RESUMEN
Aberrantly truncated immature O-glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly associated with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immature O-glycans, no anti-MUC1 antibody recognises carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure analysis, epitope mapping and biochemical analysis. The X-ray crystal structure of the anti-MUC1 monoclonal antibody SN-101 complexed with the antigenic glycopeptide provides for the first time evidence that SN-101 recognises specifically the essential epitope by forming multiple hydrogen bonds both with the proximal peptide and GalNAc linked to the threonine residue, concurrently. Remarkably, the structure of the MUC1 glycopeptide in complex with SN-101 is identical to its solution NMR structure, an extended conformation induced by site-specific glycosylation. We demonstrate that this method accelerates dramatically the development of a new class of designated antibodies targeting a variety of "dynamic neoepitopes" elaborated by disease-specific O-glycosylation in the immunodominant mucin domains and mucin-like sequences found in intrinsically disordered regions of many proteins.
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[This corrects the article DOI: 10.1039/D0SC00317D.].
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Constraints on light sources that use mercury (arc lamps) are evolving with the establishment of the Minamata Convention, which has led to the proliferation of LEDs. However, no LED light source emits intense ultraviolet radiation at wavelengths below 300 nm for photolytic applications. Thus, it is necessary to develop suitable UV light sources for the decontamination of wastewater and water sterilization processing. Herein, we explore various substitute gases (e.g., N2, Ar, He and SF6) to replace mercury, which is commonly employed in arc lamps, using an EL (electroluminescence) quartz assembly platform similar to microwave-discharge electrodeless lamps. Although nitrogen is an inexpensive and safe gas, it cannot generate significant UV radiation in the UVC region of 200-300 nm. This problem in the Hg-free light source was resolved by mixing a very small quantity of sulfur hexafluoride (SF6) as an additive filler gas in a nitrogen-, argon- or helium-filled assembly. The low-pressure mercury lamp consisting of Hg/Ar filler gases is ca. 25% more efficient than the novel N2/SF6 lamp toward the photolytic decomposition of Rhodamine-B (RhB) dye-contaminated wastewater (1.66 × 10-4 mM min-1versus 1.22 × 10-4 mM min-1). Nonetheless, the latter has proven far more efficient than an LED source emitting 365 nm radiation (0.057 × 10-4 mM min-1). The addition of TiO2 to RhB-contaminated wastewater demonstrated that this Hg-free N2/SF6 light source is as efficient as the corresponding Hg/Ar electroluminescent lamp toward the photocatalytic decomposition of the RhB dye pollutant.
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Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.
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ADN/análisis , ADN/química , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN/análisis , ARN/química , Disparidad de Par Base , Secuencia de Bases , ADN/genética , Calor , Yodoacetatos/síntesis química , Yodoacetatos/química , Desnaturalización de Ácido Nucleico , Compuestos Organotiofosforados/síntesis química , Compuestos Organotiofosforados/química , ARN/genéticaRESUMEN
Enzymatic ligation methods are useful in diagnostic detection of DNA sequence. Here we describe the investigation of nonezymatic phosphorothioate-iodoacetyl DNA chemical ligation as a method for detection and identification of RNA and DNA. Specificity of ligation on DNA target is shown to yield discrimination of single point mutation as a drop in two magnitude. Although enzymatic ligation shows very low activity for RNA target, this reaction is found to be very efficient on RNA target. This chemical ligation with RNA target completes 70% within a few seconds, which equal or overcome ligase enzyme-mediated ligation with DNA target. The reaction is also shown to exhibit a significant level of signal amplification under thermal cycle for short time. Further, we found recently that ligation fidelity changed in function of chemical reactivity of probe. This trend was systematically investigated.
