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1.
Biol Pharm Bull ; 47(1): 196-203, 2024.
Article in English | MEDLINE | ID: mdl-38246645

ABSTRACT

Psoriasis is a chronic T-cell-mediated autoimmune skin disease. Tacrolimus (FK506) is commonly used treatment for psoriasis. However, since the molecular weight of FK506 is more than 500 Da, its skin penetration is limited, so that there is a need to improve the penetrability of FK506 to allow for more effective treatment. To this end, we employed iontophoresis (ItP), which is a physical, intradermal drug delivery technology that relies on the use of weak electric current. Previous findings suggest that activation of cell signaling by the weak electric current applied during ItP may affect the expression of inflammatory cytokines, leading to aggravation of psoriasis. In this study, we analyzed the effect of ItP on the expression of various inflammatory cytokines in the skin, and subsequently examined the therapeutic effect of ItP using negatively-charged liposomes encapsulating FK506 (FK-Lipo) in a rat psoriasis model induced by imiquimod. We found that ItP (0.34 mA/cm2, 1 h) did not affect mRNA levels of inflammatory cytokines or epidermis thickness, indicating that ItP is a safe technology for psoriasis treatment. ItP of FK-Lipo suppressed the expression of inflammatory cytokines induced by imiquimod treatment to a greater extent than skin treated with FK506 ointment for 1 h. Furthermore, epidermis thickening was significantly suppressed only by ItP of FK-Lipo. Taken together, results of this study demonstrate the successful development of an efficient treatment for psoriasis by combining FK-Lipo and ItP, without disease aggravation associated with the weak electric current.


Subject(s)
Iontophoresis , Psoriasis , Animals , Rats , Tacrolimus/therapeutic use , Liposomes , Imiquimod , Psoriasis/drug therapy , Cytokines
2.
Commun Biol ; 3(1): 99, 2020 03 05.
Article in English | MEDLINE | ID: mdl-32139798

ABSTRACT

LETM1 is a mitochondrial inner membrane protein that is required for maintaining the mitochondrial morphology and cristae structures, and regulates mitochondrial ion homeostasis. Here we report a role of LETM1 in the organization of cristae structures. We identified four amino acid residues of human LETM1 that are crucial for complementation of the growth deficiency caused by gene deletion of a yeast LETM1 orthologue. Substituting amino acid residues with alanine disrupts the correct assembly of a protein complex containing LETM1 and prevents changes in the mitochondrial morphology induced by exogenous LETM1 expression. Moreover, the LETM1 protein changes the shapes of the membranes of in vitro-reconstituted proteoliposomes, leading to the formation of invaginated membrane structures on artificial liposomes. LETM1 mutant proteins with alanine substitutions fail to facilitate the formation of invaginated membrane structures, suggesting that LETM1 plays a fundamental role in the organization of mitochondrial membrane morphology.


Subject(s)
Calcium-Binding Proteins/metabolism , Membrane Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , HeLa Cells , Humans , Liposomes , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mitochondrial Membranes/ultrastructure , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Mutation , Protein Domains , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
4.
Yakushigaku Zasshi ; 50(1): 89-93, 2015.
Article in Japanese | MEDLINE | ID: mdl-26427103

ABSTRACT

INUBUSHI SEIYAKU, a Japanese pharmaceutical company established in 1807, manufactures KEISHIN-TAN. This is an original drug developed by the company, and consists of 14 exotic natural medicines, spikenard, oriental bezoar, musk, agarwood, etc. It has been used for adjusting the autonomic nervous system and physical conditions. We studied the original methods of the traditional quality management techniques handed down within INUBUSHI SEIYAKU in selecting the appropriate spikenard (Nardostachys chinensis) for medicinal use. Currently, spikenards are mainly used as incense rather than medicine. KEISHIN-TAN is a rare case in that the bulk powder of the spikenards is used for pharmaceutical products in Japan. We examined the morphological characteristics and made an analysis of the component of spikenards selected by traditional methods. The raw material of the spikenards was purchased from the Japanese market, and was classified into two categories-superior, fit for medicinal use and defective, to be discarded-by traditional methods of INUBUSHI SEIYAKU. The methods of the characterization of the spikenard by INUBUSHI SEIYAKU were investigated. As a result, only thick spikenard roots over 2.0 cm in length and approximately 0.5 cm in diameter were found to be used, and the total weight of the superior was only 15% of the raw material. By comparing the weights of hexane extracts and GC-MS analyses, the content of calarene--main sedative compound in spikenards--in the superior material was 2.8 times higher than the raw material and 4.3 times higher than the defective material. The ways to devise how to enhance the pharmacological effects of spikenards may be contained in this method. These results revealed the traditional spikenard selection criteria, and may show the indications of using spikenard or its compounds for medicinal purposes.


