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1.
Biomed Pharmacother ; 150: 112996, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35462338

ABSTRACT

CXCL12 and its receptors, which are highly expressed in the skin, are associated with various cutaneous diseases, including androgenic alopecia. However, their expression and role during the hair cycle are unknown. This study aims to investigate the expression of CXCL12 and its receptor, CXCR4, in the vicinity of hair follicles and their effect on hair growth. CXCL12 was highly expressed in dermal fibroblasts (DFs) and its level was elevated throughout the catagen and telogen phases of the hair cycle. CXCR4 is expressed in the dermal papilla (DP) and outer root sheath (ORS). In hair organ culture, hair loss was induced by recombinant CXCL12 therapy, which delayed the telogen-to-anagen transition and decreased hair length. In contrast, the suppression of CXCL12 using a neutralizing antibody and siRNA triggered the telogen-to-anagen transition and increased hair length in hair organ culture. Neutralization of CXCR7, one of the two receptors for CXCL12, only slightly affected hair growth. However, inhibition of CXCR4, the other receptor for CXCL12, increased hair growth to a considerable extent. In addition, in hair organ culture, the conditioned medium from DFs with CXCL12 siRNA considerably increased the hair length and induced proliferation of DP and ORS cells. CXCL12, through CXCR4 activation, increased STAT3 and STAT5 phosphorylation in DP and ORS cells. In contrast, blocking CXCL12 and CXCR4 decreased the phosphorylation of STAT3 and STAT5. In summary, these findings suggest that CXCL12 inhibits hair growth via the CXCR4/STAT signaling pathway and that CXCL12/CXCR4 pathway inhibitors are a promising treatment option for hair growth.


Subject(s)
Chemokine CXCL12 , Hair , Receptors, CXCR4 , Alopecia/metabolism , Chemokine CXCL12/metabolism , Hair/growth & development , Hair/metabolism , Hair Follicle/growth & development , Hair Follicle/metabolism , Humans , RNA, Small Interfering/metabolism , Receptors, CXCR4/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction
2.
Cell Rep ; 37(6): 109982, 2021 11 09.
Article in English | MEDLINE | ID: mdl-34758315

ABSTRACT

Early blastomeres of mouse preimplantation embryos exhibit bi-potential cell fate, capable of generating both embryonic and extra-embryonic lineages in blastocysts. Here we identify three major two-cell-stage (2C)-specific endogenous retroviruses (ERVs) as the molecular hallmark of this bi-potential plasticity. Using the long terminal repeats (LTRs) of all three 2C-specific ERVs, we identify Krüppel-like factor 5 (Klf5) as their major upstream regulator. Klf5 is essential for bi-potential cell fate; a single Klf5-overexpressing embryonic stem cell (ESC) generates terminally differentiated embryonic and extra-embryonic lineages in chimeric embryos, and Klf5 directly induces inner cell mass (ICM) and trophectoderm (TE) specification genes. Intriguingly, Klf5 and Klf4 act redundantly during ICM specification, whereas Klf5 deficiency alone impairs TE specification. Klf5 is regulated by multiple 2C-specific transcription factors, particularly Dux, and the Dux/Klf5 axis is evolutionarily conserved. The 2C-specific transcription program converges on Klf5 to establish bi-potential cell fate, enabling a cell state with dual activation of ICM and TE genes.


Subject(s)
Blastocyst Inner Cell Mass/cytology , Blastocyst , Cell Lineage , Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental , Kruppel-Like Transcription Factors/metabolism , Trophoblasts/cytology , Animals , Blastocyst Inner Cell Mass/metabolism , Cell Differentiation , Embryonic Stem Cells/metabolism , Female , Kruppel-Like Transcription Factors/genetics , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , RNA-Seq , Transcription Factors/genetics , Transcription Factors/metabolism , Trophoblasts/metabolism
3.
Microbiol Resour Announc ; 9(37)2020 Sep 10.
Article in English | MEDLINE | ID: mdl-32912909

ABSTRACT

Gordonia ajoucoccus strain A2, isolated from crude oil-contaminated soils, synthesizes yellow keto-γ-carotene from various n-alkanes as the sole carbon source. Its complete genome sequence consists of a single circular chromosome (5,090,254 bp, 67.3% G+C content). Seven putative genes were identified supporting the proposed keto-γ-carotene pathway of G. ajoucoccus A2.

