RESUMEN
Cattle grazing wheat pasture in the southern Great Plains are sometimes fed an energy supplement; however, the benefits of supplementation on nutrient balance, energy metabolism, and greenhouse gas emissions have not been elucidated. Therefore, we used 10 British crossbred steers (206 ± 10.7 kg initial BW) in a respiration calorimetry study to evaluate the effects of energy supplementation on energy losses, N balance, and nutrient digestibility of steers fed green-chopped wheat forage. The study design was an incomplete replicated 4 × 4 Latin square with treatments in a 2 × 2 factorial arrangement. Steers ( = 8) were assigned to 1 of 2 BW blocks (4 steers per block) with dietary factors consisting of 1) no supplementation (CON) or supplemented with a steam-flaked corn-based energy supplement (that also contained monensin sodium) at 0.5% of BW daily (SUP) and 2) NEm intakes of 1 times (1x) or 1.5 times (1.5x) maintenance. Wheat forage was harvested daily and continuously fed as green-chop to steers during the 56-d study. There were no differences ( ≥ 0.32) between CON and SUP for OM (78.3 vs. 80.7%, respectively) or NDF (68.3 vs. 64.8%, respectively) digestibility. At the 1.5x level of intake, there was no difference ( ≥ 0.16) in energy lost in feces (4.27 vs. 3.92 Mcal/d) or urine (0.58 vs. 0.55 Mcal/d), heat production (8.69 vs. 8.44 Mcal/d), or retained energy (3.10 vs. 3.46 Mcal/d) between supplementation treatments. Oxygen consumption (1,777 vs. 1,731 L/d; = 0.67) and CO production (1,704 vs. 1,627 L/d; = 0.56) of CON and SUP steers, respectively, were not different; however, SUP steers tended to have ( = 0.06) lower CH production (115 vs 130 L/d) than CON steers. Methane, as a proportion of GE intake, was similar for CON (6.87%) and SUP (6.07%; = 0.18), as was the ME:DE ratio ( = 0.24; 86.3% for CON and 87.9% for SUP). Fractional N excretion in urine and feces, as a proportion of total N excreted ( ≥ 0.84) or N intake ( ≥ 0.63), was not different between treatments. Calculated NEm and NEg values for CON were 1.76 and 1.37 Mcal/kg DM, respectively, whereas the NEm and NEg values for the SUP treatment were 2.32 and 1.61 Mcal/kg DM, respectively. Calculated NE values for steers fed additional energy were approximately 17.5% greater than the expected difference in energy content. This was probably the result of the inconsistent response at the 1x DMI level. Under these circumstances, energy supplementation did appear to enhance NEm and NEg value of the supplemented wheat forage diet.
Asunto(s)
Alimentación Animal/análisis , Bovinos/fisiología , Suplementos Dietéticos , Metabolismo Energético , Metano/metabolismo , Nitrógeno/metabolismo , Animales , Calorimetría/veterinaria , Dieta/veterinaria , Digestión , Heces/química , Masculino , Vapor , Triticum , Zea maysRESUMEN
Roughage is fed in finishing diets to promote ruminal health and decrease digestive upset, but the inclusion rate is limited because of the cost per unit of energy and feed management issues. Rumination behavior of cattle may be a means to standardize roughage in beef cattle finishing diets, and increasing the particle size of roughage could modulate the ruminal environment and aid in maintaining ruminal pH. Therefore, this experiment was conducted to determine the effects of corn stalk (CS) inclusion rate and particle size in finishing diets on digestibility, rumination, and ruminal fermentation characteristics of beef steers. Four ruminally cannulated steers were used in a 4 × 4 Latin square experiment. Treatments were arranged as a 2 × 2 factorial with treatments consisting of 5% inclusion of a short-grind roughage (5SG), 10% inclusion of a short-grind roughage (10SG), 5% inclusion of a long-grind roughage (5LG), and 10% inclusion of a long-grind roughage (10LG). Differences in particle size were obtained by grinding corn stalks once (LG) or twice (SG) using a commercial tub grinder equipped with a 7.6-cm screen and quantified using the Penn State Particle Separator (PSPS) to estimate physically effective NDF (peNDF). Each period included 14 d for adaptation and 4 d for diet, fecal, and ruminal fluid collections. Animals were outfitted with rumination monitoring collars to continuously measure