RESUMEN
The amine-donor substrate specificity of tissue-type transglutaminase has been studied in a series of recombinant alpha A-crystallin mutants. These mutant proteins have been provided with a potential substrate lysine residue, flanked by different amino acid residues, in the C-terminal extended arm of alpha A-crystallin. A biotinylated amine-acceptor hexapeptide was used as a probe for labelling the amine-donor sites. Wild-type bovine alpha A-crystallin does not function as an amine-donor substrate for tissue-type transglutaminase. Yet, upon introduction of a lysine residue at the C-terminal or penultimate position, all mutant alpha A-crystallins act as amine-donor substrates, although to different extents. This shows that accessibility is the primary requirement for a lysine residue to function as an amine-donor substrate for transglutaminase and that the enzyme has a broad tolerance towards the neighbouring residues. However, the nature of the flanking amino acid residues does clearly affect the reactivity of the substrate lysine residue. Notably, we found that a proline or glycine residue in front of the substrate lysine has a strong adverse effect on the substrate reactivity as compared to a preceding leucine, serine, alanine or arginine residue.
Asunto(s)
Cristalinas/metabolismo , Transglutaminasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Cristalinas/química , Lisina/química , Datos de Secuencia Molecular , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
An existing arterio-venous shunt thrombosis model in the rat has been modified to increase its usefulness for the testing of anti-thrombotic agents and characterised using morphological and radiometric techniques. The thrombus formed on the cotton thread held in the shunt was found to be composed of platelet aggregates surrounded by red thrombus. After 30 min of blood flow there was a 15-fold increase in the platelet content of the thrombus and a 4-fold increase in the fibrin(ogen) content compared with an equivalent weight of whole blood. Use of the anticoagulants recombinant desulphatohirudin (CGP 39393, 4 mg/kg, s.c.) and unfractionated heparin (800 IU/kg, s.c.) showed that approx. 90% inhibition of thrombus weight and approx. 80% inhibition of fibrin(ogen) content could be achieved without significant effect on the platelet content. Conversely, using the platelet inhibitor Iloprost (1 microgram kg-1 min-1), a reduction in thrombus weight of 50% was associated with 75% inhibition of platelet content and only 20% inhibition of fibrin(ogen). These observations suggest that the growth of this type of thrombus is largely the result of continued fibrin formation rather than continued platelet recruitment and activation.
Asunto(s)
Derivación Arteriovenosa Quirúrgica/efectos adversos , Fibrinolíticos/uso terapéutico , Heparina/uso terapéutico , Hirudinas/análogos & derivados , Iloprost/uso terapéutico , Trombosis/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Pruebas Hematológicas , Terapia con Hirudina , Masculino , Radiometría , Ratas , Ratas Endogámicas , Proteínas Recombinantes/uso terapéutico , Trombosis/etiologíaRESUMEN
The role of thrombin in the formation of arterial thrombi is poorly understood. With the new availability of the specific thrombin inhibitor, recombinant desulphatohirudin, in large quantities this is now under investigation. A new model of arterial thrombosis in rats is described where a thrombus is formed on a mechanically injured vessel in vivo. Both platelet and fibrin deposition is inhibited by a recombinant hirudin (CGP 39393) at doses which prolong the activated partial thromboplastin time (APTT) to no more than 3-4 times the control level. In contrast, both unfractionated heparin and Fragmin only inhibit thrombosis when the APTT is excessively prolonged (i.e. to greater than 15 times the control value). It is concluded that CGP 39393 is an effective antithrombotic in arterial thrombosis at lower levels of anticoagulation than either heparin or Fragmin.
