RESUMEN
Silver fir (Abies alba) bark extract contains a mixture of bioactive polyphenols. We tested their effectiveness in the treatment of psoriasis in order to further investigate the potential topical anti-inflammatory activity of polyphenols by means of a randomized, double-blind, placebo-controlled add-on clinical trial, after having examined their ability to downregulate the expression of IL-1ß cytokine in monocyte/macrophage primary cell culture. 61 patients with mild psoriasis met the inclusion criteria and were willing to comply with protocol requirements, were enrolled in the study. The severity of the disease was measured by psoriasis area severity index (PASI). Treatment efficacy was evaluated by assessing erythema (E, 0 to 4-point scale), desquamation (D, 0 to 4-point scale) and induration (I, 0 to 4-point scale) of lesions before and after the treatment. All patients enrolled in the study had symmetrical psoriasis plaques on the skin. All patients received O/V ointment with 2% of silver fir bark extract and/or placebo, respectively. We compared medications by right/left intra-patient comparison, so that the control group was always contralateral of the tested one. Location of the tested or control site was randomised, using a computer-generated randomisation schedule. Silver fir extract was well-tolerated. A superiority of active treatment above placebo, based on the clinical investigational PASI score system was observed by 15 % in all volunteers and in 40% regarding the improvement of psoriasis on elbows. However, statistical analysis showed no significant differences between placebo and active treatment with the extract from silver fir bark (p < 0.05).
Asunto(s)
Abies/química , Extractos Vegetales/farmacología , Polifenoles/farmacología , Psoriasis/tratamiento farmacológico , Administración Cutánea , Adulto , Anciano , Anciano de 80 o más Años , Antiinflamatorios/administración & dosificación , Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/farmacología , Células del Cúmulo , Fármacos Dermatológicos/administración & dosificación , Fármacos Dermatológicos/aislamiento & purificación , Fármacos Dermatológicos/farmacología , Método Doble Ciego , Femenino , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Pomadas , Corteza de la Planta , Extractos Vegetales/administración & dosificación , Polifenoles/administración & dosificación , Polifenoles/aislamiento & purificación , Psoriasis/patología , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Adulto JovenRESUMEN
Identification of allergen epitopes is a key component in proper understanding of the pathogenesis of type I allergies, for understanding cross-reactivity and for the development of mimotope immunotherapeutics. Phage particles have garnered recognition in the field of molecular allergology due to their value not only in competitive immunoscreening of peptide libraries but also as immunogenic carriers of allergen mimotopes. They integrate epitope discovery technology and immunization functions into a single platform. This article provides an overview of allergen mimotopes identified through the phage display technique. We discuss the contribution of phage display peptide libraries in determining dominant B-cell epitopes of allergens, in developing mimotope immunotherapy, in understanding cross-reactivity, and in determining IgE epitope profiles of individual patients to improve diagnostics and individualize immunotherapy. We also discuss the advantages and pitfalls of the methodology used to identify and validate the mimotopes.
Asunto(s)
Alérgenos/inmunología , Técnicas de Visualización de Superficie Celular , Mapeo Epitopo , Hipersensibilidad/inmunología , Hipersensibilidad/terapia , Inmunoterapia , Biblioteca de Péptidos , Alérgenos/química , Animales , Reacciones Cruzadas/inmunología , Mapeo Epitopo/métodos , Humanos , Hipersensibilidad/diagnóstico , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Inmunoglobulina E/inmunologíaRESUMEN
In the recent decades, great progress has been made in the development of ghrelin receptor ligands. The discovery of the first in vitro only active peptide growth hormone secretagogue derived from Met-enkephalin was the foundation for later discoveries of the receptor and the endogenous ligand ghrelin. Since then, the scope of peptides, peptidomimetics, and small-molecules targeting the ghrelin receptor, GHS-R1a, has expanded dramatically. Numerous agonists have been tested in animals and several in humans, and a handful have progressed to clinical trials for indications such as growth hormone release, gastric emptying, and cachexia. However, with the exception of the approval of GHRP-2 for diagnostic purposes in Japan, none of the candidates have been successfully introduced into the market. More recently, the attention of researchers has been concentrated on developing antagonists and inverse agonists for pharmacological treatment of the ever-expanding obese and overweight population. In this review, we describe the development of GHS-R1a targeting agonists, antagonists, and inverse agonists. We focus on current and completed clinical trials and the therapeutic potential of currently available ligands.
