RESUMEN
Point-of-care ultrasound in acute care medicine is a prerequisite for diagnosis and therapy monitoring of critically ill patients. There is currently no uniform education strategy for medical intensive care and emergency medicine. As part of the basic level, the trainee takes theoretical and clinical training covering abdominal and thoracic ultrasonography and focused cardiovascular ultrasound. In a second step, special knowledge and skills can be acquired at an expert level. This two-stage concept is intended to guarantee quality assurance in ultrasound education in medical intensive care and emergency medicine.
Asunto(s)
Cuidados Críticos , Enfermedad Crítica/terapia , Medicina de Emergencia/educación , Sistemas de Atención de Punto , Ultrasonografía , Curriculum , Alemania , Humanos , Sociedades MédicasRESUMEN
Ultrasound imaging has attained great significance as a tool for diagnostics in emergency and intensive care medicine. The major advantages of this technique are its instantaneous bedside availability and the possibility to perform repeatable examinations. These advantages are based on recent developments, such as portable ultrasound devices offering excellent imaging quality as well as a quick-start-function. Ultrasound imaging in critically ill patients is frequently performed under pressure of time depending on the current acute physical state. All standard examinations in echocardiography, vascular, abdominal and thoracic ultrasound scanning can be applied in these patients. Based on the clinical scenario the duration of examinations may vary from seconds during cardiopulmonary resuscitations to time-consuming repeated scanning. The transition from basic to subject-specific detailed examinations is flowing and has to be adjusted to local conditions. In the field of emergency and intensive care medicine the technique used is whole-body sonography. The goal is to classify the patient's present physical state and to define a targeted therapeutic approach. The characteristics of whole-body sonography are similar to the field of anesthesiology which is an interdisciplinary one. Currently, these characteristics deserve more attention in training in sonography.
Asunto(s)
Anestesiología/tendencias , Cuidados Críticos/métodos , Servicios Médicos de Urgencia/métodos , Ultrasonografía/métodos , Abdomen/diagnóstico por imagen , Anestesiología/educación , Enfermedad Crítica , Ecocardiografía , Cardiopatías/diagnóstico por imagen , Humanos , Enfermedades Pulmonares/diagnóstico por imagen , Médicos , Ultrasonido , Enfermedades Vasculares/diagnóstico por imagen , Imagen de Cuerpo EnteroRESUMEN
The 26S proteasome is a non-lysosomal multicatalytic protease complex for degrading intracellular proteins by ATP/ubiquitin-dependent proteolysis. Tightly ordered proteasomal degradation of proteins critical for cell cycle control implies a role of the proteasome in maintaining cell proliferation and cell survival. In this study, we demonstrate that cell-permeable proteasome inhibitors, lactacystin, benzyloxycarbonyl(Z)-leucyl-leucyl-leucinal (ZLLLal; MG-132) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucyl-leucyl-leucine vinyl sulfone (NLVS), induce apoptosis abundantly in p53-defective leukemic cell lines CCRF-CEM, U937 and K562 as well as in myelogenic and lymphatic leukemic cells obtained from adult individuals with relapsed acute leukemias. Leukemic cell apoptosis induced by the proteasome inhibitors was dependent on activation of caspase-3 and related caspase family proteases, because caspase-3 inhibitor N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartal (Ac-DEVD-cho) and, more effectively, the general caspase-inhibitor N-benzyloxycarbonyl-L-valyl-L-alanyl-L-aspartate fluoromethylketone (Z-VAD-fmk) were capable of blocking apoptosis induced by lactacystin, ZLLLal or NLVS. Induction of apoptosis by lactacystin or ZLLLal was accompanied by cell cycle arrest at G2/M phase and by accumulation and stabilization of cyclin-dependent kinase inhibitor p21WAF1/Cip and tumor suppressor protein p53. A role of p53 in mediating apoptosis or induction of p21WAF1/Cip1 was ruled out since CCRF-CEM and U937 cells express non-functional mutant p53, and K562 cells lack expression of p53. Viability and hematopoietic outgrowth of human CD34+ progenitor cells treated with lactacystin were slightly reduced, whereas treatment of CD34 + cells with ZLLLal or the cytostatic drugs doxorubicin and gemcitabine resulted in markedly reduced viability and hematopoietic outgrowth. These results demonstrate a basic role of the proteasome in maintaining survival of human leukemic cells, and may define cell-permeable proteasome inhibitors as potently anti-leukemic agents which exhibit a moderate hematopoietic toxicity in vitro.