RESUMEN
Methods to improve plasmid-mediated transgene expression are needed for gene medicine and gene vaccination applications. To maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. We report herein the development of potent minimal, antibiotic-free, high-manufacturing-yield mammalian expression vectors incorporating rationally designed additive combinations of expression enhancers. The SV40 72 bp enhancer incorporated upstream of the cytomegalovirus (CMV) enhancer selectively improved extrachromosomal transgene expression. The human T-lymphotropic virus type I (HTLV-I) R region, incorporated downstream of the CMV promoter, dramatically increased mRNA translation efficiency, but not overall mRNA levels, after transient transfection. A similar mRNA translation efficiency increase was observed with plasmid vectors incorporating and expressing the protein kinase R-inhibiting adenoviral viral associated (VA)1 RNA. Strikingly, HTLV-I R and VA1 did not increase transgene expression or mRNA translation efficiency from plasmid DNA after genomic integration. The vector platform, when combined with electroporation delivery, further increased transgene expression and improved HIV-1 gp120 DNA vaccine-induced neutralizing antibody titers in rabbits. These antibiotic-free vectors incorporating transient expression enhancers are safer, more potent alternatives to improve transgene expression for DNA therapy or vaccination.
Asunto(s)
Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Plásmidos , Transgenes , Animales , Línea Celular , Humanos , ARN Mensajero/metabolismo , eIF-2 Quinasa/antagonistas & inhibidoresRESUMEN
The developmental pattern of slow myosin expression has been studied in mouse embryos from the somitic stage to the period of secondary fiber formation and in myogenic cells, cultured from the same developmental stages. The results obtained, using a combination of different polyclonal and monoclonal antibodies, indicate that slow myosin is coexpressed in virtually all the cells that express embryonic (fast) myosin in somites and limb buds in vivo as well as in culture. On the contrary fetal or late myoblasts (from 15-d-old embryos) express in culture only embryonic (fast) myosin. At this stage, muscle cells in vivo, as already shown (Crow, M.T., and F.A. Stockdale. 1986. Dev. Biol. 113:238-254; Dhoot, G.K. 1986. Muscle & Nerve. 9:155-164; Draeger, A., A.G. Weeds, and R.B. Fitzsimons. 1987. J. Neurol. Sci. 81:19-43; Miller, J.B., and F.A. Stockdale. 1986. J. Cell Biol. 103:2197-2208), consist of primary myotubes, which express both myosins, and secondary myotubes, which express preferentially embryonic (fast) myosin. Under no circumstance neonatal or adult fast myosins were detected. Western blot analysis confirmed the immunocytochemical data. These results suggest that embryonic myoblasts in mammals are all committed to the mixed embryonic-(fast) slow lineage and, accordingly, all primary fibers express both myosins, whereas fetal myoblasts mostly belong to the embryonic (fast) lineage and likely generate fibers containing only embryonic (fast) myosin. The relationship with current models of avian myogenesis are discussed.
Asunto(s)
Extremidades/embriología , Músculos/embriología , Miosinas/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Diferenciación Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Edad Gestacional , RatonesRESUMEN
The dwarf mutation in mice interferes with the development of those anterior pituitary cells responsible for production of thyroid stimulating hormone, growth hormone, and prolactin. Myosin isozyme transitions in both cardiac and skeletal muscle were also found to be affected in this mutant. Electrophoresis of native myosins demonstrated that the fetal (V3) to adult (V1) ventricular cardiac isozyme transition was completely blocked in dwarf mice; in contrast, the neonatal to adult fast myosin transition in hind limb skeletal muscle was slowed but not totally inhibited. The persistence of neonatal myosin heavy chain for up to 55-75 d after birth in dwarf mice, as compared with 16 d in normal mice, was directly demonstrated by polypeptide and immunopolypeptide mapping. Morphological examination of 18-36-d-old dwarf skeletal muscles by optical and electron microscopy revealed a relative immaturity, but no signs of gross pathology were evident. Immunocytochemical analysis showed that the abnormal persistence of neonatal myosin occurs in most of the fibers. Multiple injections of thyroxine restored a normal isozyme complement to both cardiac and skeletal muscles within 11-15 d. Therefore, the effects of the dwarf mutation on myosin isozymes can be explained by the lack of thyroid hormone in these animals. Because the synthesis of growth hormone is not stimulated by thyroid hormone in dwarf mice as it would be in normal animals, these results demonstrate that thyroid hormone promotes myosin isozyme transitions independent of growth hormone production.
