RESUMEN
INTRODUCTION: The COVID-19 pandemic caused by SARS-CoV-2 has created serious health problems worldwide. The most effective way to prevent the occurrence of new epidemic outbreaks is vaccination. One of the modern and effective approaches to vaccine development is the use of virus-like particles (VLPs). The aim of the study is to develop a technology for production of VLP based on recombinant SARS-CoV-2 proteins (E, M, N and S) in insect cells. MATERIALS AND METHODS: Synthetic genes encoding coronavirus proteins E, M, N and S were used. VLP with various surface proteins of strains similar to the Wuhan virus, Delta, Alpha and Omicron were developed and cloned into the pFastBac plasmid. The proteins were synthesized in the baculovirus expression system and assembled into VLP in the portable Trichoplusia ni cell. The presence of insertion in the baculovirus genome was determined by PCR. ELISA and immunoblotting were used to study the antigenic activity of VLP. VLP purification was performed by ultracentrifugation using 20% sucrose. Morphology was assessed using electron microscopy and dynamic light scattering. RESULTS: VLPs consisting of recombinant SARS-CoV-2 proteins (S, M, E and N) were obtained and characterized. The specific binding of antigenic determinants in synthesized VLPs with antibodies to SARS-CoV-2 proteins has been demonstrated. The immunogenic properties of VLPs have been studied. CONCLUSION: The production and purification of recombinant VLPs consisting of full-length SARS-CoV-2 proteins with a universal set of surface antigens have been developed and optimized. Self-assembling particles that mimic the coronavirus virion induce a specific immune response against SARS-CoV-2.
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Baculoviridae , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Vacunas de Partículas Similares a Virus , Animales , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Humanos , COVID-19/virología , COVID-19/inmunología , Baculoviridae/genética , Baculoviridae/metabolismo , Vacunas contra la COVID-19/inmunología , Anticuerpos Antivirales/inmunología , Proteínas M de Coronavirus/genética , Proteínas M de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología , FosfoproteínasRESUMEN
INTRODUCTION: In Russia, almost half of the cases of acute intestinal infections of established etiology in 2022 are due to rotavirus infection (RVI). There is no specific treatment for rotavirus gastroenteritis. There is a need to develop modern, effective and safe vaccines to combat rotavirus infection that are not capable of multiplying (replicating) in the body of the vaccinated person. A promising approach is to create vaccines based on virus-like particles (VLPs). OBJECTIVE: Study of the safety and immunogenicity of a vaccine against rotavirus infection based on virus-like particles of human rotavirus A in newborn minipigs with multiple intramuscular administration. MATERIALS AND METHODS: Newborn minipigs were used as an animal model in this study. The safety of the tested vaccine was assessed based on thermometry data, clinical examination, body weight gain, clinical and biochemical blood parameters, as well as necropsy and histological examination. When studying the immunogenic properties of the Gam-VLP-rota vaccine in doses of 30 and 120 µg, the cellular, humoral and secretory immune response was studied. RESULTS: The results of assessing the general condition of animals during the immunization period, data from clinical, laboratory and pathomorphological studies indicate the safety of the vaccine against human rotavirus infection based on VLP (Gam-VLP-rota) when administered three times intramuscularly. Good local tolerance of the tested vaccine was demonstrated. The results of the assessment of humoral immunity indicate the formation of a stable immune response after three-time immunization with Gam-VLP-rota, stimulation of the production of antigen-specific IgG antibodies and their functional activity to neutralize human rotavirus A. It was shown that following the triple immunization with the minimum tested concentration of 30 µg/dose, animals developed a cell-mediated immune response. The results of the IgA titer in blood serum and intestinal lavages indicate the formation of both a systemic immunological response and the formation of specific secretory immunity to human rotavirus A. CONCLUSION: Thus, three-time intramuscular immunization of minipigs with the Gam-VLP-rota vaccine forms stable protective humoral and cellular immunity in experimental animals. Evaluated vaccine is safe and has good local tolerability.
