[Show abstract][Hide abstract] ABSTRACT: Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases, produces a unique lysine-specific cysteine proteinase (Lys-gingipain, Kgp) implicated in the virulence of this organism. Our observations show the expression of a catalytically active recombinant Kgp in a P. gingivalis Kgp-null mutant and the restoration of its functions by the use of a shuttle plasmid vector stable in P. gingivalis. The Kgp-expressing mutant exhibited a similar catalytic activity to that of the wild-type strain. This mutant also restored the ability to form black-pigmented colonies on blood agar plates and to generate a 19-kDa haemoglobin receptor protein responsible for haemoglobin binding. In order to establish the importance of the active-site Cys residue and elucidate its role in bacterial black pigmentation we constructed three Kgp mutants with changed potential active-site Cys residues. The cells expressing a single mutation (C476A) showed the high Kgp activity and the black pigmentation. In contrast, the cells expressing the single mutant (C477A) and the double mutant (C476A/C477A) exhibited neither Kgp activity nor black pigmentation. These results indicate that the 477th Cys residue is essential for both the Kgp activity and the black pigmentation of P. gingivalis.
[Show abstract][Hide abstract] ABSTRACT: Berberine, an isoquinoline alkaloid isolated from several medicinal plants, has been reported to possess anti-bacterial, anti-inflammatory and antitumor properties. Although berberine also inhibits osteoclastogenesis and bone resorption, the molecular machinery for its inhibitory effects remains unknown. This study focused on the suppressive effects of berberine on receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL)-induced osteoclastogenesis and survival. Berberine inhibited RANKL-mediated osteoclast formation and survival while having no cytotoxic effects on bone marrow macrophages or osteoblastic cells. Berberine attenuated RANKL-induced activation of NF-kappaB through inhibiting phosphorylation at the activation loop of IkappaBalpha kinase beta, phosphorylation and degradation of IkappaBalpha, and NF-kappaB p65 nuclear translocation. RANKL-induced Akt phosphorylation was strongly inhibited by berberine; however, neither monocyte/macrophage-colony stimulating factor (M-CSF)-induced nor insulin-induced Akt activation was inhibited by the drug. Under M-CSF- and RANKL-deprived condition, berberine increased the active form of caspase-3 in osteoclasts. By contrast, berberine did not potentiate the activation of caspase-3 in M-CSF-deprived bone marrow macrophages. These findings indicate that berberine inhibits osteoclast formation and survival through suppression of NF-kappaB and Akt activation and that both pathways in the osteoclast lineage are highly sensitive to berberine treatment.
No preview · Article · Mar 2008 · European Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: The present experiments were carried out to examine feeding effects on hypocalcemia induced by salicylic acid derivatives. When a large dose of aspirin was administered orally to rats of different ages (up to 45 weeks of age), hypocalcemia was observed at all ages. Because hypocalcemia was induced in both the fasting and non-fasting rats by intravenous injection of salicylic acid (SA, main metabolite of aspirin), hunger sensation was unlikely to be the cause of hypocalcemia induced by SA derivatives. Aspirin-induced hypocalcemia was maintained under fasting conditions and recovered to a normal calcium range by refeeding a normal diet. On the other hand, hypocalcemia was not resolved but strengthened by refeeding a calcium-deficient diet, indicating that hypocalcemia induced by SA derivatives is not due to the inhibition of calcium absorption from the intestine. Thus, local hormones from the gastrointestinal tract and/or calcitonin secreted by feeding stimulation appear to be involved in hypocalcemia, which can be masked by a sufficient supply of dietary calcium from the intestine. In conclusion, the present study suggests that taking calciumenriched meals before administrating a large dose of aspirin is necessary to prevent or minimize hypocalcemia.
No preview · Article · Dec 2007 · Journal of Oral Biosciences
[Show abstract][Hide abstract] ABSTRACT: We investigated the relationship between the membrane potential of frog taste cells in the fungiform papillae and the tonic
discharge of parasympathetic efferent fibers in the glossopharyngeal (GP) nerve. When the parasympathetic preganglionic fibers
in the GP nerve were kept intact, the mean membrane potential of Ringer-adapted taste cells was −40 mV but decreased to −31
mV after transecting the preganglionic fibers in the GP nerve and crushing the postganglionic fibers in the papillary nerve.