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Análisis Mutacional de ADN/métodos , Sondas de ADN/química , Reacción en Cadena de la Polimerasa/métodos , ARN/análisis , Yodoacetatos/química , Cinética , Oligonucleótidos Fosforotioatos/química , Mutación Puntual , Temperatura , Moldes GenéticosRESUMEN
We have studied a possible evolution process permitting a 'primitive' membrane to evolve towards a membrane structure with an outer wall, similar to that of bacteria. We have investigated whether a polysaccharide bearing hydrophobic phytyl or cholesteryl chains coats giant vesicles made of single- or double-chain lipids. Phytyl-pullulan 5b was found to bind to the surface of vesicles made of either single- or double-chain lipids. In contrast, cholesteryl-pullulan 5a only coated the surface of vesicles made of double-chain lipids. These results indicate that there must be a close match between the size and shape of membrane constituents and the hydrophobic molecules to be inserted. This process could, thus, provide a selection mechanism of lipid-membrane constituents during the course of biomembrane evolution. The presence of the above 'hydrophobized' polysaccharides on the surface of different giant vesicles was identified by lectin binding. Both concanavalin A and annexin V were shown by fluorescence microscopy to bind spontaneously to vesicles made of double-chain lipids. Our experiments exemplify that self-organization of amphiphiles into closed vesicles in aqueous solution automatically leads to the coating of vesicles by 'hydrophobized' polysaccharides, which then permit lectin binding. This is a possible mechanism for the evolution of primitive membranes towards 'proto-cells'.
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Membrana Celular/química , Evolución Molecular , Lípidos/química , Animales , Interacciones Hidrofóbicas e Hidrofílicas , Lectinas/química , Polisacáridos/químicaRESUMEN
A 4-months-old calf of Japanese black cattle was diagnosed with orotic aciduria by gas-chromatography/mass-spectrometry (GC/MS). Until now orotic aciduria had not been reported in Japanese black cattle. The animal showed repeated diarrhea. The hematocrit was low, and microcytes and acanthocytes were observed in blood smears. The calf had lower serum total protein concentrations with a higher blood ammonia concentration. Needle-shaped crystals of orotic acid were observed in urinary sediments. Sequence homologous analysis with cattle uridine monophosphate synthase DNA indicated silent mutation in the affected calf.
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Enfermedades de los Bovinos/orina , Complejos Multienzimáticos/deficiencia , Orotato Fosforribosiltransferasa/deficiencia , Ácido Orótico/orina , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Animales , Bovinos , Análisis Mutacional de ADN/veterinaria , Enfermedades Carenciales/orina , Enfermedades Carenciales/veterinaria , Resultado Fatal , Cromatografía de Gases y Espectrometría de Masas/veterinaria , Masculino , Complejos Multienzimáticos/genética , Orotato Fosforribosiltransferasa/genética , Ácido Orótico/sangre , Orotidina-5'-Fosfato Descarboxilasa/genética , LinajeRESUMEN
Mammalian annexins are implicated in several physiological mechanisms based on their calcium-dependent phospholipid/membrane binding and carbohydrate-binding activities. In this study, we investigated gene expression profiles of all four Caenorhabditis elegans annexins, nex-1, -2, -3 and -4, throughout the development, and compared phospholipid- and carbohydrate-binding properties of their protein products, NEX-1, -2, -3 and -4. We found that nex-1 and -3 are transcribed continuously during the developmental stages, while expression of nex-2 and -4 appeared to be temporal, peaking at the L1 stage followed by a gradual decrease toward the adult stage. NEX-1 and -3 were detected as single protein band in total worm extracts by immunoblotting, but NEX-2 was heterogenic in size. NEX-1, -2, and -3 showed the binding activities to phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine, but not to phosphatidylcholine. In contrast to their uniform phospholipids-binding properties, their glycosaminoglycan-binding activities were distinctive. NEX-2 bound to heparan sulfate and chondroitin, NEX-3 bound only to heparan sulfate, and NEX-1 showed no lectin activities under tested conditions. NEX-4 had neither phospholipids- nor carbohydrate-binding properties. Differentiated expression profiles and ligand-binding properties of NEX-1, -2, -3 and -4, shown in our study, may represent distinctive roles for each C. elegans annexins.
