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The Advisory Board chaired by the chief specialist in infectious diseases of the Ministry of Health of Russian Federation, Professor V.P. Chulanov was held on June 18, 2022 in Saint Petersburg. Aim. The main purpose of the Board was following discussion: the analysis of the real-world data of levilimab as an anticipatory therapy for COVID-19 in hospitalized patients; the review of the experience and perspectives of levilimab as an anticipatory anti-inflammatory option for outpatient patients who meet defined clinical and laboratory criteria. Results. The analyzed data on clinical efficacy and safety formed the basis of recommendations proposed by experts for the use of levilimab in the inpatient and outpatient medical care for COVID-19.
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Tratamiento Farmacológico de COVID-19 , Humanos , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antiinflamatorios , Receptores de Interleucina-6RESUMEN
AIM: Determine whether bone mineral density (BMD) assessed by dual-energy x-ray absorptiometry can be used as predictor of increased risk of death in hemodialysis patients. MATERIALS AND METHODS: A prospective study was performed of 516 patients with chronic kidney disease treated with hemodialysis (men 265, women 251, mean age 44.811.4 years) who were observed for 5.73.2 years. Before inclusion in the study, in all patients was analyzed bone mineral density using dual-energy X-ray absorptiometry in three standard departments: lumbar vertebrae, proximal femur and distal forearm. The probability analysis of the outcome was carried out using the KaplanMeier method and Cox. RESULTS: During follow-up period 111 (21.5%) patients died, 50.5% from cardiovascular events. Survival analysis by KaplanMeier method allowed to prove the increased risk of death from cardiovascular pathology in hemodailysis patients with low bone mineral density of all evaluated areas. Step-by-step multivariate Cox regression analysis showed that the T score of the femur, showing the difference of BMD of the patient with normal value of BMD for young adult, had the greatest prognostic significance. CONCLUSION: Reduced bone mineral density in patients receiving hemodialysis is associated with an increased risk of death from cardiovascular disease. Dual energy x-ray absorptiometry can be used for assessment of this risk.
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Enfermedades Cardiovasculares , Osteoporosis , Absorciometría de Fotón , Adulto , Densidad Ósea , Enfermedades Cardiovasculares/diagnóstico por imagen , Femenino , Humanos , Vértebras Lumbares/diagnóstico por imagen , Masculino , Osteoporosis/diagnóstico por imagen , Estudios Prospectivos , Diálisis Renal , Adulto JovenRESUMEN
In this paper, micro-Raman mapping and conductive atomic force microscopy (C-AFM) were jointly applied to investigate the structural and electrical homogeneity of quasi-free-standing monolayer graphene (QFMLG), obtained by high temperature decomposition of 4H-SiC(0001) followed by hydrogen intercalation at 900 °C. Strain and doping maps, obtained by Raman data, showed the presence of sub-micron patches with reduced hole density correlated to regions with higher compressive strain, probably associated with a locally reduced hydrogen intercalation. Nanoscale resolution electrical maps by C-AFM also revealed the presence of patches with enhanced current injection through the QFMLG/SiC interface, indicating a locally reduced Schottky barrier height (ΦB). The ΦB values evaluated from local I-V curves by the thermionic emission model were in good agreement with the values calculated for the QFMLG/SiC interface using the Schottky-Mott rule and the graphene holes density from Raman maps. The demonstrated approach revealed a useful and non-invasive method to probe the structural and electrical homogeneity of QFMLG for future nano-electronics applications.
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Structural changes of eye chorioretinal complex were investigated in 40 adult male outbred albino rats after total transient cerebral ischemia using electron microscopy and morphometric analysis. Furthermore, the influence of a new sterically hindered phenolic antioxidant dibornol on these processes was estimated. Our studies demonstrated that total transient cerebral ischemia in rats resulted in the capillary thrombosis of the choriocapillary lamina of the uvea, structural disturbances of the blood-retinal barrier, degeneration of the retinal neurons and radial glia. Course administration of dibornol was shown to improve the microcirculation and to protect the retinal neuronal structures, pigment epithelium, and radial glia.
