Biomedical subjects

A Palmon

Publications and source records attributed to A Palmon.

At least 19 recordsLinked to original sources

PubMed2005

Proteomic analysis of protein components in periodontal ligament fibroblasts.

BACKGROUND: Characterization of periodontal ligament (PDL) fibroblast proteome is an important tool for understanding PDL physiology and regulation and for identifying disease-related protein markers. PDL fibroblast protein expression has been studied using immunological methods, although limited to previously identified proteins for which specific antibodies are available. METHODS: We applied proteomic analysis coupled with mass spectrometry and database knowledge to human PDL fibroblasts. RESULTS: We detected 900 spots and identified 117 protein spots originating in 74 different genes. In addition to scaffold cytoskeletal proteins, e.g., actin, tubulin, and vimentin, we identified proteins implicated with cellular motility and membrane trafficking, chaparonine, stress and folding proteins, metabolic enzymes, proteins associated with detoxification and membrane activity, biodegradative metabolism, translation and transduction, extracellular proteins, and cell cycle regulation proteins. CONCLUSIONS: Most of these identified proteins are closely related to the extensive PDL fibroblasts' functions and homeostasis. Our PDL fibroblast proteome map can serve as a reference map for future clinical studies as well as basic research.

Adolescent↗

PubMed2004

Pressure simulation of orthodontic force in osteoblasts: a pilot study.

OBJECTIVES: To elucidate the RUNX2 gene expression induction in human osteoblasts after mechanical loading. DESIGN: Using a stringent pulse-chase protocol human osteoblasts were exposed to centrifugal pressure force for 30 and 90 min. Untreated control cells were processed in parallel. Before, and at defined times after centrifugation, total RNA was isolated. RUNX2 gene expression was measured using real-time quantitative reverse transcriptase polymerase chain reaction. The stress/control ratio was used to illustrate possible stimulatory or diminishing effects of force application. RESULTS: Immediately after 30 min of force application the RUNX2 gene expression was induced by a factor of 1.7 +/- 0.14 as compared with the negative control. This induction decreased rapidly and reached its pre-load levels within 30 min. Longer force applications (up to 90 min) did not change the RUNX2 gene expression. CONCLUSION: In mature osteoblasts centrifugal pressure force stimulates RUNX2 gene expression within a narrow time frame: loading of mature cells results in a temporary increase of RUNX2 expression and a fast downregulation back to its pre-load expression level. With this pilot study the gene expression behavior after mechanical stimuli could be determined with a simple laboratory setup.

Cells, Cultured↗

PubMed2004

The effect of centrifugal force on mRNA levels of collagenase, collagen type-I, tissue inhibitors of metalloproteinases and beta-actin in cultured human periodontal ligament fibroblasts.

BACKGROUND: The aim of orthodontic treatment is to relocate teeth abnormally positioned in the jaws. This is achieved by application of continuous force on the tooth, which is immediately being sensed by the periodontal ligament (PDL), bone and the gingiva. Since the bony response is mediated by the PDL, tooth movement is primarily a PDL phenomenon. OBJECTIVES: Thus, the purpose of the present study was to evaluate the direct effect of force (excluding the in vivo tissue response) on the molecular level of matrix metalloproteinase-1 (MMP-1) and collagen type-I (Col-I) in human PDL fibroblasts. METHODS: PDL cell culture flasks were centrifuged for 10, 20, 30, 60, 90 and 120 min by horizontal microplate rotor. The effect of force on mRNA levels of beta-actin, MMP-1, Col-I, tissue inhibitors-1 and -2 (TIMPs) genes was analyzed by RT-PCR. RESULTS: The results showed that force had no effect on the mRNA levels of beta-actin during the first 90 min of application of force, indicating for the first time the use of beta-actin gene as an internal invariant control. It increased the mRNA levels of MMP-1 while almost no effect on Col-I and TIMPs was observed. CONCLUSIONS: The results indicate that PDL remodeling following application of orthodontic force could be partly attributed to the direct effect of the force on MMP-1 gene expression in fibroblasts.

