RESUMO
Microorganisms residing within a biofilm become more tolerant to antibiotics and other types of adverse impact, and biofilm formation by pathogenic bacteria is an important problem of current medicine. Polysaccharides that prevent biofilm formation are among the promising candidates to help tackle this problem. Earlier we demonstrated the ability of a potato polysaccharide galactan to inhibit biofilm formation by a Pseudomonas aeruginosa clinical isolate. Here we investigate the effect of potato galactan on P. aeruginosa biofilms in more detail. Microscopic analysis indicated that the galactan did not interfere with the adhesion of bacterial cells to the substrate but prevented the build-up of bacterial biomass. Moreover, the galactan not only inhibited biofilm formation, but partially destroyed pre-formed biofilms. Presumably, this activity of the galactan was due to the excessive aggregation of bacterial cells, which prohibited the formation and maintenance of proper biofilm architecture, or due to some other mechanisms of biofilm structure remodeling. This led to an unexpected effect, i.e., P. aeruginosa biofilms treated with an antibiotic and the galactan retained more viable bacterial cells compared to biofilms treated with the antibiotic alone. Galactan is the first polysaccharide demonstrated to exert such effect on bacterial biofilms.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Galactanos/farmacologia , Pseudomonas aeruginosa/fisiologia , Aderência Bacteriana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microscopia de Fluorescência , Pseudomonas aeruginosa/crescimento & desenvolvimento , Solanum tuberosum/metabolismoRESUMO
The aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (EPO) with additional protein domains: 6His-s-tag-EPO protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) at the N-terminus and HBD-EPO and EPO-HBD proteins containing heparin-binding protein domains (HBD) of the bone morphogenetic protein 2 from Danio rerio at the N- and C-termini, respectively. The commercial preparation Epostim (LLC Pharmapark, Russia) produced by synthesis in Chinese hamster ovary cells was used for comparison. The EPO variant with the C-terminal HBD domain connected by a rigid linker (EPO-HBD) possesses the best properties as compared to HBD-EPO with the reverse domain arrangement. It was ~13 times more active in vitro (i.e., promoted proliferation of human erythroleukemia TF-1 cells) and demonstrated a higher rate of association with the erythropoietin receptor. EPO-HBD also exhibited the greatest binding to the demineralized bone matrix (DBM) and more prolonged release from the DBM among the four proteins studied. Subcutaneous administration of EPO-HBD immobilized on DBM resulted in significantly more pronounced vascularization of surrounding tissues in comparison with the other proteins and DBM alone. Therefore, EPO-HBD displayed better performance with regard to all the investigated parameters than other examined EPO variants, and it seems promising to study the possibility of its medical use.
Assuntos
Eritropoetina/genética , Escherichia coli/genética , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Animais , Matriz Óssea/metabolismo , Proteína Morfogenética Óssea 2/genética , Proliferação de Células/efeitos dos fármacos , Eritropoetina/biossíntese , Escherichia coli/metabolismo , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Ligação Proteica , Proteínas Recombinantes/biossíntese , Peixe-ZebraRESUMO
Using mouse model of regeneration of critical size cranial defects, we studied combined effect of 1 and 10 µg of BMP-2 of prokaryotic origin and recombinant erythropoietin (Epostim) injected subcutaneously in the area of bone defect in a total dose of 6000 U/kg. Erythropoietin considerably improved quantitative and qualitative characteristics of the bone tissue in the site of implantation when used in combination with BMP-2 in both concentrations.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea/fisiologia , Eritropoetina/farmacologia , Crânio/crescimento & desenvolvimento , Animais , Regeneração Óssea/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos ICR , Crânio/anormalidadesRESUMO
BACKGROUND: Restriction-modification (R-M) systems protect bacteria and archaea from attacks by bacteriophages and archaeal viruses. An R-M system specifically recognizes short sites in foreign DNA and cleaves it, while such sites in the host DNA are protected by methylation. Prokaryotic viruses have developed a number of strategies to overcome this host defense. The simplest anti-restriction strategy is the elimination of recognition sites in the viral genome: no sites, no DNA cleavage. Even a decrease of the number of recognition sites can help a virus to overcome this type of host defense. Recognition site avoidance has been a known anti-restriction strategy of prokaryotic viruses for decades. However, recognition site avoidance has not been systematically studied with the currently available sequence data. We analyzed the complete genomes of almost 4000 prokaryotic viruses with known host species and more than 17,000 restriction endonucleases with known specificities in terms of recognition site avoidance. RESULTS: We observed considerable limitations of recognition site avoidance as an anti-restriction strategy. Namely, the avoidance of recognition sites is specific for dsDNA and ssDNA prokaryotic viruses. Avoidance is much more pronounced in the genomes of non-temperate bacteriophages than in the genomes of temperate ones. Avoidance is not observed for the sites of Type I and Type IIG systems and is very rarely observed for the sites of Type III systems. The vast majority of avoidance cases concern recognition sites of orthodox Type II restriction-modification systems. Even under these constraints, complete or almost complete elimination of sites is observed for approximately one-tenth of viral genomes and a significant under-representation for approximately one-fourth of them. CONCLUSIONS: Avoidance of recognition sites of restriction-modification systems is a widespread but not universal anti-restriction strategy of prokaryotic viruses.
