RESUMO
Mast cells (MCs) are primary effector cells involved in immediate allergic reactions. Mas-related G protein-coupled receptor-X2 (MrgX2), which is highly expressed on MCs, is involved in receptor-mediated drug-induced pseudo-anaphylaxis. Many small-molecule drugs and peptides activate MrgX2, resulting in MC activation and allergic reactions. Although small-molecule drugs can be identified using existing MrgX2 ligand-screening systems, there is still a lack of effective means to screen peptide ligands. In this study, to screen for peptide drugs, the MrgX2 high-affinity endogenous peptide ligand substance P (SP) was used as a recognition group to design a fluorescent peptide probe. Spectroscopic properties and fluorescence imaging of the probe were assessed. The probe was then used to screen for MrgX2 agonists among peptide antibiotics. In addition, the effects of peptide antibiotics on MrgX2 activation were investigated in vivo and in vitro. The environment-sensitive property of the probe was revealed by the dramatic increase in fluorescence intensity after binding to the hydrophobic ligand-binding domain of MrgX2. Based on these characteristics, it can be used for in situ selective visualization of MrgX2 in live cells. The probe was used to screen ten types of peptide antibiotics, and we found that caspofungin and bacitracin could compete with the probe and are hence potential ligands of MrgX2. Pharmacological experiments confirmed this hypothesis; caspofungin and bacitracin activated MCs via MrgX2 in vitro and induced local anaphylaxis in mice. Our research can be expected to provide new ideas for screening MrgX2 peptide ligands and reveal the mechanisms of adverse reactions caused by peptide drugs, thereby laying the foundation for improving their clinical safety.
Assuntos
Anafilaxia , Hipersensibilidade a Drogas , Camundongos , Animais , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/metabolismo , Ligantes , Bacitracina/metabolismo , Bacitracina/farmacologia , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Caspofungina/metabolismo , Caspofungina/farmacologia , Peptídeos/farmacologia , Antibacterianos/farmacologia , Mastócitos/metabolismo , Degranulação Celular/fisiologiaRESUMO
In this study, the antifungal mechanism of secondary metabolites from the WB strain against Fusarium oxysporum f. sp. niveum (Fon) was investigated. The WB strain induced the accumulation of reactive oxygen species in Fon hyphae and caused morphological changes, including surface subsidence and shrinkage deformation. The cell-free supernatants (CFSs) from WB treatment caused a significant increase in superoxide dismutase, catalase, peroxidase and glutathione reductase activities and the contents of soluble protein and malondialdehyde. Additionally, CFSs from WB decreased the fusaric acid concentration in Fon. Transcriptome analysis revealed that the expression of some antioxidant-related genes was upregulated and that the expression of mycotoxin-related genes was downregulated. Four polypeptide compounds from the WB strain, including iturin A, fengycin, surfactin and bacitracin, were identified by ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis and complete genome mining. RT-qPCR and a quantitative analysis confirmed that the presence of Fon induced the expression of polypeptide genes and elevated polypeptide production. The combined minimum inhibitory concentration and quantitative analysis of four polypeptides revealed that iturin A, fengycin, surfactin and bacitracin might be responsible for inhibiting the growth of Fon. In conclusion, secondary metabolites from strain WB exhibited antifungal effects on Fon by triggering oxidative stress and decreasing toxin levels.
Assuntos
Citrullus , Fusarium , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Bacillus , Bacitracina/metabolismo , Bacitracina/farmacologia , Citrullus/genética , Citrullus/metabolismo , Citrullus/microbiologia , Estresse Oxidativo , Doenças das Plantas/microbiologiaRESUMO
SignificanceMany gram-positive organisms have evolved an elegant solution to sense and resist antimicrobial peptides that inhibit cell-wall synthesis. These organisms express an unusual "Bce-type" adenosine triphosphate-binding cassette (ABC) transporter that recognizes complexes formed between antimicrobial peptides and lipids involved in cell-wall biosynthesis. In this work, we provide the first structural snapshots of a Bce-type ABC transporter trapped in different conformational states. Our structures and associated biochemical data provide key insights into the novel target protection mechanism that these unusual ABC transporters use to sense and resist antimicrobial peptides. The studies described herein set the stage to begin developing a comprehensive molecular understanding of the diverse interactions between antimicrobial peptides and conserved resistance machinery found across most gram-positive organisms.
