Dentin resorption and cementum-like tissue formation by bone morphogenetic protein application.

BACKGROUND AND OBJECTIVE: Recent studies have shown that bone morphogenetic protein-2 (BMP-2) stimulates mineralization and osteoclast differentiation. Osteoclastic resorption by BMP-2 application may play an important role in the regulation of new cementum-like tissue formation on the dentin surfaces. Therefore, this study aimed to examine the effect of BMP-2 application on dentin resorption and cementum-like tissue formation at the dentin surfaces. MATERIAL AND METHODS: Seventy-two flat dentin blocks were prepared from rat roots and treated with 24% EDTA. Each block was assigned to group 0, group 100, or group 400, and immersed correspondingly in 0, 100, or 400 microg/ml BMP-2. The dentin blocks were then implanted into palatal connective tissue of rats, and specimens were prepared 2, 4 and 8 wk after surgery for histologic and histomorphometric analyses. RESULTS: BMP-2 caused a dose-dependent increase in dentin resorption by osteoclastic cells. New cementum-like tissue was randomly formed on parts of the nonresorbed and resorbed dentin surfaces in groups 100 and 400. Dentin resorption in groups 100 and 400 was significantly greater than group 0 (p < 0.01). However, at 8 wk, new cementum-like tissue formed in 41.8% of group 100, as compared with 16.2% of group 400 (p < 0.05). CONCLUSION: Dentin resorption was stimulated by a high dose of BMP-2, and cementum-like tissue was induced by a low dose of BMP-2, effectively suggesting that BMP-2 application, at an appropriate dose, to a dentin surface may enhance periodontal regeneration.

Outbreak of Legionnaires' disease on a cruise ship linked to spa-bath filter stones contaminated with Legionella pneumophila serogroup 5.

In January 2003, two cases of Legionnaires' disease associated with a ship's cruise were registered in the database of National Epidemiological Surveillance of Infectious Diseases. A 70-year-old male heavy smoker with mild emphysema contracted the disease during a cruise. Legionella pneumophila serogroup (sg) 5 was isolated from the patient's sputum and the ship's indoor spa. The isolate from the spa matched the patient's isolate by genotyping performed by pulsed-field gel electrophoresis (PFGE). The second case was in a 73-year-old female. During epidemiological investigation, a third case of Legionnaire's disease in a 71-year-old male was subsequently diagnosed among passengers on the same ship on the following cruise. Environmental investigation revealed that porous natural stones (Maifanshi) in the filters of the spas had harboured L. pneumophila, a phenomenon which has not been reported except in Japan. This is the first documented evidence of L. pneumophila sg 5 infection on a ship and of porous stones as a source of Legionella infection.

Radical scavenging glycoprotein inhibiting cyclooxygenase-2 and thromboxane A2 synthase from aloe vera gel.

An active glycoprotein fraction containing 58 % protein was isolated from Aloe vera gel by precipitation with 55 % ammonium sulfate followed by gel permeation using DEAE-Sephacel A-25, Sepharose 6B and Sephadex G-50 columns in a yield of 3 x 10 -3 %. The glycoprotein fraction showed a single band corresponding to a subunit of verectin at the same position when stained with both Coomassie brilliant blue and periodic acid-Schiff reagents on 18 % SDS-PAGE. The molecular weight (14 kDa) was confirmed by Sephadex G-50 column chromatography. The glycoprotein fraction showed a radical scavenging activity against superoxide anion generated by the xanthine-xanthine oxidase system as well as inhibition of cyclooxygenase-2 and reduction of thromboxane A 2 synthase level in vitro.

Preliminary examinations for applying a carbazole-degrader, Pseudomonas sp. strain CA10, to dioxin-contaminated soil remediation.