The same result occurred after blocking the nicotinic acetylcholine receptors on parasympathetic ganglion cells in the tongue
and blocking the substance P neurokinin-1 (NK-1) receptors in the gustatory efferent synapses. This indicates that the parasympathetic
nerve (PSN) hyperpolarizes the membrane potential of frog taste cells by −9 mV. Repetitive stimulation of a transected GP
nerve revealed that a −9-mV hyperpolarization of taste cells maintained under the intact GP nerve derives from an ∼10-Hz discharge
of the PSN efferent fibers. The mean frequency of tonic discharges extracellularly recorded from PSN efferent fibers of the
taste disks was 9.1 impulses/s. We conclude that the resting membrane potential of frog taste cells is continuously hyperpolarized
by on average −9 mV by an ∼10-Hz tonic discharge from the parasympathetic preganglionic neurons in the medulla oblongata.
[Show abstract][Hide abstract] ABSTRACT: Pepstatin A is well known to be an inhibitor of aspartic proteinases such as pepsin, cathepsins D and E. Except for its role
as a proteinase inhibitor, however, the pharmacological action of pepstatin A upon cells remain unclear. In this study, we
found that pepstatin A suppressed receptor activator of NF-κB ligand (RANKL)–induced osteoclast differentiation. Pepstatin
A suppressed the formation of multinuclear osteoclasts dose-dependently. This inhibition of the formation only affected osteoclast
cells, i.e., not osteoblast-like cells. Furthermore, pepstatin A also suppressed differentiation from pre-osteoclast cells to mononuclear
osteoclast cells dose-dependently. This inhibition seems to be independent of the activities of proteinases such as cathepsin
D, because the formation of osteoclasts was not suppressed with the concentration that inhibited the activity of cathepsin
D. Cell signaling analysis indicated that the phosphorylation of ERK was inhibited in pepstatin A-treated cells, while the
phosphorylation of IκB and Akt showed almost no change. Furthermore, pepstatin A decreased the expression of nuclear factor
of activated T cells c1 (NFATc1). These results suggest that pepstatin A suppresses the differentiation of osteoclasts through
the blockade of ERK signaling and the inhibition of NFATc1 expression.
No preview · Article · Apr 2006 · Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Glycosphingolipids are thought to play important roles in the development and function of several tissues, although the function of glycolipids in osteoclastogenesis has not been clearly demonstrated. In the present study, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glucosylceramide synthase inhibitor, completely inhibited osteoclastogenesis induced by macrophage-colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Following treatment with D-PDMP, nearly all glycosphingolipid expression was dramatically reduced on the surface of bone marrow cells, which suggests that glycosphingolipids are necessary for osteoclastogenesis. To determine which kinds of glycolipids are important for osteoclastogenesis, we added several types of purified glycolipids to D-PDMP treated bone marrow cells, as the precursor of osteoclasts is known to express glucosylceramide (GlcCer) and lactosylceramide (LacCer). Following treatment with RANKL, ganglioside GM3 and GM1 were increased in the treated bone marrow cells, whereas other types were not detected using thin layer chromatography analysis. In cells cultured with those glycolipids, exogenously added LacCer rescued osteoclastogenesis blocking by D-PDMP. Furthermore, receptor activator of nuclear factor kappaB (RANK) induced the recruitment of tumor necrosis factor (TNF)-associated factors 2 and 6 (TRAF2 and 6, respectively) to the cytoplasmic tail of RANKL with activated IkappaB kinase and IkappaB phosphorylation, while D-PDMP treatment inhibited RANKL and induced IkappaB phosphorylation, and that inhibition was recovered by LacCer. In addition, RANK, TRAF2, TRAF6, and LacCer were found localized in lipid rafts on the cell surfaces. These results suggest that glycosphingolipids, especially LacCer, are important for the initial step of RANKL-induced osteoclastogenesis via lipid rafts.
