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1.
Nephron ; 144 Suppl 1: 7-12, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33232963

RESUMO

Complement has been considered to be a factor that protects the host against invading microorganisms during infection. However, in recent years, complement-related protein deficiency has been found to be involved in the onset of various diseases, such as autoimmune and inflammatory diseases. In Japan, C3, C4, and CH50 tests were generally performed only when a complement system examination was necessary and there were not enough examinations for other complement factors. Since the complement system has a very complicated activation pathway, at present, it is not well known which molecule must be measured to understand the pathological condition or pathogenesis in complement-related diseases. Furthermore, since the frequency of complement factor gene alleles also differs depending on race, data from foreign countries cannot be directly applied to Japanese populations. Under these circumstances, the Japanese Association for Complement Research (JACR) has prepared approximately 20 items for complement-related examinations, including the 5 categories of functional analysis, complement factors, complement regulators, activation products, and autoantibodies.

2.
Methods Mol Biol ; 2132: 99-109, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306318

RESUMO

Native and recombinant collectins are purified by using mannan-agarose and an anti-collectin antibody column. The use of sandwich enzyme-linked immunosorbent assay (ELISA) with two antibodies against human mannan-binding lectin (MBL) enables elucidation of the collectin concentration in the blood, serum, and plasma. The collectin sugar specificity is demonstrated by determining the concentration of saccharide required to inhibit sugar binding by 50% in a saccharide-binding assay. Biological analyses including the complement-dependent hemolysis test and several other methods are used to evaluate collectin.

3.
Intern Med ; 59(1): 93-99, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31902910

RESUMO

Atypical hemolytic uremic syndrome (aHUS) is an extremely rare condition caused by an excessive activation of the complement pathway based on genetic or acquired dysfunctions in complement regulation, leading to thrombotic microangiopathy (TMA). A complement-amplifying condition (CAC) can trigger aHUS occurrence along with complement abnormality. We herein report a case of severe TMA after laparoscopic myomectomy in a healthy woman. This case was eventually diagnosed as complement-mediated TMA secondary to surgical invasive stress as a CAC, with no definitive diagnosis of aHUS despite a genetic test. The patient fully recovered after several eculizumab administrations.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Laparoscopia/efeitos adversos , Hemorragia Pós-Operatória/complicações , Microangiopatias Trombóticas/tratamento farmacológico , Miomectomia Uterina/efeitos adversos , Adulto , Inativadores do Complemento/uso terapêutico , Feminino , Humanos , Doenças Raras , Microangiopatias Trombóticas/diagnóstico , Microangiopatias Trombóticas/etiologia
4.
Brain Nerve ; 71(6): 555-564, 2019 Jun.
Artigo em Japonês | MEDLINE | ID: mdl-31171752

RESUMO

The complement was named after "a complement", a protein molecule that supports an antibody. It was considered previously that the complement mainly participates in protecting against microbial infections. But later, as research on biological functions in complement activation advanced drastically, it was elucidated that the complement could be involved in the onset of various diseases. In 2007, eculizumab (ECZ), an anti-C5 (complement factor 5) monoclonal antibody, was approved as a drug for paroxysmal nocturnal hemoglobinuria (PNH) in the United States. In Japan, ECZ was approved for PNH and atypical hemolytic uremic syndrome (aHUS) in 2010 and 2013, respectively. The success of ECZ created an opportunity for drug companies to develop new therapeutics targeting the complement system; development of complement therapeutics is now a major venture of pharmaceutical companies worldwide. Here, I will provide an outline of the approved complement therapeutics and those that are in development and clinical trial phase currently.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Inativadores do Complemento/uso terapêutico , Proteínas do Sistema Complemento , Hemoglobinúria Paroxística/tratamento farmacológico , Ensaios Clínicos como Assunto , Complemento C5/antagonistas & inibidores , Humanos , Japão
5.
Clin Appl Thromb Hemost ; 25: 1076029618821189, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30808212

