RESUMEN
An improved chromatographic method was developed to isolate and purify polypeptides and proteins from the crude venom of the Taiwan cobra Naja naja atra. The procedure devised is simple, easy to reproduce, and enables large scale isolation of almost all polypeptides and proteins in this cobra venom. Six pure polypeptide fractions of the venom were isolated and characterized using gel filtration on Sephadex G50 (medium), ion exchange chromatography on SP-Sephadex C25, desalting on Sephadex G25 (fine) and preparative HPLC on a RPC 18 column. The neuromuscular activity of these fractions was tested on the chick biventer cervicis nerve-muscle preparation and their toxicity (LD(50)) was determined after i.v. administration in mice. Their antinociceptive activity was tested in the mouse abdominal test by i.v. application. Two of these polypeptide samples had major physiological effects: one acted as a cardiotoxin causing reversible myocardial contractures with no effect on muscle twitches elicited by nerve stimulation (NS); another was a neurotoxin that blocked muscle contractions in response to NS and exogenously added acetylcholine. The cardiotoxic fraction was identified as CTX I, a well-known cardiotoxin present in this venom, and the neurotoxin was identified as neurotoxin-α with an LD50 in mice of 0.075 mg/kg.
Asunto(s)
Cromatografía/métodos , Proteínas Cardiotóxicas de Elápidos/aislamiento & purificación , Proteínas Neurotóxicas de Elápidos/aislamiento & purificación , Venenos Elapídicos/metabolismo , Péptidos/aislamiento & purificación , Analgésicos/aislamiento & purificación , Analgésicos/farmacología , Animales , Fraccionamiento Químico/instrumentación , Fraccionamiento Químico/métodos , Pollos , Proteínas Cardiotóxicas de Elápidos/química , Proteínas Cardiotóxicas de Elápidos/toxicidad , Proteínas Neurotóxicas de Elápidos/química , Proteínas Neurotóxicas de Elápidos/toxicidad , Venenos Elapídicos/química , Venenos Elapídicos/toxicidad , Elapidae/fisiología , Femenino , Corazón/efectos de los fármacos , Dosificación Letal Mediana , Masculino , Ratones , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/fisiopatología , Dolor/inducido químicamente , Dolor/tratamiento farmacológico , Umbral del Dolor/efectos de los fármacos , Péptidos/química , Péptidos/toxicidad , RatasRESUMEN
Comparative investigation concerning gelfiltration as well as haemostaseologic analysis of venoms and venom fractions of some snakes (elapidae and viperidae) have shown that in elapidae an inhibition of coagulation is dominant whilst in viperidae the stimulation of coagulation is of importance. Our investigations produce a basis to select substances for activation of coagulation and substances for inhibition of coagulation. Under pharmacological viewpoints the data may produce information to use snake fractions for anticoagulation or for procoagulant therapy in bleeding tendency.
Asunto(s)
Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Venenos de Serpiente/farmacología , Animales , Tiempo de Sangría , Cromatografía en Gel , Venenos Elapídicos/aislamiento & purificación , Venenos Elapídicos/farmacología , Hemorragia/inducido químicamente , Humanos , Venenos de Serpiente/aislamiento & purificaciónRESUMEN
BACKGROUND: Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n-Nonanoyl-CC chemokine ligand 14 (NNY-CCL14), a modified analog of the naturally occurring chemokine CCL14(9-74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5. AIM OF THE STUDY: As CCL2 and its receptor CCR2 have been shown to play important roles in the development of Th2 inflammation, we further evaluated the effects of NNY-CCL14 treatment on CCL2-mediated activation of CCR2. METHODS: Effects of NNY-CCL14 treatment were studied on cell lines transfected with human CCR2 and primary leukocytes. Functional effects were assessed by calcium efflux assays, flow cytometry and chemotaxis. RESULTS: Prestimulation with NNY-CCL14 desensitized CCR2-mediated responses to further stimulation with its selective ligand CCL2. No significant internalization of CCR2 was observed when the cells were stimulated with NNY-CCL14, even at concentrations eliciting maximal [Ca(2+)]i mobilization. Above all, NNY-CCL14 pretreatment blocked CCL2-induced chemotaxis of monocytes. CONCLUSIONS: This study demonstrates that NNY-CCL14 is a partial agonist of CCR2, inhibiting responses of monocytes to the CCR2-selective ligand CCL2. NNY-CCL14 attenuates CCR2-mediated responses by rapidly desensitizing the receptor and preventing chemotaxis, although it is able to induce calcium mobilization but does not lead to CCR2 internalization. Hence this study provides further insights into the possible mechanisms of action of NNY-CCL14, which interacts with multiple chemokine receptors inhibiting the migration and activation of different cell populations involved, thus acting as a potential therapeutic compound to alleviate allergic inflammation.