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Isquemia Encefálica/patología , Canfanos/farmacología , Coriorretinopatía Serosa Central/patología , Coroides/ultraestructura , Cresoles/farmacología , Retina/ultraestructura , Animales , Barrera Hematorretinal/metabolismo , Coroides/patología , Masculino , Neuronas/ultraestructura , Ratas , Retina/fisiopatología , Trombosis/patologíaRESUMEN
Lattice and surface impurity reactions and structural changes induced by them in slightly carbonated hydroxyapatite (SCHA) treated at 25-1100 degrees C were comprehensively studied. The SCHA was processed by a conventional wet synthesis at a high possible temperature(96 degrees C) using ammonium containing parent reagents. IR-spectroscopy, XRD, TG-DTA technique and mass spectrometric thermal analysis (MSTA) were employed for characterization of the samples. NH4+ with H3O+ in cationic-and CO3(2-) (A- and B-positions) with HPO4(2-) in anionic sites, and H2O, CO3(2-)(HCO3(-)) NO3(-), NxHy on the surface of particles were found and considered as impurity groups. Complicated changes in lattice constants of theSCHA stepwise annealed in air (for 2 h) were revealed; the changes were associated with reactions of the impurity groups. Filling the hexed sites with hydroxyl ions above 500 degrees C was shown to happen partly due to lattice reactions but was mainly owing to hydrolysis of the SCHA by water molecules in air. Decomposition of CO3(2-) groups proceeded through both thermal destruction and reactions with some of the impurity ions. The decarbonation in A-sites occurred at much lower temperatures (450-600 degrees C) than in B-sites (700-950 degrees C) and was first revealed to happen in two stages: due to an impurity reaction around 500 degrees C, and then through thermal destruction at 570 degrees C. A redistribution of CO3(2-) ions, decreasing in amount on the whole, was observed upon annealing above 500 degrees C. To avoid possible erroneous conclusions from TG-data, a sensitive method was shown to be required for monitoring gaseous decomposition products (such as the MSTA in this study), in case several impurity groups were present in a SCHA.
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Materiales Biocompatibles/química , Carbono/química , Durapatita/química , Ensayo de Materiales , Modelos Químicos , Modelos Moleculares , Materiales Biocompatibles/análisis , Carbono/análisis , Durapatita/análisis , Calor , Conformación MolecularRESUMEN
Epsilon sequence (UUAACUUUA) has originally been found in the bacteriophage T7 gene 10 leader region. It enhances translation in Escherichia coli via base pairing with nucleotides 458-466 located in the helical domain #17 of 16S rRNA. We have recently reported that when the complementarity to 16S rRNA is extended, the epsilon is converted from an enhancer to an independent initiator of translation. Here we report the effect of two other structural parameters, positioning in mRNA and the degree of complementarity to 16S rRNA on the translation initiation activity of epsilon in E. coli cells. Our results show that epsilon displays maximal activity as a translational initiator at its natural 9-nucleotide-long complementarity to 16S rRNA and at a 16-nucleotide-long distance to the initiation codon. Under these conditions its efficiency is comparable with that of the consensus Shine-Dalgarno sequence.
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Cloranfenicol O-Acetiltransferasa/genética , Escherichia coli/genética , Iniciación de la Cadena Peptídica Traduccional/genética , ARN Bacteriano/genética , ARN Mensajero/genética , Secuencia de Bases , Codón Iniciador/genética , Secuencia de Consenso , Genes Reporteros , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Mensajero/química , ARN Ribosómico 16S/genéticaRESUMEN
Epsilon (epsilon) sequence is a bacterial enhancer of translation found in the bacteriophage T7 gene 10. It is believed that its enhancing effect of epsilon is due to a base-pairing with the nucleotides 458-467 from the helical domain 17 of Escherichia coli 16S rRNA. To prove this we have taken advantage of the difference of this domain in Agrobacterium tumefaciens and E. coli. To evaluate the significance of nucleotide complementarity for the enhancing activity of epsilon, a series of nucleotide sequences matching either E. coli or A. tumefaciens domain 17 are cloned in a binary expression vector in front of the chloramphenicol acetyltransferase (CAT) gene. The CAT assay shows that: (i) the epsilon in combination with an SD consensus sequence increases the yield of CAT in both microorganisms over that obtained with the SD alone; (ii) the epsilon sequence complementary to the A. tumefaciens domain 17 leads to a 2.71-fold increase in the yield of CAT in homologous cells but not in E. coli cells; (iii) the yield of CAT correlates with the free energy of base-pairing with the helical domain 17 in both microorganisms.
