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In 2015 and 2016, two Barramundi (Lates calcarifer) farms in Singapore reported a disease outbreak characterized by lethargic behavior, pronounced inappetence, generalized skin lesions, erosions of the fins and tail, and ultimately high mortality in their fish. Next-generation sequencing and PCR confirmed presence of a novel virus belonging to the Alloherpesviridae family, Lates calcarifer herpesvirus (LCHV), which was subsequently isolated and cultured. We characterize, for the first time, the complete genome of two cultured LCHV isolates. The genome contains a long unique region of approximately 105,000 bp flanked by terminal repeats of approximately 24,800 bp, of which the first 8.2 kb do not show any similarity to described genomes in the Alloherpesviridae family. The two cultured isolates share 89% nucleotide identity, and their closest relatives are the viruses belonging to the genus Ictalurivirus. Experimental infections using one of the cultured LCHV isolates resulted in identical clinical signs as originally described in the index farm, both in intraperitoneal-injection infected fish and cohabitant fish, with mortality in both groups. Histopathological analysis showed pronounced abnormalities in the gills. Virus culture and PCR analysis confirmed the replication of LCHV in the infected fish, and thus Koch's postulates were fulfilled.
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Perciformes , Animales , Perciformes/genética , Genoma , Peces/genéticaRESUMEN
A commercial pig farm with no history of porcine circovirus 2 (PCV2) or porcine reproductive and respiratory syndrome virus (PRRSV) repeatedly reported a significant reduction in body weight gain and wasting symptoms in approximately 20-30% of the pigs in the period between three and six weeks after weaning. As standard clinical interventions failed to tackle symptomatology, viral metagenomics were used to describe and monitor the enteric virome at birth, 3 weeks, 4 weeks, 6 weeks, and 9 weeks of age. The latter four sampling points were 7 days, 3 weeks, and 6 weeks post weaning, respectively. Fourteen distinct enteric viruses were identified within the herd, which all have previously been linked to enteric diseases. Here we show that wasting is associated with alterations in the enteric virome of the pigs, characterized by: (1) the presence of enterovirus G at 3 weeks of age, followed by a higher prevalence of the virus in wasting pigs at 6 weeks after weaning; (2) rotaviruses at 3 weeks of age; and (3) porcine sapovirus one week after weaning. However, the data do not provide a causal link between specific viral infections and the postweaning clinical problems on the farm. Together, our results offer evidence that disturbances in the enteric virome at the preweaning stage and early after weaning have a determining role in the development of intestinal barrier dysfunctions and nutrient uptake in the postweaning growth phase. Moreover, we show that the enteric viral load sharply increases in the week after weaning in both healthy and wasting pigs. This study is also the first to report the dynamics and co-infection of porcine rotavirus species and porcine astrovirus genetic lineages during the first 9 weeks of the life of domestic pigs.
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Enterovirus Porcinos/aislamiento & purificación , Intestinos/virología , Rotavirus/aislamiento & purificación , Sapovirus/aislamiento & purificación , Enfermedades de los Porcinos/virología , Viroma/fisiología , Síndrome Debilitante/veterinaria , Animales , Astroviridae/aislamiento & purificación , Femenino , Masculino , Metagenómica , Porcinos , Síndrome Debilitante/virología , DesteteRESUMEN
Early in the SARS-CoV-2 pandemic concerns were raised regarding infection of new animal hosts and the effect on viral epidemiology. Infection of other animals could be detrimental by causing clinical disease, allowing further mutations, and bares the risk for the establishment of a non-human reservoir. Cats were the first reported animals susceptible to natural and experimental infection with SARS-CoV-2. Given the concerns these findings raised, and the close contact between humans and cats, we aimed to develop a vaccine candidate that could reduce SARS-CoV-2 infection and in addition to prevent spread among cats. Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus, known to be safe and efficacious in a variety of animal species, could induce neutralizing antibody responses in guinea pigs and cats. The design of the SARS-CoV-2 spike immunogen was critical in developing a strong neutralizing antibody response. Vaccination of cats was able to induce high neutralizing antibody responses, effective also against the SARS-CoV-2 B.1.1.7 variant. Interestingly, in contrast to control animals, the infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after SARS-CoV-2 challenge. Correspondingly, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit the virus. The results show that the RP vaccine induces protective immunity preventing SARS-CoV-2 infection and transmission. These data suggest that this RP vaccine could be a multi-species vaccine useful to prevent infection and spread to and between animals should that approach be required.
