Effect of methyl mercaptan on synthesis and degradation of collagen.

Measurements of the conversion of [14C]-proline to [14C]-hydroxyproline were employed to assess the effect of methyl mercaptan on intra- and extracellular metabolism of collagenous proteins in human gingival fibroblast cultures. Following a 30-min pulse, 10 ng of methyl mercaptan per ml of 95% air/5% CO2 head-space suppressed collagen synthesis by 39% and increased the intracellular degradation of newly synthesized collagen from 26% to 42%. Parallel cultures assayed for proline transport demonstrated a 29% inhibition of [14C]-proline uptake. A similar analysis of cultures exposed to methyl mercaptan for 12 h revealed an increase in intracellular degradation (20% control vs. 30% test) and a marked increase in extracellular collagenolysis (4% control vs. 55% test). While pulsing, collagen synthesis was decreased by 39%. Slab gel electrophoresis also demonstrated that treatment with methyl mercaptan caused reductions both in mature alpha 1 and alpha 2 chains of type I collagen and in type III procollagen. Identities of the procollagen species were confirmed by pepsin digestion. Reverse transcribed polymerase chain reaction was utilized to compare expression of alpha 1 chains of type I procollagen with type III procollagen and indicated suppression of mRNA synthesis for type III procollagen in cultures exposed to methyl mercaptan.

Stimulation of enzyme and cytokine production by methyl mercaptan in human gingival fibroblast and monocyte cell cultures.

The volatile sulphur compound methyl mercaptan (CH3SH) is a by-product of protein metabolism and a principal component of oral malodour. This investigation examines the effect of CH3SH on the enzymatic activities of cathepsins B and G and elastase, and on the production by human gingival fibroblasts of two key factors, prostaglandin E (PGE) and cAMP, of the PGE2-cAMP-dependent pathway, which may contribute to the increased production of collagenase and tissue destruction in human periodontal disease. The results demonstrate that CH3SH alone, or in combination with interleukin-1 (IL-1) or lipopolysaccharide, can significantly enhance the secretion of PGE2, cAMP and procollagenase by human gingival fibroblasts. CH3SH also stimulated mononuclear cells to produce IL-1, which can increase cAMP production, and act in synergism with the direct effect of CH3SH on cAMP. CH3SH also significantly enhanced the activity of cathepsin B, moderately suppressed that of cathepsin G, but did not significantly affect elastase. These results provide evidence that CH3SH could be a contributing factor in the enzymatic and immunological cascade of events leading to tissue degradation in periodontal diseases.

Evaluation of a model for post-partum arthritis and the role of oestrogen in prevention of MRL-lpr associated rheumatic conditions.

Sixty-eight percent of female MRL-lpr mice developed a post-partum exacerbation of their mild spontaneous arthritis within 30 days of parturition. The flare became evident between 5 and 15 days after delivery. Histologically it was characterized by a significant increase of subsynovial inflammation and synovial hyperplasia without changes in the level of cartilage and bone erosion. Immunohistologically, marked subsynovial and frequent synovial staining of MHC class II bearing cells was noted, along with the sporadic presence of CD3, CD4, and CD43 receptor-bearing cells in the subsynovium. Injection of physiological levels (0.08 mg/kg) of estradiol on days 2, 3, 9, 15 and 20 post-partum delayed and reduced the flare to 23% of the animals. Administration of pharmacological amounts (0.4 mg/kg per day for 2 weeks following Freund's complete adjuvant injection) prevented adjuvant-enhanced arthritis, reducing the incidence from 67% to the baseline 21% level. Deleterious changes in the underlying systemic lupus erythematosus (SLE), as demonstrated by proteinuria and mortality rate increases, were elicited only by the employed pharmacological amounts of estradiol. These results indicate that the MRL-lpr mice might serve as a model for post-partum flare of arthritis in SLE and rheumatoid arthritis by providing an approach to study the complexity of the effects of pregnancy on autoimmune diseases, and to obtain further evidence for the involvement of oestrogen in arthritis.

Lpr and MRL background gene involvement in the control of adjuvant enhanced arthritis in MRL-lpr mice.

