RESUMO
Mycoplasmas are frequent contaminants of cultured cells, leading to alterations in cellular gene expression, protein synthesis, signal transduction, and metabolic pathways. Mycoplasma hyorhinis, the major contaminant of tissue cultures, has been implicated in a variety of diseases in swine. Most human and animal mycoplasmas remain attached to the surface of epithelial cells. Nonetheless, we have recently shown that M. hyorhinis is able to invade nonphagocytic melanoma cells. In the present study, we show by confocal laser scanning microscopy, that by exposing mice splenocytes to intact M. hyorhinis, intracellular mycoplasmas were detected. Mycoplasmal components were not detected within splenocytes after exposure to heat inactivated M. hyorhinis or to a purified M. hyorhinis lipoprotein (LPP) fraction. However, incubation of the splenocytes with intact M. hyorhinis cells, heat inactivated cells or M. hyorhinis LPP fraction induced accelerated cell proliferation and the secretion of interferon gamma and interleukin 17. Thus, M. hyorhinis and its LPPs can be added to the list of infectious agents causing direct stimulation of proinflammatory responses by mammalian lymphocytes.
Assuntos
Proliferação de Células , Interferon gama/metabolismo , Interleucina-17/metabolismo , Linfócitos/imunologia , Linfócitos/microbiologia , Mycoplasma hyorhinis/imunologia , Mycoplasma hyorhinis/fisiologia , Animais , Endocitose , Camundongos , Microscopia ConfocalRESUMO
We demonstrated that when M. pneumoniae was grown on an abiotic surface of either glass or polystyrene with a serum-containing medium, the bacteria adhered to the surface and formed highly differentiated volcano-like biofilm structures. As adherence to the surface and/or biofilm formation was totally inhibited by anti-P1 polyclonal monospecific antibodies, we suggest that the adherence of M. pneumoniae to the abiotic surface and/or biofilm formation is associated with P1, the major tip organelle protein of this organism. Furthermore, adherence and/or biofilm formation was markedly inhibited by treating the serum component of the growth medium with neuraminidase or by growing the bacteria in the presence of sialyllactose, suggesting that the initial step in the adherence to and/or biofilm formation by M. pneumoniae on an abiotic surface is the interaction of the bacterium through its tip organelle with sialic acid residues of serum glycoproteins.
Assuntos
Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Mycoplasma pneumoniae/crescimento & desenvolvimento , Adesinas Bacterianas/metabolismo , Meios de Cultura/química , Glicoproteínas/química , Lactose/análogos & derivados , Lactose/química , Ácido N-Acetilneuramínico/química , Ácidos Siálicos/químicaRESUMO
Mycoplasma hyorhinis (strain MCLD) was recently isolated from a melanoma cell culture. Growth of MCLD was considerably improved by 24 serial passages in a modified Hayflick's mycoplasma medium. Transmission electron microscopy showed that MCLD exhibits a polymorphic appearance, with ovoid or elongated cells frequently harboring an electron-dense core at one of the poles. Adherence of M. hyorhinis to melanoma cells followed saturation kinetics. Furthermore, although M. hyorhinis has been considered to remain attached to the surface of the host cells, we show for the first time, qualitatively by confocal laser scanning microscopy and quantitatively by a gentamicin resistance assay, that MCLD is able to invade melanoma cells. The ingested mycoplasmas were randomly distributed in the cytoplasm, tending to concentrate near the plasma membrane. Both adherence to and invasion of melanoma cells by M. hyorhinis strain MCLD were dramatically enhanced by mild proteolytic digestion with proteinase K (2.5 microg/mg cell protein for 2.5 min at 37 degrees C) that affected the surface-exposed proteins of this organism, mainly the major 47-kDa lipoprotein. We suggest that the intracellular location of M. hyorhinis strain MCLD is a privileged niche, which may explain the survival of M. hyorhinis in tissue cultures. The enhanced binding to and invasion of melanoma cells by protease treatment may be due to either the activation or the enhanced exposure of an adhesin(s) on the mycoplasmal cell surface.