Asunto(s)
Ensayos Clínicos como Asunto , Péptidos/metabolismo , Peptidomiméticos/metabolismo , Receptores de Ghrelina/agonistas , Receptores de Ghrelina/antagonistas & inhibidores , Animales , Humanos , Ligandos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The aim of this study was to test the extract from silver fir wood (Belinal) on the reduction of the blood glucose concentrations after consumption of a standard meal. 31 healthy participants consumed 100 g of white bread 4 times (with 1 week washout period, consequently) concomitantly with a capsule of Belinal, capsule of chestnut wood extract, placebo or acarbose (active control). Glucose and insulin in the blood were measured before and after the meal. The area under the curve of glucose concentration in blood after the meal was 35 % lower when Belinal was added compared with the placebo group (p = 0.019). Acarbose lowered the area for 43 % (p = 0.002). By this, we proved that the effect of Belinal might be beneficial for prevention of diabetes. This is the first study that provides a scientific rationale for use of silver fir wood extract as food supplement for reduction of health risks connected to type 2 diabetes mellitus.
Asunto(s)
Abies/química , Glucemia/metabolismo , Hipoglucemiantes/farmacología , Extractos Vegetales/farmacología , Adulto , Estudios Cruzados , Método Doble Ciego , Femenino , Índice Glucémico , Voluntarios Sanos , Humanos , Insulina/sangre , Masculino , Periodo Posprandial/efectos de los fármacos , Adulto JovenRESUMEN
The neutralisation of circulating ghrelin, the only known peripheral orexigenic peptide hormone, is a promising approach for the pharmacological treatment of obesity. To select peptides with an affinity towards ghrelin, 4 selection procedures were carried out with random peptide phage display libraries Ph.D.-7 and Ph.D.-12. Due to the absence of a common consensus motif, a stepwise elimination approach was used. The pool of selected peptides displaying phage clones was thoroughly examined to remove any potential target-unrelated peptides. The affinity of the remaining phage clones for ghrelin was tested with ELISA. An analysis of the binding properties revealed four-phage displayed peptides that bind to ghrelin with moderate affinity, with ADTVPRH and MEMKKTHPVLGA being the most specific. Additional advantage of peptide MEMKKTHPVLGA is an indication of binding to octanoyl group on N-terminal part of ghrelin, involved in receptor interaction. Hence peptide MEMKKTHPVLGA represents the most suitable lead for further investigation.
Asunto(s)
Ghrelina/metabolismo , Hambre , Péptidos/metabolismo , Secuencia de Aminoácidos , Células Clonales , Ensayo de Inmunoadsorción Enzimática , Reacciones Falso Positivas , Humanos , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Unión ProteicaRESUMEN
Lactic acid bacteria (LAB) have become increasingly studied over the last two decades as potential delivery systems for various biological molecules to the gastrointestinal tract. This article presents an overview of characteristics of LAB as delivery systems and of the applications which have already been developed. The majority of LAB strains are able to survive the intestinal passage and some are also able to persist and colonize the intestine. Several strains were in fact described as members of the human commensal flora. They can interact with their host and are able to deliver large molecular weight biomolecules across the epithelium via M-cells or dendritic cells. The most widely applied LAB species has been Lactococcus lactis; however species from genus Lactobacillus are gaining popularity and the first examples from genus Bifidobacterium are starting to emerge. Bacteria are mostly applied live and enable continuous delivery of the biomolecules. However, killed bacteria (e.g. gram-positive enhancer matrix), with bound biomolecules or as adjuvants, are also being developed. The techniques for genetic modification of LAB are well known. This review focuses on the delivery of recombinant proteins and DNA, which can cause either local or systemic effects. We divide recombinant proteins into antigens and therapeutic proteins. Delivery of antigens for the purpose of vaccination represents the most abundant application with numerous successful demonstrations of the efficacy on the animal model. Therapeutic proteins have mostly been developed for the treatment of the inflammatory bowel disease, by the delivery of anti-inflammatory cytokines, or downregulation of proinflammatory cytokines. Delivery of allergens for the modulation of allergic disorders represents the second most popular application of therapeutic proteins. The delivery of DNA by LAB was demonstrated and offers exciting opportunities, especially as a vaccine. New discoveries may eventually lead to the transition of LAB as delivery systems in clinical practice.