Actins↗

PubMed2004

[Future therapy strategies for salivary gland impairment].

Saliva offers many modalities, mainly protecting the oral tissues by maintaining a local healthy environment. Several conditions cause salivary gland secretion impairment, in which irradiation therapy to the head and neck cancer patients is one of the most rigorous, leading to major decline in quality of life. At present, conventional therapy provides a limited answer for this situation. In the last two decades, several strategies had been proposed to overcome this problem. These approaches can be divided into three branches: 1. Preventing salivary gland damage before irradiation therapy. 2. Protecting the secretory parenchymal tissue during the course of irradiation and, 3. Trying to regenerate salivary gland function after irradiation damage has already occurred. These attitudes may provide future beneficial modalities to salivary gland dysfunction especially in patients were salivary gland function is diminished.

Amifostine↗

PubMed2002

Interferon-gamma deficiency attenuates local P. gingivalis-induced inflammation.

Infection with the periodontal pathogen Porphyromonas gingivalis causes a strong local inflammatory reaction. Using IFNgamma-deficient mice, we tested the hypothesis that the absence of IFNgamma would result in a reduction of the local pro-inflammatory response to P. gingivalis. Cytokine secretion by macrophages from IFNgamma(-/-) animals was significantly attenuated. Addition of IFNgamma restored cytokine secretion. In vivo injection of P. gingivalis into subcutaneous chambers increased the intra-chamber leukocyte counts and TNFalpha and IL-1beta levels. This increase was significantly lower in the IFNgamma(-/-) mice. Local reconstitution of IFNgamma(-/-) mice at the site of inflammation with the IFNgamma gene increased the levels of TNFalpha and decreased the IL-10 levels. Anti-P. gingivalis IgG1 levels, a marker of Th2 response, were higher in immunized IFNgamma(-/-) than in IFNgamma(+/+) mice. The results suggest that lack of IFNgamma reduced the amplitude of the local pro-inflammatory response without decreasing the humoral protective response. The higher IgG1/IgG2a ratio observed supports the possibility of a Th2-dominant response in IFNgamma-deficient animals.

Animals↗

PubMed2001

Imaging transgene expression in live animals.

Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assessing the effectiveness of systems of gene therapy delivery. We applied a new light-detection cooled charged-coupled device (CCCD) camera for continuous in vivo assessment of commonly used gene therapy delivery systems (such as ex vivo manipulated cells, viral vectors, and naked DNA), without the need to kill animals. We examined a variety of criteria related to real-time monitoring of luciferase (luc) gene expression in tissues including bone, muscle, salivary glands, dermis, liver, peritoneum, testis, teeth, prostate, and bladder in living mice and rats. These criteria included determination of the efficiency of infection/transfection of various viral and nonviral delivery systems, promoter specificity, and visualization of luciferase activity, and of the ability of luciferin to reach various organs. The exposure time for detection of luc activity by the CCCD camera is relatively short (approximately 2 minutes) compared with the intensified CCD camera photon-counting method (approximately 15 minutes). Here we transduce a variety of vectors (such as viruses, transfected cells, and naked DNA) by various delivery methods, including electroporation, systemic injection of viruses, and tail-vein, high-velocity-high-volume administration of DNA plasmids. The location, intensity, and duration of luc expression in different organs were determined. The distribution of luciferin is most probably not a barrier for the detection of in vivo luciferase activity. We showed that the CCCD photon detection system is a simple, reproducible, and applicable method that enables the continuous monitoring of a gene delivery system in living animals.

Adenoviridae↗

PubMed2001

Basic fibroblast growth factor suppresses tropoelastin gene expression in cultured human periodontal fibroblasts.