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/farmacología , Ciclinas/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Leucemia/fisiopatología , Complejos Multienzimáticos , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Enfermedad Aguda , Adulto , Antígenos CD34 , Caspasa 3 , Caspasas/metabolismo , Caspasas/fisiología , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Inhibidores de Cisteína Proteinasa/fisiología , Inhibidores Enzimáticos/metabolismo , Citometría de Flujo , Fase G2/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Humanos , Células K562 , Leucemia/enzimología , Leucemia/patología , Leupeptinas/farmacología , Mitosis/efectos de los fármacos , Complejos Multienzimáticos/antagonistas & inhibidores , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/fisiología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/farmacología , Células U937RESUMEN
A fusion protein (bFGF-rMLA), containing the mitogen basic fibroblast growth factor (bFGF) and the cytotoxic component of rViscumin (recombinant mistletoe lectin), the enzymatic A-chain (rMLA), was expressed in Escherichia coli, purified, and functionally characterized. bFGF-rMLA is cytotoxic for mouse B16 melanoma cells expressing the FGF receptor with an IC(50) value of approximately 1 nM. rMLA shows no significant effect on the viability of the B16 cells up to a concentration of 141 nM. Additionally, bFGF-rMLA was associated with the rViscumin B-chain (rMLB) in an in vitro folding procedure. The IC(50) value of bFGF-rMLA/rMLB to B16 cells in the presence of lactose-to block rMLB lectin activity-was 134 pM. Thus, it was possible to enhance the efficacy of a rViscumin A-chain mitotoxin through addition of rMLB. We conclude that rViscumin fusion proteins may be generally applicable for the receptor-specific inactivation of target cells and point out their potential in drug development.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Preparaciones de Plantas , Proteínas de Plantas , Proteínas Recombinantes de Fusión/farmacología , Toxinas Biológicas/farmacología , Animales , Muerte Celular/efectos de los fármacos , Clonación Molecular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Concentración 50 Inhibidora , Cinética , Lectinas/química , Melanoma Experimental , Ratones , Oligonucleótidos/metabolismo , Plásmidos/metabolismo , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Inactivadoras de Ribosomas Tipo 2 , Factores de Tiempo , Toxinas Biológicas/química , Toxinas Biológicas/metabolismo , Células Tumorales CultivadasRESUMEN
Recombinant mistletoe lectin (rML) belongs to the class of type II ribosome-inactivating proteins (RIP) composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding properties. To investigate the contribution of the enzymatic activity of the rML A-chain to the observed cytotoxic and apoptotic effects, an rMLA E166Q R169Q molecule was developed by means of site-specific mutagenesis. Following heterologous expression, the activity of mutant rMLA was measured in a cell-free assay for rRNA-N-glycosidase activity. Moreover, after generation of heterodimer, the activities of mutant rML E166Q R169Q and rML wild type were determined in a cytotoxicity and apoptosis assay. Although the reduction of activity as measured in the cell-free RIP assay was more pronounced (factor 237) than in both cellular assays (factors 20-22), the data clearly indicate a close correlation between cytotoxicity, apoptosis, and the enzymatic activity of the rML A-chain. Thus, RIP activity is an essential feature of rML and therefore a prerequisite for its biological function as an anticancer agent.