4.
Science ; 355(6325)2017 02 10.
Article in English | MEDLINE | ID: mdl-28082412

ABSTRACT

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) efficiently generate all embryonic cell lineages but rarely generate extraembryonic cell types. We found that microRNA miR-34a deficiency expands the developmental potential of mouse pluripotent stem cells, yielding both embryonic and extraembryonic lineages and strongly inducing MuERV-L (MERVL) endogenous retroviruses, similar to what is seen with features of totipotent two-cell blastomeres. miR-34a restricts the acquisition of expanded cell fate potential in pluripotent stem cells, and it represses MERVL expression through transcriptional regulation, at least in part by targeting the transcription factor Gata2. Our studies reveal a complex molecular network that defines and restricts pluripotent developmental potential in cultured ESCs and iPSCs.


Subject(s)
Cell Differentiation/genetics , Embryonic Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , MicroRNAs/physiology , Animals , Cell Lineage/genetics , Cells, Cultured , Endogenous Retroviruses/genetics , Endogenous Retroviruses/physiology , Female , GATA2 Transcription Factor/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Male , Metabolic Networks and Pathways , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Transcription, Genetic , Virus Activation/genetics
5.
Chem Commun (Camb) ; 51(30): 6659-62, 2015 Apr 18.
Article in English | MEDLINE | ID: mdl-25782606

ABSTRACT

Hierarchical AuZn dendrites revealed electrocatalytic properties towards the glucose oxidation and the four-electron O2 reduction. The micro fuel cell using AuZn electrodes generated a power density of 2.07 and 0.29 mW cm(-2) for glucose and human whole blood. The micro film fuel cell implanted into the rat brain produced ∼0.52 V continuously operating for more than 18 days.


Subject(s)
Bioelectric Energy Sources , Electrodes, Implanted , Glucose/chemistry , Gold/chemistry , Oxygen/chemistry , Zinc/chemistry , Animals , Electrochemistry , Humans , Rats , Rats, Wistar
6.
J Nanosci Nanotechnol ; 15(10): 7582-5, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26726376

ABSTRACT

We investigate the intrinsic electrical characteristics and source/drain parasitic resistance in p-type SnO TFTs fabricated using Ni electrodes based on the gated-four-probe method. Because of the relatively high work function and inexpensive price, Ni has been most frequently used as the source/drain electrode materials in p-type SnO TFTs. However, our experimental data shows that the width normalized parasitic resistances of SnO TFT with Ni electrodes are around one to three orders of magnitude higher than those in the representative n-type oxide TFT, amorphous indium- gallium-zinc oxide TFT, and are comparable with those in amorphous silicon TFTs with Mo electrodes. This result implies that the electrical performance of the short channel SnO TFT can be dominated by the source/drain parasitic resistances. The intrinsic field-effect mobility extracted without being influenced by source/drain parasitic resistance was ~2.0 cm2/Vs, which is around twice the extrinsic field-effect mobility obtained from the conventional transconductance method. The large contact resistance is believed to be mainly caused from the heterogeneous electronic energy-level mismatch between the SnO and Ni electrodes.