RESUMO

In sepsis, systemic coagulation activation frequently causes disseminated intravascular coagulation (DIC), and the uncontrolled activation of the complement system can induce multiple organ dysfunction and poor prognosis. This study aimed to examine the association of DIC with levels of collectin kidney 1 (CL-K1), a novel collectin of the complement system, and mannose-binding lectin (MBL), a classical-type collectin in patients with sepsis. We collected blood samples prospectively from adult patients with sepsis admitted to the intensive care unit (ICU) from day 1 (admission) to day 5. The CL-K1 and MBL levels were measured by enzyme-linked immunosorbent assay, and DIC was diagnosed by using a scoring algorithm. The correlation of CL-K1 and MBL levels with other coagulation markers was analyzed. There were 37 patients with DIC (DIC group) and 15 without DIC (non-DIC group). Compared to the non-DIC group, the DIC group had more severe conditions and higher mortality. During the 5 days after ICU admission, plasma CL-K1 levels were similar between the groups, but plasma MBL levels were significantly lower in the DIC group. Plasma CL-K1 levels were weakly correlated with prothrombin time, activated partial thromboplastin time, and antithrombin levels; plasma MBL levels were weakly correlated with fibrin/fibrinogen degradation product levels and DIC score. In conclusion, during the first 5 days of ICU admission, plasma CL-K1 levels were similar between the DIC and non-DIC groups. However, plasma MBL levels were lower in the DIC group compared to the non-DIC group, and the significance of this difference grew gradually over time.


Assuntos
Biomarcadores/sangue , Colectinas/metabolismo , Coagulação Intravascular Disseminada/sangue , Lectina de Ligação a Manose/metabolismo , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
6.
J Innate Immun ; 9(2): 217-228, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28068663

RESUMO

Collectins are C-type lectins that are involved in innate immunity as pattern recognition molecules. Recently, collectin kidney 1 (CL-K1) has been discovered, and in vitro studies have shown that CL-K1 binds to microbes and activates the lectin complement pathway. However, in vivo functions of CL-K1 against microbes have not been elucidated. To investigate the biological functions of CL-K1, we generated CL-K1 knockout (CL-K1-/-) mice and then performed a Streptococcus pneumoniae infection analysis. First, we found that recombinant human CL-K1 bound to S. pneumoniae in a calcium-dependent manner, and induced complement activation. CL-K1-/- mice sera formed less C3 deposition on S. pneumoniae. Furthermore, immunofluorescence analysis in the wild-type (WT) mice demonstrated that CL-K1 and C3 were localized on S. pneumoniae in infected lungs. CL-K1-/- mice revealed decreased phagocytosis of S. pneumoniae. Consequently, less S. pneumoniae clearance was observed in their lungs. CL-K1-/- mice showed severe pulmonary inflammation and weight loss in comparison with WT mice. Finally, the decreased clearance and severe pulmonary inflammation caused by S. pneumoniae infection might cause higher CL-K1-/- mice lethality. Our results suggest that CL-K1 might play an important role in host protection against S. pneumoniae infection through the activation of the lectin complement pathway.


Assuntos
Colectinas/metabolismo , Complemento C3/metabolismo , Pulmão/imunologia , Infecções Pneumocócicas/imunologia , Pneumonia/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Streptococcus pneumoniae/fisiologia , Animais , Carga Bacteriana , Colectinas/genética , Lectina de Ligação a Manose da Via do Complemento/genética , Humanos , Imunidade Inata , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Receptores de Reconhecimento de Padrão/genética , Transgenes/genética
7.
Biochim Biophys Acta Gen Subj ; 1861(2): 1-14, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27864148

RESUMO

BACKGROUND: Pentraxins (PTXs) are a superfamily of multifunctional conserved proteins involved in acute-phase responses. Recently, we have shown that collectin placenta 1 (CL-P1) and C-reactive protein (CRP) mediated complement activation and failed to form terminal complement complex (TCC) in normal serum conditions because of complement factor H inhibition. METHODS: We used CL-P1 expressing CHO/ldlA7 cells to study the interaction with PTXs. Soluble type CL-P1 was used in an ELISA assay for the binding, C3 and TCC deposition experiments. Furthermore, we used our previously established CL-P1 expressing HEK293 cells for the C3 fragment and TCC deposition assay. RESULTS: We demonstrated that CL-P1 also bound serum amyloid p component (SAP) and pentraxin 3 (PTX3) to activate the classical pathway and the alternative pathway using factor B. CRP and PTX3 further amplified complement deposition by properdin. We found that CRP and PTX3 recruit CFH, whereas SAP recruits C4 binding protein on CL-P1 expressing cell surfaces to prevent the formation of TCC in normal serum conditions. In addition, depletion of CFH, C4BP and complement factor I (CFI) failed to prevent TCC formation both in ELISA and cell experiments. Furthermore, soluble complement receptor 1, an inhibitor of all complement pathways prevents PTX induced TCC formation. CONCLUSION: Our current study hypothesizes that the interaction of pentraxins with CL-P1 is involved in complement activation. GENERAL SIGNIFICANCE: CL-P1 might generally inhibit PTX induced complement activation and host damage to protect self-tissues.