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In recent years, refined molecular methods coupled with powerful high throughput sequencing technologies have increased the potential of virus discovery in clinical samples. However, host genetic material remains a complicating factor that interferes with discovery of novel viruses in solid tissue samples as the relative abundance of the virus material is low. Physical enrichment processing methods, although usually complicated, labor-intensive, and costly, have proven to be successful for improving sensitivity of virus detection in complex samples. In order to further increase detectability, we studied the application of fast and simple high-throughput virus enrichment methods on tissue homogenates. Probe sonication in high EDTA concentrations, organic extraction with Vertrel™ XF, or a combination of both, were applied prior to chromatography-like enrichment using Capto™ Core 700 resin, after which effects on virus detection sensitivity by the VIDISCA method were determined. Sonication in the presence of high concentrations of EDTA showed the best performance with an increased proportion of viral reads, up to 9.4 times, yet minimal effect on the host background signal. When this sonication procedure in high EDTA concentrations was followed by organic extraction with Vertrel™ XF and two rounds of core bead chromatography enrichment, an increase up to 10.5 times in the proportion of viral reads in the processed samples was achieved, with reduction of host background sequencing. We present a simple and semi-high-throughput method that can be used to enrich homogenized tissue samples for viral reads.
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A double construct vaccine of turkey herpesvirus (HVT) was prepared that contains the fusion (F) gene from Newcastle disease virus (NDV) and the viral protein 2 (VP2) gene from infectious bursal disease virus (IBDV). Safety of the vaccine (HVT-ND-IBD) was confirmed and efficacy was evaluated after subcutaneous (SC) vaccination at 1 day of age or the in ovo route of vaccination. Challenges were performed with velogenic NDV strains (Texas GB and Herts Weybridge 33/56), with different strains of IBDV (classical strain STC; very virulent strain CS89 and variant E strain) and with Marek's disease virus (MDV) strain RB1B. Vaccination with HVT-ND-IBD induced a high level of protection against these challenges. Vaccination with HVT is often combined with Rispens CVI988 vaccine and live ND vaccines for higher and earlier, MD and ND protection, respectively. HVT-ND-IBD vaccination in combination with these vaccines showed MD protection as early as 4 days post vaccination and ND protection as early as 2 weeks post vaccination. The long protection as seen with HVT vaccination was confirmed by demonstrating protection against NDV up to 60 weeks. Finally, to evaluate the performance of the vaccine in commercial birds with maternally-derived antibodies, two field trials were performed, using in ovo vaccination in broilers and SC vaccination in combination with Rispens CVI988 vaccine in layer-type birds. The efficacy was confirmed for all components by challenges. These results demonstrate that HVT-ND-IBD is a safe and highly efficacious vaccine for simultaneous control of ND, IBD and MD. RESEARCH HIGHLIGHTS A double construct HVT vaccine with the NDV F and the IBDV VP2 genes was prepared. The vaccine protects against three important diseases: MDV, NDV and IBDV. In ovo and sub-cutaneous vaccination was evaluated in the field in commercial chickens.
