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1.
Biotechniques ; 73(6): 273-279, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36398847

RESUMO

There are various approaches in which one can isolate microglia from murine brains, such as immunomagnetic, density gradient, FACS and differential adhesive methods. In this procedure a modified flask-tapping approach was used due to its simplicity and reproducibility. Our protocol requires only a single step to isolate the microglia from the mixed cell population. Once the microglia were isolated, we characterized cell purity, microglial morphology and phagocytic activity. The single-step protocol, without the need for additional astrocyte or oligodendrocyte separation, allows microglial cells to be used immediately for experimental purposes. The protocol is low-cost and can be performed in any lab with standard cell-culture equipment.


Assuntos
Técnicas de Cultura de Células , Microglia , Animais , Camundongos , Separação Celular/métodos , Reprodutibilidade dos Testes , Técnicas de Cultura de Células/métodos , Encéfalo , Citometria de Fluxo/métodos , Células Cultivadas
2.
Biotechniques ; 70(5): 278-284, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33969703

RESUMO

Cellular redox changes are common in apoptosis, immune function, signaling pathways and cancer. The authors aimed to develop a single-wavelength method using the superior fluorescence sensitivity of a flow cytometer for measuring redox-sensitive green fluorescent protein signal during oxidative stress in cell lines. The single-wavelength method was able to discern small differences in oxidative stress between cell lines and between the cytoplasmic and mitochondrial compartments within the same cell line. In Chinese hamster ovary cells, the mitochondrial matrix compartment was more sensitive to oxidative stress compared with MDA-MB-231 cells, and the rapid changes in redox state were followed by a slow recovery phase. The authors conclude that this simplified method is useful and preferred for studies where alterations in overall redox-sensitive green fluorescent protein expression are controlled.


Assuntos
Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Mitocôndrias , Oxirredução , Animais , Células CHO , Cricetinae , Cricetulus , Mitocôndrias/metabolismo
3.
Int J Pharm Compd ; 25(1): 48-51, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33503009

RESUMO

Water activity refers to the amount of water in a system that is available for microbial growth. Commercial water activity meters are precision instruments with the ability to determine water activity to within 0.001 units and carry prices of $10,000 or more. The purpose of the study was to build a robust water activity meter from commercially available components and measure the water activity of liquids commonly used in compounded formulations. SHT-85 sensors were connected to an Adafruit Feather HUZZAH microcontroller. Standard salt slurries and common oral liquid vehicles were monitored in airtight containers until equilibrium was reached. Standard curves were used to convert sensor outputs to water activity values. The standard curves were linear with R2 >0.99. Oral liquid vehicles showed water activity values between 0.62 and 0.99. Samples equilibrated within 9.5 hours in 16-ounce jars or 2.5 hours in 20-mL vials. Stirring the sample during measurement reduced equilibration time in 16-ounce jars, but not in 20-mL vials. The inexpensive meter was accurate and precise in measuring the water activity of standards and selected oral vehicles. An accurate and precise water activity meter was constructed at a cost of approximately $150. Common oral formulation components have a water activity of >0.6, the United States Pharmacopeial threshold for requiring a preservative. Pharmacists should use caution when diluting preserved vehicles.


Assuntos
Farmacêuticos , Água , Humanos
4.
PLoS One ; 12(12): e0189589, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29240845

RESUMO

IQGAP1 interacts with a number of binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP-related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 is known to play a role in actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. In addition, phosphorylation near the CT domain is thought to control IQGAP1 activity through regulation of intramolecular interaction. In a previous study, we discovered that IQGAP1 preferentially localizes to retracting areas in B16F10 mouse melanoma cells, not areas of membrane ruffling and lamellipodium protrusion. Nothing is known of the domains needed for retraction localization and very little is known of IQGAP1 function in the actin cytoskeleton of melanoma cells. Thus, we examined localization of IQGAP1 mutants to retracting areas, and characterized knock down phenotypes on tissue culture plastic and physiologic-stiffness hydrogels. Localization of IQGAP1 mutants (S1441E/S1443D, S1441A/S1443A, ΔCHD, ΔGRD or ΔCT) to retracting and protruding cell edges were measured. In retracting areas there was a decrease in S1441A/S1443A, ΔGRD and ΔCT localization, a minor decrease in ΔCHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion just behind the lamellipodium leading edge, we surprisingly observed both ΔGRD and ΔCT localization, and increased number of microtubules. IQGAP1 knock down caused loss of cell polarity on laminin-coated glass, decreased proliferation on tissue culture polystyrene, and abnormal spheroid growth on laminin-coated hydrogels. We propose that the GRD and CT domains regulate IQGAP1 localization to retracting actin networks to promote a tumorigenic role in melanoma cells.


