Dentistry Watch

Intra Individual Variation Repeated Measures

There is evidence of a variation in salivary flow rate over a 10-month interval in children. The relation between salivary flow rate and salivary constituents suggests that this variation might have a secondary effect on the measures of inorganic ions and buffering capacity. A fluctuation independent of salivary flow rate might also be present. For example, in unstimulated submandibular saliva, circadian variation in calcium and phosphate is observed. Yet, even when salivary flow rate is controlled, there is evidence that calcium varies throughout the day in both parotid and submandibular saliva.

Variation in salivary constituents over longer intervals of time has also been reported; Shannon and Segreto (1968)observed that calcium varied haphazardly from day to day and from week to week in stimulated parotid saliva. Phosphate increased throughout the week and also showed a week to week fluctuation. However, when salivary flow rate was controlled, no significant difference in the amounts of inorganic ions was observed from day to day in parotid saliva.

Variation of salivary constituents over time could simply reflect other external influences. Hormonal factors might influence salivary constituents over longer intervals. For instance, salivary ionic calcium is higher in the mid-menstrual cycle than pre- and postmenstrually.

If a variation of salivary constituents does exist, comparison of single salivary measurements collected under the same conditions, even in a cohort, may not be appropriate. We have now examined the variation of salivary calcium, phosphate and buffering capacity over a 10-month interval. The study was carried out in North Wales where a group of 43 children, 11–12 years of age, provided a mid-afternoon saliva sample once a month between September 1990 and June 1991 inclusive. The dates of salivary collection were separated by between 28 and 33 days except on one occasion when collections were 23 days apart. The sample was collected 1 hr after eating and the dates of commencement of menstruation of female participants (n=19) were recorded each month. Details of the timetable and method of collection of the unstimulated saliva sample are as described by Kavanagh et al. (1998).

Buffering capacity was assessed by the Dentobuff® strip method (Orion Diagnostica, Espoo, Finland) immediately after the collection of each saliva sample. Results were categorized as low (endpoint pH<4.6), medium (endpoint pH between 4.6 and 5.5) or high (endpoint pH between 5.6 and 7.0) according to the colour chart provided.

The remainder of the saliva samples were stored at ?18°C and then, after thawing, analysed in the Department of Chemistry, University College Cork, Republic of Ireland. Total calcium was measured by atomic absorption spectrophotometry in a Perkin–Elmer Model 2380. Spectrophotometry was used to measure inorganic phosphorus. The coefficients of variation of reproducibility were <8% for calcium and <5% for phosphorus.

Eighteen individuals attended at every occasion throughout the study. Although saliva was collected from each individual who attended, not all of the samples were of an adequate volume to analyse buffering capacity, calcium and phosphate. Where there were missing values for buffering capacity, these were substituted by the series modal value and with regard to calcium and phosphate a series mean was used. Intra-individual variation in buffering capacity and inorganic ions was assessed by Friedman two-way ANOVA and repeated-measures ANOVA. When such variation was statistically significant, multiple pair-wise comparisons were made using the Bonferroni modification of the significance level of a t-test to identify if the variability was restricted to a few comparisons. Multiple-comparison tests were only used for continuous data. The analysis was done with SPSS software (SPSS Inc., Chicago, Illinois, U.S.A.).

The percentage of children recording high readings from Dentobuff® strips was never less than 27%. Less than 17% of individuals were in the low Dentobuff® class except in October and June when over 20% were in this category. Intra-individual variation in this total group was statistically significant (Friedman two-way ANOVA, p<0.001). There was no statistical evidence that this was related to sex.

The numbers of samples analysed for calcium ranged from 28 in February to a maximum of 37 during the months of March, April, May and June. The group average ranged from 1.38 mmol/l in December to 1.83 mmol/l in January. Intra-individual variation throughout the investigation was statistically significant (repeated-measures ANOVA, p<0.001). When segregated according to sex, no statistically significant difference was observed.

Multiple pair-wise comparisons revealed that the paired comparisons November vs December, December vs January and November vs February were statistically significant (paired t-tests Bonferroni method, p<0.001). Yet, when these 3 months were excluded individually from repeated-measures ANOVA, intra-individual variation remained statistically significant (repeated-measures ANOVA, p<0.05). When both November and February were removed from analysis, intra-individual variation remained significant (repeated-measures ANOVA, p=0.002). Only when the two months of the paired comparisons, November vs December (repeated-measures ANOVA, p=0.086) and December vs January (repeated-measures ANOVA, p=0.081), were excluded was the intra-individual variation no longer statistically significant.

The number of samples available for phosphate analysis varied between 27 collected during the month of February to a maximum of 37 in March and April. The group’s means were highest in September and April, 5.41 mmol/l and 5.40 mmol/l, respectively. The lowest group average was in December, 4.96 mmol/l. A repeated-measures ANOVA did not detect a significant intra-individual variation for the duration of the study (p=0.14).

Intra-individual variation of salivary buffering capacity and calcium was here observed over a 10-month interval. While intra-individual biological variation is not unusual, predictive tests of disease based on salivary composition assume that these variables are reproducible on the majority of occasions within an individual. This variation may reflect salivary flow-rate variation, primary fluctuation of salivary constituents and external influences such as hormonal influences. The methods used to analyse the various constituents could also have contributed to the variability observed.

There was some evidence from multiple-comparison tests that the contrast between individuals’ salivary calcium in December compared with November and January was within the time when the majority of intra-individual variation occurred. This echoed the group means, where December had the lowest mean calcium (1.38 mmol/l; n=35) recorded and the adjacent months, November and January, corresponded to the two highest means (1.82 mmol/l; n=35 and 1.83 mmol/l; n=35). When comparisons of December with its neighbouring months were excluded from the repeated-measures ANOVA, intra-individual variation was no longer statistically significant. However, this loss of significance could have been confounded by a reduced sample size. November vs February pair-wise comparisons were also statistically significant in multiple-comparison tests, yet series of measures for both these months were excluded from repeated-measures ANOVA, statistical significance remained (p=0.002). February had the least number of samples available for paired intra-individual analysis and so its elimination would have had a smaller effect on sample size. Furthermore, the maintenance of statistical significance despite the removal of November, December and February individually from repeated-measures ANOVA suggests that intra-individual variation cannot unequivocally be assumed to be wholly restricted to a few months.

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Short Period Lines in Human Dentine

There are no comprehensive data on the expected range of measurements for the spacing of short-period lines in ground sections of human dentine, or for that of other animals, at precise locations within the teeth. Kawasaki et al. (1980)found that concentric calcospheritic lines were 1.7 to 2.0 ?m apart in demineralized human dentine, and that other short-period markings were 4 ?m apart in both ground and demineralized sections of human incisors and premolars; the spacings of the calcospheritic markings were measured in dentine that had been block-stained with silver by the Bielschowsky technique before sectioning. Kobayashi (1984)measured the interval between ‘laminar lines’ in human dentine as between 2 and 4 ?m. Kodaka and Higashi (1995)described a new method of preparing dentine for scanning electron microscopy by etching a fractured surface with EDTA after treatment with NaOCl; they reported that the spacing between both spherical (calcospheritic) and ‘laminar lines’ was of the order of 1 ?m, and therefore considerably closer together than reported by Kawasaki et al. and Kobayashi. It is hard to relate any of these data to, for example, the earliest formed mantle dentine or root dentine, or to dentine that is forming fastest in cuspal regions, or to slower forming circumpulpal dentine, as little information about location is provided.

There is also some information on the average spacing of short-period markings in the dentine of other animals. Schour and Hoffman (1939a)made measurements of incremental markings in the dentine of 17 species and concluded that the average spacing of all of them was 16 ?m (range 11.25–19.25 ?m). However, these estimates were presumably based on measurements made from wherever possible in the roots or crowns of teeth. While Schour and Hoffman (1939b)also provide experimental evidence for the daily rate of dentine formation in some animals, including humans and macaque, Kawasaki et al. (1980)make the point that they never measured the spacing between the short-period incremental markings in more slowly forming, calcospheritic dentine in either human or non-human primates, nor in any of their experimental animals.

Some confusion exists in the literature on human dentine as short-period (von Ebner’s) lines have also been reported to be between 14 and 20 ?m apart, and this has become established in textbooks on dental histology suggest that such observations might have resulted from the misidentification of wider spaced, long-period Andresen lines in human dentine as short-period von Ebner’s lines in the sections used for making the measurements. The biggest problem, in fact, is that eponymous designations are an inappropriate way of describing any structural feature unless there is an anatomical definition of what they actually represent. Dental histology is replete with eponymous terms such as these that are not well defined. Frank and Nalbandian (1989)state that the eponymous descriptions of incremental markings in dentine are inadequate to distinguish between the numerous striae that can be revealed by various techniques. Part of the present purpose was to help clarify the description of short-period lines in dentine.