Asunto(s)
Enanismo Hipofisario/enzimología , Isoenzimas/metabolismo , Músculos/enzimología , Miocardio/enzimología , Miosinas/metabolismo , Envejecimiento , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Modelos Animales de Enfermedad , Enanismo Hipofisario/genética , Enanismo Hipofisario/fisiopatología , Electroforesis en Gel de Poliacrilamida , Histocitoquímica , Ratones , Ratones Mutantes , Desarrollo de Músculos , Músculos/ultraestructura , Miosinas/análisis , Tiroxina/administración & dosificaciónRESUMEN
When adult mouse muscle fibers are co-cultured with embryonic mouse spinal cord, the muscle regenerates to form myotubes that develop cross-striations and contractions. We have investigated the myosin heavy chain (MHC) isoforms present in these cultures using polyclonal antibodies to the neonatal, adult fast, and slow MHC isoforms of rat (all of which were shown to react specifically with the analogous mouse isoforms) in an immunocytochemical assay. The adult fast MHC was absent in newly formed myotubes but was found at later times, although it was absent when the myotubes myotubes were cultured without spinal cord tissue. When nerve-induced muscle contractions were blocked by the continuous presence of alpha-bungarotoxin, there was no decrease in the proportion of fibers that contained adult fast MHC. Neonatal and slow MHC were found at all times in culture, even in the absence of the spinal cord, and so their expression was not thought to be nerve-dependent. Thus, in this culture system, the expression of adult fast MHC required the presence of the spinal cord, but was probably not dependent upon nerve-induced contractile activity in the muscle fibers.
Asunto(s)
Músculos/análisis , Miosinas/análisis , Médula Espinal/análisis , Factores de Edad , Animales , Animales Recién Nacidos , Bungarotoxinas/farmacología , Inducción Embrionaria , Ratones , Morfogénesis , Contracción Muscular/efectos de los fármacos , Músculos/ultraestructura , Técnicas de Cultivo de Órganos , Médula Espinal/embriologíaRESUMEN
We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using S1-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated lambda 10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac beta-MHC), cardiac (alpha-MHC), and EOM (lambda 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type. The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors.
Asunto(s)
Regulación de la Expresión Génica , Miosinas/genética , Músculos Oculomotores/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Histocitoquímica , Isoenzimas/genética , Isoenzimas/inmunología , Miosinas/inmunología , Músculos Oculomotores/análisis , Especificidad de Órganos , ARN Mensajero/análisis , Ratas , Ratas EndogámicasRESUMEN
P-Chlorophenylalanine methyl ester and the ester plus pargyline have been reported to facilitate sexual mounting behavior in animals, but these studies have shown only a facilitation of homosexual mounting. The present study indicates that these agents do not enhance the probability or frequency of heterosexual interactions in rats.
