RESUMO
MUC4 is a transmembrane mucin, which is aberrantly expressed in pancreatic adenocarcinoma with no detectable expression in the normal pancreas. Here, we present a novel mechanism of IFN-gamma-induced expression of MUC4 in pancreatic cancer cells. Our studies highlight the upregulation of STAT-1 as a basis for MUC4 induction and demonstrate that its activation and upregulation by IFN-gamma are two distinct, albeit temporally integrated, signalling events that drive the selective induction of IRF-1 and MUC4, respectively, within a single cell system. The profile of interferon regulatory factor (IRF)-1 gene induction by IFN-gamma is consistent with its rapid transactivation by phospho-Y701-STAT-1. In contrast, the induction of the MUC4 mucin gene expression is relatively delayed, and occurs only in response to an increase in STAT-1 expression. A progressive binding of STAT-1 to various gamma-interferon-activated sequences (GAS) in the MUC4 promoter is observed in chromatin immunoprecipitation assay, indicating its direct association. Stimulation of STAT-1 expression by double-stranded polynucleotides or ectopic expression is shown to induce MUC4 expression, without Y701 phosphorylation of STAT-1. This effect is abrogated by short interfering RNA (siRNA)-mediated inhibition of STAT-1 expression, supporting further the relevance of STAT-1 in MUC4 regulation. In conclusion, our findings identify a novel mechanism for MUC4 regulation in pancreatic cancer cells and unfold new perspectives on the foundation of IFN-gamma-dependent gene regulation.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Mucinas/genética , Neoplasias Pancreáticas/metabolismo , Fator de Transcrição STAT1/fisiologia , Regulação para Cima , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Mucina-4 , Mucinas/biossíntese , Neoplasias Pancreáticas/patologia , Fosforilação , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação TranscricionalRESUMO
The major tyrosine kinase from platelets was purified as a 51 kDa active enzyme which was shown to be a degradation product of the protooncogene product p60c-src. Immuno-depletion experiments using a monoclonal antibody recognizing p60c-src failed to remove band 3 phosphorylating activity from red blood cell membranes. The erythrocyte tyrosine kinase was not at all immunoprecipitated by this antibody under conditions where the platelet enzyme was immunoprecipitated.
Assuntos
Plaquetas/enzimologia , Eritrócitos/enzimologia , Proteínas Tirosina Quinases/sangue , Proteínas Proto-Oncogênicas/sangue , Proteína 1 de Troca de Ânion do Eritrócito/análise , Eletroforese em Gel de Poliacrilamida , Humanos , Hidrólise , Imunoquímica , Proteínas Proto-Oncogênicas pp60(c-src)RESUMO
Studies on host-pathogen interactions have led to the discovery of various cell surface associated and secretory molecules. Mucins and mucin-like molecules have recently been described in several protozoan parasites, at different stages of the life cycle. These share many structural and compositional features with mammalian mucins, but vary in several other aspects. It is now becoming evident that mucins in parasite are involved in cell-cell interaction and cell surface protection, thus helping the parasite to establish infection. A large number of mucin like genes from the parasite genome have been reported, and their expression differ during the developmental stages of the parasite. In this review, we describe the structure and functions of mucin and mucin-like molecules in parasitic protozoa.
Assuntos
Eucariotos/fisiologia , Mucinas/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Eucariotos/genética , Mucinas/química , Mucinas/genética , Proteínas de Protozoários/genéticaRESUMO
Many cancer and diseased cells are distinguished from their normal counterparts by an altered expression of cell-surface epitopes. One family of molecules that show altered expression on tumor cells is mucins (MUC). Unlike normal tissue where MUC exists as heavily glycosylated form, the disease- or tumor-associated MUC molecules are underglycosylated. Such underglycosylation of the core protein in cancer tissues exposes new epitopes on the cell surface that are unique to cancer tissues. Several monoclonal antibodies (Mabs) have been generated against these normal and tumor-associated mucins. Enzymatic fragments of Mabs like F(ab')2 and Fab have shown improved clinical utility for diagnosis, imaging, and therapy of cancer. Genetic-engineering methods have been used to design antibody fragments exhibiting high functional affinity, good tumor localization, and rapid clearance from the blood stream thus minimizing radiation exposure to the normal tissues. Such recombinant fragments have shown encouraging results in preclinical studies using xenografted tumor bearing mice and present a whole new avenue for radioimmunotherapy and diagnosis of cancer.