Asunto(s)
Apoptosis/genética , Lectinas/genética , Preparaciones de Plantas , Proteínas de Plantas , Toxinas Biológicas/genética , Lectinas/metabolismo , Muérdago , Mutagénesis Sitio-Dirigida , Lectinas de Plantas , Plantas Medicinales , Inhibidores de la Síntesis de la Proteína/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleósidos/genética , Ribonucleósidos/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 2 , Ribosomas , Toxinas Biológicas/metabolismoRESUMEN
Mistletoe lectin I (pML) and its isoforms ML II and III constitute the active principle in extract preparations from mistletoe, commonly used as immunomodulator in adjuvant tumour therapy. The heterodimeric disulfide-linked cytotoxic protein is classified as type II ribosome inactivating protein (RIP). Recently, the sequence coding for the mistletoe lectin prepro-protein was identified and the existence of a single intron-free gene was shown [Eck, J., Langer, M., Möckel, B., Baur, A., Rothe, M., Zinke, H. & Lentzen, H. (1999) Eur. J. Biochem. 264, 775-784]. The aim of this study was to prepare pure and homogeneous rMLB-chain as well as rML heterodimer for studying the carbohydrate binding specificity of recombinant versus natural protein and its contribution to the observed cytotoxic effect. Expression in E. coli resulted in the production of insoluble protein (inclusion bodies). A procedure for generating correctly folded, biochemically and biologically active rMLB was established starting from the insoluble single chain. Carbohydrate binding and specificity of pMLB and rMLB were analysed by a competitive enzyme linked lectin assay (ELLA). Asialofetuin was able to compete with binding of both chains (50% at 0.8 microM). The specificity of the B-chains to lactose was more distinct with halfmaximal competition at 4.9 mM (pMLB) and > 90 mM (rMLB), respectively. Furthermore, in a coassociation process rMLA- and rMLB inclusion bodies were associated in one step by defined dilution yielding active rML-heterodimer. The activities of recombinant (rML) and plant derived mistletoe lectin (pML) were compared. Cytotoxicity was determined using MOLT-4 cells and enzymatic rRNA N-glycosidase activity was measured in a coupled transcription/translation assay. The IC50 values of the two heterodimers were similar in both assays; rMLB-chain did not show any cytotoxic effect. In the ELLA with lactose as a competitor 50% competition of binding to asialofetuin was achieved at 1.6 mM (rML) and 1.8 mM (pML). Hence, using three different assays we found no significant differences between the recombinant protein and the glycosylated form of ML. Comparing the biological activities of the single chains with those of the heterodimer we conclude, that both, lectin activity and the rRNA N-glycosidase activity, are prerequisites for the cytotoxic effects on target cells.
Asunto(s)
Lectinas/química , Muérdago/química , Proteínas de Plantas/química , Plantas Medicinales , Asialoglicoproteínas/química , Unión Competitiva , Clonación Molecular , Dimerización , Escherichia coli , Fetuínas , Lactosa/metabolismo , Lectinas/genética , N-Glicosil Hidrolasas/metabolismo , Lectinas de Plantas , Unión Proteica , Pliegue de Proteína , Isoformas de Proteínas , ARN Ribosómico 28S/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Inactivadoras de Ribosomas , alfa-Fetoproteínas/químicaRESUMEN
Mistletoe lectin I (MLI) is the major active constituent of mistletoe extracts, which are widely used for adjuvant tumour therapy. The 66-kDa heterodimeric disulphide-linked glycoprotein is classified as type II ribosome-inactivating protein (RIP) due to the rRNA-cleaving enzyme activity of the A-subunit, also referred to as toxic entity. MLI and the close relative ricin both belong to the family of the two-chain plant type II RIP proteins. Isolation of the glycosylated proteins from plant material yield inhomogeneous material probably due to post-translational modifications. The aim of this study was to prepare pure and homogeneous protein as a prerequisite for structural and mechanistic studies in order to gain insight into the mode of action of this cytotoxic plant protein on tumour and immune cells. Of particular interest was to explain whether the differences in toxicity of ML and ricin are the result of variations of their enzymatic activities. By investigating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific amplification of the mistletoe lectin gene. Applying this PCR strategy the full-length 1923 nucleotide DNA sequence coding for the prepro-protein was obtained showing the existence of a single intron-free gene. In order to elucidate the molecular basis for the observed differences in cytotoxicity within the family of RIP the enzymatic A-subunit was expressed in a heterologous system. Expression of the A-chain in E. coli BL21/pT7 resulted in production of insoluble inclusion bodies constituting 20-30% of total protein. Refolding led to a pure and homogeneous protein species with an apparent molecular mass of 27 kDa and a pI value of 6.4. The ribosome-inactivating activity of the unglycosylated recombinant A-chain (IC50 20.5 pM) protein was in the same range as that of the glycosylated plant-derived ML A-chain (IC50 3.7 pM), which was very similar to that of ricin A-chain (IC50 4.9 pM). Thus, the higher cytotoxicity of ricin cannot be accountable for differences in the enzymatic activities of the type II RIP A-chains.