7.
Cell Transplant ; 24(8): 1571-84, 2015.
Article in English | MEDLINE | ID: mdl-25008576

ABSTRACT

The purpose of this study was to investigate the effects of diabetes on mesenchymal stem cells (MSCs) in terms of their angiogenic and therapeutic potential for repairing tissue ischemia. We culture-isolated MSCs from streptozotocin-induced diabetic rats (D-MSCs) and compared their proliferation, differentiation, and angiogenic effects with those from normal rats (N-MSCs). The angiogenic effects of MSCs were evaluated by real-time PCR, in vitro tube formation assay, and transplantation of the MSCs into a hindlimb ischemia model followed by laser Doppler perfusion imaging. The number of MSCs derived from diabetic rats was smaller, and their proliferation rate was slower than N-MSCs. Upon induction of differentiation, the osteogenic and angiogenic differentiation of D-MSCs were aberrant compared to N-MSCs. The expression of angiogenic factors was lower in D-MSCs than N-MSCs. D-MSCs cocultured with endothelial cells resulted in decreased tube formation compared to N-MSCs. D-MSCs were ineffective to improve hindlimb ischemia and showed lower capillary density and angiogenic gene expression in ischemic limbs than N-MSCs. D-MSCs have defective proliferation and angiogenic activities and are ineffective for repairing hindlimb ischemia. Newer measures are needed before MSCs can be employed as a source for autologous cell therapy.


Subject(s)
Ischemia/therapy , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Neovascularization, Physiologic , Animals , Blood Vessels/physiology , Capillaries/physiopathology , Cell Differentiation , Cell Proliferation , Coculture Techniques , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Disease Models, Animal , Endothelial Cells/cytology , Hindlimb/blood supply , Hindlimb/metabolism , Humans , Ischemia/pathology , Male , Mesenchymal Stem Cells/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rats
8.
Cell Cycle ; 11(11): 2063-72, 2012 Jun 01.
Article in English | MEDLINE | ID: mdl-22580472

ABSTRACT

The p53 pathway plays an essential role in tumor suppression, regulating multiple cellular processes coordinately to maintain genome integrity in both somatic cells and stem cells. Despite decades of research dedicated to p53 function in differentiated somatic cells, we are just starting to understand the complexity of the p53 pathway in the biology of pluripotent stem cells and tissue stem cells. Recent studies have demonstrated that p53 suppresses proliferation, promotes differentiation of embryonic stem (ES) cells and constitutes an important barrier to somatic reprogramming. In addition, emerging evidence reveals the role of the p53 network in the self-renewal, proliferation and genomic integrity of adult stem cells. Interestingly, non-coding RNAs, and microRNAs in particular, are integral components of the p53 network, regulating multiple p53-controlled biological processes to modulate the self-renewal and differentiation potential of a variety of stem cells. Thus, elucidation of the p53-miRNA axis in stem cell biology may generate profound insights into the mechanistic overlap between malignant transformation and stem cell biology.


Subject(s)
MicroRNAs/metabolism , Stem Cells/metabolism , Tumor Suppressor Protein p53/metabolism , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Cellular Reprogramming , Humans , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Stem Cells/cytology
9.
Nat Cell Biol ; 13(11): 1353-60, 2011 Oct 23.
Article in English | MEDLINE | ID: mdl-22020437

ABSTRACT

Somatic reprogramming induced by defined transcription factors is a low-efficiency process that is enhanced by p53 deficiency. So far, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation, indicating that additional p53 targets may regulate this process. Here, we demonstrate that miR-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. Mir34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of Mir34a promoted iPSC generation without compromising self-renewal or differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (also known as N-Myc). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three miR-34 miRNAs acted cooperatively in this process. Taken together, our findings identified miR-34 miRNAs as p53 targets that play an essential role in restraining somatic reprogramming.


Subject(s)
Cellular Reprogramming/genetics , Gene Expression Regulation, Developmental , Induced Pluripotent Stem Cells/metabolism , MicroRNAs/metabolism , Animals , Cell Differentiation , Cells, Cultured , Coculture Techniques , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Female , Genes, myc , Homeodomain Proteins/genetics , Kinetics , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Mice, Transgenic , MicroRNAs/genetics , Nanog Homeobox Protein , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , RNA Interference , SOXB1 Transcription Factors/genetics , Teratoma/genetics , Teratoma/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
10.
Circ Res ; 107(5): 602-14, 2010 Sep 03.
Article in English | MEDLINE | ID: mdl-20634489