Assuntos
Anticorpos Monoclonais/imunologia , Mucinas/imunologia , Animais , Humanos , Fragmentos de Imunoglobulinas/imunologia , Mucina-1/sangue , Mucina-1/imunologia , Mucinas/sangue , Neoplasias/diagnóstico , Neoplasias/imunologia , Neoplasias/radioterapia , Isoformas de Proteínas/imunologia , RadioimunoterapiaRESUMO
A simple microassay and computer program are described for determining the erythrocyte hemolytic potency of drugs in vitro. This microassay is sensitive for both micro as well as macro ranges of hemoglobin concentration. An ELISA reader has been adapted to read erythrocyte lysis (hemolysis), which reduces the number and culture of replicates. A computer program was developed that calculates parameters such as C50 (concentration of drug causing 50% hemolysis), C100 (concentration of drug causing 100% hemolysis) and beta (slope of the curve) and graphically expresses the hemolytic patterns of various drugs simultaneously. The program can obtain optical densities directly from a 96-well plate ELISA reader by interfacing the microplate reader to the computer or by using a keyboard. This method is useful for screening a large number of hemolytic drugs and requires lower amounts of test compounds. It may also be applicable to quantitative functional assays, such as complement-mediated hemolysis and enumeration of antibody-secreting cells. The program can be obtained from the authors on request.
Assuntos
Processamento Eletrônico de Dados , Eritrócitos/efeitos dos fármacos , Hemólise , Análise Numérica Assistida por Computador , Animais , Ácido Flufenâmico/efeitos adversos , Ibuprofeno/efeitos adversos , Indometacina/efeitos adversos , Camundongos , Sensibilidade e EspecificidadeRESUMO
Identification of the antigenic changes in mycobacteria-infected macrophage may be important in understanding the mechanisms responsible for the intracellular survival of the bacteria. In the present study, Mycobacterium microti-infected macrophages were utilized to investigate the possibility of differentiating the infected cells from normal cells, based on the antigenic changes occurring in the membranes. Antisera were generated against bacterial extract, heat-killed bacteria and crude preparation of M. microti-infected homologous macrophage membrane. The reactivity of these antisera, towards in vitro infected macrophages, was compared by flow cytometry. Unlike anti-bacterial extract antiserum or anti-heat-killed bacterial antiserum, anti-infected macrophage membrane antiserum reacted with infected macrophage surface. This reactivity increased with the increase in post-infection time. However, it was not observed with uninfected macrophages, PMA- or lipopolysaccharide-activated macrophages and those harboring Mycobacterium tuberculosis H37Ra, heat-killed M. microti and Leishmania donovani. Interestingly, anti-infected macrophage membrane antiserum identified a 63-kDa antigen in M. microti-infected macrophage membranes which was not present in the membranes of normal macrophages, activated macrophages and of those infected with M. tuberculosis H37Ra, heat-killed M. microti and L. donovani. Thus, membranes of M. microti-infected macrophages differ antigenically from those of the normal macrophages and infected homologous macrophage membrane antiserum provides a useful tool in studying such changes.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Superfície/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium/imunologia , Animais , Anticorpos Antibacterianos/sangue , Western Blotting , Membrana Celular/imunologia , Citometria de Fluxo , Imunização , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium/crescimento & desenvolvimento , Infecções por Mycobacterium/imunologiaRESUMO
The possibility of using specific polyclonal antibodies for effective site specific drug targeting to malaria infected erythrocytes has been examined. For this purpose, rabbit polyclonal antiserum was raised against Plasmodium berghei infected mouse erythrocytes (IRBC) and extensively absorbed with normal erythrocytes (NRBC). Absorbed antiserum specifically recognized IRBC. F(ab')2-fragments of these antibodies were coupled to chloroquine (chq) laden liposomes. These immunoliposomes when tested in vivo significantly suppressed malarial infection in mice.
