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Based on the efficacy of intravenous immunoglobulin (IVIg) for the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), we developed a recombinant single-chain-fragment variable clone, VasSF, therapeutic against AAV in a mouse model (SCG/Kj mice). VasSF is thought to bind to vasculitis-associated apolipoprotein A-II (APOA2) as a target molecule. VasSF is a promising new drug against AAV, but difficulties in the yield and purification of VasSF remain unresolved. We produced monomers of new VasSF molecules by modifying the plasmid structure for VasSF expression and simplifying the purification method using high-performance liquid chromatography. We compared the therapeutic effects between 5-day continuous administration of the monomers, as in IVIg treatment, and single shots of 5-day-equivalent doses. We also evaluated the life-prolonging effect of the single-shot treatment. Two-dimensional western blots were used to examine the binding of VasSF to APOA2. Our improved manufacturing method resulted in a 100-fold higher yield of VasSF than in our previous study. Monomerization of VasSF stabilized its efficacy. Single shots of a small amount (1/80 000 of IVIg) produced sufficient therapeutic effects, including decreased glomerular crescent formation, a decreasing trend of serum ANCA against myeloperoxidase (MPO-ANCA), decreases in multiple proinflammatory cytokines, and a trend toward prolonged survival. Two-dimensional western blots confirmed the binding of VasSF to APOA2. The newly produced pure VasSF monomers are stable and therapeutic for AAV with a single low-dose injection, possibly by removing vasculitis-associated APOA2. Thus, the new VasSF described herein is a promising drug against AAV.
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Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Anticorpos Anticitoplasma de Neutrófilos , Animais , Camundongos , Imunoglobulinas Intravenosas/uso terapêutico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , PeroxidaseRESUMO
BACKGROUND AND HYPOTHESIS: Using a mouse model of MPA with microvascular lesion with a clone (VasSF) of recombinant single chain fragments of the variable region of human IgG, we previously showed that vasculitis-associated apolipoprotein A2 (VAP2) may be a therapeutic target for vasculitis. The present study estimated the target molecules for VasSF and the association between VAP2 and cytokine levels in patient sera in terms of microvascular lesion severity. METHODS: Sera and clinical information were collected from patients with microscopic polyangiitis and granulomatosis with polyangiitis (MPA/GPA) and infectious disease. Neutrophil counts, levels of C-reactive protein (CRP), creatinine, total cholesterol associated with microvascular lesion, HDL cholesterol, low-density lipoprotein cholesterol, triglycerides, glomerular filtration rate (eGFR), and cytokines were estimated. Serum VAP2 signals were determined with Western blotting. RESULTS: VasSF bound to a 24â¯kDa molecule in the serum of active MPA/GPA patients. Anti-AP2 antibody also bound with the same 24â¯kDa molecule, named VAP2, because of size difference from normal APOA2. The VAP2 signal was significantly stronger in the active-disease group but significantly weakened in remission. The signal correlated positively with eGFR but not with the Birmingham Vasculitis Activity Score, CRP, MPO-ANCA, or PR3-ANCA levels. It correlated negatively with MPO activity, IL-16, MIF, and IL-1Ra. Moreover, VasSF bound to a 17â¯kDa molecule in the remission phase. CONCLUSION: The 24â¯kDa VAP2 molecule may be associated with neutrophil functions because of its inverse correlation with MPO activity, IL-16, MIF, and IL-1Ra, suggesting that VAP2-APOA1 formation in HDL triggers microvascular injury. VasSF may reverse the injury by removing APOA1-VAP2 heterodimers from peripheral blood vessels.
