RESUMEN
In the last two decades we have witnessed sizable progress in defining the role of gastrointestinal signals in the control of glucose and energy homeostasis. Specifically, the molecular basis of the huge metabolic benefits in bariatric surgery is emerging while novel incretin-based medicines based on endogenous hormones such as glucagon-like peptide 1 and pancreas-derived amylin are improving diabetes management. These and related developments have fostered the discovery of novel insights into endocrine control of systemic metabolism, and in particular a deeper understanding of the importance of communication across vital organs, and specifically the gut-brain-pancreas-liver network. Paradoxically, the pancreatic peptide glucagon has reemerged in this period among a plethora of newly identified metabolic macromolecules, and new data complement and challenge its historical position as a gut hormone involved in metabolic control. The synthesis of glucagon analogs that are biophysically stable and soluble in aqueous solutions has promoted biological study that has enriched our understanding of glucagon biology and ironically recruited glucagon agonism as a central element to lower body weight in the treatment of metabolic disease. This review summarizes the extensive historical record and the more recent provocative direction that integrates the prominent role of glucagon in glucose elevation with its under-acknowledged effects on lipids, body weight, and vascular health that have implications for the pathophysiology of metabolic diseases, and the emergence of precision medicines to treat metabolic diseases.
Asunto(s)
Encéfalo/metabolismo , Tracto Gastrointestinal/metabolismo , Glucagón/metabolismo , Hígado/metabolismo , Enfermedades Metabólicas/metabolismo , Páncreas/metabolismo , Animales , Encéfalo/efectos de los fármacos , Tracto Gastrointestinal/efectos de los fármacos , Glucagón/farmacología , Homeostasis/fisiología , Humanos , Hígado/efectos de los fármacos , Páncreas/efectos de los fármacosRESUMEN
With their ever-growing prevalence, obesity and diabetes represent major health threats of our society. Based on estimations by the World Health Organization, approximately 300 million people will be obese in 2035. In 2015 alone there were more than 1.6 million fatalities attributable to hyperglycemia and diabetes. In addition, treatment of these diseases places an enormous burden on our health care system. As a result, the development of pharmacotherapies to tackle this life-threatening pandemic is of utmost importance. Since the beginning of the 19th century, a variety of drugs have been evaluated for their ability to decrease body weight and/or to improve deranged glycemic control. The list of evaluated drugs includes, among many others, sheep-derived thyroid extracts, mitochondrial uncouplers, amphetamines, serotonergics, lipase inhibitors, and a variety of hormones produced and secreted by the gastrointestinal tract or adipose tissue. Unfortunately, when used as a single hormone therapy, most of these drugs are underwhelming in their efficacy or safety, and placebo-subtracted weight loss attributed to such therapy is typically not more than 10%. In 2009, the generation of a single molecule with agonism at the receptors for glucagon and the glucagon-like peptide 1 broke new ground in obesity pharmacology. This molecule combined the beneficial anorectic and glycemic effects of glucagon-like peptide 1 with the thermogenic effect of glucagon into a single molecule with enhanced potency and sustained action. Several other unimolecular dual agonists have subsequently been developed, and, based on their preclinical success, these molecules illuminate the path to a new and more fruitful era in obesity pharmacology. In this review, we focus on the historical pharmacological approaches to treat obesity and glucose intolerance and describe how the knowledge obtained by these studies led to the discovery of unimolecular polypharmacology.