Given the similarity in the spacing of long-period lines in human dentine to that of short-period lines described for so many other animals by Schour, I. and Hoffman, M.M., 1939. Studies in tooth development. I. The sixteen microns calcification rhythm in the enamel and dentin from fish to man. J. Dent. Res. 18, pp. 91–102Schour and Hoffman (1939a), it is easy to see how confusion might arise in comparative studies. In fact, the short-period lines can sometimes be seen to occur between wider spaced, long-period lines in many sections of human and non-human primate dentine. Lines spaced between 14 and 20 ?m apart in human dentine are not likely, therefore, to be short-period lines at all.

Fig. 7.Both short- and long-period incremental lines in human dentine visible together in (A) a ground section viewed in polarized light, and (B) a demineralized silver-stained section viewed in transmitted light only. Both micrographs are of intercuspal circumpulpal dentine low in the crown. The long-period lines are approx. 20 ?m apart and there are seven or eight short-period lines between them in both of these sections (although they vary in number between seven and ten most commonly in human teeth). Fieldwidth 225 ?m. Original magnification ×500.

The principle aim now was to present some comparative data on the spacing of short-period (von Ebner’s) lines in human dentine and in the dentine of three other primates (a gibbon, a siamang, and a great ape, as well as in the dentine of a pig canine [as there are some good experimental data on the periodicity, but not the spacing, of von Ebner’s lines in pig dentine]. The exact locations of measurements made in dentine have not been well documented. Another aim, therefore, was to compare these data-sets at known distances from the root surface in order to describe the spacing of short-period lines close to the start of dentine formation here and, where possible, again in the axial plane of the tooth cusp, where rates of formation are likely to be maximal. A further aim was to compare data for the spacing of short-period lines in sections of demineralized human dentine with those from ground sections in order to judge the likelihood that any preparation artefact or shrinkage might affect measurements of the spacing of lines.

Several investigators have observed that incremental markings in dentine can be enhanced by either making ground sections anorganic, by demineralizing them (e.g. by observing sections of carious teeth), by heating them sufficiently to drive off CO₂ from carbonate-rich regions, by staining demineralized blocks or sections with silver, or by treating fractured blocks with NaOCl and then etching them with EDTA. In this study, sections of permanent human canine and premolar teeth were chosen from a large collection that showed good short-period markings. Two ground sections of permanent canines (made anorganic with NaOCl just before mounting with DPX) were chosen that showed good short-period markings close to the root surface and in the axial plane of the tooth, respectively. A further demineralized section of a premolar tooth stained with silver by the Bielschowsky technique, which showed good short-period markings close to the root surface and in the axial plane of the tallest cusp, was chosen.

Ground sections of some non-human primate teeth belonging to a gibbon (Hylobates moloch), a siamang (Hylobates (Syndactyles) symphalangus) and a great ape (Pongo pygmaeus), and of a pig canine (Sus) were also included in this study, primarily because they showed good short-period calcospheritic markings in the dentine close to the root surface. However, the data obtainable from these sections were much more limited than from the human sections. None of these sections had been made anorganic or was demineralized, nor had they been specially prepared to enhance lines in the dentine in any way but were simply routine ground sections from a large collection of comparative material in the Department of Anatomy and Developmental Biology at University College London. While the lack of special specimen preparation may account for their limited usefulness in this study, the data were included to broaden its scope.

Montages of photomicrographs were prepared from along the root surfaces of each of these sections that included the granular layer of Tomes and root dentine some 300 ?m deep to this layer. Sections of root were chosen in the cervical portion of the sections and the montages were constructed to extend at least 1000 ?m along the root. Montages of dentine in the axial plane of the tallest cusp were also prepared in regions where good short-period lines were present. Micrographs were taken with a ×50 objective in transmitted light using a Ziess Jenamed microscope (sections were not viewed in polarized light as this did not improve the quality of the image of the lines and if anything made them worse).

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Antifungal Agents Dissolved in Distilled Water

Oral candidosis is a common opportunistic infection in elderly denture wearers. For instance, 60% of denture wearers over the age of 60 years in Denmark suffer from Candida-associated denture stomatitis. Although these superficial infections are the most prevalent form of the disease, systemic candidal infections are increasingly seen particularly in the compromised, such as those with AIDS.

Acrylic dentures are an important predisposing factor for oral candidosis as these appliances, which are usually ill fitting and with suboptimal hygiene, act as reservoirs of infection. For instance, high salivary yeast counts are much more common in full-denture wearers than in dentate individuals, and yeasts are demonstrable in 78–100% of patients with denture-induced stomatitis as contrasted with 30–60% of the non-denture-wearing general population. It is thought that the ability of Candida spp., especially C. albicans, to adhere to acrylic surfaces may be important in the pathogenesis of the disease as adherence is apparently the initial step in microbial colonization and subsequent invasion of host surfaces.

Candida-induced denture stomatitis is most commonly treated with the polyene antifungal agents nystatin and amphotericin B, which are used topically and sometimes applied to the fitting surface of the denture before its insertion. Imidazoles such as ketoconazole and the newer triazoles are used for treating the condition in recalcitrant cases. With the increasing number of patients compromised by diseases such as HIV infection, haematological malignancy, and by treatment protocols, including aggressive cytotoxic and broad-spectrum antibiotic therapy, the use of other agents such as the DNA analogue 5-fluorocytosine has also become important in the management of candidosis.

Despite the availability of a range of antifungal agents for the treatment of oropharyngeal candidosis, failure of therapy is not uncommon, as the efficacy of treatment is dependent upon many factors. In the mouth the diluent effect of saliva and the cleansing action of the oral musculature often tend to reduce the viability of the agents to below that of the effective therapeutic concentration. Thus the organisms experience only a limited exposure to the antifungal agent during treatment and the concentration of the drug may vary in different niches of the mouth. Candida biofilms on oral surfaces and prosthetic devices may also contribute to failure of drug therapy.

Though there have been studies on candidal adhesion to denture acrylic pretreated with antifungal agents, there is limited information on the adhesion of the drug-exposed yeasts to denture acrylic. Thus, our main aim now was to compare the adhesion of seven oral C. albicans isolates to denture acrylic after limited exposure (1 h) of the isolates to subcidal concentrations of five antifungal agents, nystatin, amphotericin B, 5-fluorocytosine, ketoconazole and fluconazole.

Seven isolates of oral C. albicans were studied. BM 20617, BS 742, BU 1010, BU 47204 and CA 0202 were all oral isolates from HIV-infected patients attending the Oral HIV Research Clinic at the Faculty of Dentistry, Prince Philip Dental Hospital, University of Hong Kong. These patients were not on any antimycotic therapy. The isolates designated 128981 LA and 106083 were from HIV-negative patients. The organisms were identified by the germ-tube test and the commercially available API 20 (API System, Vercieu, France) identification kits. Stock cultures were maintained at ?20°C. After recovery these were maintained on Sabouraud dextrose agar, stored at 4–6°C, during the experimental period.

Nystatin (Sigma, USA) and amphotericin B (Sigma) were dissolved in dimethylsulphoxide and absolute ethanol (3:2 ratio). 5-Fluorocytosine (Sigma) was dissolved in sterile distilled water. Ketoconazole (Janssen, Beerse, Belgium) was dissolved in dimethylsulphoxide, and fluconazole (Pfizer, Groton, CT, USA) in absolute methanol. All agents were prepared initially as 10,000 ?g/ml solutions and stored at ?20°C before use. The antifungals were suspended/diluted in the following medium during the exposure (1 h) of yeasts: RPMI 1640 medium buffered with 0.165 M MOPS containing -glutamine and lacking sodium bicarbonate (Sigma) was dissolved in 1 litre of sterile distilled water, adjusted to pH 7.2 and filter-sterilized. This liquid RPMI 1640 was stored at 2–8°C for 2–3 months.

As the antifungal agents used were dissolved in dimethylsulphoxide and absolute methanol, equivalent amounts of these chemicals were tested initially to ascertain whether they had an effect on the isolates tested. The minute volumes of the chemicals used did not have any effect on yeast survival/growth when compared with the controls.

The MIC for all seven strains for all the five drugs were determined by the broth-dilution technique, by performing 2-fold serial dilutions of the drug in microtitre plates using an inoculum of 1–5×10⁵ c.f.u./ml (a cell suspension was made at 520 nm to obtain an OD of 0.450 in sterile PBS and a 10-fold dilution was made to obtain an inoculum of 1–5×10⁵ c.f.u./ml). The MIC was determined visually and spectrophotometrically at 595 nm, after 24 h of incubation at 37°C. The MIC was defined as the lowest concentration of the drug which inhibited growth of yeast cells, as indicated by the absence of turbidity (optically clear). In determining the MIC for ketoconazole and fluconazole a slight modification of the above technique was used as these drugs exhibited a phenomenon known as trailing. Trailing occurs when the turbidity continually decreases as the concentration of the drug increases but the suspension fails to become optically clear. Thus, for ketoconazole and fluconazole the MIC was considered as the lowest concentration of the drug with a slight haziness in the well. Subcultures were made from the turbid/clear wells to confirm the turbidity results. All experiments were repeated on two separate occasions with duplicate determinations on each occasion.