Assuntos
Endopeptidase K/metabolismo , Melanoma/microbiologia , Infecções por Mycoplasma/enzimologia , Mycoplasma hyorhinis/patogenicidade , Linhagem Celular Tumoral , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mycoplasma hyorhinis/ultraestruturaRESUMO
By serial passages through media containing decreasing concentrations of horse serum, the sterol-requiring Mycoplasma penetrans strain HF-2 was adapted to grow in a serum-free medium supplemented with bovine serum albumin, cholesterol and free fatty acids. Chemical analysis of membrane preparations obtained from the native and adapted strains revealed two major differences. (1) The polar lipid fraction of the native strain contains, in addition to the de novo-synthesized phospholipids, exogenous lipids incorporated unchanged from the growth medium, whereas exogenous lipids were not detected in the adapted strain. (2) Protein analyses of the native and adapted strains showed that upon adaptation, the 42-kDa membrane lipoprotein, one of the two major lipoproteins of this organism, was missing. Studies on the adhesion to, and invasion of HeLa cells by the native and adapted strains revealed that whereas the adherence to HeLa cells of the adapted strain was almost the same as that of the native strain, the invasiveness of the adapted strain into HeLa cells was very low or nonexistent.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Infecções por Mycoplasma/microbiologia , Mycoplasma penetrans/fisiologia , Animais , Proteínas de Bactérias/química , Meios de Cultura/metabolismo , Células HeLa , Humanos , Lipoproteínas/química , Peso Molecular , Mycoplasma penetrans/químicaRESUMO
Exposing skin to ultraviolet (UV) radiation contributes to photoaging and to the development of skin cancer by DNA lesions and triggering inflammatory and other harmful cellular cascades. The present study tested the ability of unique lipid molecules, polyhydroxylated fatty alcohols (PFA), extracted from avocado, to reduce UVB-induced damage and inflammation in skin. Introducing PFA to keratinocytes prior to their exposure to UVB exerted a protective effect, increasing cell viability, decreasing the secretion of IL-6 and PGE(2), and enhancing DNA repair. In human skin explants, treating with PFA reduced significantly UV-induced cellular damage. These results support the idea that PFA can play an important role as a photo-protective agent in UV-induced skin damage.
Assuntos
Álcoois Graxos/farmacologia , Queratinócitos/efeitos dos fármacos , Persea/química , Pele/efeitos dos fármacos , Anti-Inflamatórios , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA , Reparo do DNA , Dinoprostona/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/metabolismo , Queratinócitos/efeitos da radiação , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase , Dímeros de Pirimidina/análise , Pele/efeitos da radiação , Raios UltravioletaRESUMO
Tissue-type plasminogen activator (tPA) regulates vascular contractility through the low-density lipoprotein-related receptor (LRP), and this effect is inhibited by plasminogen activator inhibitor type 1 (PAI-1). We now report that tPA-mediated vasocontraction also requires the integrin alphavbeta3. tPA-induced contraction of rat aortic rings is inhibited by the Arg-Gly-Asp (RGD) peptide and by monoclonal anti-alphavbeta3 antibody. tPA induces the formation of a complex between LRP and alphavbeta3 in vascular smooth muscle cells. The three proteins are internalized within 10 min, causing the cells to become refractory to the readdition of tPA. LRP and alphavbeta3 return to the cell surface by 90 min, restoring cell responsiveness to tPA. PAI-1 and the PAI-1-derived hexapeptide EEIIMD abolish the vasocontractile activity of tPA and inhibit the tPA-mediated interaction between LRP and alphavbeta3. tPA induces calcium mobilization from intracellular stores in vascular smooth muscle cells, and this effect is inhibited by PAI-1, RGD, and antibodies to both LRP and alphavbeta3. These data indicate that tPA-mediated vasocontraction involves the coordinated interaction of LRP with alphavbeta3. Delineating the mechanism underlying these interactions and the nature of the signals transduced may provide new tools to regulate vascular tone and other consequences of tPA-mediated signaling.
Assuntos
Integrina alfaVbeta3/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/fisiologia , Vasoconstrição/efeitos dos fármacos , Animais , Cálcio/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Integrina alfaVbeta3/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Músculo Liso Vascular/metabolismo , Oligopeptídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Ligação Proteica , Ratos , Vasoconstrição/fisiologiaRESUMO
Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37 degrees C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans. The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with beta-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry.