Assuntos
Proteína C-Reativa/metabolismo , Colectinas/metabolismo , Ativação do Complemento/fisiologia , Componente Amiloide P Sérico/metabolismo , Reação de Fase Aguda/metabolismo , Animais , Células CHO , Linhagem Celular , Fator H do Complemento/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/fisiologia , Cricetulus , Células HEK293 , Humanos , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia
8.
Immunobiology ; 221(10): 1058-67, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27377710

RESUMO

Both the complement system and collectins play important roles in our innate immune system. The collectins, which are characterized by their inclusion of a collagen-like region and a calcium-dependent carbohydrate recognition domain, are pattern recognition molecules and include the well characterized proteins mannan-binding lectin (MBL) and the surfactant proteins SP-A/-D. Collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1) and collectin placenta 1 (CL-P1) are the most recently discovered collectins. Although their function is still under investigation, accumulating information suggests that CL-L1, CL-K1 and CL-P1 play important roles in host defense by recognizing a variety of microorganisms and interacting with effector proteins, including complement components. The recent establishment of the existence of CL-K1 in the circulation in form of heteromeric complexes with CL-L1 (known as CL-LK) and its activation of the lectin pathway via MASPs, drew new attention in the complement biology, which was further strengthened by the observed interactions between CL-P1 and CRP-C1q-factor H or properdin. Deficiency of either CL-K1 or MASP-3 has been demonstrated in 3MC syndrome patients with developmental abnormalities, showing that lectin pathway components, regulation and/or activation are essential during the embryonic development; another feature that they most likely share CL-P1. Herein, we discuss the recent characteristics and roles of the collectins CL-L1, CL-K1 and CL-P1 in the complement system, in innate immunity and their possible association with disease development and pathogenesis.


Assuntos
Colectinas/fisiologia , Ativação do Complemento , Proteínas do Sistema Complemento/fisiologia , Imunidade Inata , Animais , Colectinas/química , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Humanos , Ligantes , Família Multigênica , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Polimorfismo Genético , Relação Estrutura-Atividade
9.
Biochim Biophys Acta ; 1860(6): 1118-28, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26922829

RESUMO

BACKGROUND: C-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown. METHODS: We prepared CHO/ldlA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1. RESULTS: Here, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions. GENERAL SIGNIFICANCE: The interaction of collectin CL-P1 with CFH might be key for preventing attack on "self" as a result of complement activation induced by the CL-P1 and CRP interaction.


Assuntos
Proteína C-Reativa/química , Colectinas/química , Ativação do Complemento , Receptores Depuradores/química , Animais , Proteína C-Reativa/fisiologia , Células CHO , Colectinas/fisiologia , Fator H do Complemento/química , Cricetulus , Células HEK293 , Humanos , Receptores Depuradores/fisiologia
10.
PLoS One ; 10(7): e0132692, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26173080

RESUMO

Understanding the molecular components of immune recognition of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, can help designing novel strategies to combat TB. Here, we identify collectin CL-LK as a novel soluble C-type lectin able to bind M. tuberculosis, and characterize mycobacterial mannose-capped lipoarabinomannan as a primary ligand for CL-LK. Mice deficient in CL-K1, one of the CL-LK subunits, do not display altered susceptibility to M. tuberculosis. However, we found that the amount of CL-LK in the serum of patients with active TB is reduced, compared to that in controls, and correlates inversely to the magnitude of the immune response to the pathogen. These findings indicate that CL-LK might be of interest for future diagnostic and treatment monitoring purposes.


