[Show abstract][Hide abstract] ABSTRACT: Diabetes mellitus disrupts wound repair and leads to the development of chronic wounds, likely due to impaired angiogenesis. We previously demonstrated that human proinsulin C-peptide can protect against vasculopathy in diabetes; however, its role in impaired wound healing in diabetes has not been studied. We investigated the potential roles of C-peptide in protecting against impaired wound healing by inducing angiogenesis using streptozotocin-induced diabetic mice and human umbilical vein endothelial cells. Diabetes delayed wound healing in mouse skin and C-peptide supplement using osmotic pumps significantly increased the rate of skin wound closure in diabetic mice. Furthermore, C-peptide induced endothelial cell migration and tube formation in dose-dependent manners, with maximal effect at 0.5 nM. These effects were mediated through activation of extracellular signal-related kinase 1/2 and Akt, as well as nitric oxide formation. C-peptide enhanced angiogenesis in vivo was demonstrated by immunohistochemistry and Matrigel plug assays. Our findings highlight an angiogenic role of C-peptide and its ability to protect against impaired wound healing, which may have significant implications in reparative and therapeutic angiogenesis in diabetes. Thus, C-peptide replacement is a promising therapy for impaired angiogenesis and delayed wound healing in diabetes.Journal of Investigative Dermatology accepted article preview online, 09 July 2014; doi:10.1038/jid.2014.285.
[Show abstract][Hide abstract] ABSTRACT: Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed 'cis-silenced' (or 'occluded') genes, which remain silent even in the presence of trans-acting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes (or 'occludome' map) in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease.
Genome Research 12/2013; 24(2). DOI:10.1101/gr.143891.112 · 14.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aims:
Human C-peptide has a beneficial effect on the prevention of diabetic neuropathy, nephropathy, and vascular complications; however, its role in protection against increased vascular permeability in diabetes remains unclear. Our purpose was to explore the potential protective role of C-peptide against microvascular permeability mediated by vascular endothelial growth factor (VEGF)-induced reactive oxygen species (ROS) generation in diabetes.
Methods and results:
Generation of intracellular ROS, real-time changes in intracellular Ca(2+), ROS-dependent stress fibre formation, and the disassembly of the adherens junctions were studied by a confocal microscopy in human umbilical vein endothelial cells (HUVECs). VEGF-induced vascular leakage was investigated in the skin of diabetic mice using a Miles vascular permeability assay. Microvascular leakage in the retina of streptozotocin diabetic mice was investigated using a confocal microscopy after left ventricle injection of fluorescein isothiocyanate (FITC)-dextran. C-peptide inhibited the VEGF-induced ROS generation, stress fibre formation, disassembly of vascular endothelial cadherin, and endothelial permeability in HUVECs. Intradermal injection of C-peptide prevented VEGF-induced vascular leakage. Consistent with this, intravitreal injection of C-peptide prevented the extravasation of FITC-dextran in the retinas of diabetic mice, which was also prevented by anti-VEGF antibody and ROS scavengers in diabetic mice. Conclusions/interpretation C-peptide prevents VEGF-induced microvascular permeability by inhibiting ROS-mediated intracellular events in diabetic mice, suggesting that C-peptide replacement is a promising therapeutic strategy to prevent diabetic retinopathy.
Cardiovascular Research 10/2013; 101(1). DOI:10.1093/cvr/cvt238 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The intermediate filament protein, nestin, is a widely employed marker of multipotent neural stem cells (NSCs). Recent in vitro studies have implicated nestin in a number of cellular processes, but there is no data yet on its in vivo function. Here, we report the construction and functional characterization of Nestin knockout mice. We found that these mice show embryonic lethality, with neuroepithelium of the developing neural tube exhibiting significantly fewer NSCs and much higher levels of apoptosis. Consistent with this in vivo observation, NSC cultures derived from knockout embryos show dramatically reduced self-renewal ability that is associated with elevated apoptosis but no overt defects in cell proliferation or differentiation. Unexpectedly, nestin deficiency has no detectable effect on the integrity of the cytoskeleton. Furthermore, the knockout of Vimentin, which abolishes nestin's ability to polymerize into intermediate filaments in NSCs, does not lead to any apoptotic phenotype. These data demonstrate that nestin is important for the proper survival and self-renewal of NSCs, and that this function is surprisingly uncoupled from nestin's structural involvement in the cytoskeleton.
[Show abstract][Hide abstract] ABSTRACT: Atomic force microscopy (AFM) is an emerging technique for a variety of uses involving the analysis of cells. AFM is widely applied to obtain information about both cellular structural and subcellular events. In particular, a variety of investigations into membrane proteins and microfilaments were performed with AFM. Here, we introduce applications of AFM to molecular imaging of membrane proteins, and various approaches for observation and identification of intracellular microfilaments at the molecular level. These approaches can contribute to many applications of AFM in cell imaging.