Asunto(s)
Genes de Plantas , Lectinas/genética , Muérdago/genética , Preparaciones de Plantas , Proteínas de Plantas , Plantas Medicinales , Toxinas Biológicas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN de Plantas/genética , Escherichia coli/genética , Expresión Génica , Focalización Isoeléctrica , Lectinas/química , Datos de Secuencia Molecular , Lectinas de Plantas , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Inactivadoras de Ribosomas Tipo 2 , Toxinas Biológicas/químicaRESUMEN
A plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to increase the number and cytotoxic activity of natural killer (NK) cells in animal models, but the mechanisms underlying these effects are poorly understood. We investigated the effects of the recombinant form of this lectin (rVAA) on secretion of interleukin (IL)-12 and on NK-mediated cytotoxicity against K562 target cells in cultures of human peripheral blood mononuclear cells (PBMC) as well as against YAC-1 target cells in cultured rat spleen cells. In 24-hour cultures of PBMC, 10 ng/ml plant VAA-I and 50 ng/ml rVAA induced significant increases in the secretion of total IL-12. Its biologically active heterodimeric form, p70, was also significantly induced by rVAA. Preincubation of PBMC or splenocytes for 48 h with rVAA in concentrations ranging between 10 pg/ml and 100 pg/ml resulted in moderate enhancements of NK-mediated cytotoxicity. However, coincubation of a low dose of rVAA (100 pg/ml) together with IL-2 and IL-12 (60 U/ml and 2 U/ml, respectively) led to additive stimulation of NK activity. In in vivo experiments, rVAA showed an enhancing effect on NK activity with a bell-shaped curve of efficacy. Forty-eight hours after a single intravenous injection of its most effective doses, 0.5 and 1 ng/kg, into Wistar rats, the NK cytotoxicity of splenocytes against YAC-1 targets doubled, and the frequency of large granular lymphocytes in peripheral blood showed 2.1- and 3-fold increases as compared to control animals. Twenty-four hours following these low lectin doses, the number of large granular lymphocytes was also significantly elevated. After 48 h, 0.5 ng/kg rVAA induced a significant augmentation in the percentage of peripheral Mac-1+ mononuclear cells, including activated monocytes and NK cells. The present results suggest that rVAA augments the secretion of an active form of IL-12 and potentiates the cytokine-induced NK activation. These effects of rVAA may be related to its stimulatory effects on MHC-unrestricted cytotoxicity in vivo.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Lectinas/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Muérdago , Preparaciones de Plantas , Proteínas de Plantas , Plantas Medicinales , Toxinas Biológicas/farmacología , Animales , Células Cultivadas , Citotoxicidad Inmunológica/efectos de los fármacos , Femenino , Humanos , Interleucina-12/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Lectinas/genética , Masculino , Lectinas de Plantas , Ratas , Proteínas Recombinantes/farmacología , Proteínas Inactivadoras de Ribosomas , Proteínas Inactivadoras de Ribosomas Tipo 2 , Bazo/citología , Bazo/efectos de los fármacos , Toxinas Biológicas/genéticaRESUMEN
Subtractive cloning procedures led to the identification of a variety of transcripts expressed in mammalian brain. However, little is known about the encoded proteins and the regulation of gene expression. Here, we describe the isolation and characterisation of a single-copy gene (83.5) of 21.7 kb which is specifically expressed in porcine brain. In situ hybridisation and immunohistochemistry experiments showed a distinct pattern of gene expression in neuronal cell types in different parts of the brain. The gene contains two mini exons, confirming neural-specific expression. cDNA cloning experiments revealed two species of mRNA differing in their 5'-regions. These transcripts are generated by two distinct transcription start sites that are under the control of different potential promoter regions as shown by primer-extension experiments. The amino acid sequences of the deduced proteins predict that one mRNA species encodes a novel type-I transmembrane protein, whereas the other transcript encodes only a part of its cytoplasmic domain. In Western-blot experiments, we detected two proteins of the predicted size and cellular localisation in porcine brain. The precise function of these proteins remains to be determined. However, our findings suggest that they may be generated by alternative promoter usage, leading to the expression of a membrane protein and its truncated cytoplasmic isoform.