Assuntos
Bacitracina , Farmacorresistência Bacteriana , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/metabolismo , Bacitracina/metabolismo , Bacitracina/farmacologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Endogenous Staphylococcus aureus sortase A (SrtA) covalently incorporates cell wall anchored proteins equipped with a SrtA recognition motif (LPXTG) via a lipid II-dependent pathway into the staphylococcal peptidoglycan layer. Previously, we found that the endogenous S. aureus SrtA is able to recognize and process a variety of exogenously added synthetic SrtA substrates, including K(FITC)LPMTG-amide and K(FITC)-K-vancomycin-LPMTG-amide. These synthetic substrates are covalently incorporated into the bacterial peptidoglycan (PG) of S. aureus with varying efficiencies. In this study, we examined if native and synthetic substrates are processed by SrtA via the same pathway. Therefore, the effect of the lipid II inhibiting antibiotic bacitracin on the incorporation of native and synthetic SrtA substrates was assessed. Treatment of S. aureus with bacitracin resulted in a decreased incorporation of protein A in the bacterial cell wall, whereas incorporation of exogenous synthetic substrates was increased. These results suggest that natural and exogenous synthetic substrates are processed by S. aureus via different pathways.
Assuntos
Peptidoglicano , Staphylococcus aureus , Amidas , Aminoaciltransferases , Bacitracina/metabolismo , Bacitracina/farmacologia , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases , Fluoresceína-5-Isotiocianato , Peptidoglicano/metabolismoRESUMO
Islet amyloid deposition is increasingly seen as a pathogenic feature of type 2 diabetes mellitus (T2DM), with the deposits containing the unique amyloidogenic peptide islet amyloid polypeptide (IAPP, also known as amylin). The fibril precursors of IAPP contribute to its cytotoxicity on pancreatic ß cells and be important in causing ß-cell dysfunction in T2DM. However, the development of effective this study, inhibitors against the toxicity of IAPP has been extremely challenging. We have found that pre-incubation with geniposide dose-dependently prevented human IAPP (hIAPP)-induced cell damage in INS-1E cells, and bacitracin, an inhibitor of IDE activity, prevented significantly the protective effects of geniposide in pancreatic INS-1E cells significantly. Geniposide induced the expression of insulin-degrading enzyme (IDE), a key degrading protein of hIAPP, but had no significant effect on the aggregation of hIAPP. These findings indicate that geniposide prevents hIAPP-induced cytotoxicity in INS-1E cells involving upregulation of IDE expression.
Assuntos
Apoptose/efeitos dos fármacos , Insulisina/metabolismo , Iridoides/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Substâncias Protetoras/farmacologia , Animais , Bacitracina/metabolismo , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , RatosRESUMO
In the low-G+C-content Gram-positive bacteria, resistance to antimicrobial peptides is often mediated by so-called resistance modules. These consist of a two-component system and an ATP-binding cassette transporter and are characterized by an unusual mode of signal transduction where the transporter acts as a sensor of antimicrobial peptides, because the histidine kinase alone cannot detect the substrates directly. Thus, the transporters fulfill a dual function as sensors and detoxification systems to confer resistance, but the mechanistic details of these processes are unknown. The paradigm and best-understood example for this is the BceRS-BceAB module of Bacillus subtilis, which mediates resistance to bacitracin, mersacidin, and actagardine. Using a random mutagenesis approach, we here show that mutations that affect specific functions of the transporter BceAB are primarily found in the C-terminal region of the permease, BceB, particularly in the eighth transmembrane helix. Further, we show that while signaling and resistance are functionally interconnected, several mutations could be identified that strongly affected one activity of the transporter but had only minor effects on the other. Thus, a partial genetic separation of the two properties could be achieved by single amino acid replacements, providing first insights into the signaling mechanism of these unusual modules.