Asunto(s)
Obesidad/tratamiento farmacológico , Animales , Femenino , Humanos , Masculino , Pérdida de PesoRESUMEN
Obesity and its comorbidities, such as type 2 diabetes, are pressing worldwide health concerns. Available anti-obesity treatments include weight loss pharmacotherapies and bariatric surgery. Whilst surgical interventions typically result in significant and sustained weight loss, available pharmacotherapies are far less effective, typically decreasing body weight by no more than 5-10%. An emerging class of multi-agonist drugs may eventually bridge this gap. This new class of specially tailored drugs hybridizes the amino acid sequences of key metabolic hormones into one single entity with enhanced potency and sustained action. Successful examples of this strategy include multi-agonist drugs targeting the receptors for glucagon-like peptide-1 (GLP-1), glucagon and the glucose-dependent insulinotropic polypeptide (GIP). Due to the simultaneous activity at several metabolically relevant receptors, these multi-agonists offer improved body weight loss and glucose tolerance relative to their constituent monotherapies. Further advancing this concept, chimeras were generated that covalently link nuclear acting hormones such as oestrogen, thyroid hormone (T3 ) or dexamethasone to peptide hormones such as GLP-1 or glucagon. The benefit of this strategy is to restrict the nuclear hormone action exclusively to cells expressing the peptide hormone receptor, thereby maximizing combinatorial metabolic efficacy of both drug constituents in the target cells whilst preventing the nuclear hormone cargo from entering and acting on cells devoid of the peptide hormone receptor, in which the nuclear hormone might have unwanted effects. Many of these multi-agonists are in preclinical and clinical development and may represent new and effective tools in the fight against obesity and its comorbidities.
Asunto(s)
Hormonas Gastrointestinales/agonistas , Obesidad/tratamiento farmacológico , Péptidos/uso terapéutico , Animales , Hormonas Gastrointestinales/fisiología , Glucagón/agonistas , Glucagón/fisiología , Péptido 1 Similar al Glucagón/agonistas , Péptido 1 Similar al Glucagón/fisiología , Humanos , Obesidad/fisiopatología , Péptidos/farmacologíaRESUMEN
C-peptide, a cleavage product from the processing of proinsulin to insulin, has been considered to possess little if any biological activity other than its participation in insulin synthesis. Injection of human C-peptide prevented or attenuated vascular and neural (electrophysiological) dysfunction and impaired Na+- and K+-dependent adenosine triphosphate activity in tissues of diabetic rats. Nonpolar amino acids in the midportion of the peptide were required for these biological effects. Synthetic reverse sequence (retro) and all-D-amino acid (enantio) C-peptides were equipotent to native C-peptide, which indicates that the effects of C-peptide on diabetic vascular and neural dysfunction were mediated by nonchiral interactions instead of stereospecific receptors or binding sites.
Asunto(s)
Péptido C/química , Péptido C/uso terapéutico , Angiopatías Diabéticas/prevención & control , Neuropatías Diabéticas/prevención & control , Secuencia de Aminoácidos , Animales , Circulación Sanguínea/efectos de los fármacos , Glucemia/metabolismo , Péptido C/farmacología , Permeabilidad Capilar/efectos de los fármacos , Dicroismo Circular , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/fisiopatología , Humanos , Masculino , Datos de Secuencia Molecular , Conducción Nerviosa/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Estructura Secundaria de Proteína , Ratas , Ratas Sprague-Dawley , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , EstereoisomerismoRESUMEN
BACKGROUND: The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent ß-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. SCOPE OF REVIEW: In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. MAJOR CONCLUSIONS: Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders.
Asunto(s)
Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Inhibidor Gástrico/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Obesidad/metabolismo , Receptores de Glucagón/metabolismoRESUMEN
In this study, human insulin was compared with two of its analogs--LysB28ProB29-human insulin and AspB10LysB28ProB29-human insulin-with respect to bioavailability and metabolic effectiveness. Absorption from the subcutaneous site was determined using kinetic parameters from the washout curve, following intravenous infusion of insulin or analog. Absorption was found to be more rapid for the two analogs, with 90% absorption by 100 min for the analogs and by 180 min for insulin. Total absorption was 97 +/- 10% for insulin, 99 +/- 7% for LysB28ProB29-human insulin, and 93 +/- 12% for AspB10LysB28ProB29-human insulin. Bioactivity was assessed from the glucose infusion and using tracer-determined metabolic clearance rates (MCRs) and glucose production rates. The fractional glucose requirements (relative to the total amount infused) increased more rapidly for the two analogs than for insulin, with 50% of the glucose infused by 105 min for both analogs vs. 145 min for insulin. The total amount of glucose required was, however, significantly less (19.7 +/- 1.5 mmol/kg) for AspB10LysB28ProB29-human insulin than for either LysB28ProB29-human insulin (25.9 +/- 3.0 mmol/kg) or human insulin (27.8 +/- 2.6 mmol/kg). The glucose requirements were reflected in a lower MCR for AspB10LysB28ProB29-human insulin but equivalent decreases in the rates of glucose production. Thus both analogs demonstrated more rapid rates of absorption, onset, and termination of action, but were not completely bioequivalent, with AspB10LysB28ProB29-human insulin demonstrating a 25% decrease in bioactivity.
