Downregulation of Max dimerization protein 3 is involved in decreased visceral adipose tissue by inhibiting adipocyte differentiation in zebrafish and mice.

BACKGROUND: The diet-induced obesity model of zebrafish (DIO-zebrafish) share a common pathophysiological pathway with mammalian obesity. OBJECTIVES: We aimed to investigate the role of Max dimerization protein 3 (MXD3) in visceral fat accumulation and adipocyte differentiation, by conducting knockdown experiments using zebrafish and mouse preadipocytes. METHODS: To identify genes related to visceral adiposity, we conducted transcriptome analyses of human and zebrafish obese populations using the Gene Expression Omnibus and DNA microarray. We then intraperitoneally injected morpholino antisense oligonucleotides (MO-mxd3) to knockdown mxd3 gene expression in DIO-zebrafish and measured several parameters, which reflected human obesity and associated metabolic diseases. Finally, lentiviral Mxd3 shRNA knockdown in mouse 3T3-L1 preadipocytes was conducted. Quantitative PCR analyses of several differentiation markers were conducted during these gene knockdown experiments. RESULTS: We found that MXD3 expression was increased in the obese population in humans and zebrafish. Intraperitoneal MO-mxd3 administration to DIO-zebrafish suppressed the increase in body weight, visceral fat accumulation and the size of mature adipocytes. Subsequently, dyslipidemia and liver steatosis were also ameliorated by MO-mxd3. In mouse adipocytes, Mxd3 expression was drastically increased in the early differentiation stage. Mxd3 shRNA inhibited preadipocyte proliferation and adipocyte maturation. Quantitative PCR analyses showed that the early differentiation marker, CCAAT/enhancer-binding protein delta (Cebpd) and late differentiation markers (CCAAT/enhancer-binding protein, alpha and peroxisome proliferator-activated receptor gamma) were downregulated by Mxd3 knockdown in 3T3-L1 cells and DIO-zebrafish. Subsequently, mature adipocyte markers (adiponectin and caveolin 1 for zebrafish, and fatty acid binding protein 4 and stearoyl-coenzyme A desaturase 1 for mouse adipocytes) were also decreased. CONCLUSION: Mxd3 regulates preadipocyte proliferation and early adipocyte differentiation via Cebpd downregulation in vitro and in vivo. Integrated analysis of human and zebrafish transcriptomes allows identification of a novel therapeutic target against human obesity and further associated metabolic disease.

Differential impacts of CYP2C19 gene polymorphisms on the antiplatelet effects of clopidogrel and ticlopidine.

We examined the influence of CYP2C19 polymorphisms on the antiplatelet effects of clopidogrel and ticlopidine. The platelet aggregation induced by 20 µmol/l adenosine diphosphate (ADP) and CYP2C19 single-nucleotide polymorphisms (*2 and *3) was determined in patients with coronary artery disease (CAD) who were taking aspirin alone (n = 21), aspirin plus clopidogrel (n = 97), or aspirin plus ticlopidine (n = 47). The degree of platelet aggregation in the clopidogrel group, although not in the ticlopidine group, depended on the CYP2C19 polymorphism, and the maximal platelet aggregation in poor metabolizers (PMs) taking clopidogrel was equivalent to that in the group taking aspirin alone. After being switched from clopidogrel to ticlopidine, all seven of the PMs showed markedly lower platelet aggregation. These results suggest that CYP2C19 polymorphisms have a profound impact on the antiplatelet effect of clopidogrel but not on that of ticlopidine. Ticlopidine may be an effective therapeutic option for CYP2C19 PMs.

Vasoactive intestinal peptide and inflammatory cytokines enhance vascular endothelial growth factor production from epidermal keratinocytes.

BACKGROUND: Overexpression of vascular endothelial growth factor (VEGF) in epidermal lesions of psoriasis is well documented; however, its underlying mechanisms are largely unknown. We have recently demonstrated that vasoactive intestinal peptide (VIP) induces the production of cytokines such as interleukin-6 and stem cell factor from keratinocytes, thereby contributing to the development of inflammatory dermatoses such as psoriasis. OBJECTIVES: In this study, we attempted to determine whether VIP could increase the production of VEGF in human keratinocytes. METHODS: We examined the expression of VEGF using reverse transcription-polymerase chain reaction, immunocytochemistry, enzyme-linked immunosorbent assay and immunoblotting in normal human epidermal keratinocytes and human epidermal keratinocyte cell line DJM-1 cultured in the absence or presence of VIP and/or inflammatory cytokines. RESULTS: We demonstrate that human keratinocytes produced VEGF in a steady state at both mRNA and protein levels. VIP significantly upregulated the production of VEGF in keratinocytes in a dose- and time-dependent manner. The VIP-mediated production of VEGF was further enhanced by inflammatory cytokines such as interferon-gamma, tumour necrosis factor-alpha and interleukin-4, with maximum enhancement being observed with the combination of VIP and interferon-gamma. CONCLUSIONS: VIP and other cytokines from nerve endings, mast cells and local inflammatory cells are capable of enhancing VEGF production from epidermal keratinocytes, which may underlie excessive angiogenesis and vasodilation in skin lesions of psoriasis.

Localization of human airway trypsin-like protease in the airway: an immunohistochemical study.