rumination activity. The 10LG treatment had a greater ( < 0.01) percentage of large particles (retained on the top 3 sieves of the PSPS) compared to the other treatments. This resulted in a greater ( < 0.01) percentage of estimated peNDF for the 10LG diet compared to the others. Feeding diets containing 5% roughage tended to increase ( ≤ 0.09) DM, NDF, and starch total tract digestibility compared to diets containing 10% roughage. Cattle consuming LG treatments had greater ( < 0.01) rumination time and greater ( < 0.01) ruminal pH than cattle consuming diets containing SG roughage. Cattle receiving the 5% inclusion rate of roughage tended to have greater ( = 0.09) time (h/d) under a ruminal pH of 5.6 and a larger ( = 0.03) area under the threshold compared to cattle receiving the 10% roughage treatments. Overall, feeding a lower inclusion of roughage with a larger particle size may stimulate rumination and aid in ruminal buffering similar to that of a higher inclusion of roughage with a smaller particle size, without negatively impacting digestibility and fermentation.
Asunto(s)
Alimentación Animal/análisis , Bovinos/fisiología , Animales , Bovinos/crecimiento & desarrollo , Dieta/veterinaria , Fibras de la Dieta/administración & dosificación , Digestión , Fermentación , Masculino , Tamaño de la Partícula , Tallos de la Planta , Rumen/metabolismo , Zea maysRESUMEN
An indirect calorimetry trial examined energy metabolism, apparent nutrient digestibility, C retention (CR), and N retention (NR) of cattle supplemented with zilpaterol hydrochloride (ZH). Beef steers ( = 20; 463 ± 14 kg) blocked ( = 5) by weight and source were individually fed and adapted to maintenance energy intake for 21 d before allotment to ZH (90 mg/steerâd) or no ß-adrenergic agonist treatment (control [CONT]) for 20 d (455 ± 14 kg at the start of treatment). Respiration chambers = 4 were used to quantify heat production (HP) during maintenance (d 12 to 16 of the ZH period) and fasting heat production (FHP; d 19 to 20 of ZH period; total 4 d of fast). Steers were harvested after a 6-d ZH withdrawal and carcasses were graded 24 h after harvest. Control cattle lost more BW ( < 0.01; 9 kg for CONT and 2 kg for ZH-treated) during maintenance whereas the BW loss of ZH-treated steers was greater ( < 0.01; 9 kg for ZH-treated and vs. 4 kg, for CONT) during FHP; no differences ( ≥ 0.76) were detected for G:F, ADG, and end BW. No differences in DMI, apparent nutrient digestibility, O consumption, or CH production ( ≥ 0.12) were detected; however, ZH-treated cattle had greater CO production during maintenance ( = 0.04; 23.6 L/kgBW for ZH-treated and 22.4 L/kg BW for CONT). Digestible energy and ME did not differ ( ≥ 0.19); however, urinary energy was greater ( = 0.05; 0.091 Mcal for CONT and 0.074 Mcal for ZH-treated) in CONT cattle. Steers treated with ZH tended to have greater HP ( = 0.09; 12.44 Mcal for ZH-treated and 11.69 Mcal for CONT), but the effect was reduced on a BW basis ( = 0.12; 0.126 Mcal/kg BW0.75 for ZH-treated and 0.120 Mcal/kg BW0.75 for CONT vs. 0.120 Mcal/kg BW). No treatment difference in FHP was observed ( ≥ 0.32) although CO production (L/steer) increased with ZH treatment ( = 0.04; 1,423 L/steer for ZH-treated and 1,338 L/steer for CONT). Control cattle excreted more ( = 0.05) N in urine (39.8 g/d for CONT and 32.4 g/d for ZH-treated); therefore, NR ( = 0.07; 22.14 g/d for ZH-treated and 14.12 g/d for CONT steers) tended to be greater for ZH-fed steers. Steers treated with ZH lost more C via CO ( = 0.04; 1,036.9 g/d for ZH-treated and 974.3 g/d for CONT) although total CR did not differ ( ≥ 0.23). Empty BW, HCW, and harvest yields (g/kg empty BW) were not different ( ≥ 0.13), whereas ZH increased dressed yield ( = 0.02; 62.12 % for ZH-treated and 60.65% for CONT) and LM area ( = 0.02; 77.81 cm for ZH-treated and vs. 70.90 cm for CONT). Separable carcass lean and actual skeletal muscle protein (SMP) were increased with ZH ( ≤ 0.04; 201.6 and 41.2 kg, respectively for ZH-treated and 196.0 and 38.4 kg, respectively for CONT). Results from this trial indicate that ZH treatment increased ( = 0.03) SMP and tended ( ≥ 0.07) to increase NR and modify HP during maintenance by increasing CO production.
