Transcriptomic analysis of immunoglobulin novel antigen receptor (IgNAR) heavy chain constant domains of brownbanded bamboo shark (Chiloscyllium punctatum).

Cartilaginous fish are the evolutionarily oldest group of animals which possess antibodies, T cell receptors and major histocompatibility complex (MHC). The immunoglobulin novel antigen receptor (IgNAR) found in cartilaginous fish is a heavy chain homodimer which lacks light chain. The presence of non-canonical cysteine molecules and lack of CDR2 region make it more significant. To synthesize active binding domains based on variable region of IgNAR (VNAR), knowledge on the constant region dynamics play a significant role. The IgNAR exhibit species variations in its primary sequence features; hence, this study was conducted to determine the IgNAR heavy chain constant domain of the brownbanded bamboo shark (Chiloscyllium punctatum). Peripheral blood leukocytes (PBL) isolated from adult bamboo sharks were used to synthesize a cDNA library. A total of four billion residues of two million sequences (average length 218.41 bp) were obtained. Assembled sequences were aligned with published cartilaginous fish IgNAR constant region sequences. Transcriptome analysis revealed two distinct types of IgNAR in the brownbanded bamboo shark. Also, constant-1 domain sequences displayed 13 unique sequences which may reflect the least number of IgNAR gene clusters. The phylogenetic analysis revealed the closest relationship with the nurse shark (Ginglymostoma cirratum) followed by the wobbegong shark (Orectolobus maculatus) which belong to the same order Orectolobiformes. Analysis of the constant domains of the brownbanded bamboo shark IgNAR revealed an evolutionarily conserved nature and this knowledge can be used to design primers for VNAR cloning. Furthermore, knowledge on the structural features in IgNAR constant domains that increase the stability could be useful in the process of stabilizing human immunoglobulins.

Changes in free and total plasma cortisol levels in juvenile haddock (Melanogrammus aeglefinus) exposed to long-term handling stress.

We measured changes in free and total plasma cortisol levels, plasma glucose, gill hsp70 levels, and growth in haddock (Melanogrammus aeglefinus) subjected to a long-term handling stress (15 s out of water, each day, for 4 weeks), and the effect of this long-term stress on the ability of haddock to respond to an acute stressor. The acute stressor was a single handling stress, and fish were sampled at 1, 6, and 12 h post-stress. During the long-term stress study, free and total plasma cortisol levels increased significantly (10-fold) in the stressed group after the second week. However, the percentage of free cortisol was already significantly elevated by the first week (control 17%, stressed 55%), and remained high during the second week (control 35% and stressed 65%). After 3 and 4 weeks of handling, both free and total cortisol declined in stressed fish to levels that were not significantly different from pre-stress values. Control fish grew significantly more than stressed fish (by 32% and 18%, respectively) over the 4 week study, and condition factor only increased in control fish. Although fish from the control group showed elevated total plasma cortisol levels (to 47 ng mL(-1)) 1 h after the acute stress, and the levels in stressed fish were comparable to those for the control fish, no significant increase in plasma cortisol was measured in the group subjected to the long-term stress. Free plasma cortisol levels did not increase significantly in either group following the acute stress. However, free plasma cortisol levels were significantly higher in long-term stress group, as compared with the control group, at 6 h post-stress. Plasma glucose and gill hsp70 levels were not altered by either the long-term stress or acute stressor. Our data indicate that cortisol (free and total), but not glucose or hsp70, appears to be adequate to assess short- and long-term stress in haddock.

A putative ATP-binding cassette transporter YbdA involved in sporulation of Bacillus subtilis.

Insertional mutagenesis with mini-Tn10 was performed to identify new genes involved in sporulation of Bacillus subtilis. Here, we report on the characterization of the ybdA locus, which encodes a putative ATP-binding cassette transporter. The ybdA gene is the 6th cistron of the putative ybcOPQST-ybdABDE operon. A deletion mutation in ybdA and an insertional mutation in ybdB exhibited highly oligosporogenous phenotypes and led to a decrease in the transcription controlled by Spo0A, which is a key response regulator required for the initiation of sporulation. We further observed that the transcription of this operon was strongly induced after the end of the exponential growth phase in the wild-type strain, but not in a spo0A null mutant. Our data suggest that the YbdA and YbdB proteins are able to affect incorporation of nutrient signals during initiation of sporulation and may act as components of positive feedback systems of Spo0A activation.

Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS).

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

GFAT as a target molecule of methylmercury toxicity in Saccharomyces cerevisiae.

Using a genomic library constructed from Saccharomyces cerevisiae, we have identified a gene GFA1 that confers resistance to methylmercury toxicity. GFA1 encodes L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) and catalyzes synthesis of glucosamine-6-phosphate. Transformed yeast cells expressing GFA1 demonstrated resistance to methylmercury but no resistance to p-chloromercuribenzoate, a GFAT inhibitor. The cytotoxicity of methylmercury was inhibited by loading excess glucosamine 6-phosphate into yeast. Considering that GFAT is an essential cellular enzyme, our findings suggest that GFAT is the major target molecule of methylmercury in yeasts. This report is the first to identify the target molecule of methylmercury toxicity in eukaryotic cells.

Overexpression of L-glutamine:D-fructose-6-phosphate amidotransferase provides resistance to methylmercury in Saccharomyces cerevisiae.

To identify novel genes that confer resistance to methylmercury (MeHg), a yeast genomic DNA library was transfected into Saccharomyces cerevisiae. Two functional plasmids were isolated from transfected yeast clones D1 and H5 that exhibited resistance to MeHg. The yeast transfected with plasmid isolated from clone H5 was several-fold more resistant than yeast transfected with plasmid from clone D1. Functional characterization of the genomic DNA fragment obtained from clone H5 determined that the GFA1 gene conferred resistance to MeHg. GFA1 was reported to encode L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) which catalyzes the synthesis of glucosamine-6-phosphate from glutamine and fructose-6-phosphate. Accumulation of mercury in yeast clone W303B/pGFA1, which contains the transfected GFA1 gene, did not differ from that in control yeast clone W303B/pYES2. The W303B/pGFA1 strain did not show resistance to mercuric chloride, zinc chloride, cadmium chloride or copper chloride, suggesting that the resistance acquired by GFA1 gene transfection might be specific to MeHg. This is the first report of a gene involved in MeHg resistance in eukaryotic cells identified by screening a DNA library.

Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts.

The purpose of the present study was to investigate the effect of laser irradiation on calcified nodule formation in human dental pulp (HDP) cells. HDP cells were irradiated once with a Ga-Al-As laser for 5 and 10 min, and calcified nodule formation was determined by von Kossa staining. The laser irradiation increased the number of calcified nodules in a time-dependent manner. The activity of alkaline phosphatase and production of collagen and osteocalcin in conditioned medium were measured. Both were higher in the irradiated group than in the nonirradiated group. These results suggested that formation of calcified nodules in HDP cells, as well as in alkaline phosphatase activity, the production of collagen and osteocalcin were enhanced by laser irradiation.

Alkaline phosphatase and type I collagen gene expressions were reduced by hydroxyl radical-treated fibronectin substratum.

The influence of fibronectin (FN) substratum treated with hydroxyl radicals (.OH) generated by the H2O2-Cu2+ systems on osteoblast cells was studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that FN was degradated and/or modified by .OH treatment. The calcified bone nodule formation of osteoblast cells cultured on .OH treated FN-coated wells was significantly lower compared to those on intact FN-coated wells. Alkaline phosphatase (ALP) activity and the secretion of type I collagen were reduced by .OH-treated FN. By RT-PCR mRNA levels of ALP and type I collagen genes were also diminished. These findings suggested that the .OH damaged FN molecules and reduced the bone formation of osteoblast cells via inhibition of proliferation and/or differentiation of osteoblast progenitors and the calcification process.

Stimulatory effect of interleukin-6 on plasminogen activator activity from human dental pulp cells.

The plasminogen activator (PA)-plasmin proteolytic system has recently received considerable attention because of its participation in a wide variety of biological activities and in pathological conditions involving tissue destruction. We examined the effects of interleukin-6 (IL-6) on PA activity and the gene expressions of tissue type (t) PA and PA inhibitor-1 (PAI-1) in human dental pulp (HDP) cells. IL-6 treatment induced significantly high PA activity in the HDP cells in a time- and dose-dependent manner, compared with nontreated controls. Western-blot analysis showed that tPA protein in the conditioned medium was stimulated by IL-6, compared with the control. The tPA and PAI-1 mRNA levels were increased in HDP cells treated with IL-6, as shown by reverse transcriptase-polymerase chain reaction. These results suggest that IL-6 stimulated PA activity through an enhancement of tPA gene expression and may be involved in extracellular matrix degradation through the stimulation of the PA-plasmin system of HDP cells.

