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1.
Nat Commun ; 9(1): 682, 2018 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-29445209

RESUMEN

With more than 240 million people infected, hepatitis B virus (HBV) is a major health concern. The inability to mimic the complexity of the liver using cell lines and regular primary human hepatocyte (PHH) cultures pose significant limitations for studying host/pathogen interactions. Here, we describe a 3D microfluidic PHH system permissive to HBV infection, which can be maintained for at least 40 days. This system enables the recapitulation of all steps of the HBV life cycle, including the replication of patient-derived HBV and the maintenance of HBV cccDNA. We show that innate immune and cytokine responses following infection with HBV mimic those observed in HBV-infected patients, thus allowing the dissection of pathways important for immune evasion and validation of biomarkers. Additionally, we demonstrate that the co-culture of PHH with other non-parenchymal cells enables the identification of the cellular origin of immune effectors, thus providing a valuable preclinical platform for HBV research.


Asunto(s)
Virus de la Hepatitis B/fisiología , Hepatitis B/virología , Hígado/virología , Microfluídica/métodos , Adulto , Anciano , Animales , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo/métodos , Femenino , Células Hep G2 , Hepatocitos/citología , Hepatocitos/virología , Interacciones Huésped-Patógeno , Humanos , Lactante , Macrófagos del Hígado/citología , Macrófagos del Hígado/virología , Hígado/citología , Masculino , Ratones , Persona de Mediana Edad , Células 3T3 NIH , Reproducibilidad de los Resultados , Replicación Viral
2.
Sci Rep ; 8(1): 672, 2018 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-29330423

RESUMEN

Zika virus (ZIKV) Infection has several outcomes from asymptomatic exposure to rash, conjunctivitis, Guillain-Barré syndrome or congenital Zika syndrome. Analysis of ZIKV immunity is confounded by the fact that several related Flaviviruses infect humans, including Dengue virus 1-4, West Nile virus and Yellow Fever virus. HLA class II restricted T cell cross-reactivity between ZIKV and other Flaviviruses infection(s) or vaccination may contribute to protection or to enhanced immunopathology. We mapped immunodominant, HLA class II restricted, CD4 epitopes from ZIKV Envelope (Env), and Non-structural (NS) NS1, NS3 and NS5 antigens in HLA class II transgenic mice. In several cases, ZIKV primed CD4 cells responded to homologous sequences from other viruses, including DENV1-4, WNV or YFV. However, cross-reactive responses could confer immune deviation - the response to the Env DENV4 p1 epitope in HLA-DR1 resulted in IL-17A immunity, often associated with exacerbated immunopathogenesis. This conservation of recognition across Flaviviruses, may encompass protective and/or pathogenic components and poses challenges to characterization of ZIKV protective immunity.


Asunto(s)
Flavivirus/inmunología , Epítopos Inmunodominantes/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas no Estructurales Virales/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Reacciones Cruzadas , Virus del Dengue/inmunología , Mapeo Epitopo , Genes MHC Clase II , Ratones , Ratones Transgénicos , Virus del Nilo Occidental/inmunología , Virus de la Fiebre Amarilla/inmunología
3.
J Viral Hepat ; 23(5): 320-9, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26762605

RESUMEN

Hepatitis C virus is a major global health concern with 170 million people chronically infected. Despite the availability of potent antiviral agents targeting multiple HCV proteins and cure rates above 90%, global treatment availability, the likelihood of emerging drug-resistant viral variants and the unavailability of a protective vaccine underline the many unresolved questions remaining to be answered. Model systems allowing the dissection of individual HCV life cycle steps have previously been developed and span noninfectious and infectious means of assessing HCV entry and replication, multiple cellular systems enabling host/pathogen interaction studies as well as in vivo model systems for basic as well as translational HCV research. This review provides an overview of available systems and a comparative summary of assays and models.


Asunto(s)
Investigación Biomédica/métodos , Hepacivirus/patogenicidad , Hepatitis C/patología , Interacciones Huésped-Patógeno , Animales , Investigación Biomédica/tendencias , Modelos Animales de Enfermedad , Humanos , Modelos Biológicos
4.
Analyst ; 138(20): 6154-62, 2013 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-23971072

RESUMEN

This article describes a novel bioluminescence assay for detecting the proteolytic activity of Botulinum NeuroToxins (BoNT) in complex matrices. The assay is capable of detecting traces of BoNT in blood samples as well as in food drinks. The assay was responsive to BoNT/A subtypes 1 to 5, and serotype E3 in buffered solutions. It was responsive to filtered Clostridium botulinum supernatants and BoNT/A1 in complex with neurotoxin associated proteins in bouillon and milk (3.8% fat) down to 400 fM after 4 h RT incubation and in bouillon at concentrations down to 120 fM after 21 h RT incubation. In combination with an immunocapture/enrichment step it could detect BoNT/A1 in citrated plasma at concentrations down to 30 fM (1.2 mouse LD50 per mL). The simplicity of the assay, combined with a demonstrated ability to lyophilize the reagents, demonstrates its usefulness for detection of BoNT in non-specialised analytical laboratories.


