RESUMEN
Bisphosphoglycerate mutase (BPGM) catalyses the formation of 2,3 bisphosphoglycerate (BPG) a ligand of haemoglobin. BPG facilitates liberation of oxygen from haemoglobin at low oxygen tension enabling efficient delivery of oxygen to tissues. We describe expression of BPGM in mouse labyrinthine trophoblasts, located at the maternal-placental interface. Expression is lower in placentae of igf2(+/-) knockout mice, a widely used model of growth restriction, compared to wild type placentae. Circulating maternal BPG increased throughout gestation but this increase was less in wt mothers carrying igf2(+/-) pups than in those carrying exclusively wt pups. This reduction was observed well before term and may contribute to the low birth weight of igf2(+/-) pups. Strikingly, we also measured reductions of fetal and placental weight in wt littermates of igf2(+/-) pups compared to pups developing in an exclusively wt environment. These data suggest that placental expression of BPGM can influence maternal BPG concentrations and supports a hypothesis under which BPG synthesized in the placenta may act on maternal haemoglobin to enhance delivery of oxygen to the developing fetus.
Asunto(s)
2,3-Difosfoglicerato/sangre , Bisfosfoglicerato Mutasa/metabolismo , Desarrollo Fetal/genética , Factor II del Crecimiento Similar a la Insulina/deficiencia , Placenta/metabolismo , Animales , Bisfosfoglicerato Mutasa/genética , Femenino , Peso Fetal/genética , Eliminación de Gen , Expresión Génica/genética , Edad Gestacional , Factor II del Crecimiento Similar a la Insulina/genética , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Tamaño de los Órganos , Placenta/citología , Placenta/enzimología , Placenta/patología , Embarazo , Trofoblastos/metabolismoRESUMEN
CONTEXT: Visceral adipose tissue (AT) is known to confer a significantly higher risk of type 2 diabetes and cardiovascular disease. Epicardial AT has been shown to be related to cardiovascular disease and myocardial function through unidentified mechanisms. Epicardial AT expresses an inflammatory profile of proteins; however, the mechanisms responsible are yet to be elucidated. OBJECTIVES: The objectives of the study were to: 1) examine key mediators of the nuclear factor-kappaB (NFkappaB) and c-Jun N-terminal kinase (JNK) pathways in paired epicardial and gluteofemoral (thigh) AT from coronary artery disease (CAD) and control patients and 2) investigate circulating endotoxin levels in CAD and control subjects. DESIGN: Serums and AT biopsies (epicardial and thigh) were obtained from CAD (n = 16) and non-CAD (n = 18) patients. Inflammation was assessed in tissue and serum samples through Western blot, real-time PCR, ELISAs, and activity studies. RESULTS: Western blotting showed epicardial AT had significantly higher NFkappaB, inhibitory-kappaB kinase (IKK)-gamma, IKKbeta, and JNK-1 and -2 compared with thigh AT. Epicardial mRNA data showed strong correlations between CD-68 and toll-like receptor-2, toll-like receptor-4, and TNF-alpha. Circulating endotoxin was elevated in patients with CAD compared with matched controls [CAD: 6.80 +/- 0.28 endotoxin unit(EU)/ml vs. controls: 5.52 +/- 0.57 EU/ml; P<0.05]. CONCLUSION: Epicardial AT from patients with CAD shows increased NFkappaB, IKKbeta, and JNK expression compared with both CAD thigh AT and non-CAD epicardial AT, suggesting a depot-specific as well as a disease-linked response to inflammation. These studies implicate both NFkappaB and JNK pathways in the inflammatory profile of epicardial AT and highlight the role of the macrophage in the inflammation within this tissue.
Asunto(s)
Tejido Adiposo/fisiología , Enfermedad de la Arteria Coronaria/complicaciones , Inflamación/etiología , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , FN-kappa B/fisiología , Pericardio/metabolismo , Anciano , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Endotoxinas/sangre , Femenino , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/análisis , Masculino , Persona de Mediana Edad , FN-kappa B/análisis , Fosforilación , ARN Mensajero/análisis , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
2,3-Bisphosphoglycerate mutase (2,3-BPGM), an erythroid-expressed enzyme, synthesises 2,3-bisphosphoglycerate (2,3-BPG), the allosteric modulator of haemoglobin. This ligand has a higher affinity for adult haemoglobin than for fetal haemoglobin and differential binding of it facilitates transfer of oxygen between adult and fetal blood by lowering the affinity of adult haemoglobin for oxygen. This paper reports the discovery that 2,3-BPGM is synthesised in non-erythroid cells of the human placenta. Western blot analysis of placental extracts revealed high levels of 2,3-BPGM in the human placenta. Immunohistochemical staining and in situ hybridisation experiments indicated that abundant 2,3-BPGM is present in the syncytiotrophoblast layer of the placental villi at the feto-maternal interface. A cytochemical staining technique showed that the placental 2,3-BPGM is active, indicating that 2,3-BPG is synthesised in the outermost cells of the placenta. These observations demonstrate an unexpected and abundant presence of an enzyme key to oxygen release from adult haemoglobin, at the interface between maternal and fetal circulations.