The acrylic strips for the adhesion assay were prepared as described by Samaranayake and MacFarlane (1980), with some modifications. In brief, transparent self-polymerizing acrylic powder (1.5 g polymethyl methacrylate powder) was spread on an aluminium foil-covered glass slide (2.5×7.5 cm). Monomer liquid (1 ml) was poured on to the surface of the slide and immediately a second slide similar to the first was placed on top of the polymerizing mixture, and the slides were firmly secured at both ends with two binder clips. After bench-curing for 30 min the glass slides were separated. The resultant acrylic strips were cut into 5×5 mm squares, immersed in distilled water for 1 week to leach excess monomer, disinfected by dipping in 70% alcohol for 1 min, and washed with sterile distilled water. The strips were then ultrasonicated for 20 min (Branson 2200 E3 ultrasonic cleaner, CT, USA), washed again in sterile distilled water, dried and used for adhesion assay after checking their sterility.

Yeast cells, maintained on Sabouraud dextrose agar, were inoculated on to fresh plates and incubated overnight for 24 h before use. The organisms were harvested and a cell suspension prepared in sterile PBS at 520 nm to an OD of 1.5. From this cell suspension, 0.5 ml was added to tubes containing 2 ml of RPMI broth (control) and 2 ml of RPMI/drug solution (test) in which the drug concentrations varied from four–eight times the MIC. The drug concentrations used were subcidal concentrations of nystatin (×6 MIC), amphotericin B (×8 MIC), 5-fluorocytosine (×8 MIC), ketoconazole (×4 MIC) and fluconazole (×4 MIC). This gave a suspension of 10⁶–10⁷ cells/ml in each assay tube.

The tubes were then incubated at 37°C for 1 h in a rotary incubator. Following this limited exposure the drugs were removed by two cycles of centrifugation for 10 min at 3000×g. Afterwards the supernatant was completely decanted and the pellets were resuspended in 2.5 ml of sterile PBS. Viable counts of the control and the test were performed by spiral plating after drug removal, and control suspensions were reconstituted as needed to obtain a cell concentration similar to the test.

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Periodontal Ligament and Mechanical Stress

The periodontal ligament is a dense connective tissue between the root cementum and the alveolar bone that anchors the tooth and maintains the structural integrity of these mineralized tissues. Fibroblasts in the periodontal ligament, the major cell type, are regarded as multipotential, or form a heterogeneous population that can differentiate into either cementoblasts or osteoblasts, depending on needs and conditions. Thus, the ligament has been regarded as the source of cementoblasts and osteoblasts. Indeed, cultured fibroblasts from the ligament exhibit bone cell-like properties in vitro, including a relatively large amount of alkaline phosphatase activity, and the expression of bone-matrix proteins such as osteopontin and secreted protein, acidic and rich in cysteine (SPARC). Similar to bone-forming cultures, cells of the periodontal ligament undergo osteoblastic differentiation in response to dexamethasone, a synthetic glucocorticoid widely used to induce differentiation of osteoblasts, as characterized by elevated alkaline phosphatase activity, increased expression of osteopontin and bone sialoprotein, and the formation of mineralized nodules.

The mechanisms by which cells of the periodontal ligament maintain their phenotype and differentiate into mineralized tissue-forming cells remain unclear. In attempts to examine the involvement of polypeptide growth factors, we have earlier investigated the role of EGF and its receptor in dexamethasone-induced in vitro differentiation of rat periodontal-ligament cells. We found that EGF antagonized differentiation and up-regulated EGF-R expression, whereas EGF-R was down-regulated in the course of differentiation. Therefore, the EGF/EGF-R system appears to be important as a phenotype stabilizer by functioning as a negative regulator of osteoblastic differentiation in these cells. Although in vivo observations on the rat supported these in vitro results, a detailed study on the role of EGF/EGF-R in cells of the human periodontal ligament cultured in conditions closer to the physiological, without dexamethasone, is still needed.

Physiologically, the periodontal ligament is continuously subjected to mechanical stress caused by occlusal forces. Furthermore, remodelling of the ligament and alveolar bone occurs in response to orthodontic forces. These facts led us to speculate that responses of the ligament to mechanical stress are involved in its cell proliferation and differentiation. In fact, it has been shown that a variety of cells respond to mechanical stress, such as tension force, compression and fluid shear stress, by demonstrating significant changes in their structure and function. In bone, Raab-Cullen et al. (1994)have reported, using the rat tibia 4-point bending model, that external mechanical loading induces a rapid and transient increase in mRNA expression for c-fos, a gene associated with proliferation and/or differentiation in bone development and fracture repair. Several in vitro studies on cultured osteoblastic cells have also demonstrated elevated amounts of bone-related molecules, such as alkaline phosphatase, osteopontin and osteocalcin, in response to mechanical stretching. Therefore, it appears that essential functions of osteoblasts in bone remodelling are affected by experimentally loaded mechanical stress and that cellular responses to mechanical stress are crucial in homeostasis, adaptation to the environment, and regeneration of bone. In contrast, only a small number of studies have addressed the responsiveness of cells of the periodontal ligament to mechanical stress, in which the ability of cultured cells to proliferate in response to tension force was demonstrated. As we have shown that such cells maintain their phenotype and differentiate into osteoblastic cells through mechanisms involving the EGF/EGF-R system, it was of interest to investigate how they and the EGF/EGF-R system respond to mechanical stress. Furthermore, the interaction between mechanical stress and EGF/EGF-R has not, we believe, been reported elsewhere.

Our aim now was to elucidate the role of EGF and EGF-R in the proliferation and differentiation of cells of the human periodontal ligament in mechanical stress-loaded conditions in vitro. For this purpose, cultured ligament cells were loaded with cyclic stretch using flexible-bottomed culture plates and their responses were monitored. Then the effect of EGF on these responses was examined. Finally, we evaluated changes in the amount and autophosphorylation of EGF-R in response to cyclic stretching.

Fibroblastic cells were obtained from explant cultures of human healthy periodontal ligament taken from a third molar that had been extracted for orthodontic reasons, as described by Matsuda et al. (1996b). The tissues were minced, put in culture dishes and incubated in DMEM (Gibco Laboratories, Grand Island, NY) supplemented with 10% FBS (Intergen Company, Purchase, NY), non-essential amino acids, 10 mM sodium pyruvate, vitamins (Gibco) and an antibiotic mixture (1 U/ml penicillin, 1 U/ml streptomycin, 1 U/ml gentamycin; Sigma Chemical Co., St. Louis, MO) at 37°C in a humidified atmosphere of 5% CO₂–95% air. When the outgrowing cells reached confluency, they were trypsinized with 0.05% trypsin (1:250)–0.53 mM EDTA 4 Na (Gibco) in PBS for secondary culture. Cultures were maintained until confluency and passed at a 1:4 split ratio. All the experiments were done on cells of between three and seven passages.

As an experimental model of mechanical stretch, tension force was loaded on to cells cultured on a flexible substratum (25 mm dia., Flex I culture plate; Flexcell International Corporation, McKeesport, PA) by applying vacuum-operated negative pressure using the Flexercell Strain Unit Model FX-2000, which is capable of controlling the magnitude as well as the frequency of cell deformation. Although this system has been used in many investigations, one problem with the apparatus is that the substrate of the strain well is strained in a non-uniform manner; deformation is greatest at its periphery and least at its centre. Therefore, we aimed to examine the net response of the total number of cells to mechanical strain. Cells were subjected to 9 or 18% of maximum strain for 5 s followed by 5-s relaxation (6 cycles/min). According to the manufacturer, in these conditions the strains are distributed inhomogeneously such as ?5–9% or ?4–18%.

Cells were plated on to a Flex I culture plate at concentrations of 2.5×10⁴, 5.0×10⁴, or 1.0×10⁵ per well and incubated in DMEM/10% FBS for 24 h. After replacement of the medium with fresh DMEM/10% FBS, Flex I plates were placed on the Flexercell strain unit to apply cyclic stretch to the cells. At 1, 3, or 5 days of incubation under cyclic stretching, cells were harves ted by trypsinization and counted in a haemocytometer.

After 5 days of culture with or without stretch, cells were rinsed with PBS twice and fixed by incubation with 10% neutral-buffered formalin for 30 min. The flexible substrate of 25 mm dia. was then removed from the plate and cut to 20×20 mm square. Actin stress fibres were stained with 33 nM of rhodamine phalloidin (Molecular Probes, Inc., Eugene, OR) for 30 min. Stained cells were viewed on an LSM410 invert laser-scan microscope (Carl Zeiss, Jena, Germany). For rhodamine visualization, fluorescence was excited at 543 nm and emitted light was detected at 590 nm.