Growth factors are known to play a major role in the regeneration of the periodontium. Basic fibroblast growth factor (bFGF) is a polypeptide growth factor considered to have a role in chemotaxis and mitogenesis of periodontal ligament (PDL) cells. The aim of this study was to assess the effect of bFGF on the transcription level of tropoelastin. As known controls, we assessed the transcription levels of collagen type I, collagen type II and the housekeeping gene, actin. Initially, PDL cells were cultured without bFGF for 3, 7 and 14 days. At each time point. total RNA was extracted and the levels of transcription were assessed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay. The results showed that tropoelastin mRNA is transcribed in PDL cells and its levels increased from minimal amounts by day 3 to maximal amounts by day 14 of culture. We further examined the effect of the addition of 10 ng/ml bFGF to the culture media by day 14. The results showed that the addition of bFGF suppressed the transcription level of tropoelastin. At that time, as expected, a decrease in collagen type I transcription level was shown, while the transcription level of collagen type III was not affected. The findings that elastin is transcribed in vitro by PDL cells, but only negligibly in vivo, imply mechanisms that downregulate or even shut down the expression of the elastin gene in the functioning PDL. Basic FGF might be one of the cytokines involved in control of elastin expression in vivo.

Actins↗

PubMed2001

The effect of mechanical force on mRNA levels of collagenase, collagen type I, and tissue inhibitors of metalloproteinases in gingivae of dogs.

Orthodontic force causes an injury to and subsequent degradation of the attachment apparatus, thus leading to the transposition of the tooth. The gingiva, however, is compressed and sometimes becomes hypertrophic with tooth movement and often shrinks after treatment. To study the effect of force on the gingiva, we applied orthodontic force in dogs and analyzed gingival tissues 1, 2, 3, 7, 14, and 28 days later as well as after removing the force. The effect of force on mRNA levels of collagen type I (col-I), matrix metalloproteinase-1 (MMP- 1), and tissue inhibitors 1 and 2 (TIMPs) genes was analyzed by RT-PCR, and MMP-1 activity was determined by zymography. The results showed that force significantly increased both the mRNA levels of MMP-1 and its interstitial activity. After the removal of force, MMP-1 gene expression was significantly decreased. The results could partly explain the clinically observed shrinkage and adaptation of the gingiva during tooth movement.

Adaptation, Physiological↗

PubMed2000

Development of a highly sensitive quantitative competitive PCR assay for the detection of murine cytomegalovirus DNA.

Viral persistence and molecular latency are characteristic of infection by the human cytomegalovirus (HCMV). Using the murine cytomegalovirus (MCMV) as a model for human infection, a quantitative-competitive polymerase chain reaction (QC-PCR) assay was developed to detect and quantify MCMV-DNA in the salivary glands of infected mice. The QC-PCR detected high numbers of MCMV DNA copies in the absence of infectious virus. By comparing the DNA content and the results obtained from a standard semiquantitative plaque assay, it is concluded that 1 plaque-forming unit (pfu) is the equivalent of approximately 1500 viral genomes. By day 42-post infection (pi) 4x10(3) copies of DNA/1 mg tissue were sufficient to reactivate infectious virions after cyclophosphamide immunosupression. By day 90 pi, however, when the DNA load was decreased to <1.2x10(2), reactivation was not observed. These results indicate that viral reactivation will occur when the number of infectious DNA copies is equivalent about 2-3 pfu. This quantitative test may therefore help to detect CMV and the risk of reactivation in immunosupressed patients.

Animals↗

PubMed2000

Changes in cardiac mechanics with heat acclimation: adrenergic signaling and SR-Ca regulatory proteins.