Asunto(s)
Antialérgicos/metabolismo , Antiinflamatorios no Esteroideos/uso terapéutico , Quimiocina CCL11/uso terapéutico , Quimiocinas CC/uso terapéutico , Mediadores de Inflamación/uso terapéutico , Receptores CCR2/agonistas , Hipersensibilidad Respiratoria/tratamiento farmacológico , Animales , Antialérgicos/química , Antialérgicos/uso terapéutico , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Línea Celular , Inhibición de Migración Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL11/química , Quimiocina CCL11/fisiología , Quimiocinas CC/química , Quimiocinas CC/fisiología , Humanos , Mediadores de Inflamación/fisiología , Ratones , Receptores CCR2/antagonistas & inhibidores , Receptores CCR2/biosíntesis , Hipersensibilidad Respiratoria/patologíaRESUMEN
OBJECTIVE: Critical size defects (CSDs) of bone are defined as defects that do not heal spontaneously to new bone during the lifetime of an adult individual. In contrast, immature animals are capable to heal defects of identical size. It was our hypothesis that age-related paracrine effects are relevant for this difference in regeneration. METHODS: The pooled supernatant of primary rat calvarial osteoblast-like cell cultures (POBC) derived from prenatal or postnatal donors was concentrated and applied into CSDs of adult recipient organisms (n = 10). In addition, the supernatant of POBC derived from prenatal donors was pooled and purified by reverse-phase chromatography. Each pre-purified fraction was tested in a proliferation indicating bioassay. Peptide fractions containing proliferative activities were re-chromatographed and re-tested in a bioassay. Finally, a proliferative activity was purified, identified by sequence analysis and applied into CSDs of adult recipients. RESULTS: The application of POBC derived from prenatal donors resulted in osseous regeneration of a CSD in adult recipients, while the supernatant of postnatal donors had much smaller effects. The morphologic features resembled the spontaneous osseous healing of calvarial defects of the same size in immature organisms. The polypeptide "tissue inhibitor of metalloproteinases type II"(TIMP-2) was isolated from the supernatant of cultures of POBC derived from prenatal donors by measuring the induction of their proliferation. Additionally, the application of human TIMP-2 injected into calvarial CSDs of adult organisms resulted in osseous healing. CONCLUSION: We conclude that one component responsible for the healing effect of CSDs of POBC supernatants derived from prenatal donors is TIMP-2.
Asunto(s)
Huesos/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/biosíntesis , Envejecimiento , Secuencia de Aminoácidos , Animales , Proliferación Celular , Células Cultivadas , Cromatografía/métodos , Modelos Biológicos , Datos de Secuencia Molecular , Osteoblastos/metabolismo , Péptidos/química , Ratas , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The circulating hormonal form of human parathyroid hormone (hPTH-1-37) has been assessed in vitro as well as in vivo in the ovariectomized rat, a model for postmenopausal osteoporosis. In vitro, hPTH-1-37 induces a dose-dependent cAMP formation and increases vitality as well as alkaline phosphatase activity in UMR106 osteosarcoma cells. Differentiation and proliferation of osteoclasts in rat bone marrow-derived stem cell preparations are decreased. Daily hPTH-1-37 s.c. administration in ovariectomized rats for 60 days results in augmented formation of new bone, in amplified femural bone density, and in thickening of the calvaria.