Assuntos
Aderência Bacteriana , Mycoplasma fermentans/metabolismo , Mycoplasma fermentans/patogenicidade , Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Colesterol/metabolismo , Filipina/metabolismo , Células HeLa , HumanosRESUMO
Ascorbic acid (AA) and its derivatives participate in vitro in oxidative-reductive reactions both as antioxidants and as prooxidants. The physiological relevance of these prooxidant effects of AA and its derivatives remains unclear. There is little evidence that AA can initiate formation of reactive oxygen species (ROS) or lipid peroxidation in cells or tissue. In order to examine the effect of AA and its derivative palmitoyl ascorbate on in situ intracellular ROS production and lipid peroxidation, 2('),7(')-dichlorofluorescin diacetate (DCFH-DA) and cis-parinaric acid were used as fluorescent probes in cultural neonatal foreskin fibroblasts. The results demonstrated that the effect of AA depended on the in vitro growth conditions. AA induced augmentation of the intracellular ROS concentration in newly plated (24 hours) cells. However, in cells cultured for 72 hours, AA had a different effect: it moderately reduced intracellular ROS concentration but stimulated lipid peroxidation in the cytoplasmic membrane. Palmitoyl ascorbate demonstrated significant inhibition of intracellular DCFH-DA oxidation presumably caused by inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.
RESUMO
We have previously identified alpha-defensin in association with medial smooth muscle cells (SMCs) in human coronary arteries. In the present paper we report that alpha-defensin, at concentrations below those found in pathological conditions, inhibits phenylephrine (PE)-induced contraction of rat aortic rings. Addition of 1 microM alpha-defensin increased the half-maximal effective concentration (EC(50)) of PE on denuded aortic rings from 32 to 630 nM. The effect of alpha-defensin was dose dependent and saturable, with a half-maximal effect at 1 microM. alpha-Defensin binds to human umbilical vein SMCs in a specific manner. The presence of 1 microM alpha-defensin inhibited the PE-mediated Ca(++) mobilization in SMCs by more than 80%. The inhibitory effect of alpha-defensin on contraction of aortic rings and Ca(++) mobilization was completely abolished by anti-low-density lipoprotein receptor-related protein/alpha(2-)macroglobulin receptor (LRP) antibodies as well as by the antagonist receptor-associated protein (RAP). alpha-Defensin binds directly to isolated LRP in a specific and dose-dependent manner; the binding was inhibited by RAP as well as by anti-LRP antibodies. alpha-Defensin is internalized by SMCs and interacts with 2 intracellular subtypes of protein kinase C (PKC) involved in muscle contraction, alpha and beta. RAP and anti-LRP antibodies inhibited the binding and internalization of alpha-defensin by SMCs and its interaction with intracellular PKCs. These observations suggest that binding of alpha-defensin to LRP expressed in SMCs leads to its internalization; internalized alpha-defensin binds to PKC and inhibits its enzymatic activity, leading to decreased Ca(++) mobilization and SMC contraction in response to PE.
Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , alfa-Defensinas/farmacocinética , Animais , Aorta , Sinalização do Cálcio/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Radioisótopos do Iodo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Músculo Liso Vascular/citologia , Fenilefrina/antagonistas & inibidores , Ligação Proteica , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Veias Umbilicais , Vasoconstritores/antagonistas & inibidores , alfa-Defensinas/metabolismo , alfa-Defensinas/farmacologiaRESUMO
Tissue type plasminogen activator (tPA) is a key enzyme in the fibrinolytic cascade. In this paper we report that tPA contains 2 independent epitopes that exert opposite effects on blood vessel tone. Low concentrations of tPA (1 nM) inhibit the phenylephrine (PE)-induced contraction of isolated aorta rings. In contrast, higher concentrations (20 nM) stimulate the contractile effect of PE. The 2 putative vasoactive epitopes of tPA are regulated by the plasminogen activator inhibitor-1 (PAI-1) and by a PAI-1-derived hexapeptide that binds tPA. TNK-tPA, a tPA variant in which the PAI-1 docking site has been mutated, stimulates PE-induced vasoconstriction at all concentrations used. The stimulatory, but not the inhibitory, effect of tPA on the contraction of isolated aorta rings was abolished by anti-low-density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor (LRP) antibodies. Administering tPA or TNK-tPA to rats regulates blood pressure and cerebral vascular resistance in a dose-dependent mode. In other in vivo experiments we found that the vasopressor effect of PE is more pronounced in tPA knockout than in wild-type mice. Our findings draw attention to a novel role of tPA and PAI-1 in the regulation of blood vessel tone that may affect the course of ischemic diseases.