Assuntos
Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bacillus subtilis/metabolismo , Farmacorresistência Bacteriana , Proteínas de Membrana Transportadoras/metabolismo , Transdução de Sinais , Bacillus subtilis/genética , Bacitracina/metabolismo , Bacteriocinas/metabolismo , Transporte Biológico Ativo , Análise Mutacional de DNA , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Mutagênese , Peptídeos/metabolismo , Transporte ProteicoRESUMO
Cell invasion by human papillomavirus type 16 (HPV16) is a complex process relying on multiple host cell factors. Here we describe an investigation into the role of cellular protein disulfide isomerases (PDIs) by studying the effects of the commonly used PDI inhibitor bacitracin on HPV16 infection. Bacitracin caused an unusual time-dependent opposing effect on viral infection. Enhanced cellular binding and entry were observed at early times of infection, while inhibition was observed at later times postentry. Bacitracin was rapidly taken up by host cells and colocalized with HPV16 at late times of infection. Bacitracin had no deleterious effect on HPV16 entry, capsid disassembly, exposure of L1/L2 epitopes, or lysosomal trafficking but caused a stark inhibition of L2/viral DNA (vDNA) endosomal penetration and accumulation at nuclear PML bodies. γ-Secretase has recently been implicated in the endosomal penetration of L2/vDNA, but bacitracin had no effect on γ-secretase activity, indicating that blockage of this step occurs through a γ-secretase-independent mechanism. Transient treatment with the reductant ß-mercaptoethanol (ß-ME) was able to partially rescue the virus from bacitracin, suggesting the involvement of a cellular reductase activity in HPV16 infection. Small interfering RNA (siRNA) knockdown of cellular PDI and the related PDI family members ERp57 and ERp72 reveals a potential role for PDI and ERp72 in HPV infection.
Assuntos
Antivirais/farmacologia , Bacitracina/farmacologia , Endossomos/efeitos dos fármacos , Papillomavirus Humano 16/efeitos dos fármacos , Secretases da Proteína Precursora do Amiloide/metabolismo , Antivirais/metabolismo , Bacitracina/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Linhagem Celular , Nucléolo Celular/metabolismo , Endocitose , Endossomos/virologia , Epitopos/imunologia , Genoma Viral , Papillomavirus Humano 16/imunologia , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/imunologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética , Substâncias Redutoras/farmacologia , Internalização do Vírus/efeitos dos fármacosRESUMO
Leishmaniasis is a major health problem worldwide and tools available for their control are limited. Effective vaccines are still lacking, drugs are toxic and expensive, and parasites develop resistance to chemotherapy. In this context, new antimicrobials are urgently needed to control the disease in both human and animal. Here, we report the enzymatic and functional characterization of a Leishmania virulence factor, Leishmania major Protein disulfide isomerase (LmPDI) that could constitute a potential drug target. LmPDI possesses domain structure organization similar to other PDI family members (a, a', b, b' and c domains), and it displays the three enzymatic and functional activities specific of PDI family members: isomerase, reductase and chaperone. These results suggest that LmPDI plays a key role in assisting Leishmania protein folding via its capacity to catalyze formation, breakage, and rearrangement of disulfide bonds in nascent polypeptides. Moreover, Bacitracin, a reductase activity inhibitor, and Ribostamycin, a chaperone activity inhibitor, were tested in LmPDI enzymatic assays and versus Leishmania promastigote in vitro cultures and Leishmania amastigote multiplication inside infected THP-1-derived macrophages. Bacitracin inhibited both isomerase and reductase activities, while Ribostamycin had no effect on the chaperone activity. Interestingly, Bacitracin blocked in vitro promastigote growth as well as amastigote multiplication inside macrophages with EC(50) values of 39 µM. These results suggest that LmPDI may constitute an interesting target for the development of new anti-Leishmania drugs.
Assuntos
Leishmania major/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Fatores de Virulência/metabolismo , Animais , Antiprotozoários/metabolismo , Bacitracina/metabolismo , Linhagem Celular , Dissulfetos/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Leishmania major/efeitos dos fármacos , Leishmania major/crescimento & desenvolvimento , Monócitos/parasitologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Dobramento de Proteína , Ribostamicina/metabolismo , Fatores de Virulência/antagonistas & inibidoresRESUMO
Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI inhibitors.
