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1.
Eur Rev Med Pharmacol Sci ; 24(16): 8251-8262, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32894531

RESUMO

OBJECTIVE: Intervertebral disc (IVD) degeneration (IDD) is a well-known consequence of low back pain, as characterized by aberrant cell proliferation and apoptosis of nucleus pulposus (NP) cells. In the present study, we aimed to investigate the effect of lncRNA small nucleolar RNA host gene 6 (SNHG6) on deregulated functions of degenerative NP cells. MATERIALS AND METHODS: After the establishment of rat IDD models, the mRNA and protein levels of collagen-I (Col-I) and collagen II (Col-II), and mRNA level of SNHG6 were detected by using reverse transcription quantitative Real Time-PCR (RT-qPCR) and Western blot. We further investigated the role and molecular mechanisms of SNHG6 by overexpressing or silencing it in degenerative NP cells. Cell proliferation was measured by MTT assay and EdU staning, and apoptosis was measured by flow cytometry. The target of SNHG6 was identified by starBase and Dual-Luciferase reporter assay. RESULTS: Upregulation of SNHG6 was found in IDD NP cells than in normal cells, associated with higher level of Col-I and lower level of Col-II. Overexpression of SNHG6 inhibited cell proliferation and enhanced apoptosis, accompanied by increased expression of Bax, caspase-3, and p21, as well as decreased expression of Bcl-2, which was in reverse to the treatment of SNHG6 silencing. Moreover, miR-101-3p was indicated as a target of SNHG6, and inhibition of miR-101-3p reversed the effects on proliferation and apoptosis induced by SNHG6. CONCLUSIONS: SNHG6 suppressed cell proliferation and induced apoptosis by increasing expression of Bax, caspase-3, p21 and decreasing Bcl-2 through targeting miR-101-3p, which suggested that SNHG6 could be a potential target in the treatment of IDD.


Assuntos
Apoptose , MicroRNAs/metabolismo , Núcleo Pulposo/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Proliferação de Células , MicroRNAs/genética , Núcleo Pulposo/patologia , RNA Longo não Codificante/genética , Ratos , Ratos Wistar
2.
Oncogene ; 36(50): 6895-6905, 2017 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-28846110

RESUMO

Hepatitis B virus (HBV)-induced liver necrosis takes great part in liver cirrhosis progression. However, less is known about whether hepatitis B virus X protein (HBx) has effect on liver fibrosis. Here, we report that HBV leads to liver fibrosis and hepatocarcinogenesis through miR-30e targeting P4HA2. HBV transgenic mouse was treated by CCl4 to generate a model of liver fibrosis. A crucial enzyme catalyzing collagen formation, prolyl 4-hydroxylase subunit α2 (P4HA2) was evaluated by immunohistochemistry, western blotting or quantitative reverse transcription-PCR analysis. The function of HBV-modulated P4HA2 in hepatoma cell growth in vitro and in vivo was analyzed by EdU, MTT, colony-forming assay and animal transplantation assay. HBV transgenic mice exhibited more collagen deposition in liver after intraperitoneal injection of CCl4. P4HA2 was dramatically augmented in liver samples of HBV transgenic mice, clinical liver cirrhosis and liver cancer patients. Mechanistically, HBx was capable of inducing P4HA2 through suppressing miR-30e, in which miR-30e could target P4HA2 mRNA 3' untranslated region in liver cancer cells. HBx inhibited the miR-30e expression through increasing methylation of CpG islands in its promoter mediated by EZH2-formed complexes. Functionally, HBx-elevated P4HA2 enhanced the collagen deposition in the liver in vivo and in vitro, leading to liver fibrosis and liver cancer progression. In conclusion, HBx promotes the development of liver fibrosis and hepatocellular carcinoma through miR-30e targeting P4HA2 mRNA. We provide novel perspective on how HBx induces liver fibrosis.


