RESUMEN
Blood sample transport via pneumatic tube systems (PTS) reduces the turnaround time of laboratories, but it might influence analysis results. Its effect on platelet concentrates (PCs) is not known. Platelet function was investigated after single and multiple PTS transport in comparison with storage and irradiation. Optical and impedance aggregation, CD-62, and microparticles changed as a result of storage, but not due to transport. Irradiation lowered platelet function independently. Multiple transport impaired thrombin receptor-activating peptide-induced aggregation. This investigation demonstrates the feasibility of PTS transport. As platelet function depends on storage, it may be more important to transfuse fresh PCs.
Asunto(s)
Plaquetas/química , Manejo de Especímenes/métodos , Conservación de la Sangre , Estudios de Factibilidad , Humanos , Activación Plaquetaria , Agregación Plaquetaria , Transfusión de Plaquetas , Factores de TiempoRESUMEN
BACKGROUND: Intranasal (IN) midazolam is a potential alternative to rectal diazepam for the acute treatment of epileptic seizures. OBJECTIVE: The purpose of this pilot study was to investigate the pharmacokinetics and tolerability of IN midazolam (50 mg/mL) compared with intravenous (IV) midazolam (2.5 mg) in healthy adult volunteers. METHODS: In this single-dose, randomized-sequence, open-label, 2-period crossover pilot study subjects were randomly assigned to receive IN or IV midazolam, with a washout period of at least 5 days between treatments. The 50-mg/mL IN midazolam formulation consisted of 5 mg midazolam base per 0.1 mL (1 spray) and was administered once in 1 nostril. The IV midazolam solution (2.5 mg) was infused over 10 seconds. Blood samples were taken before and at regular intervals up to 240 minutes after dosing. Pharmacokinetic data (ie, C(max), T(max), t(½), and AUC) were analyzed using a 2-compartment model. RESULTS: Of 9 volunteers screened and enrolled, 7 completed the study (mean age 34.1 [9.0] years; mean weight, 68.6 [10.4] kg, range 53-89 kg; 6 men, 3 women; all white). The mean C(max) of 78 (40) ng/mL was reached 44 minutes after IN administration, whereas the mean C(max) was 51 (5) ng/mL after IV administration. The mean estimated C(t=5 min) was 31.4 (28.1) ng/mL after IN administration. The elimination t(½) was 1.9 (0.41) hours for IN midazolam and 2.3 (0.19) hours for IV midazolam. The bioavailability of IN midazolam was 82%. There were few adverse events, with a local burning feeling in the nose being the most reported event (6 of 7 subjects). CONCLUSIONS: In this select group of healthy volunteers, concentrations of midazolam >30 ng/mL were reached within 5 minutes of IN administration at a dose of 5 mg/0.1 mL. A burning feeling in the nostril was the main adverse effect. Additional research is needed to evaluate the safety profile, convenience, satisfaction, and efficacy of nasal midazolam in the treatment of adults with seizures. This trial is registered at www.isrctn.org, No. ISRCTN79059168.