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Infecciones por Rotavirus , Vacunas contra Rotavirus , Rotavirus , Recién Nacido , Animales , Humanos , Porcinos , Infecciones por Rotavirus/prevención & control , Porcinos Enanos , Anticuerpos Antivirales , Vacunas contra Rotavirus/efectos adversosRESUMEN
INTRODUCTION: Rotavirus infection is one of the main concerns in infectious pathology in humans, mammals and birds. Newborn piglets or rodents are usually being used as a laboratory model for the evaluation of immunogenicity and efficacy for all types of vaccines against rotavirus A (RVA), and the use of ELISA for the detection of virus-specific antibodies of specific isotype is an essential step of this evaluation. OBJECTIVE: Development of indirect solid-phase ELISA with VP2/VP6 rotavirus VLP as an antigen to detect and assess the distribution of RVA-specific IgG, IgM and IgA in the immune response to rotavirus A. MATERIALS AND METHODS: VP2/VP6 rotavirus VLP production and purification, electron microscopy, PAGE, immunoblotting, ELISA, virus neutralization assay. RESULTS: The study presents the results of development of a recombinant baculovirus with RVA genes VP2-eGFP/VP6, assessment of its infectious activity and using it for VLP production. The morphology of the VP2/VP6 rotavirus VLPs was assessed, the structural composition was determined, and the high antigenic activity of the VLP was established. VLP-based ELISA assay was developed and here we report results for RVA-specific antibody detection in sera of different animals. CONCLUSION: The developed ELISA based on VP2/VP6 rotavirus VLP as a universal antigen makes it possible to detect separately IgG, IgM and IgA antibodies to rotavirus A, outlining its scientific and practical importance for the evaluation of immunogenicity and efficacy of traditional vaccines against rotavirus A and those under development.
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Rotavirus , Humanos , Recién Nacido , Animales , Porcinos , Rotavirus/genética , Proteínas Recombinantes , Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G , Inmunoglobulina A , Inmunidad , Inmunoglobulina M , Antígenos Virales/genética , MamíferosRESUMEN
INTRODUCTION: Rotavirus infection is the leading cause of acute gastroenteritis among infants. The development of new vaccines against rotavirus A is urgent because the virus has many genotypes, some of which have regional prevalence. Virus-like particles (VLP) is a promising way to create effective and safe vaccine preparations.The purpose of the study is to develop the technology for the production of VLP, containing VP2, VP4, VP6 and VP7 of viral genotypes prevalent on the territory of the Russian Federation, and to give its molecular genetic and virological characteristics. MATERIAL AND METHODS: The virulent strain Wa G1P[8] of human RV A adapted to MARC-145 cell culture has been used. It was cultured and purified according to the method described by the authors earlier. Standard molecular genetic and cytological methods were used: gene synthesis; cloning into transfer plasmids; recombinant baculoviruses production in Bac-to-Bac expression system; VLP production in the insect cells; centrifugation in sucrose solution; enzyme-linked immunosorbent assay (ELISA); electron microscopy (EM); polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. RESULTS: VP4 and VP7 of the six most represented in Russia genotypes: G1, G2, G4, G9, P4, P8, as well as VP2 and VP6 were selected for VLP production. Recombinant baculoviruses were obtained with codon frequencies optimized for insect cells. Cabbage loopper (Trichoplusia ni) cell culture was coinfected with different combinations of baculoviruses, and VLP consisting of 2-4 proteins were produced. VLP were purified by centrifugation. The size and morphology of the particles matched the rotavirus A virion (by EM). The presence of rotavirus A proteins in VLP was confirmed by the ELISA, SDS-PAGE and western blot analysis. CONCLUSION: The technology for the synthesis of three-layer VLP consisting of VP2, VP4, VP6 and VP7 has been developed and optimized. The resulting VLP composition represents 6 serotypes of VP4 and VP7, which are most represented on the territory of Russia, and can be used for vaccine development.