Assuntos
Anticorpos , Cloroquina/administração & dosagem , Eritrócitos/parasitologia , Fragmentos Fab das Imunoglobulinas , Malária/tratamento farmacológico , Plasmodium berghei , Animais , Especificidade de Anticorpos , Cloroquina/uso terapêutico , Portadores de Fármacos , Eritrócitos/imunologia , Lipossomos , Malária/sangue , Camundongos , Camundongos Endogâmicos BALB CRESUMO
MUC4 is a type-1 transmembrane glycoprotein and is overexpressed in many carcinomas. It is a heterodimeric protein of 930 kDa, composed of a mucin-type subunit, MUC4alpha, and a membrane-bound growth factor-like subunit, MUC4beta. MUC4 mRNA contains unique 5' and 3' coding sequences along with a large variable number of tandem repeat (VNTR) domain of 7-19 kb. A direct association of MUC4 overexpression has been established with the degree of invasiveness and poor prognosis of pancreatic cancer. To understand the precise role of MUC4 in pancreatic cancer, we engineered a MUC4 complementary DNA construct, mini-MUC4, whose deduced protein (320 kDa) is comparable with that of wild-type MUC4 (930 kDa) but represents only 10% of VNTR. Stable ectopic expression of mini-MUC4 in two human pancreatic cancer cell lines, Panc1 and MiaPaCa, showed that MUC4 minigene expression follows a biosynthesis and localisation pattern similar to the wild-type MUC4. Expression of MUC4 resulted in increased growth, motility, and invasiveness of the pancreatic cancer cells in vitro. Ultra-structural examination of MUC4-transfected cells showed the presence of increased number and size of mitochondria. The MUC4-expressing cells also demonstrated an enhanced tumorigenicity in an orthotopic xenograft nude mice model, further supporting a direct role of MUC4 in inducing the cancer properties. In conclusion, our results suggest that MUC4 promotes tumorigenicity and is directly involved in growth and survival of the cancer cells.
Assuntos
Mucinas/fisiologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/ultraestrutura , Divisão Celular , Linhagem Celular Tumoral , DNA Complementar , Humanos , Microscopia Eletrônica de Transmissão , Mucina-4 , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/metabolismo , Frações Subcelulares/metabolismoRESUMO
Protein kinase activity (EC 2.7.1.37) of buffalo spermatozoa is distributed in the head (22%) and midpieces + tails (74%). Extraction of sperm heads with 0.1% Triton X-100 solubilized 35--40 of the protein kinase activity and the remaining 60--65% was associated tightly with the sperm chromatin. That the sperm chromatin preparation was pure was established by recording its spectrum at 320/260 nm (0.07), determining its composition (protein:DNA ratio, 0.79), electron microscope examination and through the assay of marker enzymes. Extraction of the chromatin preparation with 1 M-NaCl only partly solubilized the protein kinase activity while treatment with DNase in the presence of dithiothreitol inactivated the nuclear protein kinase. The chromatin-associated protein kinase activity had a broad pH optimum (7.6--8.4), an essential requirement for Mg2+ and ATP as a phosphate donor. Histones and non-histone proteins served as substrates, the preferred substrate being arginine rich histone VIII followed by casein and phosvitin. Nuclear protein kinase activity was neither stimulated by cyclic AMP nor inhibited by purified muscle protein kinase inhibitor. It is suggested that chromatin-associated protein kinase (Type III protein kinase) may be involved in the control of DNA-template activity.
Assuntos
Cromatina/metabolismo , Proteínas Quinases/metabolismo , Espermatozoides/enzimologia , Acrossomo/enzimologia , Animais , Búfalos , Concentração de Íons de Hidrogênio , Masculino , Ligação Proteica , Espermatozoides/ultraestrutura , Especificidade por Substrato , TemperaturaRESUMO
Phosphorylation of buffalo sperm chromatin proteins under optimum conditions (8 mM Mg2+, pH 8.0, and at 30 degrees C) using [gamma-32P]ATP and endogenous protein kinase activity was linear for 15 min incubation time and up to 330 micrograms protein. The 32P transferred from [gamma-32P]ATP was located in protein as a phosphoester bond. Fractionation with 1.2 M NaCl-4 M urea-0.2 M 2-mercaptoethanol-1 mM PMSF followed by acid treatment solubilized 87% of the total chromatin proteins termed "sperm histones." The remaining 21% nonhistone protein was tightly bound to DNA. Follow-up of the label showed 91% of the 32P in sperm histone and 9% with DNA-associated proteins. Histone kinase activity was solubilized with 0.35 M NaCl, which extracted 70% of the initial enzyme activity associated with chromatin. Of the different histones tested as substrates, histone kinase phosphorylated only histone H3 and, therefore, is highly specific for arginine rich histone. It also phosphorylates the acidic protein, casein. Cyclic AMP at concentrations up to 50 microM had no effect on the phosphorylation of histone H3. Phosphoamino acid analysis using histone H3 as the substrate showed serine to be the acceptor amino acid for phosphoester link.