Asunto(s)
Fibrinolíticos/uso terapéutico , Hirudinas/análogos & derivados , Terapia Trombolítica , Trombosis/tratamiento farmacológico , Animales , Aorta Abdominal/lesiones , Constricción , Fibrina/análisis , Heparina/farmacología , Heparina de Bajo-Peso-Molecular/farmacología , Terapia con Hirudina , Tiempo de Tromboplastina Parcial , Agregación Plaquetaria/efectos de los fármacos , Ratas , Proteínas Recombinantes/uso terapéutico , Trombosis/prevención & controlRESUMEN
The effects of the newly available biotechnology product, recombinant desulphatohirudin (CGP 39393) have been investigated in rats. This highly potent and selective thrombin inhibitor exhibited marked anticoagulant properties with controllable titration of anticoagulant effect, as measured by activated partial thromboplastin time (APTT), up to nearly four times control values. Furthermore, CGP 39393 exhibited impressive antithrombotic activity in vivo. In an arteriovenous shunt model of thrombus formation on a cotton-thread, the compound was capable of complete inhibition of thrombus development (ED50 = 0.3 mg/kg i.v. and 1.0 mg/kg s.c.). Venous stasis thrombosis was also highly susceptible to inhibition by CGP 39393 (ED50 = 0.01 mg/kg i.v. and 0.45 mg/kg s.c.). Comparison of the anticoagulant and antithrombotic activities of the compound shows that potent antithrombotic effects (83-97% inhibition in the rat shunt model) are achieved within the generally acceptable range of anticoagulation. These results suggest a clear potential for this new agent in the clinical treatment of thrombotic disease.
Asunto(s)
Anticoagulantes , Antifibrinolíticos , Hirudinas/análogos & derivados , Animales , Derivación Arteriovenosa Quirúrgica , Hirudinas/farmacología , Ligadura , Masculino , Tiempo de Tromboplastina Parcial , Ratas , Ratas Endogámicas , Proteínas Recombinantes/farmacología , Trombosis/etiología , Trombosis/prevención & control , Vena Cava InferiorRESUMEN
Sequential changes in skin metabolism have been studied in a model system of epidermal hyperplasia and hyperkeratinization induced by the application of n-hexadecane to shaved rat skin. The epidermal accumulation of glycogen typical of the hyperplastic response has been correlated with an increase in glycogenesis and a decrease in glycogenolysis. DNA synthesis was increased by 6 h after the start of hexadecane treatment and reached a maximum after one day. The concentration of skin cyclic AMP fell immediately after hexadecane application and subsequently rose to give a prolonged increase. Use of the combined topical application of hexadecane and the anti-inflammatory drugs triamcinolone acetonide, hydrocortisone and indomethacin showed that the hexadecane-induced changes in DNA synthesis and glycogen metabolism were linked to the initial fall in cyclic AMP concentration. The significance of the biphasic change in cyclic AMP levels is discussed as a possible system of control for the development and maintenance of hyperplasia.
Asunto(s)
AMP Cíclico/metabolismo , Epidermis/patología , Piel/metabolismo , Administración Tópica , Alcanos , Animales , Antiinflamatorios/farmacología , ADN/biosíntesis , Epidermis/metabolismo , Glucógeno/metabolismo , Glucógeno Sintasa/metabolismo , Hidrocortisona , Hiperplasia/inducido químicamente , Hiperplasia/metabolismo , Técnicas In Vitro , Indometacina/farmacología , Masculino , Ratas , Piel/efectos de los fármacos , Piel/enzimología , Triamcinolona Acetonida/farmacologíaRESUMEN
The existence of a glyconeogenic pathway in rat skin has been demonstrated by measurement of three of the key glyconeogenic enzymes, fructose 1,6-bisphosphatase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase, and by studies on the incorporation in vitro of carbon from pyruvate and alanine into skin glycogen.