No preview · Article · Apr 2006 · Journal of Pharmacological Sciences
[Show abstract][Hide abstract] ABSTRACT: We studied the anatomical properties of parasympathetic postganglionic neurons in the frog tongue and their modulatory effects on taste cell responses. Most of the parasympathetic ganglion cell bodies in the tongue were found in extremely small nerve bundles running near the fungiform papillae, which originate from the lingual branches of the glossopharyngeal (GP) nerve. The density of parasympathetic postganglionic neurons in the tongue was 8000-11,000/mm(3) of the extremely small nerve bundle. The mean major axis of parasympathetic ganglion cell bodies was 21 microm, and the mean length of parasympathetic postganglionic neurons was 1.45 mm. Electrical stimulation at 30 Hz of either the GP nerve or the papillary nerve produced slow hyperpolarizing potentials (HPs) in taste cells. After nicotinic acetyl choline receptors on the parasympathetic ganglion cells in the tongue had been blocked by intravenous (i.v.) injection of D-tubocurarine (1 mg/kg), stimulation of the GP nerve did not induce any slow HPs in taste cells but that of the papillary nerve did. A further i.v. injection of a substance P NK-1 antagonist, L-703,606, blocked the slow HPs induced by the papillary nerve stimulation. This suggests that the parasympathetic postganglionic efferent fibers innervate taste cells and are related to a generation of the slow HPs and that substance P is released from the parasympathetic postganglionic axon terminals. When the resting membrane potential of a taste cell was hyperpolarized by a prolonged slow HP, the gustatory receptor potentials for NaCl and sugar stimuli were enhanced in amplitude, but those for quinine-HCl and acetic acid stimuli remained unchanged. It is concluded that frog taste cell responses are modulated by activities of parasympathetic postganglionic efferent fibers innervating these cells.
[Show abstract][Hide abstract] ABSTRACT: Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.
[Show abstract][Hide abstract] ABSTRACT: Osteoclastic bone resorption has recently been implicated in the tooth formation and eruption in alveolar bone. Cathepsin K (CK) is a cysteine proteinase expressed predominantly in osteoclasts and is believed to play a critical role in degradation of bone matrix proteins. Here we present evidence that the alveolar bone resorption is essential for the tooth formation and that eruption proceeds normally in CK-deficient (CK-/-) mice. Radiographic and histological analyses revealed that the alveolar bone from these animals had no significant abnormalities during the tooth development between 5 and 28 days after birth. The tooth crown was normally erupted through the alveolar bone layer at 28 days after birth. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the alveolar bone around the tooth germ was apparently increased in 5-day-old CK-/- mice compared with age-matched littermates. More important, however, the immunohistochemical localization of matrix metalloproteinase-9 (MMP-9) was clearly increased in the CK-/- osteoclasts. In contrast, no significant difference in the immunoreactivity for cathepsin D was observed between the CK-/- osteoclasts and the wild-type ones. These results indicate that CK-/- osteoclasts are fully differentiated and are capable of degrading the organic phase of alveolar bone during the tooth formation and eruption, which may result from the compensatory action by MMP-9 increasingly expressed in the osteoclasts.
No preview · Article · May 2003 · Journal of Pharmacological Sciences
[Show abstract][Hide abstract] ABSTRACT: alpha 2-Macroglobulin (alpha 2M) is an abundant glycoprotein with the intrinsic capacity for capturing diverse proteins for rapid delivery into cells. After internalization by the receptor- mediated endocytosis, alpha 2M-protein complexes were rapidly degraded in the endolysosome system. Although this is an important pathway for clearance of both alpha 2M and biological targets, little is known about the nature of alpha 2M degradation in the endolysosome system. To investigate the possible involvement of intracellular aspartic proteinases in the disruption of structural and functional integrity of alpha 2M in the endolysosome system, we examined the capacity of alpha 2M for interacting with cathepsin E and cathepsin D under acidic conditions and the nature of its degradation. alpha 2M was efficiently associated with cathepsin E under acidic conditions to form noncovalent complexes and rapidly degraded through the generation of three major proteins with apparent molecular masses of 90, 85 and 30 kDa. Parallel with this reaction, alpha 2M resulted in the rapid loss of its antiproteolytic activity. Analysis of the N-terminal amino-acid sequences of these proteins revealed that alpha 2M was selectively cleaved at the Phe811-Leu812 bond in about 100mer downstream of the bait region. In contrast, little change was observed for alpha 2M treated by cathepsin D under the same conditions. Together, the synthetic SPAFLA peptide corresponding to the Ser808-Ala813 sequence of human alpha 2M, which contains the cathepsin E-cleavage site, was selectively cleaved by cathepsin E, but not cathepsin D. These results suggest the possible involvement of cathepsin E in disruption of the structural and functional integrity of alpha 2M in the endolysosome system.