Assuntos
Colectinas/imunologia , Mycobacterium tuberculosis/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Animais , Estudos de Casos e Controles , Colectinas/sangue , Colectinas/deficiência , Colectinas/genética , Feminino , Humanos , Técnicas In Vitro , Ligantes , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/patogenicidade , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/imunologia
11.
BMC Biol ; 13: 27, 2015 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-25912189

RESUMO

BACKGROUND: Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca(2+)-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly(204)Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals. RESULTS: In this study, we show that CL-K1 primarily targets a subset of high-mannose oligosaccharides present on both self- and non-self structures, and provide the structural basis for its ligand specificity. We also demonstrate that three disease-associated mutations prevent secretion of CL-K1 from mammalian cells, accounting for the protein deficiency observed in patients. Interestingly, none of the mutations prevent folding or oligomerization of recombinant fragments containing the mutations in vitro. Instead, they prevent Ca(2+) binding by the carbohydrate-recognition domains of CL-K1. We propose that failure to bind Ca(2+) during biosynthesis leads to structural defects that prevent secretion of CL-K1, thus providing a molecular explanation of the genetic disorder. CONCLUSIONS: We have established the sugar specificity of CL-K1 and demonstrated that it targets high-mannose oligosaccharides on self- and non-self structures via an extended binding site which recognises the terminal two mannose residues of the carbohydrate ligand. We have also shown that mutations associated with a rare developmental disorder called 3MC syndrome prevent the secretion of CL-K1, probably as a result of structural defects caused by disruption of Ca(2+) binding during biosynthesis.


Assuntos
Anormalidades Múltiplas/genética , Carboidratos/química , Colectinas/genética , Colectinas/metabolismo , Mutação/genética , Animais , Células CHO , Cálcio/metabolismo , Bovinos , Colectinas/química , Ativação do Complemento , Cricetinae , Cricetulus , Cristalografia por Raios X , Dissacarídeos/metabolismo , Glicoproteínas/metabolismo , Humanos , Cinética , Ligantes , Modelos Moleculares , Proteínas Mutantes/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Síndrome
12.
Biochim Biophys Acta ; 1840(12): 3345-56, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25199873

RESUMO

BACKGROUND: Collectins are considered to play a role in host defense via complement activation and opsonization, and are composed of a collagen-like domain and a carbohydrate recognition domain (CRD). Collectin placenta 1 (CL-P1) showed scavenger receptor activity as functions in vitro, and has three candidate domains: a coiled-coil domain, a collagen-like domain and CRD. METHODS: We constructed seven types of CL-P1 deletion mutants to determine the site of each ligand binding domain, and observed whether the specific binding to sugar ligand, microbes, or oxidized LDL decreases or not in cells with CL-P1 deletion mutants and CL-P1 containing mutations of amino acid, respectively. RESULTS: CL-P1 mainly interacted with ligands of microbes through the collagen-like domain and it binds a sugar ligand through the CRD. Additionally it could bind oxidized low density lipoprotein (OxLDL) due to the coiled-coil domain as well as the collagen-like domain. This binding study using mutants at three positively charged sites in the collagen-like domain reveals that the site of R496 K499 K502 plays the most important role in ligand binding functions for microbes and OxLDL. CONCLUSIONS: CL-P1 has three unique functional domains: the collagen-like domain mainly acts against most negatively charged ligands, and the CRD specifically does against sugar substances, while the coiled-coil domain additionally acts on modified LDL. GENERAL SIGNIFICANCE: We considered that the binding activity for various ligands due to the association of a coiled-coil domain, a collagen-like domain and/or a CRD in CL-P1, might play a role in physiological functions in the animal body.

13.
Biochim Biophys Acta ; 1840(11): 3226-37, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25109811

RESUMO

BACKGROUND: Scavenger receptor CL-P1 (collectin placenta 1) has been found recently as a first membrane-type collectin which is mainly expressed in vascular endothelial cells. CL-P1 can endocytose OxLDL as well as microbes but in general, the endocytosis mechanism of a scavenger receptor is not well elucidated. METHODS: We screened a placental cDNA library using a yeast two-hybrid system to detect molecules associated with the cytoplasmic domain of CL-P1. We analyzed the binding and endocytosis of several ligands in CL-P1 transfectants and performed the inhibition study using tyrphostin A23 which is a specific inhibitor of tyrosine kinase, especially in µ2-dependent endocytosis and the site-directed mutagenesis in the endocytosis YXXΦ motif in CL-P1 cytoplasmic region. Furthermore, the SiRNA study of clathrin, adaptor AP-2 and dynamin-2 during the endocytosis of OxLDL in CL-P1 transfectant cells was carried out. RESULTS: We identified µ2 subunit of the AP-2 adaptor complex as a molecule associated with the cytoplasmic region of CL-P1. We demonstrated that AP-2µ2 was essential for CL-P1 mediated endocytosis of OxLDL in CL-P1 transfectant cells and its endocytosis was also mediated by clathrin, dynamin and adaptin complex molecules. CONCLUSIONS: Tyrosine-based YXXΦ sequences play an important role in CL-P1-mediated OxLDL endocytosis associated with AP-2µ2. GENERAL SIGNIFICANCE: This might be the first finding of the clear endocytosis mechanism in scavenger receptor CL-P1.

