RESUMEN
BACKGROUND: We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases. METHODS: Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-alpha (ERalpha)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERalpha or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed. RESULTS: Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2alpha and CAIX, but not to HIF-1alpha or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERalpha or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model. CONCLUSION: We conclude that in breast cancer, Mb is co-expressed with ERalpha and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours.
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Neoplasias de la Mama/metabolismo , Carcinoma Intraductal no Infiltrante/metabolismo , Mioglobina/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Estudios de Cohortes , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Mioglobina/genética , Invasividad Neoplásica , Neoplasias Hormono-Dependientes/metabolismo , Fenotipo , Pronóstico , ARN Mensajero/análisisRESUMEN
We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence-like growth arrest after about 4 wk and shortened postmitotic life span from 1-1/2 years down to 3 months. During the first 8 wk of hyperoxia-induced 'aging', overall protein degradation (breakdown of [(35)S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin-specific activity (using the fluorogenic substrate z-FR-MCA) and cytoplasmic proteasome-specific activity (measured with suc-LLVY-MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid-loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by inhibiting the proteasome.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Ceroide/farmacología , Lipofuscina/farmacología , Mitosis/efectos de los fármacos , Complejos Multienzimáticos/antagonistas & inhibidores , Catepsinas/metabolismo , Línea Celular , Ceroide/metabolismo , Quimotripsina/antagonistas & inhibidores , Quimotripsina/metabolismo , Cisteína Endopeptidasas/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Humanos , Lipofuscina/metabolismo , Pulmón , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Complejos Multienzimáticos/metabolismo , Oxígeno/metabolismo , Oxígeno/farmacología , Complejo de la Endopetidasa Proteasomal , Biosíntesis de Proteínas , Proteínas/metabolismoRESUMEN
BACKGROUND: Myocardial infarction can lead to electrical abnormalities and rhythm disturbances. However, there is limited data on the electrophysiological basis for these events. Since regional contraction abnormalities feature prominently in infarction, we investigated whether stretch of myocardium from the infarction borderzone can modulate the electrophysiological properties of cardiomyocytes via mechanoelectric feedback providing a mechanism for post-infarction arrhythmia. METHODS: Five weeks after experimental myocardial infarction (MI) in rats due to ligation of the left coronary artery (n = 26) or after sham operation (SO, n = 16), action potentials (AP) were measured in left ventricular preparations from the infarction borderzone. Sustained stretch was applied via a micrometer. RESULTS: Preparations from MI generated spontaneous electrical and contractile activity. Cardiomyocytes from MI had a comparable AP amplitude, a more negative resting membrane potential, and a prolonged AP duration (APD) when compared to SO. In SO, stretch of 150 microns increased the APD90. This was associated with stretch activated depolarizations near APD90 (SAD-90). In MI, significantly lower stretch, of only 20 microns, elicited SAD-90s, or SADs near APD50 (SAD-50). Stretch-induced events were suppressed by gadolinium, at a concentration (40 microM) normally used to inhibit stretch-activated channels. CONCLUSION: After MI, SADs are generated in the infarction borderzone at lower degrees of stretch. Increased sensitivity of the membrane potential of cardiac myocytes to mechanical stimuli may contribute to the high risk of arrhythmia after infarction. These SADs may involve the opening of stretch-activated channels.
