RESUMEN
One hope to maintain the benefits of antiviral therapy against the human immunodeficiency virus type 1 (HIV-1), despite the development of resistance, is the possibility that resistant variants will show decreased viral fitness. To study this possibility, HIV-1 variants showing high-level resistance (up to 1,500-fold) to the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS have been characterized. Active-site mutations V32I and I84V/A were consistently observed in the protease of highly resistant viruses, along with up to six other mutations. In vitro studies with recombinant mutant proteases demonstrated that these mutations resulted in up to 10(4)-fold increases in the Ki values toward BILA 1906 BS and BILA 2185 BS and a concomitant 2,200-fold decrease in catalytic efficiency of the enzymes toward a synthetic substrate. When introduced into viral molecular clones, the protease mutations impaired polyprotein processing, consistent with a decrease in enzyme activity in virions. Despite these observations, however, most mutations had little effect on viral replication except when the active-site mutations V32I and I84V/A were coexpressed in the protease. The latter combinations not only conferred a significant growth reduction of viral clones on peripheral blood mononuclear cells but also caused the complete disappearance of mutated clones when cocultured with wild-type virus on T-cell lines. Furthermore, the double nucleotide mutation I84A rapidly reverted to I84V upon drug removal, confirming its impact on viral fitness. Therefore, high-level resistance to protease inhibitors can be associated with impaired viral fitness, suggesting that antiviral therapies with such inhibitors may maintain some clinical benefits.
Asunto(s)
Resistencia a Medicamentos , VIH-1/genética , Inhibidores de Proteasas/farmacología , ADN Recombinante , ADN Viral/genética , VIH-1/efectos de los fármacos , Humanos , MutaciónRESUMEN
The catalytic domain of herpes simplex virus protease was expressed in baculovirus-infected cells and purified in milligram quantities by ion-exchange and size-exclusion chromatography. The usefulness of this material was limited by the presence of a contaminating proteolytic activity, which caused time-dependent degradation of the protease. As a result we decided to explore an alternative approach to purification. Specific monoclonal antibodies were produced and evaluated by surface plasmon resonance as ligands for immunoaffinity chromatography. One monoclonal antibody, 6H4, was chosen for coupling to an affinity support, and the resulting column allowed us to obtain a pure and stable enzyme. Immunoaffinity chromatography of herpes simplex virus type 1 protease resulted in successful elimination of the contaminating protease activity. Moreover the immunoaffinity column permitted the isolation of stable and pure enzyme in a one-column procedure.
Asunto(s)
Cápside/inmunología , Cápside/aislamiento & purificación , Herpesvirus Humano 1/enzimología , Estructura Terciaria de Proteína , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/aislamiento & purificación , Proteínas Virales , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Sitios de Unión , Sitios de Unión de Anticuerpos , Técnicas Biosensibles , Cápside/metabolismo , Catálisis , Cromatografía de Afinidad , Humanos , Técnicas de Inmunoadsorción , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Serina Endopeptidasas/metabolismoRESUMEN
Renin is a highly specific aspartyl protease of the renin-angiotensin system initially synthesized as preprorenin. Recombinant human prorenin was produced in cell factories from stably transfected DAMP cells, a dog epithelial cell line. The equivalent of 10-15 mg of recombinant human renin was secreted in the supernatant from each cell factory. Following a single affinity chromatography step using a renin inhibitor as the ligand, a 181-fold purification was achieved with 81% recovery of the renin activity. This highly pure recombinant enzyme having a specific activity of 3.44 mg angiotensin I.mg protein-1.h-1 was used for kinetic analysis. The kinetic parameters were determined with the natural substrate angiotensinogen and a tetradecapeptide substrate corresponding to the amino terminus of angiotensinogen, Asp1-Asn14, at their respective optimum pH of 5.5 and 6.8. Although there was a six-fold increase in both Km and kcat values for the peptidic substrate (13.3 microM and 8.1 s-1, respectively), when compared with values for the natural substrate (2.04 microM and 1.41 s-1), the catalytic efficiency (0.69 microM-1.s-1) of the enzyme for both substrates was the same. However, the kcat/Km value with angiotensinogen at the physiological pH 7.4 was 30% lower than that observed at the optimum pH 5.5. The recombinant human renin displayed similar optimum pH and kinetic parameters with angiotensinogen and the tetradecapeptide substrate when compared with human kidney renin.