Asunto(s)
Química Encefálica/genética , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Exones , Dosificación de Gen , Expresión Génica , Inmunohistoquímica , Intrones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Solubilidad , Porcinos , Distribución TisularRESUMEN
A serious retroperitoneal bleeding occurred in a 56-year-old male patient receiving unfractionated heparin due to multiple pulmonary embolism. After reducing the heparin dose, the patient developed a new pulmonary embolism and a large thrombus in the right atrium. Concomitantly, the platelet count dropped to a value of 29 g/l. Heparin-induced thrombocytopenia (HIT) was confirmed by a functional assay, the heparin-induced platelet activation (HIPA) assay, whereas the results of a platelet factor 4/heparin complex ELISA were repeatedly negative. This indicated that the patient's HIT antibodies were directed towards an antigen other than platelet factor 4/heparin complexes. For treatment of the atrial thrombus, an ultra-low-dose lysis with rt-PA (2 mg/h, intravenously) was administered for a period of 52 h, overlapping with systemic treatment with recombinant hirudin (Lepirudin, Refludan, 0.06-0.14 mg/kg/h intravenously). The aim was to enhance lysis of the thrombus without increasing the haematoma, and at the same time keep the risk of fulminant pulmonary embolism due to thrombus fragmentation as low as possible. The cardiac thrombus disappeared within 48 h, without new signs of pulmonary embolism. Platelet counts normalized within nine days.
Asunto(s)
Anticoagulantes/uso terapéutico , Enfermedades Autoinmunes/inducido químicamente , Cardiopatías/complicaciones , Hemorragia/inducido químicamente , Heparina/efectos adversos , Hirudinas/análogos & derivados , Activadores Plasminogénicos/uso terapéutico , Trombocitopenia/inducido químicamente , Terapia Trombolítica , Trombosis/complicaciones , Activador de Tejido Plasminógeno/uso terapéutico , Anticoagulantes/efectos adversos , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/tratamiento farmacológico , Enfermedades Autoinmunes/complicaciones , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Atrios Cardíacos , Terapia con Hirudina , Humanos , Masculino , Persona de Mediana Edad , Fenprocumón/uso terapéutico , Activadores Plasminogénicos/administración & dosificación , Embolia Pulmonar/complicaciones , Embolia Pulmonar/tratamiento farmacológico , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/uso terapéutico , Espacio Retroperitoneal , Trombocitopenia/complicaciones , Trombocitopenia/tratamiento farmacológico , Trombocitopenia/inmunología , Activador de Tejido Plasminógeno/administración & dosificación , Filtros de Vena CavaRESUMEN
The effects of mistletoe lectin I (ML I) on the human T-cell leukemia line MOLT-4, the monocytic line THP-1 and on human peripheral blood mononuclear cells (PBMC) were investigated with regard to general cell viability and induction of apoptosis. Using a sensitive serum-free cytotoxicity assay, the time- and concentration-dependent direct toxicity towards MOLT-4 cells was determined with IC50-values ranging from 20-40 pg/ml (300-600 fmol/l). Investigations on the time course of the toxic effect using selected concentrations of ML I revealed distinct response curves for concentrations of high, low and intermediate toxicity, respectively. The ratio of apoptotic to viable MOLT-4 cells was determined after treatment with ML I for 24 h. Apoptosis and cytotoxicity were correlated at low and intermediate concentrations, whereas at long intervals and high concentrations of ML I mostly necrotic effects were observed. The data showed that in the concentration range of low cytotoxicity ML I-induced cell death is quantitatively due to apoptotic processes. The immunomodulatory activity of ML I was investigated in vitro by measuring cytokine release. At concentrations of low cytotoxicity ML I showed immunostimulatory activity on PBMC and THP-1. RT-PCR with THP-1 cells confirmed that cytokine induction by ML I is regulated on the transcriptional level. These findings suggest that in the blood cells investigated both apoptosis and cellular signalling are induced by the same concentration range of ML I.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Sanguíneas/efectos de los fármacos , Citocinas/biosíntesis , Preparaciones de Plantas , Proteínas de Plantas , Toxinas Biológicas/farmacología , Células Sanguíneas/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Humanos , Técnicas In Vitro , Lectinas , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Inactivadoras de Ribosomas Tipo 2RESUMEN
