Establishment of dental epithelial cell line (HAT-7) and the cell differentiation dependent on Notch signaling pathway

Rat incisors grow continuously throughout life. Producing a variety of dental epithelial cells is performed by stem cells located in the cervical loop of the incisor apex. To study the mechanisms for cell differentiation, we established a dental epithelial cell line (HAT-7) originating from a cervical loop epithelium of a rat incisor. Immunochemical studies showed that HAT-7 produced the cells expressing amelogenin, ameloblastin, or alkaline phosphatase (ALP). To illustrate a role of Notch signaling in the determinant of the cell fate, we examined expression patterns of Notch1 and Jagged1 in HAT-7 density dependently. At lower cell density, Notch1- or Jagged1-expressing cells were not seen. However, when they were fully confluent, cells began to express Notch1 or Jagged1 strongly. Some ALP-positive cells were almost consistent with Notch1-expressing cells but not Jagged1-expressing cells. These results suggested that the determinant of direction of differentiation was associated with Notch signaling pathway.     

Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping.

We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacrylamide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, FokI, MboII and Mn1I can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, FokI, DdeI, BsaJI, BssHII, Cfr13I, RsaI, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.     

Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection.

Both Pseudomonas aeruginosa elastase and Bacillus thermoproteolyticus thermolysin are zinc metalloproteases. On the basis of the high homology of the P. aeruginosa elastase with the Bacillus thermolysin, we hypothesized that Glu-141 and His-223 are the key residues for catalytic activity of the Pseudomonas elastase. To test this possibility, we replaced Glu-141 with Asp, Gln, and Gly and His-223 with Gly, Glu, and Leu by site-directed mutagenesis. These substitutions dramatically diminished the proteolytic activities of the mutant elastases when they were expressed in Escherichia coli cells. Although these mutant elastase precursors (proelastases) were produced, no appreciable processing was observed with these mutants. The possibility that autocatalysis is involved in both the processing and activation of elastase is discussed. Furthermore, by immunizing mice with vaccines made from these mutant elastase, we were able to obtain good protection against an intraperitoneal P. aeruginosa challenge.     

A human recessive neurosensory nonsyndromic hearing impairment locus is a potential homologue of the murine deafness (dn) locus

A locus for recessive neurosensory nonsyndromic hearing impairmentmaps to chromosome 9q13–q21 in two regionally separateconsanguineous families from India. Each family demonstratesa LOD score greater than 4.5 to this region. D9S15, tightlylinked to the Friedreich's ataxia locus, a region that has beendefined with over 1 Mb of YAC contig information and severalexpressed sequences, is one of the flanking markers. In mice,the deafness (dn) locus maps to mouse chromosome 19 and flankingloci are syntenic to human chromosome 9q11–q21. The dnmouse is a potential model for the hearing impairment foundin both these families.     

Expression enhancement of the Tn5 neomycin-resistance gene by removal of upstream ATG sequences and its use for probing heterologous upstream activating sequences in yeast

We have constructed a series of promoter or upstream activating sequence (UAS)-probe plasmids carrying the Tn5-derived neomycin resistance gene whose seven additional ATG codons in the 5-untranslated region were completely or partially removed. When the deleted version of the neo sequence retaining only one additional ATG (NeoD) was expressed under the control of a TDH3 promoter whose UAS was deleted, the transformed cells were unable to grow at a low concentration of the antibiotic G418. In contrast with this, yeast cells expressing the NeoC sequence and having no additional ATG exhibited a high level of G418-resistance. Moreover, the UAS-probe system using NeoD has been successfully applied for the identification of several E. coli DNA sequences that clearly function as UASs in yeast cells. Two of these prokaryotic sequences with UAS activity were identified as a part of the coding region of the tgt and the hydG gene, respectively.     

Mutation analysis of two Japanese patients with Fanconi-Bickel syndrome

Fanconi-Bickel syndrome (FBS), or glycogen storage disease type XI, is a rare autosomal recessive disorder characterized by hepatorenal glycogen accumulation, Fanconi nephropathy, and impaired utilization of glucose and galactose. Recently, this disease was elucidated to link mutations in the glucose transporter 2 (GLUT2) gene. Only three mutations in three FBS families have been reported. Therefore, it is important to elucidate mutations in the GLUT2 gene in FBS by answering the question of whether the syndrome is a single gene disease. In this report, we describe two patients in two unrelated families clinically diagnosed with FBS. No mutation in the entire protein coding region of the GLUT2 gene was detected in patient 1, which suggested that no mutation existed in the GLUT 2 gene, or that some mutations had affected the expression of the GLUT 2 gene. In patient 2, a novel homozygous nonsense mutation (W420X, Trp at codon 420 to stop codon) was detected. These results support the correlation between GLTU2 gene mutation and FBS syndrome. However, many patients must be analyzed to determine whether other genes are involved in FBS. Received: July 16, 1999 / Accepted: September 3, 1999     

Presynaptic opioid kappa-receptor and regulation of the release of Met-enkephalin in the rat brainstem

Opioid kappa-agonists had much more potent inhibitory effects on the high K+-evoked Met-enkephalin release from rat brain slices than did the mu- or delta-agonists. The opioid kappa- antagonist, MR2266 enhanced the evoked release of Met-enkephalin to a greater extent than did mu- or delta-antagonists in vitro and had a potent analgesia in mice in vivo. These findings suggest that the release of Met-enkephalin may be regulated in vitro and in vivo, mainly by presynaptic kappa-receptor-mediated mechanisms.     

Comparison of the inhibitory effects of glucocorticoids on the expression of eotaxin in airway epithelial cell line BEAS-2B]

OBJECTIVE: Inhaled corticosteroids play a pivotal role in the treatment of asthma. To observe the mechanisms of glucocorticoids, we focused our study on the comparison of several glucocorticoids' effects on eotaxin expression in the airway epithelial cells. METHODS: Airway epithelial cell line BEAS-2B was cultured in vitro. Cells were preincubated with or without glucocorticoids (becromethasone dipropionate; BDP, budesonide; BUD, fluticasone propionate; FP) and stimulated with TNFalpha and/or IL-4. Protein levels of eotaxin in the supernatants of the cultured cells were determined by ELISA. RESULTS AND CONCLUSIONS: TNFalpha and IL-4 increased the levels of eotaxin in BEAS-2B cells. Combination of these cytokines synergistically upregulated the eotaxin expression as reported previously. Each glucocorticoid significantly inhibited the expression of eotaxin protein induced with TNFalpha and IL-4 and the compared efficacy was in order of FP>BUD>BDP. FP seemed most potent and the inhibitory effect was also observed with relatively low concentration such as 10 (-10)M. Taken together, the comparison of the potency of each glucocorticoid using airway epithelial cells may reflect the efficacy of these drugs in asthmatics.