Inhibitory effect of statins on inflammatory cytokine production from human bronchial epithelial cells.

Statins are 3-hydroxy-3-methylglutaryl-co-enzyme A reductase inhibitors of cholesterol biosynthesis, and have been reported to exert pleiotropic effects on cellular signalling and cellular functions involved in inflammation. Recent reports have demonstrated that previous statin therapy reduced the risk of pneumonia or increased survival in patients with community-acquired pneumonia. However, the precise mechanisms responsible for these effects are unclear. In the present study, we examined the effects of statins on cytokine production from lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Interleukin (IL)-6 and IL-8 mRNA expression and protein secretion in LPS-stimulated cells were inhibited significantly by the lipophilic statin pitavastatin and the hydrophilic statin pravastatin. As these inhibitory effects of statin were negated by adding mevalonate, the anti-inflammatory effects of statins appear to be exerted via the mevalonic cascade. In addition, the activation levels of Ras homologue gene family A (RhoA) in BEAS-2B cells cultured with pitavastatin were significantly lower than those without the statin. These results suggest that statins have anti-inflammatory effects by reducing cytokine production through inhibition of the mevalonic cascade followed by RhoA activation in the lung.

Pseudo-outbreak of Mycobacterium paragordonae in a hospital: possible role of the aerator/rectifier connected to the faucet of the water supply system.

BACKGROUND: Pseudo-outbreaks of non-tuberculous mycobacteria (NTM) in association with the water supply system in hospitals have been previously reported. We found that the frequency of NTM isolation in clinical samples increased after the reconstruction and renovation of a hospital in Japan in 2014. AIM: To analyse NTM, their possible relationship with the hospital water supply system, and outcomes of preventive measures. METHODS: Environmental samples obtained from the water supply in hospital wards were tested for NTM. On obtaining positive results, the bacteria were further analysed using polymerase chain reaction (PCR). FINDINGS: The PCR products of NTM showed that most samples tested positive for Mycobacterium paragordonae. Because none of the analysed patients developed any disease due to these bacteria, this event was considered a pseudo-outbreak. Investigation of the water supply system revealed that samples obtained from the recently attached aerators/rectifiers during hospital renovation tested positive for these bacteria. Therefore, measures to remove aerators/rectifiers and prevent patients from drinking tap water in the hospital were introduced. Thereafter, the frequency of NTM-positive samples significantly decreased in the hospital. CONCLUSION: This study is one of the few reports which reveal the possibility of pseudo-outbreaks of M. paragordonae in hospitals, hence raising the question whether aerators/rectifiers should be used in hospitals at all, because their mesh structure may promote NTM proliferation in supplied water. The importance of surveillance of bacteria derived from the environment, particularly after hospital reconstruction/renovation, is re-emphasized.

Cloning and characterization of a novel RNA-binding protein SRL300 with RS domains.

AT-rich element binding factor 1 (ATBF1) mRNA encodes a transcription factor implicated in neuronal differentiation. A cDNA for the protein that can bind the 5'-noncoding sequence of the ATBF1 mRNA was cloned. The deduced protein, termed SRL300, contains a unique RNA-binding region, two large RS domains and many phosphorylation sites. SRL300 protein was detected in both human and rat cells.

Cloning and characterization of the rat p130, a member of the retinoblastoma gene family.

A cDNA clone encoding rat p130, a member of the retinoblastoma (Rb) gene family, was isolated based on the sequence homology of the E1A-binding domain. The 4.87 kb cDNA contained an 1135-amino acid open reading frame with high homologies to the human and mouse p130 and a partial homology to the pRb protein. p130 showed difference in distribution of potential phosphorylation sites from pRb in the N-terminal and the B pocket regions. p130 mRNA was detected in most rat tissues. The p130 gene was mapped to rat chromosome 19p11-13 by fluorescence in situ hybridization.

A novel keratin K5 gene mutation in Dowling-Meara epidermolysis bullosa simplex.