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Anexinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Secuencia de Aminoácidos , Animales , Anexinas/biosíntesis , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/biosíntesis , Condroitín/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Heparitina Sulfato/metabolismo , Immunoblotting , Liposomas/metabolismo , Datos de Secuencia Molecular , Fosfolípidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
Since free apoptotic cells are not detected in normal tissues, it is generally believed that apoptotic cells are removed as soon as they appear in vivo. A fluorescent derivative of phosphatidylserine, 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phospho-L-serine (NBD-PS) is known to be incorporated into living cells, and thereafter gradually absorbed into either fatty acid-free bovine serum albumin or fetal calf serum from the outer leaflet of the cell membrane. When thymocytes were irradiated with X-ray and cultured in the presence of NBD-PS, cells became less fluorescent as apoptosis advanced, but early apoptotic cells were still positive for NBD-PS. We then co-cultured such early apoptotic thymocytes with resident peritoneal macrophages. Upon examination under a time-lapse fluorescence microscope, it was found that the attachment of early apoptotic cells to macrophages does not cause rapid phagocytosis, as compared with late apoptotic cells, suggesting the possibility that, in contrast to the widely held view, early apoptotic cells may not be quickly removed by phagocytes in vivo.
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Apoptosis/fisiología , Benzoxazoles , Macrófagos Peritoneales/fisiología , Fagocitosis/fisiología , Fosfatidilserinas , Animales , Anexina A5/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente/métodos , Coloración y Etiquetado , Linfocitos T/efectos de la radiación , Factores de Tiempo , Rayos XRESUMEN
Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed.
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Amino Azúcares/metabolismo , Metabolismo de los Hidratos de Carbono , Glucuronidasa/metabolismo , Lactosa/metabolismo , Hígado/enzimología , Amino Azúcares/química , Animales , Carbohidratos/farmacología , Bovinos , Cromatografía de Afinidad/métodos , Cromatografía en Agarosa/métodos , Cromatografía Líquida de Alta Presión/métodos , DEAE-Celulosa/farmacocinética , Glucuronidasa/aislamiento & purificación , Glicoproteínas/metabolismo , Lactosa/química , Metabolismo de los Lípidos , Modelos Biológicos , Unión Proteica , Sefarosa/farmacocinética , Especificidad por SustratoRESUMEN
OBJECTIVE: To examine influences of gut ischemia/reperfusion (I/R) on gut-associated lymphoid tissue (GALT) mass and function. DESIGN: Prospective, randomized controlled study. SETTING: Research laboratory. SUBJECTS: Male Institute of Cancer Research mice. INTERVENTIONS: Ninety mice were randomized to three groups: I/R (60-min gut ischemia), sham (laparotomy only), and control (no operation). On days 1, 2, 4, 7, and 10, mice were killed to harvest lymphocytes from Peyer patches, the intraepithelial space, and the lamina propria (LP) of the small intestine. Respiratory tract and small intestinal washings were also obtained. MEASUREMENTS AND MAIN RESULTS: Gut I/R significantly reduced lymphocyte numbers in Peyer patches, the intraepithelial space, and the LP. The reduction was prominent in GALT effector sites, that is, the intraepithelial space and LP, but numbers recovered quickly in LP. Changes in cell numbers in Peyer patches, GALT inductive sites, were subtle but persistent. Gut I/R reduced B cell numbers in Peyer patches; alphabeta T cell receptor (TCR)+, gammadeltaTCR+, CD8+, and B cell numbers in the intraepithelial space; and gammadeltaTCR+, CD8+, and B cell numbers in the LP, in comparison with the sham or control group. There were no significant differences in respiratory tract immunoglobulin A levels between the I/R and sham groups. Intestinal immunoglobulin A was elevated on day 1 in the I/R group, with no significant difference after day 2 in comparison with the sham group. CONCLUSIONS: Despite the maintained mucosal immunoglobulin A level, gut I/R markedly reduces GALT cell numbers, with changes in lymphocyte phenotypes. These alterations may be associated with increased morbidity due to infectious complications after severe surgical insults.