Asunto(s)
Fenilalanina/farmacología , Conducta Sexual Animal/efectos de los fármacos , Animales , Afrodisíacos , Ésteres/farmacología , Femenino , Homosexualidad , Masculino , Pargilina/farmacologíaRESUMEN
Predatory arthropods can be important for preventing insect pests from reaching damaging levels in soybean. However, the predator community can be compromised when pest control strategies include the application of broad-spectrum insecticides. The use of selective insecticides such as diamides could conserve predators while still providing necessary pest control. We evaluated two selective diamide insecticides, chlorantraniliprole and flubendiamide, and a broad-spectrum insecticide, lambda-cyhalothrin in combination with chlorantraniliprole, for impact on predators in soybean. We applied insecticides to field plots and documented predator abundance prior to and up to 3 wk postapplication using sticky card, beat sheet, and sweep net sampling methods. In sweep net samples, total predator abundance in plots treated with the selective insecticides was not significantly different from untreated control plots. For beat sheet samples, there were no significant differences in the abundance of total predators on any day postapplication between the selective diamide insecticides or the untreated control, but abundance decreased after application of lambda-cyhalothrin + chlorantraniliprole and did not recover. For sticky cards, there were no differences in predator abundance among treatments on any day postapplication. Over all, results showed that there were no significant differences in the abundance of total predators, Anthocoridae, Araneae, or Geocoridae after application of flubendiamide or chlorantraniliprole compared with the untreated control for up to 3 wk after application. All insecticides significantly decreased populations of lepidopteran pests compared with the untreated control, but only lambda-cyhalothrin + chlorantraniliprole reduced predatory arthropod abundance.
Asunto(s)
Benzamidas/toxicidad , Insectos/efectos de los fármacos , Insecticidas/toxicidad , Nitrilos/toxicidad , Conducta Predatoria/efectos de los fármacos , Piretrinas/toxicidad , Sulfonas/toxicidad , ortoaminobenzoatos/toxicidad , Animales , Control Biológico de Vectores , Glycine max/crecimiento & desarrolloRESUMEN
The ability of embryonic chicken muscle initiation factors to translate rabbit globin messenger RNA in an efficient, fractionated cell-free system has been examined. Although muscle factors stimulate leucine incorporation to only 15--35% the levels achieved with rabbit reticulocyte initiation factors, they synthesize more than one globin chain per mRNA molecule and both alpha and beta globin are produced. Increasing the ribosome concentration and adding the polyamine spermidine to the system produce stimulatory effects which are quantitatively and qualitatively similar for both factor preparations. The lower efficiency of synthesis of muscle factors relative to reticulocyte factors is also apparent when mRNA from encephalomyocarditis virus or embryonic chicken muscle polysomes are used in the cell-free system. These results do not support a specific restriction in the capacity of muscle factors to translate globin mRNA. Furthermore, the similarity of the effects of presumed non-specific components on the activity of muscle and reticulocyte factors suggests that globin synthesis in the cell-free system may be controlled in a similar fashion for both preparations.
Asunto(s)
Globinas/biosíntesis , Músculos/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Factores de Iniciación de Péptidos , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Sistema Libre de Células , Embrión de Pollo , Virus de la Encefalomiocarditis/metabolismo , Cinética , Magnesio/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Viral/metabolismo , Conejos , Reticulocitos/metabolismo , Espermidina/farmacologíaRESUMEN
Previous studies from our laboratory have investigated messenger RNA metabolism in calf muscle cells in tissue culture. The analysis of mRNA was based on its poly(A) content. We have now examined directly the proportion of mRNA which contains poly(A) in these cells. After separation of poly(A)+ -and poly(A) - -RNA on oligo(dT) -cellulos, the two fractions were translated in a reconstituted, heterologous cell-free protein-synthesizing system and the products were compared with those from the translation of total RNA. The great majority of mRNA form either prefusion or postfusion cultures was poly(A)- containing; quantitative determinations show that about 70-90% of the actin mRNA is poly(A)-containing. In order to determine if a large fraction of the calf muscle mRNA can be translated by a heterologous cell-free system, [3H]-POLY(A)+ -RNA was added to reticulocyte lysates and the formation of initiation complexes was followed. These experiments suggest that the bulk of calf muscle cell mRNA would be utilized in such a system and justify the use of cell-free systems to examine the poly(A) content of total mRNA. Thus, differential polyadenylation does not seem to be an important aspect of mRNA metabolism in cultured muscle cells. The previous study of mRNA in these cells, based on poly(A) content, is apparently a valid measure of overall mRNA metabolism.