Asunto(s)
Bisfosfoglicerato Mutasa/análisis , Placenta/enzimología , Bisfosfoglicerato Mutasa/genética , Bisfosfoglicerato Mutasa/metabolismo , Western Blotting , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , ARN Mensajero/análisisRESUMEN
Pre-eclampsia is a common and serious complication of pregnancy characterised by hypertension and proteinuria. Genetic and environmental factors influence the occurrence and progression of the disease. Emerging experimental systems and increasingly specific analytical methods for the study of differences between normal and pre-eclamptic placentae are close to identifying specific indicators of disease, which may allow early diagnosis and intervention and reveal targets against which therapeutic agents can be developed.
Asunto(s)
Manejo de Caso , Preeclampsia/terapia , Adulto , Animales , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/genética , Fraccionamiento Celular/instrumentación , Fraccionamiento Celular/métodos , Biología Computacional , Inhibidor p57 de las Quinasas Dependientes de la Ciclina , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Hipertensión/genética , Factor II del Crecimiento Similar a la Insulina/deficiencia , Factor II del Crecimiento Similar a la Insulina/genética , Isquemia/fisiopatología , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Placenta/irrigación sanguínea , Placenta/metabolismo , Placenta/fisiopatología , Preeclampsia/sangre , Preeclampsia/epidemiología , Preeclampsia/genética , Preeclampsia/fisiopatología , Embarazo , Proteómica , Sistema Renina-Angiotensina/fisiología , Factores de Riesgo , VasoconstricciónRESUMEN
Short synthetic influenza virus-like RNAs derived from influenza virus promoter sequences were examined for their ability to stimulate the endonuclease activity of recombinant influenza virus polymerase complexes in vitro, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. An extensive set of point mutants of the 5' arm of the influenza A virus viral RNA (vRNA) was constructed to determine the cis-acting elements which influenced endonuclease activity. Activity was found to be dependent on three features of the conserved vRNA termini: (i) the presence of the 5' hairpin loop structure, (ii) the identity of residues at positions 5 and 10 bases from the 5' terminus, and (iii) the presence of base pair interactions between the 5' and 3' segment ends. Further experiments discounted a role for the vRNA U track in endonuclease activation. This study represents the first mutagenic analysis of the influenza virus promoter with regard to endonuclease activity.
Asunto(s)
Endonucleasas/metabolismo , Virus de la Influenza A/genética , Regiones Promotoras Genéticas , ARN Viral/química , ARN Polimerasa Dependiente del ARN/metabolismo , Virión/genética , Células HeLa , Humanos , Mutación Puntual , ARN Complementario/químicaRESUMEN
The poly(A) tail of influenza virus mRNA is thought to be synthesized by reiterative copying of the U track near the 5' end of the virion RNA template. This has been widely accepted as a plausible hypothesis, but until now there has been no direct experimental evidence for it. Here, we report such direct evidence based on the fact that (i) replacing the U track with an A track directs synthesis of products with poly(U) tails, both in vitro and in vivo, and (ii) interrupting the U track abolishes polyadenylation in vitro.
Asunto(s)
Virus de la Influenza A/genética , ARN Mensajero/genética , ARN Viral/genética , Humanos , Virus de la Influenza A/metabolismo , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Moldes Genéticos , Virión/genéticaRESUMEN
We present evidence, based on extensive mutagenesis, that a hairpin loop at the 5' end of influenza A virus virion RNA (vRNA) is required for the synthesis of polyadenylated mRNA from model vRNA templates in vitro. The hairpin loop, which we term the vRNA 5' hook, contains a stem of 2 bp formed by the second and third residues pairing with the ninth and eighth residues, respectively, and a 4-nucleotide loop composed of the intervening residues 4 to 7. Disruption of the base pairs of the vRNA 5' hook by introducing point mutations prevented polyadenylation, except in two mutants where a G-U base pair reformed. The polyadenylation activity of point mutants could be rescued by constructing double mutants with reformed base pairs in the stem of the vRNA 5' hook. These results suggest that base pairing rather than a particular nucleotide sequence was critical. We also show that mutation of the analogous region in the 3' arm of vRNA did not interfere with the synthesis of polyadenylated mRNA, suggesting that a hook structure in the 3' arm is not required for transcription of polyadenylated mRNA in vitro.