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Mucosa Intestinal/inmunología , Intestino Delgado/irrigación sanguínea , Intestino Delgado/inmunología , Tejido Linfoide/inmunología , Daño por Reperfusión/inmunología , Animales , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/fisiopatología , Células Cultivadas , Modelos Animales de Enfermedad , Citometría de Flujo , Inmunoglobulina A/inmunología , Mucosa Intestinal/fisiopatología , Intestino Delgado/patología , Laparotomía/efectos adversos , Recuento de Linfocitos , Tejido Linfoide/patología , Masculino , Ratones , Ratones Endogámicos , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/patología , Complicaciones Posoperatorias/inmunología , Complicaciones Posoperatorias/microbiología , Probabilidad , Distribución Aleatoria , Valores de Referencia , Daño por Reperfusión/fisiopatologíaRESUMEN
Annexins (Anx) are a family of structurally related proteins that all bind to anionic phospholipids in a Ca(2+)-dependent manner. Some biological properties of beta-2-glycoprotein I (beta(2)-GPI) are similar to those of Anx IV and Anx V. Urinary trypsin inhibitor (UTI) helps to maintain normal pregnancy and prevent preterm delivery by inhibiting uterine contraction. However, plasma beta(2)-GPI and UTI levels have not been measured in normal pregnancy. The aim of this study is to clarify the levels of these parameters. Subjects were nonpregnant women (n=50), 120 pregnant women, and maternal subjects just after delivery (n=53) or postpartum (n=67). All of the subjects were healthy. Plasma levels of beta(2)-GPI, UTI, Anx IV, Anx V and other coagulation and fibrinolysis markers were measured by ELISA. The mean plasma level of beta(2)-GPI was significantly increased during the third trimester of pregnancy and 3 to 5 days after delivery. The mean plasma level of UTI was unchanged from the first trimester of pregnancy to the postpartum period. The mean plasma UTI level in vaginal delivery group was significantly higher than that in cesarean section group. beta(2)-GPI protein was expressed in some of the syncytiotrophoblasts. These data suggest that beta(2)-GPI might act to prevent blood clotting on the placental surfaces and also prevents disseminated intravascular coagulation in the microcirculation and maternal plasma. UTI levels might be kept constant by increased urinary excretion despite overproduction during pregnancy.
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Glicoproteínas/sangre , Anexina A5/química , Anexinas/química , Coagulación Sanguínea , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibrinólisis , Humanos , Inmunohistoquímica , Modelos Estadísticos , Placenta/metabolismo , Periodo Posparto , Embarazo , Tercer Trimestre del Embarazo , Valores de Referencia , Factores de Tiempo , beta 2 Glicoproteína IRESUMEN
We have previously reported that significantly higher levels of Keratin 14 (Ker-14) was observed in oral squamous cell carcinoma (OSCC) and severely dysplastic tissues, whereas this expression was reversed in hyperplasia and in mild to moderate dysplasia. In this study, the mechanism of Keratin 14 activation in oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3 and Ca9-22) was investigated. Reporter analysis demonstrated that an upstream region (-1759/-1629) accounted for efficient promoter activity. Furthermore, electromobility sift and supershift assay demonstrated that interactions of the SP-1/SP-3 complex at the elements resided in -1737/-1702 and -1680/-1652 and may be essential for this activation in OSCC cells.
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Queratinas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Sitios de Unión/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Regulación Neoplásica de la Expresión Génica , Humanos , Queratina-14 , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Mutación , Sondas de Oligonucleótidos/genética , Sondas de Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , TransfecciónRESUMEN
T cell progenitors in the adult thymus (AT) are not well characterized. In the present study, we show that the earliest progenitors in the murine AT are, like those in fetal thymus (FT), unable to generate B or myeloid cells, but still retain the ability to generate NK cells and dendritic cells. However, AT progenitors are distinct from those in FT or fetal liver, in that they are able to produce approximately 100 times larger numbers of T cells than progenitors in fetuses. Such a capability to generate a large number of T cells was mainly attributed to their potential to extensively proliferate before the TCRbeta chain gene rearrangement. We propose that the AT is colonized by T/NK/dendritic cell tripotential progenitors with much higher potential to form diversity in TCRbeta chains than FT progenitors.