Asunto(s)
Proteínas Musculares/genética , Músculos/metabolismo , Poli A/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Línea Celular , Sistema Libre de Células , Histonas/genética , Proteínas Musculares/biosíntesis , ARN Mensajero/genéticaRESUMEN
Hemodynamic and clinical evaluations of 123 patients with acute myocardial infarction were performed during the first hour of admission to the hospital. In the 123 patients, the right atrial pressure was less than 10 mm Hg in 49 patients, the right atrial oxygen saturation was less than 70% in 97 patients, the arteriovenous oxygen difference was greater than 5.0 vol% in 78 patients. The arterial Po2 was less than 90 mm Hg in 101 of 107 patients who could be evaluated while breathing room air. The cardiac index was depressed below 3.0 L/min/m2 in 65 of 98 patients. The hemodynamic findings generally correlated with the clinical status of the patient; however, within each clinical class of patients there was a wide spectrum of values for each measurement evaluated. There was also considerable overlap of the values found within each clinical classification. It is concluded that hemodynamic evaluation of patients with acute myocardial infarction presents a profile of the patient which is frequently different from the profile that clinical evaluation presents. An objective hemodynamic classification of patients with acute myocardial infarction may provide a more useful index for the evaluation of the patient's prognosis and for the assessment of preventative therapy.
Asunto(s)
Infarto del Miocardio/fisiopatología , Adulto , Anciano , Femenino , Hemodinámica , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/diagnósticoRESUMEN
The formation of human myotubes in culture is accompanied by the induction of developmentally regulated, muscle-specific genes. We have studied the expression of human myosin light chain proteins and mRNAs during myogenesis in culture, in particular the skeletal embryonic myosin light chain 1 (MC1emb), which is indistinguishable from MLC1 of adult atrial cardiac muscle (MLC1A) as has been shown for rodent and bovine MLC1emb. We have identified distinct MLC1emb/MLC1A mRNAs in cultured human skeletal muscle cells that differ in their 5' and 3' untranslated regions but contain identical protein-coding regions. The alternative 3' untranslated region is detectable also in RNA of human atria. The different MLC1emb RNAs are likely to be encoded by one gene. It appears that the two MLC1emb 5' untranslated regions of the human gene are specific for man. In the mouse, only one 5' untranslated region of the MLC1emb gene has been detected.
Asunto(s)
Miosinas/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , Electroforesis en Gel Bidimensional , Embrión de Mamíferos , Regulación de la Expresión Génica , Genes , Humanos , Datos de Secuencia MolecularRESUMEN
We have previously characterized the proximal promoter of the mouse IIB myosin heavy chain (MyHC) gene, which is expressed only in fast-contracting glycolytic skeletal muscle fibers. We show here that the substitution into this promoter of a non-canonical TATA sequence from the IgH gene results in inactivity in muscle cells, even though TATA-binding protein (TBP) can bind strongly to this mutated promoter. Chemical foot-printing data show, however, that TBP makes different DNA contacts on this heterologous TATA sequence. The inactivity of such a non-canonical TATA motif in the IIB promoter context appears to be caused by a non-functional conformation of the bound TBP-DNA complex that is incapable of sustaining transcription. The conclusions imply that the precise sequence of the promoter TATA motif needs to be matched with the specific functional class of upstream activator proteins present in a given cell type in order for the gene to be transcriptionally active.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Cadenas Pesadas de Miosina/genética , Regiones Promotoras Genéticas , TATA Box , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia Conservada , ADN/química , ADN/genética , ADN/metabolismo , Huella de ADN , Proteínas de Unión al ADN/química , Evolución Molecular , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Estructura Molecular , Músculo Esquelético/metabolismo , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Proteína de Unión a TATA-Box , Factores de Transcripción/química , Transcripción GenéticaRESUMEN
Some patients with small cell lung cancer (SCLC) or neuroblastoma develop an immune response against HuD, a human homologue of the Drosophila protein, elav, which is expressed in the nucleus and to a lesser degree the cytoplasm of neurons and tumor cells. This immune response is characterized by antibodies (anti-Hu) that at high titers are associated with a disease called paraneoplastic encephalomyelitis/sensory neuronopathy, in which infiltrates of T cells are found in the tumor and nervous system. Although all SCLCs express HuD, anti-Hu antibodies are identified in only 17% of patients with SCLC, usually at low titers, and are associated with indolent tumor growth. To determine whether the anti-Hu immune response causes indolent tumor growth, we developed an animal model using HuD DNA immunization. We found that a plasmid coding for a secreted form of HuD induced a strong and specific anti-Hu response. Immunized animals were challenged by s.c. implantation of a neuroblastoma cell line that constitutively expresses HuD. When compared with controls, mice immunized with the secreted HuD showed significant tumor growth inhibition (51% reduction volume; P = 0.0012), and 14% of them had complete tumor rejection. Tumors from these animals showed three times more CD3+ lymphocytic infiltrates than those from control mice and had a higher CD8+:CD4+ ratio. None of the animals developed neurological deficits or neuropathological evidence of nervous system pathology. In this mouse model of neuroblastoma, DNA immunization with HuD resulted in tumor growth inhibition but did not induce neurological disease. This model closely mimics the clinical course of more indolent tumor growth seen in patients with the anti-Hu immune response.