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Pattern recognition receptors recognize RNA viruses and trigger type I and III interferon (IFN) production and apoptosis to limit viral replication and spread. Some innate immune cells produce oxidants in response to viral infection to protect against invasion. Recent studies have demonstrated the virucidal activity of hypothiocyanous acid (HOSCN), an oxidant generated by the peroxidase-catalyzed reaction of thiocyanate with hydrogen peroxide. However, the effects of HOSCN on host antiviral responses are still unknown. In this study, we aimed to clarify the role of HOSCN in host antiviral responses against RNA viruses in airway epithelial cells using polyinosinic-polycytidylic acid (polyI:C), a mimic of viral RNA. Our results show that HOSCN repressed antiviral responses in NCI-H292 human airway epithelial cells. HOSCN decreased polyI:C-induced apoptosis and the expression levels of IFNB1, IFNL1, IFNL2 and IFNL3 mRNAs. In addition, the induction of other interferon regulatory factor 3 (IRF3)-dependent genes was also suppressed by HOSCN. Further analyses focused on IRF3 revealed that HOSCN inhibited the phosphorylation of IRF3 at Ser386 and Ser396 as well as its dimerization and nuclear translocation by inhibiting the phosphorylation of TANK-binding kinase 1 (TBK1). Furthermore, HOSCN led to the phosphorylation of IRF3 at residues other than Ser386 and Ser396, implying that HOSCN may cause a conformational change in IRF3 to impair its function. Collectively, these results suggest that HOSCN plays a novel signaling role in the antiviral response, acting as a negative regulator of apoptotic and TBK1-IRF3 signaling pathways and limiting IRF3-dependent gene expression.
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Antivirais/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Fator Regulador 3 de Interferon/metabolismo , Poli I-C/farmacologia , Tiocianatos/farmacologia , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismoRESUMO
Vibrio fluvialis is a bacterium that can be found in both seawater and freshwater, and it is responsible for causing gastroenteritis and cholangitis. V. fluvialis bacteremia has rarely been reported. We report a case of V. fluvialis bacteremia due to cholangitis in an immunocompetent adult who was exposed to seawater regularly as a sushi chef. The increased risk of V. fluvialis entry into the body resulting from frequent consumption of raw fish and regular exposure to seawater, bile outflow impairment caused by transient inflammation of the bile duct, and the presence of multiple bile acid resistance-related genes in V. fluvialis may lead to the development of acute cholangitis and subsequent bacteremia in immunocompetent patients.
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BACKGROUND: During the fourth COVID-19 wave in Japan, marked differences became apparent in the scale of the epidemic between metropolitan Tokyo in eastern Japan and Osaka prefecture in western Japan. METHODS: Public epidemic data were analyzed, with performance of mathematical simulations using simplified SEIR models. RESULTS: The increase in the number of infected persons per 100,000 population during the fourth wave of expansion was greater in Osaka than in Tokyo. The basic reproduction number in Osaka was greater than in Tokyo. Particularly, the number of infected people in their 20 s increased during the fourth wave: The generation-specific reproduction number for people in their 20 s was higher than for people of other generations. Both Tokyo and Osaka were found to have strong correlation between the increase in the number of infected people and the average number of people using the main downtown stations at night. Simulations showed vaccination of people in their 60 s and older reduced the number of infected people among the high-risk elderly population in the fourth wave. However, age-specific vaccination of people in their 20 s reduced the number of infected people more than vaccination of people in their 60 s and older. CONCLUSIONS: Differences in the epidemic between Tokyo and Osaka are explainable by different behaviors of the most socially active generation. When vaccine supplies are adequate, priority should be assigned to high-risk older adults, but if vaccine supplies are scarce, simulation results suggest consideration of vaccinating specific groups among whom the epidemic is spreading rapidly.
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We previously reported that IgA vasculitis and cutaneous arteritis could be dependently associated with the presence of the antiphosphatidylserine/prothrombin complex (anti-PS/PT) antibody and lysosomal-associated membrane protein-2 (LAMP-2). The copy number of LAMP-2 mRNA in skin tissue samples from rats with cutaneous vasculitis induced by intravenous administration of anti-PS/PT antibody after subcutaneous histone injection was significantly higher than in those without cutaneous vasculitis. We found LAMP-2 protein overexpression in neutrophils and vascular endothelial cells of the affected blood vessels in rats with cutaneous vasculitis induced by intravenous administration of anti-PS/PT antibody after cutaneous priming by histones. We found typical cutaneous small-vessel vasculitis in the skin of rats given intravenous injection of both anti-PS/PT antibody and anti-LAMP-2 antibody after cutaneous priming by histones. We suggested that the introduction of skin local histones and anti-PS/PT antibody in serum could move LAMP-2 to the cell surface of neutrophils and vascular endothelial cells, and that anti-LAMP-2 antibody could bridge these cells through antigen-specific binding in typical cutaneous small-vessel vasculitis.