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Reoviridae , Infecciones por Rotavirus , Rotavirus , Humanos , Rotavirus/genética , Desarrollo de Vacunas , ViriónRESUMEN
The review presents the state-of-the-art on the problem of diagnosis of prion diseases (PD) in humans and animals with a brief description of their etiology and pathogenesis. We pointed out that understanding the nature of the etio logical agent of PD determined their zoonotic potential and led to the development of highly specific immunological diagnostic methods aimed at identifying the infectious isoform of prion protein (PrPd) as the only marker of the disease. In this regard, we briefly summarize the results of studies, including our own, concerning the conversion of normal prion protein molecules (PrPc) to PrPd, the production of monoclonal antibodies and their application as immunodiagnostic reagents for the post-mortem detection of PrPd in various formats of immunoassay. We also emphasize the issues related to the development of methods for ante mortem diagnostics of PD. In this regard, a method for amplifying amino acid sequences using quacking-induced conversion of PrPc to PrPd in real time (RTQuIC) described in details. The results of recent studies on the assessment of the sensitivity, specificity and reproducibility of this method, carried out in various laboratories around the world, are presented. The data obtained indicate that RT-QuIC is currently the most promising laboratory assay for detecting PrPd in biological material at the preclinical stage of the disease. The significant contribution of US scientists to the introduction of this method into clinical practice on the model of diagnosis of chronic wasting disease of wild Cervidae (CWD) is noted. The possible further spread of CWD in the population of moose and deer in the territories bordering with Russia, as well as the established fact of alimentary transmission of CWD to macaques, indicate the threat of the appearance of PD in our country. In conclusion, the importance of developing new hypersensitive and/or selective components of known methods for PrPd identification from the point of view of assessing the risks of creating artificial infectious prion proteins in vivo or in vitro, primarily new pathogenic isoforms ("strains") and synthetic prions, was outlined.
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Autopsia , Enfermedades por Prión/diagnóstico , Proteínas Priónicas/genética , Enfermedad Debilitante Crónica/genética , Secuencia de Aminoácidos/genética , Animales , Ciervos/genética , Humanos , Enfermedades por Prión/genética , Enfermedades por Prión/patología , Proteínas Priónicas/aislamiento & purificación , Federación de Rusia , Enfermedad Debilitante Crónica/patologíaRESUMEN
The results obtained using the diagnostic kit based on real-time polymerase chain reaction to detect the DNA of the African Swine Fever in the pathological material, as well as in the culture fluid, are presented. A high sensitivity and specificity for detection of the DNA in the organs and tissues of animals was shown to be useful for detection in the European Union referentiality reagent kits for DNA detection by real time PCR of ASFV. More rapid and effective method of DNA extraction using columns mini spin Quick gDNA(TM) MiniPrep was suggested and compared to the method of DNA isolation on the inorganic sorbent. High correlation of the results of the DNA detection of ASFV by real-time PCR and antigen detection results ASFV by competitive ELISA obtained with the ELISA SEROTEST/INGEZIM COMRAC PPA was demonstrated. The kit can be used in the veterinary services for effective monitoring of ASFV to contain, eliminate and prevent further spread of the disease.
Asunto(s)
Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/diagnóstico , ADN Viral/genética , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Fiebre Porcina Africana/virología , Animales , Antígenos Virales/inmunología , Cartilla de ADN/síntesis química , Sondas de ADN/síntesis química , ADN Viral/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Sensibilidad y Especificidad , PorcinosRESUMEN
Five hybridomas secreting monoclonal antibodies (MAbs) for the nucleocapsid protein of the rabies virus were obtained through the fusion of the SP2/0 murine myeloma cells with splenocytes of BALB/c mice immunized with fixed rabies virus (CVS strain). All hybridomas secret MAbs of the IgG class that display different specificity to the nucleocapsids of rabies and rabies-related viruses. MAbs 2ell showed the specificity for the prevalent in Russia rabies viruses that are similar to commercially available anti-rabies conjugate.