Experimental and Molecular Medicine 09/2010; 42(9):597-605. DOI:10.3858/emm.2010.42.9.064 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: While adaptive immunity genes evolve rapidly under the influence of positive selection, innate immune system genes are known
to evolve slowly due to strong purifying selection. Among the sensors of the innate immune system, Toll-like receptors (TLRs)
are particularly important due to their ability to recognize and respond to pathogen-associated molecular patterns (PAMP),
such as lipopolysaccharides, peptidoglycans, and nucleic acids from bacteria or viruses. In the present study, we examine
the evolutionary process that has operated on the TLR7 family genes TLR7, TLR8, and TLR9. The results demonstrate that the
average Ka/Ks (the ratio between nonsynonymous and synonymous substitution rates) of each TLR family gene is far lower than
one regardless of estimating methods, supporting previous observations of strong purifying selection in this gene family.
Interestingly, however, analysis of Ka/Ks ratios along the coding regions of TLR7 family genes by sliding-window analysis
reveals a few narrow high peaks (Ka/Ks > 1). The most prominent peak corresponds to a specific region in the ectodomain, which
exists only in the TLR7 family, suggesting that this unique structure of the TLR7 family might have been a target of positive
selection in a variety of lineages. Furthermore, maximum likelihood model tests suggest that positive selection is the best
explanation for a certain fraction of the amino acid substitutions in the TLR9.
KeywordsTLR-Toll-like receptor-PAMP-Pathogen-associated molecular pattern-ECD-Ectodomain-LRR-Leucine-rich receptor-TIR-Toll-IL-1 receptor-MRCA-Most recent common ancestor-LRT-Likelihood ratio test
[Show abstract][Hide abstract] ABSTRACT: Transglutaminase 2 (TG2) is a multifunctional protein that can function as a transglutaminase, G protein, kinase, protein disulfide isomerase, and as an adaptor protein. These multiple biochemical activities of TG2 account for, at least in part, its involvement in a wide variety of cellular processes encompassing differentiation, cell death, inflammation, cell migration, and wound healing. The individual biochemical activities of TG2 are regulated by several cellular factors, including calcium, nucleotides, and redox potential, which vary depending on its subcellular location. Thus, the microenvironments of the subcellular compartments to which TG2 localizes, such as the cytosol, plasma membrane, nucleus, mitochondria, or extracellular space, are important determinants to switch on or off various TG2 biochemical activities. Furthermore, TG2 interacts with a distinct subset of proteins and/or substrates depending on its subcellular location. In this review, the biological functions and molecular interactions of TG2 will be discussed in the context of the unique environments of the subcellular compartments to which TG2 localizes.
[Show abstract][Hide abstract] ABSTRACT: While photodynamic therapy (PDT) has been recognized as a promising therapeutic modality for the treatment of various cancers and diseases, developments of effective photosensitizers are highly desired to improve the prospect for the use of PDT. In this study, we evaluated DH-II-24, a new photosensitizer, for antitumor PDT in vitro and in vivo. Loaded into human colorectal carcinoma cells (HCT116), DH-II-24 was primarily accumulated in mitochondria, lysosomes, and endoplasmic reticula. Administration of DH-II-24 followed by light exposure induced necrotic cell death in a dose-dependent manner, whereas DH-II-24 in the absence of light induced minimal cell death. In order to investigate the distribution and phamacokinetics of the photosensitizer in vivo, DH-II-24 was intravenously injected to female BALB/c nude mice. Fluorescence imaging in vivo showed that DH-II-24 was rapidly distributed across the entire body and then mostly eliminated at 24 h. Next, effectiveness of DH-II-24-mediated PDT was examined on colorectal carcinoma xenografts established subcutaneously in BALB/c nude mice. DH-II-24 (1 mg/kg, i.v. administration) followed by light exposure significantly suppressed growth of xenograft tumors, compared to light exposure or DH-II-24 alone. Histological examination revealed necrotic damage in PDT-treated tumors, concomitantly with severe damage of tumor vasculature. These results suggest that DH-II-24 is a potential photosensitizer of photodynamic therapy for cancer.
Cancer Science 08/2009; 100(12):2431-6. DOI:10.1111/j.1349-7006.2009.01326.x · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein evolutionary rates have been presumed to be mostly determined by the density of functionally important amino acids in a given protein. They have been shown to correlate with variables intuitively related to functional importance of proteins, such as protein dispensability and protein-protein interactions. Surprisingly, the best correlate of the evolutionary rates has turned out to be not the functional importance of a protein, but the expression level of the protein. Drummond and Wilke suggest that the dominant role of expression levels in slowing the rate of protein evolution stems from a selection pressure against mistranslation-induced protein misfolding. We will review current evidence for and against different hypotheses on determining evolutionary rates.