Asunto(s)
Renina/aislamiento & purificación , Animales , Secuencia de Bases , Perros , Activación Enzimática , Escherichia coli/genética , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Renina/genética , Renina/metabolismo , TransfecciónRESUMEN
Fractions obtained by gel filtration or ultrafiltration of dog serum were tested for their mitogenic activity on canine prostatic epithelial cells: two prostatic growth factor (PGF) entities were found, a major one of 150 kDa (PGF-I) and a minor one of 1.5-2.0 kDa (PGF-II). Treatment and/or extraction with acetic acid, hydrochloric acid, or acidified-ethanol or preparations enriched in PGF-I obtained either by ion-exchange chromatography, acetone precipitation, or retention by ultrafiltration membrane (cut-off 30 kDa) resulted, upon gel filtration, in the detection of a mitogenic activity eluting mainly at the position of PGF-II. Acid hydrolysis and proteolysis of PGF-II led to a loss of activity. It is proposed that, in dog serum, mitogenic peptides for prostatic epithelial cells of 1.5 kDa (PGF-II) are found in their free form and/or in association with proteins of 150 kDa (PGF-I).
Asunto(s)
Proteínas Sanguíneas/fisiología , Sustancias de Crecimiento/sangre , Próstata/citología , Animales , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , División Celular , Perros , Células Epiteliales , Sustancias de Crecimiento/química , Sustancias de Crecimiento/fisiología , Hidrólisis , MasculinoRESUMEN
A growth factor-like effect has been observed on canine prostatic epithelial cells when cultured in the presence of their homologous serum and prostatic extracts; the mitogenic activities of both preparations were dose-dependent and not altered by charcoal treatment. The effect of dog serum decreased when the density of the epithelial cell cultures increased and was minimal on canine prostatic fibroblasts. Trace amounts of intracellular sex steroids did not contribute to epithelial cell proliferation since the presence of sex steroid action inhibitors did not alter growth rate; in those conditions, cycloheximide completely prevented cell division. When various hormones and known mitogenic agents were tested alone or in combination with steroids, none elicited an increase in the number of epithelial cells cultured in serum-free medium or altered the proliferative effect of dog serum observed in parallel cultures. On gel filtration, dog serum or tissue cytosol showed a major mitogenic activity at an apparent molecular mass of 150 kDa and a minor one of 1.5 kDa as evaluated by gel filtration of dog serum ultrafiltrate. Acidic extraction of prostatic tissue followed by chromatography on a hydrophobic C-18 column and subsequent gel filtration also led to the detection of the low Mr component. Thus, humoral and/or tissular factors present in vivo and different from known mitogens may be of importance as direct modulators of the basal epithelial cell growth in the adult canine prostate.
Asunto(s)
Sustancias de Crecimiento/farmacología , Próstata/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Perros , Células Epiteliales , Epitelio/efectos de los fármacos , Hormonas Esteroides Gonadales/antagonistas & inhibidores , Sustancias de Crecimiento/sangre , Hormonas/farmacología , Masculino , Mitógenos/farmacología , Próstata/citología , Esteroides/farmacología , Extractos de Tejidos/farmacologíaAsunto(s)
Próstata/citología , Esteroides/metabolismo , Andrógenos/metabolismo , Animales , Diferenciación Celular , División Celular , Células Cultivadas , Perros , Sustancias de Crecimiento/fisiología , Humanos , Técnicas In Vitro , Masculino , Metribolona/metabolismo , Orquiectomía , Receptores Androgénicos/fisiologíaRESUMEN
A mailed questionnaire was used to assess the time demands and relative stress burden of tasks performed by caregivers to patients attending a geriatric day hospital. Eighty per cent (43 of 54) of the questionnaires were completed. Most (84%) of caregivers were women, and 38% were older than 65 years of age. Fifty-three per cent of care receivers were women; 54% were 80 years of age or older. Most care receivers were related to the caregiver, and 84% lived with them. Activities causing the most stress to caregivers were bathing, bladder control, walking about the home, and travelling outside the home. A significant number of caregivers did not have access to alternate caregivers for relief during the day (18%) or night (26%), or for a weekend (28%) or a week (40%). Sixty-eight per cent of caregivers felt some mental strain as a result of their caregiver role.