Assuntos
Carcinoma Hepatocelular/etiologia , Cirrose Hepática/etiologia , Neoplasias Hepáticas/etiologia , MicroRNAs/genética , Prolil Hidroxilases/genética , Transativadores/farmacologia , Animais , Linhagem Celular Tumoral , Colágeno/metabolismo , Regulação para Baixo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/fisiologia , Proteínas Virais Reguladoras e Acessórias
3.
Zhonghua Gan Zang Bing Za Zhi ; 25(4): 317-320, 2017 Apr 20.
Artigo em Chinês | MEDLINE | ID: mdl-28494557

RESUMO

Vanishing bile duct syndrome (VBDS) manifests as progressive destruction and disappearance of the intrahepatic bile duct caused by various factors and cholestasis. VBDS associated with drug-induced liver injury (D-VBDS) is an important etiology of VBDS, and immune disorder or immune imbalance may be the main pathogenesis. According to its clinical symptoms, serological markers, and course of the disease, D-VBDS is classified into major form and minor form, and its clinical features are based on various pathomorphological findings. Its prognosis is associated various factors including regeneration of bile duct cells, number of bile duct injuries, level and range of bile duct injury, bile duct proliferation, and compensatory shunt of bile duct branches. This disease has various clinical outcomes; most patients have good prognosis after drug withdrawal, and some patients may experience cholestatic cirrhosis, liver failure, and even death. Due to the clinical manifestation and biochemical changes are similar to the primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), it need to identify by clinical physician.


Assuntos
Ductos Biliares Intra-Hepáticos/patologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colestase/induzido quimicamente , Bile , Colangite Esclerosante , Colestase/diagnóstico , Humanos , Cirrose Hepática Biliar , Falência Hepática , Prognóstico
4.
Heredity (Edinb) ; 114(6): 601-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25669608

RESUMO

Rapid genomic change has been demonstrated in several allopolyploid plant systems; however, few studies focused on animals. We addressed this issue using an allotetraploid lineage (4nAT) of freshwater fish originally derived from the interspecific hybridization of red crucian carp (Carassius auratus red var., ♀, 2n=100) × common carp (Cyprinus carpio L., ♂, 2n=100). We constructed a bacterial artificial chromosome (BAC) library from allotetraploid hybrids in the 20th generation (F20) and sequenced 14 BAC clones representing a total of 592.126 kb, identified 11 functional genes and estimated the guanine-cytosine content (37.10%) and the proportion of repetitive elements (17.46%). The analysis of intron evolution using nine orthologous genes across a number of selected fish species detected a gain of 39 introns and a loss of 30 introns in the 4nAT lineage. A comparative study based on seven functional genes among 4nAT, diploid F1 hybrids (2nF1) (first generation of hybrids) and their original parents revealed that both hybrid types (2nF1 and 4nAT) not only inherited genomic DNA from their parents, but also demonstrated rapid genomic DNA changes (homoeologous recombination, parental DNA fragments loss and formation of novel genes). However, 4nAT presented more genomic variations compared with their parents than 2nF1. Interestingly, novel gene fragments were found for the iqca1 gene in both hybrid types. This study provided a preliminary genomic characterization of allotetraploid F20 hybrids and revealed evolutionary and functional genomic significance of allopolyploid animals.


Assuntos
Carpas/genética , Carpa Dourada/genética , Hibridização Genética , Poliploidia , Animais , Quimera , Evolução Molecular , Amplificação de Genes , Biblioteca Gênica , Íntrons , Dados de Sequência Molecular , Análise de Sequência de DNA
5.
IUBMB Life ; 51(6): 337-44, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11758800

RESUMO

Myosin light chain kinase (MLCK) is a regulatory protein for smooth muscle contraction, which acts by phosphorylating 20-kDa myosin light chain (MLC20) to activate the myosin ATPase activity. Although this mode of action is well-established, there are numerous reports of smooth muscle contraction that is not associated with MLC20 phosphorylation. The kinase activity for the phosphorylation is localized at the central part of MLCK, which is also furnished with actin-binding activity at its N terminal and myosin-binding activity at its C terminal. This article overviews as to how such multifunctional properties of MLCK modify the actin-myosin interaction and presents our observations that the phosphorylation is not obligatory in induction of smooth muscle contraction.