As one of the differentiation markers of the ligament cells, their alkaline phosphatase activity was examined. Ten thousand cells were plated on Flex I culture plates in DMEM/10% FBS and incubated until they reached confluency. After further incubation for 24 h in DMEM/1% FBS containing 50 mg/ml ascorbic acid and 10 mM ?-glycerophosphate (mineralizing medium) with or without 10 nM dexamethasone (Wako Pure Chemical Industries, Osaka, Japan), cells were then subjected to cyclic stretching for 2, 4 or 6 days. Alkaline phosphate activity of cell lysates was determined by using p-nitrophenyl phosphate (Wako) as a substrate. The enzyme activity was expressed as U/mg protein. For enzyme staining, cells were washed with PBS and fixed in 10% cold neutral-buffered formalin for 15 min. After washing with distilled water, cells were incubated for 45 min at room temperature in alkaline-phosphatase substrate solution consisting of 0.1 mg/ml naphthol AS MX-PO₄ (Dojin Chemical, Kumamoto, Japan), 0.4% N,N-dimethyl-formamide (Wako) and 0.6 mg/ml Fast red-violet LB salt (Sigma) in 0.1 M Tris–HCl, pH 8.3. Cells were then washed with distilled water and with 0.5 M HCl. Finally, they were stained with 0.25% alcian blue 8GX (Sigma) in 0.5 M HCl.

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Cell Line of Arachidonic Acid

Host–tumor interactions lead to a non-specific inflammatory response mediated in part by the chronic production and release of pro-inflammatory cytokines, including IFN-?, interleukins, and tumor necrosis factor. Cytokines are critical for tumor regression. IFN-? is a cofactor in the activation of macrophages, which kill tumor cells, and also enhances cytotoxicity by natural killer cells. Interferon inhibits the growth of melanoma, renal-cell carcinoma, acquired immune deficiency syndrome-related Kaposi’s sarcoma, and other malignant carcinoid tumors. Interferon therapy can induce remission in human malignancies and is established as a treatment of choice in several diseases. The effect of IFN-? on oral cancer cells was investigated here.

EPA is derived from linolenic acid (designated n-3); it forms trienoic prostaglandins and pentaene leukotriene derivatives within several cell systems. EPA can serve as a competitive inhibitor of the biosynthesis of dienoic prostaglandins and tetraene leukotrienes from arachidonic acid. Diets containing large amounts of n-3 polyunsaturated fatty acids inhibit tumorigenesis. Diets providing high concentrations of n-3 fatty acids suppress tumour progression. Inhibition of human breast-cancer cell growth by EPA may be by the suppression of tumour eicosanoid biosynthesis. EPA may also have a suppressive effect on other cancer cells, including oral. This report will examine the biosynthesis of eicosanoids by oral cancer cells.

A number of inhibitors of cyclo-oxygenase and lipoxygenase pathways of arachidonic acid are important tools for the study of the role of arachidonic acid metabolites in various biological or pathological processes. Indomethacin is an inhibitor of the cyclo- oxygenase pathway of arachidonic acid metabolism. NDGA is an inhibitor of the lipoxygenase pathways. ETYA, the tetraacetylenic analog of arachidonic acid, is recognized as a potent inhibitor of both pathways. Dexamethasone, an anti-inflammatory steroid, exerts its effect by inhibiting the release of arachidonic acid from phospholipids. Using these enzyme inhibitors can help to confirm that this eicosanoid biosynthesis is via enzymatic pathways, as can high-temperature incubation, which would inactivate the synthesizing enzymes and thereby suppress the biosynthesis.

The possibility that oral cancer cells may be affected by IFN-?, EPA, and inhibitors of cyclo- or lipoxygenase is worthy of in vitro examination. In our laboratory, we have established an OEC-M1 cell line from the gingival tissues of a Chinese patient with oral carcinoma. We have now investigated the effects of IFN-?, EPA, indemethacin, NDGA, ETYA, and dexamethasone on the biosynthesis of two eicosanoid-like substances in the OEC-M1 cell line in vitro. We have also tested the specificity of the results by comparing the above-mentioned effects with those of a normal fibroblast cell line derived from human buccal mucosa and another human oral cancer cell line (KB).

The OEC-M1 cell line was established in our laboratory from the gingival epidermal carcinoma of a Chinese patient. The study protocol was approved by the Ethics Committee of the Tri-Service General Hospital, and written informed consent was granted by the patient. Human buccal-mucosa fibroblast cell lines were obtained and cultured according to the method of Van Wyk et al. (1994). Another human oral epidermoid-carcinoma cell line (KB) was obtained from the American Type Culture Collection (Rockville, MD). The fibroblasts and KB cell line were cultured to confluence and harvested for the detection of eicosanoid synthesis as control cells. The culture flasks contained 25 ml of RPMI-1640 medium with 10% FBS, 2 mM -glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 ?g/ml streptomycin, and 0.1% Fungizone (Gibco Labs., Grand Island, N.Y.). The cells were incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. The culture medium was changed twice per week. Confluent cells were trypsinized with 0.05% trypsin in 0.02% EDTA. The trypsinized cells were seeded into more flasks and used in the following experiments until confluent.

Confluent cells were incubated with 0.4% FBS for 1–2 days until quiescence. After 1–2 days in the quiescent stage, the medium was removed and replaced with a serum-free medium containing different additives (IFN-?, Genzyme Corp., Cambridge, MA; EPA, NDGA, ETYA, Biomol Research Lab. Inc., Plymouth Meeting, PA; indomethacin, dexamethasone, Sigma Chemical Co., St. Louis, MO) for 12 h. Control groups were incubated in media containing no additives.

After this preincubation, cells from each group were harvested and resuspended in 4 ml of sodium phosphate buffer (10 mM NaH₂PO₄, 10 mM Na₂HPO₄, 100 ?M dithiothreitol, pH 7.4), and then homogenized with a Polytron operated at full speed for 2 min. The homogenates were centrifuged at 400×g for 5 min at 4°C to remove the unminced large fragments. The supernatant was removed and incubated with 10 ?M unlabelled arachidonic acid (Sigma) and 370 kBq [³H]-arachidonic acid (Amersham Corp., Arlington Heights, IL) in an equal volume of 0.1 M Tris–HCl buffer (pH 8.5), containing 2 mM glutathione, 1 mM hydroquinone, and 2?M hemin, at 37°C for 10 min. The reaction was terminated by the addition of 2 volumes of ethanol.

The precipitates from the cell homogenates were centrifuged at 800×g for 10 min at 4°C and the supernatant layer was evaporated to the aqueous phase. Methanol was added at a final concentration of 20%. An ODS–silica column (Sep-Pak C18 cartridge, Waters Associates, Milford, MA) was attached to a 20-ml polypropylene Luerlok syringe and washed successively with 5 ml of methanol and 5 ml of water, and the biological samples were then applied. The column was washed sequentially with 6 ml of 20% methanol and 6 ml of 80% methanol. The eicosanoids were collected and evaporated to dryness under a stream of nitrogen. The residues were then reconstituted in 1 ml of methanol and filtered through a 0.45 ?m filter (Millipore). The samples were again evaporated to dryness under nitrogen and reconstituted in 50 ?l of methanol/water (60:40, v/v), then separated by RP–HPLC.

The purified eicosanoids were separated by RP–HPLC in a Waters Dual Pump System equipped with a reverse-phase ultrasphere ODS column (Inertsil–ODS, 10?, 3.9 mm×30 cm; Vercotech Inc. Taipei, Taiwan). The products were eluted with an isocratic solvent system of methanol/water/acetic acid (60:40:0.01, v/v, pH 5.7) for 60 min at a flow rate of 1 ml/min. Column effluents were monitored with a Waters UV-VIS spectrophotometric detector (486 Turnable Absorbance Detector) at 280 nm. Tritium-labelled eicosanoid-like compounds in the eluate were simultaneously detected with a Radiomatic HPLC radioactivity monitor (Flo-one/Beta) attached to the Waters HPLC unit.

Samples eluted from the HPLC were simultaneously collected with an on-line fraction collector and ultraviolet absorbance spectra were analysed with a Hewlett-Packard 8450-A ultraviolet/visible spectrophotometer at 280 nm.

Statistical analyses used one-way ANOVA (Tukey posthoc test) by SPSS package. Probability values less than 0.05 were considered significant.

After incubation of OEC-M1 cells with different concentrations of IFN-?, EPA, indomethacin, NDGA, ETYA, and dexamethasone for 12 h, we observed the state of cell growth and detected the production of eicosanoids. There were two predominant peaks in the HPLC results which coincided with the radioactivity of [³H]-arachidonic acid. One compound (peak 1; P-1) eluted before prostaglandin B₂ (more polar than prostaglandin B₂) and the other compound (peak 2; P-2) eluted before leukotriene D₄ and just after leukotriene C₄ (more polar than D₄ and less polar than C₄). P-1 had ultraviolet absorption at a ?_max of 278 nm with shoulders at 272 and 284 nm. P-2 had ultraviolet absorption at a ?_max of 284 nm and shoulders at 278 and 290 nm. The front peak may contain a number of mixed polar metabolites and the last peak is arachidonic acid itself. These findings suggest that these compounds are possible leukotriene-like substances with conjugate triene-structure.