Asunto(s)
Bacterias/metabolismo , Factores Biológicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Ácido Láctico/metabolismo , Factores Biológicos/uso terapéutico , ADN/administración & dosificación , ADN/uso terapéutico , Terapia Genética/métodos , Humanos , Intestinos/microbiología , Lactobacillus/metabolismo , Lactococcus/metabolismo , Proteínas/administración & dosificación , Proteínas/uso terapéutico , Vacunas/administración & dosificaciónRESUMEN
The aim of the present study was to evaluate the effect of long-term swimming exercise in conjunction with a high fat mixed lipid (HFML) diet on colon cancer (CC) development and lipid peroxidation in the large bowel. We used forty male Wistar rats, which were randomly divided into one control group and four cancer groups: sedentary and swimming groups fed a standard diet (LFCO) and sedentary and swimming groups fed an HFML diet. Corticosterone was determined during the experiment. After 6 months of swimming, the rats were sacrificed and blood, heart, liver, muscle and large bowel were taken for determining the activity of serum enzymes, antioxidant capacity and CC development. The results demonstrate that exercise has a protective role in CC development. Attenuated development of CC and increased levels of malondialdehyde (MDA) in the large bowel of exercised rats show that one of the protective effects of exercise on developing CC is induction of oxidative stress. However, in terms of the combined effects of dietary fat and exercise, our results indicate that the protective role of exercise on CC development is significantly depressed by an HFML diet. An HFML diet significantly reduced the protective influence of exercise on colon carcinogenesis in rats and affected the degree of peroxidation in the large bowel during exercise, as well as concentrations of serum enzymes (LDH, α-HBDH, CK, ALT and AST). Our results indicate that an HFML diet, which reflects the composition of a Western style diet, is a significant modifier of the protective effects of exercise on CC development in rats.
Asunto(s)
Antioxidantes/metabolismo , Neoplasias del Colon/metabolismo , Dieta Alta en Grasa , Estrés Oxidativo , Natación , 1,2-Dimetilhidrazina , Animales , Peso Corporal , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/patología , Peroxidación de Lípido , Masculino , Tamaño de los Órganos , Ratas , Ratas Wistar , Carga TumoralRESUMEN
AIMS: To evaluate brazzein production in Lactococcus lactis using the nisin-controlled expression (NICE) system. The approach is through analysis of different plasmid/strain combinations. METHODS AND RESULTS: Two plasmid/strain combinations of the NICE system were used in brazzein expression: L. lactis NZ9000 harbouring plasmid pNZ8148, and L. lactis IL1403 harbouring plasmid pMSP3545. The former combination proved superior, with a >800-fold increase in His-tagged brazzein expression (to 1.65 mg l(-1) of fermentation broth), comparable to expression levels in Escherichia coli. Improved expression resulted in a minor increase in secretion to the medium with the use of the Usp45 signal peptide. The yield of wild-type brazzein corresponded to that of His-tagged brazzein. Wild-type brazzein was partially soluble and low-intensity sweetness was detected. CONCLUSIONS: The plasmid/strain combination of the NICE system has a significant impact on the expression of brazzein where a >800-fold increase was achieved. The greatly increased expression of brazzein resulted in minor improvement in secretion and low-intensity sweetness. SIGNIFICANCE AND IMPACT OF THE STUDY: The choice of the plasmid/strain combination of the NICE system was shown to be of extreme importance in brazzein expression.
Asunto(s)
Expresión Génica , Ingeniería Genética/métodos , Lactococcus lactis/genética , Nisina/genética , Proteínas de Plantas/genética , Plásmidos/genética , Lactococcus lactis/metabolismo , Nisina/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Several animal models of breast cancer have been developed to study various aspects of breast cancer biology. Substantial evidence suggests that the N-methylnitrosourea (MNU) animal model mimics human breast cancer in many respects. It has therefore been used extensively to evaluate preventive and therapeutic agents for human breast cancer. Chemically induced rodent models are also suitable for studying malignant progression. Recently, Liska et al. [7] established two protocols of MNU administration depending on the animal's age and number of applications of carcinogen, with the aim of investigating the advanced stages of mammary gland tumours. We used the same protocol as Liska but have obtained substantially different results. These results are presented and discussed in the frame of suggested key drawbacks of the MNU induced breast cancer rat model, as a contribution to the debate about the suitability of that model for evaluating preventive and therapeutic agents.
