Search

Melanin plays an important role in the protection of the skin from ultraviolet irradiation. However, excessive melanin deposition leads to hyperpigmentation and freckles, which are recognized as skin problems, and signs of aging. Tyrosinase, a copper-containing protein, is the rate-limiting enzyme in melanin biosynthesis and first catalyzes the hydroxylation of l-tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and the further oxidization to dopaquinone. To assist the proper regulation of melanin production, we screened compounds and found that 5,6,7,8-tetrahydro-4H-furo[3,2-c]azepine-4-thione (T4FAT), a thioamide derivative, inhibited melanogenesis in B16F10 mouse melanoma cells. T4FAT was not toxic to cells and was stable in water; in addition, it inhibited the activity of tyrosinase derived from mushroom and B16F10 cells in a non-competitive manner. T4FAT downregulated tyrosinase protein expression in B16F10 cells without affecting mRNA expression. As copper binding to the tyrosinase protein is required for both enzymatic activity, correct folding, and maturation, we examined the metal-chelating activities of T4FAT. Equimolar amount of T4FAT resulted in almost complete chelation of copper ions. The thioamide group of T4FAT is essential for copper chelation and tyrosinase inhibition, which subsequently resulted in melanogenesis inhibition in B16F10 cells. Although T4FAT has similar in vitro properties to kojic acid, which is also a copper chelator and approved as a component of cosmetic formulations, T4FAT inhibited melanogenesis in B16F10 cells 30 times more efficiently than kojic acid. These results suggested that T4FAT, a novel copper chelator, may be helpful for the development of new cosmetics for skin whitening., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Senescence marker protein-30 (SMP30) plays an important role in intracellular Ca(2+) homeostasis. The aim of the present study was to investigate the effects of estrogens on liver apoptotic damage and changes in SMP30 expression induced by a high saturated fatty acid diet (HSFD). Ovariectomized mice (OVX) and sham-operated mice (SHAM) were randomly divided into five groups: SHAM fed a normal diet (SHAM/ND), SHAM fed HSFD (SHAM/HSFD), OVX fed ND (OVX/ND), OVX fed HSFD (OVX/HSFD) and OVX fed HSFD with 17β-estradiol (E2) supplementation using an implanted slow-release pellet (OVX/HSFD+E2). After 8 weeks, markers of endoplasmic reticulum (ER) stress and apoptosis, and levels of tumor necrosis factor-α (TNFα and SMP30 expression were investigated. Compared with SHAM/ND, OVX/HSFD mice showed significantly increased spliced X-box protein-1 (s-XBP1), phosphorylated eukaryotic initiation factor-2α (p-eIF2α), glucose-regulated protein 78 (GPR78), C/EBP homologous protein (CHOP), cytosolic cytochrome c, caspase-3 activity, and TNFα, and significantly decreased SMP30. These differences in OVX/HSFD mice were restored to the levels of SHAM/ND mice by E2 supplementation. These results suggest that E2 supplementation attenuates HSFD-induced liver apoptotic death in ovariectomized mice by up-regulating SMP30., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet beta-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in beta-cells. These mice exhibited abnormal islet morphology with reduced beta-cell proliferation after 1 week of age and glucose intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.

Gene trapping is a powerful method for identifying novel genes and for analyzing their functions. It is, however, difficult to select trapped genes on the basis of their function. To identify genes regulated by transcription factors that are important in the mesodermal formation, we selected trapped ES clones by infection of adenoviral vectors expressing Pax1, Brachyury, and Foxa2. Among 366 trapped genes, seven seemed to be controlled by these transcription factors in the first screening. The trapped genes were identified by 5' RACE, and a Northern blotting revealed that expressions of three trapped genes were regulated by these transcription factors. Expression patterns of Cx43 and HP1gamma implicated their functional relationships to Foxa2 in the formation of the notochord and the neural tube. Furthermore, Wtap mutant mice derived from the trapped clone showed defects in the mesendoderm formation. Our results indicate that trapped ES clones could be selected effectively using transcription factors.