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Cementum–ivory Junction Dark Bands of the Tusk

Elephants are characterized by a highly pneumatic skull with a premaxilla that descends vertically, bearing the roots of the paired tusks, which represent enormously enlarged maxillary incisors. The growth of the tusk is continuous throughout life and its size at any age is dependent on the sex of the animal, the rate of attrition and breakage of the tooth, as well as genetic and environmental factors. The record length of a single tusk is 3.51 m and record weight 117 kg. The bulk of the tusk consists of ivory (or dentine), ensheathed by a layer of cementum.

The unique chequered pattern of polished ivory, which has not been described in great detail in the literature, has made it a sought-after product in the manufacturing of jewellery and other works of art. Our purpose now was to propose a hypothesis on the histogenesis of the unique chequered pattern of elephant ivory.

Twenty fragments of ivory and five tusks of African elephant (Loxodonta Africana) and a developing tusk of a 22-month fetus were obtained through the National Parks Board of South Africa. The ivory was harvested from animals that had died as a result of natural causes or as part of the population-control programme of the National Parks Board. The fetal tissue was obtained within 20 min of death. The biopsies were taken with a sharp scalpel from the peripulpal ivory of the proximal part of the tusk, cut in thin slices and then fixed in 10% phosphate-buffered formalin at 21°C.

The five tusks, with masses between 0.7 and 9.3 kg, were sectioned and polished in sagittal and coronal planes. The morphological characteristics of the chequered pattern were described on polished surfaces prepared in both planes. Irregularities affecting the pattern were noted.

Ground sections were prepared parallel and perpendicular to the long axis of 12 different fragments of ivory by cutting 300-?m thick slices of ivory from the respective planes with a rotating diamond saw. The thick slices were fixed to glass slides with methylmethacrylate cement and ground to a thickness of approx. 40 ?m on a rotating disc utilizing different grades of diamond pastes. The ground sections were covered with glass cover-slips after debridement with a weak acid solution (0.1 M HCl) and a soft brush, and examined by transmission light microscopy under standard illumination.

Standardized techniques for the preparation of scanning electron micrographs of hard tissues were employed to visualize the morphology and distribution of dentinal tubules of six different tusks when viewed from the pulpal surface of the ivory. The presence of light or dark bands was noted during the preparation of an additional 10 sections at different levels perpendicular to the long axis of the tubules. The levels were selected so that five sections passed through light bands and five through dark bands. All surfaces were cleaned before scanning with a 0.1 M HCl solution and a soft brush, and flushed with deionized water to remove debris. The distance between tubules was expressed as the smallest linear measurement between two adjacent tubules and the tubule density as the number of tubules per mm². At least 10³ tubules were counted for each measurement. An image analyser was used (Flexible Image Processing System, CSIR, Pretoria) and findings were subjected to the Student’s t-test for unequal variances.

The entire outer surfaces of the tusks were covered by a layer of cellular cementum. Ivory was exposed on the working surfaces of erupted tusks through cemental abrasion. The cementum–ivory junction was visible as a dark, concentric ring demarcating the peripheral border of the ivory. In cross-sections through the tusk, the cementum–ivory junction followed an undulating circular course, forming irregular excrescences alternating with shallow convexities or concavities. The excrescences were more pronounced in tusks with smaller diameters. Occasional invaginations of cementum, which were 2–3 mm deep, were present in the outer layer of the ivory. The external contour of the cementum followed the morphology of the cementum–ivory junction, resulting in longitudinal and parallel ridges and troughs had been visible upon external examination of the tusks. These ridges and troughs had been abraded away on the anterior and more exposed surface of the tusk. The pulpal surface of the ivory was smooth and the pulpal cavity conical in shape, with the tip of the tusk composed of solid ivory and the apical foramen wide open.

Fig. 1.Cross-section through the solid part of a tusk. Note the undulating course the cementum–ivory junction follows and the geometric chequered pattern (bar=4 cm).

Fig. 2.Tusk of an elephant bull (top) and cow (bottom). Note the parallel ridges and troughs on the posterior surface of the bull’s tusk (arrows). The broken line in the lower tusk represents the wall of the conical pulpal cavity (bar=10 cm).

The unique chequered pattern was evident on polished surfaces of cross-sections through the tusk. The pattern consisted of two systems of alternating light and dark lines which started adjacent to the cementum. One system swept clockwise and the other anticlockwise towards the centre of the tusk, intersecting at regular intervals. The chequered pattern corresponded to parallel alternating light and dark lines evident on polished surfaces prepared in the sagittal plane. The cementum–ivory junction was straight when viewed in this plane. Planes of fracture through ivory tended to follow the contours of the dark bands.

Fig. 3.Sagittal section through a tusk. Note the conical pulpal cavity and parallel light and dark bands in the ivory, the latter of which are evident in the outer third (bar=4 cm).

The outermost layer of ivory (mantle ivory) was between 40 and 80 ?m thick and consisted of irregularly spaced dentinal tubules which were slanted apically at a sharp angle to the cementum–ivory junction. These tubules appeared to branch extensively, a feature which was best observed when focusing at different planes in the section. Morphologically, the mantle ivory resembled the granular layer of human radicular dentine. After passing through the mantle ivory, the tubules became more regularly spaced and gradually changed their course by curving towards the tip of the tusk. This curvature was the beginning of the regular, sinusoidal course followed by the dentinal tubules in a pulpal direction and was evident only in sections prepared in the sagittal plane. The convexities and concavities of this sinusoidal pattern corresponded to the alternating light and dark bands seen macroscopically on surfaces prepared in the sagittal plane. The dark bands correlated with that part of the dentinal tubules that curved apically. On high-power magnification, many dentinal tubules appeared to end blind and scattered tubules appeared to fuse forming one tubule. The blind-ending tubules were more frequent in the dark bands in ivory (16 blind-ending tubules per 100 tubules, SD 7 tubules) than in the light bands (6 blind-ending tubules, SD 3 tubules) as measured over 2500 tubules in each of the dark and light bands, respectively (p<0.001). The process of apparent fusion involved the main dentinal tubule and was distinct from the fine lateral branches that seemed to anastomose with those of adjacent tubules. Demineralized sections of newly formed ivory in the fetal tissue showed crowding of distally slanted odontoblasts and scattered pyknotic cells, highly suggestive of individual cell death.

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2-Mercaptoethanol Amino Acid Derived from Proteins

?-Adrenergic stimulation of the rat parotid gland induces exocytotic amylase secretion from the acinar cells through a cAMP-mediated pathway involving cAMP-dependent protein kinase. During a search of endogenous protein substrates for the kinase, we observed that ?-adrenergic stimulation induces not only phosphorylation of several endogenous proteins but also dephosphorylation of two 17-kDa soluble phosphoproteins. We identified those two proteins as destrin- and cofilin-like proteins from their partial amino acid sequences and further observed that cholinergic stimulation also induces their dephosphorylation. Both destrin and cofilin are actin-binding proteins that depolymerize F-actin; they are structurally related (sequence identity, about 70%), and destrin is also called actin-depolymerizing factor. Each protein exists in both phosphorylated and unphosphorylated forms, and the unphosphorylated form of each is active in depolymerizing F-actin. As ?-adrenergic or cholinergic stimulation of parotid cells also induces disruption of the cortical actin layers in parallel with amylase secretion, dephosphorylation of the destrin- and/or cofilin-like protein(s) might be involved in the disruption of the cortical actin layers. At present, little is known about the exact regulatory mechanisms for their phosphorylation. As a step toward understanding better the mechanism as well as their physiological roles in parotid cells, we here determined their complete amino acid sequences and phosphorylation sites.