Asunto(s)
Modelos Animales de Enfermedad , Neoplasias Mamarias Experimentales/patología , Metilnitrosourea/toxicidad , Animales , Carcinógenos/toxicidad , Femenino , Neoplasias Mamarias Experimentales/inducido químicamente , Invasividad Neoplásica , Ratas , Ratas Sprague-DawleyRESUMEN
Anthracycline antibiotics are among the most effective and commonly used anticancer drugs. Unfortunately, their clinical use is restricted by dose-dependent toxicity. Doxorubicin is an anthracycline antibiotic and cytotoxic (antineoplastic) agent. It is commonly used against ovarian, breast, lung, uterine and cervical cancers, Hodgkin's disease, soft tissue and primary bone sarcomas, as well against in several other cancer types. It has been shown that free radicals are involved in doxorubicin-induced toxicity. Doxorubicin causes the generation of free radicals and the induction of oxidative stress, associated with cellular injury. This review illustrates recent applications of different natural products, drugs, drug delivery systems, and approaches for protection against doxorubicin-induced toxicity (2006-present).
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Antineoplásicos/toxicidad , Doxorrubicina/toxicidad , Neoplasias/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos/uso terapéutico , Antineoplásicos/uso terapéutico , Antioxidantes/uso terapéutico , Sistemas de Liberación de Medicamentos , Radicales Libres , Humanos , Liposomas/química , Nanopartículas/química , Estrés OxidativoRESUMEN
AIMS: To improve the production of sweet-tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions. METHODS AND RESULTS: The nisin-controlled expression system was used for brazzein expression. The concentration of nisin for induction and the optical density (OD) at induction were therefore optimized, together with growth conditions (medium composition, pH, aerobic growth in the presence of hemin). Brazzein was assayed with ELISA on Ni-NTA plates and Western blot. Use of the M-17 medium, containing 2.5% glucose, anaerobic growth at pH 5.9 and induction with 40 ng ml(-1) nisin at OD 3.0 led to an approx. 17-fold increase in brazzein per cell production compared to non-optimized starting conditions. Aerobic growth in the presence of hemin did not increase the production. CONCLUSIONS: Considerable increase in brazzein per cell production was obtained at optimized fermentation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimized growth conditions could be used in application of brazzein expression in L. lactis. The importance of pH and OD at induction contributes to the body of knowledge of optimal recombinant protein expression in L. lactis. The new assay for brazzein quantification was introduced.
Asunto(s)
Fermentación , Lactococcus lactis/metabolismo , Proteínas de Plantas/metabolismo , Plásmidos/metabolismo , Western Blotting , Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Concentración de Iones de Hidrógeno , Lactococcus lactis/efectos de los fármacos , Lactococcus lactis/genética , Nisina/farmacología , Proteínas de Plantas/genética , Plásmidos/genética , Edulcorantes/metabolismoRESUMEN
The therapeutic utility of the anthracycline antibiotic doxorubicin is limited due to its cardiotoxicity. Our aim was to investigate the efficacy of fullerenol C(60)(OH)(24) in preventing single, high-dose doxorubicin-induced cardiotoxicity in rats with malignant neoplasm. Experiment was performed on adult female Sprague Dawley rats with chemically induced mammary carcinomas. The animals were sacrificed two days after the application of doxorubicin and/or fullerenol, and the serum activities of CK, LDH and alpha-HBDH, as well as the levels of MDA, GSH, GSSG, GSH-Px, SOD, CAT, GR, and TAS in the heart, were determined. The results obtained from the enzymatic activity in the serum show that the administration of a single dose of 8 mg/kg in all treated groups induces statistically significant damage. There are significant changes in the enzymes of LDH and CK (p < 0.05), after an i.p. administration of doxorubicin/fullerenol and fullerenol. Comparing all groups with untreated control group, point to the conclusion that in the case of a lower alpha-HBDH/LDH ratio, results in more serious the liver parenchymal damage. The results revealed that doxorubicin induced oxidative damage and that the fullerenol antioxidative influence caused significant changes in MDA, GSH, GSSG, GSH-Px, SOD, CAT, GR, and TAS level in the heart (p < 0.05). Therefore, it is suggested that fullerenol might be a potential cardioprotector in doxorubicin-treated individuals.