Variant Creutzfeldt-Jakob disease (vCJD) appears to be caused by infection with the bovine spongiform encephalopathy (BSE) agent. To date, all patients with vCJD are homozygous for methionine at codon 129 of the PrP gene. To investigate the relationship between polymorphism at codon 129 and susceptibility to BSE or vCJD prions, we performed splenic follicular dendritic cell assay with humanized knock-in mice through peripheral infection. All humanized knock-in mice showed little or no susceptibility to BSE prions. Only the subset of humanized knock-in mice with codon 129 Met/Met genotype showed weak susceptibility by Western blotting. Surprisingly, we succeeded in the transmission of vCJD prions to humanized knock-in mice not only with codon 129 Met/Met but also with codon 129 Met/Val. Humanized knock-in mice with codon 129 Val/Val were not susceptible. The results suggest that human heterozygotes at codon 129 are also at risk for secondary infection with vCJD.

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, is important in embryonic stem (ES) cells during neurogenesis. In that study, we also observed the altered expression of mesodermal marker genes, which indicated that JSAP1 is involved in the differentiation of mesodermal lineages. Here, we investigated the function of JSAP1 in cardiomyocyte development using JSAP1-null ES cells, and found that cardiomyogenesis was impaired in the JSAP1-null mutant. The JSAP1 deficiency resulted in lower gene expression of the cardiac transcription factor Nkx2.5 and contractile proteins. In contrast, the mutant showed a significantly higher expression of mesoderm-related markers other than those of the cardiomyocyte lineage. Together, these results suggest that JSAP1 may be important for the differentiation of the mesodermal lineages, functioning as a positive factor for cardiomyocyte differentiation, and as an inhibitory factor for differentiation into other lineages.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Transcription factor STAT3 is essential for the self-renewal of ES cells. In this study, we searched for downstream molecules of STAT3 in ES cells. Using DNA chip analysis, we obtained zinc finger protein (Zfp)-57. The expression of Zfp-57 was restricted to undifferentiated ES cells and activation of STAT3 led to expression of Zfp-57. We also found that forced expression of a dominant-negative mutant of STAT3 or repression of Oct-3/4 expression led to down-regulation of Zfp-57. Targeted disruption of Zfp-57 resulted in no gross phenotypical defects, including expression of undifferentiated-state-specific genes. These data suggest that Zfp-57 is a downstream molecule of STAT3 and Oct-3/4 in ES cells, although dispensable for their self-renewal.

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for fibroblasts, chondrocytes, and vascular endothelial cells. In the present study, we established transgenic (Tg) mice that overproduce CTGF/Hcs24 under the control of mouse type XI collagen promoter. Tg mice could develop and their embryonic and neonatal growth occurred normally. But they showed dwarfism within a few months of birth. X-ray analysis revealed that their bone density was decreased compared with normal mice. The femurs in the hindlimbs in particular showed an apparent low density. These results indicated that overexpression of CTGF/Hcs24 affects certain steps of endochondral ossification. In addition, the testes were much smaller than normal and fertility was affected in Tg mice, indicating that CTGF/Hcs24 may also regulate the embryonic development of the testis., (Copyright 2001 Academic Press.)

TAG-1 is a neural recognition molecule in the immunoglobulin superfamily that is predominantly expressed in the developing brain. Several lines of evidence suggest that TAG-1 is involved in the outgrowth, guidance, and fasciculation of neurites. To directly assess the function of TAG-1 in vivo, we have generated mice with a deletion in the gene encoding TAG-1 using homologous recombination in embryonic stem cells. Gross morphological analysis of the cerebellum, the spinal cord, and the hippocampus appeared normal in TAG-1-deficient mice. However, TAG-1 (-/-) mice showed the upregulation of the adenosine A1 receptors determined by [(3)H]cyclopentyl-1,3-dipropylxanthine in the hippocampus, and their greater sensitivity to convulsant stimuli than that in TAG-1 (+/+) mice. We suspect that the subtle changes in neural plasticity induced by TAG-1 deficiency during development cause the selective vulnerability of specific brain regions and the epileptogenicity in TAG-1 (-/-) mice.