Subject(s)
Drug Industry/standards , Plants, Medicinal , Japan
5.
Proc Natl Acad Sci U S A ; 111(24): 8877-82, 2014 Jun 17.
Article in English | MEDLINE | ID: mdl-24889612

ABSTRACT

Paired Ig-like type 2 receptor α (PILRα) recognizes a wide range of O-glycosylated mucin and related proteins to regulate broad immune responses. However, the molecular characteristics of these recognitions are largely unknown. Here we show that sialylated O-linked sugar T antigen (sTn) and its attached peptide region are both required for ligand recognition by PILRα. Furthermore, we determined the crystal structures of PILRα and its complex with an sTn and its attached peptide region. The structures show that PILRα exhibits large conformational change to recognize simultaneously both the sTn O-glycan and the compact peptide structure constrained by proline residues. Binding and functional assays support this binding mode. These findings provide significant insight into the binding motif and molecular mechanism (which is distinct from sugar-recognition receptors) by which O-glycosylated mucin proteins with sTn modifications are recognized in the immune system as well as during viral entry.


Subject(s)
Membrane Glycoproteins/chemistry , Mucins/chemistry , Peptides/chemistry , Polysaccharides/chemistry , Receptors, Immunologic/chemistry , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Glycosylation , HEK293 Cells , Humans , Immune System , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/chemistry , Sequence Homology, Amino Acid , Surface Plasmon Resonance
6.
PLoS Pathog ; 10(4): e1004059, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24722332

ABSTRACT

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.


Subject(s)
Arthropod Proteins/immunology , Astacoidea/immunology , Caspase 1/immunology , Catechol Oxidase/immunology , Enzyme Precursors/immunology , Immunity, Innate/physiology , Peptides/immunology , Proteolysis , Animals , Arthropod Proteins/metabolism , Astacoidea/enzymology , Caspase 1/metabolism , Catechol Oxidase/metabolism , Enzyme Precursors/metabolism , Peptides/metabolism
7.
Hum Immunol ; 74(4): 433-8, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23276819

ABSTRACT

HLA-G, a natural immunosuppressant present in the human placenta during pregnancy, prevents fetal destruction by the maternal immune system. The immunosuppressive effect of HLA-G is mediated by the immune cell inhibitory receptors, LILRB1 and LILRB2. HLA-G forms disulfide-linked dimers by natural oxidation, and the dimer associates with LILRB1/B2 much more strongly than the monomer. Furthermore, the dimer formation remarkably enhanced the LILRB-mediated signaling. In this report, we studied the in vivo immunosuppressive effect of the HLA-G dimer, using the collagen-induced arthritis model mouse. Mice were treated with the HLA-G monomer or dimer intracutaneously at the left foot joint, once or for 5 days, and the clinical severity was evaluated daily in a double-blind study. The HLA-G monomer and dimer both produced excellent anti-inflammatory effects with a single, local administration. Notably, as compared to the monomer, the dimer exhibited significant immunosuppressive effects at lower concentrations, which persisted for about two months. In accordance with this result, a binding study revealed that the HLA-G dimer binds PIR-B, the mouse homolog of the LILRBs, with higher affinity and avidity than the monomer. The HLA-G dimer is expected to be quite useful as an anti-rheumatoid arthritis agent, in small amounts with minimal side effects.