Male Wistar rats (about 200 g, fed ad libitum) were anaesthetized with diethyl ether and killed by decapitation; parotid glands were collected and stored at ?80°C. The two proteins were partially purified from the glands as described previously for preparation of the partially purified pp17 fraction from non-stimulated rat parotid slices, and subjected to the two-dimensional PAGE procedure of O’Farrell et al. (1977)with modifications. The first-dimension gels were 4 mm dia. and loaded with samples each containing about 0.8 mg of protein. In some cases, proteins were electrotransferred after two-dimensional PAGE on to PVDF membranes (Immobilon-P^SQ; Millipore, Bedford, MA, U.S.A.) by the method of Kyhse-Andersen (1984)with modifications. The spots of the phosphorylated and unphosphorylated forms of the two proteins were excised from Coomassie blue-stained gels or PVDF membranes. Unless otherwise stated, experiments were done with their unphosphorylated forms that predominate even in non-stimulated parotid cells. (From the data obtained upon amino acid sequencing of the peptides derived from the two proteins, it is now known that the one spot of the unphosphorylated destrin-like protein contained at least 40 pmol of the protein, and that one spot of the unphosphorylated cofilin-like protein contained at least 20 pmol of the protein. Data are not shown.) The 6 to 12 pieces of gel containing the same protein (at least 200 pmol) were then placed on an agarose (Agarose 421F; Funakoshi, Tokyo, Japan) tube gel (10×35 mm). Each protein in the gels was electrophoretically transferred to the agarose gel with the stacking buffer system in the SDS–PAGE procedure of Fling and Gregerson (1986). (Electrophoresis was at 3 mA/gel until the Coomassie-blue band reached 25 mm below the gel top. At this point, the SDS front and the band of the tracking dye bromophenol blue were slightly below and above, respectively, the Coomassie-blue band. Proteins were stacked between the SDS front and the bromophenol-blue band.) The gels were cut 1 mm below the SDS front and 1 mm above the bromophenol-blue band. The middle segments were placed in tubes each containing nine volumes of 20 mM Tris–HCl (pH 7.5) supplemented with 0.1% SDS, 0.1% 2-mercaptoethanol and 0.1 mM EDTA, and melted at about 100°C. The resultant solutions were frozen, kept for at least 2 h at ?80°C, thawed, and centrifuged at 4°C at 15,000 g for 20 min. The supernatant solutions were dialysed against two changes of 60 volumes of 10 mM NH₄HCO₃ containing 0.1% SDS and 0.1% 2-mercaptoethanol for a total of 24 h at room temperature and lyophilized. Each dried sample was dissolved in one gel volume of H₂O and mixed with nine volumes of ethanol. The mixtures were stored overnight at ?30°C and centrifuged at 2000 g for 20 min at room temperature. The precipitates were washed with 90% ethanol and dried.

In the initial experiments, the purified proteins were reduced on membranes with tributylphosphine (Kanto Chemicals, Tokyo, Japan) and alkylated with 4-vinylpyridine (Wako, Osaka, Japan) or reduced in solution with Tris(2-carboxyethyl)phosphine (Boehringer Mannheim, Mannheim, Germany) (see below) and alkylated with 4-vinylpyridine. However, the amino acid sequencing and mass spectrometry of the peptides from the resultant proteins revealed that most Cys residues in them reacted with residual unpolymerized acrylamide during the preparative two-dimensional PAGE and were converted to Cys-S-?-propionamide residues (data not shown); similar observations have been reported. In later experiments, we therefore treated the reduced proteins with acrylamide (see below) to ensure their complete homogeneous alkylation. Acrylamide was purified with the mixed-bed resin AG 501-X8 (Bio-Rad, Hercules, CA, U.S.A.).

For the proteins recovered on the PVDF membranes, Coomassie blue was first removed with chloroform:methanol (9:1, v/v), as described by Wong et al. (1993). Each protein (3 to 6 spots) was then reduced in a tube containing 200 ?l of 0.2 M Tris–HCl (pH 7.5) supplemented with 7 M guanidine HCl, 4 mM EDTA, and 2 mM Tris(2-carboxyethyl)phosphine for 2 h at 37°C in the dark under N₂. Each tube thereafter received 2 ?l of 2-mercaptoethanol and 8 ?l of 3.5 M acrylamide and was incubated for 2 h at 37°C in the dark under N₂. The membrane pieces in each tube were then washed with H₂O (5×1 ml), dried, washed with 1-chlorobutane (2×150 ?l), and dried.

For the proteins extracted from gels, each protein derived from 6 to 12 spots was dissolved in 100 ?l of 0.35 M Tris–HCl (pH 7.5) containing 1% SDS, 4 mM EDTA, and 2 mM Tris(2-carboxyethyl)phosphine and incubated as above. Each sample received 1 ?l of 2-mercaptoethanol and 4 ?l of 3.5 M acrylamide, and was incubated as above. At the end of incubation, each sample received 100 ?l of 2 M dithiothreitol in 0.5 M Tris–HCl (pH 7.5) and was incubated for 30 min at 37°C in the dark under N₂. The resultant samples were dialysed against two changes of 1000 volumes of 10 mM NH₄HCO₃ containing 0.1% SDS and 0.1% 2-mercaptoethanol for a total of 24 h at room temperature and lyophilized. Each dried sample was dissolved in 100 ?l of H₂O, and the proteins were precipitated with ethanol as described above.

The membrane pieces carrying alkylated protein spots were wetted with methanol, rinsed with H₂O, incubated for 20 min at room temperature in a solution (70 ?l/piece) consisting of 0.1% acetic acid, 0.25% polyvinylpyrrolidone (M_r 40,000; Sigma, St Louis, MO, U.S.A.) and 0.1% methionine, and washed with H₂O (5×300 ?l/piece) and then with 10% acetonitrile (2×50 ?l/piece). The proteins on the membranes were digested in a solution (20 ?l/piece) consisting of 0.1 M NH₄HCO₃, 10% acetonitrile, and 4 ?g/ml of trypsin (modified trypsin; Promega, Madison, WI, U.S.A.) or endoproteinase Asp-N (Boehringer Mannheim) at 37°C for 20 h, and the supernatants were collected. The membrane pieces were washed with 10% acetonitrile (20 ?l/piece) and then with dimethylsulphoxide (10 ?l/piece) (dimethylsulphoxide extraction was not done for tryptic peptides). The supernatant and the washings from each tube were mixed, and the mixtures were dried.

For Asp-N digestion in solution, each alkylated protein derived from 6 to 12 spots was incubated for 18 h at 37°C in 100 ?l of 0.1 M NH₄HCO₃ containing 10% acetonitrile and 4 ?g/ml Asp-N.

For CNBr cleavage in solution, each alkylated protein derived from 6 to 12 spots was preincubated in 30 ?l of 0.5 M NH₄HCO₃ containing 5% 2-mercaptoethanol for 24 h at room temperature under N₂ (methionine sulphoxide residues were reduced during this preincubation), lyophilized after addition of 720 ?l of H₂O, and incubated in 50 ?l of 70% HCOOH containing 1% CNBr (Wako) for 20 h at 20°C in the dark under N₂. The samples thus prepared were dried.

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Dental Pulp Sympathetic Nerves in Human

The flow of blood through the pulp of mandibular canines in cats and dogs is reduced when the inferior alveolar nerve supplying these teeth is stimulated electrically through bipolar electrodes. This diminution in blood flow following electrical stimulation is inhibited in the presence of the -receptor antagonist phentolamine, which implies that sympathetic nerves and -receptor-mediated vasoconstriction are involved in the control of blood flow in dental pulp. However, the site of release of the endogenous transmitter following depolarization of the nerve membranes cannot be determined from these experiments, as the vasoconstriction might have occurred in either the vessels supplying the pulp or the microvascular bed within the pulp, or both. Recently though, Kerezoudis, N.P, Olgart, L and Edwall, L, 1992. Activation of sympathetic fibres in the pulp by electrical stimulation of rat incisor teeth. Archs oral Biol. 37, pp. 1013–1019 Abstract Kerezoudis et al. (1992)demonstrated that the application of a current of low intensity directly to rat mandibular incisor teeth induced a frequency-dependent vasoconstriction, which was inhibited by intravenous administration of phentolamine. These results are in accord with a proposal that, when stimulated electrically, sympathetic nerves within dental pulp influence the flow of blood through this tissue. Several studies confirm the presence of noradrenaline, the principal transmitter in sympathetic nerves, in dental pulp, while Parker et al. (1994a, b) report that [³H]-noradrenaline was released from sympathetic nerves in human dental pulp by electrical stimulation following incubation of this tissue in [³H]-noradrenaline.

Activation of -adrenoceptors on sympathetic nerves by various agonists, including endogenous noradrenaline itself, inhibits the release of noradrenaline through an inhibitory feedback loop. Parker et al. (1994a, b) report that presynaptic ₂-adrenoceptors are present on sympathetic nerves in human dental pulp and when activated inhibit the release of noradrenaline. Presynaptic dopaminergic receptors in human pulp also inhibit release, although it is suggested that they do not appear to be of physiological significance, in contrast to the presynaptic ₂-adrenoceptors.

Several investigators have described a positive feedback mechanism in sympathetic nerves in which the release of noradrenaline is enhanced by activation of presynaptic ?-adrenoceptors: Adler-Graschinsky and Langer (1975)demonstrated facilitation in isolated guinea-pig atria, Hedqvist and Moawad (1975)in human oviduct, Stjärne and Brundin (1976) in human omental arteries and veins, Molderings et al. (1988)in human saphenous vein and Göthert and Hentrich (1985) in human pulmonary artery. While three subtypes of the ?-receptor have been described, ?₁, ?₂ and ?₃, the ?₃ have been reported only at postsynaptic sites. Although in most tissues the ?-presynaptic receptor is of the ?₂-subtype, the ?₁-subtype has been shown to modulate the release of noradrenaline in rat portal vein.