Assuntos
Contração Muscular , Músculo Liso/fisiologia , Quinase de Cadeia Leve de Miosina/fisiologia , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Sítios de Ligação , Calmodulina/metabolismo , Catálise , Músculo Liso/enzimologia , Miofibrilas/metabolismo , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo
6.
Cancer J ; 6(3): 162-8, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10882332

RESUMO

A rhodacyanine dye called MKT-077 has shown a highly selective toxicity toward several distinct human malignant cell lines, including bladder carcinoma EJ, and has been subjected to clinical trials for cancer therapy. In the pancreatic carcinoma cell line CRL-1420, but not in normal African green monkey kidney cell line CV-1, it is selectively accumulated in mitochondria. However, both the specific oncogenes responsible for its selective toxicity toward cancer cells, and its target proteins in these cancer cells, still remain to be determined. This study was conducted using normal and ras-transformed NIH 3T3 fibroblasts to determine whether oncogenic ras mutants such as v-Ha-ras are responsible for the selective toxicity of MKT-077 and also to identify its targets, using its derivative called "compound 1" as a specific ligand. We have found that v-Ha-ras is responsible for the selective toxicity of MKT-077 in both in vitro and in vivo. Furthermore, we have identified and affinity purified at least two distinct proteins of 45 kD (p45) and 75 kD (p75), which bind MKT-077 in v-Ha-ras-transformed cells but not in parental normal cells. Microsequencing analysis has revealed that the p45 is a mixture of beta- and gamma-actin, whereas the p75 is HSC70, a constitutive member of the Hsp70 heat shock adenosine triphosphatase family, which inactivates the tumor suppressor p53. MKT-077 binds actin directly, bundles actin filaments by cross-linking, and blocks membrane ruffling. Like a few F-actin-bundling proteins such as HS1, alpha-actinin, and vinculin as well as F-actin cappers such as tensin and chaetoglobosin K (CK), the F-actin-bundling drug MKT-077 suppresses ras transformation by blocking membrane ruffling. These findings suggest that other selective F-actin-bundling/capping compounds are also potentially useful for the chemotherapy of ras-associated cancers.


Assuntos
Actinas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Proteína Oncogênica p21(ras)/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Células 3T3 , Actinas/química , Actinas/farmacologia , Actinas/ultraestrutura , Animais , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Membrana Celular/metabolismo , Cromatografia de Afinidade , Reagentes de Ligações Cruzadas/farmacologia , Citoesqueleto/metabolismo , Citosol/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Feminino , Fibroblastos/efeitos dos fármacos , Camundongos , Camundongos Nus , Microscopia Eletrônica , Miosinas/metabolismo , Ligação Proteica , Sarcoma Experimental/tratamento farmacológico , Fatores de Tempo
7.
J Biochem ; 125(6): 1055-60, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10348907

RESUMO

The catalytic domain of myosin light chain kinase (MLCK) not only exerts kinase activity to phosphorylate the 20 kDa light chain but also inhibits the actin-myosin interaction. The site of action of this novel role of the domain has been suggested to be myosin [Okagaki et al. (1999) J. Biochem. 125, 619-626]. In this study, we have analyzed the amino acid sequences of MLCK and myosin that are involved in the inhibition. The ATP-binding peptide of Gly526-Lys548 of chicken gizzard MLCK exerted the inhibitory effect on the movement of actin filaments on a myosin-coated glass surface. However, the peptide that neighbors the sequence failed to inhibit the movement. The inhibition of the ATP-binding peptide was confirmed by measuring ATPase activities of the myosin. The inhibition by parent MLCK of the movement was relieved by the 20 kDa light chain, but not by the 17 kDa myosin light chain. The peptide of the 20 kDa light chain sequence of Ser1-Glu29 also relieved the inhibition. Thus, the interaction of the ATP-binding sequence with the 20 kDa light chain sequence should cause the inhibition of the actin-myosin interaction. Concerning the regulation of the inhibition, calmodulin relieved the inhibitory effect of MLCK on the movement of actin filaments. The calmodulin-binding peptide (Ala796 Ser815) prevented the relief, suggesting the involvement of this sequence. Thus, the mode of regulation by Ca2+ and calmodulin of the novel role of the catalytic domain is similar, but not identical, to the mode of regulation of the kinase activity of the domain.