Stimulation of interleukin-6 production in human dental pulp cells by peptidoglycans from Lactobacillus casei.

Interleukin-6 (IL-6), which is a multifunctional cytokine, has an important role in acute and chronic inflammation. The peptidoglycan (PG) was purified from Lactobacillus casei, which was a Gram-positive bacteria frequently isolated from deep carious lesions and suspected to be a pathogen of pulpitis. In this study, the effects of PG on the production of IL-6 in human dental pulp cells were examined. PG stimulated IL-6 production in a time- and dose-dependent manner. Reverse transcriptase-polymerase chain reaction experiments showed that the increase was dependent on the enhancement of IL-6 mRNA levels. These findings suggest that Gram-positive bacteria, such as L. casei, from carious lesions, might be involved in developing pulpitis through the stimulation of IL-6 production.

HsMCM6: a new member of the human MCM/P1 family encodes a protein homologous to fission yeast Mis5.

BACKGROUND: The tight regulatory mechanism that prevents more than one round of chromosomal DNA replication per cell cycle is thought to require the function of Mcm/P1 proteins. We report here the structural and functional analyses of HsMcm6, a human homologue of the Mis5 of Schizosaccharomyces pombe. RESULTS: We demonstrate here that the transcription of the HsMCM6 gene was repressed in quiescent cells but was rapidly induced at the G1/S phase by growth factor stimulation. The 5' regulatory region of the HsMCM6 gene was found to harbour four putative E2F binding motifs, and these were responsible for the promoter activity. The HsMcm6 protein level oscillated during the cell cycle, with a peak at the G1/S phase. We also showed that the cell-cycle dependent change of subcellular localization of HsMcm6 resembles those of other Mcm/P1 proteins. HsMcm6 consists of two forms, a form extractable by Nonidet P-40 and the nucleus-bound form. A demonstration of the association of HsMcm6 with HsMcm2 and HsMcm7 in vivo supports the idea that they behave as a heteromeric complex. We mapped the HsMCM6 gene at 2q12-14. CONCLUSION: The results indicate that the behaviour of HsMcm6 is reminiscent of replication licensing factor like other Mcm/P1 family members.

Stimulation of interleukin-1 beta production of human dental pulp cells by Porphyromonas endodontalis lipopolysaccharide.

IL-1 beta is synthesized as an inactive precursor, which is subsequently processed by IL-1 beta converting enzyme (ICE) and found extracellularly as a mature biologically active polypeptide. Also, IL-1 beta has been detected in necrotic and inflamed dental pulp. We examined the IL-1 beta production in human dental pulp (HDP) cells treated with lipopolysaccharide (LPS) from Porphyromonas endodontalis (P. e.) isolated from root canals and radicular cyst fluids. We demonstrated that P. e. LPS stimulated IL-1 beta release from HDP cells in a time- and dose-dependent manner. However, ICE activity was not increased by P. e. LPS. Northern blot hybridization analysis revealed that the IL-1 beta mRNA level in HDP cells was increased by P. e. LPS. These results suggest that stimulation of IL-1 beta release from HDP cells by P. e. LPS may have an important role in the progression of inflammation in pulpal and periapical disease.

Stimulation of interleukin-1beta-independent interleukin-6 production in human dental pulp cells by lipopolysaccharide.

Dental pulpal infection is most commonly caused by extensive dental caries. A principal driving force behind pulpal disease response appears to lie in the immune system's response to bacteria. However, the production of interleukin (IL)-1beta and IL-6 in human dental pulp (HDP) cells in response to lipopolysaccharide (LPS) has not been well characterized. We examined IL-1beta and IL-6 production in HDP cells by challenging with LPS from Porphyromonas endodontalis, which is a Gram-negative bacteria found in root canals. Our results presented here showed that when HDP cells were stimulated by LPS, the production of IL-6 always preceded that of IL-1beta. Since the IL-6 production was observed even in the presence of the IL-1beta receptor antagonist, we concluded IL-6 production was independent of the IL-1beta molecule in LPS-stimulated HDP cells. This idea was further supported by the results obtained from RT-PCR experiments, in which IL-6 mRNA, but not IL-1beta mRNA, was present in the RNA preparation isolated from the early stage of cells.