A method for bioremediation of chlorinated dibenzo-p-dioxins (CDDs) and dibenzofurans (CDFs) by a carbazole-utilizing bacterium, Pseudomonas sp. strain CA10, was developed. CA10 cells transferred to carbon- and nitrogen-free mineral medium supplemented with 1 mg carbazole (CAR)/ml grew rapidly during the first 2 days; and the cells at the end of this rapid growth period showed the highest 2,3-dichlorodibenzo-p-dioxin (2,3-Cl2DD)-degrading activity. The CA10 cells pregrown for 2 days efficiently degraded 2,3-Cl2DD in aqueous solution at either 1 ppm or 10 ppm. The effect of inoculum density on the efficiency of 2,3-Cl2DD degradation was investigated in a soil slurry microcosm [ratio of soil:water = 1:5 (w/v)]. The results showed that a single inoculation with CA10 cells at densities of 10(7) CFU/g soil and 10(9) CFU/g soil degraded 46% and 80% of 2,3-Cl2DD, respectively, during the 7-day incubation. The rate of degradation of each CDD congener, 2-ClDD, 2,3-Cl2DD, and 1,2,3-Cl3DD (1 ppm each) by strain CA10 in the soil slurry system was not significantly influenced by the coexistence of the other congeners. Using this soil slurry system, we tried an experimental bioremediation of the actual dioxin-contaminated soil, which contained mainly tetra- to octochlorinated dioxins. Although the degradation rate of total CDD and CDF congeners by a single inoculation with CA10 cells was 8.3% after a 7-day incubation, it was shown that strain CA10 had a potential to degrade tetra- to hepta-chlorinated congeners including the most toxic compound, 2,3,7,8-tetrachlorinated dibenzo-p-dioxin.

[Mitogen-induced lymphocyte transformation in patients with Yusho].

To evaluate chronic immune effects of polychlorinated biphenyl (PCB), in vitro lymphocyte transformation in response to nonspecific mitogens were studied in 74 patients with Yusho in 2000. Elevated response in phytohemagglutinin (PHA)-induced lymphocyte transformation was detected in fifteen (20.3%) patients, while decreased PHA response was found in six (8.1%) patients. Concanavalin A (Con A)-induced lymphocyte transformation was increased in six (8.1%) patients, and decreased in eleven (14.9%) patients. There were no significant correlations between blood PCB concentrations and lymphocyte transformation in response to PHA, Con A, or pokeweed mitogen (PWM). We conclude that abnormality of lymphocyte transformation in patients with Yusho is frequent, but it may not be associated with blood PCB concentration.

Distribution of verectin in Aloe vera leaves and verectin contents in clonally regenerated plants and the commercial gel powders by immunochemical screening.

Verectin antiserum raised in white rabbits was immunochemically applied to examine verectin distribution in Aloe vera leaves during growth and flowering seasons, and to quantify verectin in clonally regenerated plants and commercial A. vera gel products.

[Immune effects of endocrine disruptors].

To evaluate chronic immune effects of endocrine disruptors, serum autoantibodies and immunoglobulin concentrations were studied in patients with Yusho. Autoantibodies were present in some patients of Yusho; 45.6% for antinuclear antibody, 12.7% for rheumatoid factor and 11.1% for thyroglobulin antibody. LE factor was not detected. Serum levels of immunoglobulin A(IgA), immunoglobulin G(IgG) and immunoglobulin M(IgM) were elevated in 12.7%, 24.1% and 8.9%, respectively. Thyroglobulin antibody was detected in 19.5% of Yusho patients with high PCB concentration(higher than 3.0 ppb), and in only 2.5% of patients with low PCB concentration (lower than 2.9 ppb). Furthermore, the mean absolute density of CD4 positive lymphocytes was higher in patients with high PCB concentration than in patients with low PCB concentration. We conclude that autoantibodies in patients with Yusho are frequent, and it may be associated with the increase of helper/inducer T cells. Effects of lactational exposure to chlorinated dioxins and related chemicals on lymphocyte subpopulations in Japanese babies were also studied. Estimated total TEQ intakes of chlorinated dioxins and related chemicals from the breast milk correlated negatively with the percentages of CD8 positive lymphocytes. It is showed that exposure to background levels of chlorinated dioxins and related chemicals through breast milk may cause some immunologic disorder.

[Serum immunoglobulin concentrations and autoantibodies in patients with Yusho].

To evaluate chronic immune effects of polychlorinated biphenyl (PCB), serum immunoglobulin concentrations and autoantibodies were studied in 79 patients with Yusho in 1997. Serum levels of immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) were elevated in 10 cases (12.7%), 19 cases (24.1%) and 7 cases (8.9%), respectively. Autoatibodies were present in some patients of Yusho; 10 cases (12.7%) for rheumatoid factor and 36 cases (45.6%) for antinuclear antibody. LE factor was not detected. There were no significant correlations between blood PCB concentrations and serum immunoglobulin concentrations, or presence of autoantibodies. We conclude that antinuclear antibody in patients with Yusho is frequent, although it may not be associated with blood PCB concentration.

