Biomedical subjects

Masatoshi Kataoka

Publications and source records attributed to Masatoshi Kataoka.

At least 19 recordsLinked to original sources

PubMed2006

Usefulness of the 5' region of the cDNA encoding acidic ribosomal phosphoprotein P0 conserved among rats, mice, and humans as a standard probe for gene expression analysis in different tissues and animal species.

Housekeeping genes are often used as internal standards for gene expression analysis. When steady-state transcript levels of 4 typically used housekeeping genes, i.e., beta-actin, glyceraldehyde 3-phosphate dehydrogenase, cyclophilin, and acidic ribosomal phosphoprotein P0 (36B4), were evaluated in various rat tissues, the 36B4 gene seemed to be the most suitable as a standard to compare the expression levels of genes among different tissues. Next, for possible quantitative comparison of the expression level of this gene among different animal species, we compared the nucleotide sequence of the cDNA of 36B4 among rats, mice, and humans. As a result, highly conserved regions showing more than 97.5% identities were observed in the 5' portion of its open reading frame. When samples of synthesized mRNA encoding rat, mouse, and human 36B4 were hybridized with the entire cDNA encoding rat 36B4 as a probe, hybridization signals of mRNAs of mouse and human 36B4 were much weaker than those of mRNA encoding rat 36B4. However, when they were hybridized with an oligonucleotide probe corresponding to the highly conserved regions, they showed similar signal intensities. Thus, these highly conserved regions of the cDNA encoding 36B4 were concluded to be an effective standard for use in gene expression analysis.

Animals↗

PubMed2006

The structure of the second cytosolic loop of the yeast mitochondrial ADP/ATP carrier AAC2 is dependent on the conformational state.

To detect structural changes in the second cytosolic loop of the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae AAC2, we prepared 20 single cysteine mutants by replacing each amino acid in the S213 to L232 region. All single cysteine mutants were fully functional, because they could restore growth on glycerol of a yeast strain lacking functional ADP/ATP carriers. First, these single-Cys mutants were treated with carboxyatractyloside to lock the carrier in the cytosolic state or with bongkrekic acid to generate the matrix state, and then with the membrane-impermeable SH reagent eosin-5-maleimide (EMA) to probe accessibility. The amino acid residues S213C, L214C, F231C and L232C were not labeled, indicating that these 4 residues must have been buried in the membrane, whereas the region between residues K215 and S230 is accessible to labeling and must, therefore, have protruded into the aqueous phase. Residue L218C showed strong resistance against EMA labeling regardless of the state of the carrier, but the reason for such behavior is unclear. On the contrary, the labeling of the residues between F227C and S230C was strongly dependent on the state of the carrier. Thus, the C-terminal region of the second cytosolic loop in AAC2 changes its environment when the carrier cycles between the matrix and cytosolic state.

Amino Acid Substitution↗

PubMed2006

High-turnover osteoporosis is induced by cyclosporin A in rats.

Cyclosporin A (CsA) is used widely as an immunosuppressive agent, but it induces osteoporosis as a prominent side effect. To elucidate the mechanisms involved in CsA-induced osteoporosis, the effects of CsA on bone metabolism were investigated in a rat experimental model. Fifteen-day-old rats were fed a powdered diet containing or lacking CsA for 8-30 days. Analysis was performed by micro-computed tomography (muCT) and light microscopy to examine histomorphometric changes in rat tibiae on days 8, 16, and 30. Plasma parathyroid hormone (PTH) and osteocalcin (OCN) levels were determined by enzyme-linked immunosorbent assay (ELISA) on days 8, 16, and 30. The expression of OCN, osteopontin (OPN), and cathepsin K mRNAs in tibial bone marrow was examined by Northern blot analysis on days 8 and 16. Although no significant differences were observed in tibial length during the experimental periods, or in histomorphometric parameters on day 8, an apparent decrease in bone volume was observed in the CsA-treated group after day 16. Histologic analysis showed that the number of osteoblasts and osteoclasts on the surface of trabecular bone in the CsA-treated group had increased significantly on day 16. Plasma PTH and OCN levels in CsA-treated rats were significantly higher than those in control animals on day 8. Northern blot analysis revealed that the CsA-treated group showed an increase in the expression of OCN, OPN, and cathepsin K mRNAs on day 8 compared with the controls. These findings suggest that bone resorption in CsA-treated rats is induced by high-turnover osteoporosis and that bone remodeling activity may be activated by PTH.

