A micro-Raman spectroscopic study of hydrazine-treated human dental calculus

Hydrazine has been used to remove organic components and to isolate the mineral(s) from human calculus. Micro-Raman measurements were performed on the mineral phase. After the hydrazine-treatment, not only a large reduction in fluorescence but also an increase in Raman signal was observed. The treatment was essential in minimizing thermally-induced chemical changes which could otherwise occur to the original calculus mineral due to the intense laser light. The Raman spectral features of the mineral were nearly all identical among the Raman spectra obtained at many randomly-selected sites by the micro-Raman microbe with a lateral resolution of approximately 1 micron, and were consistent with those of impure hydroxyapatite containing CO3(2-) and HPO4(2-). The spectra contained typical hydroxyapatite bands including PO4(3-)bands of the v1, v2, v3 and v4 modes and one OH- stretch band. Other minor bands due to the CO3(2-) v1 and v3 modes and bands possibly due to the HPO4(2-) v1, v2 and v4 modes were observable by the technique despite the hydrazine-treatment that could in principle remove the HPO4 and CO3 ions from the mineral. In comparison with pure synthetic hydroxyapatite, the intensity of the OH- stretch band relative to that of the PO4(3-) v1 band was approximately 70% weaker, and the bandwidth of the phosphate v1 band was 200% broader, reflecting various crystal imperfections presumably present in the calculus mineral.     

Fluorescence of dental calculus from cats, dogs, and humans and of bacteria cultured from dental calculus

Recently we reported that feline and canine dental calculus fluoresced pink to red under long wavelength ultraviolet light due to the presence of porphyrin. Here we report the observation of such fluorescence in 30 of 30 cats, 30 of 30 dogs, and 8 of 13 supragingival samples and 5 of 5 subgingival samples of humans. The fluorescence spectra of the calculus dissolved in 9 M HCl show that it is due to three distinct metal-free porphyrins. Similar fluorescence is obtained from bacterial cultures grown from calculus deposits of cats and dogs and bacteria grown on blood agar containing hemin and vitamin K1. The results of the bacterial culture study suggest that the metal-free porphyrin is produced by bacteria in the mouth. The clinical observation of fluorescence can be used for diagnosis.     

Paleomicrobiological study in dental calculus: Streptococcus mutans

Morphological types of bacterial remains preserved in ancient tartar of teeth from extinct human groups, which included some communities of coastal gatherers, fishermen, hunters, and farmers, and those practicing a mixed economy, were analyzed. Previous studies have shown the presence of bacteria in ancient tartar. The aim of this work was to determine whether Streptococcus mutans was present in ancient populations (500-12,000 years old). Teeth samples were from ancient skulls obtained from different anthropological collections: the north and south of Chile (before the Spanish conquest), Palencia, Spain, and an eastern Mediterranean region (Levant). Optical microscopy showed Gram positive and Gram negative bacteria. Scanning electron microscopy identified morphological types of bacteria. Transmission electron microscopy enabled categorization of bacterial structures. Fluorescence microscopy helped label and identify S. mutans, using polyclonal antibodies. Bacterial morphotypes were related to different subsistence patterns. Hunters, fishermen, and gatherers had a less diverse flora with bacillary and coccal morphotypes. Agricultural groups showed greater diversity with additional filamentous and spiral morphotypes. The best preserved ultrastructural feature was the cell wall. The existence and colonization capacity of the mutans-like streptococci preserved in tartar was established for the ancient populations studied, with the exception of Cerro Sotta (south of Chile). Hence, their occurrence could not be related to diet or subsistence pattern.     

Gingival state and dental calculus in early-onset periodontitis

This study was undertaken to 1) compare the prevalence of gingival inflammation and dental calculus in adolescents with early-onset periodontitis and their matched controls and 2) assess and compare the relationship between the presence of dental calculus and the extent of gingival bleeding and attachment loss in these subjects. The study group consisted of 1,285 13 to 20 year-old individuals, 651 males and 634 females, selected from a national survey of the oral health of U.S. adolescents in 1986/1987. It included 709 (55.2%) Blacks, 224 (17.4%) Hispanics, and 352 (27.4%) Whites. Eighty-nine subjects had localized or generalized juvenile periodontitis (JP), 218 had incidental attachment loss (IAL), and 978 were without clinical attachment loss (controls). The controls were matched to cases on gender, race, age, and geographic location. The subjects were examined clinically to assess the percentage of sites with gingival bleeding and supragingival calculus only and subgingival calculus with or without supragingival calculus. The IAL and JP groups had significantly more gingival bleeding and subgingival calculus than the controls. Also, the JP group had significantly higher prevalence of both conditions than the IAL group. The percentage of sites with supragingival calculus was not different between the groups, but varied by ethnicity. Hispanics with JP had the highest percentage of sites with gingival bleeding and subgingival calculus, and the lowest percentage of sites with only supragingival calculus. The results demonstrate that gingival inflammation and subgingival calculus are associated with early periodontal breakdown, and contradict earlier reports of early-onset periodontitis not being associated with these factors.     