Asunto(s)
Hipoglucemiantes/farmacocinética , Insulina/análogos & derivados , Insulina/farmacocinética , Absorción , Animales , Disponibilidad Biológica , Femenino , Técnica de Clampeo de la Glucosa , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/sangre , Bombas de Infusión Implantables , Inyecciones Subcutáneas , Insulina/administración & dosificación , Insulina/sangre , Insulina Lispro , Tasa de Depuración Metabólica , Porcinos , Factores de TiempoRESUMEN
Plasma insulin-like growth factor-I (IGF-I) concentrations and the effects of exogenous IGF-I administration were determined in 26 rhesus monkeys; each animal was well characterized regarding its degree of obesity, plasma glucose and insulin levels, and glucose tolerance (KG). Five separate groups were identified: lean normal, obese normoinsulinemic and normoglycemic, obese hyperinsulinemic with normal glucose tolerance, impaired glucose tolerant, and spontaneously diabetic (type II, non-insulin-dependent diabetes mellitus [NIDDM]). Basal plasma IGF-I levels in all monkeys ranged from 249 to 1,093 ng/mL and were strongly associated with age (r = -.66; P less than .001) and KG (r = .59; P less than .001), but not with body weight, body fat, or fasting plasma glucose or insulin levels. In addition, the acute insulin-like effects of exogenously administered IGF-I on glucose disappearance were studied in vivo in a dose-response comparison to insulin (subcutaneous administration of IGF-I at doses of 50, 100, or 200 micrograms/kg v insulin at 0.3 U/kg). Five hyperinsulinemic normoglycemic monkeys (fasting plasma glucose, 67 +/- 2 mg/dL; insulin, 163 +/- 42 microU/mL) and overt type II diabetic monkeys (fasting plasma glucose, 201 +/- 13 mg/dL; insulin, 38 +/- 6 microU/mL) each underwent a series of three to five experiments to determine the time course and degree of hypoglycemia induced by IGF-I as compared with insulin or with control (saline) injection.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Factor I del Crecimiento Similar a la Insulina/análisis , Animales , Glucemia/análisis , Diabetes Mellitus/sangre , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/farmacología , Macaca mulatta , Obesidad/sangre , Obesidad/genética , Obesidad/fisiopatología , Concentración OsmolarRESUMEN
OBJECTIVE: To discuss the optimal role of insulin in the treatment of patients with diabetes mellitus. METHODS: We review the complications of diabetes, highlight the attempts to improve control of plasma glucose, and summarize current recommendations for use of insulin in clinical practice. RESULTS: With the strict new guidelines for the diagnosis of diabetes issued by the American Diabetes Association in July 1997--a plasma glucose level of 126 mg/dL (7 mmol/L) rather than 140 mg/dL (7.8 mmol/L)--an additional 8 million persons will be diagnosed this year, and diabetes and its complications will be at the forefront of public health concerns. Strong evidence indicates that with reduction of plasma glucose levels and tight control of glycohemoglobin, the rate of complications can be considerably decreased. Although insulin replacement therapy is well known to be both the best and the most cost-effective way to control glucose levels in patients with type 1 diabetes, studies have no shown that those with type 2 diabetes can likewise benefit from appropriate insulin therapy. Other investigations have indicated that coronary events are as likely to occur in patients with recently discovered impaired glucose tolerance as in patients with known diabetes (at a 2-hour postprandial glucose threshold of 96 mg/dL [5.3 mmol/L]). Such finding suggest that there may be no such thing as "borderline diabetes" and give impetus to the search for improved types of insulin to treat all patients with diabetes. A recent candidate is the new insulin analogue lispro, which was developed by recombinant DNA techniques, with its design influenced by the structural analogy to another endogenous hormone, insulin-like growth factor I. Lispro is a fast-acting, rapidly dissipating insulin formulation. This profile allows lispro to be given less than 15 minutes before a meal, yet with little risk of postprandial hypoglycemia because its high mealtime peak is followed by rapid disappearance from the bloodstream. CONCLUSION: Lispro has been shown to improve postprandial control of plasma glucose and to decrease the occurrence of hypoglycemia episodes in comparison with regular insulin. Because of its stability and pharmacokinetic properties, lispro can also be used in insulin pump therapy.