Assuntos
Actinas/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/metabolismo , Domínio Catalítico/genética , Bovinos , Galinhas , Técnicas In Vitro , Dados de Sequência Molecular , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/genética , Miosinas/química , Miosinas/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo
8.
Proc Natl Acad Sci U S A ; 96(12): 6666-71, 1999 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-10359769

RESUMO

Myosin light-chain kinase (MLCK) of smooth muscle is multifunctional, being composed of N-terminal actin-binding domain, central kinase domain, and C-terminal myosin-binding domain. The kinase domain is the best characterized; this domain activates the interaction of smooth-muscle myosin with actin by phosphorylating the myosin light chain. We have recently shown that the Met-1-Pro-41 sequence of MLCK binds to actin to inhibit this interaction. However, it is not known whether the myosin-binding domain modifies the actin-myosin interaction. We designed MLCK.cDNA to overexpress the Asp-777-Glu-972 sequence in Escherichia coli. The purified Asp-777-Glu-972 fragment, although devoid of the kinase activity, exerted a stimulatory effect on the ATPase activity of dephosphorylated myosin (Vmax = 7.36 +/- 0.44-fold, Km = 1.06 +/- 0. 20 microM, n = 4). When the N-terminal 39 residues of the fragment were deleted from the fragment, the resultant fragment, Met-816-Glu-972, lost the stimulatory activity. We synthesized the Ala-777-Ser-815 peptide that was deleted from the fragment and confirmed its stimulatory effect of the peptide (Vmax = 3.03 +/- 0. 22-fold, Km = 6.93 +/- 1.61 microM, n = 3). When this peptide was further divided into Asp-777-Met-795 and Ala-796-Ser-815 peptides, the stimulatory activity was found in the latter. We confirmed that the myosin phosphorylation did not occur during the experiments with the above fragments and peptides. Therefore, we suggest that phosphorylation is not obligatory for smooth-muscle myosin not to be active.


Assuntos
Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Animais , Bovinos , Galinhas , Ativação Enzimática , Fosforilação , Proteínas Recombinantes/metabolismo
9.
Biochim Biophys Acta ; 1450(1): 12-24, 1999 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-10231551

RESUMO

In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.


Assuntos
Proteínas dos Microfilamentos/química , Músculo Liso/enzimologia , Quinase de Cadeia Leve de Miosina/química , Citoesqueleto de Actina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calmodulina/química , Galinhas , Brometo de Cianogênio , Moela das Aves , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas Recombinantes/química , Tiocianatos
10.
Mol Cell Biochem ; 190(1-2): 85-90, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10098974

RESUMO

Myosin light chain kinase (MLCK) has been purified from various muscles as an enzyme to phosphorylate myosin light chains. While the regulatory role of smooth muscle MLCK is well understood, the role of skeletal muscle MLCK in the regulation of contraction has not been fully characterized. Such characterization of skeletal muscle MLCK is difficult because skeletal muscle myosin interacts with actin whether or not the myosin is phosphorylated. Taking the hint from our recent finding that smooth muscle MLCK inhibits the actin-myosin interaction by binding to actin (Kohama et al., Biochem Biophys Res Commun 184: 1204-1211, 1992), we investigated the regulatory role of the actin-binding activity of MLCK from chicken breast muscle in the actin-myosin interaction. The amount of MLCK that bound to actin increased with increases in the concentration of MLCK. However, MLCK hardly bound to myosin. The actin-binding activity of MLCK was affected when Ca2+ and calmodulin (Ca2+ -CaM) were present. The effect of MLCK on the actin-myosin interaction was examined by an in vitro motility assay; the movement of actin-filaments on a myosin-coated glass surface was inhibited by increasing the concentration of MLCK. When CaM was present, the inhibition was overcome in a Ca2+ -dependent manner at microM levels. The inhibition of the movement by MLCK and the recovery from the inhibition by Ca2+ -CaM were not altered whether we use phosphorylated or unphosphorylated myosin for the assay, ruling out the involvement of the kinase activity of MLCK.