Assuntos
Enzimas de Restrição-Modificação do DNA/genética , Células Procarióticas/virologia , Vírus/genética , Bacteriófagos/genética , Composição de Bases/genética , Enzimas de Restrição do DNA/metabolismo , Bases de Dados Genéticas , Genoma ViralRESUMO
Many proteins need recognition of specific DNA sequences for functioning. The number of recognition sites and their distribution along the DNA might be of biological importance. For example, the number of restriction sites is often reduced in prokaryotic and phage genomes to decrease the probability of DNA cleavage by restriction endonucleases. We call a sequence an exceptional one if its frequency in a genome significantly differs from one predicted by some mathematical model. An exceptional sequence could be either under- or over-represented, depending on its frequency in comparison with the predicted one. Exceptional sequences could be considered biologically meaningful, for example, as targets of DNA-binding proteins or as parts of abundant repetitive elements. Several methods to predict frequency of a short sequence in a genome, based on actual frequencies of certain its subsequences, are used. The most popular are methods based on Markov chain models. But any rigorous comparison of the methods has not previously been performed. We compared three methods for the prediction of short sequence frequencies: the maximum-order Markov chain model-based method, the method that uses geometric mean of extended Markovian estimates, and the method that utilizes frequencies of all subsequences including discontiguous ones. We applied them to restriction sites in complete genomes of 2500 prokaryotic species and demonstrated that the results depend greatly on the method used: lists of 5% of the most under-represented sites differed by up to 50%. The method designed by Burge and coauthors in 1992, which utilizes all subsequences of the sequence, showed a higher precision than the other two methods both on prokaryotic genomes and randomly generated sequences after computational imitation of selective pressure. We propose this method as the first choice for detection of exceptional sequences in prokaryotic genomes.
Assuntos
Genoma Arqueal , Genoma Bacteriano , Genômica/métodos , Células Procarióticas/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Bases de Dados Genéticas , Cadeias de MarkovRESUMO
Recombinant human erythropoietin (EPO) with additional N-terminal heparin-binding protein domain (HBD) from bone morphogenetic protein 2 was synthesized in Escherichia coli cells. A procedure for HBD-EPO purification and refolding was developed for obtaining highly-purified HBD-EPO. The structure of recombinant HBD-EPO was close to that of the native EPO protein. HBD-EPO contained two disulfide bonds, as shown by MALDI-TOF mass spectrometry. The protein demonstrated in vitro biological activity in the proliferation of human erythroleukemia TF-1 cell test and in vivo activity in animal models. HBD-EPO increased the number of reticulocytes in the blood after subcutaneous injection and displayed local angiogenic activity after subcutaneous implantation of demineralized bone matrix (DBM) discs with immobilized HBD-EPO. We developed a quantitative sandwich ELISA method for measuring HBD-EPO concentration in solution using rabbit polyclonal serum and commercial monoclonal anti-EPO antibodies. Pharmacokinetic properties of HBD-EPO were typical for bacterially produced EPO. Under physiological conditions, HBD-EPO can reversibly bind to DBM, which is often used as an osteoplastic material for treatment of bone pathologies. The data on HBD-EPO binding to DBM and local angiogenic activity of this protein give hope for successful application of HBD-EPO immobilized on DBM in experiments on bone regeneration.