Assuntos
Melanoma Experimental/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Hidrogéis , Melanoma Experimental/patologia , Camundongos , Microtúbulos/metabolismo , Mutação , Pseudópodes/metabolismo , Proteínas Ativadoras de ras GTPase/genética
5.
Biochem Biophys Res Commun ; 448(1): 39-44, 2014 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-24747073

RESUMO

IQGAP1 has emerged as a key component in the regulation of cytoskeleton dynamics during cell migration, maintenance of adherens junctions, microbial pathogenesis and intracellular trafficking. IQGAP1 is known to localize to the protruding edge of lamellipodia in a variety of cell types and interact with regulators of actin dynamics. Here, we provide evidence suggesting a novel role of IQGAP1 in cell motility through cell edge retraction. In some of the cell lines examined, IQGAP1 was markedly separated from WAVE localization suggesting IQGAP1 may localize to retracting edges. B16F10 mouse melanoma cells exhibited the most restricted separation in which the appearance of GFP-IQGAP1 correlated with cell edge retraction velocity and the disappearance of mCherry-Arp3. These results demonstrate that in some cell types IQGAP1 may function to promote cell retraction not lamellipodium edge protrusion. In addition, we examined co-localization of IQGAP1 with adhesion site markers, myosin IIA, calmodulin and IQGAP2. In areas rich in IQGAP1 there was decreased immunofluorescence staining of vinculin, paxillin and phosphorylated-tyrosine indicating adhesion site disassembly. Interestingly, calmodulin, but not myosin IIA or IQGAP2, co-localized with IQGAP1 in areas of cell retraction. Overall these results suggest a new role of IQGAP1, distinct form IQGAP2, in cell migration through up regulation of contractility and downregulation of adhesion sites potentially through calmodulin interaction.


Assuntos
Movimento Celular/fisiologia , Animais , Calmodulina/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Camundongos , Miosina não Muscular Tipo IIA/metabolismo , Pseudópodes/fisiologia , Proteínas Ativadoras de ras GTPase/metabolismo
6.
Biochem Biophys Res Commun ; 417(1): 67-72, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22120625

RESUMO

The plus-ends of microtubules target the cell cortex to modulate actin protrusion dynamics and polarity, but little is known of the molecular mechanism that couples the interaction. EB1 protein associates with the plus-ends of microtubules, placing EB1 in an ideal spatial position to mediate microtubule-actin cross talk. The objective of the current study was to further understand intracellular signaling involved in EB1-dependent cell polarity and motility. B16F10 mouse melanoma cells were depleted of EB1 protein using short hair-pin RNA interference. Correlative live cell-immunofluorescence microscopy was performed to determine localization of WAVE2 and IQGAP1 to protruding versus retracting edges. EB1 knock down caused poor subcellular separation of WAVE2 and IQGAP1, and overall decreased localization. Activation of PKC corrected defects in WAVE2 and IQGAP1 localization, cell spreading and cell shape to levels observed in control cells, but did not correct defects in cell migration. Consistent with these findings, decreased PKC phosphorylation was observed in EB1 knock down cells. These findings support a model where EB1 protein links microtubules to actin protrusion and cell polarity through signaling pathways involving PKC.


Assuntos
Polaridade Celular , Melanoma Experimental/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Quinase C/biossíntese , Animais , Linhagem Celular Tumoral , Movimento Celular , Ativação Enzimática , Técnicas de Silenciamento de Genes , Melanoma Experimental/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo
7.
Mol Biol Cell ; 21(15): 2661-73, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20519438

RESUMO

Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150(Glued) (J. Cell Biol. 2004: 166, 1003-1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150(Glued) as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150(Glued). We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.