Asunto(s)
Carbono/metabolismo , Bovinos/fisiología , Suplementos Dietéticos , Metabolismo Energético , Nitrógeno/metabolismo , Compuestos de Trimetilsililo/farmacología , Agonistas Adrenérgicos beta/farmacología , Alimentación Animal/análisis , Animales , Composición Corporal , Peso Corporal , Dieta/veterinaria , Ingestión de Alimentos , Ingestión de Energía , Masculino , Músculo Esquelético/efectos de los fármacosRESUMEN
One experiment was conducted to evaluate the influence of glycerin (GLY) on animal performance and health when used as a partial replacement for roughage in receiving diets. The second experiment was conducted using ruminally and duodenally cannulated steers in a 4 × 4 Latin square to determine the site of nutrient digestion and ruminal fermentation characteristics when GLY replaced roughage at 0%, 2.5%, 5%, and 10% of diet DM. In Exp. 1, steers (initial BW = 245 ± 2.3 kg) were fed treatment diets over a 42-d period that consisted of a control diet based on steam-flaked corn with GLY inclusion in replacement of dietary roughage at 0%, 5%, and 10% of diet DM. A linear reduction in DMI was observed as GLY increased (P = 0.01). Glycerin incorporation tended to improve G:F in a linear manner (P = 0.07); efficiency was improved 5.4% and 4.7% at 5% and 10% GLY. The number of animals receiving treatment for bovine respiratory disease did not differ among treatments. Furthermore, there were no differences among treatments for mortality or the frequency of steers that were seropositive for serum antibody titers to infectious bovine rhinotracheitis on d 28. In Exp. 2, apparent OM and apparent and true starch digestibility increased linearly (P < 0.05) as GLY concentration increased, whereas true OM digestibility responded in a quadratic (P < 0.01) manner. Bacterial OM and bacterial starch flow responded quadratically (P ≤ 0.02), and flow increased from 0% to 5% GLY inclusion and decreased thereafter. Feed OM flow responded quadratically (P ≤ 0.05), where it decreased from 0% to 2.5% GLY and increased from 2.5% to 10% GLY inclusion. Feed starch (P = 0.02) and total starch (P = 0.02) flow from the duodenum decreased linearly as the concentration of GLY increased in the diet. Bacterial N flow to the duodenum responded quadratically (P < 0.01); it increased with increasing GLY in the diet up to 5% and then decreased from 5% to 10%. The acetate to propionate (A:P) ratio in the ruminal fluid decreased (P < 0.05) as the concentration of GLY in the diet increased, which could have implications on improved G:F. The decrease in the A:P ratio as GLY increased in the diet, coupled with the linear decrease in DMI and improvement in G:F with GLY addition up to 5% of DM in place of roughage, implies that GLY is a viable dietary ingredient in growing and receiving diets.