Human airway trypsin-like protease (HAT) has been isolated from mucoid sputum of patients with chronic airway diseases. In order to clarify the cellular source of this novel protease in the human airway, we examined the localization of immunoreactive HAT in bronchial tissues obtained at surgery and fixed in 4% paraformaldehyde using an extremely sensitive immunohistochemical technique called a catalyzed signal amplification method and a monoclonal antibody against recombinant HAT. HAT immunoreactivity was demonstrated in cytoplasm of ciliated cells of bronchial epithelium and/or at the basal part of cilia. No positive reaction was found in submucosal glands or mast cells. The heterogeneous distribution of HAT immunoreactivity within the bronchial epithelium indicates that its expression might be changeable and that it might be closely related to the physiological status of the airway epithelium. Non-specific but intense reaction caused by endogenous avidin-binding activity (EABA) was selectively detected in submucosal glands, but was effectively blocked by successive treatments with avidin and biotin. These results indicate that HAT may be synthesized in the ciliated cells and that it may play some physiological roles within the epithelial layer and on the airway surface. It is necessary to keep in mind that some cells show strong EABA, especially when a highly sensitive immunohistochemical technique is applied.

Diversity of the HO gene encoding an endonuclease for mating-type conversion in the bottom fermenting yeast Saccharomyces pastorianus.

Two types of HO gene were cloned, sequenced and characterized from the bottom fermenting yeast Saccharomyces pastorianus. The HO gene present on the 1500 kb chromosome was designated Sc-HO (S. cerevisiae-type HO), because the nucleotide sequence of its promoter region and the open reading frame (ORF) was almost identical to that of the S. cerevisiae laboratory strain HO gene (Lab-HO). The other HO gene, designated Lg-HO (Lager-fermenting-yeast specific HO), showed 64% and 83% homology with the promoter and ORF of the Lab-HO at the nucleotide sequence level, respectively, and was located on the 1100 kb chromosome. Analysis of the 4 kb DNA fragment amplified from S. bayanus type strain indicated that the nucleotide sequence of S. bayanus-HO is almost identical to that of the Lg-HO. The SSB1 gene located downstream of the HO gene in S. cerevisiae was also found in the 3' distal region of the Sc-HO, Lg-HO and S. bayanus HO genes. These results showed that the genetic arrangement around the HO loci both of S. pastorianus and S. bayanus is identical to S. cerevisiae. Southern analysis has revealed that Saccharomyces sensu stricto contain four types of HO genes; S. paradoxus-type HO, the Sc-HO, the Lg-HO and S. uvarum-type HO genes. This HO gene diversity provides useful information for the classification of strains belonging to Saccharomyces sensu stricto. The S. pastorianus Sc-HO, Lg-HO and S. bayanus-HO Accession Nos in the DDBJ Nucleotide Sequence Database are AB027449, AB027450 and AB027451, respectively.

Prevalence of childhood and adolescent atopic dermatitis in a Japanese population: comparison with the disease frequency examined 20 years ago.

To ascertain the prevalence of childhood and adolescent atopic dermatitis in a Japanese population, we clinically observed the total body of 5 to 6-year-old children (994 cases), 7 to 9-year-old children (1,240 cases), 10 to 12-year-old children (1,152 cases), 13 to 15-year-old children (1,670 cases), and 16 to 18-year-old adolescents (2,159 cases). The examination was performed in the spring of 1994-96, when exacerbation of childhood and adolescent atopic dermatitis most frequently occurs in Japan. Atopic dermatitis was observed in 24% of the 5 to 6-year group, in 19% of the 7 to 9-year group, in 15% of the 10 to 12-year group, in 14% of the 13 to 15-year group, and in 11% of the 16 to 18-year group. The prevalence of atopic dermatitis in 9 to 12-year-old children was two times and in 18-year-old adolescents five times as high as in similar age groups examined 20 years ago.

Cloning and characterization of the cDNA for human airway trypsin-like protease.

Previously we isolated a trypsin-like enzyme designated human airway trypsin-like protease from the sputum of patients with chronic airway diseases. This paper describes the cDNA cloning, characterization of the primary protein structure deduced from the cDNA, and gene expression of this enzyme in various human tissues. We obtained an entire 1517-base pair sequence of cDNA with an open reading frame encoding a polypeptide with 418-amino acid residues. The polypeptide consisted of a 232-residue catalytic region and a 186-residue noncatalytic region with a hydrophobic putative transmembrane domain near the NH2 terminus. The polypeptide was suggested to be a type II integral membrane protein in which the COOH-terminal catalytic region is extracellular. Therefore, this protein is thought to be synthesized as a membrane-bound precursor and to mature to a soluble and active protease by limited proteolysis. It showed 29-38% identity in the sequence of the catalytic region with human hepsin, enteropeptidase, acrosin, and mast cell tryptase. The noncatalytic region had little similarity to other known proteins. In Northern blot analysis a transcript of 1.9 kilobases was detectable most prominently in the trachea among 17 human tissues examined.

Ultraviolet light is an environmental factor aggravating facial lesions of adult atopic dermatitis.

An increasing number of adult patients with atopic dermatitis (AD) in Japan are distressed by persistent eczematous lesions of the face (so-called atopic red face). Phototests were carried out in 28 patients with the atopic red face to test a possibility that ultraviolet (UV) light could be an aggravating factor. Contact and photocontact dermatitis had been ruled out by repeated patch and photopatch tests. All of the patients had a normal response to a screening dose of UVA (10 J/sq cm) and a normal minimal erythema dose (MED) of UVB. Ten of these patients, however, showed an abnormal papular response to a single or 3-times consecutive UVB radiation above the MED (90 mJ/sq cm).