O- and N-glycans included in erythrocyte membrane glycoproteins from beta1,4-galactosyltransferase I (GalT-I) knockout mice were analyzed to examine how this enzyme deficiency affects glycosylation of proteins in erythroid cells. The results indicated that greater than 80% of core 2 O-glycans from GalT-I-/- mice are not galactosylated by beta1,4 linkage, resulting in the expression of Neu5Acalpha2 --> 3Galbeta1 --> 3(GlcNAcbeta1 --> 6)GalNAc, while core 2 O-glycans from GalT-I+/+ mice are fully galactosylated and occur as Neu5Acalpha2 --> 3Galbeta1 --> 3(Neu5Acalpha2 --> 3Galbeta1 --> 4GlcNAcbeta1 --> 6)GalNAc. On the other hand, beta1, 4-galactosylation of N-glycans of the mutant was approximately 60% that of the wild type. Thus, it is suggested that GalT-I is predominantly responsible for beta1,4-galactosylation of the core 2 O-glycan branch in erythroid cells., (Copyright 1999 Academic Press.)

8-Hydroxyguanine (8-OH-G) is the site of a frequent mutagenic lesion of DNA, produced by oxidative damage. MutM of E. coli and OGG1 of Saccharomyces cervisiae are known to possess 8-OH-G glycosylase activity and apurinic (AP) site lyase activity to repair 8-OH-G lesions. Recently, cDNA clones of human OGG1 homologues (hMMH) of four isoforms (type 1a, type 1b, type 1c, and type 2) were isolated. However, it is unknown whether expression of endogenous hMMH proteins actually occurs in mammalian cells. Here using hMMH type 1a-specific antibody and cells overexpressing tag-fused hMMH type 1a, we show the expression of hMMH type 1a protein in many types of human cells and show that endogenous hMMH type 1a protein has 8-OH-G glycosylase/AP lyase activity. Furthermore, we show that upon depletion of hMMH type 1a protein in a whole cell extract by its antibody, most of the AP lyase activity is lost, indicating that hMMH type 1a protein is a major enzyme for repair of 8-OH-G lesions in human cells., (Copyright 1999 Academic Press.)

The presence of receptors specific for connective tissue growth factor (CTGF) was demonstrated on a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8. The binding of 125I-labeled recombinant CTGF to HCS-2/8 cells was inhibited by unlabeled CTGF but not by PDGF-BB or bFGF. Scatchard analysis revealed the presence of two classes of binding sites with Kd values of 18.6 and 259 nM on cells. A cross-linking study revealed the formation of 125I-CTGF-receptor complex with an apparent molecular weight of 280 kDa. The 125I-CTGF-receptor complex disappeared almost completely on the addition of unlabeled CTGF but not PDGF-BB or bFGF. In addition, the 125I-CTGF-receptor complex was immunoprecipitated with anti-CTGF antiserum but not with anti-PDGF receptor antiserum. These findings suggest that CTGF directly binds to specific receptor molecules on HCS-2/8 cells.

Polysialic acid and HNK-1 carbohydrate are expressed on Gal beta 1-->4GlcNAc outer chains of N-linked sugar chain of neural cell recognition molecules at certain developmental stages and involved in neural tissue formation. Targeted inactivation of the mouse beta-1,4-galactosyltransferase (beta-1,4-GalT) gene resulted in short life of the mice which supposedly do not have such carbohydrate antigens but have no defects in neural tissue formation. Analysis of the mutant mouse brain glycoproteins revealed that polysialic acid and HNK-1 carbohydrate are normally expressed in an age-dependent manner. In support of this, protein bands reacted with Ricinus communis agglutinin-I, which interacts with oligosaccharides terminated with the Gal beta 1-->4GlcNAc group, and beta-1,4-GalT activity toward GlcNAc beta-S-pNP were detected in the mutant mouse brain, indicating that brain contains another functional beta-1,4-GalT important for the expression of the carbohydrate antigens.