RESUMEN
The aim of this study was to compare growth hormone (GH) response of barrows and gilts to porcine growth hormone-releasing hormone (pGRH) at the pituitary level. Anterior pituitary cells from barrows and gilts responded to pGRH in a dose-dependent manner. The median effective pGRH concentration (EC50) which stimulated GH release from cells of barrows was greater (P less than .05) than that for cells obtained from intact female siblings. Maximal pGRH mediated GH secretion from barrows was not different (P greater than 0.05) than that from gilt stimulated cells. These data demonstrate that somatotrophs of growing peripubertal gilts are more responsive to pGRH stimulation than are cells from their castrated male siblings. This difference could be caused by castration of the neonatal male.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/farmacología , Hormona del Crecimiento/metabolismo , Adenohipófisis/efectos de los fármacos , Caracteres Sexuales , Porcinos/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Orquiectomía/veterinaria , Adenohipófisis/citología , Adenohipófisis/metabolismoRESUMEN
A good correlation exists between specific neutralising antibody titre and protection against challenge with foot-and-mouth disease virus (FMDV) in infected or virus-vaccinated cattle, but not in the case of animals immunised with synthetic FMDV peptides. Therefore, mechanisms other than simple neutralisation are likely to be important in vivo. Antibody affinity may influence the protective capacity of sera from immunised animals and experiments were carried out to measure the functional affinity for synthetic FMDV peptide of sera from guinea pigs and cattle given various synthetic vaccines. In guinea pigs given a single dose of synthetic vaccine, antibody affinity increased with time after immunisation. In cattle, however, administration of a second dose of peptide 21 days after the first markedly retarded the process of affinity maturation. For guinea pig sera of equivalent neutralising activity, those of higher functional affinity had higher protective indices than those of lower functional affinity. Knowledge of the importance of antibody affinity in protection against FMD is important for an improved understanding of the mechanisms of protection and for the design of novel vaccines.
Asunto(s)
Afinidad de Anticuerpos , Aphthovirus/inmunología , Enfermedades de los Bovinos/prevención & control , Fiebre Aftosa/prevención & control , Vacunas Virales , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Bovinos , Ensayo de Inmunoadsorción Enzimática , Femenino , Cobayas , Sueros Inmunes/inmunología , Inmunización Secundaria/veterinaria , Pruebas de Neutralización , Vacunación/veterinaria , Vacunas SintéticasRESUMEN
Two photolabile multi-detachable alkoxybenzyl alcohol resins, 2-[4-(oxymethyl)phenoxy]propionyl-resin 4 and 4-[4-(oxymethyl)phenoxymethyl]-3-nitrobenzamidomethyl-resin 5 have been synthesized. Bpoc-peptide attached to resin 4 or 5 when treated with 50% trifluoroacetic acid provided the free, unprotected peptide, but on photolysis gave Bpoc-peptide p-hydroxybenzyl ester. Removal of the p-hydroxybenzyl ester in aqueous base or by oxidative work up gave a protected Bpoc-peptide suitable for fragment synthesis at its C-terminus. However, methylation of the ester to Bpoc-peptide p-methoxybenzyl ester followed by removal of the Bpoc-group gave a protected peptide p-methoxybenzyl ester suitable for fragment coupling at its N-terminus. The efficacies of these resins were evaluated in the syntheses of a model tetrapeptide and an octapeptide by using N alpha-Bpoc-, Fmoc- and Nps-amino acids. The use of 2-thiopyridine with pyridinium hydrochloride as a new and efficient thiolytic reagent for the deprotection of the Nps-group was studied.