Asunto(s)
Potenciales de Acción , Contracción Miocárdica , Infarto del Miocardio/fisiopatología , Potenciales de Acción/efectos de los fármacos , Animales , Retroalimentación , Gadolinio/farmacología , Técnicas In Vitro , Canales Iónicos/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Estrés Mecánico , Remodelación VentricularRESUMEN
Little is known about pharmacological interventions with thiophosphates or lazaroids in endothelial cells injured by hypoxia/reoxygenation with respect to membrane lipid peroxidation (LPO) caused by reactive oxygen species. Therefore, a cell line of bovine aortic endothelial cells was studied after 120-min hypoxia followed by 30-min reoxygenation, resulting in moderate and predominantly reversible injury (energy depression/cytosolic Ca2+-accumulation during hypoxia, which almost normalized during reoxygenation; membrane blebs, an increasing amount of lysosomes, vacuolization, lipofuscin formation, alterations in mitochondria size, some lyzed cells). 18.9 +/- 4.3% of the cells died. Radical-induced LPO measured as malondialdehyde continuously increased to 2.18 +/- 0.17 nmol/mg of protein after reoxygenation vs control (0.41 +/- 0.13, P < 0.05). Simultaneously, the content of 4-hydroxynonenal, a novel indicator of LPO, increased from 0.02 +/- 0.01 to 0.11 +/- 0.02 nmol/mg of protein (P < 0.01). The results support the assumption that reoxygenation injury is accompanied by an increase in membrane LPO, causing structural and functional disturbances in the monolayer. The thiophosphate WR 2721 [S-2-(3-aminopropylamino) ethylphosphorothioic acid] and the lazaroid U83836E [(-)-2-[[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl] methyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol (dihydrochloride)] were effective scavengers of .OH, being more efficient than trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbon acid) used as standard (EC50: 12, 5 and 15 microM, respectively, measured by electron spin resonance spectroscopy). One mM WR 2721, 10 microM U83836E, and 5 microM trolox C reduced formation of malondialdehyde during hypoxia/reoxygenation to 53 +/- 7, 51 +/- 10 and 48 +/- 6%, respectively (P < 0.05 each, versus control). In general, WR 2721 and U83836E prevent radical-induced membrane LPO in a model of endothelial cells injured by hypoxia/reoxygenation. The use of these two agents is a new approach to protect the endothelium against oxidative stress.
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Amifostina/farmacología , Cromanos/farmacología , Citoprotección , Endotelio Vascular/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Peroxidación de Lípido/efectos de los fármacos , Piperazinas/farmacología , Animales , Antioxidantes/farmacología , Aorta/citología , Aorta/efectos de los fármacos , Bovinos , Hipoxia de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Endotelio Vascular/citología , Oxígeno/farmacologíaRESUMEN
We have synthesized a linear, bifunctional peptide that comprises an integrin-targeting domain containing an arginine-glycine-aspartic acid tripeptide motif and a DNA-binding moiety consisting of a short stretch of 16 lysine residues. This peptide can form distinctive, condensed complexes with DNA and is capable of mediating its delivery and expression in a variety of mammalian cells in culture. Internalization is mediated by cell surface integrin receptors via a mechanism that is known to be phagocytic. We have analyzed the relationship between DNA and peptide and have investigated the conditions suitable for optimal gene delivery. The formation of condensed peptide DNA complexes leads to resistance to nuclease degradation. The level of reporter gene expression obtained is dependent on the peptide-to-DNA ratio and is enhanced in the presence of the endosomal buffer chloroquine, polyethyleneimine, and deactivated adenovirus during gene delivery. Under optimal conditions the levels of reporter gene expression obtained approach or even exceed those obtained with DNA delivered with the commercial liposome Lipofectamine. The ability to produce an efficient gene delivery system using small, easily modified, and well-defined constructs that have no constraint of particle size demonstrates the advantages of integrin-targeting peptides for gene transfer.
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Proteínas de Unión al ADN , Técnicas de Transferencia de Gen , Vectores Genéticos , Integrinas/metabolismo , Oligopéptidos , Polilisina , Células 3T3 , Animales , Células CACO-2 , Cloroquina , Proteínas de Unión al ADN/síntesis química , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Luciferasas/genética , Ratones , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Plásmidos/metabolismo , Polietileneimina , Polilisina/síntesis química , Polilisina/metabolismo , TransfecciónRESUMEN
Up- and downstream processing of human monoclonal IgM is known to bring about problems with respect to clone stability and quantity of antibodies produced. A human B cell hybridoma producing a natural polyreactive IgM antibody (CB03) was adapted to growth in serum-free medium and scaled-up using a hollow fiber bioreactor system. The process of fermentation has been carried out continuously over a period of 4 months. In comparison to stationary culture conditions in the presence of 10% fetal calf serum, antibody concentrations in hollow fiber bioreactor supernatants were found to be significantly increased. Semicontinuously harvested supernatants contained up to 400 mg/liter immunoreactive IgM antibody. During the last weeks of fermentation, a markedly reduced number of viable cells was observed, whereas antibody production seemed to remain stable. Furthermore, we detected formation of cell clusters in the fermentor system. These clusters carried IgM on the surface and secreted immunoreactive IgM antibodies. Clusters were found to represent fusions of hybridoma cells using electron microscopy. Cluster formation was accompanied by decreased glucose consumption and lactate accumulation and was not seen during growth of other human hybridomas. We discuss these results in the content of the polyreactive binding properties of this particular antibody.
