in percentage of cells in the anterior pituitary gland that accumulated 3H-estradiol. Also, these data suggest that there are functionally heterogeneous cell types among the LH and PRL cells in hamsters, guinea pigs and gerbils as has been previously demonstrated in rats and baboons.

Animals↗

A new method of inhalation challenge with propranolol: comparison with methacholine-induced bronchoconstriction and role of vagal nerve activity.

To establish a safe procedure for examining propranolol-induced bronchoconstriction, we have developed a new method for performing inhalation challenge with propranolol. Monitoring respiratory resistance during tidal breathing with continuous inhalation of propranolol in 1.5-fold increasing concentrations from 0.78 to 30 mg/ml for 1 minute at each concentration, we tested 43 subjects with stable asthma and 10 normal subjects. We also compared bronchial responsiveness with responsiveness to inhaled methacholine on separate days. In addition, to determine the role of vagal nerve activity in propranolol-induced bronchoconstriction, we studied the effect of atropine. Inhaled propranolol caused dose-related bronchoconstriction in all subjects with asthma but not in normal subjects. None of the subjects suffered severe asthmatic attack during the test, which was performed in 15 minutes or less. The minimum cumulative dose of methacholine and of propranolol, at the point where respiratory conductance began to decrease, was not significantly correlated. Increased respiratory resistance was reversed by atropine in 70% of the subjects with asthma with marked individual differences. These data suggest that, although in most subjects with asthma, vagal nerve activity contributes in varying degree to bronchoconstriction, other constricting factors may contribute in the remaining subjects. It is also suggested that the mechanism of bronchial response to propranolol differs from that of the nonspecific airway reactivity estimated by methacholine challenge.

Adolescent↗

Possible sensory receptor of nonadrenergic inhibitory nervous system.

To determine the sensory receptor of the nonadrenergic inhibitory nervous system (NAIS), 22 cats were anesthetized and serotonin was continuously administered (50-250 micrograms.kg-1.min-1 iv) to increase pulmonary resistance (RL) to 377 +/- 57% (SE) of the control value. We then 1) mechanically irritated the trachea, 2) intravenously administered capsaicin (5 micrograms/kg), or 3) induced hypoxia (arterial PO2 30-40 Torr) to stimulate irritant and bronchial C-fiber receptors, pulmonary C-fiber receptors, or the carotid body (chemoreceptors), respectively. After treatment with atropine (3 mg/kg iv) and propranolol (2 mg/kg iv), the serotonin-induced change in RL was reduced by 58.6 +/- 14.3% by mechanical irritation and 63.3 +/- 12.1% by intravenous capsaicin. However, hypoxia produced no dilatation of the airways. In further experiments, we employed capsaicin inhalation to stimulate bronchial C-fiber receptors. Inhaled capsaicin (0.1%, for 5 breaths) also reduced RL by 79.2 +/- 9.2% of the elevated value, after atropine and propranolol. Treatment with a ganglionic blocking agent, hexamethonium (2 mg/kg iv), abolished bronchodilator responses, implying that a reflex pathway through vagal nerves is involved in this phenomenon. These results suggest that pulmonary and bronchial C-fiber receptors may be involved as sensory receptors in NAIS reflex bronchodilatation.

Airway Resistance↗

Deficiency of density-dependent regulation of cell growth in the culture of skin fibroblasts from patients with mucolipidosis III.

Cultured skin fibroblast cells were prepared from two patients with mucolipidosis III (ML III), which is a genetic disorder characterized by low activities of multiple lysosomal enzymes in fibroblasts. Genetic complementation analysis of fused fibroblast hybrids revealed that the patients were classified in different complementation groups. Growth curves of fibroblasts of ML III patients in culture were compared with those of fibroblasts of Sanfilippo's syndrome patients as well as of the normal fibroblasts. Normal and Sanfilippo fibroblasts gave essentially the same sigmoid curve of cell growth. However, although both ML III cell lines grew at the normal rate in the initial logarithmic phase, they continued to proliferate actively even after the cultures reached confluency. This is the first report to demonstrate the deficiency of density-dependent regulation of cell growth in the culture of nontransformed cell types. Therefore, the culture of skin fibroblasts of ML III patients may serve as a useful experimental model for investigating the regulation of cell proliferation in vitro.

Adult↗

Levels of creatine kinase activity in cartilage of tubular and nontubular bone in relation to pathogenesis of achondroplasia.

For the purpose of investigating events of energy metabolism in growing cartilages of human tubular and nontubular bones, measurements of creatine kinase (CK), lactate dehydrogenase (LDH), and aldolase of freshly excised cartilages in normal and pathologic conditions were performed. The level of cartilage CK of the normal phalanges (tubular bone) and exostosis was significantly less than those of the normal iliac bones (nontubular bone) and the achondroplasia tubular bone. A similar difference was also found between the tubular and the nontubular bone of an autopsy subject with achondroplasia. There was no statistical difference in the levels of LDH and aldolase in the cartilage between the tubular and the nontubular bone. These observations together with results obtained from iliac and limb cartilages of the newborn rat suggest the hypothesis that CK activity of cartilage of the tubular and the nontubular bone is different; cartilage of achondroplasia tubular bone has CK activity similar to that of normal nontubular bone cartilage.

Achondroplasia↗