Assuntos
Ácidos/química , Proteínas de Fluorescência Verde/química , Isomerases de Dissulfetos de Proteínas/metabolismo , Bacitracina/química , Bacitracina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Desnaturação Proteica , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por SubstratoRESUMO
Peptide antibiotics are produced by a wide range of microorganisms. Most of them target the cell envelope, often by inhibiting cell wall synthesis. One of the resistance mechanisms against antimicrobial peptides is a detoxification module consisting of a two-component system and an ABC transporter. Upon the detection of such a compound, the two-component system induces the expression of the ABC transporter, which in turn removes the antibiotic from its site of action, mediating the resistance of the cell. Three such peptide antibiotic-sensing and detoxification modules are present in Bacillus subtilis. Here we show that each of these modules responds to a number of peptides and confers resistance against them. BceRS-BceAB (BceRS-AB) responds to bacitracin, plectasin, mersacidin, and actagardine. YxdJK-LM is induced by a cationic antimicrobial peptide, LL-37. The PsdRS-AB (formerly YvcPQ-RS) system responds primarily to lipid II-binding lantibiotics such as nisin and gallidermin. We characterized the psdRS-AB operon and defined the regulatory sequences within the P(psdA) promoter. Mutation analysis demonstrated that P(psdA) expression is fully PsdR dependent. The features of both the P(bceA) and P(psdA) promoters make them promising candidates as novel whole-cell biosensors that can easily be adjusted for high-throughput screening.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Farmacorresistência Bacteriana , Peptídeos/farmacologia , Transdução de Sinais , Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacitracina/metabolismo , Bacitracina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Bacteriocinas/farmacologia , Técnicas Biossensoriais , Regulação Bacteriana da Expressão Gênica , Óperon , Peptídeos/metabolismo , CatelicidinasRESUMO
The extracellular presence of antibiotics is a common threat in microbial life. Their sensitive detection and subsequent induction of appropriate resistance mechanisms is therefore a prerequisite for survival. The bacitracin stress response network of Bacillus subtilis consists of four signal-transducing systems, the two-component systems (TCS) BceRS, YvcPQ and LiaRS, and the extracytoplasmic function (ECF) sigma factor sigma(M). Here, we investigated the mechanism of bacitracin perception and the response hierarchy within this network. The BceRS-BceAB TCS/ABC transporter module is the most sensitive and efficient bacitracin resistance determinant. The ABC transporter BceAB not only acts as a bacitracin detoxification pump, but is also crucial for bacitracin sensing, indicative of a novel mechanism of stimulus perception, conserved in Firmicutes bacteria. The Bce system seems to respond to bacitracin directly (drug sensing), whereas the LiaRS TCS and sigma(M) respond only at higher concentrations and indirectly to bacitracin action (damage sensing). The YvcPQ-YvcRS system is subject to cross-activation via the paralogous Bce system, and is therefore only indirectly induced by bacitracin. The bacitracin stress response network is optimized to respond to antibiotic gradients in a way that maximizes the gain and minimizes the costs of this stress response.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacitracina/farmacologia , Proteínas de Bactérias/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Antibacterianos/metabolismo , Bacillus subtilis/genética , Bacitracina/análogos & derivados , Bacitracina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico/efeitos dos fármacos , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Teste de Complementação Genética , Hidrólise , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacosRESUMO
We described the main pathways of bacitracin (Bc) decomposition, chromatographically set the position of its major degradation products and evaluated microbiological activity of isolated components of Bc and its degradation products. All processes of Bc decomposition under stress and accelerated test conditions were monitored with HPLC, performed mainly on a new type reversed-phase (RP-18e) monolithic silica column (Chromolith) enabling fast separation times and some of them also on conventional HPLC columns. Diode array detection, preparative HPLC and FAB mass spectrometry were used for identification of individual Bc components. We found that the major decomposition mechanism in water solutions of Bc is oxidation, and in alkaline solutions, deamidation. In oxidation process the components B1, B2 and B3 and A are oxidized into their corresponding oxidative products H1, H2, H3 and F respectively by the same mechanism. A detailed study of oxidative degradation products revealed that HPLC separation with an acid mobile phase caused splitting of peaks of components H2, H3 and F into two peaks but the peak of component H1 did not split due to its special structural properties. For the component A we confirmed gradual formation of desamido product through an intermediate. We found oxidative degradation products of Bc to be relatively stable, and desamido degradation products to be rather unstable. The estimation of kinetics of Bc decomposition was presented with a semi-quantitative model. Microbiological activity of individual isolated active components of Bc was established and the negligible antimicrobial activity of the degradation products was confirmed.