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Infecciones por Birnaviridae/veterinaria , Pollos/inmunología , Herpesvirus Gallináceo 2/inmunología , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Enfermedad de Marek/prevención & control , Enfermedad de Newcastle/prevención & control , Virus de la Enfermedad de Newcastle/inmunología , Enfermedades de las Aves de Corral/prevención & control , Animales , Infecciones por Birnaviridae/prevención & control , Infecciones por Birnaviridae/virología , Femenino , Masculino , Enfermedad de Marek/virología , Enfermedad de Newcastle/virología , Enfermedades de las Aves de Corral/virología , Organismos Libres de Patógenos Específicos , Vacunación/veterinaria , Vacunas Atenuadas/inmunología , Vacunas Virales/inmunologíaRESUMEN
Atypical porcine pestivirus (APPV) is a single-stranded RNA virus from the family Flaviviridae, which is linked to congenital tremor (CT) type A-II in newborn piglets. Here, we retrospectively investigated the molecular evolution of APPV on an affected herd between 2013 and 2019. Monitoring was done at regular intervals, and the same genotype of APPV was found during the entire study period, suggesting no introductions from outside the farm. The nucleotide substitutions over time did not show substantial amino acid variation in the structural glycoproteins. Furthermore, the evolution of the virus showed mainly purifying selection, and no positive selection. The limited pressure on the virus to change at immune-dominant regions suggested that the immune pressure at the farm might be low. In conclusion, farms can have circulation of APPV for years, and massive testing and removal of infected animals are not sufficient to clear the virus from affected farms.
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Evolución Molecular , Infecciones por Pestivirus/veterinaria , Pestivirus/genética , Enfermedades de los Porcinos/virología , Animales , Animales Recién Nacidos , Brotes de Enfermedades/veterinaria , Variación Genética , Genoma Viral/genética , Pestivirus/aislamiento & purificación , Infecciones por Pestivirus/congénito , Infecciones por Pestivirus/epidemiología , Infecciones por Pestivirus/virología , Filogenia , Estudios Retrospectivos , Selección Genética , Porcinos , Enfermedades de los Porcinos/congénito , Enfermedades de los Porcinos/epidemiología , Temblor/congénito , Temblor/epidemiología , Temblor/veterinaria , Temblor/virología , Proteínas Virales/genéticaRESUMEN
The increase of the human population is accompanied by growing numbers of livestock to feed this population, as well as by an increase of human invasion into natural habitats of wild animals. As a result, both animals and humans are becoming progressively vulnerable to infections with known (zoonotic) pathogens, but are also increasingly exposed to novel viruses. Global trade as well as climate changes can contribute to pathogen transmission, e.g. through import of infected vectors or expansion of habitats for arthropod vectors such as mosquitoes and midges. Infectious disease outbreaks, especially those by novel viruses, are generally unexpected, and therefore we should be prepared with tools and abilities for immediate action, including the identification of the causative agent, the evaluation of its pathogenic potential for animals and humans, and the fast development of diagnostic assays to allow contact tracing and quarantine measures. HONOURs is a Marie Sklodowska-Curie Actions Innovative Training Network (MSCA-ITN), teaching 15 talented young researchers to become "preparedness-experts". HONOURs, initiated in April 2017, involves 11 laboratories from 6 different European countries, all at the forefront of novel virus investigations and characterizations. The network includes surveillance experts in both the veterinary and the human health sector, who have developed and utilize highly sensitive virus discovery techniques, e.g. next generation sequencing based genomics and universal primers based PCR, to allow identification and characterization of novel viruses. Production of pure viral proteins, providing high-resolution structures, aids in the design of novel, fast and easy-to-use diagnostics. Organotypic in vitro cell cultures systems (e.g. pseudostratified human airway epithelia) provide tools for virus replication, if needed via a reverse genetics platform, and the production of virus stocks permits inoculation in animal models to examine disease, evaluate candidate vaccines, and fulfilment of the Koch's postulates. Scientists of the various institutes will provide training in the HONOURs network through specialized courses and workshops, combined with challenging research projects. The final aim of the network is to deliver 15 expert scientists, ready to act in case of the emergence of an epidemic.