Asunto(s)
Anticuerpos/inmunología , Proteínas del Tejido Nervioso , Neuroblastoma/terapia , Proteínas de Unión al ARN/inmunología , Vacunas de ADN/administración & dosificación , Animales , Anticuerpos/sangre , Proteínas ELAV , Proteína 4 Similar a ELAV , Femenino , Vectores Genéticos/genética , Humanos , Inmunoterapia , Subgrupos Linfocitarios/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Trasplante de Neoplasias , Neuroblastoma/sangre , Neuroblastoma/inmunología , Neuroblastoma/prevención & control , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Células Tumorales Cultivadas , Vacunas de ADN/uso terapéuticoRESUMEN
Muscle strength has been shown to predict bone mineral density (BMD) in women. We examined this relationship in 50 healthy men who ranged in age from 28 to 51 years (average 38.3 years). BMD of the lumbar spine, proximal femur, whole body, and tibia were measured by dual-energy x-ray absorptiometry (Hologic QDR 1000W). Dynamic strength using one repetition maximum was assessed for the biceps, quadriceps, and back extensors and for the hip abductors, adductors, and flexors. Isometric grip strength was measured by dynamometry. Daily walking mileage was assessed by 9 week stepmeter records and kinematic analysis of video filming. Subjects were designated as exercisers and nonexercisers. Exercisers participated in recreational exercise at least two times each week. The results demonstrated that BMD at all sites correlated with back and biceps strength (p < 0.01 to p = 0.0001). Body weight correlated with tibia and whole-body BMD (p < 0.001); age negatively correlated with Ward's triangle BMD (p < 0.01). In stepwise multiple regressions, back strength was the only independent predictor of spine and femoral neck density (R2 = 0.27). Further, back strength was the most robust predictor of BMD at the trochanter, Ward's triangle, whole body, and tibia, although biceps strength, age, body weight, and leg strength contributed significantly to BMD at these skeletal sites, accounting for 35-52% of the variance in BMD. Exercisers and nonexercisers were similar for walking (3.97 versus 3.94 miles/day), age (37.8 versus 38.5) years, and weight (80.0 versus 77.7 kg). However, BMD and muscle strength were significantly greater in exercises than in nonexercisers.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Densidad Ósea/fisiología , Ejercicio Físico , Músculos/fisiología , Absorciometría de Fotón , Adulto , Peso Corporal , Fémur , Humanos , Vértebras Lumbares , Masculino , Persona de Mediana EdadRESUMEN
The particulate form of the major envelope or surface (S) protein of hepatitis B virus (HBV) can be taken up by antigen-presenting cells and processed for class I presentation as an exogenous protein. We have used several DNA plasmid vectors expressing the HBV envelope proteins to determine whether these sequences are able to induce cytotoxic T lymphocyte (CTL) responses in BALB/c mice after intramuscular DNA injection. A potent and specific induction was obtained, which can be detected ex vivo using either specific or nonspecific (interleukin-2) stimulation in cell culture, and the DNA-primed CTL responses are stronger than those obtained with protein injection with either stimulation protocol. The CTL response induced by DNA-based immunization is both canonical and highly specific as indicated by the nature of the epitope presented (amino acids 28-39), the class I allele used (Ld), and the T lymphocytes involved (CD8+). The CTL response is initiated between 3 and 6 days after DNA injection. By 6-12 days after a single DNA injection, ex vivo cytolytic activity is nearly maximal, and similar high levels of activity can still be detected 4 months after injection. The possibility is discussed that the unusual mode of delivery of the antigen to the immune system provided by in situ expression might allow HBV envelope antigen to be taken up and processed for class I presentation by in situ expression might allow HBV envelope antigen to be taken up and processed for class I presentation as an exogenous protein in addition to activating potentially the classical endogenous pathway.