Asunto(s)
Osteoporosis Posmenopáusica/tratamiento farmacológico , Hormona Paratiroidea/uso terapéutico , Fragmentos de Péptidos/uso terapéutico , Fosfatasa Alcalina/metabolismo , Análisis de Varianza , Animales , Línea Celular Tumoral , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Fémur/diagnóstico por imagen , Fémur/efectos de los fármacos , Fémur/crecimiento & desarrollo , Humanos , Ovariectomía , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Radiografía , Ratas , Ratas Wistar , Cráneo/efectos de los fármacos , Cráneo/crecimiento & desarrolloRESUMEN
BACKGROUND: CC chemokine ligand 11 (CCL11) is the outstanding member of all described CC chemokine receptor 3 (CCR3) ligands and is shown to be selective for this receptor. However, it also activates CCR5 but only in the micromolar range. The in vivo activity of CCL11 is expected to be temporally restricted, as it is degraded by specific proteases such as the dipeptidyl-peptidase IV (DP4), also termed CD26. Based on the approach to inactivate chemokine receptors in allergic disease models as has been demonstrated for DP4-resistant n-nonanoyl (NNY)-CCL14 and for amino-oxypentane (AOP)-CCL5, it is tempting to study similar compounds derived from CCL11. METHODS: Synthesis of NNY-CCL11 was performed and it was characterized for biological functions in human and mouse eosinophils as well as in cell lines stably transfected either with human CCR3 or CCR5. Resistance to DP4 treatment was also investigated. RESULTS: The functional activities of NNY-CCL11 mediated via CCR3 show an almost identical pattern to CCL11 with respect to intracellular calcium mobilization and CCR3 internalization. N-terminal cleavage of CCL11 by preincubation with DP4 results in a reduced capacity to internalize CCR3, while preincubation of NNY-CCL11 shows no influence. In contrast to CCL11, NNY-CCL11 also activates CCR5+ cell lines and human monocytes in the nanomolar range, being about 100 times more potent than CCL11. CONCLUSIONS: n-Nonanoyl-CCL11 represents a compound with dual activity restricted to CCR3 and CCR5. Because of its receptor-inactivating capacity and stability against DP4 degradation, NNY-CCL11 is a suitable tool for the decoding of the pathophysiological mechanisms of allergic diseases.
Asunto(s)
Quimiocinas CC/química , Quimiocinas CC/metabolismo , Hipersensibilidad/metabolismo , Receptores CCR5/metabolismo , Receptores de Quimiocina/metabolismo , Adenosina Desaminasa/fisiología , Animales , Calcio/metabolismo , Quimiocina CCL11 , Dipeptidil Peptidasa 4/fisiología , Eosinófilos/metabolismo , Femenino , Glicoproteínas/fisiología , Humanos , Ratones , Ratones Endogámicos BALB C , Monocitos/metabolismo , Receptores CCR3 , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Vesículas Transportadoras/metabolismoRESUMEN
Mounting evidence suggests that urodilatin, not atrial natriuretic peptide (ANP) is the responsible peptide in regulation of renal Na superset+- and water homeostasis. Following the discovery of ANP this peptide was thought to be responsible for the induction of natriuresis and diuresis in the mammalian kidney. However, the isolation of urodilatin from human urine and substantial work contributed to a better understanding of the renal physiology of these two natriuretic peptides. Indeed, subsequent elucidation supported that urodilatin rather than ANP seems to be the natriuretic peptide responsible for the regulation of Na superset+- and water homeostasis in the kidney. Urodilatin - synthesized and secreted from the distal tubules of the kidney - may act as a paracrine mediator when secreted into the lumen. In contrast, while the role of ANP as regulator of the cardiovascular system is established, its physiological regulatory role on transport processes in the nephron is questionable. This review attempts to analyze the roles of both ANP and urodilatin and to discuss new potential candidates which may also play a role in electrolyte and water handling in the kidney.
Asunto(s)
Factor Natriurético Atrial/fisiología , Riñón/fisiología , Humanos , Túbulos Renales/fisiología , Fragmentos de Péptidos/fisiología , Sodio/metabolismoRESUMEN
BACKGROUND: Several skin diseases and atopic disorders including Netherton syndrome and atopic dermatitis have been associated with mutations and deviations of expression of SPINK5, the gene encoding the human 15-domain serine proteinase inhibitor LEKTI. The biochemical mechanisms underlying this phenomenon have not yet been fully clarified. OBJECTIVES: To identify target proteinases of LEKTI important for processes of desquamation and inflammation of the skin which will enable the development of specific drugs. METHODS: The inhibitory activities of LEKTI domains 6 and 15 were tested on a number of commercially available serine proteinases and also on the purified kallikreins hK5 and hK7. In addition, recombinant hK5 was used. RESULTS: LEKTI domain 6 is a potent inhibitor of hK5 and hK7, whereas LEKTI domain 15 exhibits inhibitory activity on plasmin. hK5 and hK7 in particular are relevant to skin disorders. CONCLUSIONS: The inhibition of hK5 and hK7 by LEKTI domain 6 indicates an important regulatory role of LEKTI in processes of skin desquamation and inflammation, which may explain the severe pathological symptoms associated with abnormalities of SPINK5 and/or its expression. Thus, LEKTI represents a potential drug for the treatment of these disorders.