Assuntos
Escherichia coli/metabolismo , Domínios Proteicos/genética , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 2/química , Eritropoetina/química , Eritropoetina/genética , Eritropoetina/metabolismo , Feminino , Meia-Vida , Heparina/metabolismo , Humanos , Peptídeos/análise , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains were s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4). The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity of s-tag-BMP-2 in vivo was shown in various experimental animal models.
Assuntos
Proteína Morfogenética Óssea 2 , Escherichia coli , Expressão Gênica , Proteínas Recombinantes de Fusão , Animais , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/farmacologia , Bovinos , Linhagem Celular , Humanos , Camundongos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.
Assuntos
Eritropoetina/biossíntese , Eritropoetina/isolamento & purificação , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Cromatografia , Enteropeptidase/metabolismo , Eritropoetina/genética , Escherichia coli/genética , Expressão Gênica , Histidina , Humanos , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Oligopeptídeos , Fragmentos de Peptídeos , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Ribonuclease Pancreático/químicaRESUMO
Recombinant human bone morphogenetic protein-2 with an additional s-tag domain (s-tag-BMP-2) synthesized in E. coli is characterized by higher solubility and activity than the protein without additional s-tag domain, which increases the yield during purification and simplifies protein introduction into the osteoplastic materials. The high osteoinductivity of the demineralized bone matrix with s-tag-BMP-2 was shown on the model of regeneration of cranial defects of a critical size in mice and on the model of implantation of porous titanium matrix into defects of femoral and tibial bones in rabbits.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea/efeitos dos fármacos , Fêmur/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Crânio/efeitos dos fármacos , Tíbia/efeitos dos fármacos , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fêmur/lesões , Expressão Gênica , Implantes Experimentais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Crânio/lesões , Tíbia/lesões , Engenharia Tecidual , Alicerces Teciduais , Titânio/química , Titânio/farmacologiaRESUMO
Comparison of structures of homological proteins often helps to understand functionally significant features of these structures. This concerns not only structures of separate protein chains, but also structures of macromolecular complexes. In particular, a comparison of complexes of homologous proteins with DNA is significant for analysis of the recognition of DNA by proteins. We present program LCore for detecting geometrical cores of a family of structures; a geometrical core is a set of amino acid residues and nucleotides that disposed similarly in all structures of the family. We describe the algorithm of the program, its web interface, and an example of its application to analysis of complexes of homeodomains with DNA.
Assuntos
Substâncias Macromoleculares/química , Software , Interface Usuário-Computador , Algoritmos , DNA/química , InternetRESUMO
Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Lisostafina/farmacologia , Staphylococcus/genética , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/metabolismo , Biomassa , Clonagem Molecular , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Lisostafina/biossíntese , Lisostafina/isolamento & purificação , Peptidoglicano/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Staphylococcus/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus haemolyticus/efeitos dos fármacos , TemperaturaRESUMO
Osteoinductive characteristics of new osteoplastic materials based on demineralized bone matrix of xenogenic origin with high and controlled degree of purification were studied on the model of regeneration of critical-size cranial defects in rats using modern approaches, including histological analysis, evaluation of morphological parameters of the bone tissue obtained by micro-computed tomography, and estimation of bone tissue growth rate using in vivo fluorochrome label. Demineralized bone matrix and, to a much greater extent, its activated form containing modified recombinant growth factor rhBMP-2 with high content of the dimeric form exhibited osteoinductive activity.