The present study was undertaken to test for the presence of presynaptic ?-adrenoceptors on sympathetic nerves in human dental pulp and, if present, to investigate the subtype present.

The method was described in detail by Parker et al., (1994a)and involves the removal of dental pulp from teeth (usually third permanent molars) extracted from healthy, young adults of both sexes, aged between 18 and 30 years. Pulp was cut into small segments (approx. 4 mg) and two to four pieces were weighed and equilibrated at 37°C in Krebs solution gassed with 95% O₂ : 5% CO₂ for at least 45 min before incubation in Krebs solution containing tritiated noradrenaline ([³H]-noradrenaline; 0.6 ?mol/l). After incubation for 30 min, pulp was rinsed and placed in a small chamber and superfused at 1 ml/min with warm, gassed Krebs solution. Portions of superfusate were collected at 5- or 10-min intervals and their [³H] contents were assayed by liquid-scintillation spectrometry.

Sympathetic nerves were stimulated through platinum field electrodes by square-wave pulses for 100 sec at 5 Hz (1.0 msec duration, 40 V) at 30 min, 80 min (S₁) and 140 min (S₂) after superfusion commenced. As required, agonists and antagonists of ?-adrenoceptors were added 30 min before S₂; other agents were added to the superfusate 30 min before S₁ and remained throughout the experiment. At the conclusion of an experiment the residual [³H] content in the pulp was extracted in 0.4 mol/l HClO₄ (containing EDTA, 3.0 mmol/l and Na₂SO₃, 10 mmol/l) at 4°C for at least 16 h and assayed. From this data the fractional overflow of [³H] during each collection period was computed and the overflow per min plotted. The few experiments in which the resting overflow declined rapidly throughout or fluctuated were rejected.

Fig. 1.The fractional overflow of [³H] per min in a typical experiment in which the sympathetic nerves in human dental pulp were stimulated for 100 sec (5 Hz, 1.0 msec duration) at the arrows. One pulp (?) was untreated and the other (?) was exposed to isoprenaline (1.0 ?mol/l) for the period represented by the bar. The overflow increased following the onset of stimulation and declined to the resting level within 15 min. Isoprenaline increased the stimulation-induced overflow of [³H] at S₂ relative to that in untreated pulp.

The resting overflow of [³H] (R₁ or R₂) is defined as the fractional overflow in the 10 min preceding a period of stimulation (S₁ or S₂). The effects of agonists/antagonists on this overflow were determined by comparing the R₂/R₁ overflow ratio with that in appropriate control pulp. The stimulation-induced overflows, SIO₁ and SIO₂, were calculated by subtracting the resting overflow from the fractional overflow in the 10 min following the onset of stimulation at S₁ and S₂, respectively. The effects of agonists or antagonists on the release of [³H]-noradrenaline were determined by comparing the SIO₂/SIO₁ ratio with that in control tissue.

Krebs solution was of the following composition (mmol/l): NaCl (120), KCl (4.7), NaHCO₃ (25), KH₂PO₄ (1.0), CaCl₂ (2.5), MgCl₂ (1.0), glucose (5.5), EDTA (0.01).

The incubation medium comprised [³H]- noradrenaline (0.3 ?mol/l) and noradrenaline (0.3 ?mol/l) in Krebs solution containing ascorbic acid (0.3 ?mol/l).

The significance of the effects of agents were assessed by paired or unpaired Student’s t-tests, with significance levels at p<0.05.

(-)-2,5,6-[³H]-noradrenaline, spec. act. 1.5 TBq/mmol (New England Nuclear, Boston, MA, U.S.A.); atenolol, corticosterone, desipramine hydrochloride, (-) isoprenaline hydrochloride, (-) noradrenaline bitartrate, propranolol hydrochloride, salbutamol, ICI-188,551 hydrochloride (Sigma, St Louis, MO, U.S.A.); rauwolscine hydrochloride (Roth, Karlsruhe, Germany); UK 14,304 (Pfizer, CT, U.S.A.); BRL 37344 (a gift from SmithKline Beecham, Surrey, U.K.). Stock solutions of atenolol (10 mmol/l) and corticosterone (10 mmol/l) were dissolved in ethanol before addition to the Krebs perfusion medium. Ethanol, at the concentration used in the experiments, had no effect on the resting or stimulation-induced overflows.

As reported by Parker et al. (1994a), the overflow of [³H] from human dental pulp following incubation in [³H]-noradrenaline was rapid initially, but declined to a near steady-state within 60–70 min. Stimulation of the sympathetic nerves at S₁ caused a rapid rise in the overflow of [³H], which declined to its resting level within 20 min. The second period of stimulation produced a similar effect, although the magnitude of the increase was diminished slightly. The overflow of [³H] from untreated pulp was similar to that reported by Parker et al. (1994a)and resulted in an R₂/R₁ ratio of 0.81±0.01 and a SIO₂/SIO₁ ratio of 0.91±0.01 (n=28).

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Calculus Deposition of Periodontal Disease of Teeth

Dental calculus or tartar used to be regarded as the most common cause of periodontal disease but it is now recognized that it is probably of secondary importance in the development and progression of the disease and that bacterial irritants in dental plaque are more directly implicated. Calculus may be defined as mineralized plaque and whilst there is a positive relation between the amount of calculus and gingivitis it is not as marked as that between plaque and gingivitis. The presence of calculus favours the deposition of dental plaque and periodontal disease in elderly people appears to be more related to the extent of calculus deposition than it is in juveniles. Calculus presents an increased roughened surface area on the tooth crown and root, thus favouring the deposition of plaque. Whilst its relation to periodontal disease may be considered as secondary, clinicians believe it to have an important role in the maintenance and progression of the disease.

Contemporary epidemiological studies reveal a remarkable similarity in the prevalence of periodontal disease across continents and nations, although a relation to systemic disease is believed to exist.

The most commonly used clinical indicator of periodontal disease activity is gingivitis but in those studies there is no clear statement on the relation between gingivitis and periodontitis. A practical method of determining whether or not chronic adult periodontitis is active is to monitor changes in alveolar bone height. In the living person this can be done by using a probe or inspecting radiographs. Advantages in using skeletal material for research into periodontal disease were reviewed by Hildebolt and Molnar (1991); they include visibility, measurement and the ability to study fine detail such as fenestrations and dehiscences. Various indices for recording the distance between the cervical tooth margin and the alveolar crest have been devised; the influence of continuing eruption on these measurements is discussed by Whittaker et al. (1982); Whittaker et al. (1985) and Whittaker et al. (1990).

Some studies on skeletal material have quantified the distribution and extent of calculus; others have noted the presence of calculus but with no detail as to how it was recorded. Trace-element analysis of ancient calculus has been done by Parsche et al. (1991) and glycosaminoglycan content was investigated by Langley et al. (1989). Lussi et al. (1992) suggested that calculus deposition might be related to sugar intake but not to social class, and Littleton, J. and Frohlich, B., 1993. Fish-eaters and farmers: dental pathology in the Arabian Gulf. Littleton and Frohlich (1993)related calculus experience to an existence based on pastoralism or fishing and agriculture rather than on the practice of intensive gardening. Our aims now were to study calculus deposition in two skeletal populations with known differences in attrition rates and caries prevalence suggesting differences in dietary habits. In addition, the relation between calculus deposition and alveolar bone loss was studied.

Skulls were selected from fourth-century Romano-British (Poundbury) specimens and eighteenth-century Londoners (Christ Church, Spitalfields) held in the British Museum of Natural History, Cromwell Road, London. Selection was made on the basis of skulls bearing more than 17 teeth and, in the case of the Spitalfields sample, of known age at death. Forty skulls from Poundbury and 88 from Spitalfields were examined, the number of teeth being 1074 and 2081, respectively. For each specimen the sex, age (estimated or known), number of teeth present, number of teeth lost ante and post mortem were recorded. The Poundbury estimated ages were based on dental-attrition scores.

The number of teeth with any degree of both supra- and subgingival calculus were recorded for each specimen in the following 12 groups: upper or lower anterior, premolars and molars of right- and the left-hand sides of the arch. Supra- and subgingival calculus were distinguished by the darker colour of the subgingival and its position on the tooth. The number of teeth with either sub- or supragingival calculus in each segment of the mouth was recorded. There were therefore 12 scores recorded for each individual specimen. A calculus score, based on the scoring pattern of Ramjford (1967), was recorded as a mean figure for the 12 groups previously indicated.

Fig. 1.Supragingival (S) and subgingival calculus (arrow) are distinguished by position and colour.

For each tooth in each quadrant a score (averaged from the four surfaces of each tooth) was made as follows:

0=No calculus present.

1=Calculus not more than 1 mm wide below the gingival margin.

2=Moderate amount of sub- and supragingival calculus.

3=Abundant amounts of both types of calculus.