We examined keratin K14 and K5 genes mutation in a Japanese Dowling-Meara epidermolysis bullosa simplex patient with severe generalized blistering and erosions at birth. The patient had a C to T transition at the first position of codon 174 in the keratin K5 gene, which resulted in a Leu->Phe substitution at the highly conserved 1A domain in keratin K5. Thus, our results revealed a novel mutation in the helix initiation peptide of keratin K5.

Detection of autoantibodies to thyroid peroxidase in autoimmune thyroid diseases by micro-ELISA and immunoblotting.

Serum autoantibodies to thyroid peroxidase (TPO) in patients with thyroid autoimmune diseases were studied by micro-ELISA and immunoblotting. Twenty-four patients, 15 with Graves' disease and 9 with Hashimoto's thyroiditis, whose serum titers were greater than 3200 on the microsomal hemagglutination test (except for 1 patient with a titer of 800) had autoantibodies to TPO. Both immunoglobulin G and M classes of autoantibodies were detected, with the former being more prominent. When TPO and thyroid microsomes were used as a target in a competitive binding inhibition test, the results suggested that TPO was a major thyroid microsomal antigen. On the other hand, immunoblotting analysis showed 3-4 bands in the 45-60K region stained by patients' sera in addition to human TPO with mol wt of 100K and 107K; only the latter 2 bands stained with antiporcine TPO antibody. In the majority of sera, TPO bands were clearer than others, although some sera showed the clearest band with a mol wt of 55K. These results indicate that patients with autoimmune thyroid disease often have autoantibodies to TPO that can be detected by micro-ELISA and immunoblotting, and that TPO is a major component of the thyroid microsomal antigen.

Nucleotide sequence of the cDNA encoding mouse thyroid peroxidase.

The nucleotide (nt) sequence of the cDNA encoding mouse thyroid peroxidase (TPO) has been determined. The TPO cDNA is 3281 nt long and its open reading frame encodes a protein composed of 914 amino acids (aa), including a putative initiation Met. The mouse TPO cDNA shows 75.3, 70.8, and 93.5% homology in the coding nt sequence with human, porcine, and rat TPO cDNAs, respectively. In the aa sequence, mouse TPO shows 73.4, 68.4, and 93.9% homology with human, porcine, and rat TPOs, respectively. Specifically, the aa sequence surrounding the proximal His residue, probably essential for TPO activity, is well conserved among these four organisms.

A monoclonal antibody to rat liver arylsulfatase C and its application in immunohistochemistry.

We purified arylsulfatase C from rat liver microsomes and prepared a monoclonal antibody (P42C2) to the purified enzyme. By SDS-PAGE and immunoblotting analysis using P42C2, the molecular weight of the purified enzyme and of the enzyme in liver and kidney microsomes were estimated at 62,000 daltons. P42C2 caused little inhibition of arylsulfatase C activity, and was bound only slightly to liver microsomes. Localization of arylsulfatase C was studied at the light and electron microscopic level by the indirect immunoperoxidase method using P42C2. In rat liver, arylsulfatase C was detected mainly in the hepatocytes, and less frequently in endothelial cells, Kupffer's cells, and Ito's cells. In rat kidney, strong staining was observed in the straight portions of the proximal tubules. The podocytes, interstitial cells, endothelial cells, and epithelial cells of Henle's thin limbs were stained faintly. By electron microscopy, arylsulfatase C was found localized on the membranes of the endoplasmic reticulum and nuclear envelopes in these cells. These immunohistochemical findings agree with the localization demonstrated by an enzyme-histochemical method which we had previously developed.

Epitopic difference among rat thyroglobulins.

Epitopes on thyroglobulin (Tg) were examined using mouse monoclonal antibodies (mAbs). Two out of 6 mAbs indicated a remarkable difference in binding to three rat Tgs. Namely, they bound to one Tg as well as immunized Tg but could not bind to another at all. They could hardly bind to the third Tg. Another mAb was similar, to some extent, in binding to three rat Tgs as described above, although three other mAbs bound almost equally to three rat Tgs. Since competitive binding inhibition by Tgs supported the result of the binding study, the epitopic difference on rat Tgs was confirmed. Furthermore, competitive binding inhibition by unlabeled mAbs revealed that six mAbs recognized different epitopes individually. Therefore, there were at least two unique epitopes located on one rat Tg but not located on another. The relation between unique epitopes and thyroiditis in the rats used in the present study is also discussed.