14.
J Thromb Thrombolysis ; 38(3): 331-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24474086

RESUMO

Collectin kidney 1 (CL-K1) is a recently identified collectin that is synthesized in most organs and circulates in blood. CL-K1 is an innate immune molecule that may play a significant role in host defense. As some collectins also play a role in coagulation, we hypothesized that an effect of CL-K1 may be apparent in disseminated intravascular coagulation (DIC), a gross derangement of the coagulation system that occurs in the setting of profound activation of the innate immune system. DIC is a grave medical condition with a high incidence of multiple organ failure and high mortality and yet there are no reliable biomarkers or risk factors. In our present study, we measured plasma CL-K1 concentration in a total of 659 specimens, including 549 DIC patients, 82 non-DIC patients and 27 healthy volunteers. The median plasma CL-K1 levels in these cohorts were 424, 238 and 245 ng/ml, respectively, with no significant difference in the latter two groups. The incidence of elevated plasma CL-K1 was significantly higher in the DIC patients compared to the non-DIC patients, resulting in an odds ratio of 1.929 (confidence interval 1.041-3.866). Infection, renal diseases, respiratory diseases, and cardiac diseases were more frequently observed in the DIC group than in the non-DIC group. In the DIC group, vascular diseases were associated with elevated plasma CL-K1 levels while age and acute illness had little effect on plasma CL-K1 levels. Independent of DIC, elevated plasma CL-K1 levels were associated with respiratory disease and coagulation disorders. These results suggest that specific diseases may affect CL-K1 synthesis in an organ dependent manner and that elevated plasma CL-K1 levels are associated with the presence of DIC. Further investigations in cohorts of patients are warranted. We propose that elevated plasma CL-K1 may be a new useful risk factor and possibly biomarker for the prediction of developing DIC.


Assuntos
Colectinas/sangue , Coagulação Intravascular Disseminada/sangue , Fatores Etários , Idoso , Biomarcadores/sangue , Transtornos da Coagulação Sanguínea/sangue , Estudos de Coortes , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Respiratórias/sangue , Fatores de Risco
16.
J Pharmacol Exp Ther ; 344(1): 124-32, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23042952

RESUMO

Resveratrol (trans-3,5,4'-trihydroxystilbene; RSV), a natural polyphenol, exerts a beneficial effect on health and diseases. RSV targets and activates the NAD(+)-dependent protein deacetylase SIRT1; in turn, SIRT1 induces an intracellular antioxidative mechanism by inducing mitochondrial superoxide dismutase (SOD2). Most RSV found in plants is glycosylated, and the effect of these glycosylated forms on SIRT1 has not been studied. In this study, we compared the effects of RSV and two glycosyl RSVs, resveratrol-3-O-ß-d-glucoside (3G-RSV; polydatin/piceid) and resveratrol-4'-O-ß-d-glucoside (4'G-RSV), at the cellular level. In oxygen radical absorbance capacity and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays, the antioxidant activity of 3G-RSV was comparable to that of RSV, whereas the radical-scavenging efficiency of 4'G-RSV was less than 50% of that of RSV. However, 4'G-RSV, but not 3G-RSV, induced SIRT1-dependent histone H3 deacetylation and SOD2 expression in mouse C2C12 skeletal myoblasts; as with RSV, SIRT1 knockdown blunted these effects. RSV and 4'G-RSV, but not 3G-RSV, mitigated oxidative stress-induced cell death in C2C12 cells and primary neonatal rat cardiomyocytes. RSV and 4'G-RSV inhibited C2C12 cell proliferation, but 3G-RSV did not. RSV was found in both the intracellular and extracellular fractions of C2C12 cells that had been incubated with 4'G-RSV, indicating that 4'G-RSV was extracellularly deglycosylated to RSV, which was then taken up by the cells. C2C12 cells did not deglycosylate 3G-RSV. Our results point to 4'G-RSV as a useful RSV prodrug with high water solubility. These data also show that the in vitro antioxidative activity of these molecules did not correlate with their ability to protect cells from oxidative stress-induced apoptosis.