Assuntos
Búfalos/metabolismo , Cromatina/enzimologia , Protamina Quinase/metabolismo , Espermatozoides/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Arginina/metabolismo , AMP Cíclico/fisiologia , Histonas/metabolismo , Técnicas In Vitro , Masculino , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Proteínas Quinases/isolamento & purificação , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Espermatozoides/ultraestruturaRESUMO
Surface antigens on Leishmania promastigotes and infected macrophages are obvious targets in immunoprophylaxis for leishmanial infection. We have recently demonstrated that the polyclonal antiserum and monoclonal antibodies generated by homologous immunizations with the crude membranes of parasite-infected cells react effectively with the 'neo-antigenic' determinants on the infected cell surface. In the present study, we investigated the utility of such polyclonal antisera for identifying 'minor' surface components of promastigotes. The reactivity of anti-Leishmania donovani-(strain RMRI68) infected macrophage membrane (anti-IMm) antiserum was compared with that of anti-promastigote (anti-Pr) antiserum towards the infected macrophage surface and promastigotes of three Indian strains of L. (donovani, RMRI68, AG83 and DD8. While anti-Pr antiserum showed no reactivity with the infected macrophage surface but reacted strongly with air dried and live promastigotes of all three strains, anti-IMm antiserum reacted with the infected cell surface and, interestingly, specifically recognized live promastigotes of the strain used for infection, i.e., strain RMRI68. The reactivity patterns of the two antisera with the immunodominant components of the L. donovani promastigote surface, i.e., purified LPG-KMP11 complex and gp63 molecules, indicated that unlike anti-Pr antiserum, the specificities in anti-IMm antiserum were mainly directed towards molecules other than the LPG-KMP11 complex and gp63. Antiserum generated in a similar fashion against the macrophage membrane of cells infected in vitro with strain AG83 also contained antibodies specific to strain AG83 promastigotes. The present approach may therefore greatly help in identifying specific antigen(s) important in clinical and epidemiological control of leishmaniasis.
Assuntos
Anticorpos Antiprotozoários/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Macrófagos/parasitologia , Animais , Anticorpos , Anticorpos Monoclonais/imunologia , Membrana Celular/parasitologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Soros Imunes , Camundongos , Camundongos Endogâmicos BALB C , Especificidade da EspécieRESUMO
The B-subunit of phosphate-specific transporter (PstB) is an ABC protein. pstB was polymerase chain reaction-amplified from Mycobacterium tuberculosis and overexpressed in Escherichia coli. The overexpressed protein was found to be in inclusion bodies. The protein was solubilized using 1.5% N-lauroylsarcosine and was purified by gel permeation chromatography. The molecular mass of the protein was approximately 31 kDa. The eluted protein showed ATP-binding ability and exhibited ATPase activity. Among different nucleotide triphosphates, ATP was found to be the preferred substrate for M. tuberculosis PstB-ATPase. The study of the kinetics of ATP hydrolysis yielded K(m) of approximately 72 microm and V(max) of approximately 0.12 micromol/min/mg of protein. Divalent cation like manganese was inhibitory to the ATPase activity. Magnesium or calcium, on the other hand, had no influence on the functionality of the enzyme. The classical ATPase inhibitors like sodium azide, sodium vanadate, and N-ethylmaleimide were without any effect but an ATP analogue, 5'-p-fluorosulfonylbenzoyl adenosine, inhibited the ATPase function of the recombinant protein with a K(i) of approximately 0.40 mm. Furthermore, there was hardly any ATP hydrolyzing ability of the PstB as a result of mutation of the conserved aspartic acid residue to lysine in the Walker motif B, confirming the recombinant protein is an ATPase. Interestingly, analysis of the recombinant PstB revealed that it is a thermostable ATPase; thus, our results highlight for the first time the presence of such an enzyme in any mesophilic bacteria.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias , Mycobacterium tuberculosis/química , Sarcosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Southern Blotting , Western Blotting , Cálcio/farmacologia , Cátions , Clonagem Molecular , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Hidrólise , Cinética , Luz , Magnésio/farmacologia , Manganês/farmacologia , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Proteínas Recombinantes/metabolismo , Sarcosina/metabolismo , Espalhamento de Radiação , Espectrometria de Fluorescência , Temperatura , Fatores de TempoRESUMO