Subject(s)
Arthritis, Experimental/drug therapy , Arthritis, Rheumatoid/drug therapy , HLA-G Antigens/immunology , Immunosuppressive Agents/immunology , Joints/drug effects , Receptors, Immunologic/antagonists & inhibitors , Animals , Arthritis, Experimental/chemically induced , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/chemically induced , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Collagen Type II , Disulfides/chemistry , HLA-G Antigens/administration & dosage , HLA-G Antigens/chemistry , Immune Tolerance/drug effects , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/chemistry , Injections , Joints/immunology , Joints/pathology , Male , Mice , Mice, Inbred DBA , Protein Binding , Protein Multimerization , Receptors, Immunologic/immunology , Severity of Illness Index
8.
Immunobiology ; 218(2): 159-68, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22459272

ABSTRACT

An important characteristic of the innate immune systems of crayfish and other arthropods is the activation of a serine proteinase cascade in the hemolymph, which results in the activation of prophenoloxidase and subsequently leading to the formation of toxic quinones and melanin. Although no true complement homologues have been detected in crayfish or crustaceans, several proteins with similarities to vertebrate pattern recognition receptors (PRRs), which are involved in the lectin pathway of complement activation in vertebrates, are present. One is a C-type lectin, a mannose-binding lectin (Pl-MBL), which is secreted from granular hemocytes. Here we report that Pl-MBL has LPS-binding capacity and is dependent upon high Ca(2+) for its solubility and Pl-MBL interferes with proPO activation in vitro when HLS is prepared at high Ca(2+). The proPO-activating system is efficiently activated by microbial polysaccharides and it has to be neatly regulated to avoid activation in places where it is inappropriate and the active enzyme PO should be prevented from spreading throughout the body of the animal. This may be particularly important during molting when proPO is involved in hardening of a new cuticle and the animal is vulnerable to microbes. The presence of high amount of Pl-MBL in the granular hemocytes may play a role in this process. Since a hemocyte lysate supernatant (HLS) prepared at 100 mM Ca(2+) could become activated when the concentration of LPS was increased up to 3 mg/ml, this may indicate that Pl-MBL acts as a scavenger for LPS to prevent spreading of LPS in the hemolymph to avoid further activation of the proPO-system.


Subject(s)
Catechol Oxidase/metabolism , Enzyme Precursors/metabolism , Hemocytes/immunology , Infections/immunology , Mannose-Binding Lectins/metabolism , Mannose-Binding Protein-Associated Serine Proteases/metabolism , Animals , Astacoidea/immunology , Complement Pathway, Mannose-Binding Lectin/immunology , Hemolymph/immunology , Immunity, Innate , Lipopolysaccharides/metabolism , Mannose-Binding Lectins/genetics , Mannose-Binding Lectins/immunology , Molting/immunology , Phylogeny , Receptors, Pattern Recognition/immunology
9.
Phytother Res ; 25(9): 1328-35, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21308824

ABSTRACT

Cherry blossom flowers are familiar to the Japanese, and some species of the flowers soaked in salty vinegar are used as processed foods. The constituents of aqueous ethanol extract from cherry blossom (Prunus lannesiana) flowers (CBE) were examined and cinnamoyl and flavonol glucosides were isolated. To elucidate the pharmacological functions of CBE and its constituents, their effects on the production of advanced glycation end products (AGEs) and on AGE-induced fibroblast damage were examined. CBE and 1-O-(E)-caffeoyl-ß-D-glucopyranoside (CaG), a principal compound in CBE, significantly suppressed the production of AGEs derived from glucose and albumin at 100 µg/mL. Among the flavonol glucosides, quercetin 3-O-ß-D-glucopyranoside (QG) exhibited potent suppressive activity (IC50 : 30 µg/mL). CBE and CaG suppressed glyoxal-induced AGE production in fibroblasts at 10 µg/mL, but QG did not. In addition, CBE and CaG recovered collagen lattice formation consisting of collagen and glycated fibroblasts at 10 µg/mL. Moreover, CBE and its constituents, except kaempferol 3-O-(6″-malony)-ß-D-glucopyranoside, significantly suppressed fibroblast apoptosis induced by carboxymethyl lysine-collagen at 10 µg/mL. These results show that cinnamoyl and flavonol glucosides of cherry blossom flowers suppress AGE production and AGE-induced fibroblast apoptosis. Cherry blossom flowers may be effective against skin AGE production and fibroblast damage by AGEs.