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Dermatopatias Vasculares , Vasculite Leucocitoclástica Cutânea , Ratos , Animais , Protrombina , Proteína 2 de Membrana Associada ao Lisossomo , Células Endoteliais , Histonas , Autoanticorpos , PeleRESUMO
BACKGROUND: Kawasaki disease (KD) is usually treated with high-dose intravenous immunoglobulin (IVIg) as severe infectious and other diseases. Due to issues that are associated with immunoglobulin preparation, such as the risk of possible contamination by infectious agents and limited blood banking resources, recombinant immunoglobulins are required. We developed a novel recombinant antibody drug candidate, "VasSF," based on the therapeutic effects it exerted on a mouse spontaneous crescentic glomerulonephritis model (SCG/Kj). Apolipoprotein A-2 (ApoA2) has been identified as one of VasSF's target molecules. METHODS: Here, we tested the potential of anti-apolipoprotein A-2 antibodies (anti-ApoA2) as a new therapeutic drug against KD by examining its effect on a mouse model, in which KD was induced via Candida albicans water-soluble fraction (CAWS). CAWS (2 mg/mouse) was injected intraperitoneally into C57BL/6NCrSlc mice for five consecutive days. The incidence and histological severity of vasculitis in CAWS-induced coronary arteritis in mice administered anti-ApoA2 was examined. The following experimental groups were tested: solvent (only PBS (-) injection); anti-ApoA2 antibodies at dosages of 0.05 mg, 0.1 mg, and 0.5 mg/kg/day; human IgG at 0.1 mg/kg/day. RESULTS: The group treated with anti-ApoA2 0.5 mg/kg/day showed a lower incidence of panvasculitis induced by CAWS, less inflammation of the coronary arteries and aortic roots, and lower levels of serum IL-6, M-CSF, and MIP-1α and 32 cytokines/chemokines compared with those in the solvent group. CONCLUSIONS: The anti-ApoA2 treatment suppressed the development of coronary arteritis in an animal KD model and anti-ApoA2 shows potential as an effective therapeutic candidate for the treatment of KD vasculitis. The use of specific antibodies that display higher vasculitis-suppressing effects, such as anti-ApoA2, may attenuate KD as well as other infectious diseases, with less severe adverse side effects than treatment with IVIg.
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Arterite , Doença da Artéria Coronariana , Síndrome de Linfonodos Mucocutâneos , Vasculite , Humanos , Camundongos , Animais , Síndrome de Linfonodos Mucocutâneos/complicações , Síndrome de Linfonodos Mucocutâneos/tratamento farmacológico , Imunoglobulinas Intravenosas/uso terapêutico , Camundongos Endogâmicos C57BL , Vasculite/etiologia , Doença da Artéria Coronariana/complicações , Modelos Animais de Doenças , Vasos Coronários/patologia , Arterite/tratamento farmacológico , Arterite/etiologia , Solventes/efeitos adversosRESUMO
Rapidly dried onion (Allium cepa L. cv. Momiji No. 3) powder (OP) prepared from the outer layers (from second to fourth scale leaves from the surface) of onion bulbs was analysed for its quercetin and polyuronide contents, the effects of enzymatic treatment and the anti-atherogenic effect on rats fed a high-fat diet. Quercetin 4'-glucoside (50%), free quercetin (30%) and quercetin 3,4'-diglucoside (20%) were identified as quercetin derivatives, and boiling-water extraction was effective in extracting these compounds. OP contained 12.9% of polyuronides, the basic skeleton of pectin. Enzymatic degradation (cellulase and pectinase, 50°C for 12h, pH 6.0) of OP was effective in obtaining a slurry of smaller particle sizes. The free quercetin increased and the glucosides decreased with enzyme treatment. In Wistar rats fed an OP-added high-fat diet, the total cholesterol, HDL-cholesterol and triglyceride concentrations were not significantly different from the rats fed a high-fat diet without OP. However, the atherogenic index (AI) of Wistar rats fed an OP-added high-fat diet was lower (AI=3.3) than rats fed the diet without OP (AI=4.1). The incremental elastic modulus (IEM) of the aorta from rats fed the OP-added diet was also significantly lower than that of the rats fed the diet without OP. The AI and IEM values of the rats fed the OP-added diet were quite similar to the values of rats fed the diet without OP but were allowed spontaneous exercise. These results suggest that OP intake is effective for decreasing the risk of arteriosclerosis.