Asunto(s)
Alcoholes Bencílicos/síntesis química , Compuestos de Bencilo/síntesis química , Resinas de Plantas/síntesis química , Secuencia de Aminoácidos , Aminoácidos , Fragmentos de Péptidos , Péptidos , Fotólisis , Ácido TrifluoroacéticoRESUMEN
A protected tridecapeptide of the sequence Boc-Lys(2CIZ)-Arg(Tos)-Leu-Glu (OcHex)-Trp(For)-Ile-Ala-Ala-Ser(Bzl)-Arg(Tos)-Asn-Lys(2CIZ)-Gly-OH, representing residues 43-55 of the variable region of the heavy chain of mouse myeloma protein M603, was synthesized. It was assembled by a stepwise solid phase method designed to give a fully protected peptide in high yield and purity with minimal side reactions. Thus, the peptide chain was attached as an alpha-methyl phenacyl ester to a 2-bromopropionyl-resin. After the synthesis the protected peptide fragment was obtained in 89% yield by photolytic cleavage from the resin. The peptide was purified by multiple precipitation and column chromatography. It was shown to be homogeneous by reverse phase high pressure liquid chromatography, and it had the correct amino acid composition and sequence. In the course of this work it was shown that tert.-butyloxycarbonyl-amino acids caused the formation of significant amounts of pyrrolidone carboxylic acid residues during the coupling reaction when a gamma-benzyl glutamyl residue was NH2-terminal. Other weak-acid additives also caused this chain terminating side reaction. The cyclization was markedly suppressed by protection of the glutamyl side chain as a cyclohexyl ester. With this protecting group, no evidence of pyrrolidone carboxylic acid formation could be detected in the tridecapeptide 43-55.
Asunto(s)
Inmunoglobulinas , Proteínas de Mieloma , Fragmentos de Péptidos/síntesis química , Secuencia de Aminoácidos , Animales , Cadenas Pesadas de Inmunoglobulina , Región Variable de Inmunoglobulina , Indicadores y Reactivos , RatonesRESUMEN
An ELISA to measure bovine serum immunoglobulin isotypes (IgG1, IgG2, IgM and IgA) specific for foot-and-mouth disease virus (FMDV) or for synthetic FMDV peptides is described. Sera from cattle infected by FMDV, vaccinated with conventional inactivated virus vaccines or vaccinated with synthetic peptides were examined using this assay. Generally IgG subclasses dominated the antibody responses of all groups after an early IgM response had waned. An exception to this pattern was seen in the case of a group of immature calves given multiple or high doses of synthetic peptide and in which levels of IgM continued to rise until the end of the experimental period. Both infected animals and those vaccinated with inactivated virus mounted antibody responses in which IgG1 titres tended to predominate over those of IgG2. In some infected animals, an early IgG2 response was evident but resolution of lesions and clinical recovery did not occur until IgG1 antibody appeared in the serum some days later. In synthetic-peptide immunized animals the response was more variable but IgG1:IgG2 ratios at 21 days postvaccination were significantly lower than those of virus-vaccinated animals. It is proposed that differences in the isotype profiles induced by conventional FMD vaccines and those resulting from vaccination of cattle with synthetic FMDV peptides may in part account for the lower protective index of peptide-induced antibodies.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Aphthovirus/inmunología , Isotipos de Inmunoglobulinas/biosíntesis , Vacunas Virales/farmacología , Animales , Bovinos , Enfermedades de los Bovinos/prevención & control , Fiebre Aftosa/prevención & control , Pruebas de Neutralización , Péptidos/inmunología , Vacunas de Productos Inactivados/farmacología , Vacunas Sintéticas/farmacologíaRESUMEN
Amylin, an islet amyloid peptide secreted by the pancreatic beta cell, has been proposed as a humoral regulator of islet insulin secretion. Four separate preparations of amylin were tested for effects on hormone secretion in both freshly isolated and cultured rat islets and in HIT-T15, hamster insulinoma cells. With all three experimental models, exposure to human amylin acid and human and rat amylin at concentrations as high as 100 nM had no significant effect on rates of insulin or glucagon secretion. These observations suggest that amylin, even at concentrations appreciably higher than those measured in peripheral plasma, is not a significant humoral regulator of islet hormone secretion.