Asunto(s)
Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/farmacocinética , Midazolam/administración & dosificación , Midazolam/farmacocinética , Administración Intranasal , Adulto , Anticonvulsivantes/efectos adversos , Estudios Cruzados , Femenino , Humanos , Infusiones Intravenosas , Masculino , Midazolam/efectos adversos , Persona de Mediana Edad , Rociadores Nasales , Países Bajos , Proyectos PilotoRESUMEN
BACKGROUND: Although acetaminophen is used to reduce pain after breast reduction or augmentation surgery, pain during the removal of the surgical drains is typically not specifically treated. Intranasally administered fentanyl may be suitable for pain control during removal of drains. The reported therapeutic window of fentanyl is between 0.2 and 1.2 ng/mL. OBJECTIVE: The aim of this study was to evaluate the analgesic effect, tolerability, and pharmacokinetics of a single preprocedural dose of intranasal fentanyl administered before removal of surgical drains in patients who had undergone breast reduction or augmentation surgery. METHODS: This was a randomized, double-blind, prospective study in healthy women (American Society of Anesthesiologists physical status I or II) between the ages of 18 and 65 years who were scheduled to undergo removal of surgical drains 1 to 4 days after breast reduction or augmentation surgery. A single dose of fentanyl nasal spray 0.05 mg/0.1 mL or placebo (preserved normal saline) 0.1 mL was administered 10 minutes before removal of drains. Because drain removal is generally carried out without specific analgesia, no rescue medication was provided. Pain intensity was measured on a visual analog scale (VAS) from 0 = no pain at all to 100 = worst pain possible. Pain intensity was evaluated immediately before administration of study medication (t = 0), at the time of drain removal (t = 10), and at 15, 20, 25, 40, and 70 minutes after administration of study medication. Safety measures included oxygen saturation, respiratory rate, heart rate, and blood pressure. Local and systemic adverse events were elicited by direct questioning throughout the study. Blood samples for pharmacokinetic analysis were collected at baseline and at 5, 10, 15, 30, 60, and 120 minutes after administration of study medication. The population pharmacokinetic parameters of fentanyl were calculated according to a 1-compartment open model with an iterative 2-stage Bayesian fitting procedure. RESULTS: Thirty-six women were randomized to treatment, and 33 completed the study. Their mean (SD) age was 39.2 (13.0) years, and their mean weight was 68.9 (10.7) kg. Mean VAS scores at baseline were 14.8 (17.8) for the fentanyl group and 6.0 (9.7) for the placebo group (P = NS); at the time of drain removal, the corresponding VAS scores were 31.0 (20.6) and 33.8 (25.7) (P = NS). Analysis of a random-effects model with mean VAS scores as a function of time as the dependent variable indicated a significant difference in mean VAS scores between the fentanyl and placebo groups (P = 0.006). The overall incidence of adverse events was 39.4% (13/33). Among the 17 patients in the fentanyl group, 8 reported > or =1 adverse event; among the 16 patients in the placebo group, 9 reported > or =1 adverse event. A mean estimated C(max) of 0.184 (0.069) ng/mL was reached at 13.76 (3.56) minutes after administration of intranasal fentanyl. The mean measured C(max) was 0.22 (0.088) ng/mL. CONCLUSIONS: In these women who had undergone breast reduction or augmentation surgery, a single preprocedural dose of intranasal fentanyl was significantly more effective than placebo in reducing pain intensity over the hour after removal of surgical drains. However, there was no significant difference in pain intensity between fentanyl at the time of drain removal and placebo. Intranasal fentanyl was generally well tolerated. At the dose used (0.05 mg), plasma fentanyl concentrations were below the reported therapeutic window.