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Agrobacterium tumefaciens/genética , Elementos de Facilitación Genéticos , Escherichia coli/genética , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , ADN Bacteriano , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genéticaRESUMEN
Although Mg2+ is an important cofactor for the specific degradation of RNA by ribozymes, it is not considered as a typical chemical nuclease. In this study we show that in combination with common buffers such as tris(hydroxymethyl)aminomethane and sodium borate, Mg2+ is a powerful catalyst for the degradation of RNA. pH and temperature are found to be the principal factors for the efficient degradation of RNA. Whereas in Tris-HCl/Mg2+ the efficient cleavage starts at pH values higher than 7.5 and temperatures higher than 37 degrees C, in sodium borate RNA degradation begins at pH 7.0 and at 37 degrees C. RNA hydrolysis promoted under the combined catalytic activity of buffer/Mg2+ results in partially degraded RNA and negligible amounts of acid-soluble material. Reaction is insensitive to the concentration of monovalent cations but is completely prevented by chelating agents (EDTA and citrate) at concentrations exceeding that of Mg2+. Borate-magnesium reaction is inhibited also by some polyvalent alcohols (glycerol) and sugars.
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Magnesio/química , ARN/química , Tampones (Química) , Catálisis , HidrólisisRESUMEN
The efficiency of a novel non-Shine-Dalgarno translational initiator (ACCUACUCGAGUUAG, denoted PL) to promote translation in Escherichia coli was compared with that of the Shine-Dalgarno (SD) consensus sequence (AAGGAGGU) using four reporter genes. The obtained results showed that the genes of pokeweed antiviral protein (PAP I) and human calcitonin (CT) were poorly expressed under the conventional SD and were better expressed under the PL sequence. On the contrary, the genes of human interferon gamma (hIFN gamma) and chloramphenicol acetyltransferase (CAT) were highly expressed under SD and poorly expressed under the PL sequence. Computer search revealed a great diversity between the four reporter genes in respect to their complementarity to E. coli 16S rRNA. PAP I and CT genes were rich in nucleotides matching 16S rRNA (called downstream boxes) whereas the complementary domains in the other two (hIFN-gamma and CAT) genes were much shorter. The different behavior of the four reporter genes when placed under the translational control of SD and PL sequences was explained by the different binding energy of their mRNAs to the 30S ribosomal subunit.
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Secuencia de Consenso/fisiología , Escherichia coli/metabolismo , Regulación de la Expresión Génica/genética , N-Glicosil Hidrolasas , Biosíntesis de Proteínas/genética , Calcitonina/biosíntesis , Calcitonina/genética , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/genética , Secuencia de Consenso/genética , Escherichia coli/genética , Genes Reporteros/genética , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Inactivadoras de Ribosomas Tipo 1RESUMEN
Some physics during heating and sintering of powder pressings of hydroxyapatite (HA) under conventional (usual) conditions have been studied. It is revealed that heating and firing of the pressings of a middle-dispersity powder are accompanied by release of gases. The gas release hinders and can stop the shrinkage (sintering). The microhardness is low and has a complicated distribution on the surface and in the volume of ceramics. A slight degassing (drying) of the pressing before sintering heightens the density and improves the microhardness characteristics of the ceramics. The shrinkage stop effect is eliminated in pressings of a fine powder. On the basis of the results, a technique and some methods for quality improvement of ceramics were proposed and approbated. They consisted of the manufacture of samples of a mixture of two powders with different dispersity, use of a press technique with two male dies, thermal treatment of pressing before sintering, and the choice of moderate sintering conditions. The resulting ceramics had a density close to the theoretical, almost homogeneous microhardness distribution in the sample and much higher values of microhardness and compressive strength.
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Materiales Biocompatibles , Cerámica , Durapatita , Materiales Biocompatibles/química , Materiales Biocompatibles/aislamiento & purificación , Cerámica/química , Cerámica/aislamiento & purificación , Durapatita/química , Durapatita/aislamiento & purificación , Dureza , Tamaño de la Partícula , Polvos , Espectrofotometría InfrarrojaRESUMEN
Inhomogeneous ceramics of hydroxyapatite (HA) were prepared by sintering briquettes in which an inhomogeneous distribution of density was made by pressing HA powder into a die with rough walls. The resulting sample of such a ceramic is a hard thin shell with a loose core, and it is characterized by an inhomogeneous macro- and microstructure. It is a sintered conglomerate from HA grains containing grain boundary macropores in contact with the surface and micropores located inside the grains, part of which are also associated with the free surface. The highest value of microhardness is fixed on the surface of the sample. The radial distribution of microhardness in the (cylindrical) sample has an axisymmetric, nonmonotonic character and, on the whole, shows the decrease of microhardness (the increase of porosity) from the surface to the center. The highest values of microhardness, crushing strength, and fracture strength are close to those known for ceramics of moderate strength.