Asunto(s)
Bovinos/fisiología , Fibras de la Dieta/análisis , Digestión , Duodeno/fisiología , Glicerol/metabolismo , Rumen/fisiología , Alimentación Animal/análisis , Crianza de Animales Domésticos , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Bovinos/crecimiento & desarrollo , Dieta/veterinaria , Suplementos Dietéticos/análisis , Relación Dosis-Respuesta a Droga , Fermentación , Glicerol/administración & dosificación , Masculino , Distribución Aleatoria , Estados UnidosRESUMEN
Two hundred sixty-four crossbred heifers (initial BW = 354 kg ± 0.5) were used to determine effects of corn processing method and wet distillers grains plus solubles (WDGS) inclusion in finishing diets on animal performance, carcass characteristics, and manure characteristics. The study was conducted as a randomized complete block with a 2 × 2 factorial arrangement of treatments. Dietary treatments included steam-flaked corn (SFC)- and dry-rolled corn (DRC)-based finishing diets containing 0 or 20% WDGS (0SFC, 20SFC, 0DRC, and 20DRC, respectively). Heifers averaged 154 d on feed and were marketed in 3 groups. There were no interactions between corn processing method and WDGS detected (P ≥ 0.29) for any performance or carcass response variables. Heifers fed diets containing WDGS tended to have greater final BW (P = 0.10) and increased G:F (P = 0.08) compared with heifers fed diets without WDGS. Heifers fed SFC-based diets consumed 7% less feed (P < 0.01) and were 9% more efficient (P < 0.01) than heifers fed DRC-based diets. Carcass characteristics were not affected by corn processing method or WDGS inclusion (P ≥ 0.16). Intakes of OM, N, P, and K were greater (P ≤ 0.05) for heifers fed DRC-based diets than those fed SFC-based diets, which resulted in greater net accumulation of the nutrients in the manure (P ≤ 0.04). Heifers fed diets containing WDGS had greater (P < 0.01) intakes of N, P, and K than heifers fed diets without WDGS. As a result, a greater net accumulation of P and K (P ≤ 0.03) and N (P = 0.10) were present in the manure from cattle fed diets containing WDGS compared with those fed diets without WDGS. There was no interaction (P ≥ 0.16) between corn processing and WDGS on N volatilization losses. Nitrogen volatilization losses from manure (expressed as a percentage of intake and g·heifer(-1)·d(-1)) were greater (P < 0.01) for heifers fed SFC-based diets than heifers fed DRC-based diets. Feeding DRC-based finishing diets to heifers resulted in increased manure production and nutrient excretion and decreased N volatilization. Both corn processing method and WDGS inclusion affected animal performance and manure characteristics.
Asunto(s)
Alimentación Animal/análisis , Bovinos/fisiología , Dieta/veterinaria , Estiércol/análisis , Nitrógeno/análisis , Fósforo/análisis , Zea mays/química , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Bovinos/crecimiento & desarrollo , Suplementos Dietéticos , Grano Comestible , Ambiente , Femenino , Carne/normas , Nitrógeno/metabolismo , Fósforo/metabolismoRESUMEN
The human immunodeficiency virus type 1 Vpr protein is both packaged into virions and efficiently localized to the nucleus. In this report, we show that a significant fraction of Vpr also accumulates in the cytoplasm of virus-producing cells. Although Vpr shuttles between the nucleus and the cytoplasm, studies with an export-deficient Vpr mutant reveal that nuclear export is not required for virion incorporation.
Asunto(s)
Núcleo Celular/metabolismo , Productos del Gen vpr/metabolismo , VIH-1/metabolismo , Virión/metabolismo , Ensamble de Virus/fisiología , Transporte Activo de Núcleo Celular , Citoplasma/metabolismo , Productos del Gen vpr/genética , Células HeLa , Humanos , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
HIV-1 is able to infect nondividing cells productively in part because the postentry viral nucleoprotein complexes are actively imported into the nucleus. In this manuscript, we identify a novel nuclear localization signal (NLS) in the viral integrase (IN) protein that is essential for virus replication in both dividing and nondividing cells. The IN NLS stimulates the efficient nuclear accumulation of viral DNA as well as virion-derived IN protein during the initial stages of infection but is dispensable for catalytic function. Because this NLS is required for infection irrespective of target cell proliferation, we suggest that interactions between uncoated viral nucleoprotein complexes and the host cell nuclear import machinery are critical for HIV-1 infection of all cells.