Asunto(s)
Analgésicos Opioides/uso terapéutico , Fentanilo/uso terapéutico , Mamoplastia/métodos , Dolor/tratamiento farmacológico , Administración Intranasal , Adolescente , Adulto , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/efectos adversos , Remoción de Dispositivos/métodos , Método Doble Ciego , Drenaje , Femenino , Fentanilo/administración & dosificación , Fentanilo/efectos adversos , Humanos , Persona de Mediana Edad , Dolor/etiología , Dimensión del Dolor , Estudios Prospectivos , Factores de Tiempo , Adulto JovenAsunto(s)
Compensación de Dosificación (Genética) , Proteínas de Drosophila/genética , Silenciador del Gen , Células Madre/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Epigénesis Genética , Femenino , N-Metiltransferasa de Histona-Lisina , Histonas/química , Histonas/metabolismo , Humanos , Masculino , Modelos Genéticos , Proteína de la Leucemia Mieloide-Linfoide , Neoplasias/etiología , Nucleosomas/metabolismo , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Proto-Oncogenes/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina/metabolismoRESUMEN
Aberrant expression of platelet-derived growth factor and its receptor (PDGFR) has been implicated in various human disorders, including cardiovascular disease and certain types of cancer. Inhibitors of the tyrosine kinase activity of PDGFR are leads in the development of novel agents to combat these diseases. We describe here a novel, potent inhibitor of PDGFR tyrosine kinase, 3-(4-dimethylamino-benzylidenyl)-2-indolinone (DMBI). The compound also inhibits signal transduction through fibroblast growth factor receptor 1 (FGFR1), but is not active towards epidermal growth factor receptor (EGFR) or c-Src tyrosine kinase. The activity of DMBI and other tyrosine kinase inhibitors was compared in a cell-based assay as well as in an assay based on purified recombinant platelet-derived growth factor beta-receptor (beta-PDGFR) lacking the transmembrane and ligand-binding domain. We showed that this truncated beta-PDGFR could dimerize, and that dimerization was required for tyrosine kinase activity. Tyrosine kinase activity was modulated by inhibitors of beta-PDGFR autophosphorylation in cells, but not by specific inhibitors of EGFR or c-Src tyrosine kinase. We conclude that beta-PDGFR lacking the transmembrane and ligand-binding domain retains the essential properties of the full-length receptor tyrosine kinase.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Sustancias de Crecimiento/farmacología , Indoles/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células 3T3 , Animales , Becaplermina , Carcinoma de Células Escamosas , División Celular/efectos de los fármacos , Línea Celular , Citoplasma/enzimología , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Cinética , Ratones , Músculo Liso Vascular , Fosforilación , Fosfotirosina/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Arteria Pulmonar , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/aislamiento & purificación , Receptor beta de Factor de Crecimiento Derivado de Plaquetas , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The three-dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 A resolution by X-ray crystallography. The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55-residue protease inhibitor. However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two beta-strands, and does not inhibit factor Xa. The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations. Consequently, the domain shapes are different, the N-terminal domain being wedge-shaped and the C-terminal domain flat. Docking studies suggest that differences in domain shape enable the N-terminal, but not C-terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site. Furthermore, a putative exosite binding region could be defined in the N-terminal domain of antistasin, comprising residues 15-17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa. In the C-terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain.
Asunto(s)
Anticoagulantes/química , Factor Xa/química , Hormonas de Invertebrados/química , Inhibidores de Proteasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Humanos , Sanguijuelas , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
Tissue Factor Pathway Inhibitor (TFPI) is a 36 kDa glycoprotein that helps maintain haemostasis by inhibiting Factor Xa and the Factor VIIa/Tissue Factor (TF) complex. TFPI contains three tandemly linked Kunitz inhibitor domains, of which the second inhibits factor Xa. We have undertaken a multidisciplinary approach to study the structure and function of the second Kunitz domain of TFPI, with a view towards the rational design of factor Xa inhibitors. Amino acid residues 93 to 154 of the mature TFPI protein, corresponding to the second Kunitz domain (TFPI-kII), were expressed in Escherichia coli. The protein was purified to near homogeneity by ion exchange, hydrophobic interaction, and size exclusion chromatography, respectively. TFPI-kII is a potent factor Xa inhibitor with a Ki of 1.5 x 10(-10) M, a value that does not differ significantly from that of intact TFPI. The three-dimensional structure of TFPI-kII in aqueous solution was determined by 1H nuclear magnetic resonance spectroscopy (NMR). A set of 30 conformers was calculated with the program DIANA using 906 distance constraints derived from nuclear Overhauser effects and 23 dihedral angle constraints. This set, representing the solution structure of TFPI-kII, has an average root-mean-square deviation of 0.78 A for the backbone atoms and 1.38 A for all heavy atoms of residues 1 to 58. The structure of TFPI-kII has also been determined in complex with porcine trypsin using X-ray crystallographic techniques. The complex has been solved to a resolution of 2.6 A, with a final R-factor of 16.2%. Comparison of the NMR derived structure with that of TFPI-kII in complex with trypsin reveals little divergence of the two structures, with the exception of residue Tyr17. Superposition of the trypsin:TFPI-kII complex on factor Xa provides insights into macromolecular determinants for the inhibition of factor Xa. Complexation would require a degree of reorganisation of factor Xa residues, in particular of TyrF99, but also perhaps of the F148-loop. The interaction was further investigated using restrained molecular dynamics. Electrostatic interactions would appear to play a major role. The reorganisation of factor Xa is in contrast to the proposed factor Xa:TAP interaction, where TAP would bind to the "ground state" structure of factor Xa.