Assuntos
Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dineínas/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Ácido Okadáico/farmacologia , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteoma/metabolismo
8.
Cancer Lett ; 284(1): 30-6, 2009 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-19427113

RESUMO

Remodeling of actin and microtubule cytoskeletons is thought to be coupled; however, the interplay between these two systems is not fully understood. We show a microtubule end-binding protein, EB1, is required for formation of polarize morphology and motility of melanoma cells. EB1 depletion decreased lamellipodia protrusion, and resulted in loss of opposed protruding and retracting cell edges. Lamellipodia attenuation correlated with mis-localization of filopodia throughout the cell and decreased Arp3 localization. EB1-depleted cells displayed less persistent migration and reduced velocity in single-cell motility experiments. We propose EB1 coordinates melanoma cell migration through regulating the balance between lamellipodial and filopodial protrusion.


Assuntos
Actinas/fisiologia , Movimento Celular/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Pseudópodes/fisiologia , Animais , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Técnicas de Silenciamento de Genes , Melanoma , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/ultraestrutura , Pseudópodes/ultraestrutura
9.
J Cell Biol ; 184(5): 691-706, 2009 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-19255245

RESUMO

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Células CHO , Diferenciação Celular/fisiologia , Cricetinae , Cricetulus , Dimerização , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/ultraestrutura , Multimerização Proteica , Estrutura Terciária de Proteína
10.
J Cell Sci ; 120(Pt 7): 1235-44, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17356063

RESUMO

Interaction between the microtubule system and actin cytoskeleton has emerged as a fundamental process required for spatial regulation of cell protrusion and retraction activities. In our current studies, analysis of digital fluorescence images revealed targeting of microtubules to filopodia in B16F1 melanoma cells and fibroblasts. We investigated the functional consequence of targeting on filopodia reorganization and examined mechanisms by which microtubules may be guided to, or interact with, filopodia. Live cell imaging studies show that targeting events in lamellipodia wings temporally correlated with filopodia turning toward the lamellipodium midline and with filopodia merging. Rapid uncoupling of targeting with nocodazole decreased filopodia merging events and increased filopodia density. Total internal reflection fluorescence microscopy identified microtubules near the ventral surface and upward movement of targeted filopodia. The role of adhesion sites and microtubule plus-end proteins in targeting was investigated. Correlation of adhesion sites with microtubule targeting to filopodia was not observed and depletion of microtubule plus-end proteins did not significantly alter targeting frequency. We propose that microtubules target filopodia, independent of focal adhesions and plus-end proteins, causing filopodia movement and microtubules regulate filopodia density in lamellipodia wings through filopodia merging events.


Assuntos
Adesões Focais/metabolismo , Microtúbulos/metabolismo , Pseudópodes/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Células Cultivadas , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes , Adesões Focais/efeitos dos fármacos , Imuno-Histoquímica , Cinética , Proteínas Luminescentes/metabolismo , Melanoma Experimental/patologia , Camundongos , Microscopia de Fluorescência , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Paclitaxel/farmacologia , Faloidina , Pseudópodes/efeitos dos fármacos , Interferência de RNA , Transfecção/métodos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologia
11.
J Biol Chem ; 278(28): 25808-15, 2003 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-12736251