Asunto(s)
Toxinas Botulínicas Tipo A/análisis , Toxinas Botulínicas Tipo A/química , Mediciones Luminiscentes/métodos , Animales , Clostridium botulinum/química , Humanos , Ratones , Ratones Endogámicos BALB C , Estructura Secundaria de Proteína
5.
J Med Primatol ; 42(4): 220-4, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23617545

RESUMEN

Few lethal pathogens in wild-living primates have been described, and little is known about infectious diseases of the reproductive tract and their possible impact on health and reproduction. This report describes the pathology and isolation of an alpha-toxin producing strain of Clostridium septicum in a case of necrotizing endometritis in a wild sooty mangabey found dead in a tropical rainforest of West Africa.


Asunto(s)
Toxinas Bacterianas/biosíntesis , Cercocebus atys , Infecciones por Clostridium/veterinaria , Clostridium septicum/metabolismo , Endometritis/veterinaria , Enfermedades de los Monos/microbiología , Animales , Clostridium septicum/aislamiento & purificación , Côte d'Ivoire , Endometritis/microbiología , Endometritis/patología , Femenino , Necrosis
6.
Oncogene ; 29(32): 4588-98, 2010 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-20514021

RESUMEN

Endemic Burkitt's lymphoma (BL) is considered to preferentially develop in equatorial Africa because of chronic co-infection with Epstein-Barr virus (EBV) and the malaria pathogen Plasmodium falciparum. The interaction and contribution of both pathogens in the oncogenic process are poorly understood. Earlier, we showed that immune activation with a synthetic Toll-like receptor 9 (TLR9) ligand suppresses the initiation of EBV lytic replication in primary human B cells. In this study we investigate the mechanism involved in the suppression of EBV lytic gene expression in BL cell lines. We show that this suppression is dependent on functional TLR9 and MyD88 signaling but independent of downstream signaling elements, including phosphatidylinositol-3 kinase, mitogen-activated protein kinases and nuclear factor-kappaB. We identified TLR9 triggering resulting in histone modifications to negatively affect the activation of the promoter of EBV's master regulatory lytic gene BZLF1. Finally, we show that P. falciparum hemozoin, a natural TLR9 ligand, suppresses induction of EBV lytic gene expression in a dose-dependent manner. Thus, we provide evidence for a possible interaction between P. falciparum and EBV at the B-cell level and the mechanism involved in suppressing lytic and thereby reinforcing latent EBV that has unique oncogenic potential.


Asunto(s)
Linfoma de Burkitt/patología , Herpesvirus Humano 4/genética , Histonas/metabolismo , Receptor Toll-Like 9/metabolismo , Transactivadores/genética , Transcripción Genética , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfocitos B/patología , Linfocitos B/virología , Secuencia de Bases , Linfoma de Burkitt/virología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Islas de CpG/genética , Hemoproteínas/metabolismo , Hemoproteínas/farmacología , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/fisiología , Humanos , Ligandos , Factor 88 de Diferenciación Mieloide/genética , Plasmodium falciparum/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/farmacología , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Activación Viral/efectos de los fármacos
7.
Poult Sci ; 88(2): 281-90, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19151341