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Brotes de Enfermedades/prevención & control , Investigadores/educación , Enseñanza/organización & administración , Zoonosis/prevención & control , Academias e Institutos , Animales , Control de Enfermedades Transmisibles , Enfermedades Transmisibles/microbiología , Enfermedades Transmisibles/virología , Modelos Animales de Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Zoonosis/virologíaRESUMEN
In the last few years, increased emphasis has been devoted to understanding the contribution of mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) to human pathology in general, and neurodegenerative diseases in particular. A major reason for this is the central role that this subdomain of the ER plays in metabolic regulation and in mitochondrial biology. As such, aberrant MAM function may help explain the seemingly unrelated metabolic abnormalities often seen in neurodegeneration. In the specific case of Alzheimer disease (AD), besides perturbations in calcium and lipid homeostasis, there are numerous documented alterations in mitochondrial behavior and function, including reduced respiratory chain activity and oxidative phosphorylation, increased free radical production, and altered organellar morphology, dynamics, and positioning (especially perinuclear mitochondria). However, whether these alterations are primary events causative of the disease, or are secondary downstream events that are the result of some other, more fundamental problem, is still unclear. In support of the former possibility, we recently reported that C99, the C-terminal processing product of the amyloid precursor protein (APP) derived from its cleavage by ß-secretase, is present in MAM, that its level is increased in AD, and that this increase reduces mitochondrial respiration, likely via a C99-induced alteration in cellular sphingolipid homeostasis. Thus, the metabolic disturbances seen in AD likely arise from increased ER-mitochondrial communication that is driven by an increase in the levels of C99 at the MAM.
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Enfermedad de Alzheimer/metabolismo , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Retículo Endoplásmico/genética , Humanos , Mitocondrias/genéticaRESUMEN
Congenital tremor type A-II in piglets has been regarded as a transmissible disease since the 1970s, possibly caused by a very recently-described virus: atypical porcine pestivirus (APPV). Here, we describe several strains of APPV in piglets with clinical signs of congenital tremor (10 of 10 farms tested). Piglets on a farm with no history of congenital tremor were PCR-negative for the virus. To demonstrate a causal relationship between APPV and disease, three gilts were inoculated via intramuscular injection at day 32 of pregnancy. In two of the three litters, vertical transmission of the virus occurred. Clinical signs of congenital tremor were observed in APPV-infected newborns, yet also two asymptomatic carriers were among the offspring. Piglets of one litter were PCR-negative for the virus, and these piglets were all without congenital tremors. Long-term follow up of farm piglets born with congenital tremors showed that the initially high viremia in serum declines at five months of age, but shedding of the virus in feces continues, which explains why the virus remains present at affected farms and causes new outbreaks. We conclude that trans-placental transmission of APPV and subsequent infection of the fetuses is a very likely cause of congenital tremor type A-II in piglets.
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Infecciones por Pestivirus/veterinaria , Pestivirus/patogenicidad , Enfermedades de los Porcinos/congénito , Temblor/veterinaria , Animales , Animales Recién Nacidos , Heces/virología , Pestivirus/aislamiento & purificación , Infecciones por Pestivirus/congénito , Porcinos , Factores de Tiempo , Temblor/congénito , Viremia/veterinaria , Viremia/virología , Esparcimiento de VirusRESUMEN
From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch's postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.
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Enfermedades de los Peces/virología , Iridoviridae , Animales , Lubina , Enfermedades de los Peces/patología , Filogenia , Reacción en Cadena de la PolimerasaRESUMEN
Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by γ-secretase, via an unknown mechanism. We recently showed that presenilin-1 and -2, the catalytic components of γ-secretase, and γ-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER-mitochondrial communication-as measured by cholesteryl ester and phospholipid synthesis, respectively-are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and γ-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER-mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD.