Fluoride profiles in dental calculus from Japanese, Chinese and British residents

Whether the fluoride concentrations and profiles differ in human dental calculus obtained from different countries was investigated. A total of 203 dental calculus deposits on 203 permanent teeth from residents (mean age, 52.1 years) of Nagoya (Japan), Shanghai (China), Leeds (Great Britain) and the Wuhan mountainous area (China, fluoridated area) were analysed. An abrasive microsampling procedure was used to examine fluoride distribution. There were five types of fluoride profiles in dental calculus in each area/country (designated types L, J, U, T, W). In supragingival calculus, type L (highest in the outermost layers) and type J (highest in the innermost layers) both had significantly higher values than type U (high in the surface and innermost layers) but were relatively identical. In subgingival calculus, type W (high in the outermost, mid and innermost layers) was characteristics. Calculus from the Wuhan mountainous area (fluoridated) had the highest fluoride concentration, followed by Leeds (non-fluoridated), and Nagoya and Shanghai (non-fluoridated) calculus had the lowest. Fluoride concentrations in supragingival calculus were higher in teeth extracted because of periodontal diseases than dental caries. It is concluded that fluoride concentrations and distribution in dental calculus differ from country to country, probably due to different fluoride environments.     

Light-microscopical investigation of the distribution of extracellular matrix molecules and calcifications in human dental pulps of various ages

Human glioma cell line KG-1C contains GM3 ganglioside as its sole glycolipid. The degree of M2590 antibody binding to GM3 was found to be regulated by the cell density; the percentage of positive cells in FACS analysis decreased from approximately 20% to close to none as the cells increased their density from sparse to confluent. The contents of GM3 with different cell densities were consistent, being more than 0.4 micromol/g of the cellular weight, which was high enough to be recognized by the antibody. Trypsin treatment of the cells did not increase antibody reactivity. The extracted GM3 retained its antigenicity, being intensely stained with M2590 on a TLC plate; there was no change in chromatographic mobility either, indicating no modification of its chemical structure. The fluorescent microscope disclosed scattered dot-like staining of GM3, particularly at the periphery of the cells. We were able to expose cryptic GM3 fully within 12 h by dispersion of the cells to a sparse density. Surface labeling of GM3 with the use of limited sodium periodate oxidation of sialylated residue equally labeled GM3 either from the confluent cells or the sparse cells. Disassembly of actin filaments with cytochalasin B (10 microM) partially exposed cryptic GM3 of confluent cells, indicating reversibility of the crypticity. All together, the results indicate that cryptic GM3 actually exists on the cell surface, hidden from the surface not by other molecules but by other mechanisms associated with the cellular architecture. We are beginning to explore the possibility of selective localization of GM3 in small caves or folds of the cell membrane produced upon cell-to-cell contact.     

Possible relation of osteopontin to development of psammoma bodies in human papillary thyroid cancer

Fine-needle aspiration cytology (FNAC) is the most useful procedure for the evaluation of thyroid nodules. The requirement for repeated aspirations in the follow-up of benign nodular thyroid disease, however, is controversial. To determine the value of re-aspirations in benign nodular thyroid disease, we studied 457 fine-needle reaspirations performed on 216 patients (197 female, 19 male) aged 42.9+/-12 years with uninodular (n = 65) and multinodular (n = 151) thyroid disease. Two hundred fifty-seven of these were second, 137 were third, 46 were fourth, and 17 were fifth re-aspirations of the same nodule, performed in a mean follow-up time of 43.9+/-31 (3-156) months. FNAC results were benign in 407 (89%), insufficient for diagnosis in 31 (6.8%), suspicious in 16 (3.5%), and papillary carcinoma (PC) in 3 (0.7%). An initial benign diagnosis did not change after multiple aspirations in 213 (98.61%) of the cases. Three patients with initial aspirations read as benign had a diagnosis of PC from their second biopsies, (diagnosis confirmed at surgery). Re-examination of the initial FNAC revealed atypical features in 1 of the 3 patients. These 3 patients likely represent a false-negative result of the initial FNAC rather than benign nodular disease transformed to a malignant one during the follow-up period. In conclusion, a second aspiration of clinically suspicious nodules may correct a few initial false-negative results, but routine additional re-aspirations are not useful for clinically stable disease.     