RESUMEN

One of the key benefits in using chickens for immunization is the high yield of antibodies obtainable. It is known that egg production decreases over time, while animal maintenance costs remain stable. It would, however, be desirable to keep hens as long as possible to obtain maximal amounts of antibodies. To identify a suitable length of time that animals can be kept and to optimize the cost:yield ratio, we monitored the number of eggs laid, the total amount of chicken IgY, and the specific antibody titer from individually prepared eggs over a 2-yr period. The plant toxin ricin and the Clostridium botulinum neurotoxins type A and B were used to immunize 4 chickens. The number of eggs laid in 2 yr was approximately 600 per hen (about 80% of the maximum egg number), yielding about 20 to 40 g of total IgY per hen. A stable antibody titer of 1:100,000 to 1:1,000,000, as measured by ELISA, was obtained following up to 11 injections of 10 to 20 microg of immobilized native toxin. Laying capacities were found to decrease, on average, from 7 eggs/wk at the point of first immunization to 2 eggs/wk after more than 2 yr. In parallel, the yield of total and specific IgY increased over time, so that the antibody recovery remained high, even after prolonged immunization times. Using purified IgY preparations, classical immunological assays such as ELISA and Western blotting were performed. Furthermore, the IgY showed neutralizing capacity when used to block the functional activity of the toxins both in vitro and in vivo. Analysis of the total IgY content over time demonstrated a complex biological oscillation (and the antigen-specific titer), with a shorter time period of around 7 d (circaseptan rhythm). In summary, we successfully immunized chickens with ricin and botulinum neurotoxins and monitored laying capacity, IgY concentration, and specific antibody titer over an extended period of 2 yr.


Asunto(s)
Pollos/fisiología , Huevos/análisis , Inmunoglobulinas/análisis , Inmunotoxinas/sangre , Oviposición/inmunología , Animales , Toxinas Botulínicas/inmunología , Pollos/inmunología , Femenino , Inmunización/veterinaria , Ricina/inmunología
8.
Cell Mol Life Sci ; 63(5): 627-35, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16482397

RESUMEN

Cell-penetrating peptides (CPPs) have been shown to improve antigen loading of dendritic cell vaccines. Here we asked whether fusion of a CPP to a protein improves its immunogenicity when this fusion protein is directly applied as vaccine. We used the cell-penetrating translocation motif (TLM) derived from the hepatitis B virus, because no size limitation of cargos has been observed. Increased immunogenicity was observed when TLM was fused to ovalbumin (TLM-ova). TLM-ova was found to be superior to ova in inducing proliferation and cytotoxicity of ova-specific CD8+ T cells in vitro and in vivo. Using ovalbumin-expressing thymoma cells (EG7-ova), an improved anti-tumor immune response was observed for TLM-ova vaccination versus vaccination with ova. Moreover, TLM-ova vaccination induced a higher titer of anti-ovalbumin IgG2a antibodies compared to ova. These data demonstrate that CPP-protein vaccines can improve cellular as well as humoral immune responses.


Asunto(s)
Secuencias de Aminoácidos/inmunología , Virus de la Hepatitis B/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Secuencias de Aminoácidos/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/análisis , Ratones , Ovalbúmina/administración & dosificación , Ovalbúmina/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Bazo/citología , Bazo/inmunología , Linfocitos T/inmunología , Vacunas Sintéticas/administración & dosificación
9.
Mol Biol Cell ; 12(9): 2688-98, 2001 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11553708

RESUMEN

The mitochondrial tRNA gene for lysine was analyzed in 11 different marsupial mammals. Whereas its location is conserved when compared with other vertebrate mitochondrial genomes, its primary sequence and inferred secondary structure are highly unusual and variable. For example, eight species lack the expected anticodon. Because the corresponding transcripts are not altered by any RNA-editing mechanism, the lysyl-tRNA gene seems to represent a mitochondrial pseudogene. Purification of marsupial mitochondria and in vitro aminoacylation of isolated tRNAs with lysine, followed by analysis of aminoacylated tRNAs, show that a nuclear-encoded tRNA(Lys) is associated with marsupial mitochondria. We conclude that a functional tRNA(Lys) encoded in the nuclear genome is imported into mitochondria in marsupials. Thus, tRNA import is not restricted to plant, yeast, and protozoan mitochondria but also occurs also in mammals.


Asunto(s)
Marsupiales/genética , Mitocondrias/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Animales , Anticodón/genética , Secuencia de Bases , Transporte Biológico , Humanos , Lisina/genética , Lisina/metabolismo , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Edición de ARN , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/genética , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Transcripción Genética/genética
10.
Ann Oncol ; 10(3): 323-7, 1999 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10355577