Asunto(s)
Virus de la Influenza A/genética , ARN Mensajero/biosíntesis , ARN Viral/química , Virión/genética , Mutagénesis , Relación Estructura-ActividadRESUMEN
We previously demonstrated, by limited mutagenesis, that conserved sequence elements within the 5' end of influenza virus virion RNA (vRNA) are required for the polyadenylation of mRNA in vitro. To further characterize the nucleotide residues at the 5' end of vRNA which might be involved in polyadenylation, a complete set of short and long model vRNA-like templates with mutations at nucleotides 1' to 13' (prime notation denotes numbering from the 5' end) of vRNA were synthesized and transcribed in vitro. The products were assayed for mRNA production with both reverse transcription-PCR and [alpha-32P]ATP incorporation assays. Results from these independent assays showed that vRNA templates with point mutations at positions 2', 3', 7' to 9', and 11' to 13' synthesized polyadenylated transcripts inefficiently compared with those with mutations at positions 1', 4' to 6', and 10'. Positions 2', 3', 7' to 9', and 11' are known to be involved in RNA polymerase binding. Furthermore, residues at positions 11' to 13' are known to be involved in base pairing between the 3' and 5' ends of vRNA. These findings demonstrate that the RNA polymerase has to bind to the 5' end of the template vRNA, which must then interact with the 3' end of the same template for polyadenylation to occur. These results support a model in which a cis-acting RNA polymerase is required for the polyadenylation of influenza virus.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Virus de la Influenza A/enzimología , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Conformación de Ácido Nucleico , Radioisótopos de Fósforo , Mutación Puntual , Reacción en Cadena de la Polimerasa , ARN Mensajero/química , ARN Viral/químicaRESUMEN
An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-borne orthomyxovirus. Endonuclease activity relied on an interaction between the 3' and 5' termini of virion RNA (vRNA) and not those of cRNA. Evidence was obtained that cap structures are cleaved directly from cap donors and that cleavage does not occur after pyrimidines. A 5' hook structure, present in the vRNA promoter but not the cRNA promoter, was introduced into cRNA promoter mutants. These mutants stimulated endonuclease activity, although at levels slightly lower than that of vRNA. The ability of the cRNA promoter to stimulate endonuclease activity when mutated to contain a 5' hook structure indicates that this structure constitutes a switching mechanism for endonuclease activity between the vRNA and cRNA promoters.
Asunto(s)
Endorribonucleasas/metabolismo , Regiones Promotoras Genéticas , ARN Complementario , ARN Viral , Thogotovirus/enzimología , Animales , Línea Celular , Cricetinae , Conformación de Ácido Nucleico , Caperuzas de ARN , ViriónRESUMEN
The cRNA promoter of Thogoto virus, a tick-borne orthomyxovirus, was investigated using an in vitro polymerase assay based on purified viral cores and synthetic oligoribonucleotides corresponding to the 3' and 5' ends of cRNA. In vitro polymerase activity relied on an interaction between the 3' and 5' ends of cRNA and was ApG primer-dependent. Mutational analysis of the promoter showed that interstrand base-pairing of residues 11 and 12 of the 3' promoter arm with residues 10 and 11 of the 5' promoter arm, respectively, was essential for polymerase activity. These data provide the first clear evidence for a cRNA panhandle in an orthomyxovirus. No evidence was obtained for the presence of a 5' or 3' hook structure in the cRNA promoter, and transcription could not be primed with rabbit globin mRNA or synthetic cap analogues. This demonstrates that cap snatching activity relies on the presence of the vRNA terminal sequences.