The involvement of adrenergic signaling and sarcoplasmic calcium regulatory proteins in the development of heat acclimation-induced adaptations in cardiac mechanics was studied in heat-acclimated (34 degrees C) rats for 2, 5, and 30 days (AC(2), AC(5), and AC(30), respectively). Control (C) rats were held at 24 +/- 1 degrees C. Systolic pressure (LVP) and velocities of contraction (dP/dt/P) and relaxation (-dP/dt/P) were measured using a Langendorff system. For adrenergic signaling, beta-adrenoreceptor (AR) density and affinity (Scatchard plots) and cardiac inotropic response to norepinephrine (10(-7) mM, +/- 10(-6) mM propranolol) were measured. For the regulatory proteins, steady-state levels of Ca(2+)-ATPase and phospholamban (PLB) mRNAs and the encoded proteins Ca(2+)-ATPase [sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] and PLB were measured using semiquantitative RT-PCR and Western immunoblotting, respectively. Both short (STHA; AC(2) and AC(5))- and long-term heat acclimation (LTHA; AC(30)) enhanced LVP. However, dP/dt. P and -dP/dt. P in STHA hearts resembled that of the controls, whereas on LTHA, both parameters decreased (P < 0.05), implying decreased velocity of contraction and relaxation. beta-AR density remained unchanged with their affinity markedly decreased (P < 0.05). AR responsiveness, however, diminished in AC(2) but was markedly enhanced on LTHA. During STHA, PLB and sarcoplasmic reticulum Ca(2+)-ATPase transcripts were upregulated with no change in the encoded proteins except for SERCA downregulation on AC(5), leading to an increased PLB/SERCA ratio (P < 0.05). This mismatched preacclimation lusitropic state on STHA and increased PLB/SERCA ratio was evident (P < 0.05) due to downregulation of SERCA and upregulation of PLB. Our data fit a biphasic acclimation model in which desensitized adrenergic signaling is dominant during STHA, whereas on LTHA, the contractile machinery is influenced by altered expression of the calcium regulatory proteins leading to both augmented adrenergic inotropic response (via PLB elevation) and decreased velocity of relaxation. The sustained low thyroxin measured on LTHA causally associates with this response.

Adaptation, Physiological↗

PubMed2000

Inverse dose- and time-dependent effect of basic fibroblast growth factor on the gene expression of collagen type I and matrix metalloproteinase-1 by periodontal ligament cells in culture.

BACKGROUND: Growth factors are known to play a major role in the regeneration of the periodontium. Basic fibroblast growth factor (bFGF) is a polypeptide growth factor considered to have a role in chemotaxis and mitogenesis of periodontal ligament cells (PLC). The aim of this study was to assess the dose-dependent effect of bFGF administration on the levels of gene expression of collagen type I (a1) (col I), collagen type III (col III), and collagenase-1 (MMP-1) in PLC. METHODS: PLC were cultured in different concentrations of bFGF (0.1 to 10 ng of bFGF) for 14 and 21 days. At each time point, the gene expression of the examined molecules was assessed semi-quantitatively by reverse transcription-polymerase chain reaction (RT-PCR) assay. RESULTS: The results indicated that bFGF exhibits an inverse time- and dose-dependent effect on the gene expression of col I and MMP-1: it simultaneously downregulates the gene expression of col I and upregulates the gene expression of MMP-1. On the other hand, bFGF had no dose-dependent effect on col III gene expression. The effect of bFGF on the expression of the three genes was modulated by the time of incubation with bFGF. CONCLUSIONS: These results suggest that bFGF is one of the important regulators involved in the active remodeling of col I in the periodontal ligament and possibly in other connective tissues.

Cell Division↗

PubMed1999

Gingival response to orthodontic force.

Orthodontic tooth movement is brought about by prolonged application of force on the attachment apparatus. This results in cellular and extracellular changes within the periodontium. As shown in numerous studies, tooth movement is achieved after the remodeling of alveolar bone and the response of the periodontal ligament to the mechanical force. Although gingival changes have also been found to be an important factor in the overall response, the effect of orthodontic tooth movement on the gingiva has been investigated to a lesser extent. Unlike bone and periodontal ligament, which regain their original structure after removal of force, the gingival tissue does not regain its pretreatment structure, a fact on which a hypothesis has been made that tooth relapse after removal of retention may be associated with changes in the gingiva. The present review summarizes available data on the effect of orthodontic force on collagen, elastin, and collagenase in the gingiva and its relevance to understanding the mechanism of tooth relapse.