Pharmacological effects of SA96 (bucillamine) and its metabolites as immunomodulating drugs--the disulfide structure of SA-96 metabolites plays a critical role in the pharmacological action of the drug.

SA96 (generic name, bucillamine) is a disease-modifying anti-rheumatoid arthritis (RA) drug with immunological effects. This compounds has two sulfhydryl groups in its molecule, and the differences and similarities between this drug and D-penicillamine, which is also a sulfhydryl group-containing anti-rheumatic drug, have frequently been discussed. To clarify the pharmacological differences between these two drugs, we examined the concentrations of the compounds and its metabolites in serum and synovial fluid, paying special attention to the metabolites of SA96 produced in vivo. SA96 was metabolized in a very short time to SA981 which is a disulfide compound formed by intramolecular binding of two sulfhydryl groups, and transferred to synovial fluid. In addition SA981 had significant suppressive effects on IL-6 and IL-8 production by synovial cells in vitro. These results demonstrate that SA96, which has two sulfhydryl groups, exhibits anti-rheumatic effects via a pharmacological action clearly different from that of D-penicillamine.

Time course of apoptosis in collagen-induced arthritis.

Recently, apoptotic cells were discovered in the synovial cells of rheumatoid arthritis patients. Their role, however, is unknown. We thus examined the time course of apoptosis in the synovium using an animal model of rheumatoid arthritis, in which arthritis was induced by intracutaneous injection of collagen. No apoptotic cells were detected before the onset of synovitis, but they increased in parallel with the progress of arthritis in its initial period, and decreased in the later chronic stages. Apoptotic cells appear to maintain the homeostasis of a joint when synovial proliferation occurs.

Immunochemical distinction of Aloe vera, A. arborescens, and A. chinensis gels.

Verectin antiserum raised in white rabbits was immunoprecipitated with the Aloe vera nondialysable fraction. Analysis of the immunoprecipitation revealed that verectin accounted for about 1.25% of the total proteins in the nondialysable fraction of Aloe vera gel. The verectin antibody showed differential immunoreactivities against nondialysable fractions of A. arborescens, A. chinensis, and A. vera: 1) an immunopreciptin line was formed against the fraction of A. vera, but not against those of A. arborescens and A. chinensis gel in an Ouchterlony double immunodiffusion test and 2) an immunopositive band was detected in the A. vera and A. chinensis nondialysable fractions but not in that of A. arborescens in immunoblotting. These findings indicate that the verectin antibody can be used to distinguish Aloe materials.

Prediction of the surgical outcome for the treatment of cervical myelopathy by using hyperbaric oxygen therapy.

The effectiveness of hyperbaric oxygen therapy (HBO) in predicting the recovery after surgery in patients with cervical compression myelopathy was evaluated. HBO has been used to treat brain and spinal cord diseases, but the effect is generally temporary. This is the first paper to utilize HBO as a diagnostic tool to evaluate the functional integrity of the spinal cord. The study group consisted of 41 cervical myelopathy patients aged 32-78 years. Before surgery, the effect of HBO was evaluated and was categorized as four grades. The severity of the myelopathy and the recovery after surgery were evaluated by the score proposed by the Japanese Orthopaedic Association (JOA score). The correlation between many clinical parameters including the HBO effect and the recovery rate of JOA score was evaluated. The recovery rate of JOA score was found to be 75.2 +/- 20.8% in the excellent group, 78.1 +/- 17.0% in the good group, 66.7 +/- 21.9% in the fair group and 31.7 +/- 16.4% in the poor group. There was a statistically significant correlation between the HBO effect and the recovery rate of the JOA score after surgery (r = 0.641, P < 0.0001). The effect of HBO showed a high correlation with the recovery rate after surgery as compared to the other investigated parameters. HBO can be employed to assess the chance of recovery of spinal cord function after surgical decompression.

Laboratory-scale bioremediation of oil-contaminated soil of Kuwait with soil amendment materials.