Animals↗

PubMed2006

VDAC1, having a shorter N-terminus than VDAC2 but showing the same migration in an SDS-polyacrylamide gel, is the predominant form expressed in mitochondria of various tissues.

The voltage-dependent anion channel (VDAC) is a pore-forming protein expressed in the outer membrane of eukaryotic mitochondria. Three isoforms of it, i.e., VDAC1, VDAC2, and VDAC3, are known to be expressed in mammals; however, the question as to which is the main isoform in mitochondria is still unanswered. To address this question, we first prepared standard VDACs by using a bacterial expression system and raised various antibodies against them by using synthetic peptides as immunogens. Of the three bacterially expressed VDAC isoforms, VDAC3 showed faster migration in SDS-polyacrylamide gels than VDAC1 and VDAC2, although VDAC2 is longer than VDAC1 and VDAC3, due to a 12-amino acid extension of its N-terminal region. Even with careful structural characterization of the expressed VDACs by LC-MS/MS analysis, serious structural modifications of VDACs causing changes in their migration in SDS-polyacrylamide gels were not detected. Next, immunoreactivities of the raised antibodies toward these bacterially expressed VDAC isoforms were evaluated. Trials to prepare specific antibodies against the three individual VDAC isoforms were not successful except in the case of VDAC1. However, using a synthetic peptide corresponding to the highly conserved region among the three VDACs, we were successful in preparing an antibody showing essentially equal immunoreactivities toward all three VDACs. When mitochondrial outer membrane proteins of various rat tissues were subjected to 2-dimensional electrophoresis followed by immunoblotting with this antibody, six immunoreactive protein spots were detected. These spots were characterized by LC-MS/MS analysis, and the signal intensities among the spots were compared. As a result, the signal intensity of the spot representing VDAC1 was the highest, and thus, VDAC1 was concluded to be the most abundantly expressed of the three VDAC isoforms in mammalian mitochondria.

Amino Acid Sequence↗

PubMed2006

Norepinephrine stimulates calprotectin expression in human monocytic cells.

BACKGROUND AND OBJECTIVE: Calprotectin is composed of two proteins, S100A8 and S100A9, which are S100 family members, and is detected in gingival crevicular fluid and gingival tissue with inflammation. The release and production of calprotectin are regulated by lipopolysaccharides of periodontopathic bacteria and cytokines. Emotional or psychological stress, a risk factor of periodontal disease, is transmitted by stress modulators including norepinephrine and cortisol. The aim of the present study was to investigate the effect of stress on calprotectin expression using norepinephrine and cortisol. METHODS: U-937 cells, a human monocytic cell line, were incubated with norepinephrine in the presence or absence of beta- or alpha-adrenergic receptor antagonists, or with cortisol. The expression of S100A8/S100A9 mRNAs was examined by northern blotting and the amount of calprotectin was measured by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPalpha (CCAAT enhancing binding protein), a transcription factor, was examined by electrophoretic mobility shift assay. RESULTS: Norepinephrine stimulated the expression of S100A8/S100A9 mRNAs via beta-adrenergic receptors in U-937 cells and significantly increased calprotectin production to about 3.6-fold that of the control. However, cortisol had no effect on calprotectin expression at the mRNA and protein levels. Norepinephrine elevated C/EBPalpha DNA binding activity, but cortisol did not increase the activity. CONCLUSION: Norepinephrine, a stress modulator, stimulated calprotectin expression in human monocytic cells. Calprotectin expression may be regulated by stress in addition to inflammatory factors.