ABSTRACT

RATIONALE: Bone marrow (BM) cells play an important role in physiological and therapeutic neovascularization. However, it remains unclear whether any specific uncultured BM cell populations have higher angiogenic and vasculogenic activities. Moreover, there has been controversy regarding the vasculogenic ability of BM cells. OBJECTIVE: Preliminary flow cytometric analysis showed that CD31, traditionally a marker for endothelial cells, is expressed in certain nonendothelial BM mononuclear cells in both human and mouse. Based on the conserved CD31 expression in the axis of hematopoietic stem/progenitor cells (HSC/HPCs) to endothelial cells, we further sought to determine the comprehensive vasculogenic and angiogenic characteristics of human and mouse BM-derived CD31(+) cells. METHODS AND RESULTS: Flow cytometric analysis demonstrated that all CD31(+) cells derived from BM were CD45(+) and expressed markers for both HSC/HPCs and endothelial cells. Comprehensive gene expression analyses revealed that BM-CD31(+) cells expressed higher levels of angiogenic genes than CD31(-) cells. Endothelial progenitor cells, as well as HSC/HPCs, were almost exclusively confined to the CD31(+) cell fraction, and culture of CD31(+) cells under defined conditions gave rise to endothelial cells. Finally, injection of CD31(+) cells into ischemic hindlimb repaired ischemia, increased expression of angiogenic and chemoattractive factors, and, in part, directly contributed to vasculogenesis, as demonstrated by both 3D confocal microscopy and flow cytometry. CONCLUSIONS: These data indicate that BM-CD31(+) cells represent highly angiogenic and vasculogenic cells and can be a novel and highly promising source of cells for cell therapy to treat ischemic cardiovascular diseases.


Subject(s)
Bone Marrow Cells/physiology , Bone Marrow Transplantation , Ischemia/surgery , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Angiogenic Proteins/metabolism , Animals , Biomarkers/metabolism , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cells, Cultured , Chemotactic Factors/metabolism , Disease Models, Animal , Flow Cytometry , Gene Expression Regulation , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Hindlimb , Humans , Immunophenotyping , Ischemia/immunology , Ischemia/metabolism , Ischemia/physiopathology , Leukocyte Common Antigens/metabolism , Mice , Mice, Inbred C57BL , Mice, Nude , Mice, Transgenic , Microscopy, Confocal , Neovascularization, Physiologic/genetics , Phenotype , Time Factors
11.
Stem Cells ; 27(7): 1686-96, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19544451

ABSTRACT

Bone marrow-derived mononuclear cells (BMNCs) have been shown to effectively treat ischemic cardiovascular diseases. Because diabetic neuropathy (DN) is causally associated with impaired angiogenesis and deficiency of angiogenic and neurotrophic factors in the nerves, we investigated whether DN can be ameliorated by local injection of BMNCs. Severe peripheral neuropathy, characterized by a significant decrease in the motor and sensory nerve conduction velocities (NCVs), developed 12 weeks after the induction of diabetes with streptozotocin in rats. The injection of BMNCs restored motor and sensory NCVs to normal levels and significantly improved vascular density and blood flow in diabetic nerves over 4 weeks. Fluorescent microscopic observation revealed that DiI-labeled BMNCs preferentially engrafted in sciatic nerves. Whole-mount fluorescent imaging and confocal microscopic evaluation demonstrated that many of the BMNCs localized following the course of the vasa nervorum in close proximity to blood vessels without incorporation into vasa nervorum as endothelial cells at a detectable level. Real-time reverse transcription-polymerase chain reaction analysis showed that the levels of angiogenic and neurotrophic factors were significantly increased in the nerves by BMNC injection. Local transplantation of BMNCs improved experimental DN by augmenting angiogenesis and increasing angiogenic and neurotrophic factors in peripheral nerves. These findings suggest that BMNC transplantation may represent a novel therapeutic option for treating DN.