Asunto(s)
Regiones Promotoras Genéticas/fisiología , ARN Complementario/fisiología , ARN Viral/fisiología , Thogotovirus/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Mutagénesis , Thogotovirus/enzimologíaRESUMEN
Here we report the development of two independent assays which demonstrate for the first time that exogenous model RNA templates based on influenza virus virion RNA (vRNA) are transcribed in vitro to produce polyadenylated mRNA. We investigated the activities of mutated templates with known polymerase binding properties to test our model that polyadenylation occurs when a polymerase complex, which is bound to conserved 5' sequences of vRNA, prevents read-through of the U track at which polyadenylation subsequently occurs by reiterative copying. Mutated templates with perturbed polymerase binding sites (i.e., a deletion mutant lacking the first 4 5' residues and a U-->A point mutant at the third residue) initiated transcription in the in vitro assay but failed to produce polyadenylated transcripts, whereas an A-->U point mutant at the fourth residue, which retained polymerase binding properties similar to those of the wild type, produced polyadenylated transcripts. Our results show that nucleotides within the conserved 5' sequence are required for polyadenylation and support the hypothesis that polymerase binding to 5' sequences of the template is required for mRNA synthesis.
Asunto(s)
Orthomyxoviridae/genética , ARN Mensajero/genética , ARN Viral/genética , Transcripción Genética , Secuencia de Bases , Secuencia Conservada , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Moldes Genéticos , Virión/genéticaRESUMEN
An in vitro transcription assay was used to study transcription from synthetic RNA corresponding to the 3' terminus of influenza A virus cRNA. Micrococcal nuclease-treated influenza virus ribonucleoprotein was used as a source of active polymerase complex. Mutations at two regions of the 13 nucleotide-long conserved cRNA 3' terminus were shown to reduce transcription templated by the short added model RNAs. The first region, at positions 1 and 2 from the 3' terminus, was shown to be affected by the exact nature of the dinucleotide primer used in the in vitro transcription reactions and may not be relevant in vivo. The second region, centred on positions 11 and 12, may be involved in base pairing with conserved nucleotides at the 5' terminus of the cRNA. Evidence for this comes from the finding that RNA corresponding to 5' conserved sequences, but mutated to restore the postulated base pairing with the mutated 3' ends, could partly restore transcription. Binding of the influenza virus polymerase complex to a set of 5'-mutated RNAs was investigated using a photochemical cross-linking assay. Specific binding to two regions of the cRNA 5' terminus was demonstrated, at positions 1 to 3 and positions 8 to 10. Together, these observations suggest that a panhandle forms from the termini of the cRNA molecule and that this structure may play a role in transcription to produce virion RNA.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Virus de la Influenza A/genética , ARN Complementario/metabolismo , ARN Viral/metabolismo , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión Proteica , ARN Viral/química , Transcripción GenéticaRESUMEN
We describe a convenient system for analyzing bunyavirus transcription using a recombinant RNA template derived from the plasmid pBUNSCAT, which comprises a negative-sense reporter gene (chloramphenicol acetyltransferase or CAT) flanked by the exact 5' and 3' untranslated regions of the Bunyamwera virus (BUN) S RNA segment. When cells which expressed bunyavirus proteins (either by recombinant vaccinia viruses or by the vaccinia virus-T7 system) were transfected with BUNSCAT RNA, CAT activity could be measured, indicating transcription of the negative-sense reporter RNA into mRNA. The system permits investigation of both the protein and RNA sequence requirements for transcription. Extensions of 2 bases at the 5' end or 11 or 35 bases at the 3' end of BUNSCAT RNA allowed transcription but a lower level than the wild-type template. Deletion of the 5 nucleotides at the 3' end of BUNSCAT RNA reduced CAT activity by > 99%. Investigation of the viral protein requirements of the system showed that only the bunyavirus L and N proteins were needed for CAT activity. The BUN L protein was also able to transcribe the reporter RNA in concert with the N proteins of closely related bunyaviruses such as Batai, Cache Valley, Maguari, Main Drain, and Northway, but only inefficiently with those of Kairi, Guaroa, or Lumbo viruses. When BUN L proteins containing specific mutations were expressed CAT activity was only observed using those mutated L proteins previously reported to be active in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, 1992, J. Gen. Virol. 73, 2235-2244). These results illustrate the utility of this system for a detailed genetic analysis of the factors involved in bunyavirus transcription.