Adrenergic alpha-Agonists↗

PubMed2005

Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry.

The mitochondrial membrane potential (DeltaPsi(m)) is an important indicator of the energetic state of both the mitochondria and the cells. To develop a sensitive, convenient, and rapid method for the measurement of DeltaPsi(m), we carried out cell fluorescence assays using the Agilent 2100 bioanalyzer system which, unlike the conventional flow cytometry, is based on microfluidic technology employing fluorescence detection with a 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)(3)) fluorescent probe. The use of DiOC(6)(3) in the fluorometer was shown to be feasible for monitoring variations in DeltaPsi(m) in the mitochondria isolated from rat liver and treated with rotenone, succinate, ADP, and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Flow cytometry analysis showed severe reduction of fluorescence intensity in Jurkat cells after treatment with 1.0 and 10 microM FCCP. However, fluorescence microscopy demonstrated obvious accumulation of fluorescence in the mitochondria and induction of diffuse cytoplasmic fluorescence not localized to the mitochondria in these cells. The dose response range of DiOC(6)(3) in the Agilent 2100 bioanalyzer system for yielding sufficient fluorescence intensity in the mitochondria of the cells was 20 nm-2.0 microM. Furthermore, significant reduction of fluorescence intensity in the cells stained with 2.0 microM DiOC(6)(3) was observed after treatment with 10 microM FCCP for 30 min. These results indicate that the Agilent 2100 bioanalyzer is potentially useful for monitoring DeltaPsi(m) in cell assays.

Adenosine Diphosphate↗

PubMed2005

Two critical factors affecting the release of mitochondrial cytochrome C as revealed by studies using N,N'-dicyclohexylcarbodiimide as an atypical inducer of permeability transition.

N,N'-dicyclohexylcarbodiimide (DCCD) was earlier reported to have stimulatory effects on mitochondrial respiration and to induce mitochondrial swelling, when it was added to mitochondrial suspensions. These data seem to imply that DCCD caused the mitochondrial permeability transition (PT), but this possibility had never been investigated. In the present study, effects of DCCD on the mitochondrial structure and function were studied in detail. DCCD was found to induce mitochondrial PT in a cyclosporine A-insensitive manner. Electron microscopic analysis also supported the induction of the mitochondrial PT by DCCD. However, different from many other PT inducers, DCCD failed to cause massive release of mitochondrial cytochrome c. To understand the relationship between the induction of mitochondrial PT and the release of mitochondrial cytochrome c, we compared the actions of DCCD on mitochondrial structure and function with those of Ca2+, known as an ordinary PT inducer. As a result, two parameters considered to be critical for controlling the release of mitochondrial cytochrome c on the induction of PT were mitochondrial volume and the velocity of mitochondrial oxygen consumption.

Animals↗

PubMed2005

Molecular basis of morphological changes in mitochondrial membrane accompanying induction of permeability transition, as revealed by immuno-electron microscopy.

The mitochondrial inner membrane typically shows a condensed structure when examined by electron microscopy. However, this typical structure is known to disappear upon induction of the mitochondrial permeability transition (PT). This change in the appearance of the mitochondrial membrane structure that accompanies the induction of PT is thought to reflect changes in the permeability of inner mitochondrial membrane; however, its molecular basis has remained uncertain. In the present study, changes in membrane status were examined by immuno-electron microscopy using antibodies against the voltage-dependent anion channel (VDAC), beta-subunit of F1-ATPase (F1beta), and cytochrome c (cyt. c). In control mitochondria, antibody against VDAC was observed at the rim of the mitochondria, whereas antibodies against F1beta and cytochrome c bound these molecules inside of the mitochondria. However, in PT-induced mitochondria, all three antibodies were observed at the mitochondrial rim. These results strongly suggest that the inner mitochondrial membrane is shoved to the rim region of mitochondria upon induction of mitochondrial PT.