The MRL-lpr mouse strain develops a mild spontaneous arthritis which can be enhanced by the intradermal injection of complete Freund's adjuvant (CFA). In this study we examined the requirement of the lpr gene and background MRL genes on CFA-enhanced murine arthritis. MRL+, MRL-lpr, AKR/J, and B6-lpr mice (experimental) and B6 mice (control) were injected intradermally with CFA containing M. tuberculosis H37 RA. The development of swelling and erythema was monitored for 1 month after the injection, when the histopathology of the joints was investigated. It was found that while 74% of both 7-month-old MRL + and 3-month-old MRL-lpr mice and 11% of AKR/J mice displayed clinically visible arthritis, B6, B6-lpr, and 3-month-old MRL+ did not develop the condition after CFA treatment. In accordance with the clinical observations, the histopathological changes were manifested only in older MRL+, AKR/J and 3-month-old MRL-lpr mice. One month after the CFA injection, milder changes were observed in the MRL+ than in the MRL-lpr mice, with the MRL+ mice developing a disease of similar severity to uninjected MRL-lpr mice. The AKR/J mice demonstrated the least severe histopathological changes. In the long term (150 days) more severe destructive changes could be demonstrated in the cartilage and bone of the MRL+ mice although the average histological scores did not show statistically significant differences from those found in the MRL+ 30 days after injection. The serological evaluation of the adjuvant-injected mice demonstrated significantly enhanced antibody production to type II collagen and M. tuberculosis, but did not correlate with the disease activity. These observations suggest that while the lpr gene causes a more severe early effect, background genes other than the lpr are more involved in the adjuvant-enhanced arthritis-afflicted mice.

Acquisition of resistance to 6-azauridine through DNA amplification in neoplastic but not normal osteoblasts.

In this communication, we have characterized the resistance to AZUrd in tumorigenic mouse C3H-OS osteosarcoma cells and non-tumorigenic MC3T3-E1 osteoblast cells. DNA and RNA blot analysis showed a 30-fold increase in UMP synthase specific DNA and a 10-fold increase in mRNA, respectively, in resistant versus non-resistant C3H-OS cells. No corresponding increases in either UMP synthetase DNA or mRNA were evident in resistant MC3T3-E1 osteoblasts. Karyotype analysis of MC3T3-E1 and C3H-OS cells revealed translocations in the resistant cells. Regardless of drug-sensitive or resistant phenotype, the normal and neoplastic cells exhibited aneuploidy which was significantly more pronounced in the non-resistant tumor cells. Additionally, the number of chromosomes decreased in all resistant cells whether normal or neoplastic. We conclude that genomic instability in neoplastic cells is a prerequisite for the generation of drug resistant variants via the process of gene amplification.

Photodynamic therapy; a comparison with other immunomodulatory treatments of adjuvant-enhanced arthritis in MRL-lpr mice.

Although numerous experimental immunomodulatory regimens have been reported to be effective in the treatment of rheumatoid arthritis, they also produce undesirable side effects. An alternative specific modality of localized treatment is photodynamic therapy (PDT). In this study we treated 13-week-old MRL-lpr mice whose spontaneous arthritis was enhanced by intradermal injection of Freund's complete adjuvant (FCA). One group received transcutaneous photodynamic therapy at days 0, 10, and 20, following the FCA injection. The other groups were injected with 1 mg/kg per day indomethacin, 40 mg/kg per day cyclosporin A (CsA), or treated with 3 Gy sublethal whole body irradiation (WBI). The development of swelling was monitored for 1 month, at which time proteinuria, lymphadenopathy and the histopathology of the joints and kidneys were assessed. The results demonstrated that PDT and the conventional treatments significantly ameliorated swelling of the hindlimbs from 70% in the untreated FCA-injected animals to below the 19% level characteristic of the unmanipulated control. Histological examination showed a reduction in pannus formation, and cartilage and bone destruction, the characteristics of adjuvant-enhanced arthritis. PDT did not affect the survival rate, lymphoproliferation, or proteinuria of the treated animals. However, indomethacin increased proteinuria, and was less effective in preventing cartilage and bone destruction. Furthermore, lower doses of CsA and WBI exacerbated arthritis activity. These results indicate that photodynamic therapy can inhibit the development of adjuvant-enhanced arthritis in MRL-lpr mice with similar effectiveness to the conventional treatments, but without their negative side effects.

Complete Freund's adjuvant induces an earlier and more severe arthritis in MRL-lpr mice.