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Materiales Biocompatibles/química , Cerámica/química , Hidroxiapatitas/química , Dureza , Porosidad , Polvos , Espectrofotometría Infrarroja , Propiedades de SuperficieRESUMEN
Pokeweed (Phytolacca americana) antiviral protein (PAP) is a highly specific ribosome-inactivating glycosidase. The PAP gene was isolated and cloned in an expression vector containing a polylinker-derived sequence (PL) but devoid of a Shine-Dalgarno (SD) sequence. Surprisingly, E. coli cells transformed with this vector produced over twice the amount of PAP than that with the consensus SD sequence. Computer analysis of the 5' terminal region of PAP mRNA revealed a nucleotide sequence (ACCUACUCGAGUUAG) which was complementary to two domains in 16S rRNA. The heptanucleotide ACCUACU (box I) is complementary to nucleotides 1434-1440 and the GAGUUAG (box II) to nucleotides 507-513 in 16S rRNA of E. coli. To examine the role of this sequence in the translation of PAP mRNA, single or both boxes were mutated and the protein yield was measured. Mutation of box I and of box II resulted in a 2.7 and 5.3 fold decrease in protein yield respectively, indicating that the PAP gene expression was dependent on the presence of both boxes. To investigate whether PL also increases expression of other genes, human calcitonin monomeric and tetrameric genes were used as reporters. It was found that the expression level was doubled compared to that by SD. These results demonstrate that the PL is an efficient translational initiator and may be used for high level expression of certain genes in E. coli. The possible mechanisms for the high level expression are discussed.
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Escherichia coli/genética , N-Glicosil Hidrolasas , Proteínas de Plantas/genética , Biosíntesis de Proteínas , Animales , Secuencia de Bases , Calcitonina/genética , Clonación Molecular , Genes Reporteros , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Mutación , Plásmidos/genética , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 16S/genética , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Inactivadoras de Ribosomas Tipo 1RESUMEN
Pokeweed (Phytolacca americana) antiviral protein (PAP) is a glycosidase which inactivates both eukaryotic and prokaryotic ribosomes. Due to this activity the wild-type PAP gene encoding mature protein has not so far been expressed in Escherichia coli. In spite of the ribosome impairing activity of the pre-PAP (containing two signal peptides at both termini) on bacterial ribosomes in vitro, the full-length PAP gene has been expressed successfully, although at a low level in E. coli under an inducible lac promoter. In this study we show that the full-length PAP gene, but not the PAP gene devoid of the N-terminal signal peptide codons, can be expressed constitutively in E. coli cells to produce a much higher yield as compared with the inducible expression. The full-length PAP is biologically active and it accumulates as inclusion bodies in bacterial cytoplasm. RNA analysis together with protein measurements show that the PAP gene is poorly transcribed and the PAP mRNA is poorly translated when a lac operator sequence is placed in front of the Shine/Dalgarno (SD) sequence. Nucleotide folding analysis of the 5' untranslated mRNA revealed that the SD sequence in the presence of a lac operator is involved in a stable secondary structure, whereas it is more relaxed in the mRNA transcribed from the constitutive vector. These results provide evidence that the low expression level of full-length PAP gene is due to inefficient transcription and translation but not to the toxicity of the expressed PAP.