Subject(s)
Bone Marrow Cells/cytology , Bone Marrow Transplantation/methods , Diabetic Neuropathies/pathology , Diabetic Neuropathies/therapy , Animals , Hemodynamics , Immunophenotyping , Male , Neovascularization, Physiologic , Rats , Rats, Inbred F344 , Reverse Transcriptase Polymerase Chain Reaction , Vasa Nervorum/metabolism , Vasa Nervorum/pathology
12.
Circulation ; 119(5): 699-708, 2009 Feb 10.
Article in English | MEDLINE | ID: mdl-19171856

ABSTRACT

BACKGROUND: Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence suggested that diabetic neuropathy is causally related to impaired angiogenesis and deficient growth factors. Accordingly, we investigated whether diabetic neuropathy could be reversed by local transplantation of EPCs. METHODS AND RESULTS: We found that motor and sensory nerve conduction velocities, blood flow, and capillary density were reduced in sciatic nerves of streptozotocin-induced diabetic mice but recovered to normal levels after hind-limb injection of bone marrow-derived EPCs. Injected EPCs were preferentially and durably engrafted in the sciatic nerves. A portion of engrafted EPCs were uniquely localized in close proximity to vasa nervorum, and a smaller portion of these EPCs were colocalized with endothelial cells. Multiple angiogenic and neurotrophic factors were significantly increased in the EPC-injected nerves. These dual angiogenic and neurotrophic effects of EPCs were confirmed by higher proliferation of Schwann cells and endothelial cells cultured in EPC-conditioned media. CONCLUSIONS: We demonstrate for the first time that bone marrow-derived EPCs could reverse various manifestations of diabetic neuropathy. These therapeutic effects were mediated by direct augmentation of neovascularization in peripheral nerves through long-term and preferential engraftment of EPCs in nerves and particularly vasa nervorum and their paracrine effects. These findings suggest that EPC transplantation could represent an innovative therapeutic option for treating diabetic neuropathy.


Subject(s)
Diabetes Mellitus, Experimental/complications , Diabetic Neuropathies/therapy , Endothelial Cells/cytology , Hematopoietic Stem Cell Transplantation , Sciatic Nerve/blood supply , Animals , Apoptosis , Bone Marrow Cells/cytology , Cell Division , Cells, Cultured , Diabetic Neuropathies/physiopathology , Hematopoietic Stem Cells/cytology , Laser-Doppler Flowmetry , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Neural Conduction , Schwann Cells/cytology , Sciatic Nerve/physiology
13.
Genes Dev ; 22(18): 2485-95, 2008 Sep 15.
Article in English | MEDLINE | ID: mdl-18794346

ABSTRACT

Axon formation is fundamental for brain development and function. TSC1 and TSC2 are two genes, mutations in which cause tuberous sclerosis complex (TSC), a disease characterized by tumor predisposition and neurological abnormalities including epilepsy, mental retardation, and autism. Here we show that Tsc1 and Tsc2 have critical functions in mammalian axon formation and growth. Overexpression of Tsc1/Tsc2 suppresses axon formation, whereas a lack of Tsc1 or Tsc2 function induces ectopic axons in vitro and in the mouse brain. Tsc2 is phosphorylated and inhibited in the axon but not dendrites. Inactivation of Tsc1/Tsc2 promotes axonal growth, at least in part, via up-regulation of neuronal polarity SAD kinase, which is also elevated in cortical tubers of a TSC patient. Our results reveal key roles of TSC1/TSC2 in neuronal polarity, suggest a common pathway regulating polarization/growth in neurons and cell size in other tissues, and have implications for the understanding of the pathogenesis of TSC and associated neurological disorders and for axonal regeneration.


Subject(s)
Axons , Tumor Suppressor Proteins/physiology , Animals , Base Sequence , Cells, Cultured , DNA Primers , Electroporation , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mutation , Protein Serine-Threonine Kinases/metabolism , Rats , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/genetics
14.
J Exp Med ; 204(13): 3257-69, 2007 Dec 24.
Article in English | MEDLINE | ID: mdl-18070934