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Anticuerpos Monoclonales/biosíntesis , Proteínas de la Nucleocápside/inmunología , Virus de la Rabia/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Gatos , Perros , Zorros , Humanos , Hibridomas , Inmunización , Ratones , Ratones Endogámicos BALB C , Mustelidae , Proteínas de la Nucleocápside/genética , Rabia/diagnóstico , Rabia/virología , Virus de la Rabia/genética , Virus de la Rabia/aislamiento & purificación , Federación de Rusia , LobosAsunto(s)
Amiloide/metabolismo , Priones/metabolismo , Proteínas Recombinantes/metabolismo , Amiloide/química , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida , Humanos , Concentración de Iones de Hidrógeno , Priones/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Factores de TiempoRESUMEN
Recombinant nucleocapsid (rN) protein N of porcine reproductive and respiratory syndrome virus (PRRSV) was prepared, by using the E. coli expressiom system. Insertion of a polyhistidine marker into the structure of the protein allowed the latter to be purified by metal-chelate affinity chromatography. The purity of protein was confirmed by PAAG electrophoresis and its immunospecificity was verified by immunoblotting using rN-specific monoclonal antibodies. The protein was used as an antigen to develop indirect ELISA of PRRSV antibodies. ELISA was shown to be highly sensitive and specific.
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Síndrome Respiratorio y de la Reproducción Porcina/diagnóstico , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Animales , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas de la Nucleocápside/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/sangre , Juego de Reactivos para Diagnóstico , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Pruebas Serológicas , PorcinosRESUMEN
Recombinant antigen ORF2 from porcine circovirus type 2 (PCV-2) was produced, by using the baculovirus expression system, with histidine tags to allow purification by metal-chelate affinity chromatography. The purity of the protein was verified by polyacrylamide gel electrophoresis; and its immunospecificity was confirmed by the immunoblotting test using reference PCV-2-positive and PCV-2-negative porcine sera and monoclonal antibodies. The protein was used as an antigen to develop an indirect enzyme immunoassay (EIA) of PCV-2 antibodies. EIA was shown to have a high sensitivity and specificity as compared with indirect immunofluorescence test. Porcine serum samples from 15 pig-breeding farms of the Russian Federation were studied. Seropositive samples were found in all age pig groups in all the farms, The number of seropositive animals was shown to be directly related to its age.
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Infecciones por Circoviridae/diagnóstico , Circovirus/inmunología , Técnicas para Inmunoenzimas/métodos , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Antígenos Virales/aislamiento & purificación , Baculoviridae/metabolismo , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Proteínas de la Cápside/aislamiento & purificación , Línea Celular , Cromatografía de Afinidad , Infecciones por Circoviridae/sangre , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Sensibilidad y Especificidad , PorcinosRESUMEN
Full-length Bos taurus PrPC protein was obtained in the eu- and prokaryotic expression systems. Immunoblotting and indirect enzyme immunoassay demonstrated high specificity and antigenic activity of full-length proteins in the reactions with monoclonal antibodies (anti-SAF-32 and VRQ-84). Membrane location of recombinant PrPC protein in insect cells was shown by immunofluorescent analysis.
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Proteínas PrPC/biosíntesis , Proteínas Recombinantes/biosíntesis , Animales , Anticuerpos Monoclonales/inmunología , Antígenos/análisis , Baculoviridae/genética , Bovinos , Membrana Celular/química , Proteínas PrPC/análisis , Proteínas PrPC/inmunología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunologíaRESUMEN
We optimized the procedure for the formation of Langmuir films of antibodies based on amphiphilic polyelectrolytes and studied the physicochemical and immunochemical properties of the films obtained. Their immunochemical properties were compared with the immunochemical activity of antibodies in Langmuir films without amphiphilic polyelectrolytes and with antibodies adsorbed on the surface of polystyrene and graphite. The efficiency of immune adsorption by the films based on amphiphilic polyelectrolytes was shown to be greater; the affinity of antibodies and surface concentration of their active conformation depended on the type of amphiphilic polyelectrolytes used to obtain the films. We investigated the structure of these films at the surface of highly oriented pyrolytic graphite using the method of atomic force microscopy. Changes in the structure of the films under study caused by the increase of surface pressure were demonstrated.