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We performed a neuraminidase sequence analysis of thirty-two pediatric patients with influenza B who visited Teikyo University Hospital from January 2016 to March 2017, and found oseltamivir-resistant samples belonging to the Yamagata and Victoria lineages. Comparison with the neuraminidase sequence of oseltamivir-susceptible B/Brisbane/60/2008 revealed 5 common amino acid substitutions in many of these samples. According to the binding free energy calculation, the N340D and E358K substitutions reduced the affinity of oseltamivir to neuraminidase. Unexpectedly, these substitutions were located distant from the oseltamivir-binding site in neuraminidase. According to the molecular dynamics simulations, the N340D substitution rearranged complicated hydrogen bond networks in an extensive surface region of neuraminidase. The E358K substitution extensively altered the electrostatic potential map of the overall neuraminidase structure. Through these novel mechanisms, the N340D and E358K substitutions indirectly influenced the affinity reduction. These results may be useful for designing drugs for the treatment of oseltamivir-resistant virus infections.Communicated by Ramaswamy H. Sarma.
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Influenza Humana , Neuraminidase/genética , Oseltamivir , Proteínas Virais/genética , Substituição de Aminoácidos , Antivirais/farmacologia , Antivirais/uso terapêutico , Domínio Catalítico , Criança , Farmacorresistência Viral/genética , Inibidores Enzimáticos/farmacologia , Humanos , Influenza Humana/tratamento farmacológico , Mutação de Sentido Incorreto , Oseltamivir/farmacologiaRESUMO
We developed a questionnaire to examine the findings of cutaneous arteritis among dermatological specialists experienced in vasculitis as certified by the Committee for guidelines for the management of vasculitis and vascular disorders of the Japanese Dermatological Association. We sent a questionnaire to 12 dermatological facilities identified through the revised Committee for guidelines for the management of vasculitis and vascular disorders of the Japanese Dermatological Association. Retrospective data obtained from 84 patients at the 12 dermatological facilities between 2012 January 2016 December were evaluated. The 84 patients were categorized into two groups, a systemic steroid treatment group (group 1, n = 52) and a no systemic steroid treatment group (group 2, n = 32). C-reactive protein in group 1 patients was significantly higher than that in group 2 patients. Frequency of fever, arthritis, myalgia- and peripheral neuropathy in group 1 was significantly higher than that in group 2. We propose that these symptoms could serve as early markers for the transfer from cutaneous arteritis to systemic polyarteritis nodosa. We further suggest that patients who are subsequently associated with cerebral hemorrhage and infarction, who are originally diagnosed as having cutaneous arteritis, could progress to systemic polyarteritis nodosa. The study demonstrated that it is important for dermatologists to detect these findings early in order to establish an accurate diagnosis and a timely treatment.
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Dermatologistas/estatística & dados numéricos , Poliarterite Nodosa/etiologia , Pele/patologia , Adulto , Biomarcadores/análise , Proteína C-Reativa/análise , Feminino , Seguimentos , Glucocorticoides/uso terapêutico , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Poliarterite Nodosa/sangue , Poliarterite Nodosa/tratamento farmacológico , Poliarterite Nodosa/patologia , Estudos Retrospectivos , Pele/irrigação sanguínea , Inquéritos e Questionários/estatística & dados numéricosRESUMO
BACKGROUND: Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated vasculitis is a pauci-immune disease with the inflammation of the small blood vessels. The efficacies of antibody drugs for induction therapies of vasculitis vary among cases. Here, we developed a novel clone of a single chain Fv region (ScFv) with vasculitis-specific therapeutic potential. MATERIALS AND METHODS: The clone, termed VasSF, was selected from our Escherichia coli expression library of recombinant human ScFv based on the therapeutic efficacy in an SCG/Kj mouse model of MPO-ANCA-associated vasculitis (MAAV), such as improvement of the urinary score and decreased crescent formation in glomeruli, granulomatous in lung, MPO-ANCA biomarkers, the anti-moesin antibody, and some cytokine levels. RESULTS: We identified vasculitis-associated apolipoprotein A-II (VAP2) as a target molecule of the clone and confirmed the independently-established VAP2 antibodies were also therapeutic in SCG/Kj mice. In MAAV, MPO-ANCA and cytokines stimulate neutrophils by facilitating heterodimer formation of VAP2 with apolipoprotein A-I in HDL. CONCLUSION: VasSF would constitute a novel antibody drug for vasculitis by suppressing the heterodimer formation of the apolipoproteins.