Assuntos
Antibacterianos/química , Bacitracina/química , Peptídeos/química , Amidas/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacitracina/metabolismo , Bacitracina/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Estabilidade de Medicamentos , Fermentação , Ácido Clorídrico/química , Ácido Clorídrico/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Espectrometria de Massas/métodos , Testes de Sensibilidade Microbiana/métodos , Micrococcus luteus/efeitos dos fármacos , Micrococcus luteus/crescimento & desenvolvimento , Micrococcus luteus/metabolismo , Estrutura Molecular , Oxirredução , Peptídeos/metabolismo , Peptídeos/farmacologia , TemperaturaRESUMO
PURPOSE: To verify or refute the mechanism of permeation enhancement with thiolated polymers via GSH by the use of NaFlu as marker for the paracellular permeation. METHODS: The capability of 0.5% polycarbophil cysteine conjugate (PCP-Cys) to reduce 0.02% oxidized glutathione (GSSG) was evaluated via iodometric titration in aqueous solution. Glutathione in its reduced form (GSH; 0.1%-0.4%) and in combination with 0.5% PCP-Cys were tested for their permeation enhancement of sodium fluorescein (NaFlu) and fluorescence labeled bacitracin (bac-FITC) used as paracellular markers. Permeation studies across guinea pig duodenum were carried out in Ussing-type chambers. Opening of the tight junctions was additionally monitored by transepithelial electrical resistance (TEER) measurements. RESULTS: PCP-Cys (0.5%) was shown to reduce 22.0%+/-8.2% of GSSG (0.02%) to GSH in aqueous solution at pH 7.0 and 37 degrees C within 3 h. Permeation of NaFlu was shown to depend on the concentration of GSH. The apparent permeability coefficient (Papp) of NaFlu in buffer only was 4.98+/-0.5*10(-6), while in the presence of 0.4% GSH a Papp of 9.31+/-0.92*10(-6) was achieved, representing an enhancement ratio (R = Papp enhancer system/Papp control) of 1.86. The combination of GSH (0.4%) with PCP-Cys (0.5%) led to a significant (p < 0.001) improvement of R for NaFlu up to 2.93 accompanied by a decrease in TEER of 20.3%+/-1.4%. Incubation of bac-FITC with the same GSH/PCP-Cys combination led to an enhancement ratio of 2.06 within 3 h. CONCLUSION: GSH plays an important role in the opening of tight junctions of intestinal epithelia. It would appear that PCP-Cys is able to reduce GSSG, prolonging the concentration of GSH at the apical membrane, resulting in significantly enhanced paracellular transport.
Assuntos
Resinas Acrílicas/farmacologia , Cisteína/farmacologia , Glutationa/metabolismo , Resinas Acrílicas/química , Animais , Bacitracina/metabolismo , Transporte Biológico , Cisteína/química , Portadores de Fármacos , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Fluoresceína/química , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Glutationa/química , Dissulfeto de Glutationa/metabolismo , Cobaias , Técnicas In Vitro , Absorção Intestinal , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Oxirredução , Permeabilidade , Junções Íntimas/fisiologiaRESUMO
In order to facilitate production and secretion of a soluble form of a small, single-chain antibody ScFv (32 kDa) in tobacco cell suspension culture, several modifications were made simultaneously to the antibody cDNA that included elements that have been shown to regulate the expression of proteins in plants. The scFv cDNA was initially ligated into a binary vector under the control of the CaMV 35S promoter and the T7 terminator for expression in tobacco suspension culture. Subsequently, modifications were engineered into the cDNA for enhancement of scFv production. These included the following: (i) the signal peptide (SP) of the tobacco pathogenesis-related protein PR1a which was added in-frame to the N-terminal end of scFv cDNA; (ii) a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence), which was fused to the N-terminal end of the SP; and (iii) the endoplasmic reticulum retention signal peptide KDEL, which was added to the C-terminal end of the scFv protein. Using a modified disruption method involving pectinase, the highest expression of total scFv (344 ng scFv/g cell) occurred when the plant leader sequence, the TEV sequence, and the KDEL peptide were all present in the expression construct. Although the addition of the KDEL sequence significantly increased the total yield of protein 5.4-fold, it did not increase the overall amount of protein secreted. These studies indicate that while the SP is very important in promoting secretion of the scFv, it had little influence on increasing scFv secretion levels even when both the TEV and the KDEL sequences significantly increased overall protein levels.
