Amino acid residues conferring the nucleotide binding properties of N-acetyl-D-glucosamine 2-epimerase (renin binding protein)

Our recent studies have demonstrated that the middle domain of N-acetyl-D-glucosamine (GlcNAc) 2-epimerase participates in the specificity for and binding of nucleotides. To identify the residue conferring nucleotide binding, amino acid substitutions were introduced in the human and rat GlcNAc 2-epimerases. The mutational analyses indicate that residue 171 of GlcNAc 2-epimerase is critical for the nucleotide binding of GlcNAc 2-epimerase.     

Aberrant B1 cell migration into the thymus results in activation of CD4 T cells through its potent antigen-presenting activity in the development of murine lupus

B1 cells have different origin and function from conventional B (B2) cells and are considered to be involved in autoantibody production in the development of autoimmune disease. We found that B1 cells preferentially accumulated in the target organs including thymus in aged BWF1 mice, a murine model for systemic lupus erythematosus, and that B lymphocyte chemoattractant (BLC/CXCL13) expression was increased in the thymus before the onset of lupus nephritis, while stromal cell-derived factor-1 (SDF-1/CXCL12) and secondary lymphoid tissue chemokine (SLC/CCL21) expression remained unchanged. Adhesion molecules such as peripheral node addressin (PNAd), ICAM-1, and VCAM-1 were also expressed on endothelial cells in the enlarged thymic perivascular space (PVS) in aged BWF1 mice. BLC protein and PNAd were co-localized on these high-endothelial-venules-like vessels in enlarged PVS. B1 cells expressed higher level of costimulatory molecules and showed a potent antigen-presenting activity in allogeneic mixed lymphocyte reaction comparable to splenic dendritic cells. Interestingly, B1 cells stimulated proliferation of autologous thymic CD4 T cells in the presence of IL-2. These results indicate that aberrant B1 cell trafficking into the thymus due to ectopic high expression of BLC may result in an activation of self-reactive T cells in the development of murine lupus.     

Clinical heterogeneity of neonatal intrahepatic cholestasis caused by citrin deficiency: case reports from 16 patients

A deficiency of citrin, which is encoded by the SLC25A13 gene, causes both adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). We analyzed 16 patients with NICCD to clarify the clinical features of the disease. Severe intrahepatic cholestasis with fatty liver was the most common symptom, but the accompanying clinical features were variable, namely; suspected cases of neonatal hepatitis or biliary atresia, positive results from newborn screening, tyrosinemia, failure to thrive, hemolytic anemia, bleeding tendencies and ketotic hypoglycemia. Laboratory data showed elevated serum bile acid levels, hypoproteinemia, low levels of vitamin K-dependent coagulation factors, and hypergalactosemia. Hypercitrullinemia was detected in 11 out of 15 patients examined. Most of the patients were given a lactose-free and/or medium chain triglycerides-enriched formula and lipid-soluble vitamins. The prognosis of the 16 patients is going fairy well at present, but we should observe these patients carefully to see if they manifest any symptom of CTLN2 in the future.     

Rapid screening of point mutations of the Neisseria gonorrhoeae parC gene associated with resistance to quinolones.

To detect quinolone resistance-associated mutations within the Asp-86, Ser-87, Ser-88, and Glu-91 codons of the Neisseria gonorrhoeae parC gene, we developed a rapid and simple assay based on amplification of the regions of the parC gene containing the mutations sites by PCR and digestion of the PCR products with restriction enzymes. By using the method of primer-specified restriction site modification, artificial SalI, PstI, EcoRI, and HinfI restriction sites were created in the regions containing the Asp-86, Ser-87, Ser-88, and Glu-91 codons, respectively. The mutations generating alterations at Asp-86, Ser-87, Ser-88, and Glu-91 were detected as failures of SalI, PstI, EcoRI, and HinfI to digest the respective PCR products. Fifty-five clinical strains of N. gonorrhoeae were examined for mutations in the parC gene by this assay. Appropriate mutations at either the Asp-86, Ser-87, Ser-88, or Glu-91 codon were detected in each of 11 strains in which a mutation had previously been observed by DNA sequencing. This rapid and simple assay could be a useful device for screening genetic alterations in the parC gene associated with resistance to quinolones in N. gonorrhoeae.     

Use of the genomic subtractive hybridization technique to develop a real-time PCR assay for quantitative detection of Prevotella spp. in oral biofilm samples

Genomic subtractive hybridization was used to design Prevotella nigrescens-specific primers and TaqMan probes. Based on this technique, a TaqMan real-time PCR assay was developed for quantifying four oral black-pigmented Prevotella species. The combination of real-time PCR and genomic subtractive hybridization is useful for preparing species-specific primer-probe sets for closely related species.     

Purification of fully activated Clostridium botulinum serotype B toxin for treatment of patients with dystonia

Clostridium botulinum serotype B toxins 12S and 16S were separated by using a beta-lactose gel column at pH 6.0; toxin 12S passed through the column, whereas toxin 16S bound to the column and eluted with lactose. The fully activated neurotoxin was obtained by applying the trypsin-treated 16S toxin on the same column at pH 8.0; the neurotoxin passed through the column, whereas remaining nontoxic components bound to the column. The toxicity of this purified fully activated neurotoxin was retained for a long period by addition of albumin in the preparation.     