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Escherichia coli/genética , Expresión Génica , N-Glicosil Hidrolasas , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , beta-Galactosidasa/genética , Secuencia de Bases , Clonación Molecular , Citoplasma/química , Escherichia coli/efectos de los fármacos , Escherichia coli/ultraestructura , Vectores Genéticos , Conformación de Ácido Nucleico , Proteínas de Plantas/farmacología , Plásmidos/genética , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas Recombinantes , Mapeo Restrictivo , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas/efectos de los fármacos , Transcripción GenéticaRESUMEN
Pokeweed antiviral protein (PAP) from Phytolacca americana is a highly specific N-glycosidase removing adenine residues (A4324 in 28S rRNA and A2660 in 23S rRNA) from intact ribosomes of both eukaryotes and prokaryotes. Due to the ribosome impairing activity the gene coding for mature PAP has not been expressed so far in bacteria whereas the full-length gene (coding for the mature 262 amino acids plus two signal peptides of 22 and 29 amino acids at both N- and C-termini, respectively) has been expressed in Escherichia coli. In order to determine: 1) the size of the N-terminal region of PAP which is required for toxicity to E. coli; and 2) the location of the putative enzymatic active site of PAP, 5'-terminal progressive deletion of the PAP full-length gene was carried out and the truncated forms of the gene were cloned in a vector containing a strong constitutive promoter and a consensus Shine-Dalgarno ribosome binding site. The ribosome inactivation or toxicity of the PAP is used as a phenotype characterized by the absence of E. coli colonies, while the mutation of PAP open reading frames in the small number of survived clones is used as an indicator of the toxicity to E. coli cells. Results showed that the native full-length PAP gene was highly expressed and was not toxic to E. coli cells although in vitro ribosome inactivating activity assay indicated it was active. However, all of the N-terminal truncated forms (removal of seven to 107 codons) of the PAP gene were toxic to E. coli cells and were mutated into either out of frame, early termination codon or inactive form of PAP (i.e., clone PAP delta107). Deletion of more than 123 codons restored the correct gene sequence but resulted in the loss of the antiviral and ribosome inactivating activities and by the formation of a large number of clones. These results suggest that full-length PAP (with N- and C-terminal extensions) might be an inactive form of the enzyme in vivo presumably by inclusion body formation or other unknown mechanisms and is not toxic to E. coli cells. However, it is activated by at least seven codon deletions at the N-terminus. Deletions from seven through to 107 amino acids were lethal to the cells and only mutated forms (inactive) of the gene were obtained. But deletion of more than 123 amino acids resulted in the loss of enzymatic activity and made it possible to express the correct PAP gene in E. coli. Because deletion of Tyr94 and Val95, which are involved in the binding of the target adenine base, did not abolish the activity of PAP, it is concluded that the location previously proposed for PAP enzymatic active site should be reassessed.
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Escherichia coli/genética , N-Glicosil Hidrolasas , Proteínas de Plantas/genética , Antivirales/química , Antivirales/toxicidad , Secuencia de Bases , Clonación Molecular , ADN Complementario , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/toxicidad , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Proteínas Inactivadoras de Ribosomas Tipo 1 , Eliminación de SecuenciaRESUMEN
A non-Shine-Dalgamo translational initiator is identified in Escherichia coli. The nucleotide sequence ACCUACUCGAGUUAG, designated as PL, is capable of initiating translation of pokeweed antiviral protein (PAP) and human calcitonin (hCT) mRNAs in E. coli cells. The yield of recombinant protein was double that obtained with the consensus Shine-Dalgarno-sequence-(SD)-driven translation. The PL sequence is composed of two heptanucleotides (ACCUACU, box I and GAGUUAG, box II) which are complementary to nucleotides 1434-1440 and 507-513, respectively, in 16S rRNA. Mutational analysis shows that the translation initiation efficiency with either box alone is much lower than that obtained with the entire PL sequence, indicating that the boxes interact simultaneously with both complementary regions in 16S rRNA during the translation initiation step. Based on these results, we propose that the two widely separated regions 507-513 (part of helical domain 18) and 1434-1440 (belonging to helical domain 44) are organized in close proximity to each other and to the ribosome decoding center on the surface of the E. coli 30S ribosomal subunit.
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Escherichia coli/genética , N-Glicosil Hidrolasas , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Secuencia de Bases , Calcitonina/biosíntesis , Calcitonina/genética , Escherichia coli/metabolismo , Expresión Génica , Genes Reporteros , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Biosíntesis de Proteínas , ARN Bacteriano/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 16S/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas/química , Ribosomas/genéticaRESUMEN
Epsilon (epsilon) sequence [UUAACUUUA, complementary to nucleotides 458-466 of the 16S ribosomal RNA (rRNA)] which is naturally occurring at the 5'-untranslated leader of phage T7 gene 10 mRNA was originally described as a powerful translational enhancer in Escherichia coli. Recent studies with this sequence led to controversial conclusions about its translational initiation and enhancing capability. In this study different sequence derivatives of epsilon were constructed to evaluate its efficiency not only to enhance translation of the chloramphenicol acetyltransferase (CAT) mRNA in E. coli, but also to function as an independent initiator of translation. It was observed that the epsilon sequence in combination with the CAT natural Shine-Dalgarno (SDn) or the SD consensus sequences enhanced, as expected, the translation of CAT mRNA. The natural epsilon sequence without an SD sequence failed to initiate or enhance the translation of CAT mRNA. However, when the complementarity of epsilon to 16S rRNA was increased from 9 to 16 nucleotides, epsilon alone (without the SD sequence) became an independent translational initiator with an efficiency of about 80% that obtained with the SD consensus sequence.