Asunto(s)
Especificidad de Anticuerpos/genética , Hibridomas/inmunología , Inmunoglobulina M/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Agregación Celular/genética , Agregación Celular/inmunología , Humanos , Hibridomas/metabolismo , Hibridomas/ultraestructura , Inmunoglobulina M/genética , RatonesRESUMEN
Reactive oxygen species are thought to be important for a variety of pathological processes in the brain. Endothelial cells have been proposed as both a significant source of oxidants and targets of oxidative damage. Therefore, lipid peroxidation (LPO) was investigated and compared to biochemical and morphological alterations in cultured pig brain capillary endothelial cells after hypoxia (120 min. 95% N2/5% CO2) and reoxygenation (30 min. 95% O2/5% CO2). The content of thiobarbituric acid reactive substances (TBARS) representing radical-induced LPO was 2.50 +/- 0.46 after hypoxia and 5.92 +/- 0.54 nmol/mg protein after reoxygenation (p < 0.05 each, vs. normoxic control 1.79 +/- 0.21). During hypoxia, ATP content decreased to 7.9 +/- 1.6 nmol/mg protein; lactate dehydrogenase activity in the incubation solution increased to 0.17 +/- 0.03 U/mg protein; (p < 0.05 vs. control 15.7 +/- 3.1 and 0.09 +/- 0.02, respectively). After hypoxia, morphological changes in lysosomes, multivesicular bodies and vacuoles were observed in contrast to normoxic cells. During reoxygenation, the ATP values were normalized; electron micrographs showed increasing amounts of lysosomes, multivesicular bodies, vacuoles, blebs and lipofuscin granula and lyzed cells. Comparing the biochemical and morphological observations, a sequence of disturbances occurred, in which energy depletion was accompanied and followed, respectively, by membrane destruction, cellular disintegration and an increase in LPO products. These results support the assumption that the damage of brain endothelial cells caused by hypoxia and reoxygenation is accompanied by peroxidation of membrane lipids.
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Circulación Cerebrovascular , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Peroxidación de Lípido , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Aerobiosis , Animales , Capilares , Hipoxia de la Célula , Membrana Celular/ultraestructura , Supervivencia Celular , Células Cultivadas , Endotelio Vascular/ultraestructura , L-Lactato Deshidrogenasa/análisis , Lactatos/metabolismo , Microscopía Electrónica , Especies Reactivas de Oxígeno/metabolismo , Porcinos , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisAsunto(s)
Circulación Cerebrovascular , Gránulos Citoplasmáticos/metabolismo , Endotelio Vascular/metabolismo , Peroxidación de Lípido , Lipofuscina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Aerobiosis , Animales , Encéfalo/irrigación sanguínea , Capilares , Hipoxia de la Célula , Células Cultivadas , Gránulos Citoplasmáticos/ultraestructura , Endotelio Vascular/ultraestructura , L-Lactato Deshidrogenasa/análisis , Microscopía Electrónica , Porcinos , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisRESUMEN
The properties of three different recombinant hepatitis B virus core proteins expressed in Escherichia coli were compared: an N-terminal fusion protein, a C-terminally truncated protein and a sequence-authentic protein. All three proteins assembled into capsid-like particles with typical HBc-antigenicity, sedimentation behavior and distinctive electron microscopical images. Apart from this, however, variant HBc proteins displayed properties different from sequence-authentic HBc protein p21.4. Unlike p21.4, the particles of the N-terminal fusion protein p22.2 were sensitive to proteolytic attack by trypsin at variable sites within its arginine-rich C-terminus but not in its extended N-terminus. We therefore conclude that the C-terminal region is located on the surface of the p22.2 particle. These particles also showed increased HBe-antigenicity, as did the C-terminally truncated core particles p17.6, and to an even greater extent p18* particles which were derived from p22.2 by tryptic digestion. This might be interpreted as evidence for an--albeit minor--structural change. All variant core particles were less stable and contained less RNA. Electron microscopic indication for DNA binding of C-terminal deleted p17.6 particles was obtained using an aqueous spreading technique.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/biosíntesis , Virus de la Hepatitis B/genética , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Western Blotting , Clonación Molecular , Escherichia coli , Genes Virales , Antígenos del Núcleo de la Hepatitis B/aislamiento & purificación , Antígenos del Núcleo de la Hepatitis B/ultraestructura , Virus de la Hepatitis B/metabolismo , Microscopía Electrónica , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Plásmidos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/ultraestructuraRESUMEN