Asunto(s)
Vectores Genéticos/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos contra la Hepatitis B/biosíntesis , Anticuerpos contra la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Humanos , Inmunización , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Músculos/fisiología , Bazo/inmunologíaRESUMEN
Striated muscle is the only tissue found to be capable of taking up and expressing reporter genes that are transferred in the form of plasmid DNA. Thus, direct gene transfer is a potential method of gene therapy for the primary inherited myopathies. However, results to date have had insufficient and too variable expression to consider using direct gene transfer in human trials. We have determined that much of the variability of expression is due to nonuniform distribution of substances injected into skeletal muscle in vivo, and have developed a model to ameliorate this. Preinjection of muscles with a relatively large volume of hypertonic sucrose improves the distribution of injected substances and results in significantly less variable expression of reporter genes for luciferase or beta-galactosidase; the coefficient of variation for mean luciferase activity was reduced from about 120% to 25%. Expression is not directly proportional to dose, but is more so if the muscles are preinjected with sucrose than not. Expression is higher and less variable if DNA is injected in a larger than a smaller volume. The choice of promoter appears to be particularly important. Luciferase reporter gene expression from the SV40 promoter was transient and low, whereas expression driven by the Rous sarcoma virus (RSV) promoter was high and sustained, such that a 1,000-fold difference in expression could be observed. The mechanism of gene uptake is still unknown, but our findings indicate that fibers damaged by the injection procedure do not take up and express plasmid DNA.
Asunto(s)
Carbono , Músculos/metabolismo , Transfección , Animales , Colorantes/farmacocinética , Colorantes/farmacología , ADN/administración & dosificación , Expresión Génica , Terapia Genética , Luciferasas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades Musculoesqueléticas/genética , Enfermedades Musculoesqueléticas/terapia , Proyectos Piloto , Regiones Promotoras Genéticas , Sacarosa/farmacología , beta-Galactosidasa/genéticaRESUMEN
Direct gene transfer into skeletal muscle offers several therapeutic possibilities. We assessed direct intramuscular injection of recombinant plasmids, adenovirus, or retrovirus in normal or regenerating muscles of mice. The incorporation and expression of reporter genes introduced by any of these three vectors is greater in regenerating than in mature muscle. In regenerating muscle, pure DNA and adenovirus result in equivalent numbers of fibers expressing reporter gene (> 10%), but adenovirus also induces considerable cellular infiltration. In mature muscle, recombinant DNA is better than adenovirus. Retrovirus failed to infect mature muscle fibers and was less effective than plasmid DNA or adenovirus in regenerating muscle. The surprisingly high relative efficiency of pure plasmid DNA suggests that this method will provide a simple, safe and viable alternative for gene therapy involving muscle tissue.