Asunto(s)
Factor Xa/metabolismo , Lipoproteínas/química , Lipoproteínas/genética , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Factor Xa/química , Humanos , Lipoproteínas/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia , Soluciones , Tripsina/química , Tripsina/metabolismoRESUMEN
Antistasin is a Factor Xa inhibitor that is present in the salivary glands of the Mexican leech Haementeria officinalis. The antistasin protein consists of 119 amino acids, of which residues 1-55 (domain I) are 56% similar to residues 56-110 (domain II). Of the nine C-terminal amino acids (residues 111-119; domain III), four are positively charged. The reactive site for Factor Xa is located in domain I. In this study we assessed the role of separate domains and of individual amino acids in the reactive site for the inhibition of Factor Xa. A series of mutants was constructed and expressed in Chinese hamster ovary (CHO) cells. In vitro chromogenic assays for Factor Xa show that domain I is sufficient for inhibition of Factor Xa. Domains II and III neither contain any intrinsic Factor Xa inhibitory activity, nor contribute to the activity of domain I. Furthermore, domain II does not become a Factor Xa inhibitor by partially adaptating its sequence towards that of the reactive site in domain I. Mutation of the cysteine at position 33 is not crucial for Factor Xa inhibition, suggesting a relatively rigid reactive site loop structure.
Asunto(s)
Anticoagulantes/aislamiento & purificación , Inhibidores del Factor Xa , Hormonas de Invertebrados/genética , Hormonas de Invertebrados/aislamiento & purificación , Sanguijuelas/química , Secuencia de Aminoácidos , Animales , Células CHO/metabolismo , Cricetinae , Análisis Mutacional de ADN , Sondas de ADN , Sanguijuelas/genética , Datos de Secuencia MolecularRESUMEN
Antithrombin is a member of the serine proteinase inhibitor (serpin) family which contain a flexible reactive site loop that interacts with, and is cleaved by the target proteinase. In cleaved and latent serpins, the reactive site loop is inserted into a large central beta-sheet in the same molecule, whereas in ovalbumin, a nonfunctional serpin, the reactive site loop is completely exposed and in an alpha-helical conformation. However, in neither conformation can the reactive site loop bind to target proteinases. Here we report the structure of an intact and cleaved human antithrombin complex. The intact reactive site loop is in a novel conformation that seems well suited for interaction with proteinases such as thrombin and blood coagulation factor Xa.