No preview · Article · Apr 2003 · European Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: α2-Macroglobulin (α2M) is an abundant glycoprotein with the intrinsic capacity for capturing diverse proteins for rapid delivery into cells. After internalization by the receptor- mediated endocytosis, α2M-protein complexes were rapidly degraded in the endolysosome system. Although this is an important pathway for clearance of both α2M and biological targets, little is known about the nature of α2M degradation in the endolysosome system. To investigate the possible involvement of intracellular aspartic proteinases in the disruption of structural and functional integrity of α2M in the endolysosome system, we examined the capacity of α2M for interacting with cathepsin E and cathepsin D under acidic conditions and the nature of its degradation. α2M was efficiently associated with cathepsin E under acidic conditions to form noncovalent complexes and rapidly degraded through the generation of three major proteins with apparent molecular masses of 90, 85 and 30 kDa. Parallel with this reaction, α2M resulted in the rapid loss of its antiproteolytic activity. Analysis of the N-terminal amino-acid sequences of these proteins revealed that α2M was selectively cleaved at the Phe811-Leu812 bond in about 100mer downstream of the bait region. In contrast, little change was observed for α2M treated by cathepsin D under the same conditions. Together, the synthetic SPAFLA peptide corresponding to the Ser808–Ala813 sequence of human α2M, which contains the cathepsin E-cleavage site, was selectively cleaved by cathepsin E, but not cathepsin D. These results suggest the possible involvement of cathepsin E in disruption of the structural and functional integrity of α2M in the endolysosome system.
[Show abstract][Hide abstract] ABSTRACT: To investigate the relationship between histological changes and distributions of medullasin, a neutrophil elastase-like serine proteinase, in phenytoin-induced gingival overgrowth, we established a rat model of gingival overgrowth. Thirty-two, 20-day-old male Fischer 344 rats were fed a diet containing phenytoin and sacrificed at 1, 2, 4 and 8 weeks. Control rats (n = 40) were fed the same diet, but without the drug and killed at the same weeks as experimental rats (n = 32) and 0 week (n = 8). The mandible specimens were resected and sectioned bucco-lingually between the first and second molars. A marked inflammatory-cell infiltration and elongated rete pegs were seen in the phenytoin-treated group. The extent of the overgrowth assessed by computer image analysis and the density of medullasin-positive cells by immunohistochemistry in the approximal gingiva showed a significant increase in the phenytoin-treated group compared to the control group. A marked infiltration of the positive cells in experimental rats was observed as early as 2 weeks when gingival overgrowth was not fully established. Medullasin-positive cells were mostly neutrophils and partly macrophage-like cells. These findings suggest that medullasin may be involved in mainly host defense and secondarily collagen metabolism in the phenytoin-induced rat model of gingival overgrowth.
No preview · Article · Aug 2002 · The Japanese Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Proteases are involved in the invasion and metastasis of carcinoma cells. In vivo, oral carcinoma cells easily invade the bone tissue and metastasize to the submandibular and neck lymph nodes. Cathepsin expression has been shown in some neoplastic tissues and serves as a prognostic indicator. The purpose of this study was to investigate the relationship between clinicopathohistologic grades and cathepsin expressions in oral squamous cell carcinoma and to investigate which cathepsin provides prognostic information for patients with oral carcinoma.
Immunohistochemical studies were performed on 78 carcinoma samples with monoclonal antibodies against cathepsins B, H, and L, and a polyclonal antibody against cathepsin D. Serial sections were stained by hematoxylin-eosin staining and classified by Anneroth's classification. Cathepsin B, H, L and D activities of blood serum were determined. Positive results indicative of the presence of cathepsin were investigated to determine any correlation between a particular cathepsin and histologic malignancy grades, tumor cell growth, serum cathepsin activities, and clinical factors.
Cathepsins B, H, L, and D were positive in every case. Although the labeling indices for cathepsins B (CB-LI), H (CH-LI), and D (CD-LI) for the cancer cases showed significant differences from those of controls, cathepsin L (CL-LI) of cancer cases showed no difference from that of controls (P <.05). A close correlation was found between CD-LI and T categories of TNM classification (P <.05), and between CD-LI and PCNA-LI (P <.05). Furthermore, a close correlation was found between CD-LI and N categories in TNM classification (P <.05). Pathologically, a close correlation was found between CB-LI or CD-LI and the pattern and/or stage of invasion (P <.05).