RESUMO

CCN1 (cysteine-rich 61) and CCN2 (connective tissue growth factor) are growth factor-inducible immediate-early gene products found in atherosclerotic lesions, restenosed blood vessels, and healing cutaneous wounds. Both CCN proteins have been shown to support cell adhesion and induce cell migration through interaction with integrin receptors. Recently, we have identified integrin alphaMbeta2 as the major adhesion receptor mediating monocyte adhesion to CCN1 and CCN2 and have shown that the alphaMI domain binds specifically to both proteins. In the present study, we demonstrated that activated monocytes adhered to a synthetic peptide (CCN1-H2, SSVKKYRPKYCGS) derived from a conserved region within the CCN1 C-terminal domain, and this process was blocked by the anti-alphaM monoclonal antibody 2LPM19c. Consistently, a glutathione S-transferase (GST) fusion protein containing the alphaMI domain (GST-alphaMI) bound to immobilized CCN1-H2 as well as to the corresponding H2 sequence in CCN2 (CCN2-H2, TSVKTYRAKFCGV). By contrast, a scrambled CCN1-H2 peptide and an 18-residue peptide derived from an adjacent sequence of CCN1-H2 failed to support monocyte adhesion or alphaMI domain binding. To confirm that the CCN1-H2 sequence within the CCN1 protein mediates alphaMbeta2 interaction, we developed an anti-peptide antibody against CCN1-H2 and showed that it specifically blocked GST-alphaMI binding to intact CCN1. Collectively, these results identify the H2 sequence in CCN1 and CCN2 as a novel integrin alphaMbeta2 binding motif that bears no apparent homology to any alphaMbeta2 binding sequence reported to date.


Assuntos
Arteriosclerose/metabolismo , Proteínas Imediatamente Precoces/química , Peptídeos e Proteínas de Sinalização Intercelular/química , Antígeno de Macrófago 1/química , Cicatrização , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Adesão Celular , Proteína Rica em Cisteína 61 , Relação Dose-Resposta a Droga , Glutationa Transferase/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Antígeno de Macrófago 1/metabolismo , Manganês/metabolismo , Manganês/farmacologia , Dados de Sequência Molecular , Monócitos/metabolismo , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo
12.
Blood ; 99(12): 4457-65, 2002 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12036876

RESUMO

Cysteine-rich 61 (Cyr61, CCN1) and connective tissue growth factor (CTGF, CCN2) are growth factor-inducible immediate-early gene products found in blood vessel walls and healing cutaneous wounds. We previously reported that the adhesion of endothelial cells, platelets, and fibroblasts to these extracellular matrix-associated proteins is mediated through integrin receptors. In this study, we demonstrated that both Cyr61 and CTGF are expressed in advanced atherosclerotic lesions of apolipoprotein E-deficient mice. Because monocyte adhesion and transmigration are important for atherosclerosis, wound healing, and inflammation, we examined the interaction of THP-1 monocytic cells and isolated peripheral blood monocytes with Cyr61 and CTGF. THP-1 cells and monocytes adhered to Cyr61- or CTGF-coated wells in an activation-dependent manner and this process was mediated primarily through integrin alpha(M)beta(2). Additionally, expression of alpha(M)beta(2) on human embryonic kidney 293 cells resulted in enhanced cell adhesion to Cyr61. Consistent with these data, a GST-fusion protein containing the I domain of the integrin alpha(M) subunit bound specifically to immobilized Cyr61 or CTGF. We have also investigated the requirement of cell surface heparan sulfate proteoglycans (HSPGs) as coreceptors for monocyte adhesion to Cyr61. Pretreatment of monocytes with heparin or heparinase I resulted in partial inhibition of cell adhesion to Cyr61. However, monocytes, but not fibroblasts, were capable of adhering to a Cyr61 mutant deficient in heparin binding activity. Collectively, these results show that activated monocytes adhere to Cyr61 and CTGF through integrin alpha(M)beta(2) and cell surface HSPGs. However, unlike fibroblast adhesion to Cyr61, cell surface HSPGs are not absolutely required for this adhesion process.


Assuntos
Substâncias de Crescimento/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Antígeno de Macrófago 1/metabolismo , Monócitos/química , Animais , Apolipoproteínas E/genética , Arteriosclerose/etiologia , Arteriosclerose/genética , Arteriosclerose/metabolismo , Adesão Celular/fisiologia , Fator de Crescimento do Tecido Conjuntivo , Proteína Rica em Cisteína 61 , Modelos Animais de Doenças , Humanos , Proteínas Imediatamente Precoces/biossíntese , Imuno-Histoquímica , Integrinas/metabolismo , Integrinas/fisiologia , Antígeno de Macrófago 1/química , Antígeno de Macrófago 1/fisiologia , Camundongos , Camundongos Knockout , Monócitos/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína
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