RESUMEN

BACKGROUND: An association between Kaposi's sarcoma associated herpesvirus (KSHV) and the pathogenesis of multiple myeloma (MM) was postulated recently. The dendritic cells of patients with MM were proposed to be infected with the virus. PATIENTS AND METHODS: Bone marrow mononuclear cells (MNC) of 23 patients, 22 with MM and one with MGUS, were cultured according to three distinct protocols for the generation of dendritic cells. One was essentially the stromal cell culture protocol described by Rettig et al. (Science 1997; 276: 1851-4), while the two other protocols comprised growth factors. Cultured cells were characterised by FACS analysis and assessed for the presence of KSHV DNA with a highly sensitive and specific nested PCR assay detecting the KS 330233 sequence of the virus genome followed by hybridisation with a KSHV specific oligonucleotide. RESULTS: FACS analysis of the cells with the specific markers CD1a, CD86 and HLA-DR, characteristic for dendritic cells, revealed differences in the expression pattern depending on the protocol used. The proportion of CD1a+ cells was very low in the stromal cell cultures (median 0.4%), while a higher percentage of CD14+ cells could be observed (median 37.8%). Growth factor containing cultures revealed a distinctly higher median percentage of CD1a+ cells of 32.5%. The proportion of CD86+ cells varied between 10.4% and 78.5% and HLA-DR+ cells between 26% and 94.4%. Examination of those cells with PCR did not reveal positivity for KSHV in any of the 34 samples assessed. Amplification of seven samples revealed PCR products of approximately the size of the KS 330(233), which, however, could not be confirmed as KSHV specific after hybridisation. CONCLUSION: We have no evidence that bone marrow derived dendritic cells from patients with MM are infected with KSHV.


Asunto(s)
Células de la Médula Ósea/virología , ADN Viral/análisis , Células Dendríticas/virología , Herpesvirus Humano 8/aislamiento & purificación , Mieloma Múltiple/virología , Adulto , Anciano , Southern Blotting , Células de la Médula Ósea/patología , Células Cultivadas/patología , Células Cultivadas/virología , Células Dendríticas/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/patología , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sarcoma de Kaposi/virología , Sensibilidad y Especificidad
11.
Plant J ; 16(1): 117-25, 1998 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9807833

RESUMEN

Rapid and efficient procedures for the detection of sequence polymorphisms are essential for chromosomal walking and mutation detection analyses. While DNA chip technology and denaturing high-performance liquid chromatography (DHPLC) are the methods of choice for large scale facilities, small laboratories are dependent on simple ready-to-use techniques. We show that heteroduplex analysis on high resolution gel matrices efficiently detects sequence polymorphism differing as little as a single base pair (e.g. single-nucleotide polymorphism, SNP) with standard laboratory equipment. Furthermore, the matrices also discerned differences between homoduplexes, a prerequisite for co-dominant markers. The markers thus generated are referred to as duplex analysis markers. We designed PCR primers for 36 Arabidopsis thaliana loci ranging in length from 230 bp to 1000 bp. Among three ecotypes, more than half (n = 19) of the loci examined were polymorphic; five of which contained three different alleles. This simple, high resolution technique can be used to rapidly convert sequence tagged sites into co-dominant PCR-based molecular markers for fine-scale mapping studies and chromosomal walking strategies as well as for the detection of mutations in particular genes.


Asunto(s)
Arabidopsis/genética , Marcadores Genéticos , Análisis Heterodúplex/métodos , Reacción en Cadena de la Polimerasa , Alelos , Cromatografía Líquida de Alta Presión , Paseo de Cromosoma/métodos , ADN de Plantas/química , Electroforesis en Gel de Poliacrilamida , Genes de Plantas , Polimorfismo Genético
12.
Ann Neurol ; 44(4): 635-40, 1998 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9778262

RESUMEN

We report seven unrelated families with mitochondrial tRNA(Ser(UCN)) gene mutations at three different loci. A novel G7497A mutation is found in two families, both of which present with progressive myopathy, ragged-red fibers, lactic acidosis, and deficiency of respiratory chain complexes I and IV. This mutation presumably affects the tertiary tRNA(Ser(UCN)) dihydrouridine interaction. Mutations 7472 insC and T7512C, found in three and two families, respectively, are associated with myoclonus epilepsy, deafness, ataxia, cognitive impairment, and complex IV deficiency. No ragged-red fibers or ultrastructural abnormalities are seen. It is interesting that 6 of our 7 index patients are apparently homoplasmic, indicating a minor pathogenetic power of the tRNA(Ser(UCN)) mutations.