Cathepsin D and B expression were closely correlated with carcinoma invasion and progression. These proteases may be useful in determining the prognoses of patients with oral carcinoma.
No preview · Article · Apr 2002 · Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology
[Show abstract][Hide abstract] ABSTRACT: Glycosphingolipids and their metabolites play important roles in a variety of biological processes. Several signal molecules
are localized in a glycolipid-enriched microdomain on the cell surface, and their signals are regulated by the glycolipid
composition. However, the function of glycolipids in osteoclastogenesis has not been clearly understood. We found thatd-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP), a glucosylceramide synthase inhibitor, completely inhibits the osteoclast formation induced by macrophage-colony-stimulating
factor and receptor activator of nuclear factor-κB ligand (RANKL) in a dose-dependent manner. Expression of RANK, the receptor
of RANKL, induced by macrophage colony-stimulating factor, was reduced markedly ind-PDMP-treated cells. d-PDMP also inhibited the phosphorylation of the inhibitor of nuclear factor-κB and extracellular signal-regulated kinase 1/2
induced by RANKL. In several experiments with the addition of glycolipids tod-PDMP-treated purified bone marrow cells, lactosylceramide (LacCer) strongly affected the differentiation into tartrate-resistant
acid phosphatase mononucleated cells, but not positive multinucleated cells. GM3 and GM1 also recovered, but less effectively
compared with LacCer. Moreover, exogenous LacCer recovered the reduced expression of RANK and the phosphorylation of inhibitor
of NF-κB and extracellular signal-regulated kinase 1/2 after stimulation by RANKL at the same level of cells without d-PDMP treatment. Our data suggest that glycosphingolipids, especially LacCer, are necessary for the initiation step of RANKL-induced
Full-text · Article · Dec 2001 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Tooth cementum, a calcified hard tissue covering the root surfaces, is an important component connecting the teeth to the collagenous fibres of the periodontal ligament. Although the overall composition of cementum may closely resemble that of bone, each part has not been fully characterized. Here, the localization of the matrix gamma-carboxyglutamic acid (Gla) protein (MGP), one of the major Gla-containing proteins in the body, in cementum was investigated using immunohistochemistry and in situ hybridization. (1) Strong MGP antigenicity was observed in the acellular cementum, but was only moderate in the cellular cementum; (2) polygonal periodontal ligament cells facing the acellular cementum and the uncalcified cellular cementum expressed MGP mRNA, indicating that these cells produced MGP and deposited it on the cementum; (3) MGP accumulated at the junction between the uncalcified and calcified cellular cementum; and (4) the distribution pattern of MGP antigenicity resembled that of osteopontin. As one function of MGP could be as a negative regulator for mineral apposition, the expression of MGP in the cells adjacent to the cementum may be important to prevent hyperapposition of minerals.
No preview · Article · Aug 2001 · Archives of Oral Biology
[Show abstract][Hide abstract] ABSTRACT: The mechanism controlling the disappearance of osteoclasts from bone surfaces after bone resorption in vivo is largely unknown. This is because there is no suitable experimental system to trace the final fate of osteoclasts. Here, we used an experimental model of tooth movement in rats to show that preexisting osteoclasts disappeared from the bone surface through apoptosis during a force-induced rapid shift from bone resorption to formation. On the distal alveolar bone surface of the maxillary molar in growing rats, many mature osteoclasts were present. When light tensional force was applied to the bone surface through an orthodontic appliance, these preexisting osteoclasts gradually disappeared. One day after the application of force, about 24% of the osteoclasts exhibited apoptotic morphology and the proportion of apoptotic cells was increased to 41% by day 2, then decreased afterward. These changes were undetectable on the control distal alveolar bone surface, which is free from tensional force. As shown by in situ hybridization, a marked increase in transforming growth factor β1 (TGF-β1) and osteoprotegerin (OPG) messenger RNA (mRNA) was observed in the stretched cells on the tensioned distal bone surface, simultaneously with the loss of osteoclasts. Both of these factors are known to have a negative effect on osteoclast recruitment and survival. As early as 2 days after force application, some of these stretched cells were identified as cuboidal osteoblasts showing intense signals for both factors. Our data suggest there may be a sequential link in tensional force applied on the bone lining cells, up-regulation of TGF-β1/OPG, and disappearance of osteoclasts.