A sensitive nonradioactive method to determine the activity of ribosome-inactivating proteins (RIPs) based on a combined transcription/translation in vitro assay was established. Using this assay we investigated the RIP activities of the heterodimeric toxic plant lectins ricin and mistletoe lectin I (ML-I). The enzymatic activities of the holoproteins were compared to that of the RIP-active chain of ML-I (ML-I A-chain) and recombinant ML-I A-chain expressed in Escherichia coli. The IC50 values determined for the plant toxins showed that the translation-inactivating activity of ML-I (39.8 pM) is about four times higher than that of ricin (176.0 pM). The plant-derived ML-I A-chain is more toxic (3.4 pM) in the cell-free translation system than the respective holoprotein. The recombinant ML-I A-chain was found to be about three times less active (IC50 10.6 pM) than the A-chain from plant. The in vitro assay described here is a convenient method for the fast determination of RIP activity with a 1000-fold lower detection limit than that of commonly used RIP assays.
Asunto(s)
Preparaciones de Plantas , Proteínas de Plantas , Proteínas Ribosómicas/análisis , Animales , Relación Dosis-Respuesta a Droga , Formicinas/farmacología , Lectinas/análisis , Muérdago , Lectinas de Plantas , Plantas Medicinales , Conejos , Proteínas Recombinantes/análisis , Ribonucleótidos/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 2 , Ricina/análisis , Toxinas Biológicas/análisisRESUMEN
Apolipoprotein (apo) A-I is the major protein component of high-density lipoproteins (HDLs), which are responsible for reverse cholesterol transport from peripheral tissues to the liver. A low level of plasma HDL is correlated with susceptibility to atherosclerosis and coronary heart disease. Mammalian apo A-I synthesis has been attributed mainly to liver and intestine. Recently, apo A-I expression has been shown in porcine brain capillaries, suggesting an independent lipid metabolism within the brain. In this study, protein synthesis and secretion were investigated in primary cultures of porcine brain microvascular endothelial cells and compared with those in large vessel endothelium. Active protein synthesis in vitro was demonstrated by metabolic labeling. Cerebral endothelial cells were shown to secrete apo A-I into the culture supernatant, whereas aortic endothelial cells were negative for apo A-I expression. Further studies of transcriptional regulation showed that cerebral endothelium was responsive to apo A-I-inducing agents, such as cholesterol, insulin, and retinoic acid, as previously shown in human hepatoma HepG2 cells. Thus, cultures of porcine cerebral endothelial cells may represent a suitable model for physiological studies of apo A-I-regulation with regard to brain lipid metabolism and blood-brain barrier function. To investigate the interspecies conservation of regulatory elements, 178 bp of the 5' flanking region of the porcine apo A-I gene was cloned using PCR techniques. Alignments of the cDNA, of the deduced apo A-I protein sequence, and of the 5' promoter region with the corresponding genomic sequences of different species show a high degree of similarity between the porcine and the primate apo A-I genes, thus indicating a similar function and possibly common regulatory mechanisms in those species. In contrast, the rodent and avian apolipoprotein A-I promoter sequences differed significantly.