Asunto(s)
Infecciones por VIH/enzimología , Integrasa de VIH/metabolismo , VIH-1 , Señales de Localización Nuclear/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Secuencia de Aminoácidos , Núcleo Celular/virología , Células Cultivadas , ADN Viral/biosíntesis , Integrasa de VIH/química , Integrasa de VIH/genética , Células HeLa , Humanos , Leucocitos Mononucleares/virología , Mutagénesis Sitio-Dirigida , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/fisiología , Estructura Terciaria de Proteína , Alineación de Secuencia , Fracciones Subcelulares/química , Linfocitos T/virologíaRESUMEN
The Vpr protein of human immunodeficiency virus type 1 (HIV-1) performs a number of functions that are associated with the nucleus. Vpr enhances the nuclear import of postentry viral nucleoprotein complexes, arrests proliferating cells in the G2 phase of the cell cycle, and acts as a modest transcriptional activator. For this paper, we have investigated the nuclear import of Vpr. Although Vpr does not encode a sequence that is recognizable as a nuclear localization signal (NLS), Vpr functions as a transferable NLS both in somatic cells and in Xenopus laevis oocytes. In certain contexts, Vpr also mediates substantial accumulation at the nuclear envelope and, in particular, at nuclear pore complexes (NPCs). Consistent with this, Vpr is shown to interact specifically with nucleoporin phenylalanine-glycine (FG)-repeat regions. These findings not only demonstrate that Vpr harbors a bona fide NLS but also raise the possibility that one (or more) of Vpr's functions may take place at the NPC.
Asunto(s)
Productos del Gen vpr/metabolismo , VIH-1/fisiología , Membrana Nuclear/metabolismo , Transporte Biológico , Células Cultivadas , VIH-1/química , Células HeLa , Humanos , Macrófagos/virología , Nucleoproteínas/metabolismo , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Human immunodeficiency virus type-1 (HIV-1) is able to infect non-dividing cells such as tissue macrophages productively because post-entry viral nucleoprotein complexes are specifically imported into the nucleus in the absence of mitosis. Although it has been proposed that an amino-terminal region of the viral matrix (MA, p17Gag) protein harbors a basic-type nuclear localization sequence (NLS) that contributes to this process, utilization of three distinct nuclear import assays failed to provide any direct supporting evidence. Instead, we found that disruption of this region (26KK-->TT) reduces the rate at which the viral Gag polyprotein (p55Gag) is post-translationally processed by the viral protease. Consistent with the fact that appropriate proteolytic processing is essential for efficient viral growth in all cell types, we also show that the 26KK-->TT MA mutation is equivalently deleterious to the replication of a primary macrophage-tropic viral isolate in cultures of non-dividing and dividing cells. Taken together, these observations suggest that proteins other than MA supply the NLS(s) that enable HIV-1 to infect non-dividing cells.