Asunto(s)
Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/fisiopatología , Miopatías Mitocondriales/genética , Mutación/fisiología , Adolescente , Adulto , Niño , Preescolar , Progresión de la Enfermedad , Transporte de Electrón/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miopatías Mitocondriales/metabolismo , Miopatías Mitocondriales/fisiopatología , Músculos/patología , Linaje , Aminoacil-ARN de Transferencia/genética
13.
Am J Physiol ; 274(4): R997-1003, 1998 04.
Artículo en Inglés | MEDLINE | ID: mdl-9575962

RESUMEN

The peripheral nervous system and the immune system were shown to have neurohumoral interactions. This study extends observations that demonstrated neuronal modulation of spontaneous interleukin-6 (IL-6) secretion in the spleen by norepinephrine (NE) and beta-endorphin. Spontaneous IL-6 secretion in vivo was markedly reduced by removal of macrophages with the clodronate technique. Furthermore, spontaneous IL-6 secretion was significantly inhibited at physiological concentrations of cortisol (10(-7) M). In the presence of 10(-7) M cortisol, addition of norepinephrine (NE; 10(-5) M) and isoproterenol (10(-6) and 10(-5) M) significantly increased spontaneous IL-6 secretion (+20%; P = 0.0280, P = 0.0005, and P = 0.0050, respectively). In contrast, addition of beta-endorphin significantly inhibited spontaneous IL-6 secretion in the presence of 10(-7) M cortisol (-40%; 10(-11) M, P = 0.0410; 10(-10) M, P = 0.0005). To study the effect of endogenously released transmitters on spontaneous IL-6 secretion, spleen slices were electrically stimulated with 1, 5, 10, 50, and 100 Hz. Spontaneous IL-6 secretion was markedly reduced at a frequency of 10 Hz with 10(-7) M cortisol present (P < 0.0001). This indicates that the combination of nerve firing at 5-10 Hz and physiological cortisol conditions inhibits spontaneous IL-6 secretion. Inhibition of spontaneous IL-6 secretion from spleen macrophages is most probably due to a net inhibitory effect of opioidergic transmission under these conditions.


Asunto(s)
Interleucina-6/antagonistas & inhibidores , Fenómenos Fisiológicos del Sistema Nervioso , Bazo/metabolismo , Transmisión Sináptica/fisiología , betaendorfina/fisiología , Animales , Sinergismo Farmacológico , Estimulación Eléctrica , Femenino , Hidrocortisona/farmacología , Técnicas In Vitro , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/fisiología , Ratones , Ratones Endogámicos , Norepinefrina/farmacología , betaendorfina/farmacología
14.
Pflugers Arch ; 434(5): 639-41, 1997 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9242729

RESUMEN

With pulse oximetry it is possible to record the pulse-synchronic variation of the oxygen saturation due to variable blood flow during systole and diastole. In the present study on chicken embryos, the pulse rate based on oximetry was compared with the heart rate recorded by means of ECG. We conclude that the pulse curve detected by means of pulse oximetry can be used to determine the heart rate in chicken embryos between day 12 and day 20 of incubation.


Asunto(s)
Embrión de Pollo/fisiología , Frecuencia Cardíaca , Oximetría , Animales , Electrocardiografía , Corazón Fetal/fisiología
15.
FEBS Lett ; 409(3): 320-4, 1997 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-9224682

RESUMEN

RNA editing subsumes a number of functionally different mechanisms which have in common that they change the nucleotide sequence of RNA transcripts such that they become different from what would conventionally be predicted from their gene sequences. RNA editing has now been found in the organelles of numerous organisms as well as in a few nuclear transcripts. Most recently, it was shown to affect tRNAs in the mitochondria of several animals. The occurrence and evolutionary persistence of RNA editing is perplexing since backmutations in the genes might be assumed rapidly to eliminate the need for 'correction' of the gene sequences at the post-transcriptional level. Here, we review the recent RNA editing systems discovered in animal mitochondria and propose that they have arisen as a mechanism counteracting the accumulation of mutations that occurs in asexual genetic system.


Asunto(s)
Mitocondrias/genética , Mitocondrias/fisiología , Edición de ARN/fisiología , Reproducción Asexuada/genética , Animales , Femenino , Masculino , ARN de Transferencia/genética
16.
FEMS Microbiol Rev ; 20(1-2): 25-46, 1997 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9276927

RESUMEN

During evolution prokaryotes have developed different envelope structures exterior to the cell wall proper. Among these surface components are regularly arranged S-layers and capsules. The structural characterization and the detailed chemical analysis of these surface molecules is a prerequisite to understand their biosynthesis and functional role(s) at the molecular level. Of particular interest are the glycosylated S-layer proteins which belong to the first prokaryotic glycoproteins ever described. Their characterization was performed on strains belonging to the thermophilic Bacillaceae and included structural studies and experiments to learn about the pathways for the glycan biosynthesis of S-layer glycoproteins. As an example for non-glycosylated S-layer proteins those of Lactobacillus helveticus strains are described in detail. Recently, a novel type of bacterial glycoconjugate was observed in the cell envelope of the extremely halophilic archaeon Natronococcus occultus which consists of a glycosylated polyglutamyl polymer. Beside the conventional biochemical techniques for the analysis new sophisticated instrumental methods such as X-ray photoelectron spectroscopy and matrix-assisted laser desorption ionization or electrospray ionization mass spectrometry have been introduced for the analysis of the protein and glycan portions of these cell surface macromolecules.