Animals↗

PubMed1999

Heat acclimation induces changes in cardiac mechanical performance: the role of thyroid hormone.

The involvement of reduced thyroxine level in the emergence of heat acclimation-induced negative lusitropic effect was examined. Experiments were carried out on 1) control rat hearts maintained at 24 +/- 1 degreesC (C); 2) rat hearts acclimated at 34 degreesC for 1 mo (AC); 3) AC-euthyroid rat hearts, via administration of thyroxine in the drinking water (AT); and 4) hypothyroid rat hearts, maintained at 24 +/- 1 degreesC, via administration of thiouracil in the drinking water (CP). Systolic pressure and velocities of contraction (dP/dt. P) and relaxation (-dP/dt. P) were measured using the Langendorff perfusion system. The steady-state levels of Ca2+-ATPase and phospholamban mRNAs and the expression of the encoded proteins Ca2+-ATPase (SERCA) and phospholamban (PLB) were measured, using semi-quantitative RT-PCR and Western immunoblotting, respectively. Rat thyroxine levels were measured using RIA. Heat acclimation, which brought about a reduced thyroxine level, led to downregulation of Ca2+-ATPase mRNA expression and translation and upregulation of phospholamban mRNA and PLB. Consequently, the PLB-to-SERCA ratio (PLB/SERCA) of the AC hearts showed a significant increase. These changes, as well as the greater pressure generation and the reduced dP/dt. P and -dP/dt. P observed in AC hearts were blunted in the AT hearts. Our data suggest that sustained heat acclimation-induced low thyroxine level has a decisive effect on the contractile machinery of the AC heart. Elevated PLB/SERCA apparently explains the negative lusitropic effect observed in these hearts.

Acclimatization↗

PubMed1999

Heat acclimation increases the basal HSP72 level and alters its production dynamics during heat stress.

It has been previously shown that heat acclimation leads to an elevated basal level of 72-kDa heat shock protein (HSP72). Augmented expression of HSP72 is considered as a cytoprotective response. This led us to hypothesize that alterations in the heat shock protein (HSP) defense pathway are an integral part of the heat acclimation repertoire. To investigate this, we studied the temporal profile of basal HSP expression upon acclimation and the dynamics of their accumulation subsequent to acute heat stress (HS). In parallel, HSP72 mRNA level before and after HS was measured. For comparison, HSC mRNA [the constitutive member of 70-kDa HSP (HSP70) family] was measured in similar conditions. Heat acclimation was attained by continuous exposure of rats to 34 degrees C for 0, 1, 2, and 30 days. HS was attained by exposure to 41 or 43 degrees C for 2 h. Thermoregulatory capacity of the rats was defined by rectal temperature, heating rate, and the cumulative heat strain invoked during HS. HSP72 and HSP70 gene transcripts were measured in the left ventricle of the heart by means of Western immunoblotting and semiquantitative RT-PCR, respectively. The resultant acclimatory change comprised a higher resting level of the encoded 72-kDa protein (Delta175%, P < 0.0001). After HS, peak HSP72 mRNA level was attained, 40 and 20 min post-HS at 41 and 43 degrees C, respectively, vs. 60 and 40 min in the nonacclimated group. The subsequent HSP synthesis, however, was dependent on the severity of the cumulative heat strain. At the initial phase of heat acclimation, augmented HSP72 transcription unaccompanied by HSP synthesis was observed. It is concluded that upon heat acclimation, the HSP defense pathway is predisposed to a faster response. At the initial phases of heat acclimation, inability to elevate the HSP cytosolic level rules out their direct cytoprotective role.

Acclimatization↗

PubMed1998

The effect of centrifugal force on the transcription levels of collagen type I and collagenase in cultured canine gingival fibroblasts.