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Enfermedad de Alzheimer/patología , Embrión de Mamíferos/patología , Fibroblastos/patología , Microdominios de Membrana/patología , Mitocondrias/patología , Membranas Mitocondriales/patología , Presenilina-1/fisiología , Presenilina-2/fisiología , Enfermedad de Alzheimer/metabolismo , Animales , Western Blotting , Células Cultivadas , Embrión de Mamíferos/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Fibroblastos/metabolismo , Humanos , Microdominios de Membrana/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Presenilina-1/antagonistas & inhibidores , Presenilina-2/antagonistas & inhibidores , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones SubcelularesRESUMEN
Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
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Complejo I de Transporte de Electrón/metabolismo , Embrión de Mamíferos/enzimología , Fibroblastos/enzimología , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Línea Celular Transformada , Complejo I de Transporte de Electrón/genética , Embrión de Mamíferos/citología , Estabilidad de Enzimas/fisiología , Fibroblastos/citología , Eliminación de Gen , Humanos , Ácido Láctico/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , NAD/genética , NAD/metabolismo , NADP/genética , NADP/metabolismo , Fosforilación/fisiología , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
Presenilin-1 (PS1) and -2 (PS2), which when mutated cause familial Alzheimer disease, have been localized to numerous compartments of the cell, including the endoplasmic reticulum, Golgi, nuclear envelope, endosomes, lysosomes, the plasma membrane, and mitochondria. Using three complementary approaches, subcellular fractionation, gamma-secretase activity assays, and immunocytochemistry, we show that presenilins are highly enriched in a subcompartment of the endoplasmic reticulum that is associated with mitochondria and that forms a physical bridge between the two organelles, called endoplasmic reticulum-mitochondria-associated membranes. A localization of PS1 and PS2 in mitochondria-associated membranes may help reconcile the disparate hypotheses regarding the pathogenesis of Alzheimer disease and may explain many seemingly unrelated features of this devastating neurodegenerative disorder.
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Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Células Cultivadas , Coenzima A Ligasas/metabolismo , Retículo Endoplásmico/ultraestructura , Humanos , Membranas Intracelulares/ultraestructura , Ratones , Mitocondrias/ultraestructura , Presenilina-1/genética , Presenilina-2/genética , Ratas , Fracciones Subcelulares/metabolismoRESUMEN
BACKGROUND: The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression. RESULTS: Spontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux increased with passage number, while cellular oxygen consumption decreased. This conversion in metabolic properties was associated with a change in mitochondrial NAD+/NADH redox, indicative of decreased mitochondrial tricarboxic acid cycle and OXPHOS activity. CONCLUSION: The high rate of oxidative metabolism in newly transformed cells is in marked contrast with the high glycolytic rate in cells in the later tumor stage. In our experimental system, with cells growing under ambient oxygen conditions in nutrient-rich media, the shift towards this Warburg phenotype occurred as a step-wise adaptation process associated with augmented tumorigenic capacity and improved survival characteristics of the transformed cells. We hypothesize that early-transformed cells, which potentially serve as founders for new tumor masses may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype.
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Transformación Celular Neoplásica , Fibroblastos/metabolismo , Glucólisis , Fosforilación Oxidativa , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/fisiología , Animales , Línea Celular Transformada , Proliferación Celular , Células Cultivadas , Fibroblastos/citología , Fibroblastos/ultraestructura , Ácido Láctico/metabolismo , Masculino , Metaboloma , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Electrónica de Rastreo , Mitocondrias/metabolismo , NAD/metabolismo , Trasplante de Neoplasias , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Consumo de Oxígeno , Retroviridae/genética , Superóxidos/metabolismo , Proteínas ras/genética , Proteínas ras/fisiologíaRESUMEN
Intracellular pathogenic organisms such as salmonellae and shigellae are able to evade the effects of many antibiotics because the drugs are not able to penetrate the plasma membrane. In addition, these bacteria may be able to transfer genes within cells while protected from the action of drugs. The primary mode by which virulence and antibiotic resistance genes are spread is bacterial conjugation. Salmonellae have been shown to be competent for conjugation in the vacuoles of cultured mammalian cells. We now show that the conjugation machinery is also functional in the mammalian cytosol. Specially constructed Escherichia coli strains expressing Shigella flexneri plasmid and chromosomal virulence factors for escape from vacuoles and synthesizing the invasin protein from Yersinia pseudotuberculosis to enhance cellular entry were able to enter 3T3 cells and escape from the phagocytic vacuole. One bacterial strain (the donor) of each pair to be introduced sequentially into mammalian cells had a conjugative plasmid. We found that this plasmid could be transferred at high frequency. Conjugation in the cytoplasm of cells may well be a general phenomenon.