A huge amount of oil-contaminated soil remains unremediated in the Kuwait desert. The contaminated oil has the potentiality to cause pollution of underground water and to effect the health of people in the neighborhood. In this study, laboratory scale bioremediation experiments were carried out. Hyponex (Hyponex, Inc.) and bark manure were added as basic nutrients for microorganisms, and twelve kinds of materials (baked diatomite, microporous glass, coconut charcoal, an oil-decomposing bacterial mixture (Formula X from Oppenheimer, Inc.), and eight kinds of surfactants) were applied to accelerate the biodegradation of oil hydrocarbons. 15% to 33% of the contaminated oil was decomposed during 43 weeks' incubation. Among the materials tested, coconut charcoal enhanced the biodegradation. On the contrary, the addition of an oil-decomposing bacterial mixture impeded the biodegradation. The effects of the other materials were very slight. The toxicity of the biodegraded compounds was estimated by the Ames test and the tea pollen tube growth test. Both of the hydrophobic (dichloromethane extracts) and hydrophilic (methanol extracts) fractions showed a very slight toxicity in the Ames test. In the tea pollen tube growth test, the hydrophobic fraction was not toxic and enhanced the growth of pollen tubes.

Analysis of oil components and hydrocarbon-utilizing microorganisms during laboratory-scale bioremediation of oil-contaminated soil of Kuwait.

A huge amount of oil-contaminated soil remains unremediated in the Kuwait desert. The contaminated oil has the potentiality to cause pollution of underground water and to effect the health of people in the neighborhood. We have been studying bioremediation of Kuwait oil-contaminated soil. Chemical analyses of biodegraded compounds and isolation of petroleum hydrocarbon-decomposing microorganisms were carried out. From the chemical analyses, it was revealed that the decomposed compounds were mainly saturated fractions from alumina column chromatography and that the aromatic fractions were not decomposed well. Isolation of bacteria was carried out for eight kinds of hydrocarbons which are components of crude petroleum (n-hexadecane, 2,6,10,14-tetramethylpentadecane, 1,4-diisopropylbenzene, naphthalene, 1-methylnaphthalene, phenanthrene, anthracene, and perylene). Many of the n-hexadecane- and 2,6,10,14-tetramethylpentadecane-decomposing bacteria were isolated, but aromatic compound-decomposing bacteria were not enriched. It was concluded that the slow decomposition of aromatic compounds was due to the low population of aromatic compound-decomposing bacteria in the Kuwait desert soil.

[Thyroid function in patients with Yusho: 28 year follow-up study].

To evaluate chronic effect of polychlorinated biphenyl (PCB) on thyroid functions, thyroid hormone levels and thyroidal autoantibodies were studied in 81 patients with Yusho in 1996. Serum level of thyroid stimulating hormone (TSH) was elevated in 7 cases (8.6%). All of them showed normal triiodothyronine (T3), thyroxine (T4) and free T4 levels, and regarded as latent hypothyroidism. There were no significant correlations between blood PCB concentrations and TSH levels, T2 levels, T4 levels or free T4 levels. Thyroglobulin antibodies were detected in 8 cases (19.5%) of 41 Yusho patients with high PBC concentration (higher than 3.0 ppb), and in only one case (2.5%) of 40 patients with low PBC concentration (lower than 2.9 ppb). We conclude that thyroglobulin antibody in patients with Yusho is not frequent and it may be associated with blood PCB concentration.

Isolation and characterization of the glycoprotein fraction with a proliferation-promoting activity on human and hamster cells in vitro from Aloe vera gel.

Fractions of leaf gel from Aloe barbadensis Mill. were prepared by gel permeation using DEAE Sephadex A-25, Sepharose 6B, and Sephadex G-50 columns. These were then tested by in vitro assays for proliferation of human normal dermal and baby hamster kidney cells. The glycoprotein fraction promoted cell growth, while the neutral polysaccharide fraction did not show any growth stimulation. Moreover, the polar-colored glycoprotein fraction strongly inhibited the in vitro assays. An active glycoprotein fraction (protein 82%, carbohydrate 11%) examined on polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE showed a single band. Its molecular weight was 29 kD on a Sephadex G-50 column and its isoelectric point was pH 6.8. Immunoblotting after SDS-PAGE showed that the glycoprotein was composed of two subunits (14 kD). Deglycosylation of glycoprotein (Pg21-2b fraction) by trifluoromethanesulphonic acid provided a protein band with a molecular weight of 13 kD on SDS-PAGE. The colored glycoprotein fraction was shown on SDS-PAGE to be a mixture with a molecular weight of 18 kD-15 kD. It was later hydrolyzed with 10% H2SO4 to produce phenolic substances.