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Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Anticorpos de Cadeia Única/uso terapêutico , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Biblioteca de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
The pandemic influenza 2009 (A(H1N1)pdm09) virus currently causes seasonal and annual epidemic outbreaks. The widespread use of anti-influenza drugs such as neuraminidase and matrix protein 2 (M2) channel inhibitors has resulted in the emergence of drug-resistant influenza viruses. In this study, we aimed to determine the anti-influenza A(H1N1)pdm09 virus activity of azithromycin, a re-positioned macrolide antibiotic with potential as a new anti-influenza candidate, and to elucidate its underlying mechanisms of action. We performed in vitro and in vivo studies to address this. Our in vitro approaches indicated that progeny virus replication was remarkably inhibited by treating viruses with azithromycin before infection; however, azithromycin administration after infection did not affect this process. We next investigated the steps inhibited by azithromycin during virus invasion. Azithromycin did not affect attachment of viruses onto the cell surface, but blocked internalization into host cells during the early phase of infection. We further demonstrated that azithromycin targeted newly budded progeny virus from the host cells and inactivated their endocytic activity. This unique inhibitory mechanism has not been observed for other anti-influenza drugs, indicating the potential activity of azithromycin before and after influenza virus infection. Considering these in vitro observations, we administered azithromycin intranasally to mice infected with A(H1N1)pdm09 virus. Single intranasal azithromycin treatment successfully reduced viral load in the lungs and relieved hypothermia, which was induced by infection. Our findings indicate the possibility that azithromycin could be an effective macrolide for the treatment of human influenza.
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Antivirais/farmacologia , Azitromicina/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Células A549 , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Antivirais/administração & dosagem , Azitromicina/administração & dosagem , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Humanos , Pulmão/virologia , Camundongos , Infecções por Orthomyxoviridae/tratamento farmacológico , Resultado do Tratamento , Carga Viral , Liberação de Vírus/efeitos dos fármacosRESUMO
Lupus nephritis (LN) is the secondary glomerulonephritis (GN) involved in systemic lupus erythematosus (SLE) and a typical immune complex-type GN. Antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease characterized by systemic vasculitis and pauci-immune-type crescentic glomerulonephritis (CrGN) with ANCA production. Human AAV causes death due to lung haemorrhage and end-stage renal disease, for which renal replacement therapies are necessary. The SLE/AAV overlap syndrome was recently reported in humans. The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse is a unique model of human AAV showing production of myeloperoxidase (MPO)-ANCA. We previously discovered seven disease susceptibility quantitative trait loci (QTL) derived from SCG/Kj mice by linkage analysis. To investigate the individual functions of each QTL, and to identify AAV susceptibility genes, we introduced them into a B6/lpr background to establish SCG/Kj interval congenic mice (SICM). B6/lpr.C1scg mice, a type of SICM, exhibited the production of autoantibodies, including MPO-ANCA. The GN in B6/lpr.C1scg mice was not pauci-immune type: deposition of immunoglobulins and complement components was observed in nephritic glomeruli, similar to that in LN. The incidence of GN in female B6/lpr.C1scg mice was 100%. Granulocyte infiltration was also observed in the glomerular tuft and crescents. B6/lpr.C1scg mice also displayed vasculitis in multiple organs, most frequently the lung and kidney. Vasculitis was characterized by the infiltration of mononuclear cells to vascular walls followed by granulocyte infiltration, resembling human lupus vasculitis. The incidence of lung vasculitis was over 90% in male and female B6/lpr.C1scg mice. Blood MPO-ANCA levels were significantly associated with histopathological disease phenotypes. MPO deposition was observed in nephritic glomeruli, and granulocytes infiltrated into inflamed vessels and glomeruli. These observations suggest that the activation of granulocytes and local MPO release contribute to the pathogenesis of GN and vasculitis. As a monocongenic mouse, B6/lpr.C1scg mice show the association between murine chromosome 1 segment and autoimmunity. This strain can be used as a model of the SLE/AAV overlap syndrome, and will be useful for elucidating the mechanism of ANCA generation and the pathogenesis of CrGN and vasculitis, as well as in the search for genetic factors related to AAV.