Ultrastructural localization of osteopontin immunoreactivity in phagolysosomes and secretory granules of cells in human intestine

A post-embedding ultrastructural immunogold method was used to detect osteopontin in human intestinal biopsies with special emphasis on secretory and phagocytic organelles. Osteopontin immunoreactivity was localized to phagolysosomes of macrophages, fibroblasts, absorptive epithelial cells of the small intestine and Paneth cells. The mucigen secretory granules and Golgi structures of mucous epithelial cells of the small intestinal epithelium contained osteopontin, but secretory granules of numerous other cells, including Paneth cells, did not. Extracellular and phagocytosed Tropheryma whippelii within macrophage phagolysosomes also bound osteopontin. These localizations are supportive of a role for osteopontin in phagocytic and some secretory cell functions in human intestine.     

A fluorescent compound in bovine dental enamel matrix compared with synthetic dityrosine

Dityrosine is a substance frequently found in structural proteins. Emission and excitation fluorescence spectra, absorption spectra, thin layer and molecular weight chromatography showed that the fluorescing enamel material isolated strongly resembled dityrosine and had at least an o,o-diphenyl bonding.     

Extracellular matrix regulates apoptosis in human neutrophils

BACKGROUND: During inflammation, polymorphonuclear neutrophils (PMNs) migrate into the affected tissue interacting with extracellular matrix (ECM) proteins. We tested the hypothesis that PMN-matrix interaction affects PMN apoptosis. METHODS: Apoptosis of human PMNs was detected by DNA-fragmentation assay and was quantitated by flow cytometry, ultraviolet and light microscopy. Cell adhesion was assessed by a toluidine blue assay, and cell spreading was detected by phase contrast microscopy. Protein tyrosine phosphorylation was studied using Western blotting and confocal microscopy. RESULTS: PMN apoptosis was not different in unstimulated cultures on either surface-adherent fibronectin or on PolyHema, a surface that prevents cell adherence. However, tumor necrosis factor-alpha (TNF alpha) treatment significantly increased apoptosis on fibronectin (37 +/- 4%) compared with PolyHema (20 +/- 3%). Tests on other matrix substances revealed that the percentage of apoptotic PMNs in the presence of TNF alpha was 8 +/- 1% on PolyHema, 26 +/- 4% on fibronectin, 17 +/- 2% on collagen I, 16 +/- 2% on collagen IV, and 16 +/- 3% on laminin (P < 0.05 for all matrices compared with PolyHema). Preincubation with genistein (50 microM) significantly inhibited TNF alpha-mediated apoptosis on fibronectin (39 +/- 4% to 21 +/- 4%) but not on PolyHema (21 +/- 4% to 16 +/- 4%). Genistein also reduced PMN spreading on fibronectin. In contrast, inhibitors of mitogen-activated protein kinase and protein kinase C showed no effect on PMN apoptosis. Fibronectin strongly increased tyrosine phosphorylation of three 102, 63, and 54 kDa proteins. Five newly tyrosine-phosphorylated 185, 85, 66, 56, and 42 kDa bands were also visible. Using confocal microscopy, highest tyrosine phosphorylation was localized to sites of cell-matrix interaction. CONCLUSIONS: ECM influences apoptosis in TNF alpha-activated, adherent, spreading PMNs. The process is regulated by tyrosine phosphorylation. Acceleration of apoptosis may shorten the PMN lifespan and thereby locally regulate inflammation.     

Identification of biologically active sites in laminin an extracellular matrix protein

Laminin-1, a major component of basement membranes, has multiple biological activities including promotion of cell adhesion, spreading, migration, growth, neurite outgrowth and tumor metastasis. Several active sites on laminin-1 have been identified previously. We modified these biologically active peptides to enhance their activities. The multimeric YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides assembled on a branched lysine core were found to strongly enhance the activity of YIGSR in inhibiting tumor growth and metastasis. We also found the all-D-configuration peptide segment containing the IKVAV (Ile-Lys-Val-Ala-Val) sequence had similar biological activities to the native all-L-peptide in vitro and in vivo. These results suggest that these modified compounds are potentially useful for clinical applications. We have identified new active sequences from the laminin alpha 1 chain carboxyl-terminal globular domain (G domain). Using a systematic screening for cell binding sites with 113 overlapping synthetic peptides, we found five peptides (AG-10, AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with cell-type specificities. AG-10 and AG-32 were found to interact with integrins. AG-73 caused metastases of B16-F10 mouse melanoma cells to the liver colonization in mice. Additionally AG-73 was found to promote neurite outgrowth. Moreover, this peptide inhibited laminin mediated acinar-like development of a human submandibular gland cell line. The AG-73 domain on laminin-1 could be one of the most important biologically active sites. These active peptides may useful for study of the molecular mechanism of laminin-receptor interactions and for development of therapeutic reagents for tumor metastasis and angiogenasis.     