Asunto(s)
Doxorrubicina/toxicidad , Fulerenos/farmacología , Corazón/efectos de los fármacos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Animales , Femenino , Glutatión/sangre , Disulfuro de Glutatión/sangre , L-Lactato Deshidrogenasa/sangre , Masculino , Malondialdehído/sangre , Neoplasias Mamarias Experimentales/metabolismo , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
We have studied a sedative tea made of Valerianae radix (Valeriana officinalis L.), Lupuli strobuli (Humulus lupulus L.), Melissae folium (Melissa officinalis L.) and Menthae piperitae folium (Mentha piperita L.). In order to identify the constituent drugs a method was established involving amplification of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA on the basis of restriction analysis and real-time PCR. ITS regions of individual drugs were amplified and sequenced. Restriction analysis was performed with selected restriction endonucleases Nae I, PshA I and Xcm I. Real-time PCR was carried out, using primers specifically designed for each individual herbal drug. Real-time PCR proved to be a method for identifying individual herbal drugs in a tea mixture with a single DNA extraction in a single PCR run, since its limit of detection is lower than that for restriction analysis.
Asunto(s)
ADN de Plantas/química , ADN de Plantas/genética , Plantas Medicinales/química , Plantas Medicinales/genética , Dermatoglifia del ADN , ADN Ribosómico/química , Humulus/química , Humulus/genética , Melissa/química , Melissa/genética , Mentha/química , Mentha/genética , Extractos Vegetales/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Valeriana/química , Valeriana/genéticaRESUMEN
Interference with fat hydrolysis results in the reduced use of ingested lipids. Inhibition of pancreatic lipase reduces the efficiency of fat absorption in the small intestine and thereby initiates modest long-term reduction in body weight. In an attempt to select peptides with affinity for the surface of pancreatic lipase and potential inhibitory activity, a random, cyclic heptapeptide phage-displayed library was used. Five independent selections, differing in elution step, were performed. In three selection protocols, a sequential elution strategy was applied in anticipation of improving the selection of high-affinity clones. Four heptapeptides with the highest affinity, seemingly for pancreatic lipase, were selected, synthesized, and characterized for their capacity to inhibit enzyme function. Although no clear consensus among the sequenced peptides was found, one of the selected peptides inhibited pancreatic lipase with an apparent inhibition constant of 16 muM.
Asunto(s)
Lipasa/antagonistas & inhibidores , Biblioteca de Péptidos , Secuencia de Aminoácidos , Técnicas Químicas Combinatorias/métodos , Grasas de la Dieta/metabolismo , Digestión/efectos de los fármacos , Páncreas/enzimología , Péptidos/aislamiento & purificaciónRESUMEN
Oxidative instability of the hydroxy methylglutaryl Co-A reductase inhibitor lovastatin in the solid state and stabilization with natural antioxidants (ascorbic acid, rutin, gallic acid, quercetin and caffeic acid) was investigated. Lovastatin in the solid state and binary mixtures with 10% (w/w), 25% (n/n), 12.5% (n/n) and 6.3% (n/n) of each of the antioxidants were prepared. Oxidation experiments were performed on the scanning calorimeter using dynamic oxygen atmosphere. The amount of non-oxidized lovastatin was determined using HPLC. The results of the experiments have shown that lovastatin is unstable to oxidation under higher temperatures and in the presence of oxygen, and that some antioxidants markedly stabilize the drug. The most significant antioxidative effect was seen with caffeic acid and rutin, followed by gallic acid and quercetin. Ascorbic acid was only moderately effective. The results prove that flavonoids do have significant antioxidative potential. This phenomenon can be used to improve oxidative stability of drugs such as lovastatin which are sensitive to the presence of oxygen.
Asunto(s)
Anticolesterolemiantes/química , Antioxidantes/química , Lovastatina/química , Estabilidad de Medicamentos , Excipientes , Lípidos/química , Oxidación-Reducción , Comprimidos , TemperaturaRESUMEN
A capillary zone electrophoretic (CZE) method for the determination of usnic acid is described for the first time. Usnic acid is an antibiotic substance from lichens. Due to its low solubility in water, a high content of methanol in CZE buffer is required. Because of the methanol in the buffer, the electroosmotic flow velocity was lower than the electrophoretic mobility of usnic acid. Accordingly, the use of reversed-polarity (with the anode on the detector side of the capillary) was necessary. The optimal buffer composition was 50 mM NaOH, 20 mM acetic acid and 5% water in methanol. The detection limit of UV detector at 290 nm for usnic acid in the injected extract was 3.5 mg/L and the relative standard deviation of the normalized peak area was 3.3% at 250 mg/L.