Assuntos
Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Músculo Esquelético/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Animais , Cálcio/metabolismo , Galinhas , Ligação Proteica
11.
Adv Exp Med Biol ; 453: 229-34, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9889833

RESUMO

Myosin light chain kinase (MLCK) plays a central role in regulating the actin-myosin interaction of smooth muscle. MLCK phosphorylates the light chain of myosin in the presence of Ca2+ and calmodulin (CaM) thereby activating myosin so that it can interact with actin. Besides this kinase activity, MLCK shows i) actin-binding activity that can assemble actin filaments into their bundles and ii) myosin-binding activity that can form myosin filaments. To localize the actin- and myosin-binding activities in the MLCK molecule and to examine their possible role in regulating the actin-myosin interaction, we expressed various fragments of cDNA encoding MLCK in Escherichia coli as recombinant proteins. We found that MLCK consists of an N-terminal actin-binding domain, a central kinase domain, and a C-terminal myosin-binding domain. The Met1-Pro41 sequence is responsible for Ca2+/CaM-sensitive binding to actin. This binding site exerts an inhibitory effect on the actin-myosin interaction only when myosin is phosphorylated. MLCK binds to myosin at the C-terminal domain, the sequence of which is identical to telokin, an abundant myosin-binding protein in smooth muscle cells. This domain itself has no regulatory role in the interaction. However, the interaction was stimulated when this domain was extended to include the sequence known to regulate the activity of the kinase domain. The stimulation was observed only when myosin was unphosphorylated.


Assuntos
Músculo Liso/química , Músculo Liso/metabolismo , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , DNA Complementar/genética , Escherichia coli , Humanos , Quinase de Cadeia Leve de Miosina/genética , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
J Biol Chem ; 272(51): 32182-9, 1997 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-9405419

RESUMO

In addition to its kinase activity, the myosin light chain kinase (MLCK) of smooth muscle has an actin binding activity through which it can regulate the actin-myosin interaction of smooth muscle (Kohama, K., Okagaki, T., Hayakawa, K., Lin, Y., Ishikawa, R., Shimmen, T., and Inoue, A. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211). In this study, we have analyzed the actin binding activity of MLCK and related it to its amino acid sequence by producing native and recombinant fragments of MLCK. Parent MLCK exhibited both calcium ion (Ca2+) and calmodulin (Ca2+/CaM)-sensitive and Ca2+/CaM-insensitive binding to actin filaments. The native fragment, which consists of the Met1-Lys114 sequence (Kanoh, S., Ito, M., Niwa, E., Kawano, Y., and Hartshorne, D. J. (1993) Biochemistry 32, 8902-8907), and the recombinant NN fragment, which contains this 1-114 sequence, showed only Ca2+/CaM-sensitive binding. An inhibitory effect of the NN fragment on the actin-myosin interaction was observed by assaying in vitro motility and by measuring the actin-activated ATPase activity of myosin. The recombinant NN/41 fragment, which is constructed without the Met1-Pro41 sequence of the NN fragment, lost both the actin binding activity and the inhibitory effect. We confirmed the importance of the 1-41 sequence by using a few synthetic peptides to compete against the NN fragment in binding to actin filaments. The experiments using recombinant fragments and synthetic peptides also revealed that the site for CaM-binding is the Pro26-Pro41 sequence. The site for the Ca2+/CaM-insensitive binding, which is shown to be localized between the Ca2+/CaM-sensitive site and the central kinase domain of MLCK, exerted no regulatory effects on the actin-myosin interaction.