Animals↗

PubMed2005

Drug-induced gingival overgrowth--a review.

Drug-induced gingival overgrowth is a side effect associated with 3 types of drugs: anticonvulsants (phenytoin), immunosuppressive agents (cyclosporine A), and various calcium channel blockers for cardiovascular diseases. Gingival overgrowth is characterized by the accumulation of extracellular matrix in gingival connective tissues, particularly collagenous components with various degrees of inflammation. Although the mechanisms of these disorders have not been elucidated, recent studies suggest that these disorders seem to be induced by the disruption of homeostasis of collagen synthesis and degradation in gingival connective tissue, predominantly through the inhibition of collagen phagocytosis of gingival fibroblasts. The integrins are a large family of heterodimeric transmembrane receptors for extracellular matrix molecules. alpha2beta1 integrin serves as a specific receptor for type I collagen on fibroblasts, and alpha2 integrin has been shown to play a crucial role in collagen phagocytosis. Actin filaments, which are assembled from monomers and oligomers, are involved in collagen internalization after binding to integrins. Furthermore, the implication of intracellular calcium in the regulation of integrin-mediated binding activity and gelsolin activity, known as a calcium-dependent actin-severing protein, is also described. In this review, we focus on collagen metabolism in drug-induced gingival overgrowth, focusing on the regulation of collagen phagocytosis in fibroblasts.

Anticonvulsants↗

PubMed2005

Calprotectin expression in human monocytes: induction by porphyromonas gingivalis lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1beta.

BACKGROUND: Calprotectin is a major cytosolic protein of monocytes and granulocytes. It is increased in inflammatory tissues and is detected at high levels in the gingival crevicular fluid (GCF) of periodontitis patients. We previously reported that lipopolysaccharide of Porphyromonas gingivalis (P-LPS) and cytokines induced the release of calprotectin from monocytes isolated from human peripheral blood. The mechanisms of calprotectin expression and presence of its regulation factors in periodontal disease are unknown. On the other hand, P-LPS and cytokines are significant etiologic factors in the initiation and progression of periodontal diseases. In this study, we investigated the expression and production of calprotectin from human monocytes by examining the effects of lipopolysaccharide of P-LPS, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). METHODS: Monocytes were isolated from the peripheral blood of healthy donors and cultured in the presence or absence of P-LPS, TNF-alpha, or IL-1beta. The expressions of calprotectin mRNAs (MRP8 and MRP14) were detected by Northern blotting. The contents of calprotectin in the cells and medium fractions were determined by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPalpha, a transcription factor of MRP14, was assessed by electrophoretic mobility shift DNA-binding assay (EMSA). RESULTS: P-LPS, TNF-alpha, and IL-1beta induced MRP8/14 mRNAs and calprotectin production in monocytes. These factors also induced DNA CEBPalpha binding activity in monocytes. P-LPS increased MRP14 mRNA expression in monocytes to the maximum level, about two times the control level after 24 hours treatment, but did not enhance the basal level of MRP8. When the effects of TNF-alpha and IL-1beta on those mRNAs were investigated, both MRP8 and MRP14 significantly increased to about 2- and 2.5-fold the control level, respectively. Increases of MRP8/14 mRNA expression were followed by their protein production at about 2-fold the basal amount. DNA binding activity of C/EBPalpha was increased in P-LPS, TNF-alpha, and IL-1beta-treated monocytes. CONCLUSIONS: These results demonstrate that P-LPS, TNF-alpha, and IL-1beta induce calprotectin production from human monocytes and that this production is associated with the activation of DNA C/EBPalpha binding complex.

Blotting, Northern↗

PubMed2004

Regulation of tenascin expression in cultured rat dental pulp cells.