A study was performed on the effect of CFA on the spontaneous arthritis of MRL-lpr mice. The development of swelling and erythema was monitored for 1 mo after injecting 13- to 14-wk-old mice intradermally with CFA, at which time the histopathology of the joints and serologic responses to extracellular matrix proteins were investigated. In a series of six experiments, 67 to 82% of mice showed early clinical evidence of arthritis in contrast to the low percentage observed in control animals. Similarly, the histopathologic analyses on the CFA-injected mice indicated a significantly higher frequency of advanced histopathologic alterations, characterized by cartilage erosion and pannus formation. The serologic evaluation of the adjuvant-injected mice demonstrated a significant enhanced antibody production to type I and type II collagens, DNA, and the Mycobacterium tuberculosis-positive control. This reproducible adjuvant-enhanced model of murine arthritis will be extremely useful in evaluating experimental therapeutic regimes as the arthritis is initiated earlier and exhibits an enhanced frequency and severity compared with the spontaneous arthritis seen in MRL-lpr mice.

Effect of volatile thiol compounds on protein metabolism by human gingival fibroblasts.

The levels of volatile sulphur compounds (VSC) in periodontal pockets and mouth air have been found to correlate with severity of the disease process. The purpose of this study was to examine the influence of hydrogen sulphide and methyl mercaptan on protein metabolism of human gingival fibroblasts. The incorporation of labelled amino acids into protein was used to evaluate effects on total protein content. Changes in collagenous protein concentration were monitored by release of radioactivity following collagenase digestion as well as direct analysis of hydroxyproline. Both thiols were found to reduce total protein synthesis, with mercaptan exerting a greater adverse effect. In cultures exposed to mercaptan, total protein was reduced by 35%. The changes in total protein were accompanied by a corresponding decrease in collagenase-digestible protein. Hydroxyproline analysis of CH3SH-exposed cultures confirmed the changes associated with collagenous proteins. It indicated that in comparison to the controls the CH3SH-exposed cultures had a 70% reduction in collagen which resulted from a combined effect of suppressed synthesis and increased rate of collagen degradation. The possibility of thiol reaction with collagen was determined using in vitro systems in which type I collagen was reacted with varying concentrations of [35S]-H2S. The carboxymethyl (CM) cellulose assays of resulting reaction mixtures indicate that [35S]-radioactivity was incorporated directly into alpha 1, alpha 2, beta 11, beta 12 peptide chains. Furthermore, upon exposure of collagen to elevated H2S concentrations, the H2S converted some of the acid-soluble collagen to a more soluble product which could be extracted in neutral salt and analyzed by CM-cellulose chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of human gingival fibroblast cell metabolism by methyl mercaptan.

Methyl mercaptan (CH3SH) is a malodorous compound whose levels are elevated in mouth and crevicular air of individuals with active periodontal disease. Since it may play a role in the disease process, its effects were evaluated using human gingival fibroblast cultures and viable porcine unkeratinized oral mucosal tissue sections. Results showed that the protein content of CH3SH-exposed cell cultures pulsed with [14C]-labelled glycine and proline was decreased by approximately 25%. Furthermore, this deleterious effect was irreversible in test cultures subsequently incubated for 24 h in a control 95% air/5% CO2 mercaptan-free environment. The supporting slab-gel electrophoresis profiles yielded evidence that exposure to CH3SH caused an alteration in collagen metabolism and a pooling of Type I procollagens. In addition, DNA synthesis was suppressed in CH3SH-exposed cultures by 44.1% at the 24 to 26 h peak of DNA synthesis. This is a true inhibition and not a shift in peak of maximum DNA synthesis as the shape and location of time-course curves of control and test systems is very similar. Proline transport study using [14C]-proline indicated a reduction in proline transport in the range of 40 to 50% in cultures exposed for 24 to 30 h to CH3SH. Significantly even 15 min exposure to 6.7 ng CH3SH/ml of incubating atmosphere suppressed proline transport by approximately 24%. This indicates that even brief exposure to low concentrations of CH3SH has a significant adverse effect on proline transport. Fluorescent staining of tissue sections exposed to mercaptan indicated that the agent elevated the number of cells stained with vital dye.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of volatile sulphur compounds production at individual gingival crevicular sites in humans.

The present investigation describes a method for collection and analysis of volatile sulphur compounds (VSC) from gingival crevicular sites in humans. Tenax-GC trapping devices were used to adsorb and concentrate VSC from crevicular air at -55 degrees C, which were then thermally desorbed at 120 degrees C. Gas chromatographic (GC) analyses were performed using a Tracor 550 GC equipped with a flame-photometric detector and a Teflon column packed with 5% polyphenyl ether and 0.05% phosphoric acid on 30-40 mesh Teflon. Sulfides identified from crevicular sites include hydrogen sulfide [H2S], methyl mercaptan [CH3SH], dimethyl sulfide [(CH3)2S], and dimethyl disulfide [(CH3S)2]. Of the seventeen patients studied, crevicular sites that were either deep (P.D. > or = 4 mm) or inflamed (BoP = 1) exhibited significantly larger CH3SH to H2S ratios than corresponding crevicular shallow (P.D. < or = 3 mm) sites (p < .10) or noninflamed (BoP = 0) sites (p < .05). Similarly, total sulphur in deep and inflamed sites was significantly higher than in corresponding shallow (p < .01) and noninflamed (p < .05) sites. This is the first known in vivo study to quantitate VSC directly from individual gingival crevices.