Full-text · Article · Sep 2000 · Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research
[Show abstract][Hide abstract] ABSTRACT: The present study demonstrates that loss of cell adhesion potently promotes apoptosis in osteoclasts, a process termed "anoikis." When purified mature rabbit osteoclasts were cultured on plastic for 18 h, about 25% of them were spontaneously committed to apoptosis. The death rate increased more than twofold, after osteoclasts were subjected to suspension culture in inverted Terasaki plates. The osteoclast anoikis was significantly prevented by bongkrekic acid, an inhibitor of mitochondrial permeability transition (PT), and z-VAD-FMK, a caspase inhibitor, suggesting involvement of mitochondrial PT and caspase activation in the death process. Colony-stimulating factor-1 (CSF-1), receptor activator of NF-kappaB ligand (RANKL), and calcitonin protected adherent osteoclasts, but not floating osteoclasts from anoikis. These data show that adhesion is a primary requirement for osteoclast survival.
No preview · Article · May 2000 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N-glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N-glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either high-mannose or complex-type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high-mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.
Full-text · Article · Jan 2000 · European Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: To clarify the possible pathophysiological role of medullasin, a neutrophil elastase-like proteinase, in nifedipine (NF)-induced gingival overgrowth, a rat model of gingival overgrowth was first established using a diet containing NF. The relation between histopathological changes and the distribution of the proteinase was then investigated. Thirty-two, specific pathogen-free 20 day-old, male, Fisher 344 rats were fed a diet containing NF and killed at 2, 8, 16 and 32 weeks. Control rats (n = 32) were fed the same diet but without the drug. The mandible of each rat was resected and sectioned at 4-microm thickness buccolingually between the first and second molars. Computer image analysis was used to evaluate the extent of overgrowth in the approximal gingiva of each sample. To examine medullasin activity, the mean percentage of medullasin-positive cells per total cells counted in the pocket epithelium and the connective tissue adjacent to the epithelium of approximal gingiva was determined immunohistochemically. The height of the mid-portion and the area in NF-treated group increased significantly with time (with the exception of area at 2 weeks) compared with the corresponding regions in the control group. A marked inflammatory-cell infiltration and elongated rete pegs, especially in the mid-portion of approximal gingiva, were seen in the NF-treated group. The mean percentages of medullasin-positive cells in the NF-treated group at 8, 16 and 32 weeks were significantly higher than those of the control. Although medullasin-positive cells were mainly neutrophils, in several samples of the NF-treated group they were recognized as macrophage-like. These findings suggest that medullasin may be involved in host defence and immunoregulation in a NF-induced rat model of gingival overgrowth.
No preview · Article · Nov 1998 · Archives of Oral Biology
[Show abstract][Hide abstract] ABSTRACT: At the maturation stage, the ameloblasts of the rat incisor incorporate iron, supplied through the bloodstream, and deposit it into the surface layer of the enamel. In this unique iron transport system, ferritin functions as a transient iron reservoir in the cells. Here the expression of ferritin mRNA and its localization in the rat enamel organ was examined. Among various tissues, the enamel organ showed the highest expression for both ferritin H- and L-chain mRNA, as quantified by reverse transcription-polymerase chain reaction. In situ hybridization using digoxigenin-labelled cRNA probes for each chain demonstrated that both ferritin H- and L-chain mRNA were abundantly expressed in presecretory and secretory ameloblasts. The intensity of the positive hybridization signal gradually decreased toward the incisal direction. Differing from the mRNA localization, ferritin protein was immunologically undetectable in presecretory or secretory ameloblasts but was found in ameloblasts at the maturation stage, into which iron is known to be incorporated from the bloodstream. Thus, the expression of ferritin mRNA precedes the protein expression in the developmental stages of rat incisor ameloblasts, and the translation of ferritin and its half-life are probably controlled by the iron entry, as has been reported for other cell types.
No preview · Article · Jun 1998 · Archives of Oral Biology