Asunto(s)
Productos del Gen gag/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , VIH-1/patogenicidad , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Transporte Biológico Activo , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Femenino , Productos del Gen gag/genética , Antígenos VIH/genética , Antígenos VIH/metabolismo , Infecciones por VIH/metabolismo , VIH-1/genética , Células HeLa , Humanos , Técnicas In Vitro , Mutagénesis Sitio-Dirigida , Oocitos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Replicación Viral , Xenopus laevis , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of late HIV-1 mRNAs. This function is dependent on the mutationally defined Rev activation domain, which also forms a potent nuclear export signal. Transcription factor IIIA (TFIIIA) binds to 5S rRNA transcripts and this interaction has been proposed to play a role in the efficient nuclear export of 5S rRNA in amphibian oocytes. Here it is reported that amphibian TFIIIA proteins contain a sequence element with homology to the Rev activation domain that effectively substitutes for this domain in inducing the nuclear export of late HIV-1 mRNAs. It is further demonstrated that this TFIIIA sequence element functions as a protein nuclear export signal in both human cells and frog oocytes. Thus, this shared protein motif may play an analogous role in mediating the nuclear export of both late HIV-1 RNAs and 5S rRNA transcripts.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Productos del Gen rev/química , Productos del Gen rev/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Bufonidae , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/biosíntesis , Femenino , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Oocitos/fisiología , ARN Mensajero/metabolismo , ARN Ribosómico 5S/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factor de Transcripción TFIIIA , Factores de Transcripción/biosíntesis , Transcripción Genética , Xenopus laevis , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
The human immunodeficiency virus type 1 Rev trans activator binds directly to unspliced viral mRNA in the nucleus and activates its transport to the cytoplasm. In additon to the sequences that confer RNA binding and nuclear localization, Rev has a carboxy-terminal region, the activation domain, whose integrity is essential for biological activity. Because it has been established that Rev constitutively exits and reenters the nucleus and that the activation domain is required for nuclear exit, it has been proposed that Rev's activation domain is a nuclear export signal (NES). Here, we used microinjection-based assays to demonstrate that the activation domain of human immunodeficiency virus type 1 Rev imparts rapid nuclear export after its transfer to heterologous substrates. NES- mediated export is specific, as it is sensitive both to inactivation by missense mutation and to selective inhibition by an excess of the wild-type, but not mutant, activation domain peptide. Examination of the Rev trans activators of two nonprimate lentiviruses, visna virus and equine infectious anemia virus, revealed that their activation domains are also potent NESs. Taken together, these data demonstrate that nuclear export can be determined by positively acting peptide motifs, namely, NESs, and suggest that Rev proteins activate viral RNA transport by providing export ribonucleoproteins with specific information that targets them to the cytoplasm.
Asunto(s)
Productos del Gen rev/metabolismo , VIH-1/metabolismo , Virus de la Anemia Infecciosa Equina/metabolismo , Señales de Clasificación de Proteína/metabolismo , Virus Visna-Maedi/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico , Núcleo Celular/metabolismo , Núcleo Celular/virología , Productos del Gen rev/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Temperatura , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
The productive infection of many susceptible human cells, including lymphocytes and macrophages derived from peripheral blood, by the pathogenic lentivirus human immunodeficiency virus type 1 requires expression of the virally encoded vif (for virion infectivity factor) gene. Interestingly, this gene appears to have been conserved among all of the lentiviruses of primates and almost all of the lentiviruses of nonprimates. Using T cells constitutively expressing vif genes derived from diverse sources and virus replication assays, we show that the vif gene of a second primate lentivirus, simian immunodeficiency virus from macaques, complements vif-defective human immunodeficiency virus type 1 but that those of three distinct nonprimate lentiviruses do not. Although the molecular basis for Vif function has yet to be defined, the potential implications of this noted restriction of vif complementarity are discussed.
Asunto(s)
Virus Defectuosos/genética , Genes vif , VIH-1/genética , Virus de la Inmunodeficiencia de los Simios/genética , Animales , Línea Celular , Prueba de Complementación Genética , Humanos , Ratones , Ratones Endogámicos BALB C , Replicación ViralRESUMEN
The HIV-1 Rev protein is a nuclear trans-activator essential for the transport of unspliced viral transcripts to the cytoplasm. In this paper we demonstrate that Rev, rather than being confined to the nucleus, is constantly shuttling between the nucleus and the cytoplasm. We also show that inactivation of Rev's leucine-rich activation domain generates mutant proteins that not only fail to induce the nuclear export of viral transcripts but are also unable to enter the cytoplasm. On the basis of this correlation, we propose that Rev activates viral mRNA transport by directly binding to these RNAs and translocating, with them, to the cytoplasm. In addition, these results also identify, for the first time, a peptide sequence that is important for nuclear export.
Asunto(s)
Núcleo Celular/metabolismo , Productos del Gen rev/metabolismo , VIH-1/genética , VIH-1/metabolismo , Secuencia de Aminoácidos , Animales , Citoplasma/metabolismo , Vectores Genéticos , Células HeLa , Humanos , Intrones , Células L , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Transcripción Genética , Activación Transcripcional , Transfección , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.