Subject(s)
Apoptosis/drug effects , Fibroblasts/drug effects , Glucosides/pharmacology , Glycation End Products, Advanced/metabolism , Plant Extracts/pharmacology , Prunus/chemistry , Caffeic Acids/pharmacology , Cell Line , Collagen/metabolism , Flavonols/pharmacology , Flowers/chemistry , Humans , Quercetin/analogs & derivatives , Quercetin/pharmacology
10.
Appl Biochem Biotechnol ; 164(6): 715-28, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21279468

ABSTRACT

A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 µg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.


Subject(s)
2,4-Dichlorophenoxyacetic Acid/immunology , Bombyx/genetics , Gene Expression , Herbicides/immunology , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics , 2,4-Dichlorophenoxyacetic Acid/analysis , Amino Acid Sequence , Animals , Base Sequence , Bombyx/growth & development , Bombyx/metabolism , Bombyx/virology , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Genetic Vectors/genetics , Genetic Vectors/metabolism , Hemolymph/chemistry , Hemolymph/metabolism , Hemolymph/virology , Herbicides/analysis , Larva/genetics , Larva/metabolism , Larva/virology , Molecular Sequence Data , Nucleopolyhedroviruses/genetics , Nucleopolyhedroviruses/metabolism , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/metabolism
11.
J Biochem ; 148(3): 335-40, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20592135

ABSTRACT

A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli.


Subject(s)
Bombyx/genetics , Cloning, Molecular/methods , Enzyme-Linked Immunosorbent Assay/methods , Ginsenosides/analysis , Single-Chain Antibodies/biosynthesis , Animals , Baculoviridae/genetics , Enzyme-Linked Immunosorbent Assay/standards , Genetic Vectors , Ginsenosides/immunology , Nucleopolyhedroviruses/genetics , Recombinant Proteins , Single-Chain Antibodies/genetics
12.
J Biol Chem ; 284(40): 27327-35, 2009 Oct 02.
Article in English | MEDLINE | ID: mdl-19654330

ABSTRACT

The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca(2+)-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K(d) approximately 7-12 microm), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1(+)NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.


Subject(s)
Cadherins/metabolism , Receptors, Immunologic/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cadherins/chemistry , Cadherins/genetics , Cattle , Cell Line, Tumor , Gene Expression Regulation , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lectins, C-Type , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Staining and Labeling , Surface Plasmon Resonance
13.
J Nat Prod ; 69(10): 1445-9, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17067159

ABSTRACT

Twenty-eight compounds related to dehydrozingerone (1), isoeugenol (3), and 2-hydroxychalcone (4) were synthesized and evaluated in vitro against human tumor cell replication. Except for isoeugenol analogues 27-35, most compounds exhibited moderate or strong cytotoxic activity against KB, KB-VCR (a multidrug-resistant derivative), and A549 cell lines. In particular, chalcone 15 showed significant cytotoxic activity against the A549 cell line with an IC50 value of 0.6 microg/mL. Furthermore, dehydrozingerone analogue 11 and chalcones 16 and 17 showed significant and similar cytotoxic activity against both KB (IC50 values of 2.0, 1.0, and 2.0 microg/mL, respectively) and KB-VCR (IC50 values of 1.9, 1.0, and 2.0 microg/mL, respectively) cells, suggesting that they are not substrates for the P-glycoprotein drug efflux pump.


Subject(s)
Antineoplastic Agents , Chalcones/pharmacology , Eugenol/analogs & derivatives , Styrenes/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Chalcones/chemical synthesis , Chalcones/chemistry , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Eugenol/chemical synthesis , Eugenol/chemistry , Eugenol/pharmacology , Humans , Inhibitory Concentration 50 , Molecular Structure , Styrenes/chemical synthesis , Styrenes/chemistry , Tumor Cells, Cultured
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