Galactosylated chitosan as a synthetic extracellular matrix for hepatocytes attachment

Galactose moiety as the hepatocyte anchorage was covalently coupled with chitosan for the development of synthetic extracellular matrix. Hepatocytes adhesion to galactosylated chitosan (GC)-coated polystyrene (PS) dish became as high as 94.7% after 2 h incubation whereas the hepatocytes adhesion to chitosan-coated PS dish was 69.1%, indication of galactose-specific recognition between GC molecules and asialoglycoprotein receptors of hepatocytes. The DNA synthesis of the hepatocytes adhered to GC-coated dish was increased in the presence of epidermal growth factor (EGF) at low concentration of GC (0.05 microg/ml) whereas the DNA synthesis of the hepatocytes adhered to GC-coated dish was decreased in the presence of EGF at high concentration of GC (5 microg/ml). The spreading shapes of the hepatocytes adhered to the surface in the presence of EGF at low concentration of GC (0.05 microg/ml) were enhanced than in the absence of EGF. The hepatocytes adhered to the surface at high concentration of GC (5 microg/ml) showed round shapes and exhibited many spheroid formation after 24 h in the presence of EGF.     

Melanoma cell adhesion molecule (MCAM/CD146) is expressed on human luteinizing granulosa cells: enhancement of its expression by hCG,interleukin-1 and tumour necrosis factor-alpha

Melanoma cell adhesion molecule (MCAM) was originally reported to be involved in the invasion and progression of melanoma. It was also shown to be responsible for the attachment of cells to endothelial cells. In this study, we demonstrated by immunohistochemistry that immunoreactive MCAM was not expressed on granulosa cells in the pre-ovulatory follicle, but it was clearly detected in large luteal cells in corpora lutea from the mid-luteal phase of the menstrual cycle. Northern blotting analysis confirmed the expression of MCAM mRNA in corpus luteum. MCAM was weakly detected by immunocytochemical staining in human luteinizing granulosa cells isolated from patients undergoing IVF treatment. Its expression was found to be increased during time in culture of these cells. Flow cytometry and Northern blot analysis revealed that MCAM expression on luteinizing granulosa cells was enhanced when the cells were cultured for 5 days in the presence of hCG (1 IU/ml) or cytokines such as interleukin-1alpha (10 ng/ml) and tumour necrosis factor-alpha (10 ng/ml). No significant difference of MCAM expression was observed between the cultures under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. These results indicate that luteinizing granulosa cells express MCAM and that MCAM expression is regulated by LH/hCG and cytokines during luteinization. Since MCAM has been reported to mediate cellular interaction with endothelial cells, this molecule may play a role in neovascularization during corpus luteum formation in the human ovary.     

Generation of velogenic Newcastle disease viruses from a nonpathogenic waterfowl isolate by passaging in chickens

A benign Newcastle disease virus (NDV) recently became highly virulent during replication in domestic chickens. It is still unclear whether NDVs circulating among wild waterfowl also have the potential to become highly pathogenic (velogenic) in chickens. To demonstrate experimentally the generation of velogenic NDV from a nonpathogenic waterfowl isolate, we passaged an avirulent goose isolate in chickens. After nine consecutive passages by air-sac inoculation, followed by five passages in chick brain, the virus became highly virulent in chickens, producing a 100% mortality rate, and demonstrating typical velogenic properties in pathogenicity tests. Sequence analysis at the fusion protein cleavage site showed that the original isolate contained the typical avirulent type sequence, E-R-Q-E-R/L, which progressed incrementally to a typical virulent type, K-R-Q-K-R/F, during repeated passage in chickens. These results demonstrate that avirulent viruses, maintained in wild waterfowl in nature and bearing the consensus avirulent type sequence, have the potential to become velogenic after transmission to and circulation in chicken populations. The results also suggest that chickens provide a mechanism for the selection of virulent viruses from an avirulent background.     

Heavy-ion-induced mutations in the gpt delta transgenic mouse: comparison of mutation spectra induced by heavy-ion,X-ray,and gamma-ray radiation

Heavy-ion radiation accounts for the major component of absorbed cosmic radiation and is thus regarded as a significant risk during long-term manned space missions. To evaluate the genetic damage induced by heavy particle radiation, gpt delta transgenic mice were exposed to carbon particle irradiation and the induced mutations were compared with those induced by reference radiations, i.e., X-rays and gamma-rays. In the transgenic mouse model, deletions and point mutations were individually identified as Spi(-) and gpt mutations, respectively. Two days after 10 Gy of whole-body irradiation, the mutant frequencies (MFs) of Spi(-) and gpt were determined. Carbon particle irradiation significantly increased Spi(-) MF in the liver, spleen, and kidney but not in the testis, suggesting an organ-specific induction of mutations by heavy-ion irradiation. In the liver, the potency of inducing Spi(-) mutation was highest for carbon particles (3.3-fold increase) followed by X-rays (2.1-fold increase) and gamma-rays (1.3-fold increase), while the potency of inducing gpt mutations was highest for gamma-rays (3.3-fold increase) followed by X-rays (2.1-fold increase) and carbon particles (1.6-fold increase). DNA sequence analysis revealed that carbon particles induced deletions that were mainly more than 1,000 base pairs in size, whereas gamma-rays induced deletions of less than 100 base pairs and base substitutions. X-rays induced various-sized deletions and base substitutions. These results suggest that heavy-ion beam irradiation is effective at inducing deletions via DNA double-strand breaks but less effective than X-ray and gamma-ray irradiation at producing oxidative DNA damage by free radicals.