Asunto(s)
Antitrombina III/química , Endopeptidasas/química , Serpinas/química , Clorometilcetonas de Aminoácidos/química , Clorometilcetonas de Aminoácidos/metabolismo , Secuencia de Aminoácidos , Antitrombina III/genética , Antitrombina III/metabolismo , Sitios de Unión , Electroquímica , Endopeptidasas/metabolismo , Humanos , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Ovalbúmina/química , Ovalbúmina/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Serpinas/metabolismo , Trombina/química , Trombina/metabolismoRESUMEN
The survival of Chlamydia pneumoniae in aerosols was investigated by using a chamber with a capacity of 114.5 liters. We injected 5 x 10(7) inclusion-forming units (IFU) of C. pneumoniae in aerosols with a droplet size of 3 to 5 microns. Samples were taken after 30 s and every 1 min thereafter. The survival of C. pneumoniae was measured at four temperatures (8.5, 15, 25, and 35 degrees C) and at three different relative humidities (RH) of 5, 50, and 95% for each temperature. The survival rates of Streptococcus pneumoniae, Streptococcus faecalis, Klebsiella pneumoniae, Chlamydia trachomatis LGV2, and cytomegalovirus were also determined at 25 degrees C and 95% RH and compared with that of C. pneumoniae. At the mentioned temperatures and RH, a rapid decrease of C. pneumoniae IFU was observed in the first 30 s. After this the decrease in the number of IFU was more gradual. The survival of C. pneumoniae in aerosols were optimal at 15 to 25 degrees C and 95% RH; it was good compared with those of other microorganisms. A lower death rate was observed only in S. faecalis. In C. trachomatis, the death rate during the first 30 s was higher than that in C. pneumoniae (85 and 53.3%, respectively). After the first 30 s, the death rates in the two organisms were identical. It was concluded that transmission of C. pneumoniae via aerosols was possible. There is probably a direct transmission from person to person, taking into account the relatively short survival period of C. pneumoniae in aerosols.
Asunto(s)
Microbiología del Aire , Infecciones por Chlamydia/transmisión , Chlamydophila pneumoniae/patogenicidad , Aerosoles , Chlamydophila pneumoniae/aislamiento & purificación , Humanos , Humedad , TemperaturaRESUMEN
The salivary gland of the Mexican leech Haementeria officinalis contains a 15 kDa protein which is a potent and selective inhibitor of factor Xa. It inhibits not only blood coagulation, but also metastasis. A gene, coding for a sequence similar to published antistasin sequences, has been synthesized and expressed in Chinese hamster ovary (CHO) cells. The recombinant protein was purified and crystallized at pH 6.0, using 31% ammonium sulfate as a precipitant. The crystals diffract at least to 2.8 A. The spacegroup is I422 with a = b = 77.7 A and c = 88.4 A. The crystals contain 42% solvent and one protein molecule in the asymmetric unit. A search for heavy atom derivatives is in progress.
Asunto(s)
Inhibidores del Factor Xa , Hormonas de Invertebrados/química , Animales , Células CHO , Cricetinae , Cristalización , Sanguijuelas , Proteínas Recombinantes/química , Difracción de Rayos XRESUMEN
Antithrombin-III (AT-III) is a heparin-dependent inhibitor of thrombin and Factor Xa, two serine proteases that are crucial for blood coagulation. In order to assess whether it would be possible to target AT-III only towards Factor Xa, we replaced parts of the reactive site, or P region, of AT-III by sequences present in prothrombin, a substrate of Factor Xa in the coagulation cascade. We show that replacement of the P3 to P3' region generates the hypothesized phenotype. In fact, point mutation of the P1' site from Ser (present in AT-III) to Ile (present in prothrombin) is sufficient to dissociate heparin-dependent thrombin and Factor Xa inhibitory activities. Interestingly, a combined mutation at P3 and P3' brings about the same dissociation. We show that besides Ile, other amino acids at P1' can lead to the dissociation in inhibitory activity. Amino acids with small side chains (Gly, Ser, Ala, and Thr) have only a marginal effect on the inhibitory activity against either protease. However, larger residues at the P1' position abolish the heparin-dependent anti-thrombin activity, whereas the heparin-dependent anti-Factor Xa activity is not at all or only moderately affected. These results can be rationalized by a comparison of the x-ray structure and a three-dimensional model of the S1' binding pockets of thrombin and Factor Xa, respectively. It appears that the S1' pocket of Factor Xa leaves much more space for the P1' residue of AT-III than the S1' pocket of thrombin.