The original classification of neuroendocrine tumours proposed by Pearse was based on a common embryologic origin in the neuroectoderm. The term, carcinoid, literally means carcinoma-like, was coined in 1907 to describe the histologic similarity of these tumors to carcinomas on the one hand and their generally indolent biologic behaviour on the other hand. Neuroendocrine tumours represent a group with complex biological, histological, ultrastructural and immunocytochemical properties. This concept was replaced by another classification based on results of modern techniques (electron microscopy, immunocytochemistry, molecular and DNA analyses). This permits a more reliable classification of tumours, that can be used to determine their biological behaviour and prognosis.
Asunto(s)
Células APUD/patología , Tumor Carcinoide/patología , Neoplasias de las Glándulas Endocrinas/patología , Sistemas Neurosecretores/patología , Células APUD/citología , Apudoma/clasificación , Apudoma/patología , Apudoma/ultraestructura , Tumor Carcinoide/clasificación , Tumor Carcinoide/ultraestructura , Neoplasias de las Glándulas Endocrinas/clasificación , Neoplasias de las Glándulas Endocrinas/ultraestructura , Humanos , Sistemas Neurosecretores/citologíaRESUMEN
The reconstitution of influenza virus haemagglutinin into liposomes from lipid/protein/detergent mixtures by detergent removal provides vesicles that are similar in structure to viral particles. The dissociation properties of haemagglutinin aggregates and the molar ratio of lipid to protein in the starting mixture are the key factors for the individual and total yield of protein incorporation into liposomes. Structural properties of the detergent used as well as special reconstitution conditions are of minor importance for the formation of haemagglutinin liposomes. As determined by radial immunodiffusion-, haemolysis- and fusion experiments, specific properties of haemagglutinin were maintained to a large extent on liposomal incorporation, but its immunogenicity is increased, if the antigen is incorporated into the lipid bilayer of liposomes.
Asunto(s)
Hemaglutininas Virales/química , Liposomas/química , Animales , Detergentes , Hemaglutininas Virales/inmunología , Lípidos/química , Ratones , Ratones Endogámicos ICR , Orthomyxoviridae , Proteínas/químicaRESUMEN
The C-reactive protein is the major acute phase protein (APP) in humans which binds lectin-like to different membraneous structures and exerts an important function in non-specific defense. Because of a pentameric molecular symmetry CRP as well as serum amyloid P component (SAP) and hamster female protein (FP) was merged into a special protein family named pentraxins. In rats a protein was found referred to as rat FP which was close related to hamster FP with respect to hormonal regulation and APP nature as well. Based on this conformity the molecular structure of rat FP was analyzed and as the results a pentameric structure could be demonstrated for rat FP, too. Furthermore, the response of rat CRP and FP on injection of adrenal hormones, agents being involved in acute phase reaction, was investigated. Epinephrine administration led to an increase in CRP and a decrease in FP serum concentration. Dexamethasone has the same effect in case of FP and changed the CRP concentration in a biphasic way with a maximum at about 0.01 mg/kg, a minimum at 0.6 mg/kg and a return to control values at 1.8 mg/kg. Thus, the results indicate a neuroendocrine control of CRP and FP but probably in a different way. Using FITC-labelled lectin the exposition of galactose-containing membraneous structures could be demonstrated in carbon tetrachloride-injured liver tissue in contrast to controls. These binding sites are in accordance with increased FP-binding shown by immunofluorescence histochemistry. Thus, lectin-like properties may be ascribed to rat FP comparable to CRP and SAP activity. The results are discussed with respect to findings from literature that also the acetylcholine receptor seems to have a pentameric structure.