Asunto(s)
Apolipoproteína A-I/metabolismo , Circulación Cerebrovascular , Endotelio Vascular/metabolismo , Secuencia de Aminoácidos , Animales , Apolipoproteína A-I/genética , Secuencia de Bases , Células Cultivadas , ADN Complementario/genética , Endotelio Vascular/citología , Exones , Técnica del Anticuerpo Fluorescente , Humanos , Intrones , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Porcinos , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Our approach to analyze molecular components of the blood-brain barrier led to the identification of additional transcripts which can be regarded as "BBB markers". Other candidates are presently analyzed in order to find hitherto unknown cell type-specific transcripts. We investigated the expression of these marker-genes in cell culture and found all genes still being transcribed after 10 days in primary cultures, although at a lower level. This is surprising, since other authors report the disappearance of BBB characteristics under such conditions. Moreover, the BBB marker gamma-GT is found to be not only expressed in BMEC, but also in the closely associated pericytes. The hitherto unknown physiological function of the enzyme, especially the abundance in pericytes is still under investigation. Since the method of subtractive cloning has been proven as a fruitful approach, we consider to establish further subtractive cDNA libraries, using different subtraction parameters. The PCR method is applicable for amplification of subtracted cDNA (Timblin et al., 1990) and we expect to find additional clones, mainly of lower abundance which are of functional importance for the BBB phenomenon. The described characterization of cultured BMEC now allows to proceed to study BBB-specific gene expression with special regard to regulatory elements. We will perform these experiments by use of enhancer trap vectors transfected into BMEC. The isolation of the corresponding genomic DNA fragments of the BBB markers is in progress.
Asunto(s)
Barrera Hematoencefálica , Encéfalo/fisiología , Transcripción Genética , Animales , Encéfalo/irrigación sanguínea , Capilares/enzimología , Células Cultivadas , Endotelio Vascular/enzimología , Marcadores Genéticos , Modelos Neurológicos , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , gamma-Glutamiltransferasa/genéticaRESUMEN
A porcine brain microvessel-derived cDNA library enriched in blood-brain-barrier-specific sequences was constructed by a subtractive cloning procedure. Two cDNA clones from this library were found to encode a glucose transport protein. These clones were used to isolate a nearly full length cDNA from a representative brain microvessel library. Analysis of the amino-acid sequence deduced from the cDNA sequence revealed 97% identity with the human HepG2 glucose carrier. Amino-acid substitutions appear to be clustered in certain regions of the polypeptide. The nucleotide sequences of the 3'-noncoding regions close to the putative polyadenylation sites are highly conserved in glucose transporter mRNAs of different species. The expression of this mRNA has been investigated in various tissues and shown to be decreased in primary cultures of brain microvascular endothelial cells.
Asunto(s)
Barrera Hematoencefálica , Clonación Molecular , ADN/genética , Proteínas de Transporte de Monosacáridos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Células Cultivadas , Endotelio Vascular/citología , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , ARN/aislamiento & purificación , ARN Mensajero/genética , Conejos , Ratas , PorcinosRESUMEN
In the present work the influence of moist air, of sand and of several solutions on the geotropic behaviour of primary roots is studied. The course of the geotropic movement is the result of a concerted action of positive and negative reactions the intensity and duration of which differ in roots of various species.In pea roots the negative movement appears only during a short stage of development. No direct relation exists between the speed of elongation and the appearance of the negative reaction.Primary roots of Zea mays and of Pisum arvense are indifferent to thigmotropic stimuli. The negative movement has, at least in pea roots a smaller mechanically effective force than the positive movement. Therefore the negative reaction does not appear in sand because it cannot overcome the mechanical resistance of the granular medium.In liquid media pea roots react in another way than in air: here the negative reaction begins later, but it has then more influence on the course of the geotropic curvature.The influence of different cations on the geotropic behaviour and on the elongation of the roots can be understood as a combined action of the osmotic effect and of the specific ionic permeability.In pea roots the negative reaction, which appears during the time from 3 to 6 hours after the induction also depends on a definite level of turgor.Primary roots of Zea mays, which grow in a relatively large angle to the vertical line do not lose their geotropic sensibility. They react like plagiotropic organs.In pea roots relations exist between the development of the positive and the negative reaction and the presence of the cotyledons and the tip of the root.Both reactions are induced at the same time by the gravitional stimulus. Their reaction times, however, are different.The root tip is necessary for the induction of both reactions. The negative curvature also appears when the tip is cut off before the end of the reaction time.The course of the geotropic movement of primary roots is compared with the geotropic behaviour of rhizomes. As a possible explanation of both kinds of reactions a two-hormone-hypothesis is discussed.