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Anticuerpos/inmunología , Anticuerpos/metabolismo , Electrólitos/metabolismo , Técnicas de Inmunoadsorción , Membranas Artificiales , Polímeros/metabolismo , Técnicas Biosensibles/métodosRESUMEN
Recombinant major surface glycoprotein E2 from virulent Shimen strain of classical swine fever virus (CSFV) has been tested for immunogenicity in animal immunization experiments. Immunization of 3-month-old piglets with 200 micrograms of recombinant protein protected the animals from lethal challenge with virulent CSFV strain. CSFV-specific antibody detection test based on competitive ELISA has been developed using the recombinant E2 protein. The test can evaluate specific antibody levels after subunit vaccination with recombinant E2 after immunization with live vaccine based on attenuated CSFV strain.
Asunto(s)
Peste Porcina Clásica/prevención & control , Proteínas Recombinantes/administración & dosificación , Proteínas del Envoltorio Viral/administración & dosificación , Vacunas Virales/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Porcinos , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/aislamiento & purificación , Vacunas Virales/inmunologíaRESUMEN
Recombinant E2 protein from vaccine strain of classical swine fever virus (CSFV) and from SCFV virulent strain Shimen was synthesized in SF-21 and High-Five cell culture with baculovirus as the expressing vector. For secretion, hydrophobic C-terminal transmembrane domain was removed and N-terminal signal polypeptide of 38 amino acids was added. Maximum accumulation of recombinant products in SF-21 cells was observed after 48 h and in medium 96 h after infection with recombinant baculovirus. In High-Five cells and in culture medium the maximum accumulation of E2 was observed after 96 h. The level of E2 expression is 5-10 micrograms/106 cells. The products of expression were purified by affinity chromatography and their specificity confirmed in immunochemical tests with a series of reference monoclonal antibodies. The product can be used for detecting antibodies to SCFV by competitive enzyme immunoassay.
Asunto(s)
Proteínas del Envoltorio Viral/genética , Animales , Anticuerpos Monoclonales/inmunología , Baculoviridae/genética , Secuencia de Bases , Línea Celular , Cromatografía de Afinidad , Cartilla de ADN , Técnicas para Inmunoenzimas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Spodoptera , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Mutual activation of reproduction of type 1 HIV and herpes simplex types 1 and 2 viruses (HSV) was observed in simultaneous infection of continuous T-cellular lymphoblastoid lines (CEM, 119, Hut-78, MT-4, Jurkat-tat) and U-937 monocytic line. Syncytium formation and cytodestructive pattern of reproduction of viruses of both families in these cell lines necessitated the use of enzyme immunoassay (EIA) to detect the antigens of these viruses in order to assess the level of reproduction. The concentration of HIV antigens in EIA increased in mixed infection by 1.4 to 2.1 times in different cultures in comparison with the culture infected with HIV-1 alone, and concentrations of HSV-1 and HSV-2 increased by 1.3-1.8 times in mixed infection, in comparison with reproduction in lymphoblastoid cultures infected with HSV alone. EIA was alone used to examine the production of IgG and IgM antibodies to Epstein-Barr virus, another representative of Herpesviridae family, in the blood sera of patients with immunodeficiency states in whose sera antibodies to proteins produced by gag HIV gene (p15/17, p24, p55) were detected. Increased concentration of IgG antibodies were revealed in 36% of these patients, whereas in healthy donors the sera with elevated concentrations of IgG to Epstein-Barr virus were far less incident (12%). A hypothesis about mutual activation of HIV and herpes viruses is put forward.
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VIH-1/fisiología , Herpesviridae/fisiología , Replicación Viral , Animales , Antígenos Virales/análisis , Línea Celular , Chlorocebus aethiops , Humanos , Técnicas para Inmunoenzimas , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Ratones , Ratones Endogámicos BALB C , Células VeroRESUMEN
Solid-phase IEA was used to measure the level of autoantibodies to somatostatin in the blood serum of 44 schizophrenics and 24 healthy donors. The patients suffering from schizophrenia manifested a higher (p less than 0.01) level of immune responsiveness of the blood serum to somatostatin (0.665 +/- 0.03) as compared to the control group (0.509 +/- 0.05). The main contribution to the differences between the groups as regards the parameter measured is made by patients with malignant (0.810 +/- 0.10) and paranoid (0.773 +/- 0.08) schizophrenia whereas the patients' subgroup with slow-progressive schizophrenia did not differ from normal regarding the level of autoantibodies to somatostatin in the serum (0.504 +/- 0.03). These tentative data agree with a hypothesis of the involvement of autoimmune processes into the development of schizophrenia.