Asunto(s)
Proteínas Portadoras/farmacología , Serina Endopeptidasas/química , Inhibidores de Serina Proteinasa/farmacología , Piel/enzimología , Relación Dosis-Respuesta a Droga , Fibrinolisina/antagonistas & inhibidores , Humanos , Técnicas In Vitro , Calicreínas , Proteínas Inhibidoras de Proteinasas Secretoras , Inhibidor de Serinpeptidasas Tipo Kazal-5 , Enfermedades de la Piel/enzimologíaRESUMEN
BACKGROUND: Whereas recent studies underlie the fundamental importance of the CC chemokine receptor 3 (CCR3) for the recruitment of eosinophils in allergic diseases, controversial data exist about the relevance of CCR1 on eosinophils. Therefore, the purpose of this study was to investigate the expression and regulation of CCR1 on eosinophils. METHODS: Flow cytometric analysis of whole blood eosinophils and CD16-negative selected eosinophils from healthy nonatopic donors and from patients with atopic disorders was performed and CCR1 receptor internalization and re-expression were studied. RESULTS: Flow cytometric analysis of whole blood eosinophils revealed that 17.8% of the donors expressed high levels of CCR1 (CCR1high) and 82.2% low levels of CCR1 (CCR1low). A significant down-regulation of CCR1 was induced by 24 h preincubation of isolated eosinophils from CCR1high donors either with IL-3, CC chemokine ligand 3 (CCL3), CCL5, CCL7, or CCL13. Internalization experiments using eosinophils from CCR1high donors revealed that CCL5 is more effective to induce CCR1 internalization than CCL3. Whereas CCR1 re-expression after stimulation with CCL3 reached prestimulation levels (120 min: 81.3% relative CCR1 surface expression) CCL5 induced a prolonged CCR1 internalization (120 min: 15.7%). CONCLUSIONS: This study demonstrates a distinct pattern of CCR1 internalization and re-expression in human eosinophils between CCL3 and CCL5, as CCL5 induces a prolonged CCR1 internalization and the basic value is not reached after 24 h. Since prolonged receptor internalization plays a central role in chemokine-mediated inhibition of receptor function, CCR1 seems to be an attractive target on human eosinophils for chemokine receptor blockade besides CCR3.
Asunto(s)
Quimiocinas CC/farmacología , Eosinófilos/metabolismo , Receptores de Quimiocina/metabolismo , Quimiocina CCL5 , Humanos , Hipersensibilidad Inmediata , Receptores CCR1 , Proteínas Recombinantes/farmacología , Factores de Tiempo , Regulación hacia Arriba/inmunologíaRESUMEN
The orphan receptor ChemR23 is a G-protein coupled receptor (GPCR) with homology to neuropeptide and chemoattractant receptors. Tazarotene, a synthetic retinoid activating retinoic acid receptor (RAR), up-regulates tazarotene-induced gene-2 (TIG2). The function and molecular target of this protein are now described. By means of reverse pharmacology screening using a peptide library generated from human hemofiltrate, we have isolated and identified TIG2 as the natural ligand of ChemR23 and report the specific molecular form of the bioactive, circulating TIG2, representing the amino-acid residues 21 to 154 of the 163 amino acid-containing prepropeptide. Based on the expression pattern of ChemR23 and TIG2, the physiological role in bone development, immune and inflammatory responses and the maintenance of skin is now being investigated.