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Fenómenos Fisiológicos Bacterianos , Conjugación Genética/fisiología , Citoplasma/microbiología , Interacciones Huésped-Parásitos/fisiología , Células 3T3 , Animales , Ratones , PlásmidosRESUMEN
Genetic ablation of adenylate kinase 1 (AK1), a member of the AK family of phosphotransfer enzymes, disturbs muscle energetic economy and decreases tolerance to metabolic stress, despite rearrangements in alternative high energy phosphoryl transfer pathways. To define the mechanisms of this adaptive response, soleus and gastrocnemius muscles from AK1(-/-) mice were characterized by cDNA array profiling, Western blot and ultrastructural analysis. We demonstrate that AK1 deficiency induces fiber-type specific variation in groups of transcripts involved in glycolysis and mitochondrial metabolism and in gene products defining structural and myogenic events. This was associated with increased phosphotransfer capacities of the glycolytic enzymes pyruvate kinase and 3-phosphoglycerate kinase. Moreover, in AK1(-/-) mice, fast-twitch gastrocnemius, but not slow-twitch soleus, had an increase in adenine nucleotide translocator (ANT) and mitochondrial creatine kinase protein, along with a doubling of the intermyofibrillar mitochondrial volume. These results provide molecular evidence for wide-scale remodeling in AK1-deficient muscles aimed at preservation of efficient energetic communication between ATP producing and utilizing cellular sites.
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Adenilato Quinasa/deficiencia , Adenilato Quinasa/genética , Metabolismo Energético , Isoenzimas/deficiencia , Isoenzimas/genética , Músculo Esquelético/metabolismo , Adenilato Quinasa/metabolismo , Animales , Exones , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Homocigoto , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Músculo Esquelético/ultraestructura , Análisis de Secuencia por Matrices de Oligonucleótidos , Consumo de Oxígeno , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
Muscle function depends on an adequate ATP supply to sustain the energy consumption associated with Ca(2+) cycling and actomyosin sliding during contraction. In this regulation of energy homeostasis, the creatine kinase (CK) circuit for high energy phosphoryl transfer between ATP and phosphocreatine plays an important role. We earlier established a functional connection between the activity of the CK system and Ca(2+) homeostasis during depolarization and contractile activity of muscle. Here, we show how CK activity is coupled to the kinetics of spontaneous and electrically induced Ca(2+) transients in the sarcoplasm of myotubes. Using the UV ratiometric Ca(2+) probe Indo-1 and video-rate confocal microscopy in CK-proficient and -deficient cultured cells, we found that spontaneous and electrically induced transients were dependent on ryanodine-sensitive Ca(2+) release channels, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps, extracellular calcium, and functional mitochondria in both cell types. However, at increasing sarcoplasmic Ca(2+) load (induced by electrical stimulation at 0.1, 1, and 10 Hz), the Ca(2+) removal rate and the amount of Ca(2+) released per transient were gradually reduced in CK-deficient (but not wild-type) myotubes. We conclude that the CK/phosphocreatine circuit is essential for efficient delivery of ATP to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps and thereby directly influences sarcoplasmic reticulum refilling and the kinetics of the sarcoplasmic Ca(2+) signals.