Asunto(s)
Antitrombina III/farmacología , Inhibidores del Factor Xa , Heparina/metabolismo , Trombina/metabolismo , Secuencia de Aminoácidos , Animales , Antitrombina III/química , Antitrombina III/genética , Secuencia de Bases , Sitios de Unión , Células CHO , Clonación Molecular , Simulación por Computador , Cricetinae , Factor Xa/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , OligodesoxirribonucleótidosRESUMEN
Human antithrombin III has been crystallized from 18 to 21% (w/v) polyethylene glycol 4000 at pH 7.15. The spacegroup is P2(1) with cell parameters a = 89.8 A, b = 100.8 A, c = 70.0 A and beta = 106 degrees. The diffraction limit is 3.2 A. The asymmetric unit contains two protein molecules. Analysis of dissolved crystals for biological activity and by gel electrophoresis suggests that one protein molecule in the asymmetric unit is intact, while the other is cleaved.
Asunto(s)
Antitrombina III/química , Antitrombina III/metabolismo , Cristalización , Humanos , Conformación Proteica , Difracción de Rayos XRESUMEN
Current anti-HIV-1 screening tests combine an excellent anti-HIV-1 sensitivity with a sensitivity of only 28-93% for anti-HIV-2 positive plasma or serum samples. The reactivity of anti-HIV-2 sera in anti-HIV-1 screening tests is based mainly on the immunological cross-reactivity of the GAG and POL proteins of HIV-1 and HIV-2. We describe here a sandwich immunoassay, in which HIV-1 viral lysate is combined with an HIV-2 ENV synthetic peptide, corresponding to the immunodominant envelope epitope, as the coating antigens on microELISA plates. This immunoassay has a sensitivity of 100% for anti-HIV-1 (128 sera tested) and 100% for anti-HIV-2 (109 sera tested). Assay specificity with fresh human donor sera was 99.9% (2256 sera tested).
Asunto(s)
Anticuerpos Anti-VIH/análisis , VIH-1/inmunología , VIH-2/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Sensibilidad y EspecificidadRESUMEN
Wnt-1 (int-1) is a cellular oncogene often activated by insertion of proviral DNA of the mouse mammary tumor virus. We have mapped the 5' end and the promoter area of the Wnt-1 gene by nuclease protection and primer extension assays. In differentiating P19 embryonal carcinoma cells, in which Wnt-1 is naturally expressed, two start sites of transcription were found, one preceded by two TATA boxes and one preceded by several GC boxes. In P19 cells, a 1-kilobase upstream sequence of Wnt-1 was able to confer differentiation-specific expression on a heterologous gene. We have investigated how Wnt-1 transcription was affected by mouse mammary tumor virus proviral integrations in various configurations near the promoters of the gene. One provirus has been inserted in the 5' nontranslated part of Wnt-1, in the same transcriptional orientation, and has functionally replaced the Wnt-1 promoters. Wnt-1 transcription in this tumor starts in the right long terminal repeat of the provirus, with considerable readthrough transcription from the left long terminal repeat. Another provirus has been inserted in the orientation opposite that of Wnt-1 into a GC box, disrupting the first Wnt-1 transcription start site but not the downstream start site. Most insertions have not structurally altered the Wnt-1 transcripts and have enhanced the activity of the normal two promoters.