Asunto(s)
ADN Recombinante , ADN Viral , Técnicas de Transferencia de Gen , Vectores Genéticos , Músculos/metabolismo , Plásmidos , Adenoviridae/genética , Animales , Proteínas Cardiotóxicas de Elápidos/farmacología , Genes Reporteros , Terapia Genética , Masculino , Ratones , Ratones Endogámicos C57BL , Músculos/efectos de los fármacos , Músculos/fisiología , Regeneración/efectos de los fármacos , Retroviridae/genéticaRESUMEN
The intermediate filament (IF) composition of muscle cells of various sources is still a controversial issue. In the present study, the IF composition of bovine Purkinje fibres (PFs), atrial and ventricular myocardium, and gastric smooth muscle (SM) has been compared using biochemical and immunocytochemical methods. The Mr of the major IF subunit protein in all four tissues was 55,000. In two-dimensional (2-D) electrophoresis gels of Triton-treated ordinary atrial and ventricular myocardium and the gastric muscular wall, two or three isoelectric isoforms were seen, whereas in PFs up to seven isoforms caused by phosphorylation were observed. In immunofluorescence studies antibodies against the Mr 55,000 subunit of PFs and gastric SM, respectively, both showed identical reactivity with PFs, atrial and ventricular myocytes, gastric SM cells and some SM cells in intramyocardial and gastric muscular wall blood vessels. A small amount of vimentin (Mr 57,000) was also detected in 2-D gel electrophoresis in all four tissues as well as in immunoblotting of PFs with antibodies to vimentin. Immunofluorescence studies using both polyclonal and monoclonal antibodies to vimentin showed that vimentin was present in the endothelium and SM cells of both intramyocardial and gastric muscular wall vessels, sometimes together with desmin in the vascular SM cells, but was never seen in PF, atrial, ventricular or gastric SM cells proper. As expected, vimentin was present in interstitial tissue, i.e., fibroblasts and capillaries. However, interestingly, the monoclonal antibodies, which recognized different antigenic determinants of vimentin, did not give identical staining patterns. Especially the staining of the vascular SM cells differed. Since this staining pattern did not change upon denaturation and unmasking experiments, it seems that the organization of vimentin in different mesenchymal cell types varies. Vimentin was also detected in isolated PFs but here it was located solely in the contaminating interstitial tissue. Thus, desmin is the sole IF protein expressed in PFs, in atrial and ventricular myocytes and in gastric SM cells proper; vimentin alone being present in the interstitial tissue cells, whilst in vascular SM cells desmin and vimentin are coexpressed in various proportions. The variation in number of isoforms of desmin and the heterogeneity in staining of mesenchymal tissues with monoclonal vimentin antibodies probably indicates that the IF cytoskeletons are differently organized in various cell types, even though they contain IFs of the same class.
Asunto(s)
Sistema de Conducción Cardíaco/análisis , Proteínas de Filamentos Intermediarios/análisis , Músculo Liso/análisis , Ramos Subendocárdicos/análisis , Estómago/análisis , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Peso Molecular , Músculo Liso/citología , Especificidad de Órganos , Ramos Subendocárdicos/citología , Estómago/citologíaRESUMEN
A chronology of theoretical development in studies of the role of testicular hormones in murine aggression is presented. Evidence which brings into question the generality of current theory is reviewed, and the implications of this evidence for the direction of future research are discussed. A new method by which to characterize individual differences in aggressive behavior is described, and recent data which provide the basis for the development of a new theory are presented. It is theorized that testicular hormones reduce individual differences in the aggressive behavior of male mice, and that this behavioral "homogenization" is mediated by a testicular-hormone regulated "discrimination mechanism," possibly localized within the mouse olfactory system. The generality of this theory and the implications for other hormone/behavior systems are also discussed.
Asunto(s)
Agresión/fisiología , Individualidad , Testículo/fisiología , Testosterona/fisiología , Animales , Masculino , Ratones , OrquiectomíaRESUMEN
Over the past 20 years many investigators have shown that one can facilitate sexual receptivity in estrogen-primed rats either by giving progesterone or a drug which stimulates or inhibits a neurotransmitter system. Drugs which have been reported to substitute for progesterone include cholinergic agonists, serotonergic agonists and antagonists, dopaminergic agonists and antagonists, opiate antagonists, neurohormones, pituitary, ovarian and adrenal hormones and drugs that interact with cyclic nucleotide systems. Most of the drugs that are active are known to increase neural levels of cyclic GMP either by acting on guanylate cyclase or on phosphodiesterase. We propose that the cGMP system mediates the common behavioral effect of the wide variety of drugs that facilitate receptivity.