Assuntos
Técnica de Desmineralização Óssea/métodos , Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Crânio/efeitos dos fármacos , Alicerces Teciduais , Animais , Materiais Biocompatíveis/farmacologia , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes , Expressão Gênica , Humanos , Proteínas Imobilizadas/biossíntese , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/farmacologia , Masculino , Multimerização Proteica , Ratos , Ratos Wistar , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Crânio/lesões , Crânio/cirurgia , Engenharia Tecidual , Microtomografia por Raio-XRESUMO
Experiments were carried out on 22 albino male Wistar rats to study the morphological peculiarities of osseointegration of titanium grafts with bioactive surface stimulated additionally with bone plastic material "Gamalant-paste-FORTE Plus" containing recombinant human bone morphogenetic protein-2 (rhBMP-2). In 9 rats the implants were placed into femoral bones after local treatment of bone canal with rhBMP-2-containing material. Another 9 animals were implanted but received no treatment, 4 rats formed the group of intact control. Zone of osseointegration was studied 4, 8 and 12 weeks after graft placement using histological and morphometric methods as well as immune histochemistry to demonstrate osteonectin, CD68, MMP-9, and TIMP-1. The study showed that preliminary treatment of bone canal with rhBMP-2-containing material preceding implant placement was accompanied by an additional osteoinductive effect. More intense and outrunning bone formation in the area of osseointegration was observed, together with remodeling and compaction of the contiguous cancellous bone, thus providing the necessary balance between MMP-9 and TIMP-1 with a high level of each factor expression.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Colágeno/farmacologia , Hidroxiapatitas/farmacologia , Implantes Experimentais , Osseointegração/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/farmacologia , Titânio , Animais , Humanos , Masculino , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologiaRESUMO
Comparative analysis of structures of complexes of homologous proteins with DNA is important in the analysis of DNA-protein recognition. Alignment is a necessary stage of the analysis. An alignment is a matching of amino acid residues and nucleotides of one complex to residues and nucleotides of the other. Currently, there are no programs available for aligning structures of DNA-protein complexes. We present the program StructAlign, which should fill this gap. The program inputs a pair of complexes of DNA double helix with proteins and outputs an alignment of DNA chains corresponding to the best spatial fit of the protein chains.
Assuntos
Algoritmos , DNA/metabolismo , Proteínas/metabolismo , Interface Usuário-Computador , DNA/química , Internet , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Proteínas/químicaRESUMO
Restriction-modification (R-M) systems are able to methylate or cleave DNA depending on methylation status of their recognition site. It allows them to protect bacterial cells from invasion by foreign DNA. Comparative analysis of a large number of available bacterial genomes and methylomes clearly demonstrates that the role of R-M systems in bacteria is wider than only defense. R-M systems maintain heterogeneity of a bacterial population and are involved in adaptation of bacteria to change in their environmental conditions. R-M systems can be essential for host colonization by pathogenic bacteria. Phase variation and intragenomic recombinations are sources of the fast evolution of the specificity of R-M systems. This review focuses on the influence of R-M systems on evolution and ecology of prokaryotes.
Assuntos
Bactérias/enzimologia , Bactérias/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , Evolução Molecular , Bactérias/virologia , Bacteriófagos/fisiologia , Genoma Bacteriano/genéticaRESUMO
The content of multipotent stromal cells (MSC) in the bone marrow and efficiency of their cloning (ECF-MSC) increased by 3 times 1 day after administration of complex S. typhimurium antigens to CBA mice, while the relative content of alkaline phosphatase-positive MSC colonies (marker of osteogenesis; P(+) colonies) decreased from 14% (control) to 3%. After administration of the complex S. typhimurium antigens to CBA mice 3 h after (or 3 h before) curettage or treatment with morphogenetic protein (BMP-2), the content of MSC and ECF-MSC decreased on the next day by ~3 times in comparison with animals receiving antigens alone and approached the control level. The relative content of P(+) colonies increased to 20 and 35%, respectively, in comparison with animals receiving antigens (3%), but was significantly lower than after curettage (34%) or BMP-2 (42%) administration. Expression of IL-1ß, IL-6, IL-12, TNF-α, and IFN-γ genes in the primary cultures of stromal bone marrow cells induced by antigen administration was suppressed, while the concentrations of IL-12 and TNF-α in the culture medium sharply decreased after antigen treatment in combination with curettage or BMP-2 administration. Administration of complex S. typhimurium antigens after pretreatment with BMP-2 (3 h before) was associated with a decrease in serum levels of IL-2, IFN-γ, IL-12, and TNF-α in mice receiving BMP-2+S. typhimurium group 4 h after treatment in comparison with the animals receiving only S. typhimurium antigens alone by 1.9, 4.4, 1.5, and 6 times, respectively, i.e. to normal level or below it, while the concentration of IL-10 increased by almost 2 times, which probably reflected anti-inflammatory properties of BMP-2. These data probably attest to competitive relations between osteogenesis and immune response at the level of MSC.