ABSTRACT

Noncellular differentiation effects have emerged as important mechanisms mediating therapeutic effects of stem or progenitor cell transplantation. Here, we investigated the expression patterns and sources of humoral factors and their regional and systemic biological effects after bone marrow (BM)-derived endothelial progenitor cell (EPC) transplantation into ischemic myocardium. Although most of the transplanted EPCs disappeared within a week, up-regulation of multiple humoral factors was sustained for longer than two weeks, which correlated well with the recovery of cardiac function. To determine the source of the humoral factors, we injected human EPCs into immunodeficient mice. Whereas the expression of human EPC (donor)-derived cytokines rapidly decreased to a nondetectable level within a week, up-regulation of mouse (recipient)-derived cytokines, including factors that could mobilize BM cells, was sustained. Histologically, we observed higher capillary density, a higher proliferation of myocardial cells, a lower cardiomyocyte apoptosis, and reduced infarct size. Furthermore, after EPC transplantation, BM-derived stem or progenitor cells were increased in the peripheral circulation and incorporated into the site of neovascularization and myocardial repair. These data indicate that myocardial EPC transplantation induces humoral effects, which are sustained by host tissues and play a crucial role in repairing myocardial injury.


Subject(s)
Antibody Formation , Cell Transplantation/methods , Endothelial Cells/cytology , Heart/physiology , Myocardial Ischemia/pathology , Stem Cells/cytology , Animals , Apoptosis , Cell Proliferation , Humans , Mice , Mice, Inbred C57BL , Mice, SCID , Models, Biological , Myocardial Infarction/pathology , Stem Cells/metabolism
15.
Pflugers Arch ; 455(2): 261-71, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17503070

ABSTRACT

Calcium (Ca2+) pathways are important in cell volume regulation in many cells, but its role in volume regulatory processes in cholangiocytes is unclear. Thus, we have investigated the role of Ca2+ in regulatory volume decrease (RVD) in cholangiocytes using freshly isolated bile duct cell clusters (BDCCs) from normal mouse. No significant increase in [Ca2+]i was observed during RVD, while ionomycin and ATP showed significant increases. Confocal imaging also showed no significant changes in the levels or distributions of intracellular Ca2+ during RVD. Cell volume study by quantitative videomicroscopy indicated that removal and chelation of extracellular Ca2+ by ethylene glycol-bis (beta-aminoethyl ether)-N,N,N-tetraacetic acid (EGTA) or administration of nifedipine did not affect RVD but verapamil significantly inhibited the RVD. Moreover, Ca2+ agonists or inhibitors of Ca2+ release from intracellular stores had no significant effect on RVD. However, 1,2-bis (2-aminophenoxy) ethane-N,N,N'N'-tetraacetic acid-AM (BAPTA-AM) showed significant decreases in [Ca2+]i and significantly inhibited RVD, which was reversed with coadministration of valinomycin, suggesting that BAPTA-AM-induced inhibition is due to potassium conductance or other cellular processes requiring permissive [Ca2+](i. These findings indicate that an increase in [Ca2+]i or extracellular Ca2+ is not required for RVD but Ca2+ has a permissive role in RVD of mouse cholangiocytes.


Subject(s)
Bile Ducts, Intrahepatic/cytology , Bile Ducts, Intrahepatic/metabolism , Calcium/metabolism , Cell Size , Animals , Bile Ducts, Intrahepatic/drug effects , Calcium Channel Blockers/pharmacology , Cell Size/drug effects , Cells, Cultured , Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Male , Mice , Mice, Inbred C57BL , Nifedipine/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Verapamil/pharmacology
16.
J Biotechnol ; 127(2): 300-9, 2007 Jan 01.
Article in English | MEDLINE | ID: mdl-16919348

ABSTRACT

Improvement of thermal stability of the Bacillus stearthermophilus No. 236 endo-beta-1,4-xylanase (XynA) was tried by engineering a de novo designed disulfide bridge. Disulfide design was performed firstly using the disulfide bond design program (Disulfide by Designtrade mark) to identify residue pairs having the favorable geometric characteristics for disulfide formation. Subsequently, the selected 25 amino acid pairs were filtered with the evolutionarily conserved Cys residues identified by alignment of 34 family 11 mesophilic and thermophilic xylanases, and also by doing inspection of the molecular model of the xylanases. Only one pair (Ser100 and Asn150) was finally chosen, and the respective amino acids were substituted with cysteine residues. The newly designed disulfide bridge increased thermostability of the XynA about 5 degrees C. This improved thermal stability was supported by the increase in the energy barrier for inactivation. As expected, the mutant XynA SNC demonstrated its better use in the hydrolysis of xylan at substantially higher temperatures than permitted by its native counterpart. The mutation had little influence on the catalytic efficiency and other functional properties of the XynA. In conclusion, it is evident that the strategically placed disulfide bridge has made the XynA be more effective in resisting thermal inactivation.