Asunto(s)
Antiinfecciosos/análisis , Benzofuranos/análisis , Electroforesis Capilar/métodos , Electroforesis Capilar/normas , Estructura Molecular , Factores de TiempoRESUMEN
Thrombin is the key serine proteinase of the coagulation cascade and therefore a suitable target for inhibition of blood coagulation. A number of pharmacologically active secondary metabolites from mushrooms have already been isolated, thus providing the rationale for screening for new thrombin inhibitors in mushrooms. In this study, inhibitory activities of mushroom extracts on thrombin and trypsin were measured using the chromogenic substrates H-D-phenylalanine-L-pipecolyl-L-arginine-paranitroaniline dihydrochloride (S-2238) for thrombin and N-benzoyl-D,L-Arg-p-nitroanilide (BAPNA) for trypsin. The inhibitory activities of extracts from 95 Basidiomycete species have been determined. The majority of samples inhibited trypsin and thrombin with various potencies; however, some extracts showed no activity against one or both of the enzymes. An aqueous extract of Gleophyllum odoratum exhibited high inhibitory activity on both thrombin and trypsin (72 and 60%, respectively), while extracts of Clitocybe gibba, Amanita virosa, Cantharellus lutescens, Suillus tridentinus, Hypoloma fasciculare and Lactarius badiosanguineus considerably inhibited thrombin (49, 48, 36, 34, 32 and 31%, respectively) and showed no inhibitory activity on trypsin. The results at this point are promising for further research with the objective of finding an effective and safe thrombin inhibitor.
Asunto(s)
Agaricales/química , Basidiomycota/química , Trombina/antagonistas & inhibidores , Benzoilarginina-Nitroanilida/metabolismo , Compuestos Cromogénicos/metabolismo , Dipéptidos/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Especificidad de la Especie , Trombina/metabolismo , Tripsina/metabolismo , Inhibidores de Tripsina/aislamiento & purificaciónRESUMEN
Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine. This has led to the conclusion that a basic residue at this position, which interacts with the active-site aspartates, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p. To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pH 2 and 4.7. All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with Glu36p showed higher activation rates than the two Lys36p forms. Glu36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae. A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porcine pepsinogen A [Richter, C., Tanaka, T., Koseki, T. & Yada, R.Y. (1999) Eur. J. Biochem. 261, 746-752] can be reconciled with those for prochymosin. Lys/Arg36p is involved in stabilizing the propeptide-enzyme interaction, along with residues nearer the N-terminus of the propeptide, the sequence of which varies between species. The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former.
Asunto(s)
Quimosina/química , Precursores Enzimáticos/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Catálisis , Bovinos , Dicroismo Circular , Clonación Molecular , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Exones , Ácido Glutámico/química , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Lisina/química , Leche/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Pepsinógeno A/química , Péptidos/química , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Precursores de Proteínas/química , Proteínas Recombinantes/química , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Ovinos , Factores de TiempoRESUMEN
Equistatin (EI) is a cysteine protease inhibitor that was isolated from the sea anemone Actinia equina. It belongs to a recently discovered group of thyroglobulin type-I domain inhibitors called thyropins. Since native EI is found only in low amounts in the body of sea anemone and expression of recombinant EI in Escherichia coli yielded only 1 mg/liter of protein, we used the Pichia pastoris expression system to obtain higher yields. A cDNA encoding EI was inserted into pPIC9 vector and transformed into the P. pastoris, strain GS115. Clones expressing high levels of EI were selected from 48 transformants. Recombinant EI was produced in 2-liter shake flasks and recovered from the fermentation broth by affinity chromatography using CM-papain-Sepharose. SDS-PAGE and N-terminal sequence analysis revealed that EI was N-terminally intact and running at the expected molecular weight of 22 kDa. The equilibrium dissociation constants of EI with papain and bovine cathepsin D were determined and were found to be similar to the results for the native inhibitor. EI production was scaled up to a bench top fermentor with a 25 mg/liter yield of active EI.
Asunto(s)
Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/genética , Pichia/genética , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Reactores Biológicos , Western Blotting , Clonación Molecular , Inhibidores de Cisteína Proteinasa/biosíntesis , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Expresión Génica , Vectores Genéticos , Biosíntesis de Proteínas , Proteínas/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Transformación GenéticaRESUMEN
A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 m guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nm), cathepsin L (K(i) = 0.41 nm), cathepsin B (K(i) = 0.48 micrometer), and bromelain (K(i) = 0.16 micrometer) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).