Expression of dipeptidyl aminopeptidase IV activity in thyroid carcinoma.

Dipeptidyl aminopeptidase IV activity staining was performed in various thyroid tissues to evaluate this enzyme activity as a thyroid tumor marker. A total of 195 thyroid tissues were tested for their enzyme activity expression. All papillary and follicular carcinomas, 40 cases and 3 cases, respectively, showed enzyme activity, although two other carcinomas, one medullary and one anaplastic, were not stained. Follicular adenoma expressed enzyme activity in 4 of 26 cases. Fifty-two cases with adenomatous goiter, 54 with Graves' disease and 13 with chronic thyroiditis were judged to be negative. Five normal thyroids expressed no activity except for occasional positive staining of capillary endothelia. These data suggest that dipeptidyl aminopeptidase IV activity staining is very useful for pathological diagnosis of thyroid tumors.

A novel mutation in the human thyroid peroxidase gene resulting in a total iodide organification defect.

In this study we describe a novel mutation of the thyroid peroxidase (TPO) gene that resulted in a total iodide organification defect. TPO activity and thyroxine formation in thyroglobulin in the thyroid gland of the patient were below the limits of detection. However, TPO mRNA was detectable at a similar size and concentration as compared with normal thyroid tissues when measured by Northern blot analysis. Sequence analysis of the TPO gene showed the presence of two mutations, a missense mutation in exon 7 and C insertion in exon 14. These mutations were heterozygous and located in different alleles. The latter mutation has already been reported as one of the mutations of the TPO gene resulting in total iodide organification defect. The former mutation was further analysed by mRNA transfection studies in which mutated mRNA was transfected to CHO-K1 cells by electroporation. The results of transfection studies showed that the cells transfected with mutated mRNA expressed similar size TPO molecules to those of cells transfected with wild-type mRNA but that they lacked TPO activity. The two mutations of the TPO gene resulting in the total iodide organification defect in the patient cosegregated from her parents.

Iodide organification defects resulting from cosegregation of mutated and null thyroid peroxidase alleles.

This report describes an intriguing combination of the thyroid peroxidase (TPO) alleles resulting in an iodide organification defect. Sequence analysis of the patient's TPO gene showed the presence of T-deletion in exon 14 of the TPO gene (T2512del). From the sequencing pattern, this new mutation of the TPO gene was thought to be homozygous. mRNA transfection studies in which mutated mRNA was transfected to CHO-K(1) cells by electroporation showed that the cells transfected with mutated mRNA expressed smaller TPO molecules than those of cells transfected with wild-type mRNA and that they had TPO activity. However, the smaller TPO molecules could not translocate onto the cell surface. To investigate T2512del in the parents, their genomic DNAs were sequenced. Results showed that the mother had T2512del but the father did not. However, when seven polymorphic positions reported earlier were analyzed, the mother showed two kinds of nucleotides at four positions but the patient and father showed only one nucleotide at all seven positions. We suspected a deletion of the TPO gene (2p25) in one of two second chromosomes, and analyzed the patient's chromosomes by FISH using TPO cDNA and N-myc genomic DNA as probes. N-myc genomic DNA exhibited two signals and TPO cDNA only one signal, although the G-band showed no morphological abnormalities. T2512-deleted and 2p25-deleted null alleles cosegregated from her parents, resulting in iodide organification defect in the patient.