Antibodies to extracellular matrix proteins in the sera of MRL-lpr mice.

Autoimmune MRL-lpr mice develop a spontaneous arthritis displaying similar articular and extra-articular features to rheumatoid arthritis in humans. In this study we used an ELISA assay to evaluate the serological responses of MRL-lpr mice to select extracellular matrix proteins associated with the joint. Significant levels of antibodies to collagens types I, II, III, IV, and V were demonstrated starting between 17 and 20 weeks of age. Moreover, the sera contained a strong reactivity to fibronectin. Responses to proteoglycans and laminin were weaker but still detectable. Specificity studies on pooled sera from MRL-lpr mice suggest that the autoantibodies produced are highly cross-reactive. The results indicate that the MRL-lpr mouse strain exhibits similar anti-extracellular matrix antibody profiles to those seen in varying frequencies in the sera and synovial fluid of patients with rheumatoid arthritis.

Gas chromatographic method for trapping and detection of volatile organic compounds from human mouth air.

The present investigation describes a convenient method for collection and analysis of volatile organic compounds from 25 ml mouth air samples. Tenax-GC trapping devices coated with Teflon are used to adsorb and concentrate volatile organic compounds in mouth air at -20 degrees C, which are then thermally desorbed at 140 degrees C. Gas chromatography (GC) analyses are performed using an aluminum column coated with Teflon and packed with 2% poly-MPE on 80/100 mesh Tenax-GC, and employing a flame ionization detector. This procedure allows for amplification of peak heights and detection of compounds that may otherwise escape direct analysis. Of the six prominent peaks detected, identification based on retention times indicates the presence of methanol, acetaldehyde, ethanol and acetone. Volatiles collected using this procedure can be maintained at -20 degrees C for up to 48 hours before analysis. The compact sample tubes allow the system to be easily portable, particularly suitable for sampling breath of persons with localized oral or systemic diseases at locations away from the laboratory. The superiority of this method is that relatively small samples are required for analysis, unlike previously published methods which are based on collection of large volumes of expired air in plastic bags.

Conservatism of sites of tRNA loci among the linkage groups of several Drosophila species.

The sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization. 125I-labeled tRNA probes from Drosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from four Drosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, and Drosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA(2Arg) and tRNA(2Lys) hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution of Drosophila species.

Induced and natural break sites in the chromosomes of Hawaiian Drosophila.

gamma-Irradiation of a laboratory strain of the Hawaiian species Drosophila heteroneura yielded 310 breaks in the five major acrocentric polytene chromosomes. Their map positions conform to the Poisson distribution, unlike most of the 436 natural breaks mapped in 105 closely related species endemic to Hawaii. Genome element E is longer and has more induced breaks than the others. Both in Hawaiian and related species groups, this element shows increased polymorphism and fixation of naturally occurring inversions. The X chromosome (element A) also accumulates many natural breaks; the majority of the resulting aberrations become fixed rather than remain as polymorphisms. Although size may play a small role in initial break distribution, the major effects relative to the establishment of a rearrangement in natural populations are ascribed to the interaction of selection and drift. Nonconformance of the natural breaks to the Poisson distribution appears to be due to the tendency for breaks to accumulate both in the proximal euchromatic portion of each arm and in heterochromatic regions that are not replicated in the polytene chromosomes.

The influence of aging on lysosomal acid DNase of the rat submandibular gland.

Lysosomal and cytoplasmic fractions were prepared from rat submandibular glands for investigation of the release of lysosomal acid DNase in relation to aging. It was found that the acid DNase activity ratio for cytoplasmic/lysosomal fractions in rats aged 27 months was higher than that in three-month-old rats. The release of acid DNase from the lysosomal fraction by shaking was markedly increased in the fraction from the older animals.

Improved high-performance liquid chromatography method for quantitation of proline and hydroxyproline in biological materials.