Assuntos
Actinas/metabolismo , Músculo Liso/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/metabolismo , Galinhas , Dados de Sequência Molecular , Quinase de Cadeia Leve de Miosina/química , Miosinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
13.
J Neurosci ; 16(22): 7161-70, 1996 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-8929425

RESUMO

Morphological changes in the dendritic spines have been postulated to participate in the expression of synaptic plasticity. The cytoskeleton is likely to play a key role in regulating spine structure. Here we examine the molecular mechanisms responsible for the changes in spine morphology, focusing on drebrin, an actin-binding protein that is known to change the properties of actin filaments. We found that adult-type drebrin is localized in the dendritic spines of rat forebrain neurons, where it binds to the cytoskeleton. To identify the cytoskeletal proteins that associated with drebrin, we isolated drebrin-containing cytoskeletons using immunoprecipitation with a drebrin antibody. Drebrin, actin, myosin, and gelsolin were co-precipitated. We next examined the effect of drebrin on actomyosin interaction. In vitro, drebrin reduced the sliding velocity of actin filaments on immobilized myosin and inhibited the actin-activated ATPase activity of myosin. These results suggest that drebrin may modulate the actomyosin interaction within spines and may play a role in the structure-based plasticity of synapses.


Assuntos
Córtex Cerebral/citologia , Citoesqueleto/química , Dendritos/ultraestrutura , Neuropeptídeos/fisiologia , Actinas/análise , Actinas/metabolismo , Actomiosina/antagonistas & inibidores , Fatores Etários , Animais , Química Encefálica , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Dendritos/química , Gelsolina/análise , Gelsolina/metabolismo , Immunoblotting , Isomerismo , Microscopia Confocal , Microscopia Eletrônica , Miosinas/análise , Miosinas/metabolismo , Neurônios/química , Neurônios/ultraestrutura , Neuropeptídeos/análise , Neuropeptídeos/química , Prosencéfalo/química , Ligação Proteica/fisiologia , Ratos , Ratos Wistar , Sinapses/química , Membranas Sinápticas/química , Sinaptossomos/química , Sinaptossomos/ultraestrutura
14.
Trends Pharmacol Sci ; 17(8): 284-7, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8810874

RESUMO

Myosin light chain kinase (MLCK) is a key regulator of smooth muscle contraction. The most conspicuous form of regulation is achieved by phosphorylation of the myosin light chain, allowing myosin to interact with actin. This interaction is regulated by actin-binding proteins that modulate actin filaments. In this review Kazuhiro Kohama and colleagues consider MLCK as an actin-binding protein and attempt to shed light on the cross-talk between the different kinds of regulation of the actin-myosin interaction in smooth muscle. An understanding of these mechanisms will assist the development of compounds with therapeutic importance in muscular disorders.


Assuntos
Trifosfato de Adenosina/fisiologia , Quinase de Cadeia Leve de Miosina/fisiologia , Miosinas/fisiologia , Actinas/metabolismo , Animais , Humanos , Ligação Proteica
15.
J Biochem ; 118(1): 1-3, 1995 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8537296

RESUMO

Myosin light chain kinase (MLCK) was prepared from smooth muscle of bovine aorta. MLCK inhibited the ATP-dependent movement of actin filaments on a glass surface coated with smooth muscle myosin that had been phosphorylated. The inhibitory effect was abolished by calmodulin in the presence of Ca2+ (Ca-CaM). The abolition was also observed when the concentration of actin filaments was increased. The inhibitory effect and its abolition were related to the actin-binding activity of MLCK, that is antagonized by Ca-CaM.


Assuntos
Actinas/metabolismo , Trifosfato de Adenosina/antagonistas & inibidores , Músculo Liso Vascular/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Bovinos , Ligação Proteica , Propriedades de Superfície
16.
J Biochem ; 116(6): 1377-82, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7706232