A keratin K14 gene mutation in a Japanese patient with the Dowling-Meara type of epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is caused by an aberration of the keratin intermediate filaments and recent studies indicated causal mutations in the keratin K14 and K5 genes. In this study, we examined keratin K14/5 gene mutation in a Japanese patient with EBS Dowling-Meara (EBSDM). The patient had a C to T transition at the first position of codon 125, which resulted in Arg-->Cys at the N-terminus of the rod domain in the keratin K14 gene. The mutation position described here was identical to those reported in some other EBSDM patients. Our result revealed mutation in the peptide initiating helical structure of keratin K14 and, together with the results of other workers, suggests that the mutation in the keratin K14 gene of EBSDM sufferers occurs in virtually every ethnic group and geographical area.

Type VII collagen DNA linkage analysis in a Japanese family with dominant dystrophic epidermolysis bullosa.

Type VII collagen, a major component of anchoring fibrils in the basement membrane zone, is now considered to be a primary genetic factor in the pathogenesis of dominant dystrophic epidermolysis bullosa (DDEB). In this study, we performed genetic linkage analysis in a Japanese family with DDEB using a PvuII polymorphism in the type VII collagen gene. The pedigree consisted of 10 affected and 13 unaffected living individuals and was diagnosed as having Cockayne-Touraine type of DDEB. Electron microscopic examination of the skin demonstrated a diminished number and rudimentary structure of anchoring fibrils. PCR-based detection of PvuII polymorphism resulted in 3 genotypes and co-segregated with DDEB phenotype in this pedigree. The maximum lod score was 2.10 at recombination fraction (theta) of 0. The absence of recombination between DDEB and type VII collagen gene locus, as well as the observation of altered anchoring fibrils, suggested that type VII collagen is a candidate gene for the Japanese family with DDEB, although the lod score was statistically not significant.

Structures of multi-branched dextrins produced by saccharifyiing alpha-amylase from starch.

From the digest of beta-limit dextrin (prepared from glutinous rice starch) with saccharifying alpha-amylase of Bacillus subtilis [EC 3.2.1.1] (BSA), two extensibely branched dextrins consisting of nine (No. 6, Fig. 1) and ten (No 7, Fig.1) glucose units were isolated by paper chromatography. Structural analysis using various enzymes revealed that No. 6 and No. 7 were both mixtures of four triply branched dextrins. They had structures which were built up with 63-alpha-glucosylmaltotriose and/or 62-alpha-glucosylmaltose as a linking unit. However, the branching configuration and the minimum alpha-1, 4-glucosidic linkages existing between two branches followed one of the three structures shown below: (see article).

Structures of singly branched heptaoses produced by bacterial liquefying alpha-amylase.

1. A singly branched heptaose produced as a limit dextrin in the digest of beta-limit dextrin with liquefying alpha-amylase [EC 3.2.1.1] of Bacillus amyloliquefaciens was isolated in a paper chromatographically pure state. 2. Analysis using several enzymes revealed that the isolated branched dextrin was a mixture of six singly branched heptaoses with different ramifying points. 3. All the branched heptaoses contained a 62-alpha-maltosylmaltotriose moiety in their molecules, differing only in the mode of attachment of one maltose or two glucose residues by alpha-1,4-glucosidic bonds from this core dextrin. 4. The formation of various singly branched heptaoses (the present paper) and hexaoses (the previous paper) is discussed regarding the attack site specificity of the enzyme on beta-limit dextrin.

Phenotypic heterogeneity in bullous congenital ichthyosiform erythroderma: possible somatic mosaicism for keratin gene mutation in the mildly affected mother of the proband.

BACKGROUND: Bullous congenital ichthyosiform erythroderma (BCIE) shows phenotypic variability. An epidermal nevus may represent somatic mosaicism for keratin gene mutation, which produces generalized BCIE in the next generation. This fact provides evidence that a postzygotic mutation can be passed on to the next generation in BCIE. We hypothesized that the same phenomenon occurred in a family with BCIE whose phenotypes were extremely different. OBSERVATIONS: We studied a 19-year-old boy with severe ichthyosiform erythroderma and prominent palmoplantar hyperkeratosis with digital contracture. In contrast, the proband's mother exhibited only mild ichthyosiform skin, granular verrucous lesions, and less severe streaky palmoplantar hyperkeratosis. Mutation analysis in the proband showed a keratin K1 mutation (N187S, ie, an A-to-G transition at the second position of codon 187, resulting in an asparagine-to-serine substitution). In the mother, the same keratin gene mutation was recognized, but only faintly in the leukocyte DNA, indicating that the amount of the mutated allele in leukocyte DNA was very low compared with that from the proband. CONCLUSIONS: We speculate that the mildly affected mother showed keratin 1 gene mosaicism, and that the BCIE phenotype had been transmitted in a severe form through a mechanism that passes the keratin gene mutation to the next generation. These results suggest that mild forms of BCIE may actually represent extensive epidermal nevi/keratin gene mosaicism.