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Bacteriófago T7/genética , Cloranfenicol O-Acetiltransferasa/genética , Escherichia coli/genética , Iniciación de la Cadena Peptídica Traduccional , Secuencia de Bases , ADN Viral/genética , Regulación Bacteriana de la Expresión Génica , Regulación Viral de la Expresión Génica , ARN Mensajero/genética , ARN Ribosómico 16S/genéticaRESUMEN
The human interferon (hIFN alpha 1) gene contains 11 arginine (Arg) codons AGG or AGA, which are extremely rare for bacteria, four of which are organized in tandems. The two AGG tandems (corresponding to Arg12 Arg13 and Arg163 Arg164) are known to inhibit the translation of hIFN alpha 1 mRNA and therefore they are considered to be responsible for the poor expression of hIFN alpha 1 gene in bacterial cells. To study the effect of these two tandems on the expression of hIFN alpha 1 in E. coli, four new gene variants were designed to contain preferential Arg codons (CGT) substituted for the rare AGG codons in either the first, the second or both AGG tandems. We found that, whereas the yield of hIFN alpha 1 protein per cell remained unchanged, the level of hIFN alpha 1 mRNA decreased gradually (by a factor of two) with the consecutive substitution of the first, second and both AGG tandems. These results indicated, first, that the AGG clusters might have a stabilizing effect on the mRNA, and second, that mRNAs devoid of such clusters were translated at a higher rate in vivo. The protein products of the four genes (having the same amino acid sequence) showed different specific antiviral activity. The most active was the product of gene hIFN alpha 1(c) in which the second AGG tandem (corresponding to Arg163, Arg164) was preserved while the least active was the protein of gene hIFN alpha 1(d) (devoid of both AGG clusters). The role of the AGG tandems in folding of the gene product is discussed.
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Codón , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Interferón-alfa/genética , Familia de Multigenes , Arginina/genética , Humanos , Interferón Tipo I/biosíntesis , Interferón Tipo I/genética , Interferón-alfa/biosíntesis , Proteínas RecombinantesRESUMEN
Six new asymmetric monomethine cyanine dyes have been synthesized and their fluorescence characteristics in the presence of nucleic acids studied. The new dyes have no fluorescence of their own in water solutions upon excitation at 480 nm but they become strongly fluorescent in the presence of nucleic acids. The fluorescence maxima of the investigated dyes are found at 525-545 nm when bound to dsDNA and around 600 nm upon binding to RNA and ssDNA. Fluorescence quenching studies with increasing concentrations of NaCl indicate that the cyanine dyes have a mixed (intercalating and groove binding) type of interaction with dsDNA.
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ADN/química , Colorantes Fluorescentes/química , Sustancias Intercalantes/química , Tiazoles/química , Soluciones , Espectrometría de FluorescenciaRESUMEN
Human interferon-alpha 1 (HuIFN-alpha 1) gene containing signal peptide codons is poorly expressed in bacteria, and this is explained by the presence of clusters of rare (AGG) arginine codons in its structure. In this study, we have constructed a series of modified HuIFN-alpha 1 genes to study the effect of both residual signal peptide codons and clusters of AGG codons on gene expression in Escherichia coli cells. Our results showed that substitution of preferential for rare arginine codons in two clusters did not affect the yield, whereas deletion of the signal peptide codons led to a 10-fold increase in the yield of recombinant protein. To understand the mechanism of interference of gene structure on the expression of the HuIFN-alpha 1 gene in vivo, both the level and stability of HuIFN-alpha 1 mRNA were measured. The amount of HuIFN mRNA increased almost five times on deletion of the signal peptide codons from HuIFN-alpha 1 gene constructs (containing AGG clusters or not). The stability of mRNA obtained from all gene constructs was shown to be the same (half-life of 60 +/- 5 secs), indicating that the signal peptide codons interfere with both the efficiency of transcription of the HuIFN-alpha 1 gene and translation of its mRNA.