Asunto(s)
Amiloide/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Amiloide/síntesis química , Animales , Línea Celular , Células Cultivadas , Cricetinae , Glucagón/metabolismo , Glucosa/farmacología , Humanos , Técnicas In Vitro , Secreción de Insulina , Insulinoma , Polipéptido Amiloide de los Islotes Pancreáticos , Islotes Pancreáticos/efectos de los fármacos , Cinética , Masculino , Neoplasias Pancreáticas , Ratas , Ratas EndogámicasRESUMEN
To determine whether the serum level of IGF-I influences its hepatic synthesis through negative feedback regulation, we infused 200 micrograms/d of human IGF-I subcutaneously into young male rats eating either an energy-restricted or ad lib diet. In energy-restricted rats, a two-fold increase in serum IGF-I concentration produced a 41% increase in growth rate at the end of one week, and a 30% decrease in steady state hepatic IGF-I mRNA and 56% drop in serum GH at the end of two weeks. In ad lib fed rats, the increased serum IGF-I concentration neither enhanced growth rate nor significantly reduced hepatic IGF-I mRNA abundance or serum GH levels. These data suggest that the abundance of hepatic IGF-I mRNA in energy-restricted rats is controlled, in part, by serum IGF-I levels via negative feedback regulation.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Hígado/metabolismo , Trastornos Nutricionales/metabolismo , ARN Mensajero/metabolismo , Somatomedinas/administración & dosificación , Animales , Metabolismo Energético/efectos de los fármacos , Crecimiento/efectos de los fármacos , Hormona del Crecimiento/sangre , Bombas de Infusión , Factor I del Crecimiento Similar a la Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/metabolismo , Hígado/efectos de los fármacos , Masculino , Trastornos Nutricionales/sangre , Trastornos Nutricionales/fisiopatología , Ratas , Ratas EndogámicasRESUMEN
Modified-live, attenuated infectious bovine rhinotracheitis (IBR) hybrid virus vaccines have been constructed by inserting in the major IBRV glycoprotein gIII gene chemically synthesized deoxyribonucleotide sequences encoding the bovine growth hormone signal sequence and monomeric or dimeric forms of the foot and mouth disease virus (FMDV) VP 1 epitope sequences. The foreign DNA sequences were inserted at the N-terminal end of the IBRV gIII coding sequence and were driven by the IBRV gIII promoter. The sequences encoding the first 38 and the first 21 amino acids of the IBRV gIII were deleted from the hybrid viruses containing inserts of the monomeric and dimeric FMDV epitope sequences, respectively, to avoid redundant signal sequences. Plaque immunoassay experiments with guinea pig and bovine anti-FMDV peptide antisera, and with anti-IBRV gIII monoclonal antibodies demonstrated that IBRV-FMDV fusion proteins were expressed in virus-infected MDBK cells. Immunoelectron microscopy analyses demonstrated that the IBRV-FMDV fusion proteins were expressed as repeated structures on the surface of virus particles. Experiments showed that the recombinant IBRV-FMDV viruses protected cattle from IBRV (Cooper) challenge and induced anti-FMDV peptide antibodies, thereby demonstrating that the FMDV epitopes were expressed in vivo.
Asunto(s)
Antígenos de Superficie/inmunología , Aphthovirus/inmunología , Cápside/inmunología , Herpesvirus Bovino 1/genética , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Antígenos de Superficie/genética , Antígenos Virales/genética , Antígenos Virales/inmunología , Aphthovirus/genética , Secuencia de Bases , Cápside/genética , Bovinos , Clonación Molecular , ADN Viral , Epítopos/genética , Epítopos/inmunología , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Vacunas Virales/genéticaRESUMEN
A passive immunity test (MPT) in suckling mice for the quantification of protective anti-foot-and-mouth disease virus (FMDV) antibodies in serum is described. Comparisons with titres obtained using conventional serum neutralization tests show that for cattle given synthetic peptide vaccines this in vivo assay is a better indicator of protection, while for convalescent animals and virus-vaccinates both tests are equally valid predictors of immune status. Cleavage of Fc fragments from anti-virus or anti-peptide IgG results in a marked decrease in MPT titres although binding to virus in ELISA is unaffected, indicating that intact antibodies are required for in vivo clearance of FMDV. Cross-protection studies demonstrate that anti-peptide sera, while less potent than anti-viral sera in conferring passive immunity against FMDV challenge, have a wider protective range than anti-viral sera within the O serotype and also between O and A serotypes. Possible qualitative differences between anti-viral and anti-peptide sera are discussed in the light of these findings.