Coexistence of generalized morphea with hisotological changes in lichen sclerosus et atrophicus and lichen planus.

We reported a 44-year-old Japanese woman with generalized multiple sclerotic plaques, which showed histological findings of morphea. This patient also had an erosive lesion on her mouth; its histological findings were consistent with lichen planus. A sclerotic lesion on her thigh showed the histological findings of lichen sclerosus et atrophicus (LSA). These data suggest that similar etiologic events or closely related pathologic processes are involved in morphea, lichen planus, and LSA.

[A case of tuberculous peritonitis diagnosed by ultrasonography-guide peritoneal biopsy].

The diagnosis of tuberculous peritonitis is quite difficult because the symptoms are not specific for the disease and the incidence of occurrence are relatively rare. We report a case of tuberculous peritonitis diagnosed by ultrasonography-guided peritoneal biopsy. A 64-year-old male was admitted to our hospital because of fever, dyspnea and abdominal pain. Laboratory findings revealed an elevated ESR (53 mm/1 hr.) and positive CRP. The tuberculin skin test was negative. The chest radiograph revealed bilateral pleural effusion. Abdominal ultrasonographic examination and computed tomography showed ascitic fluid, thickening of the mesentery and peritoneum, and inflammatory pseudotumor of the omentum. Ascitic fluid was exudate with a high lymphocyte count and elevated ADA (184 IU/l). Microbiological studies with the fluid were negative. Peritoneal biopsy guided by ultrasonography was performed, and the specimens showed central caseous necrosis surrounded by epitheloid cells and acid-fast bacilli were demonstrated. The size of the pseudotumor, pleural effusion and ascites decreased after antituberculous chemotherapy with corticosteroid was given. Diagnosis of tuberculous peritonitis has often been made by laparotomy or laparoscopy. In a case of this kind, percutaneous peritoneal biopsy guided by ultrasonography is safe and useful.

[Measurement of human thyroid peroxidase autoantibodies by enzyme immunoassay using recombinant human TPO].

An EIA for measuring anti-TPO autoantibodies (rhTPO-EIA) was developed using recombinant human TPO expressed in CHO cells and was compared with MC-HA generally used in laboratory routine work. rhTPO-EIA showed a satisfactory reproducibility in the intra-assay test and did not have an accidental error of lots. Almost equal number of healthy females and males were measured for their IgG binding to TPO to define a normal range of anti-TPO autoantibodies. After setting 20 IU/ml as an upper limit of normal range, sera from patient with thyroid disorders were measured for their anti-TPO autoantibodies. Chronic thyroiditis and Graves' disease were highly positive, while adenoma, thyroid cancer, SLE, and RA were low in their positivity. The positive rate of anti-TPO autoantibodies was compatible to those of previous reports in each disorder. Seventy-two sera from patients with chronic thyroiditis or Graves' disease were measured for their autoantibodies by both rhTPO-EIA and MC-HA and the results were compared between both methods. A correlation coefficient was 0.486. Following absorption with thyroglobulin, sera were measured again and as the results, the correlation coefficient increased to 0.723. Therefore, MC-HA was thought to be influenced in the presence of anti-thyroglobulin autoantibodies. Since rhTPO-EIA is excellent in quality and not affected by anti-thyroglobulin antibodies, it is useful and applicable to clinical diagnosis and observation of thyroid disorders.