Different human tenascin-C variants in the extracellular matrix of cultured human fibroblasts

Using an immunoadsorbent prepared with a monoclonal antibody specific for the high molecular mass isoform of human tenascin-C, we purified tenascin-C molecules containing at least one large subunit from the extracellular matrix of cultured normal human fibroblasts. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting analyses have shown that both high and low molecular mass subunits are present in these tenascin-C preparations. Because the monoclonal antibody used is able to bind only the high molecular mass isoform, the present data show that part of the tenascin-C present in the fibroblast extracellular matrix is made up of heterohexameric molecules.     

Identification of algogenic substances in human erythrocytes

Lysates of human erythrocytes produce pain when applied to a human blister base. The algogenic material is not potassium, acetylcholine, bradykinin, 5-hydroxytryptamine, histamine or a prostaglandin, and is dialysable. 2. Fractionation of dialysates of freshly lysed erythrocytes by Sephadex gel filtration coupled with assays on the human blister base preparation showed that the algogenic material was a mixture of the adenyl compounds adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). 3. On the human blister base preparation ATP, ADP and AMP had comparable activity and produced threshold pain in a concentration of 2 muM. 4. The rabbit isolated jejunum preparation was found useful in these studies since ATP, ADP and AMP produced a relaxation which was proportional to their concentration in test samples obtained from dialysates. Of more limited usefulness was the rat isolated stomach strip preparation on which ATP and ADP produced contractions which also were proportional to their concentrations in text samples. 5. The possible role of adenyl compounds in the production of pain in vivo is discussed.     

Identification of myoglobin in human smooth muscle

Myoglobin (Mb) has been believed to be absent generally from mammalian smooth muscle tissue. Examination of human rectal, uterine, bladder, colon, small intestine, arterial, and venous smooth muscle by immunohistochemical techniques shows that each of these tissues is immunopositive for both smooth muscle myosin and human Mb. Mb-specific primers were used for the polymerase chain reaction to generate cDNA from smooth muscle tissues. Southern hybridization with a Mb-specific probe gave a very strong signal with the cDNA from rectum, weaker signals from small intestine and uterus, a faint signal from colon, and no signal from bladder tissue. High performance liquid chromatography analysis coupled with sequence determination has shown that contaminating heme-binding serum albumin as well as hemoglobin in extracts of smooth muscle seriously compromise any heme-based or spectrophotometric assay of Mb. Combined affinity and size exclusion chromatography, however, provide the necessary resolution. The cDNA-derived amino acid sequence of human smooth muscle Mb was found to be identical to that of Mb from striated muscle.     

Heat-treatment-induced reduction in the apparent solubility of human dental enamel

Holcomb and Young (1980) have shown a significant increase in human dental enamel (HE) structural order resulting from heat treatment in the temperature range of from 275 to 400 degrees C. Also, previous work in our laboratory had shown dramatic decreases in the initial dissolution rates (IDRs) of both carbonated apatite (CAP) heated at similar temperatures (from 300 to 500 degrees C) and HE exposed to CO2 laser irradiation for which calculated surface temperatures were in this same range. We hypothesize that thermal treatment shifts the apparent solubility distribution profile of HE toward lower apparent solubilities, paralleling the observed increased in crystal structural order and the decrease in IDRs. Powdered HE was heated in a furnace at temperatures ranging from 150 to 500 degrees C for 24 hours. The apparent solubility distributions of both heated and unheated HE powders were measured by equilibration for 24 hours in a series of partially saturated solutions simulating various amounts of HE dissolved in a pH 4.5 dissolution medium. The apparent solubility distribution for the unheated HE showed a peak at KHAP [the ion activity product based on the Ca10(PO4)6(OH)2 stoichiometry] of 10(121.0). Heat treatment shifted the apparent solubility distribution to lower solubilities. The peak KHAP values were approximately 10(124.8) at 200 degrees C; approximately 10(127.8) at 300 degrees C; and approximately 10(-129.1) from 400 to 500 degrees C. This approximately 8 orders of magnitude decrease in KHAP for HE heated at from 400 to 500 degrees C correlates with the previously observed reduction in the IDR driving force for laser-treated HE experiencing a similar surface temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incremental lines in human dental cementum in relation to age

