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.

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.

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.

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.

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.

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.

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.

[Myelodysplastic syndrome in a child which developed into megakaryocytic leukemia with late appearing Ph1 chromosome and rearrangement of breakpoint cluster region].

A 3-year-old boy was referred to our hospital in September 1985, because of pancytopenia. His bone marrow was normocellular with 18% blasts, which had Auer rod and were positive for peroxidase staining. A diagnosis of refractory anemia with excess blasts in transformation was made according to FAB criteria. Chromosome analysis of bone marrow cells showed normal male karyotype. He attained complete remission with aclarubicin and BH-AC and continued it until August 1987 when pancytopenia and hypoplastic bone marrow developed. Chromosome analysis of bone marrow cells showed normal male karyotype and gene analysis revealed germ-line configuration of breakpoint cluster region (bcr). Overt leukemia developed in May 1988 when his WBC count increased to 60, 600/microliters with 91% blasts, which were negative for peroxidase staining, positive for anti-Ia and CDw 41 by cell surface analysis, and positive for ultrastructurally demonstrable platelet peroxidase. A diagnosis of megakaryocytic leukemia was made. Chromosome analysis of bone marrow cells showed 46, XY, t(9;22) (q34;q11) and gene analysis revealed rearrangement of bcr. He died in November 1988. Our results and review of literature suggest that late appearing ph1 chromosome and rearrangement of bcr may occur in a variety of hematologic malignancies and influence the course of disease.