An orthodontically treated tooth is often destabilized in its newly corrected location and relapses towards its original position. Hitherto, the explanation for this phenomenon was that orthodontic force brings about "stretching" of gingival collagen fiber, which "pull back" the tooth towards its pretreatment position. A previous ultrastructural study showed that after force application the gingival collagen fibres were torn, laterally spaced and of increased diameter. Therefore, they could not "pull back" the tooth and be the cause of the relapse. In the present study, in order to find a more plausible explanation at the molecular level, the effect of pressure on the gene transcription of collagen type I and tissue collagenase was examined by semiquantitative, reverse transcriptase-polymerase chain reaction assay. Attached buccal gingiva was excised from anaesthetized dogs and gingival fibroblasts were grown in culture. Following application of pressure (0.167 kg/l g cell mass), the transcription of collagen type I was increased while that of tissue collagenase was decreased. These results corroborate the ultrastructural in vivo findings that orthodontic force is associated with larger amounts of collagen type I in the gingiva.

Animals↗

PubMed1998

Tuftelin--aspects of protein and gene structure.

The acidic enamel protein tuftelin has now been cDNA cloned, sequenced and characterized in a number of vertebrate species. Recently, the bovine tuftelin gene structure was elucidated. Cloning of the human tuftelin gene and partial sequencing of a number of exons have also been achieved. Immunologically, the protein has been shown to be conserved throughout 550 million years of vertebrate evolution. The gene has been localized to the long arm of the autosomal chromosome 1. The mapping of the human tuftelin gene to a well-defined cytogenetic region could be important in understanding the etiology of autosomally inherited amelogenesis imperfecta, the most common hereditary disease of enamel. The present paper reviews the primary structure, mRNA/cDNA structure, and gene structure of tuftelin. It describes its immunolocalization at the light microscope level and at the ultrastructural level in both the ameloblast cells and in the extracellular enamel matrix. The timing of tuftelin expression and its possible roles in enamel formation are discussed.

Ameloblasts↗

PubMed1998

Tuftelin mRNA is expressed in a human ameloblastoma tumor.

RT-PCR, Southern blotting and DNA sequencing have established for the first time that tuftelin mRNA is expressed in human ameloblastoma tumor. The expression of amelogenin mRNA in ameloblastoma was also established, confirming earlier reports by Snead et al. These results corroborate, on a molecular level, the enamel organ epithelial origin of ameloblastoma. In view of the present results, it is interesting that previous studies have indicated that although ameloblastoma, a non-mineralized odontogenic tumor, transcribes amelogenin mRNA, amelogenin (and enamelin) proteins are not expressed in this tissue. However, in mineralizing odontogenic tumors, both these classes of proteins are expressed.

Ameloblastoma↗

PubMed1997

Tuftelin: enamel mineralization and amelogenesis imperfecta.

Tuftelin is a novel acidic enamel protein thought to play a major role in enamel mineralization. Its identity and localization has been confirmed by amino acid composition, enzyme-linked immunosorbant assay, Western blots, indirect immunohistochemistry and high resolution protein-A gold immunocytochemistry. The deduced tuftelin protein (pI 5.2) contains 389 amino acids and has a calculated peptide molecular mass of 43,814 Da. Immunological studies suggest conservation of tuftelin structure between species throughout vertebrate evolution. The cDNA sequence encodes for several putative post-translation sites including one N-glycosylation consensus site, seven O-glycosylation sites and seven phosphorylation sites, as well as an EF-hand calcium-binding domain (with mismatch), localized towards the N-terminal region. At the C-terminal region (residues 252-345) tuftelin contains structurally relevant determinants for self assembly. We recently cloned and partially sequenced the human tuftelin gene (four exons have now been sequenced). These sequences include exon 1 and over 1000 bases of the putative promoter region. Employing fluorescent in situ hybridization, we mapped the human tuftelin gene to chromosome 1q 21-31. Localization of the human tuftelin gene to a well-defined cytogenetic region may be important in understanding the aetiology of autosomally inherited amelogenesis imperfecta, the most common enamel hereditary disease.

Amelogenesis↗