Tenascin (TN) is a glycoprotein of extracellular matrix abundantly present in embryonic mesenchymal tissues. Transforming growth factor-beta1 (TGF-beta1), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and retinoic acid (RA) are important regulators of dentinogenesis. Dental pulp cells have the capacity to differentiate into odontoblast-like cells. In this study, we investigated the effects of growth factors on TN expression and adhesive function using rat clonal dental pulp cells, RPC-C2A. Analyses of reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting revealed that RPC-C2A cells expressed TN molecules and that TGF-beta1, HGF, and RA increased expression of TN at the mRNA and protein level, while bFGF and EGF showed a weak effect. An adhesion assay revealed that treatment with TGF-beta1, HGF, and RA induced a marked reduction of cell attachment to fibronectin (FN)-coated surfaces, whereas there was no change with bFGF and EGF. Functional blocking of growth factor-stimulated TN protein by pretreating cells with anti-TN antibodies restored cell attachment to control levels. These findings suggest that TGF-beta1, HGF, and RA may regulate pulpal cell adhesion to FN-coated surfaces and that this effect is mediated by TN.

Animals↗

PubMed2004

Usefulness of microchip electrophoresis for reliable analyses of nonstandard DNA samples and subsequent on-chip enzymatic digestion.

The Hitachi SV1100 utilizes capillary electrophoresis on a microchip that is capable of rapidly sizing DNA fragments. Reproducibility of electrophoresis in different channels was shown by comparing the migration times of the internal controls, DNA fragments of 100 and 800 bp. The range of DNA sizing for this microchip is between 100 and 800 bp, and accuracy in sizing of a 322 bp DNA fragment of a pUC118 PvuII digest was observed, independent of DNA concentration. Although relatively good quantification of this fragment was observed with a DNA concentration of 1.83 ng.microL(-1), error increased in a dose-dependent manner. Furthermore, the feasibility of sequential analysis with this microchip was shown by the reproducibility of successive electrophoreses of the internal control in one channel. When the pUC118 PvuII digest was treated with endonuclease KpnI on the microchip for 10 min, sequential analysis showed that the 322 bp fragment completely disappeared and two peaks corresponding to the 130 and 192 bp fragments appeared. This analysis was performed within 4 min, and the peaks were estimated as 127 and 183 bp, respectively. These results indicate the potential of on-microchip endonuclease treatment of plasmid DNA with sequential analysis, offering high resolution in a short time.

DNA↗

PubMed2004

Multiple effects of DiS-C3(5) on mitochondrial structure and function.

3,3'-Dipropyl-2,2'-thiadicarbocyanine iodide [DiS-C(3)(5)], often used as a tracer dye to assess the mitochondrial membrane potential, was investigated in detail regarding its effects on the structure and function of isolated mitochondria. As reported previously, DiS-C(3)(5) had an inhibitory effect on NADH-driven mitochondrial electron transfer. On the contrary, in the presence of inorganic phosphate, DiS-C(3)(5) showed dose-dependent biphasic effects on mitochondria energized by succinate. At higher concentrations, such as 50 micro m, DiS-C(3)(5) accelerated mitochondrial oxygen consumption. Measurements of the permeability of DiS-C(3)(5)-treated mitochondrial membranes to poly(ethylene glycol) and analysis of mitochondrial configuration by transmission electron microscopy revealed that the accelerating effect of DiS-C(3)(5) on mitochondrial oxygen consumption reflects the induction of the mitochondrial permeability transition (PT). When the mitochondrial PT was induced by DiS-C(3)(5), release of mitochondrial cytochrome c was observed, as in the case of the PT induced by Ca(2+). On the contrary, at a low concentration such as 5 micro m, DiS-C(3)(5) showed an inhibitory effect on the latent oxygen consumption by mitochondria. This effect was shown to reflect inhibition of the PT induced by a low concentration of Ca(2+). Furthermore, in the absence of inorganic phosphate, DiS-C(3)(5) caused mitochondrial swelling. Under this condition, DiS-C(3)(5) caused changes in the membrane status of the mitochondria, but did not induce a release of mitochondrial cytochrome c.