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Linaje de la Célula , Células Dendríticas/citología , Feto/citología , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Células Madre Hematopoyéticas/citología , Células Asesinas Naturales/citología , Linfocitos T/citología , Timo/citología , Animales , Linfocitos B/citología , Diferenciación Celular , Ratones , Ratones Endogámicos C57BL , Timo/inmunologíaRESUMEN
To identify differentially expressed genes during the development of oral malignancy, differential display, northern blotting, reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analyses were undertaken using oral squamous cell carcinoma (OSCC) and leukoplakia tissues. Significantly higher levels of keratin (Ker)-14 and -17 mRNAs, combined with lower levels of Ker-4, Ker-13 and transglutaminase 3 (TG-3) transcripts, were observed in OSCC and severely dysplastic tissues, whereas this expression profile was reversed in hyperplasia and in mild to moderate dysplasia. The expression of Ker-4 and Ker-13 was elevated in density-arrested OSCC cell lines (Ca9-22, HSC-2, -3 and -4) but the expression of Ker-17 mRNA was elevated in these cells, regardless of the growth conditions. In addition, Ker-4 and Ker-13 proteins were predominantly expressed in moderate dysplasia and hyperplasia, whereas Ker-17 was markedly expressed in OSCC tissues. The expression patterns of these genes could therefore be an important determinant of the manifestation of oral malignancy.
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Proteínas de Unión al Calcio/biosíntesis , Carcinoma de Células Escamosas/metabolismo , Queratinas/biosíntesis , Leucoplasia Bucal/metabolismo , Neoplasias de la Boca/metabolismo , Transglutaminasas/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Proteínas de Unión al Calcio/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hiperplasia/metabolismo , Técnicas para Inmunoenzimas , Queratina-14 , Queratinas/genética , Leucoplasia Bucal/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transglutaminasas/genética , Células Tumorales CultivadasRESUMEN
Annexins are a family of proteins that bind to phospholipids and carbohydrates in a calcium-dependent manner. They are present in a variety of body fluids. Previous studies have shown that annexins have anti-inflammatory activities for lipid A of Gram-negative bacteria. The present study investigated the effect of annexins on interaction between Gram-positive bacteria and immune cells such as macrophages. Annexins I and IV bound to lipoteichoic acids which are surface molecules on Gram-positive bacteria. Binding of annexins I and IV to whole Staphylococcus aureus (S. aureus) were observed and these bindings were inhibited by lipoteichoic acid from S. aureus. Moreover, annexins I and IV suppressed the attachment of S. aureus to phorbol 12-myristate 13-acetate-treated THP-1 cells (human macrophages). These results suggest that annexins I and IV have ligand specificities toward foreign substances, and that the annexins might have some anti-inflammatory property for Gram-positive bacteria.
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Anexina A1/farmacología , Anexina A4/farmacología , Adhesión Bacteriana/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Anexina A1/metabolismo , Anexina A4/metabolismo , Línea Celular , Fluoresceína-5-Isotiocianato/farmacología , Humanos , Lipopolisacáridos/metabolismo , Ésteres del Forbol/farmacología , Unión Proteica , Ácidos Teicoicos/metabolismoRESUMEN
Compared to glutathione S -transferase (GST), tagging with hexahistidine residues (His) has several merits: low levels of toxicity and immunogenicity, a smaller size and no electric charge. We have constructed a novel expression vector, designated as pHisJM (EMBL/GenBank/DDJB accession no. AB116367), for producing recombinant His-fusion proteins. This vector was constructed by replacing GST and multiple cloning site (MCS) cassettes in pGEX-5X-3 with those of hexahistidine and MCS derived from pRSET C vector. Human annexin IV (Anx IV) was used as target protein. His-Anx IV fusion protein was expressed using pHisJM and gave a 40 kDa band when immuno-stained with anti-His mAb or anti-Anx IV mAb as predicted. To compare expression efficiency, a Anx IV cDNA inserted-pHisJM or pGEX-5X-3 was transformed into Escherichia coli DH5alpha, JM109, BL21 and BL21(DE3). Using pHisJM, Anx IV protein was highly expressed in all cell strains. In addition to the merits of using His-tag, pHisJM has several advantages: 1) it has high expression efficiency; 2) it can be used in any Escherichia coli strain; and 3) it can be used in a single strain of Escherichia coli in all steps from plasmid construction to the expression of the target gene.