RESUMO

Myosin light chain kinase (MLCK) is present in muscle cells including those of smooth muscle as an actin-binding protein. By avoiding complication introduced as a result of kinase activity of MLCK, we have demonstrated regulatory role of MLCK through its actin-binding activity [Kohama et al. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211]. To analyze such a regulatory role of MLCK, we compared the effects of MLCK on the velocity of the movement of actin filaments on a surface coated with smooth muscle myosin with those of another actin-binding proteins in smooth muscle, namely, caldesmon (CaD) and calponin (CaP). Both CaD and CaP stimulated movement when their concentrations were low, but they inhibited movement as their concentrations were increased. Calmodulin (CaM) in the presence of Ca2+ (Ca-CaM) antagonized the inhibition but hardly affected the stimulation. The effect of MLCK, by contrast, was simply inhibitory when Ca-CaM was not present. No stimulation was observed until Ca-CaM was added. The inhibitory ability of these actin-binding proteins increased in the following order: CaD < CaP < MLCK. The effect of MLCK and CaD on movement was further examined on surfaces coated with skeletal muscle myosin. The basic effect was similar to that observed with smooth muscle myosin. However, 10-fold greater concentrations of MLCK and CaD were required for a comparable effect. Such an increase in the required concentration was also observed when the velocity of movement was increased by elevation of the temperature during the assay with smooth muscle myosin. Thus, it is the velocity of movement itself that determines the required concentrations of MLCK and CaD.


Assuntos
Proteínas dos Microfilamentos/fisiologia , Quinase de Cadeia Leve de Miosina/fisiologia , Actinas/fisiologia , Animais , Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/farmacologia , Calmodulina/fisiologia , Proteínas de Ligação a Calmodulina/farmacologia , Galinhas , Calefação , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Músculo Esquelético/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Quinase de Cadeia Leve de Miosina/farmacologia , Miosinas/farmacologia , Miosinas/fisiologia , Calponinas
17.
Cell Motil Cytoskeleton ; 29(3): 250-8, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7895289

RESUMO

We reported previously that smooth muscle caldesmon stimulates the ATP-dependent interaction between actin and phosphorylated smooth muscle myosin, as monitored by ATPase measurement and in vitro motility assay. Furthermore, this effect changes from stimulatory to inhibitory with increasing concentrations of caldesmon [Ishikawa et al., 1991: J. Biol. Chem. 266:21784-21790]. The N-terminal (myosin-binding) fragment and the C-terminal (actin-binding) fragment were purified from digests of caldesmon. The effects of the myosin-binding fragment and the actin-binding fragment on the interaction were stimulatory and inhibitory, respectively, indicating that stimulatory and inhibitory domains are localized in the myosin-binding domain and actin-binding domain of caldesmon, respectively. The effect of the myosin-binding fragment on the interaction was exclusively stimulatory when the interaction was challenged by caldesmon, both at lower and higher concentrations. However, the actin-binding fragment had no effect on the interaction at lower concentrations and inhibited the interaction at higher concentrations. Thus, the stimulatory effect of caldesmon that is observed at lower concentrations can be explained by the hypothesis that the stimulatory effect of the myosin-binding domain predominates over the inhibitory effect of the actin-binding domain when the concentration of caldesmon is low. With uncleaved caldesmon, we also emphasized the role of the myosin-binding domain in the stimulation as follows; the stimulatory effect of caldesmon became obscured when binding of caldesmon to myosin was competed by the exogenous caldesmon-binding fragment of myosin.


Assuntos
Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a Calmodulina/farmacologia , Músculo Liso/metabolismo , Miosinas/metabolismo , Animais , Sítios de Ligação , Galinhas , Relação Dose-Resposta a Droga , Moela das Aves , Contração Muscular , Fragmentos de Peptídeos/farmacologia , Ligação Proteica
18.
J Biochem ; 113(6): 643-5, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8370658

RESUMO

Calponin, a calmodulin-binding protein of smooth muscle that inhibits the actin-myosin interaction by binding to actin, was shown to bind to myosin and to stimulate the ATPase activity of myosin to some extent. Actin abolished this myosin-linked, stimulatory effect of calponin. Ca(2+)-calmodulin affected neither the myosin-binding activity nor the stimulatory effect of calponin. We further presented a few data which suggest that calponin may exert regulatory activity toward myosin in quite a different way from caldesmon, another smooth muscle protein that binds to myosin, actin, and calmodulin.