It is unclear whether the abnormal relaxation seen in diabetes is due to decreased levels of nitric oxide (NO) and how eicosapentaenoic acid (EPA, C20:5 omega 3) affects the endothelial production of NO. We investigated the effects of EPA ethyl ester (EPA-E) and elevated glucose on NO production by human endothelial cells (HUE). EPA-E (0.3 mM) significantly enhanced [NO2] production and the intracellular concentration of free Ca2+ within 3 min after EPA-E was added to the cultures. High levels of glucose (27.5 mM) significantly increased endothelial glucose, sorbitol and fructose, and inhibited [NO2-] production. However, EPA-E (0.3 mM) prevented the inhibition of [NO2-] production due to the activation of the Ca(2+)-calmodulin system of NO synthase. EPA-E decreased the glucose-mediated inhibition of NO production by HUE. These results suggest this agent might ameliorate endothelial dysfunction associated with diabetes.

Rho proteins have been implicated in a variety of cytoskeletal functions, but it is unclear how Rho proteins regulate these cellular functions and how Rho proteins are regulated. In this study, we cloned the rhoB gene from the mouse genome and characterized its promoter region. The predicted amino acid sequence was identical to that encoded by the human rhoB gene. A site for initiation of transcription was found at position -376 relative to the site for initiation of translation. Deletion analysis of the 5'-flanking region of the rhoB gene revealed that the minimum region of the promoter was located between positions -507 and -376. Northern blotting analysis showed that the expression of the mouse rhoB gene was induced by serum, suggesting that expression of the rhoB gene might be controlled by some signal-responsive element(s).

Activin is a member of the transforming growth factor beta (TGF-beta) and possesses various activities in cellular control phenomena. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus activin receptor cDNA from Xenopus tadpole cDNA library and examined the expression of the Xenopus activin receptor gene during the course of early embryonic development. The Xenopus activin receptor has an 87% homology at the level of deduced amino acid sequence with the mouse activin receptor, and using the cDNA obtained, three bands of mRNA with different lengths were detected in Xenopus embryos throughout early embryogenesis. We synthesized activin receptor mRNA in vitro and tested the effect of the injection of the mRNA into Xenopus fertilized eggs on subsequent development. When the synthetic mRNA was injected into uncleaved fertilized eggs, embryos with reduced trunk structure were formed. However, when the mRNA was injected into the ventral blastomeres at the 16-cell stage, embryos with a secondary body axis were formed. These results indicate the importance of the function of activin receptor in the regulatory mechanism for body axis formation.

Follistatin is a specific activin-binding protein and is supposed to control activin functions. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus follistatin cDNA from Xenopus ovary cDNA library and studied the expression of Xenopus follistatin gene during the course of early embryonic development. The Xenopus follistatin has an 84% homology at the level of deduced amino acid sequence with human and porcine follistatin. Its 3.5 kb mRNA is first expressed at the gastrula stage, when the expression of activin mRNA becomes first detectable, and increased thereafter. Another species of 2 kb mRNA become detectable from early neurula and also increased dramatically in tadpole. These results suggest that the follistatin acts also as a regulator of activin in inductive interactions during amphibian embryonic development.

To define the structure-function relationship, we have made a number of mutants of human granulocyte colony-stimulating factor (hG-CSF) by in vitro mutagenesis. The results indicate that most of the mutations located in the internal and C-terminal regions of the molecule abolished the activity, whereas the mutants without N-terminal 4, 5, 7, or 11 amino acids retained the activity. N-terminal amino acids were also altered by cassette mutagenesis using a synthetic oligonucleotide mixture. Among them, KW2228, in which Thr-1, Leu-3, Gly-4, Pro-5 and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg and Ser, showed more potent granulopoietic activity than that of intact hG-CSF both in vitro and in vivo.