[Malignancies in patients with sarcoidosis].

We evaluated the occurrence and type of malignant tumors in 148 patients with sarcoidosis followed at the Okayama University Hospital. Nine patients had malignancies; in 2 of 9 patients the development of malignancy preceded that of sarcoidosis, and one patient presented with sarcoidosis and malignancy at the same time. Six patients developed six types of malignancy following the development sarcoidosis; one case each of stomach cancer, lung cancer, breast cancer, thyroid cancer, testicular tumor, laryngeal cancer, and chronic lymphocytic leukemia. There was no significant difference between sexes (3 males and 3 females). The mean age of the cancer group at the onset of sarcoidosis was 56 years, which was significantly higher (p less than 0.05) than that of the control group. In these 6 patients, the mean interval from onset of sarcoidosis to detection of cancer was 11.7 years (range 1.5 to 30.2 years). The relative risk of malignancy was calculated based on the data for 148 patients with sarcoidosis with a total of 1371 person-years. The expected incidences of cancer for all sites and specific sites were estimated by applying age- and sex-adjusted person-years. The observed incidence of cancer was significantly (p less than 0.05) greater than the expected incidence for thyroid cancer, laryngeal cancer, and leukemia. No significant difference in incidence was found for all sites or for the other sites of cancer. The increased cancer incidence in sarcoidosis may be secondary to immunological abnormalities associated with this disease.

[Release of interleukin-6 by alveolar macrophages in patients with sarcoidosis].

The supernatants from cultures of alveolar macrophages from 12 patients with sarcoidosis and 7 control subjects were assayed for interleukin-6 (IL-6) using an ELISA system. IL-6 was detectable without a stimulant in supernatants from all subjects with sarcoidosis and controls. However, the supernatants from 4 of 12 untreated patients with sarcoidosis contained significantly greater amounts of IL-6. When macrophages were stimulated by Propionibacterium acnes (P. acnes), the mean level of IL-6 in the supernatant of patients with sarcoidosis was 5.18 +/- 1.46 ng/ml, which was significantly higher than in controls (3.34 +/- 0.39) (p less than 0.05). Furthermore, in patients with sarcoidosis, the mean level of IL-6 in the supernatant was significantly correlated with the percentage of lymphocytes in bronchoalveolar lavage fluid (p less than 0.05), the level of interleukin-1 released by alveolar macrophages stimulated by P. acnes (p less than 0.05), and the phagocytic index of alveolar macrophages (p less than 0.05). The large amount of IL-6 in the supernatant after stimulation by LPS was measured in patients with sarcoidosis (24.49 +/- 13.36) and in controls (12.4 +/- 8.53), and there was no significant difference between patients with sarcoidosis and controls. Small amounts of IL-6 were detectable in bronchoalveolar fluid from only 2 of 26 patients with sarcoidosis; however, it was detected in none of 15 controls. It is suggested that the enhancement of IL-6 release by alveolar macrophages has a role in the activation of immune effector cells at sites of sarcoidosis.

Clinical features of 125 patients with sarcoidosis: Okayama University Hospital review of a recent 10-year period.

Clinical features were studied in 125 patients with sarcoidosis (72 females and 53 males) diagnosed at Okayama University Hospital during a recent 10-year period. The age distribution had two peaks in patients in their 20s and the 50s. Over half of the patients were detected at health screening check and were asymptomatic, while the remaining were symptomatic. Twelve patients were in stage 0, 41 were in stage I, 54 were in stage II, 16 were in stage III, and 2 were in stage IV according to the chest x-ray findings. Serum angiotensin converting enzyme levels and serum lysozyme levels were elevated in 60% and 76% of the patients, respectively. The bronchoalveolar lavage fluid showed lymphocytosis, especially of helper T-cells. The clinical features of sarcoidosis appear to depend on the duration of the disease.