Asunto(s)
Glucógeno/biosíntesis , Piel/metabolismo , Alanina/metabolismo , Animales , Carboxiliasas/análisis , Fructosa-Bifosfatasa/análisis , Hígado/enzimología , Músculos/enzimología , Piruvato Carboxilasa/análisis , Piruvatos/metabolismo , Ratas , Piel/enzimologíaRESUMEN
1. Five glucocorticoids, when administered daily to rats for 5-7 days at a dosage of 5mg/kg, were in the following order of effectiveness with respect to their ability to decrease the weight gain of whole animals and the vastus lateralis, vastus medialis and gluteus medius muscles: corticosteroneAsunto(s)
Glucocorticoides/farmacología
, Mitocondrias Musculares/efectos de los fármacos
, Músculos/metabolismo
, Ribosomas/efectos de los fármacos
, Aminoácidos/metabolismo
, Animales
, Betametasona/farmacología
, Peso Corporal/efectos de los fármacos
, Isótopos de Carbono
, Corticosterona/farmacología
, Dexametasona/farmacología
, Relación Dosis-Respuesta a Droga
, Leucina/metabolismo
, Masculino
, Músculos/citología
, Músculos/efectos de los fármacos
, Oxidación-Reducción
, Prednisolona/farmacología
, Ratas
, Triamcinolona/farmacología
, Triamcinolona Acetonida/farmacología
, Tritio
Asunto(s)
Mitocondrias Musculares/efectos de los fármacos , Triamcinolona Acetonida/farmacología , Envejecimiento , Animales , Isótopos de Carbono , Centrifugación por Gradiente de Densidad , Antagonismo de Drogas , Leucina/metabolismo , Masculino , Metandrostenolona , Microscopía Electrónica , Proteínas Musculares/biosíntesis , Músculos/citología , Músculos/efectos de los fármacos , Músculos/metabolismo , Enfermedades Musculares/inducido químicamente , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Ratas , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Factores de TiempoAsunto(s)
Aberraciones Cromosómicas/efectos de la radiación , Cricetinae/efectos de la radiación , Hígado/metabolismo , Genética de Radiación , Animales , Isótopos de Carbono , Cromátides/efectos de la radiación , Isótopos de Cobalto , ADN/biosíntesis , Hepatectomía , Hígado/patología , Timidina/metabolismo , Factores de TiempoAsunto(s)
Aberraciones Cromosómicas/efectos de la radiación , Mitosis/efectos de la radiación , Efectos de la Radiación , Animales , Cromátides , Isótopos de Cobalto , Colchicina , Cricetinae , Hepatectomía , Hígado/citología , Hígado/efectos de la radiación , Regeneración Hepática/efectos de la radiación , Dosis de Radiación , Factores de TiempoRESUMEN
1. The powerful anti-inflammatory glucocorticoid triamcinolone acetonide, administered to rats at 20 and 2.5mg/kg, leads to a decrease in the incorporation in vivo of [(3)H]uridine and [(32)P]orthophosphate into hind-limb skeletal muscle. 2. At the higher dose, this decrease in the rate of incorporation of precursors into RNA precedes a decrease in the incorporating ability of muscle ribosomes, which commences about 4-5h after drug administration, but is unaccompanied by any changes in the concentration of tissue ATP or free amino acids. 3. The ribosomal dysfunction extends to polyribosomes, which can only be successfully isolated from the muscle of triamcinolone-treated animals after the addition of alpha-amylase to the tissue homogenate to remove glycogen. 4. The specific radioactivity of muscle protein labelled in vivo with (14)C-labelled amino acids does not decrease progressively after triamcinolone administration. After 2h there is an apparent stimulation of incorporation which leads to an overall discrepancy between measurements of protein-synthetic activity made in vivo and in vitro. 5. There is a significant increase in muscle-glycogen concentration between 8 and 12h after the administration of triamcinolone acetonide (20mg/kg), although a significant decrease occurs after 4h. The fall in glycogen concentration may be due to a decrease in the rate of synthesis of protein essential for glucose uptake into the tissues. 6. As judged by (a) incorporation of (14)C-labelled amino acids into protein, (b) [(3)H]uridine and [(32)P]-orthophosphate incorporation into RNA, (c) the rate of induction of tryptophan pyrrolase and (d) changes in the pool sizes of taurine and tryptophan, the responses in liver followed the same time-course as those in muscle after administration of the drug.