Asunto(s)
Orthobunyavirus/genética , Orthobunyavirus/metabolismo , ARN Viral/metabolismo , Transcripción Genética , Proteínas Virales/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa/análisis , Cloranfenicol O-Acetiltransferasa/biosíntesis , Chlorocebus aethiops , Riñón , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , Plásmidos , Mutación Puntual , ARN/biosíntesis , ARN/metabolismo , ARN Viral/biosíntesis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Moldes Genéticos , Transfección , Virus VacciniaRESUMEN
We have synthesized four different 5'-diphosphorylated oligoribonucleotides, varying in length from 11 to 13 nucleotides by a new solid phase method. After deprotection and partial purification the 5'-diphosphorylated oligoribonucleotides could be converted to capped (m7Gppp) oligoribonucleotides using guanylyl transferase. Radiolabelled capped oligoribonucleotides acted as primers for the influenza A virus RNA polymerase in vitro. The solid phase method described here should also allow the addition of 5'-diphosphates to synthetic oligodeoxyribonucleotides and be capable automation.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Virus de la Influenza A/enzimología , Virus de la Influenza A/genética , Oligorribonucleótidos/síntesis química , Caperuzas de ARN/síntesis química , ARN/síntesis química , Secuencia de Bases , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Oligorribonucleótidos/química , Fosforilación , ARN/química , Caperuzas de ARN/química , ARN Viral/genéticaRESUMEN
It has been shown that both 3' and 5' conserved termini of influenza A virus virion RNA are involved in the initiation of transcription. An RNA-fork model has been proposed, according to which there is a crucial double-stranded region formed by complementary bases at positions 10 to 12 of the 3' terminus and bases at positions 11' to 13' of the 5' terminus, which are extended by 2 or 3 segment-specific base pairs. The two termini at positions 1 to 9 and 1' to 10' in the 3' and 5' termini, respectively, are in a single-stranded conformation. Here we further characterize this model, focusing on the individual roles of the proposed duplex region and the proposed two single-stranded ends. Residues within the conserved 5' terminus that are involved in the initiation of transcription were determined. Single, double, and triple mutations in the proposed duplex region provided further evidence that, for the initiation of transcription in vitro, the duplex RNA is more important than the actual sequence of these residues, although some restrictions in sequence were apparent. On the other hand, there was evidence that base pairing is not required at residues 1 to 7. We propose that the 5' terminus of virion RNA should be treated as an integral part of the virion RNA promoter and discuss a possible mechanism for the recognition of the virion RNA promoter by the influenza A virus RNA polymerase complex.
Asunto(s)
Virus de la Influenza A/genética , ARN Viral/química , Transcripción Genética , Virión/genética , Secuencia de Bases , Datos de Secuencia Molecular , Mutación , Caperuzas de ARN/metabolismoRESUMEN
The role of the influenza A virus panhandle structure formed from the 3'- and 5'-terminal nucleotides of virion RNA segments was studied in both an RNA polymerase binding assay and an in vitro transcription assay. Despite recent indications that promoter activity is simply a function of the 3'-terminal sequence of virion RNA, our results show that both 3'- and 5'-terminal sequences are involved in the initiation of transcription. We propose a new model for the initiation of transcription which has implications for the mechanisms by which influenza virus transcription, replication, and polyadenylation may be regulated in the infected cell.
Asunto(s)
Virus de la Influenza A/genética , ARN Viral/genética , Transcripción Genética , Animales , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/metabolismo , Humanos , Virus de la Influenza A/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/química , ARN Viral/metabolismoRESUMEN
Bunyaviruses have a genome comprising three segments of negative-sense RNA. The smallest RNA segment, S, encodes the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. The sequences of the S genome RNA segments of seven bunyaviruses (Batai, Cache Valley, Guaroa, Kairi, Main Drain, Northway and Lumbo) were determined from cloned cDNAs obtained using a one-step reverse transcription-PCR protocol. These sequences were compared to those of six viruses previously published, reinforcing earlier conclusions about relationships of the bunyaviruses. Sequence homologies between N proteins correlated with the subdivision of these viruses into three serogroups, Bunyamwera, California and Simbu. The encoded N proteins are either 233 or 235 amino acids in length, depending on the serogroup, whereas the NSs proteins are more variable (83 to 109 amino acids). Certain nucleotide sequence motifs are conserved in the S segments of the Bunyamwera and California serogroup viruses, including the spacing of the AUG initiation codons for the N and NSs proteins (except Guaroa virus), and a CA-rich motif in the virion-sense RNA just downstream of the predicted mRNA termination site. A duplicated sequence was observed in the 3' non-coding region of the Lumbo virus S segment, which accounts for the significantly longer S genome segment of this virus.