We have studied day-time vigilance in 31 patients (median age 49 years) with suspected sleep disorders using a new visual reaction time and performance test. The findings in the day-time vigilance test were compared with the number of desaturation events and movement arousals measured with a sensitive movement detector in the night-time. In our statistical model the high number of desaturations correlated with a high dispersion in reaction-times. The squared multiple r was 0.465 in a model where the dispersion of reaction times was the dependent variable and the number of desaturations, duration of quiet sleep and the mode of oxygen saturation were independent variables. A high amount of body movements (movement arousals, duration less than 5 seconds) correlated with gradual deterioration in the performance test. The squared multiple r was 0.447 in a model where the regression coefficient of reaction times was the dependent variable and active sleep and number of body movements less than 5 seconds in duration were the independent variables. Frequent arousals in apnoeic patients are observed in hyper-excitable responders and are known to cause sleep deprivation and hypersomnia. Our findings in desaturating patients indicate that in those with a low chemoreceptor response to hypoxia the failure in day-time regulation of vigilance may differ from the failure associated with sleep-deprivation.     

Activation of alphav beta3 integrin on human osteoclast-like cells stimulates adhesion and migration in response to osteopontin

Integrins mediate cell adhesion and can induce different cellular responses, including changes in intracellular pH, changes and oscillation in intracellular free calcium, and protein phosphorylation on tyrosine. During bone resorption, the integrin alphav beta3 regulates adhesion of osteoclasts to bone extracellular matrix proteins, such us osteopontin (Opn). Adhesion via alphav beta3 is followed by osteoclast polarization onto the bone surface and by the onset of bone resorption. To characterize these events at the molecular level, we investigated the state of activation of alphav beta3 on the human osteoclast-like cell line GCT23 using the monoclonal antibody AP5 which binds to and can induce, under low calcium conditions, activated alphav beta3. By flow cytometry, approximately 50% of alphav beta3 on the surface of the osteoclast-like cell line GCT23 was reactive with AP5 and was therefore in the activated state. Incubation with AP5 in the presence of low calcium concentrations increased activated alphav beta3 to 90-100%. Activation of alphav beta3 increased the efficiency of GCT23 adhesion to Opn by 2-fold. Furthermore, haptotactic migration on Opn was also enhanced about 40% compared to control. We propose that changes in the activation state of alphav beta3 may be a regulation point for osteoclasts during bone resorption.     

Bacteria inhibit biosynthesis of bone matrix proteins in human osteoblasts

The effect of extracts from Staphylococcus aureus and Staphylococcus epidermidis on bone matrix production were assessed by analyzing the biosynthesis of osteocalcin and Type I collagen in a human osteoblastic osteosarcoma cell line (MG-63). In MG-63 cells, extracts from Staphylococcus aureus and Staphylococcus epidermidis decreased 1,25(OH)2-vitamin D3 stimulated osteocalcin biosynthesis, and insulin-like growth factor I induced production of Type I collagen in a concentration dependent manner. The basal rate of osteocalcin and Type I collagen formation was unaffected by the bacterial extracts. The inhibitory effect of the bacteria on osteocalcin biosynthesis was seen after 24 hours of treatment and was maintained for at least 96 hours. The extracts of Staphylococcus aureus and Staphylococcus epidermidis enhanced prostaglandin E2 formation in the MG-63 cells. Abolition of the prostaglandin E2 response by treatment with indomethacin and flurbiprofen did not affect bacteria induced inhibition of osteocalcin production. Stimulation of osteocalcin biosynthesis by 1,25(OH)2-vitamin D3 was associated with a decreased rate of cell proliferation. The inhibitory action of the bacterial extracts was not linked to any inhibition of [3H]-thymidine incorporation into deoxyribonucleic acid. These data show that extracts of Staphylococcus aureus and Staphylococcus epidermidis have the ability to inhibit the biosynthesis of bone matrix proteins by a nonprostaglandin and noncytotoxic dependent mechanism and suggest that bone loss in inflammatory processes containing Staphylococcus aureus or Staphylococcus epidermidis may not be caused only by enhanced bone resorption but also by decreased bone formation.