Assuntos
Antioxidantes/farmacologia , Citoproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Animais Recém-Nascidos , Antioxidantes/isolamento & purificação , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/química , Western Blotting , Morte Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Cromanos/farmacologia , Depuradores de Radicais Livres/farmacologia , Radicais Livres/química , Glucosídeos/farmacologia , Imuno-Histoquímica , Camundongos , Mioblastos/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Phytolacca americana/química , Picratos/química , Reação em Cadeia da Polimerase , Ratos , Resveratrol , Sirtuína 1/genética , Estilbenos/isolamento & purificação
18.
J Biomed Biotechnol ; 2012: 493945, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22570530

RESUMO

Collectins are characterized by a collagen-like sequence and a carbohydrate recognition domain and are members of the vertebrate C-type lectin superfamily. Recently, "novel collectins", different from "classical collectins" consisting of mannan-binding lectin (MBL) and surfactant proteins A and D (SP-A and SP-D), have been found by reverse genetics. These "novel collectins" consist of collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1), and collectin placenta 1 (CL-P1) and are encoded by three separate genes. Experimental findings on human and animal collectins have shown that both novel collectins and classical collectins play an important role in innate immunity. Based on our recent results and those of others, in this paper, we summarize the new biological functions of these novel collectins in embryonic morphogenesis and development.


Assuntos
Colectinas/imunologia , Animais , Feminino , Humanos , Imunidade Inata/imunologia , Morfogênese , Gravidez
19.
J Biochem ; 151(1): 57-64, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21893516

RESUMO

Mannan-binding lectin (MBL) was first discovered as a collectin in animal blood, and was shown to have such unique characteristics as a collage-like domain and a carbohydrate recognition domain. We recently identified human collectin kidney 1 (CL-K1, COLEC11) from a human kidney cDNA library. To quantitate the CL-K1 concentration in blood, we developed several polyclonal and monoclonal antibodies using recombinant human CL-K1 in CHO cells and the CL-K1 fragment in Escherichia coli. Using these antibodies, we established a sandwich enzyme-linked immunosorbent assay (ELISA) system. The concentration of CL-K1 in human plasma was 0.34 ± 0.13 µg/ml and that in MBL was 1.72 ± 1.51 µg/ml. Concentrations of MBL are often low due to its single nucleotide polymorphisms (SNPs) which seem to be related to an opsonic defect. However, no low concentrations of CL-K1 were observed on testing over two hundred blood samples. We also found that the blood concentration of CL-K1 was not dependent on gender or age and did not correlate completely with that of MBL. The ELISA system developed in this study will be useful for elucidating the physiological and pathophysiological role of CL-K1 in humans.


Assuntos
Colectinas/sangue , Lectina de Ligação a Manose/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Western Blotting , Células CHO , Colectinas/genética , Colectinas/imunologia , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/imunologia , Pessoa de Meia-Idade , Coelhos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Valores de Referência , Adulto Jovem
20.
Biochim Biophys Acta ; 1810(12): 1150-9, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22001438

RESUMO

BACKGROUND: Scavenger receptors are generally expressed in macrophages and vascular endothelial cells and some scavenger receptors are thought to contribute to the development of atherosclerosis. METHODS: We cloned the cDNA of a zebrafish CL-P1 (collectin placenta 1) and performed a knockdown study using its antisense morpholino oligonucleotides (MO). RESULTS: Zebrafish CL-P1 (zCL-P1) is 51% identical to human CL-P1 in its amino acid sequence. Microbes and OxLDL bound to zCL-P1 cDNA transfected cells. zCL-P1 mRNA expression gradually increased after 6hours post-fertilization (hpf), reached its highest level at 24hpf, and then decreased, which is similar to the gene expression pattern of Tie-2. The knockdown of zCL-P1 led to an increase in the number of zebrafish embryos with severe morphological abnormalities such as short body lengths and defects in the dorsal aorta at 48hpf. Simultaneous injection of both MO and synthetic zCL-P1 or zVEGF mRNA rescued the abnormal phenotype. CONCLUSIONS: In vivo knockdown study shows that zCL-P1 is implicated in vasculogenesis and those of our in vitro study support its role as a scavenger receptor. GENERAL SIGNIFICANCE: These results suggest that zCL-P1 might be essential for vasculogenesis during the early embryonic phase in bone fish.


Assuntos
Receptores Depuradores/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , DNA Complementar , Técnicas de Silenciamento de Genes , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptores Depuradores/fisiologia , Peixe-Zebra
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