[Show abstract][Hide abstract] ABSTRACT: The RC2 antibody is widely used to label mouse radial glial cells in the developing central nervous system. While the antibody is known to recognize a 295-kDa intermediate filament proximal protein, the gene encoding the RC2 antigen remains to be identified. Here, we present evidences clearly demonstrating that Nestin encodes the RC2 antigen. First, the RC2 antigen and nestin have the same molecular weight and very similar tissue distribution. Second, genetic manipulations altering nestin expression also exert the same effect on the expression of the RC2 antigen. In particular, Nestin null mutation completely abolishes the RC2 immunoreactivity. Third, the expression of a truncated mouse nestin in Nestin-/- cells produces a small RC2 antigen whose size is the same to that of the truncated nestin. Furthermore, our data suggest that the RC2 antibody recognizes the C-terminal domain of nestin with unidentified posttranslational modification(s).
Biochemical and Biophysical Research Communications 04/2009; 382(3):588-92. DOI:10.1016/j.bbrc.2009.03.074 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An increasing body of evidence suggests that astrocytic gliomas of the central nervous system may be derived from gliotypic neural stem cells. To date, the study of these tumors, particularly the identification of originating cellular population(s), has been frustrated by technical difficulties in accessing the native niche of stem cells. To identify any hallmark signs of cancer in neural stem cells or their progeny, we cultured subventricular zone-derived tissue in a unique in vitro model that temporally and phenotypically recapitulates adult neurogenesis. Contrary to some reports, we found undifferentiated neural stem cells possess few characteristics, suggesting prototumorigenic potential. However, when induced to differentiate, neural stem cells give rise to intermediate progenitors that transiently exhibit multiple glioma characteristics, including aneuploidy, loss of growth-contact inhibition, alterations in cell cycle, and growth factor insensitivity. Further examination of progenitor populations revealed a subset of cells defined by the aberrant expression of (the pathological glioma marker) class III beta-tubulin that exhibit intrinsic parental properties of gliomas, including multilineage differentiation and continued proliferation in the absence of a complex cellular regulatory environment. As tumorigenic characteristics in progenitor cells normally disappear with the generation of mature progeny, this suggests that developmentally intermediate progenitor cells, rather than neural stem cells, may be the origin of so-called "stem cell-derived" tumors.
[Show abstract][Hide abstract] ABSTRACT: The characteristics of human disease genes were investigated through a comparative analysis with mouse mutant phenotype data. Mouse orthologs with mutations that resulted in discernible phenotypes were separated from mutations with no phenotypic defect, listing 'phenotype' and 'no phenotype' genes. First, we showed that phenotype genes are more likely to be disease genes compared to no phenotype genes. Phenotype genes were further divided into 'embryonic lethal', 'postnatal lethal', and 'non-lethal phenotype' groups. Interestingly, embryonic lethal genes, the most essential genes in mouse, were less likely to be disease genes than postnatal lethal genes. These findings indicate that some extremely essential genes are less likely to be disease genes, although human disease genes tend to display characteristics of essential genes. We also showed that, in lethal groups, non-disease genes tend to evolve slower than disease genes indicating a strong purifying selection on non-disease genes in this group. In addition, phenotype and no phenotype groups showed differing types of disease mutations. Disease genes in the no phenotype group displayed a higher frequency of regulatory mutations while those in the phenotype group had more frequent coding mutations, indicating that the types of disease mutations vary depending on gene essentiality. Furthermore, missense disease mutations in no phenotype genes were found to be more radical amino acid substitutions than those in phenotype genes.
[Show abstract][Hide abstract] ABSTRACT: The full potential of embryonic stem (ES) cells to generate precise cell lineages and complex tissues can be best realized
when they are differentiated in vivo—i.e. in developing blastocysts. Owing to various practical and ethical constraints, however, it is impossible to introduce
ES cells of certain species into blastocysts of the same species. One solution is to introduce ES cells into blastocysts of
a different species. However, it is not known whether ES cells can contribute extensively to chimerism when placed into blastocysts
of a distantly related species. Here, we address this question using two divergent species, Apodemus sylvaticus and Mus musculus, whose genome sequence differs by ∼18% from each other. Despite this considerable evolutionary distance, injection of Apodemus ES cells into Mus blastocysts led to viable chimeras bearing extensive Apodemus contributions to all major organs, including the germline, with Apodemus contribution reaching ∼40% in some tissues. Immunostaining showed that Apodemus ES cells have differentiated into a wide range of cell types in the chimeras. Our results thus provide a proof of principle
for the feasibility of differentiating ES cells into a wide range of cell types and perhaps even complex tissues by allowing
them to develop in vivo in an evolutionarily divergent host—a strategy that may have important applications in research and therapy. Furthermore,
our study demonstrates that mammalian evolution can proceed at two starkly contrasting levels: significant divergence in genome
and proteome sequence, yet striking conservation in developmental programs.
Human Molecular Genetics 02/2008; 17(1):27-37. DOI:10.1093/hmg/ddm282 · 6.39 Impact Factor