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Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Cruzamentos Genéticos , Glomerulonefrite , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/metabolismo , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Modelos Animais de Doenças , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , CamundongosRESUMO
The purpose of our research is creating a new nanocomposite material. Generally silk fibroin (SF) is regarded as a promising base material for biomedical uses. The incorporation of montmorillonite (MMT) into SF fibers would improve physical properties of the SF fibers. We investigated a new method of combining electospun SF with MMT. Specifically, electrospun silk nanofibers were treated with methanol and dipped in a MMT suspension. We could obtain a nanosheet composite of silk nanofibers and MMT. Their ultrastructures were successfully visualized by high resolution transmission electron microscopy. This compound was comprised of individual silk nanofibers surrounded by thin layers of MMT, each with a thickness of about 1.2 nm. This structure was confirmed by elemental analysis. We also performed IR, NMR and X-ray diffraction analyses in conjunction with morphological data. Conclusively we obtained a new composite of silk nanofiber and MMT, which has never been reported. Using this unique nanocomposite biological tests of its application for a scaffold for tissue engineering are under way.
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Bentonita/química , Bombyx/química , Fibroínas/química , Nanocompostos/química , Nanofibras/química , Animais , Nanocompostos/ultraestrutura , Nanofibras/ultraestruturaAssuntos
Proteínas dos Microfilamentos/metabolismo , Poliarterite Nodosa/patologia , Pele/patologia , Trombose Venosa/patologia , Adulto , Autoanticorpos/sangue , Autoanticorpos/imunologia , Biópsia , Feminino , Dosagem de Genes , Humanos , Masculino , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Poliarterite Nodosa/sangue , Poliarterite Nodosa/imunologia , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/irrigação sanguínea , Trombose Venosa/sangue , Trombose Venosa/imunologia , Adulto JovemRESUMO
We developed a hydrogel self-assembling method driven by the interaction between recombinant tax-interactive protein-1 (TIP1) with the PDZ domain in a molecule, which is fused to each end of the triangular trimeric CutA protein (CutA-TIP1), and a PDZ domain-recognizable peptide which is covalently bound to each terminus of four-armed poly(ethylene glycol) (PDZ-peptide-PEG). Genetic manipulation based on molecular-dynamic simulation generated a cell-adhesive RGD tripeptidyl sequence in the CutA loop region [CutA(RGD)-TIP1]. Spontaneous viscoelastic hydrogel formation occurred when either CutA-TIP1- or CutA(RGD)-TIP1-containing buffer solution and PDZ-peptide-PEG-containing buffer solutions were stoichiometrically mixed. Dynamic viscoelasticity measurement revealed shear stress-dependent reversible-phase transformation: a spontaneous viscoelastic hydrogel was formed at low shear stress, but it was transformed into a sol at high shear stress. Upon the cessation of shear, hydrogel was restored. When chondrocytes were pre-mixed with one of these two components containing buffer solutions, the stoichiometric mixed solution was also spontaneously gelled. Individual rounded cells and multicellular aggregates were entrapped within both hydrogels without substantial cellular impairment regardless of the presence or absence of RGD motif in the CutA-TIP1 molecule. The potential use of such a shear-sensitive hydrogel for injectable cell delivery into diseased or lost cartilage tissue is discussed.