Asunto(s)
Proteínas de Fase Aguda/fisiología , alfa-Globulinas/fisiología , Lectinas/metabolismo , alfa-Globulinas/química , Animales , Proteína C-Reactiva/química , Proteína C-Reactiva/metabolismo , Cromatografía de Afinidad , Dexametasona/farmacología , Epinefrina/farmacología , Femenino , Inmunoelectroforesis Bidimensional , Sustancias Macromoleculares , Masculino , Microscopía Electrónica , RatasRESUMEN
In the haemagglutinin (HA) detergent mixtures coexist various protein complexes. Using 125I-HA tracer, gel chromatography, and centrifugation techniques we found protein aggregates comprising up to eight HA trimers. Formation of these structures seemed to be a function of the protein storage medium only. By contrast, special properties of the detergent as well as physicochemical conditions during the protein/detergent interaction had nearly no influence.
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Detergentes/química , Hemaglutininas Virales/aislamiento & purificación , Orthomyxoviridae/química , Animales , Centrifugación por Gradiente de Densidad , Pollos , Cromatografía , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales/química , Hemaglutininas Virales/ultraestructura , Conformación ProteicaRESUMEN
Simultaneous appearance of IgM and type A retro-virus particles in endoplasmic cisternae and vesicles of human hybridomas has been demonstrated for the first time by immunogold-labeling at ultrastructural level. A suspected link between type A particle and IgM gene-expression in hybridoma cells could not be substantiated in this study.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Hibridomas/ultraestructura , Inmunoglobulina M/biosíntesis , Retroviridae/ultraestructura , Virión/ultraestructura , Animales , Anticuerpos Monoclonales/análisis , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/ultraestructura , Humanos , Hibridomas/inmunología , Hibridomas/microbiología , Inmunoglobulina M/análisis , Inmunohistoquímica , Ratones , Microscopía Electrónica , Microscopía Inmunoelectrónica , Células Plasmáticas/ultraestructuraRESUMEN
The envelope structure of human immunodeficiency virus type 1 (HIV-1) was examined using a computer image processor combined with an image rotation-averaging system. Our results indicate that the envelope of the HIV-1 particle is constructed of a T-7 laevo icosahedral surface net, and the knobs are distributed in the positions of pentamer-hexamer clustering, the total number being 72, which correspond to the results obtained by Gelderblom et al. and Ozel et al.
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VIH-1/ultraestructura , Proteínas del Envoltorio Viral/ultraestructura , Procesamiento de Imagen Asistido por Computador , Modelos MolecularesRESUMEN
Immunogold labelling and in vitro transcription of influenza virus vRNA have been used to analyse the interaction of anti-influenza polymerase antibodies with influenza-ribonucleoprotein (RNP) complexes. The polymerase proteins (P proteins) were localized exclusively at one end of the RNP segments. In the course of transcription the amount of P protein decreased significantly. The in vitro transcriptase activity y of influenza A virus RNP complexes in the presence of anti-polymerase antibodies to the strain A/PR/8/34 was inhibited by 60%. In contrast, RNP transcriptase activity of influenza B virus was not inhibited by these antibodies.