Assuntos
Proteínas de Ligação ao Cálcio/farmacologia , Miosinas/metabolismo , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , Proteínas de Ligação a Calmodulina/farmacologia , Galinhas , Técnicas In Vitro , Cinética , Proteínas dos Microfilamentos , Proteínas Musculares/farmacologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Músculos/efeitos dos fármacos , Músculos/enzimologia , Calponinas
19.
Am J Physiol ; 264(2 Pt 1): C287-95, 1993 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8447362

RESUMO

In the present work we investigated the influence of vitamin D3 metabolites on Na(+)-dependent phosphate (Pi) transport in the clonal osteoblastic cell line UMR-106. The vitamin D3 metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] dose-dependently inhibited Pi transport with a half-maximal concentration of approximately 5 x 10(-11) M. The effect of 1,25(OH)2D3 was first observed after 8 h of preincubation period. Inhibition of phosphate uptake was relatively specific for the 1,25(OH)2D3 analogue of vitamin D3. The potency order was 1,25(OH)2D3 >> 24,25-dihydroxyvitamin D3 > 25-[3H]hydroxyvitamin D3. Kinetically, 1,25(OH)2D3 decreased the maximal velocity of the phosphate uptake system, whereas the affinity for phosphate was unaffected. Activation of protein kinase C (PKC) in UMR-106 cells stimulated Na(+)-dependent Pi transport. Nonetheless, the inhibitory effect of 1,25(OH)2D3 on Pi transport was not related to downregulation of PKC. Chemical determination of intracellular Pi showed a 50% reduction after 24-h preincubation with 10(-8) M 1,25(OH)2D3. We conclude that 1,25(OH)2D3 inhibits Na(+)-dependent phosphate transport in osteoblastic cells. This in turn leads to intracellular Pi depletion. The physiological implication of this phenomenon on the effects of vitamin D on osteoblasts in situ is discussed.


Assuntos
Calcitriol/farmacologia , Osteoblastos/metabolismo , Fosfatos/metabolismo , Sódio/fisiologia , Transporte Biológico/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cinética , Osteoblastos/citologia , Proteína Quinase C/fisiologia
20.
Am J Physiol ; 263(6): E1070-6, 1992 12.
Artigo em Inglês | MEDLINE | ID: mdl-1476179

RESUMO

Cytosolic free calcium ([Ca2+]i) is an important regulator of bone cell physiology. We studied the interaction of vitamin D metabolites on the hormonal-activated Ca message system in the osteoblastic cell line UMR-106. The acute rise in [Ca2+]i induced by different calciotropic hormones [parathyroid hormone, prostaglandin E2 (PGE2)] was dose dependently blunted by 1,25-dihydroxyvitamin D [1,25(OH)2D3; half-maximal inhibitory concn approximately 5 x 10(-11) M] and was initially observed after 8 h of preincubation. The 1,25(OH)2D3 metabolite of vitamin D was two orders of magnitude more potent than 24,25(OH)2D3 and 25(OH)D3. To discern between an effect of 1,25(OH)2D3 on hormonal-induced Ca2+ entry through the plasma membrane channel vs. release of Ca2+ from internal stores, we suspended fura-2-loaded cells in Mn2+ rather than Ca2+ buffers. In cells preincubated with 1,25(OH)2D3, [Ca2+]i release (indicated by [Ca2+]i transient) was significantly blunted, whereas Mn2+ influx (indicating Ca2+ flux across the plasma membrane) was unaltered, suggesting a selective effect of 1,25(OH)2D3 on hormonal-activated release of Ca2+ from intracellular stores. 1,25(OH)2D3 also inhibited the PGE2-induced production of inositol 1,4,5-trisphosphate. We conclude that, in osteoblasts, chronic (hours) incubation with 1,25(OH)2D3 leads to attenuated stimulation of the [Ca2+]i transduction pathway by calciotropic hormones. This effect of 1,25(OH)2D3 may provide a cellular basis for the synergism between the effects of vitamin D and calciotropic hormones at the bone level.


Assuntos
Calcitriol/farmacologia , Cálcio/metabolismo , Citosol/metabolismo , Hormônios/fisiologia , Osteoblastos/metabolismo , Relação Dose-Resposta a Droga , Concentração Osmolar , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositóis/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas
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