Assuntos
Antígenos de Bactérias/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2/farmacologia , Citocinas/sangue , Osteogênese/efeitos dos fármacos , Salmonella typhimurium/imunologia , Células Estromais/efeitos dos fármacos , Animais , Curetagem , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-2/sangue , Camundongos , Camundongos Endogâmicos CBA , Fator de Necrose Tumoral alfa/sangueRESUMO
Test system ELISA-BMP-2 is developed for measuring recombinant human bone morphogenetic protein-2 in human and laboratory animal serum and plasma by sandwich ELISA. The test system has been used for studies of the kinetics of bone morphogenetic protein-2 release from collagen carrier in the presence of plasma proteins.
Assuntos
Proteínas Sanguíneas/química , Proteína Morfogenética Óssea 2/química , Colágeno/química , Portadores de Fármacos/química , Animais , Coloides , Ensaio de Imunoadsorção Enzimática , Humanos , Hidrogéis , Cinética , Coelhos , RatosRESUMO
We studied the effect of BMP-2 added to the culture medium on osteogenic and proliferative properties of multipotent stromal cells (MSC) and on the expression of cytokine genes induced by immunization of experimental animals with bacterial antigens. It is shown that the presence of BMP-2 in the culture medium stimulates proliferation of bone marrow MSC and especially spleen MSC (which was seen from enlargement of MSC colonies); improves the efficiency of MSC cloning; increases osteogenic activity of mouse bone marrow MSC; induces osteogenic differentiation of splenic MSC (osteogenesis is normally not observed in the spleen); reduces the number of macrophages in cultures; inhibits synthesis of mRNA for proinflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α) that typically occurs in cultures of the bone marrow and spleen from animals immunized with S. typhimurium or group A streptococcus antigens. Bearing in mind that proinflammatory cytokines negatively affect osteogenic activity of the bone marrow, we can hypothesize that BMP-2 not only stimulates osteogenesis, but also provides optimal conditions for its realization by suppressing the expression of genes encoding these cytokines.
Assuntos
Antígenos de Bactérias/imunologia , Células da Medula Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , RNA Mensageiro/antagonistas & inibidores , Baço/efeitos dos fármacos , Animais , Antígenos de Bactérias/administração & dosagem , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Expressão Gênica , Imunização , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/biossíntese , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos CBA , Osteócitos/citologia , Osteócitos/efeitos dos fármacos , Osteócitos/imunologia , Osteogênese/efeitos dos fármacos , Cultura Primária de Células , RNA Mensageiro/biossíntese , Baço/citologia , Baço/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Recombinant plasmids containing fusion proteins composed of two different modules were constructed and expressed in Escherichia coli. The modules encoded the lactase LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus and the cellulase CelD, a cellulose-binding module (CBM) from Anaerocellum thermophilum. The CelD CBM provides a spontaneous and strong sorption of the fusion proteins onto a cellulose carrier. The enzymatic activities of both the free LacA protein and LacA-CelD CBM fusion proteins immobilized onto the cellulose carrier were assessed. The LacA activity of the fusion protein was dependent upon its position with respect to the CBM. The highest level of lactase activity and stability was observed when the lactase domain was localized at its N terminus. A continuous-flow column reactor of lactase immobilized on a cellulose carrier was constructed, and its activity was assessed. The lactose hydrolysis rate for a 150 mM (5%) solution at a flow rate of 1 reactor volume per min was 75%, which is a value optimal for further whey transformation into glucose/galactose syrup.