Asunto(s)
ADN Viral/genética , Regulación de la Expresión Génica , Virus del Tumor Mamario del Ratón/genética , Oncogenes , Regiones Promotoras Genéticas , Provirus/genética , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Exones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN/genética , Mapeo Restrictivo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , TeratomaRESUMEN
Acquired proviruses of mouse mammary tumor virus (MMTV) in T-cell leukemias of male GR mice have rearrangements in the U3 region of their long terminal repeats (LTR). In contrast to the endogenous nonrearranged MMTV proviruses, these mutated copies are highly expressed in leukemic T cells. To investigate whether the sequence alterations in the LTR are responsible for the high expression of rearranged MMTV proviruses, we made constructs in which normal and variant LTRs drive the bacterial reporter gene chloramphenicol acetyltransferase (CAT). Two different rearranged LTRs were used, one containing a 420-base-pair (bp) deletion (L13) and another carrying a 456-bp deletion plus an 82-bp insertion (L42). These constructs were transfected into murine (GRSL) and human (MOLT-4) T-cell lines that either had or had not been treated with phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]). In GRSL cells, the L13-LTR-CAT construct showed transcriptional activity that was further enhanced by TPA. In MOLT-4 cells, both variant LTRs were active, but only after stimulation with TPA. In contrast, normal(N)-LTR-CAT constructs were not expressed, irrespective of TPA addition. In XC rat fibrosarcoma cells, neither normal nor variant LTRs gave rise to detectable CAT activity, either in the presence or in the absence of TPA, but dexamethasone strongly stimulated CAT activity driven by N and L42 LTRs. The L13 LTR was considerably less active, probably caused by the deletion of the distal part of the glucocorticoid responsive element. We conclude that the LTR rearrangements generate TPA responsiveness and contribute to T-cell-specific expression of MMTV variants.
Asunto(s)
Regulación de la Expresión Génica , Leucemia de Células T/microbiología , Virus del Tumor Mamario del Ratón/genética , Provirus/genética , Acetato de Tetradecanoilforbol/farmacología , Animales , Línea Celular , ADN Viral/genética , Reordenamiento Génico , Humanos , Ratones , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
First generation ELISA screening assays for antibodies to HTLV-III (HIV) generated between 0.1 and 1.0% false positive results. Western blot analysis in specialized reference centers is almost uniformly used as a method to confirm the specificity of the ELISA results. Yet, the high cost, time delay and lack of standardization in these systems cause a growing demand for tests that can be performed on site and that can at least reduce the number of sera that have to be sent to reference centers. Such tests thus should primarily be aimed at the detection of false positive results. Ancillary to the Vironostika anti-HTLV-III screening test, we developed a set of reagents (VERIFY) which can be used for the verification of initially or repeatedly positive screening results. The test employs a reagent specifically blocking true HTLV-III-anti HTLV-III reactions, a reagent blocking HLA-anti HLA reactions and a control reagent. Use of this test may reduce the number of sera found false positive by reference methods by more than 90%. The introduction of improved versions and second generation screening assays obviously will reduce the number of false positive results. Yet the significant results of this verification assay and the ease with which it can be integrated in the screening procedures will make it a valuable tool in the blood bank screening program.
Asunto(s)
Anticuerpos Antivirales/análisis , Donantes de Sangre , Ensayo de Inmunoadsorción Enzimática , Seropositividad para VIH , VIH/inmunología , Reacciones Falso Positivas , Anticuerpos Anti-VIH , Humanos , Inmunoensayo , Valor Predictivo de las PruebasRESUMEN
This report concerns ontogenetic aspects of the production and in vitro release of NH2-terminally acetylated forms of melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin by the pars intermedia of the pituitary gland of the mouse. In vitro biosynthetic analysis and radioimmunoassay revealed that approximately 12 h before birth most of the MSH in the fetal pars intermedia is present as des-N alpha-acetyl alpha-MSH. The same non-acetylated peptide is at this stage also the major release form of melanotropin. In 1-day-old mice the level of alpha-MSH and diacetylated alpha-MSH had increased considerably, although des-N alpha-acetyl alpha-MSH remained the major form. Five days after birth alpha-MSH and its diacetylated form constitute the major tissue and release form of the peptide, a situation very similar to that in adult mice. Acetylation of beta-endorphin appeared to occur earlier in development, N alpha-acetyl beta-endorphin (1-31) being the major form of endorphin already in the fetal pars intermedia. It is concluded that in the mouse acetylation of melanotropin and acetylation of beta-endorphin are not necessarily concomitant events. It could be established that the ability of the pars intermedia cells for cleaving N alpha-acetyl beta-endorphin (1-31) to yield C-terminally shortened forms of beta-endorphin develops after birth.