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Autoanticuerpos/análisis , Enfermedades Autoinmunes/inmunología , Esquizofrenia/inmunología , Somatostatina/inmunología , Adolescente , Adulto , Enfermedades Autoinmunes/etiología , Enfermedad Crónica , Humanos , Esquizofrenia/etiología , Esquizofrenia Paranoide/etiología , Esquizofrenia Paranoide/inmunologíaRESUMEN
Four mouse monoclonal antibodies (E11, A8, F8, H5) to alpha-endorphin have been produced. The antibodies bind 12.5, 20.6, 9.6, 6.6% of 125I-beta-endorphin and 35.5, 15.1, 12.8, 12.2% of 125I-gamma-endorphin; the binding of 125I-alpha-endorphin being taken for 100%. The binding of antibodies E11, A8, F8 and H5 to 125I-alpha-endorphin was 50% inhibited by unlabeled ligand in concentrations 5, 50, 30 and 35 nM respectively. Using tissue sections of rat pituitary it was shown that antibody E11 can be used for the localization of endorphin producing cells by immunofluorescence. The antibodies F8 and H5 effectively detected endorphin precursor proopiomelanocortin by immunoblotting.
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Anticuerpos Monoclonales/aislamiento & purificación , Endorfinas/inmunología , Immunoblotting/métodos , Inmunohistoquímica/métodos , Animales , Anticuerpos Monoclonales/análisis , Reacciones Cruzadas , Hibridomas/inmunología , Inmunización , Radioisótopos de Yodo , Ratones , Ratones Endogámicos BALB C , Proopiomelanocortina/análisis , alfa-EndorfinaRESUMEN
A combination of gel chromatography, fluorimetric analysis and polyacrylamide gel electrophoresis with immunochemical identification, the protein-peptide composition of secretory granules of the lactogenic hormone (LTH) isolated from the anterior lobe of bovine hypophysis was investigated. It was found that the peptide content of the granules is less than 3% of that of immunoreactive LTH. Using gel chromatography, the secretory granules were found to contain a hormone monomer and two immunoreactive forms with Mr 42 and 64 kD. With respect to immunoreactivity, the hormone form content was 90, 3 and 7%, respectively. Using polyacrylamide gel electrophoresis and subsequent immunochemical identification, the presence of four immunoreactive forms of LTH were identified in the secretory granules.
Asunto(s)
Gránulos Citoplasmáticos/análisis , Péptidos/análisis , Adenohipófisis/análisis , Prolactina/análisis , Proteínas/análisis , Animales , Bovinos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Masculino , Peso MolecularRESUMEN
An asymmetrical LH-RH distribution in rat hypothalamus has been found. In Wistar rats LH-RH content in the right hypothalamus exceeds that in the left one; in albino rats a contrary distribution is observed. LH-RH lateralization changes during a 24-h period. Unilateral castration or cold stress lead to a shift in LH-RH distribution in the hypothalamus.
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Dominancia Cerebral/fisiología , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Nivel de Alerta/fisiología , Castración , Masculino , Eminencia Media/metabolismo , Área Preóptica/metabolismo , Ratas , Ratas EndogámicasRESUMEN
The properties of rat heart peptidase hydrolyzing luliberin were studied. This peptidase was shown to be a sulfhydryl metalloenzyme with m.w. of about 100000. The maximal enzyme activity was observed at neutral values of pH Ca2+ (5 X 10(-6) M) increased the enzyme activity by 50%, thus being indicative of an anomalous dependence of the enzyme activity of substrate concentration. At luliberin concentrations of 10(-7)-10(-6) M the enzyme activation by Ca2+ was considerably reduced and returned to the initial level when the peptide concentration was increased up to 10(-5) M. It was assumed that the peptidase under study is a regulatory enzyme whose activity depends on concentrations of Ca2+ and of the reaction substrate, luliberin.