Asunto(s)
Aphthovirus/inmunología , Vacunas Virales/farmacología , Animales , Anticuerpos Antivirales/sangre , Estudios de Evaluación como Asunto , Fiebre Aftosa/prevención & control , Inmunización Pasiva , Ratones , Pruebas de Neutralización , Vacunas Sintéticas/farmacologíaRESUMEN
Small peptides with activity as enzyme inhibitors, hormone antagonists, or other peptide mimetics, can be identified by synthesizing and screening large numbers of peptides as defined mixtures. Several coupling reactions, each with a different amino acid, can be conducted simultaneously and then combined to generate a near equimolar mixture before coupling additional residues. The peptide mixtures are recovered free from the matrix and in quantities sufficient for screening in many assays. We describe the rapid identification of a potent peptide inhibitor of human immunodeficiency virus protease from twenty-two mixtures containing more than 240,000 tetrapeptides.
Asunto(s)
Antivirales/farmacología , Inhibidores de la Proteasa del VIH , VIH-1/enzimología , Oligopéptidos/farmacología , Secuencia de Aminoácidos , Antivirales/síntesis química , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Especificidad por SustratoRESUMEN
Formation of pyrrolidone carboxylic acid (pyroglutamic acid) residues from amino-terminal glutaminyl residues in peptides was shown to be catalyzed by weak acids, but not by strong acids. During dicyclohexylcarbodiimide-mediated coupling reactions the N alpha-protected amino acid reagent accelerated this cyclization and resulted in a significant amount of chain termination. The side reaction could be minimized by accelerating the coupling reaction and simultaneously reducing the time of exposure to weak acids. The most effective procedure was to couple in dimethylformamide with the preformed symmetric anhydride of the amino acid.
Asunto(s)
Glutamina , Oligopéptidos/síntesis química , Pirrolidinonas , Ácido Pirrolidona Carboxílico , Fenómenos Químicos , Química , Diciclohexilcarbodiimida , Dimetilformamida , Ácido TrifluoroacéticoRESUMEN
Following the development of a nondestructive synthetic procedure for rapid production of des-Val1-myoglobin in large quantities, the synthesis of a series of myoglobin derivatives varying in structure and charge in the NH2-terminal region was accomplished. In comparison to the untreated myoglobin, the des-Val1-myoglobin was found to possess at low pH a decreased stability and an increased net positive charge in the pH range 5.5-8.5. While the elevated net positive charge was no longer apparent after removal of the second residue, the instability of the molecule was found to be sharply increased. Substitutions of the first residue, directed toward elucidating its structural importance, included glutamic acid, lysine, and glycine. Addition of any of the three amino acids to the des-Val1-myoglobin was found to restore much of the acid stability, with the [Gly1]myoglobin appearing nearly identical with the native molecule. All three semisynthetic myoglobins showed potentiometric titration curves characteristic of their respective, substituted residue. Carbamylation of the NH2 terminal of myoglobin and des-Val1-myoglobin yielded two nearly identical molecules in terms of all physical properties examined. Consequently, it was concluded that the first residue primarily serves the function of maintaining the positively charged NH2 terminus a certain distance away from the beginning of the A helix and from the charge pair interaction of Lys-133 with Glu-6. In addition, through physical measurements of the des-Val1,Leu2-myoglobin prior and subsequent to carbamylation of the NH2 terminus, it was apparent that the stabilization conferred on the des-Val1-myoglobin by the second residue was dependent to a large degree upon the hydrophobic interactions of its side chain.