Asunto(s)
Ácidos Nicotínicos/genética , Receptores de Quimiocina/metabolismo , Receptores de Ácido Retinoico/sangre , Receptores de Ácido Retinoico/genética , Secuencia de Aminoácidos , Animales , Células CHO , Calcio/análisis , Calcio/metabolismo , Cricetinae , Fluorometría/métodos , Hemofiltración , Humanos , Ligandos , Datos de Secuencia Molecular , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/química , Receptores de Quimiocina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , TransfecciónRESUMEN
A large and steadily growing subfamily of antimicrobially active peptides of animals and plants is formed by the defensins, which are highly disulfide-bonded, cationic peptides with a molecular mass of about 4 kDa. The synthesis of the human beta-defensins 1 and 2 (hBD-1, hBD-2) as well as of the novel murine beta-defensins 7 and 8 (mBD-7 and mBD-8) is reported. The peptides were synthesized by solid-phase peptide synthesis using fluorenylmethoxycarbonyl chemistry. The linear products were oxidized in the presence of the cysteine/cystine redox system to the biologically active molecules. The correct disulfide connectivity of the resulting cyclic products was partly verified by mass spectrometry and sequence analysis of the fragments obtained after tryptic cleavage. In addition, the recently discovered antimicrobially active human peptide LEAP-1/hepcidin, which contains four disulfide bonds, was successfully synthesized and subsequently oxidized. For Liver-expressed anti microbial peptide (LEAP)-1/hepcidin and hBD-1, the identity of native and synthetic peptides was demonstrated by high-pressure liquid chromatography and capillary electrophoretic analysis. The general synthetic procedure is suitable to rapidly perform the total chemical synthesis of novel fully bioactive defensins, which are expected to be identified soon, as well as of structurally modified analogs.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/síntesis química , Proteínas/síntesis química , beta-Defensinas/síntesis química , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Cromatografía Líquida de Alta Presión , Disulfuros/química , Hepcidinas , Humanos , Ratones , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/química , Pliegue de Proteína , Proteínas/química , beta-Defensinas/químicaRESUMEN
Proteinases are involved in specific and non-specific proteolytic reactions, and participate in many pathophysiological processes. Normally, they are regulated by endogenously produced proteinase inhibitors which, thus, represent lead structures for the development of therapeutics. We succeeded in partially isolating and cloning a novel human serine proteinase inhibitor which, according to its structure and the expression pattern of the corresponding gene, was termed lympho-epithelial Kazal-type-related inhibitor (LEKTI). This inhibitor is of special interest because it exhibits an extraordinarily large number of 15 potentially inhibitory domains and is of pathophysiological importance for the severe congenital disease Netherton syndrome. Here, we review the as yet known data on protein structure, biochemical properties, genomic organization and gene expression. Furthermore, the relevance of LEKTI for several disorders pointing out its possible future therapeutic value, is discussed.
Asunto(s)
Proteínas Portadoras , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/fisiología , Animales , Expresión Génica , Humanos , Eritrodermia Ictiosiforme Congénita/genética , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína/fisiología , Proteínas Inhibidoras de Proteinasas Secretoras , Homología de Secuencia de Aminoácido , Inhibidor de Serinpeptidasas Tipo Kazal-5 , Inhibidores de Serina Proteinasa/aislamiento & purificación , SíndromeRESUMEN
Previous studies have shown the implication of beta-defensins in host defense of the human body. The human beta-defensins 1 and 2 (hBD-1, hBD-2) have been isolated by biochemical methods. Here we report the identification of a third human beta-defensin, called human beta-defensin 3 (hBD-3; cDNA sequence, Genbank accession no. AF295370), based on bioinformatics and functional genomic analysis. Expression of hBD-3 is detected throughout epithelia of many organs and in non-epithelial tissues. In contrast to hBD-2, which is upregulated by microorganisms or tumor necrosis factor-alpha (TNF-alpha), hBD-3 expression is increased particularly after stimulation by interferon-gamma. Synthetic hBD-3 exhibits a strong antimicrobial activity against gram-negative and gram-positive bacteria and fungi, including Burkholderia cepacia. In addition, hBD-3 activates monocytes and elicits ion channel activity in biomembranes, specifically in oocytes of Xenopus laevis. This paper also shows that screening of genomic sequences is a valuable tool with which to identify novel regulatory peptides. Human beta-defensins represent a family of antimicrobial peptides differentially expressed in most tissues, regulated by specific mechanisms, and exerting physiological functions not only related to direct host defense.
Asunto(s)
Antibacterianos/farmacología , Membrana Celular/metabolismo , Quimiotaxis/fisiología , Macrófagos/fisiología , beta-Defensinas/metabolismo , beta-Defensinas/farmacología , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Antibacterianos/metabolismo , Burkholderia cepacia/efectos de los fármacos , Línea Celular , Células Epiteliales/fisiología , Regulación de la Expresión Génica , Humanos , Canales Iónicos/metabolismo , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Oocitos , Técnicas de Placa-Clamp , Alineación de Secuencia , Xenopus laevis , beta-Defensinas/química , beta-Defensinas/genéticaRESUMEN
Defensins are cationic and cysteine-rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian beta-defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human beta-defensins, hBD-1 and hBD-2, as well as those of two novel murine defensins, termed mBD-7 and mBD-8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent-exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that beta-defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the beta-defensin-specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because beta-defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.