Animals↗

PubMed2004

Substance P enhances the inhibition of osteoblastic cell differentiation induced by lipopolysaccharide from Porphyromonas gingivalis.

BACKGROUND: Substance P (SP) is a multifunctional neuropeptide that transmits pain signals, regulates the immune system, and may modulate emotional stress. SP stimulates bone resorption activity of osteoclasts, and SP level in gingival crevicular fluid is correlated with the degree of periodontal inflammation. However, the exact roles of SP in bone metabolism and periodontal diseases are poorly understood. To elucidate the effect of stress on bone metabolism, we investigated the effect of SP on osteoblastic cell differentiation in the presence of lipopolysaccharide from Porphyromonas gingivalis (P-LPS). METHODS: The primary osteoblastic cells were isolated from fetal rat calvaria (RC) and cultured with SP, P-LPS, and an SP antagonist (SPa). The effects of SP on bone nodule (BN), alkaline phosphatase (ALPase) activity, mRNA expressions of SP receptor, bone matrix proteins, and Cbfa 1 were investigated. RESULTS: SP stimulated the expression of SP receptor mRNA in RC cells and enhanced its expression in the presence of P-LPS (50 ng/ml). SP inhibited BN formation and ALPase activity in a dose-dependent manner (10(-7) to 10(-5) M) and further suppressed mRNA expression of bone sialoprotein, osteopontin, and osteocalcin but not of type I collagen mRNA. The inhibitory effects were enhanced in the presence of P-LPS and blocked by Spantide III. Furthermore, the expression of Cbfa 1 mRNA was also markedly suppressed in the presence of SP and P-LPS. CONCLUSION: These findings suggest that SP inhibits osteoblastic cell differentiation and may be related to bone metabolism in periodontal diseases under conditions of stress.

Alkaline Phosphatase↗

PubMed2004

Identification of possible protein machinery involved in the thermogenic function of brown adipose tissue.

Brown adipose tissue (BAT) is believed to function by dissipating excess energy in mammals. It is very important to understand the energy metabolism held in BAT since disorder of its energy-dissipating function may cause obesity or lifestyle-related diseases such as hypertension and diabetes. This function in BAT is mainly attributable to uncoupling protein (UCP), specifically expressed in its mitochondria. This protein consumes excess energy as heat by dissipating the H+ gradient across the inner mitochondrial membrane that is utilized as a driving force for ATP synthesis. In this review article, in addition to providing a brief introduction to the functional properties of BAT and UCP, we also describe and discuss properties of cultured brown adipocytes and the results of our exploratory studies on protein components involved in the energy-dissipating function in BAT.

Adipose Tissue, Brown↗

PubMed2003

Calprotectin release from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide via the CD-14-Toll-like receptor-nuclear factor kappaB pathway.

OBJECTIVES: Calprotectin is a cytosolic protein with antibacterial action in leukocytes and its level increases in some inflammatory diseases, including periodontal diseases, rheumatoid arthritis and ulcerative colitis. Recently, we found that the lipopolysaccharide of Porphyromonas gingivalis (P-LPS) induced calprotectin release from human neutrophils. P-LPS, a major virulence factor of periodontal pathogens, is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor (TLR) and nuclear factor kappaB (NF-kappaB). In the present study, we investigated whether calprotectin release by P-LPS is induced via the CD14-TLR-NF-kappaB pathway and the cellular mechanism of calprotectin release in human neutrophils. MATERIAL AND METHODS: Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, TLR2 and TLR4, or several inhibitors of NF-kappaB, microtubules and microfilaments, and then incubated with P-LPS. The calprotectin amount in the culture medium was determined using ELISA, and the nuclear extracts from cells were used for the examination of NF-kappaB binding activity using electrophoretic mobility shift assays. RESULTS: P-LPS increased calprotectin release from neutrophils and its induction was inhibited by anti-CD14 and anti-TLR2 antibodies, but not by two anti-TLR4 antibodies. NF-kappaB inhibitors suppressed P-LPS-induced NF-kappaB binding activity and calprotectin release. The inhibitors of microtubule and microfilament polymerization significantly decreased P-LPS-induced calprotectin release. CONCLUSION: These results suggest that calprotectin release is induced by P-LPS via the CD14-TLR2-NF-kappaB signal pathway in human neutrophils and may be dependent on microtubule and microfilament systems.