RESUMEN
Perfusion of canine prostatic tissue with [1, 2-3H] 5 alpha-androstane-3 alpha, 17 beta-diol and [4-14C] dihydrotestosterone and the measurement of the isotopic concentrations at the steady state were used to calculate the metabolic dynamics of these steroids by prostatic tissue obtained from normal, castrated or androgen-treated dogs. From the results it was concluded that: Entry of both steroids into the tissue was similar, and no saturation was observed with increasing concentrations of these androgens in the perfusion medium. In contrast, the entry of both steroids was reduced when the perfusion buffer was replaced with diluted plasma. Dihydrotestosterone in prostatic tissue was present in a diffusible form, whereas 3 alpha,17 beta-androstanediol was not. The conversion of 3 alpha, 17 beta-androstanediol to dihydrotestosterone was always higher than the conversion of dihydrotestosterone to 3 alpha, 17 beta-androstanediol; thus, the oxidative pathway was favored. The entry and uptake of both androgens was greatly reduced in those prostates excised from castrated dogs. The uptake of dihydrotestosterone by normal prostatic tissue was 3- to 6-fold higher than the uptake of 3 alpha, 17 beta-androstanediol. However, higher intratissular concentrations of dihydrotestosterone were obtained by increasing the concentration of 3 alpha, 17 beta-androstanediol perfused over that observed by increasing the concentration of dihydrotestosterone perfused. In the hyperplastic tissue, the entry, uptake and metabolism of the two androgens were similar to those observed in the normal gland, but their intratissular concentrations were found to be higher.
Asunto(s)
Androstano-3,17-diol/metabolismo , Androstanoles/metabolismo , Dihidrotestosterona/metabolismo , Próstata/metabolismo , Animales , Transporte Biológico Activo , Proteínas Sanguíneas/metabolismo , Castración , Perros , Técnicas In Vitro , Cinética , Masculino , Perfusión , Hiperplasia Prostática/metabolismoRESUMEN
Radiolabelled methyltrienolone, dihydrotestosterone and estradiol were used as ligands to identify and quantify androgen and estrogen receptors in freshly dispersed cells from the canine prostate. Soluble extracts (cytosols) were obtained from secretory and non-secretory epithelial cells separated on the basis of their density in Percoll gradients. For both cell types, as well as for the whole prostate, Scatchard plot analyses were linear and showed a single class of high affinity binding sites: Kd values of 3.6 +/- 2.2 X 10(-9) M and 3.0 +/- 1.2 X 10(-10) M were measured for the androgen and estrogen receptors, respectively. The number of binding sites for the cytosolic androgen receptor, expressed per mg of protein or per mg of DNA, was 2.4- to 6.7-fold higher in the non-secretory cells compared to the secretory cells. However, these two cell types contained a similar number of specific sites for the estrogens. The specificities of the androgen and estrogen receptors were shown to be identical for the two cell types: the binding of [3H]R1881 was strongly inhibited by unlabelled R1881, 5 alpha-androstane-3 alpha, 17 beta-diol and dihydrotestosterone, while 5 alpha-androstane-3 beta, 17 beta-diol, estradiol and estrone did not displace bound R1881. The addition of triamcinolone acetonide did not alter the binding of R1881 in extracts of either cell type or in the whole prostate. The binding of [3H]estradiol to the estrogen receptor was highly specific since a strong displacement was only observed with estradiol (83%).
Asunto(s)
Próstata/análisis , Receptores Androgénicos/análisis , Receptores de Estrógenos/análisis , Receptores de Esteroides/análisis , Animales , Unión Competitiva , Separación Celular , Citosol/análisis , Dihidrotestosterona/metabolismo , Perros , Epitelio/análisis , Epitelio/metabolismo , Estradiol/metabolismo , Estrenos/metabolismo , Masculino , Metribolona , Receptores Androgénicos/metabolismo , Receptores de Estrógenos/metabolismoRESUMEN
Kinetic parameters of two enzymes, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, have been calculated for freshly isolated canine prostatic epithelial cells separated into secretory and non-secretory cells on the basis of their density in Percoll gradients. For 5 alpha-reductase, a Km value of 3 X 10(-6) M was obtained in both epithelial cell types, and similar Vmax values of 1.7 X 10(-12) and 1.9 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P greater than 0.50) were calculated in secretory and non-secretory cells, respectively. The Km of 3 alpha-hydroxysteroid dehydrogenase (dihydrotestosterone----3 alpha-androstanediol) varied between 2.2 and 2.8 X 10(-6) M (P greater than 0.50) with respective Vmax's of 9 and 24 X 10(-12) moles of 3 alpha-androstanediol formed/min/10(6) cells (P less than 0.005) in secretory and non-secretory cells. For the reverse reaction, that is the transformation of 3 alpha-androstanediol to dihydrotestosterone, the Km's obtained were 0.4 and 0.5 X 10(-6) M (P greater than 0.50) with Vmax's of 14 and 19 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P less than 0.50) in secretory and non-secretory cells, respectively. Vmax values for 3 alpha-hydroxysteroid dehydrogenase are 10-fold higher than the ones for 5 alpha-reductase. Moreover, Km values for the reaction 3 alpha-androstanediol----DHT are 5-fold lower than those calculated for the reverse reaction and for 5 alpha-reductase. This could explain why the major metabolic pathway in the canine prostate gland is the conversion of 3 alpha-androstanediol to dihydrotestosterone.