Asunto(s)
Transformación Celular Neoplásica/genética , Genes src/genética , Proteína Oncogénica pp60(v-src)/genética , Actinas/metabolismo , Animales , Embrión de Pollo , Citoesqueleto/metabolismo , Análisis Mutacional de ADN , Fibroblastos , Morfogénesis , Mapeo Peptídico , Fosforilación , Proteínas Tirosina Quinasas , Relación Estructura-ActividadRESUMEN
PC12 cells treated with nerve growth factor (NGF) or infected with Rous sarcoma virus differentiate into sympathetic, neuronlike cells. To compare the differentiation programs induced by NGF and v-src, we have established a PC12 cell line expressing a temperature-sensitive v-src protein. The v-src-expressing PC12 cell line was shown to elaborate neuritic processes in a temperature-inducible manner, indicating that the differentiation process was dependent on the activity of the v-src protein. Further characterization of this cell line, in comparison with NGF-treated PC12 cells, indicated that the events associated with neurite outgrowth induced by these two agents shared features but could be distinguished by others. Both NGF- and v-src-induced neurite outgrowths were reversible. In addition, NGF and v-src could prime PC12 cells for NGF-induced neurite outgrowth, and representative early and late NGF-responsive genes were also induced by v-src. However, unlike NGF-induced neurite growth, v-src-induced neurite outgrowth was not blocked at high cell density. A comparison of phosphotyrosine containing-protein profiles showed that v-src and NGF each increase tyrosine phosphorylation of multiple cellular proteins. There was overlap in substrates; however, both NGF-specific and v-src-specific tyrosine phosphorylations were observed. One protein which was found to be phosphorylated in both the NGF- and v-src-induced PC12 cells was phospholipase C-gamma 1. Taken together, these results suggest that v-src's ability to function as an inducing agent may be a consequence of its ability to mimic critical aspects of the NGF differentiation program and raise the possibility that Src-like tyrosine kinases are involved in mediating some of the events triggered by NGF.
Asunto(s)
Genes src , Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Línea Celular , Regulación de la Expresión Génica , Cinética , Mutación , Neuronas/efectos de los fármacos , Fosforilación , Temperatura , Tirosina/metabolismoRESUMEN
Increasing attention in the literature has focused on the relationship between psychosocial factors and infertility. This study presents a psychosocial profile of infertile couples over a two-year period. During this period, psychosocial data including life change, social support, and personality traits were obtained prospectively from 134 individuals participating in the In Vitro Fertilization Program of the Jones Institute of Reproductive Medicine at the Eastern Virginia Medical School. Instruments utilized included the Life Experiences Survey, the Family APGAR, the Friends APGAR, the Multidimensional Health Locus of Control, and the Eysenck Personality Inventory. Despite the presumed adverse psychological influence of their infertility, Life Experience Survey scores for both men and women did not differ significantly from normative data. Family APGAR scores indicated a high degree of satisfaction with family function. Friends APGAR scores were somewhat lower than normative data. The Multidimensional Health Locus of Control, a measure of perception regarding factors influencing health outcomes, was unremarkable. The Eysenck Personality Inventory revealed a lower score on the neuroticism scale for the men. These psychosocial results were surprisingly normal.
Asunto(s)
Infertilidad/psicología , Adulto , Femenino , Fertilización In Vitro , Salud , Humanos , Control Interno-Externo , Acontecimientos que Cambian la Vida , Masculino , Inventario de Personalidad , Pruebas Psicológicas , Apoyo SocialRESUMEN
Depressed (N = 20) and nondepressed outpatients (N = 20) and normal controls (N = 20) rated the frequency with which they used various interpersonal behaviors in different social roles and emotional circumstances. The results indicated considerable situational specificity in subjects' reports of how they actually behave; the three subject groups displayed similar patterns. Most notable were findings that subjects report relatively frequent use of sadness displays when stressed by intimates, but not when similar stresses are imposed by strangers. In the latter case, social withdrawal is reported as a relatively frequent response. The results are discussed as contradicting an invariant, trait-like interpersonal style among depressed individuals.