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Condrócitos/citologia , Hidrogéis , Oligopeptídeos/química , Proteínas/química , Sequência de Bases , Células Cultivadas , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Microscopia Eletrônica de Varredura , Modelos Moleculares , Simulação de Dinâmica MolecularRESUMO
Survival motor neuron (SMN) complex is essential for the biogenesis of the small nuclear ribonucleoprotein (snRNP) complex, although the complete role of each SMN complex component for the snRNP synthesis is largely unclear. We have identified an interaction between the two components Gemin2-Gemin7 using the mammalian two-hybrid system. In vitro stability assay revealed that the known SMN-Gemin7 interaction becomes stable in the presence of Gemin2 possibly via the identified Gemin2-Gemin7 interaction. Gemin7 knockdown revealed a decrease in snRNP assembly activity and a decrease in SmE protein, a component of snRNP, in the SMN complex, which was consistent with a previous discussion that the Gemin6-Gemin7 heterodimer may serve as a surrogate for the SmD3-SmB particle in forming a subcore, the intermediate complex for snRNP. Interestingly, we found that Unrip, but not Gemin8, can remove Gemin7 from the stable SMN-Gemin2-Gemin7 ternary complex. In an in vitro snRNP assembly assay using the Unrip knockdown and the untreated cell lysates, we revealed that there was a decrease in Gemin7 and increase in SmB/B' in the SMN complex observed in untreated cells during the assay, suggesting that the Gemin6-Gemin7 heterodimer in the subcore is exchanged by the SmD3-SmB particle to form snRNP. Surprisingly, these changes were not observed in the assay using the Unrip knockdown cell extracts, indicating the importance of Unrip in the formation of snRNP likely via removal of the Gemin6-Gemin7 from the SMN complex. Taken together, these results indicate that snRNP is synthesized by harmonization of the SMN complex components.
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Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN/metabolismo , Animais , Células CHO , Extratos Celulares , Cricetinae , Cricetulus , Técnicas de Silenciamento de Genes , Camundongos , Ligação Proteica , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Ribonucleoproteínas Nucleares Pequenas/química , Proteínas do Complexo SMN/químicaRESUMO
Self-assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self-assembling proteins, named "Nanolego." Nanolego consists of "structural elements" of a structurally stable symmetrical homo-oligomeric protein and "binding elements," which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self-assembly process. The stabilization method is mediated by disulfide bonds between Cysteine-residues incorporated into the binding elements, and the termination method uses "capping Nanolegos," in which some of the binding elements in the Nanolego are absent for the self-assembled ends. With these technologies, we successfully constructed timing-controlled and size-regulated filament-shape complexes via Nanolego self-assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.
Assuntos
Nanoestruturas , Nanotecnologia/métodos , Engenharia de Proteínas , Multimerização Proteica , Proteínas , Animais , Simulação por Computador , Dissulfetos/metabolismo , Humanos , Camundongos , Modelos Moleculares , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Ligação Proteica , Estabilidade Proteica , Proteínas/química , Proteínas/metabolismo , Proteínas/ultraestrutura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
To characterize the molecular basis of specific interactions of PDZ proteins, dynamic force spectroscopy (DFS) for the PDZ protein Tax-interacting protein-1 (TIP-1) and its recognition peptide (PDZ-pep) derived from beta-catenin was performed using an atomic force microscope (AFM), together with measurement of thermodynamic and kinetic parameters using surface plasmon resonance (SPR). The unbinding force of this pair was measured under different conditions of AFM tip-retraction velocity. The relationship between the unbinding force and the logarithmic force-loading rate, that is, the dynamic force spectrum, exhibited two different rate regimes, for each of which the forces increased linearly with the force-loading rate. On the basis of the theoretical treatment of the Bell-Evans model, the positions of two different activation barriers in the reaction coordinate and dissociation rate constants in each barrier were evaluated from slopes and x-intercepts of the two linear regimes (first barrier: 0.04 nm and 1.10 x 10 s(-1); second barrier: 0.21 nm and 2.77 x 10(-2) s(-1), respectively). Although two-step unbinding kinetics between TIP-1 and PDZ-pep was suggested from the DFS analysis, SPR results showed single-step dissociation kinetics with a rate constant of 2.89 x 10(-1) s(-1). Different shapes of the free energy profile of the unbinding process were deduced from each result of DFS and SPR. The reason for such topographic differences in the energy landscape is discussed in relation to the differences in the pathways of forced unbinding and spontaneous dissociation.