Asunto(s)
beta-Defensinas/química , Secuencia de Aminoácidos , Animales , Cromatografía , Secuencia Conservada , Cristalografía por Rayos X , Disulfuros , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
We have previously isolated from human hemofiltrate an N-terminally truncated form of the hemofiltrate CC chemokine 1 (HCC-1), and characterized HCC-1[9-74] as a strong agonist of CCR1, CCR5, and to a lower extent CCR3. In this study, we show that conditioned media from human tumor cell lines PC-3 and 143B contain proteolytic activities that convert HCC-1 into the [9-74] form. This activity was fully inhibited by inhibitors of urokinase-type plasminogen activator (uPA), including PA inhibitor-1, an anti-uPA mAb, and amiloride. Pure preparations of uPA processed HCC-1 with high efficiency, without further degrading HCC-1[9-74]. Plasmin could also generate HCC-1[9-74], but degraded the active product as well. The kinetics of HCC-1 cleavage by uPA and plasmin (Michaelis constant, K(m), of 0.76 +/- 0.4 microM for uPA, and 0.096 +/- 0.05 microM for plasmin; catalytic rate constant, k(cat): 3.36 +/- 0.96 s(-1) for uPA and 6 +/- 3.6 s(-1) for plasmin) are fully compatible with a role in vivo. The activation of an abundant inactive precursor into a broad-spectrum chemokine by uPA and plasmin directly links the production of uPA by numerous tumors and their ability to recruit mononuclear leukocytes, without the need for the transcriptional activation of chemokine genes.
Asunto(s)
Quimiocinas CC/metabolismo , Fibrinolisina/farmacología , Proteínas de Neoplasias/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Aequorina/metabolismo , Quimiotaxis de Leucocito , Medios de Cultivo Condicionados , Fibrinolisina/química , Fibrinolisina/aislamiento & purificación , Humanos , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/fisiología , Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores CCR5/agonistas , Células Tumorales Cultivadas/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/aislamiento & purificación , Activador de Plasminógeno de Tipo Uroquinasa/fisiologíaRESUMEN
The hemofiltrate CC chemokines CCL14a (formerly HCC-1), CCL14b (formerly HCC-3), and CCL15 (formerly HCC-2) are encoded by mono- as well as bicistronic transcripts from a tandem gene arrangement on human chromosome 17q11.2. The transcription and splicing into several mono- and bicistronic transcripts of this gene complex are unique for human genes. No corresponding mechanism is known in nonprimate mammalian species such as mice and rats. The extremely high concentration of CCL14a in human plasma is exceptional for chemokines and led to the identification of this chemokine. Several molecular forms of CCL14a have been isolated and investigated. The mature propeptide CCL14a(1-74) is a low-affinity agonist of CCR1 which is converted to a high-affinity agonist of CCR1 and CCR5 on proteolytic processing by serine proteases. In contrast, CCL15 is characterized using molecular forms deduced from the mRNA/cDNA and shown to activate cells via CCR1 and CCR3, also dependent on the amino-terminal length. Hemofiltrate CC chemokines are chemoattractants for different types of leukocytes including monocytes, eosinophils, T cells, dendritic cells, and neutrophils. In this review, we emphasize the genomic organization, expression patterns, and biochemical properties of CCL14a, CCL14b, and CCL15. We report results of significance for the development of therapeutic strategies, especially concerning HIV infection and inflammatory diseases.