Subject(s)
Disulfides/metabolism , Endo-1,4-beta Xylanases/chemistry , Endo-1,4-beta Xylanases/metabolism , Geobacillus stearothermophilus/enzymology , Hot Temperature , Protein Engineering/methods , Amino Acid Sequence , Disulfides/chemistry , Enzyme Stability , Hydrolysis , Kinetics , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Denaturation , Protein Structure, Secondary , Sulfhydryl Compounds/metabolism , Thermodynamics , Titrimetry , Xylans/metabolism
17.
Biochem Biophys Res Commun ; 351(1): 153-8, 2006 Dec 08.
Article in English | MEDLINE | ID: mdl-17055461

ABSTRACT

XaiF, a novel 32kDa protein encoded by the ORF located in the immediate downstream of the xynA gene of Bacillus stearothermophilus No. 236, was identified to be the xylanase-specific trans-activator. In this study, the positive effect of XaiF was confirmed to be xylanase-specific, and the results from Northern blot and in vitro transcription assays showed that the XaiF increased the xynA transcripts at post-transcriptional step. Moreover, analysis of the mRNA decay rate led to the assertion that the XaiF functions to stabilize the xynA mRNA. Intriguingly, in vitro RNA-protein binding assay and analysis using gst-xynA 3'-UTR chimeric gene constructs demonstrated that the XaiF stabilizes xynA mRNA by direct binding onto the 3'-UTR of the mRNA.


Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial/physiology , Geobacillus stearothermophilus/physiology , RNA Cap-Binding Proteins/genetics , RNA Processing, Post-Transcriptional/physiology
18.
Biosci Biotechnol Biochem ; 70(5): 1089-101, 2006 May.
Article in English | MEDLINE | ID: mdl-16717408

ABSTRACT

The Bacillus stearothermophilus no. 236 gene encoding the bifunctional enzyme HprK/P, the key regulator of carbon catabolite repression/activation (CCR/CCA) in most Gram-positive bacteria, was cloned and the (His)(6)-tagged gene product was characterized in detail. The nucleotide sequence of the hprK/P gene corresponded to an open reading frame of 951 bp that encoded a polypeptide of 316 amino acid residues with a calculated molecular mass of 35,458 Da. The deduced amino acid sequence of the B. stearothermophilus no. 236 HprK/P showed 64.5% identity with the B. subtilis enzyme, allowing us to identify two highly conserved motifs, the nucleotide binding P-loop (Walker motif A) and the HprK/P family signature sequence in the C-terminal half of the protein. Furthermore, complementation experiments showed that the cloned hprK/P gene product was functionally active in the B. subtilis cells. The purified (His)(6)-tagged B. stearothermophilus no. 236 HprK/P migrated on SDS-PAGE gel as a single species with a molecular mass of about 36 kDa, and behaved in gel filtration like a hexameric protein. The recombinant protein catalyzes the pyrophosphate (PPi)-dependent (highest activity at pH 7.0 and 40 degrees C) as well as the ATP-dependent phosphorylation of Ser46 in HPr (maximum activity at pH 8.0 and 45 degrees C). It also catalyzes the inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr, optimally at pH 6.5 and 40 degrees C. BIAcore surface resonance analysis confirmed that a divalent cation, preferentially Mg(2+), was an indispensable cofactor for the three activities of the HprK/P. Fructose-1,6-bisphosphate (FBP) was observed to stimulate ATP-dependent kinase activity, while inorganic phosophate (Pi) inhibited ATP-dependent kinase activity. Mutations in the Walker motif A simultaneously abolished both types of kinase and phosphorylase activities. On the other hand, the conserved signature residues were confirmed to be involved in the PPi-dependent kinase and phosphorylase reactions.