Numerous high-performance liquid chromatography systems have been described for the determination of hydroxyproline (Hyp) and proline (Pro) levels in biological materials. These methods are generally complicated and have shortcomings in applicability due to poor separation, low sensitivity or derivatization-associated problems. The large number of chemical components present in biological samples further complicates the analysis of Hyp which usually occurs in extremely low concentrations. The present investigation describes the development of a simple highly sensitive derivatization method which results in good separation of peaks and which is capable of quantitating less than 10 pmol of Hyp and Pro in complex test systems. The method is based on removal of o-phthalaldehyde (OPA) derivatives of primary amino acids using reversed-phase chromatography, pre-column derivatization with OPA and phenylisothiocyanate, and detection of derivatized Hyp and Pro using a UV detection system. The procedure yields good peaks and a 93% recovery of Hyp and Pro provided that the analysis is initiated within 5 min of completion of OPA derivatization. While a 93% recovery of Pro was obtained up to 100 min post-derivatization with OPA, the recovery of Hyp is decreased to approximately 80% within the same time interval.

Radiochromatographic analysis of H2S-collagen complexes in rat-tail type I collagen.

Suspensions of 15 mg lyophilized type I, acid-soluble, rat-tail tendon collagen in 0.02 M tris/0.134 M NaCl (pH 7.4) buffer were reacted for up to 4 days at 4 degrees C with air containing [35S]-H2S. Gas-chromatography showed that all the H2S was absorbed: of the 46.8 per cent in the supernatant, 2.7 per cent was associated with neutral salt-soluble protein product and 53.2 per cent was complexed with a more cross-linked product which was soluble in 0.5 M acetic acid; approximately half (26.3 per cent of this product was stable in acid. The remaining 26.9 per cent was dissociated upon solubilization of the product in acetic acid and the cleaved fragment was separated by elution of paper chromatograms with 0.02 M tris/0.134 M NaCl buffer. The H2S-collagen complex was stable in this buffer, moderately stable in acetate/ethanol and highly unstable in an acetone/2-propanol/NH4OH solvent system. Thus there are at least two different binding sites on the type I collagen which react with H2S. One covalent bond, labile in acid, can be cleaved by dialysis of supernatant fraction in acetic acid. The second linkage, stable in acid within the time studied, is cleaved by acetone/2-propanol/NH4OH.

Sulfur uptake by type I collagen from methyl mercaptan/dimethyl disulfide air mixtures.

Type I acid-soluble collagen, suspended in 0.02 M Tris/0.13 M NaCl buffer (pH 7.4), was exposed to air atmosphere admixed with 10.7 X 10(-9) moles of [35S]-labeled dimethyl disulphide/methyl mercaptan mixture in the ratio of 99.82% (CH3S)2/0.18% CH3SH. Gas chromatographic analyses of head-space following one and four days of incubation indicated that all of the CH3SH was absorbed from the head-space by the collagen-containing liquid phase, while the (CH3S)2 concentration of the head-space remained essentially unchanged. Of the total [35S]-activity initially present in the head-space, only 1.0 the total [35S]-activity initially present in the head-space, only 1.0 and 1.86% was absorbed by the collagen-containing liquid phase within one and four days, respectively. Paper chromatographic separation of the reaction mixture showed that only 0.42 and 0.60% of the radioactivity in the head-space was associated with collagen after one and four days of reaction. In comparable [35S]-H2S systems devoid of (CH3S)2, all of the H2S was absorbed within 48 hours of incubation with 18.04% (1 day) and 18.28% (4 days) of the available [35S]-H2S complexed with collagen. The results provide evidence that the reaction of collagen with H2S and CH3SH/(CH3S)2 mixture proceeded via the H2S and CH3SH thiol groups.

Effect of hydrogen sulfide and methyl mercaptan on the permeability of oral mucosa.

Hydrogen sulfide (H2S) and methyl mercaptan (CH3SH) are the volatile sulfur compounds (VSC) that were investigated for a possible role in the etiology of periodontal disease. The results show that the permeability of porcine non-keratinized sublingual mucosa is increased by up to 75% or 103% following exposure to H2S and CH3SH, respectively. The effect may be attributed to VSC reaction with tissue components resulting in alteration in the integrity of the tissue barrier. The increase in permeability of the mucosa to [35S]-Na2SO4 was dependent on both the time of exposure and concentration of VSC in the head-space. The [35S]-H2S was retained by the mucosal tissue and was able to penetrate the intact layers consisting of non-keratinized epithelium, basal membrane, and connective tissue. Treatment of the mucosa with 0.22% ZnCl2, either prior to or after exposure to CH3SH, nullified the effect of CH3SH and restored the permeability to a state similar to that observed in control 95% air/5% CO2 systems.