An average score for each of the 12 segments in upper and lower arches was computed. Finally, a score of bone loss was recorded as a mean value for each of the 12 groups of teeth. Length of exposed tooth root was measured from the cervical margin to the alveolar crest and 2 mm was subtracted from each measurement to allow for the 2 mm gap between the cervical margin and the bone in the healthy individual. Where the cervical margin of the tooth was obscured by calculus, a small section of calculus was removed by scalpel at the point of measurement.

Measurements were made at the distal, central and mesial aspects of each tooth, both palatally and buccally in upper and lower arches. The six measurements for each tooth were summed for all the teeth in that group (e.g. three upper molars) and the total figure divided by the number of countable surfaces. In this way a mean record of distance from cervical margin to alveolar crest bone was made for each of the 12 groups of teeth. Statistical significance of difference between data-sets was calculated with the student t-test for unpaired data.

The two populations studied were not entirely comparable in respect of their structure and oral condition. There were more teeth present in the Poundbury skulls and this was due to a greater ante- and post-mortem loss in the eighteenth-century material. Nevertheless the post-mortem loss was low, indicating high standards of recovery of the skeletal material by the archaeologists.

The mean number of teeth per skull that had supragingival calculus was greater in the Poundbury population than in Spitalfields and this was the case for both upper and lower arches. In both populations, the lower teeth were more frequently affected than the uppers but the difference was more marked in Poundbury than in Spitalfields. In the upper arch the posterior segments had more teeth involved than had the anterior segments but in the lower arch more anterior teeth were affected than posterior. The teeth closest to the salivary-gland outflows were therefore most likely to have deposits of supragingival calculus.

Tags: periodontal disease

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Total Secretory Response of Isoproterenol

Both motor branches of the nervous autonomic system supply secretory innervation to the salivary glands. Neurons localized in the superior cervical ganglia provide sympathetic fibres innervating the salivary glands, the pineal gland and other extra- and intracranial structures. Noradrenaline (norepinephrine) released by sympathetic nerve terminals or circulating catecholamines activate adrenergic receptors and produce secretory sympathetic responses. Adrenergic receptors have been classified into - and ?-types. The response to adrenergic agonists and antagonists led to the characterization of ?₁- and ?₂-adrenoceptors. Postsynaptic ?₁-adrenoceptors have been identified in rat submandibular glands. In vivo stimulation of these receptors elicits the secretion of protein-rich saliva. A reduction of responsiveness (desensitization) occurs when the ?-adrenergic receptors are exposed to an excess of endogenous or exogenous agonists. In both in vitro or in vivo conditions, this desensitization is expressed as a reduction of stimulated responses.

Binding analysis with labelled adrenergic agonists and antagonists distinguishes between ₁- and ₂-adrenoceptors. The ₂-adrenoceptors show high relative affinity for the agonist clonidine and the antagonist yohimbine. In the peripheral sympathetic nervous system, presynaptic ₂-adrenoceptors inhibit the impulse-dependent release of the neurotransmitter. There is evidence for the presence of submandibular ₂-adrenergic autoreceptors in the rat. The presence of submandibular postsynaptic ₂-adrenoceptors has been also proposed. The physiological role of these receptors is uncertain. In the rat, intravenous injections of clonidine partially inhibit the salivary secretion induced by noradrenaline, ₁- and ?-adrenergic agonists. Clonidine did not produce an inhibitory effect when the secretory saliva was stimulated with the cholinergic agent metacholine.

In man, the rate of resting salivary secretion can be influenced by environmental stimuli related to light–dark cycles, or by noxious stimuli (stressors) of psychological origin. The sympathetic branch of the autonomic nervous system and the adrenal medulla play an important part in homeostatic responses. During the daily light phase, the activity of sympathetic efferents to the pineal and salivary glands decreases and increases in sympathetic nerves projected to the adrenal gland. This rhythm persisted in the pineal glands of blinded rats and was abolished in constant light.

Earlier work in our laboratory has shown that chronic exposure of rats to constant light promotes increased sympathetic secretory responses such as parotid acinar degranulation and submandibular ?-adrenergic desensitization. Now we have analysed the submandibular salivary secretory response mediated by postsynaptic ?- and ₂-adrenergic receptors in rats chronically exposed to constant darkness, constant light or stress.

Groups of 20–30 male Wistar rats (300–350 g), 100–120 days old, were used. They were provided with food and water ad libitum, maintained at room temperature (24±2.0°C) and in a 14 h light–10 h dark cycle. The onset of the daily light phase (0600 hr) and daily dark phase (2000 hr) are defined as zeitgeber time 0 (ZT 0) and 14 (ZT 14), respectively. Half of the rats in each group were maintained under standard laboratory conditions of illumination and the other half (experimental) were continuously exposed to 150 lux for 20 days, or maintained in constant darkness for 20 days. In a fourth group (stress), rats were immobilized (2 hr daily) for 14 successive days and the secretory response was tested 24 h after the last immobilization. These procedures observed the norms of the Animal (Scientific Procedure) Act 1986.

In all groups of rats the ?-adrenergic secretory response was analysed during the light phase (ZT 2–ZT 7) of the light–dark cycle. Rats were anaesthetized with chloralose (100 mg/kg) through a cannula inserted into the jugular vein after induction with ether. A tracheal tube allowed free pulmonary ventilation. Body temperature was measured with a rectal thermometer and maintained at 37.5°C. Secretory ducts from both submandibular glands were exposed and cannulated with fine glass tubes that gave about 45 drops per ml of distilled water. ?-adrenergic secretory responses were obtained by injecting, through a femoral cannula, increasing doses (1.5, 2.0, 3.0, 10.0, 30.0 and 50.0 ?g/kg) of isoproterenol. The saliva was collected on aluminium foils and weighed. The secretory response to low doses (up to 10 ?g/kg) stopped within the 3 min following the injection of each dose (3-min secretory response). The 3-min response was recorded for all doses. For doses greater than 10 ?g/kg, secretion continued after 3 min and collection was prolonged until it had stopped (total secretory response).

Twenty minutes after the isoproterenol secretory response had been determined (first dose–response curve), the ₂-adrenergic agonist clonidine (20 ?g/kg) was injected (i.v.) and, 10 min later, a second secretory response to the same range of doses of isoproterenol was obtained. When all the doses had been injected and the responses recorded, submandibular glands were dissected, weighed, and dried at 110°C for 72 h. The secretory response was expressed as ?g of saliva per mg of dry tissue. In animals exposed to constant dark, ether induction was done under a red lamp and the eyes carefully covered with a black cloth and adhesive tape.

All numerical data are given as means±SEM. To calculate the average value of the secretory response for each dose of isoproterenol, the volume of saliva produced by each submandibular gland was taken separately. Student’s t-test (non-paired) was used to compare the values of the first dose–response curve from each experimental group with the control group. Student’s t-test (paired) was used to compare the values of the secretory response for each dose of isoproterenol obtained after (second dose–response curve) with those obtained before clonidine injection. Statistical significance was set at p<0.05.

Isoproterenol sulphate, clonidine hydrochloride and -chloralose were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). The solution of isoproterenol for injection was prepared immediately before use with 0.150 M NaCl.

Fig. 1 presents the dose–response curves to isoproterenol (3-min response) of submandibular glands in rats exposed to constant light, darkness, and stress compared to their respective controls. For all experimental groups, the secretory response was significantly lower than for controls. The total secretory responses to the larger doses of isoproterenol (10.0, 30.0, and 50.0 ?g/kg) were also significantly lower for all experimental groups.

Fig. 1.Effect of exposure to constant light (A), constant dark (B) and stress (C) on salivary secretion (3 min) by rat submandibular gland elicited by isoproterenol. control; experimental. The values are means±SEM. Statistical significance assessed with student t-test. With the exception of 2.0 and 3.0 ?g/kg in (B) and (C) (p<0.05), all differences were p<0.01.

Fig. 2(A) shows dose–response curves to isoproterenol before and after clonidine in control rats. Paired statistics showed that, for all doses of isoproterenol, secretory responses obtained after clonidine (second curve) were significantly lower than those before (first curve). Percentages of inhibition (3-min response) ranged from 50.32 % for the 3.0 ?g/kg dose to 22.7% for the 50.0 ?g/kg dose. Total secretory responses to the three higher doses of isoproterenol in the second dose–response curve were also significantly lower than in the first curve. Inhibition ranged from 40.99% to 33.39% for 10.0–50.0 ?g/kg isoproterenol. In animals exposed to constant darkness, clonidine also inhibited the secretory response (3-min response) to isoproterenol. With the exception of 2.0 ?g/kg, the secretory responses to all doses of isoproterenol in the second dose–response curve were significantly lower than those in the first curve. Inhibition ranged from 79.82% for the 3.0 ?g/kg dose to 17.29% for the 50.0 ?g/kg. For the total secretory response, inhibition ranged from 61.03% to 26.39% for 10.0–50.0 ?g/kg isoproterenol. Clonidine had no inhibitory effect on the secretory response of the submandibular gland elicited by isoproterenol in groups of rats exposed to constant illumination or stress.

Tags: secretory response

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