Asunto(s)
Quimiocinas CC/genética , Quimiocinas CC/fisiología , Monocinas , Secuencia de Aminoácidos , Fenómenos Fisiológicos Sanguíneos , Cromosomas Humanos Par 17 , Infecciones por VIH/terapia , Humanos , Proteínas Inflamatorias de Macrófagos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas , Homología de Secuencia de AminoácidoRESUMEN
In a model of acute pulmonary hypertension in intact rabbits, we investigated the vasodilatory potency of intravascularly administered urodilatin, a renal natriuretic peptide type A known to stimulate particulate guanylate cyclase. Urodilatin infusion was performed in the absence and presence of the phosphodiesterase (PDE) type 5 inhibitor dipyridamole. Stable pulmonary hypertension was evoked by continuous infusion of the thromboxane mimetic U46619, resulting in approximate doubling of the pulmonary artery pressure (PAP). When infused as sole agents, both urodilatin and dipyridamole dose-dependently attenuated the pulmonary hypertension, with doses for a 20% decrease in PAP being 30 ng/kg min for urodilatin and 10 microg/kg min for dipyridamole. A corresponding decrease in systemic arterial pressure (SAP) was noted to occur in response to both agents. Sequential intravenous administration of a subthreshold dose of dipyridamole (1 microg/kg min), which per se did not affect pulmonary and systemic hemodynamics, and a standard dose of urodilatin (30 ng/kg min) resulted in a significant amplification of both the PAP and the SAP decrease in response to the natriuretic peptide. At the same time, manifold enhanced plasmatic cyclic guanosine monophosphate (cGMP) levels were detected. Aerosolized dipyridamole also dose-dependently attenuated pulmonary hypertension, with only 1 microg/kg min being sufficient for a 20% decrease in PAP, with no SAP decline. Preceding administration of subthreshold aerosolized dipyridamole (50 ng/kg min) did, however, cause only a minor amplification of the pulmonary vasodilatory response to a subsequently infused standard dose of urodilatin. In conclusion, this is the first study to show that urodilatin does possess vasodilatory potency in the pulmonary circulation, and enhanced plasma levels of cGMP and synergy with the PDE5 inhibitor dipyridamole both strongly suggest that this effect proceeds via guanylate cyclase activation. The effect of infused urodilatin is, however, not selective for the pulmonary vasculature, as the systemic vascular resistance declines in a corresponding fashion.
Asunto(s)
Factor Natriurético Atrial/farmacología , Dipiridamol/farmacología , Guanilato Ciclasa/metabolismo , Hipertensión Pulmonar/prevención & control , Fragmentos de Péptidos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/toxicidad , Administración por Inhalación , Animales , Factor Natriurético Atrial/administración & dosificación , Presión Sanguínea/efectos de los fármacos , GMP Cíclico/sangre , Dipiridamol/administración & dosificación , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/fisiopatología , Infusiones Intravenosas , Fragmentos de Péptidos/administración & dosificación , Inhibidores de Fosfodiesterasa/administración & dosificación , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiopatología , Circulación Pulmonar/efectos de los fármacos , ConejosAsunto(s)
Antiinfecciosos/farmacología , beta-Defensinas/farmacología , Secuencia de Aminoácidos , Antibacterianos , Antiinfecciosos/química , Factores Quimiotácticos , ADN Complementario/química , Sinergismo Farmacológico , Células Epiteliales/metabolismo , Escherichia coli/efectos de los fármacos , Expresión Génica , Humanos , Pulmón/metabolismo , Datos de Secuencia Molecular , Monocitos , Infecciones por Pseudomonas/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/efectos de los fármacos , Staphylococcus/efectos de los fármacos , Infecciones Estreptocócicas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , beta-Defensinas/química , beta-Defensinas/genéticaRESUMEN
Endostatin, a C-terminal fragment of collagen XVIII, is a promising protein drug which is in development for cancer therapy due to its anti-angiogenic activity. Although several endogenous molecular forms of human endostatin differing in their N-terminal length and their post-translational modifications (18.5-22 kDa) have been discovered, only one recombinant form of 20 kDa is used in clinical trials. This protein, recombinantly expressed in Pichia pastoris, contains four cysteines forming two disulfide bonds (Cys1-Cys4 and Cys2-Cys3). In contrast, there are conflicting data about the disulfide pattern of endogenous material. This report presents the disulfide analyses of both the endogenous circulating endostatins isolated from human hemofiltrate and the recombinant protein. The determination of the disulfide pattern was performed by Edman degradation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap mass spectrometry (ESI-ITMS) performed in the off-line nanospray mode. All native and recombinant endostatins exhibited a Cys1-Cys4 (Cys(162)-Cys(302)) and Cys2-Cys3 (Cys(264)-Cys(294)) linkage. For a clear discussion of fragmented disulfide-bridged peptide chains obtained from MS(n) experiments, a modified general nomenclature is proposed.