Assuntos
Genes Reguladores , Região Variável de Imunoglobulina/genética , Nicotiana/genética , Animais , Bacitracina/metabolismo , Células Cultivadas , Região Variável de Imunoglobulina/biossíntese , Camundongos , Poligalacturonase/metabolismo , TransgenesAssuntos
Cisteína Endopeptidases/isolamento & purificação , Entamoeba histolytica/enzimologia , Sequência de Aminoácidos , Animais , Bacitracina/metabolismo , Western Blotting , Cromatografia de Afinidade/métodos , Cromatografia em Agarose/métodos , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Dados de Sequência Molecular , Análise de Sequência de ProteínaRESUMO
Se evaluó el efecto promotor de crecimiento de la bacitracina zinc en tilapia híbrida, para lo cual se utilizaron 40 tilapias híbridas divididas en cuatro lotes: 1, 2, 3 y el testigo. A todos los peces se les suministró alimento balanceado empastillado, al que se le adicionó 100 mg, y 150 mg de bacitracina zinc por kilogramo de alimento respectivamente para los tres primeros lotes; al lote testigo se le suministró alimento sin promotor de crecimiento. los resultados revelan que hubo diferencias significativas al menos en un tratamiento (P > .05 < .10), para los lotes con mayor ganancia de peso (lote 2 y testigo) se tuvo como resultado (P<.39). Se concluye que la inclusión de bacitracina zinc como promotor de crecimiento a las dosis antes indicadas, no tuvo efecto en la especie tratada.
Assuntos
Animais , Bacitracina/administração & dosagem , Aumento de Peso/efeitos dos fármacos , Peixes/crescimento & desenvolvimento , Bacitracina/metabolismo , Peixes/metabolismo , Ração Animal/análise , Ração Animal , Produção de Alimentos/economiaRESUMO
The degradation of the neuropeptide galanin(1-29) and its fully active synthetic N-terminal fragment galanin(1-16) in hypothalamic tissue, where these peptides potently affect feeding behaviour, is studied. Galanin(1-29) had a half-life of 100 min while galanin(1-16) had a half-life of 28 min when incubated with a hypothalamic membrane preparation. The putative sites of peptidolytic cleavage of the active N-terminal fragment galanin(1-16) were determined as being between amino acids Leu4 and Asn5, between Asn5 and Ser6, and between His14 and Ala15, respectively. The synthetic analogs of galanin(1-16) where Leu4, Asn5 or Ser6 was substituted by Ala were all more stable to peptidolysis; [Ala4]galanin(1-16) had a half-life of 55 min. Cleavage of the galanin(1-16) between His14-Ala15 yields a ligand-galanin(1-14) which binds to the receptor with high affinity (KD approximately 10(7) M), while cleavage at amino acid residues Leu4, Asn5 and Ser6 results in inactive peptide fragments with affinities for the galanin receptor below 10(-4) M. The enzyme(s) responsible for degradation of galanin were identified as endopeptidase(s), which were partially inhibited by bacitracin (1 mg/ml) by up to 50%, but not significantly by EDTA (1 mM), phosphoramidon (1 microM), phenylmethylsulfonyl fluoride, (100 microM) or aprotinin (10 micrograms/ml).
Assuntos
Hipotálamo/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Bacitracina/metabolismo , Cromatografia Líquida de Alta Pressão , Galanina , Meia-Vida , Técnicas In Vitro , Ligantes , Masculino , Dados de Sequência Molecular , Inibidores de Proteases/farmacologia , Ratos , Ratos Endogâmicos , SuínosRESUMO
The elimination in cow's milk of polypeptidic antibiotic residues (colistin sulfate, colistin methanesulfonate and bacitracin) was investigated following intramuscular and intramammary administrations of eight drugs marketed in France. The quantitative analysis was performed according to the microbiological method of agar diffusion. The mean elimination periods lasted between three and six milkings for injected preparations, between four and six milkings for intramammary ones. These results were used as a basis to determine the withdrawal times required on these drugs. The problem of the diffusion of the residues into milk from a quarter treated by the intramammary route, to the untreated quarters was also addressed.
Assuntos
Bacitracina/metabolismo , Bovinos/metabolismo , Colistina/metabolismo , Resíduos de Drogas/metabolismo , Leite/análise , Animais , Bacitracina/administração & dosagem , Bacitracina/análise , Colistina/administração & dosagem , Colistina/análogos & derivados , Colistina/análise , Feminino , Injeções Intramusculares , Glândulas Mamárias Animais/metabolismoRESUMO
A selective medium was developed on which both major subgroups of Strep. mutans (c/e/f and d/g) can be cultured and recognized on the basis of colonial morphology, Trypticase, yeast, cystine (TYC) agar was modified by adding separately autoclaved sucrose to a final concentration of 20 per cent and 0.1 unit/ml bacitracin (TYCSB). This medium yielded significantly higher counts of Strep. mutans than the widely used mitis-salivarius bacitracin (MSB) medium.