Reduction of the allergenicity of soybean by treatment with proteases.

Soybeans were treated with proteases to reduce allergenicity. By immunoblotting with a monoclonal antibody against a major soybean allergen (Gly m Bd 30K), two of eight proteases so far tested were selected to achieve a reduction in allergenicity. Both antigenicity to the monoclonal antibody and allergenicity to the sera from soybean-sensitive patients proved to be markedly reduced by processing with either protease. Thus, soybeans treated with an appropriate protease may possibly be supplied as a hypoallergenic foodstuff for patients.

Cloning and sequencing of cDNA encoding 4-aminobenzoate hydroxylase from Agaricus bisporus.

A cDNA clone encoding 4-aminobenzoate hydroxylase (EC 1.14.13.27) has been isolated using a probe prepared by PCR on the basis of partially determined amino acid sequences of the enzyme. The cDNA contained 1380-base pair open reading frame encoding 460 amino acid residues (M(r) 50974), 14-base pair 5'-untranslated region and 123-base pair 3'-untranslated region including a poly(A) tail of 20 nucleotides. All of the partially determined amino acid sequences were shown to be included in the deduced amino acid sequence. Homology analyses showed that the two regions on the enzyme share other flavoproteins such as salicylate hydroxylase and p-hydroxybenzoate hydroxylase.

Abnormal modulation of serum osteocalcin by dietary phosphate and 1,25-dihydroxyvitamin D3 in the hypophosphatemic mouse.

We evaluated in normal and hypophosphatemic (Hyp) mice whether changes in serum levels of osteocalcin in response to dietary phosphate supplementation, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) administration were related to perturbations in calcium phosphate homeostasis. In normal mice, serum osteocalcin levels were not altered by phosphate supplementation. In contrast, phosphate supplementation in Hyp mice led to a 2-fold decrease in serum osteocalcin to normal levels after 3 days and to an increase in osteocalcin levels after 14 days. The decrease in osteocalcin was associated with normophosphatemia, severe hypocalcemia, and marked increases in circulating 1,25(OH)2D3 levels, whereas the increase in osteocalcin levels was associated with normophosphatemia and no change in serum calcium and 1,25(OH)2D3. Administration of PTH decreased serum osteocalcin in both genotypes. Infusion of 1,25(OH)2D3 for 3 days elicited increases in serum osteocalcin and calcium levels in normal mice, whereas in Hyp mice it produced significant decreases in osteocalcin levels and no change in serum calcium. However, with a more prolonged infusion of 1,25(OH)2D3, hypercalcemia and increases in serum osteocalcin were induced in mutant mice. Our results suggest that the abnormal osteocalcin response of Hyp mice is not directly attributable to an osteoblast dysfunction but is secondary, at least in part, to perturbations in factors that modulate the osteoblast activity, especially serum calcium and/or PTH.

Cytokinin induces a rapid decrease in the levels of mRNAs for catalase, 3-hydroxy-3-methylglutaryl CoA reductase, lectin and other unidentified proteins in etiolated cotyledons of cucumber.

Large-scale differential hybridization was performed to examine rapid changes in gene expression caused by a phytohormone, cytokinin, in etiolated cotyledons of cucumber (Cucumis sativus L.). We isolated 86 cDNA clones for mRNAs whose levels decreased within 2 h of the start of treatment with N6-benzyladenine (BA). Partial nucleotide sequences showed that some of the cDNAs were homologous to those for catalase, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and a lectin. This is the first report that the levels of the mRNAs for those proteins are regulated by a cytokinin in darkness. Together with previous results [Teramoto et al. (1993) Physiol. Plant. 87: 584, (1994) Planta 193: 573, (1995) Planta 196: 387], the present study suggests that the cytokinin act to lower the levels of mRNAs transcribed from various genes in etiolated cotyledons.