Identification of neo-antigenic determinant(s) on parasite infected cell surface is important to control intracellular infections. Such determinant(s) on the surface of intact Plasmodium berghei infected erythrocytes have not been conclusively demonstrated. To generate polyclonal antiserum selectively recognizing the parasite infected cell surface determinant(s), in natural state, we have examined the efficacy of the homologous immunizations, in BALB/c mice, with the membrane rich preparation of: i) erythrocytes in vivo infected with Plasmodium berghei and, ii) macrophages in vitro infected with Leishmania donovani. Anti-infected erythrocyte membrane antiserum specifically recognized, albeit at low level, the infected cell surface as determined by flow cytometry and immunoelectron microscopy. Immunoprecipitation of radiolabeled antigens revealed at least three parasite proteins of > 205 kDa, 160 kDa and 100 kDa specifically present on infected erythrocyte surface. Normal uninfected erythrocytes did not react with the antiserum. Anti-L. donovani-infected macrophage membrane antiserum also recognized only infected macrophage surface and not the normal macrophages. Thus, the approach may find wide application in delineating disease specific determinant(s) on the infected cell surface, particularly to those where animal models are available.
Assuntos
Anticorpos/imunologia , Antígenos de Superfície/imunologia , Eritrócitos/parasitologia , Leishmania donovani/imunologia , Macrófagos/imunologia , Plasmodium berghei/imunologia , Animais , Extratos Celulares/imunologia , Membrana Celular/imunologia , Eritrócitos/citologia , Eritrócitos/imunologia , Feminino , Citometria de Fluxo , Humanos , Macrófagos/citologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Imunoeletrônica , Testes de PrecipitinaRESUMO
The suitability of liposomes as drug carriers in the treatment of drug-resistant rodent malaria was examined after covalently attaching F(ab')2 fragments of a mouse monoclonal antibody (MAb), MAb F10, raised against the host cell membranes isolated from the Plasmodium berghei-infected mouse erythrocytes, to the liposome surface. The antibody-bearing liposomes thus formed specifically recognized the P. berghei-infected mouse erythrocytes under both in vitro and in vivo conditions. No such specific binding of the liposomes with the infected cells was observed when MAb F10 was replaced by another mouse monoclonal antibody, MAb D2. Upon loading with the antimalarial drug chloroquine, the MAb F10-bearing liposomes effectively controlled not only the chloroquine-susceptible but also the chloroquine-resistant P. berghei infections in mice. The chloroquine delivered in these liposomes intravenously at a dosage of 5 mg/kg of body weight per day on days 4 and 6 postinfection completely cured the animals (75 to 90%) of chloroquine-resistant P. berghei infections. These results indicate that selective homing of chloroquine to malaria-infected erythrocytes may help to cure the chloroquine-resistant malarial infections with low doses of chloroquine.
Assuntos
Cloroquina/administração & dosagem , Eritrócitos/efeitos dos fármacos , Malária/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Animais , Anticorpos Monoclonais/efeitos dos fármacos , Anticorpos Monoclonais/imunologia , Portadores de Fármacos , Resistência a Medicamentos , Eritrócitos/imunologia , Lipossomos/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Standardized calcium oxalate monohydrate (COM) crystal growth assay system was employed to study the ability of various test samples to influence growth rates of COM crystals. The inhibitory activity (IA) of various samples was expressed in terms of inhibitory units. Urine samples obtained from normal persons and kidney stone patients were found to have IA of 3.18 +/- 0.62 and 1.02 +/- 0.08, respectively. A potent inhibitor having molecular weight between 14.2 and 16.2 kDa was found to be primarily responsible for the differences observed in the urinary IAs between normal persons and kidney stone patients. The potent inhibitor was found to be tightly associated with a chromophore resembling Urobilirubin. An ELISA based assay system, using monoclonal antibodies against the above most potent inhibitor confirmed the difference observed in the urinary IA between the normal persons and kidney stone patients. This assay system has the potential to be routinely used to screen human beings for potential stone formers.