Actin Cytoskeleton↗

PubMed2003

Decreased expression of alpha2 integrin in fibroblasts isolated from cyclosporin A- induced gingival overgrowth in rats.

OBJECTIVES: Cyclosporin A (CsA), an immunosuppressive agent, induces fibrous gingival overgrowth through reduction of collagen phagocytosis by fibroblasts. Distinct receptors are involved in the binding of collagen to fibroblasts in collagen phagocytosis, and alpha2beta1 integrin serves as a specific receptor for type I collagen on fibroblasts. To elucidate the role of alpha2beta1 integrin in CsA-induced gingival overgrowth, we investigated collagen phagocytosis and alpha2beta1 integrin expression in rat gingival fibroblasts. MATERIALS AND METHODS: Fibroblats were isolated from gingiva of rats fed a powdered diet containing or lacking CsA for 30 d. Flow cytometric analysis were performed to measure the collagen phagocytosis and the alpha2 integrin expression in fibroblasts. Furthermore, total RNAs were isolated from fibroblasts, and the reverse transcriptase-polymerase chain reaction was employed to investigate the mRNA levels of alpha2 integrin. RESULTS: In vitro collagen phagocytosis assay revealed that CsA-treated and control fibroblasts contained a mean of 13.5% and 36.1% phagocytic cells, respectively. CsA-treated fibroblasts had 28% lower expression of alpha2 integrin than that of control. and mRNA expression of alpha2 integrin in CsA-treated fibroblasts was apparently lower than in the controls, but the mRNA expression of beta1 integrin was not affected. CONCLUSION: These findings suggest that one etiological factor of gingival overgrowth may be inhibition of collagen phagocytosis by reducing alpha2 integrin expression in gingival fibroblasts.

Animals↗

PubMed2003

Effect of Porphyromonas gingivalis lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1beta on calprotectin release in human monocytes.

BACKGROUND: Calprotectin is a major cytosolic protein of monocytes, granulocytes, and epithelial cells. It is known that calprotectin is released in inflammatory tissues and detected in gingival crevicular fluid (GCF) of periodontitis patients at high levels. The origin of calprotectin in GCF and its regulation in periodontal disease are unknown. In this study, we investigated the distribution of calprotectin in gingival tissue with inflammation and the induction of calprotectin release from human monocytes by lipopolysaccharide of Porphyromonas gingivalis (P-LPS), tumor necrosis factor-alpha (TNF-alpha), or interleukin-1beta (IL-1beta). METHODS: Gingival tissues were obtained from a healthy donor and a periodontitis patient and calprotectin in gingival tissues was examined by immunohistochemical staining. Monocytes were isolated from the peripheral blood of healthy donors and cultured with P-LPS, TNF-alpha or IL-1beta for 30 minutes to 4 hours. The content of calprotectin in the cell and medium fractions was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Calprotectin was markedly detected at the epithelial and adjacent connective tissue with many inflammatory cells in the gingival tissue from the periodontitis patient. P-LPS increased calprotectin release from monocytes to the maximum level after 30 minutes of treatment and its level was elevated to about 2- to 3-fold of the control level in a dose-dependent manner (1 to 1,000 ng/ml). When the effect of TNF-alpha and IL-1beta on calprotectin release was investigated, calprotectin release significantly increased to about 2.2- and 1.5-fold that of the control level, respectively. CONCLUSION: These results demonstrate that calprotectin release from monocytes is induced by P-LPS, TNF-alpha, and IL-1beta, which in turn, cause and aggravate periodontal disease.

Adult↗