Subject(s)
Bacterial Proteins/chemistry , Geobacillus stearothermophilus/enzymology , Protein Serine-Threonine Kinases/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Base Sequence , Cations, Divalent/chemistry , Cloning, Molecular , Fructosediphosphates/chemistry , Geobacillus stearothermophilus/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , Molecular Weight , Phosphates/chemistry , Phosphorylases/chemistry , Phosphorylases/genetics , Phosphorylases/isolation & purification , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/isolation & purification , Sequence Analysis, Protein
19.
Biotechnol Lett ; 27(13): 949-53, 2005 Jul.
Article in English | MEDLINE | ID: mdl-16091891

ABSTRACT

A glass bead biofilm reactor was operated using H2 as an electron donor to remove nitrate at 150 mg NO3-N l(-1) to below detection level. The microbial community in the glass beads biofilm reactor was investigated by using denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. In DGGE analysis of the biofilm, five bands were dominant and indicated the presence of eight beta-proteobacteria, one gamma-proteobacteria and twelve clostridia. An unculturable Hydrogenophaga sp., which is a new genus of hydrogen-oxidizing bacterium was dominant in microbial community of the biofilm reactor.


Subject(s)
Bacteria/metabolism , Biofilms , Bioreactors/microbiology , Nitrates/metabolism , Bacteria/genetics , Bacteria/growth & development , Betaproteobacteria/genetics , Betaproteobacteria/growth & development , Betaproteobacteria/metabolism , Biodiversity , Clostridium/genetics , Clostridium/growth & development , Clostridium/metabolism , Comamonadaceae/genetics , Comamonadaceae/growth & development , Comamonadaceae/metabolism , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Electrophoresis, Polyacrylamide Gel/methods , Gammaproteobacteria/genetics , Gammaproteobacteria/growth & development , Gammaproteobacteria/metabolism , Glass , Hydrogen/metabolism , Microspheres , Nitrites/metabolism , Phylogeny , RNA, Ribosomal, 16S/genetics , Sewage/microbiology
20.
Biosci Biotechnol Biochem ; 68(7): 1414-23, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15277745

ABSTRACT

The gene encoding the catabolite control protein A (CcpA) of Bacillus stearothermophilus No. 236, a strong xylanolytic bacterium, was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the ccpA gene corresponded to an open reading frame of 1,005 bp that encodes a polypeptide of 334 amino acid residues with a calculated molecular mass of 36,902 kDa. The CcpA protein belonging to the LacI/GalR family of transcriptional regulators was produced by a recombinant E. coli strain expressing the B. stearothermophilus No. 236 ccpA gene and purified to apparent homogeneity. The transcription start site was mapped at a position 63 nucleotides upstream of the translation initiation codon, and a presumed promoter sequence was also identified. The deduced amino acid sequence of the ccpA gene product contained the helix-turn-helix motif found in many DNA-binding proteins, and showed the highest identity (62%) with CcpA from B. subtilis. The B. stearothermophilus No. 236 ccpA gene was demonstrated to be able to complement a B. subtilis ccpA mutant that exhibited two distinct mutant phenotypes: a growth defect and a release of carbon catabolite repression (CCR). These results indicate that the ccpA gene product of B. stearothermophilus No. 236 is functionally active also in B. subtilis. Electrophoretic mobility shift assay with the purified CcpA revealed that the CcpA of B. stearothermophilus No. 236 bound specifically to the xynA creB (catabolite responsive element B) sequence. Taken together, these results strongly suggest that the CcpA protein participates in CCR of B. stearothermophilus No. 236 xynA gene.


Subject(s)
Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Geobacillus stearothermophilus/genetics , Repressor Proteins/genetics , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , Blotting, Northern , Chromatography, Agarose , Cloning, Molecular , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , Escherichia coli/genetics , Genetic Complementation Test , Geobacillus stearothermophilus/metabolism , Molecular Sequence Data , Molecular Weight , Open Reading Frames , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Transcription, Genetic/physiology
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