Asunto(s)
Virus de la Influenza A/inmunología , ARN Nucleotidiltransferasas/inmunología , ARN Polimerasa Dependiente del ARN/inmunología , Ribonucleoproteínas/inmunología , Replicación Viral , Animales , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Embrión de Pollo , Inmunohistoquímica , Virus de la Influenza A/crecimiento & desarrollo , Microscopía Electrónica , ARN Viral/biosíntesis , Transcripción GenéticaRESUMEN
Cis-dichlorodiamineplatinum(II) (cis-DDP) was encapsulated in reverse phase evaporation vesicles (REV, 0.6 mg cis-DDP/ml lipid solution) and multilayered liposomes (MLV, 0.3 mg cis-DDP/ml lipid solution) with different cholesterol content. The identity of cis-DDP in free and encapsulated form was checked by various techniques. Particle size, homogeneity of liposomes and distribution of cis-DDP in REVs were shown by electron microscopy. The examination of entrapped cis-DDP in REVs relating to buffer and serum stability, in vitro and in vivo antitumour activity and nephrotoxicity proved that all points are strongly influenced by the cholesterol (CH) content. Enclosed cis-DDP in phosphatidyl (PC)-REV has the same, and in PC: CH-REV, a lower effect in vitro and in vivo compared to treatment with the free drug. Irrespective of the application of tumour cells and substance (i.v., i.p.) in optimal therapeutic doses, an equal increase in life-span (ILS) was registered with the free drug and with PC-cis-DDP-REV, while cis-DDP in PC: CH-REV had a significantly reduced effectiveness. Liposomal encapsulation of cis-DDP also influenced body weight change and leucocyte counts. The blood urea nitrogen (BUN) level, as an indicator of renal toxicity, was only moderately increased after very high doses of cis-DDP (24 mg/kg) in PC: CH-REV.
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Cisplatino/administración & dosificación , Liposomas , Animales , Colesterol/análisis , Cisplatino/uso terapéutico , Cisplatino/toxicidad , Portadores de Fármacos , Femenino , Liposomas/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
It has been found that the microvillous membrane of rat enterocytes contains an aminopeptidase P which is one of the few enzymes capable to hydrolyze the peptide imido bond on the N-terminal side of proline residues. The enzyme was enriched 9-fold in chromatographically purified brush border membrane vesicles of the small intestine. Various extraction procedures and proteinase treatments yielded strong evidence that it is an integral part of the membrane without a stalked hydrophilic head exposed to the outer surface. It was solubilized by detergent and further enriched by ion exchange chromatography up to 73-fold.
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Aminopeptidasas/metabolismo , Intestino Delgado/enzimología , Microvellosidades/enzimología , Animales , Fraccionamiento Celular , Mucosa Intestinal/enzimología , Microscopía Electrónica , Microvellosidades/ultraestructura , Prolina , Ratas , Ratas EndogámicasRESUMEN
Molecular events on the human erythrocyte membrane subsequent to influence virus binding were investigated by electron spin resonance (ESR) measurements after spin labeling of the cell membrane at different positions. Virus binding affected the glycocalyx structure as well as the physical state of the cytoskeleton at the inner leaflet, but not the lipid phase. A lateral reorganization of spin-labeled glycophorin was not indicated after virus attachment.
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Membrana Eritrocítica/ultraestructura , Orthomyxoviridae/fisiología , Absorción , Sitios de Ligazón Microbiológica , Fusión Celular , Óxidos N-Cíclicos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Espectroscopía de Resonancia por Spin del Electrón , Glicoproteínas/análisis , Hemólisis , Humanos , Concentración de Iones de Hidrógeno , Fluidez de la Membrana , Microscopía Electrónica , Orthomyxoviridae/metabolismo , Orthomyxoviridae/ultraestructura , Polisacáridos/análisis , Marcadores de Spin , TemperaturaRESUMEN
Rabbits were immunized with 10 nm filaments of a mixture of cytokeratins which has been isolated from human heel callus material and reconstituted to filaments in vitro. The antisera to keratins (ASK) have been tested histologically at fixed and unfixed tissue samples by means of the indirect immunofluorescence and PAP technique. The ASK recognized specifically only the epithelial cells of skin, of the mucous membranes of mouth and digestive tract, of salivary glands, sweat gland and mammary gland, but did not react with hepatocytes or kidney cells. The following tumors, tested till now, reacted with the antikeratin antisera: epithelial and lymphoepithelial carcinomas of skin, mouth and digestive tract, carcinomas of salivary glands, mammary gland and thyroid gland, adamantinoma, basalioma of skin, and metastases from carcinomas.