Asunto(s)
Bacterias/química , Proteínas Bacterianas/química , Membrana Celular/química , Glicoconjugados/química , Glicoproteínas de Membrana/química , Archaea/química , Archaea/ultraestructura , Bacillaceae/química , Bacillaceae/ultraestructura , Bacterias/ultraestructura , Evolución Biológica , Conformación de Carbohidratos , Secuencia de Carbohidratos , Pared Celular/química , Pared Celular/ultraestructura , Glicosilación , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Ácido Poliglutámico/química , Polisacáridos/química
17.
Mol Biochem Parasitol ; 85(1): 67-76, 1997 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-9108549

RESUMEN

The spliced leader (SL) RNA plays a key role in mRNA maturation in trypanosomatid protozoa by providing the SL sequence, which is joined to the 5' end of every mRNA. As a first step towards a better understanding of the biogenesis and function of the SL RNA, we expressed a tagged SL RNA gene in a cell-free system of procyclic Trypanosoma brucei cells. Transcription initiates at + 1 can be detected as early as 1 min after addition of extract. Transcription of the SL RNA gene in vitro, as well as in permeable cells, is mediated by an alpha-amanitin/tagetitoxin resistant complex, suggesting a promoter that is intermediate between a classical RNA polymerase II and RNA polymerase III promoter. An analysis of the promoter architecture of the SL RNA gene revealed that regulatory elements are located upstream of the coding region and that the SL sequence, in contrast to the nematode SL sequence, is not required for T. brucei SL RNA gene transcription.


Asunto(s)
Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Protozoario/genética , Transcripción Genética , Trypanosoma brucei brucei/genética , Amanitinas/farmacología , Animales , Secuencia de Bases , Ácidos Dicarboxílicos/farmacología , Genes Protozoarios , Datos de Secuencia Molecular , Mutación , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Compuestos Organofosforados/farmacología , Empalme del ARN , ARN Mensajero/biosíntesis , ARN Protozoario/biosíntesis
18.
Nucleic Acids Res ; 23(17): 3380-4, 1995 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-7567446

RESUMEN

In marsupial mitochondria, the nucleotide residue at the second position of the anticodon of the tRNA for aspartic acid is changed post-transcriptionally such that the translational machinery recognizes it as a uracil rather than the cytosine residue encoded in the gene. By postlabeling nucleotide analysis, we show here that the cytosine residue is converted to a conventional uracil residue in an RNA editing event that affects approximately half of the tRNA molecules under steady state conditions. Furthermore, we have identified three different tRNA(Asp) species which all carry three pseudouridines and two methylations but have the anticodons GCC, GUC and QUC respectively, the latter representing a rare example of queuine incorporation into a mitochondrial tRNA. This allows us to describe a likely sequential order of modification of the tRNA(Asp), where methylations and conversions of uridines to pseudouridines precede the editing event, while the exchange of guanine by queuine takes place after the C to U editing event.


Asunto(s)
Zarigüeyas/genética , Edición de ARN , ARN de Transferencia de Aspártico/metabolismo , ARN/metabolismo , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , ARN Mitocondrial
19.
Mol Biol Evol ; 12(5): 944-8, 1995 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-7476141

RESUMEN

L1 (LINE-1) elements, previously described in several placental species, were shown to exist in several thousands of copies dispersed in the genome of a marsupial (the North American opossum, Didelphis virginiana). A restriction fragment containing the partial nucleotide sequence of the reverse transcriptase gene (ORF-2) from the marsupial L1 element was cloned and sequenced. The element was shown to evolve in concert within the marsupial when compared to placental mammals. When the marsupial L1 element is used as an outgroup to root a phylogeny of placental mammals, the results support the view that humans more recently shared a common ancestor with ungulates than with rodents.


Asunto(s)
Evolución Biológica , Elementos Transponibles de ADN , Mamíferos/genética , Zarigüeyas/genética , Filogenia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Bovinos , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Femenino , Genoma , Humanos , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , Placenta , Embarazo , Probabilidad , Ratas , Mapeo Restrictivo
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