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Anexina A4/biosíntesis , Escherichia coli/metabolismo , Vectores Genéticos/genética , Histidina/biosíntesis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Anexina A4/genética , Clonación Molecular/métodos , Escherichia coli/genética , Histidina/genética , Humanos , Datos de Secuencia MolecularRESUMEN
An 18 month-old, intact female American Shorthair cat was presented for evaluation of stunted growth and postprandial depression. Fasting serum ammonia and serum bile acid concentrations were above reference ranges at 396 microg/dl and 6.5 micromol/ l and their postprandial concentrations were 785 microg/dl and 9.5 micromol/l, respectively. The initial tentative diagnosis of a portosystemic shunt was excluded by mesenteric portography and histopathology of the liver. The cat was then suspected of a urea cycle enzyme deficiency and its urine was analyzed by gas chromatography-mass spectrometry. A presumptive diagnosis of ornithine transcarbamylase deficiency was made on the basis of the detection of orotic acid and uracil.
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Enfermedades de los Gatos/enzimología , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/veterinaria , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Ácidos y Sales Biliares/sangre , Enfermedades de los Gatos/diagnóstico , Gatos , Femenino , Cromatografía de Gases y Espectrometría de Masas/veterinaria , Hiperamonemia/veterinaria , Ornitina Carbamoiltransferasa/biosíntesis , Ornitina Carbamoiltransferasa/metabolismo , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/diagnóstico , Ácido Orótico/orina , Derivación Portosistémica Quirúrgica , Portografía/veterinaria , Periodo Posprandial , Uracilo/orina , Orina/químicaRESUMEN
Annexin V is a calcium-dependent phospholipid-binding protein that exhibits anticoagulant activity on binding to phosphatidylserine exposed on the activated surfaces of endothelial cells and platelets, inhibiting activation of factor X and prothrombin in the blood coagulation cascade. Sulfatide (galactosylceramide I(3)-sulfate), one of the glycosphingolipids of the platelet cell membrane, is thought to be involved in blood coagulation systems via activation of factor XII. In this study, we examined whether or not annexin V binds to sulfatide and affects the coagulant activity of sulfatide. Solid phase assaying of annexin V revealed that it binds specifically to sulfatide, i.e. not to galactosylceramide or gangliosides, in the presence of calcium ions. Affinity analysis by means of surface plasmon resonance showed that the K(D) of the interaction between annexin V and sulfatide is 1.2 micro M. Kinetic turbidometric assaying of plasma coagulation initiated by CaCl(2) revealed that the coagulation rate in the presence of sulfatide or phosphatidylserine was decreased by annexin V. These results suggest that annexin V regulates coagulability in the blood stream by binding not only to phosphatidylserine but also to sulfatide.
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Anexina A5/química , Sulfoglicoesfingolípidos/química , Anexina A5/metabolismo , Anexina A5/farmacología , Coagulación Sanguínea , Calcio/metabolismo , Cloruro de Calcio/farmacología , Cromatografía en Capa Delgada , Relación Dosis-Respuesta a Droga , Factor X/antagonistas & inhibidores , Galactosilceramidas/metabolismo , Gangliósidos/metabolismo , Glutatión Transferasa/metabolismo , Glucolípidos/química , Humanos , Iones , Cinética , Lectinas/química , Unión Proteica , Protrombina/antagonistas & inhibidores , Proteínas Recombinantes/química , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
Annexin (Anx) V is pivotal in the maintenance of pregnancy by preventing the activation of blood coagulation. The homology of the amino acid sequence between Anx IV and Anx V is highest in Anx family proteins. However, little is known about the roles of Anx IV in pregnancy. The aim of this study is to clarify the roles of circulating Anx IV and Anx V in normal pregnancy. Subjects were non-pregnant women (n = 50), 120 pregnant women, and maternal subjects just after delivery (n = 53) or postpartum (n = 67). Anx IV in the plasma of non-pregnant women was at a concentration 20 times that of Anx V. The plasma levels of Anx IV suddenly increase after delivery, but Anx V levels remain low during this period. Anx IV and Anx V exert similar levels of anticoagulant activity. Anx IV protein was expressed on the basal surface of syncytiotrophoblasts; Anx V protein, on the apical surface of syncytiotrophoblasts. These results suggest that Anx IV enters the maternal bloodstream just after delivery and might play a role in preventing disseminated intravascular coagulopathy, and that Anx V helps to prevent clotting in the placenta during pregnancy.