RESUMO
AIM: The purpose of this study was to clarify the mechanism of the antibacterial action of two high potential and natural food additives, epigallocatechin gallate (EGCg) and theaflavin-3,3'-digallate (TF3), on Clostridium perfringens. METHODS AND RESULTS: Minimal inhibitory concentrations were determined by the serial dilution method. Afterwards, the cells were treated with 250 or 1000 mg l-1 of EGCg and 125 or 500 mg l-1 of TF3 and morphological changes were observed and cell sizes were also measured under fluorescence microscopy. Our results showed that TF3 had a twice stronger antibacterial activity than EGCg against C. perfringens. Phase-contrast and fluorescence microscopy confirmed that the bacterial cells elongated without DNA segregation and septum formation in the presence of 250 mg l-1 EGCg. While in the higher concentration of EGCg and TF3, cell growth was suppressed. Bacterial cells reached to around 12 µm after the 24 h incubation with 250 mg l-1 EGCg, but the cells were shorter than the control at 1000 mg l-1 of EGCg. After washing and incubating the elongated cells in fresh medium, DNA segregated at 2 h of incubation. The average cell length decreased gradually and reached the normal size at 8 h. CONCLUSION: It seems that EGCg at a low concentration affected the proteins involved in the septum formation, DNA segregation and cell division. Furthermore, the high concentration of EGCg and TF3 seemed to cause stronger cellular damage to C. perfringens. SIGNIFICANCE AND IMPACT OF THE STUDY: These polyphenols are widely distributed in all higher plants especially in tea plants, and people tend to use natural food additives rather than synthetic ones. EGCg and TF3, as natural food additives, can prevent C. perfringens food poisoning along with other potential health benefits.
Assuntos
Antibacterianos/farmacologia , Biflavonoides/farmacologia , Catequina/análogos & derivados , Clostridium perfringens/efeitos dos fármacos , Catequina/farmacologiaRESUMO
A cryoprotective protein, HIC6, was expressed transgenically in tobacco, a cold-sensitive plant, and the localization of the protein within the cell as well as freezing tolerance of the transgenic tobacco was investigated. For constitutive expression of HIC6 in tobacco, its corresponding gene was subcloned into pBI121. Through the transformation with pBI121/hiC6, fifteen transgenic tobacco lines were acquired, out of which twelve lines expressed the HIC6 protein. None of the transgenic tobacco lines, however, showed significant differences in freezing tolerance from the control plants (wild-type and transformed with pBI121) at -1, -3, and -4 degrees C, with the exception that their freezing temperature was -2 degrees C. In order to increase the accumulation level of HIC6, pBE2113 with a stronger promoter was used. Eight lines expressed the protein out of thirteen lines transformed with pBE2113/hiC6. The accumulation levels of the protein were clearly higher in the tobacco plants transformed with pBE2113/hiC6 than in those with pBI121/hiC6. The HIC6 protein seemed to be localized in mitochondria of the transgenic tobacco plants. Freezing-tolerance tests at -1 - -4 degrees C showed that the degree of electrolyte leakage was significantly lower in the plants with pBE2113/hiC6 than in the control plants. A leaf browning observation also showed that high accumulation of HIC6 significantly suppressed injury caused by freezing to the transgenic tobacco at -3 degrees C.
Assuntos
Nicotiana/genética , Nicotiana/fisiologia , Proteínas de Algas/biossíntese , Proteínas de Algas/genética , Southern Blotting , Chlorella/enzimologia , Eletrólitos/metabolismo , Congelamento , Hibridização In Situ , Folhas de Planta/química , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Plasmídeos/genética , Frações Subcelulares/metabolismoRESUMO
The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC61) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1lambdaT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins in E. coli, then purified. The two HIC proteins were found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1 x 10(6) times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.
Assuntos
Chlorella/química , Crioprotetores/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Crioprotetores/química , Crioprotetores/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , L-Lactato Desidrogenase/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Conformação Proteica , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17 x b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp-Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-AspAsp-Val-Leu -Leu-Val- Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.
Assuntos
Bacillus cereus/enzimologia , Bacillus cereus/genética , IMP Desidrogenase/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , IMP Desidrogenase/biossíntese , IMP Desidrogenase/química , Dados de Sequência Molecular , Peso Molecular , Mutação , Fases de Leitura Aberta , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato , TemperaturaRESUMO
Monoclonal antibodies (MAbs) raised against Escherichia coli O6:H16 were screened against 15 strains of E. coli and 19 non-E. coli bacteria. A MAb-luminescence assay using MAb-5.8, which shows no cross-reactions with non-E. coli bacteria, and a photon-counting television camera were developed for rapid enumeration of E. coli O6:H16 in water. The membrane filter that retained bacteria was boiled for 5 min in a buffer and incubated with biotinylated MAb-5.8. After incubation with streptavidin-peroxidase conjugate, it was reacted with luminol-based reaction mixture. Luminous image and light intensity of the filter was recorded with a Biocell Counter. Levels of E. coli O6 higher than 7 x 10(3) CFU were detected by the MAb-luminescence assay when E. coli O6 was spotted onto the membrane filter. The sample that contained E. coli O6:H16 was filtered through a membrane filter, and the filter that retained bacteria was incubated on a filter paper soaked with nutrient broth supplemented with 0.5% NaCl at 37 degrees C for 6 h. The number of light emission points on the filter correlated well with initial E. coli O6:H16 counts within the range of 1 to 3 x 10(2) CFU. The correlation coefficient was 0.89.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Enterotoxinas/isolamento & purificação , Escherichia coli , Microbiologia da Água , Anticorpos Monoclonais , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Filtração , Técnicas Imunoenzimáticas , Luminol , Fótons , TelevisãoRESUMO
A bioluminescence assay carried out with a photon-counting TV camera was evaluated for rapid enumeration of viable bacterial counts. The test sample was filtered through a membrane filter, and the membrane filter retaining bacteria was incubated at 37 degrees C for 6 h on a filter paper soaked with nutrient broth supplemented with 0.5% NaCl. The membrane filter was then subjected to a bioluminescence reaction, and the intensity of light and numbers of light emission points on the filter were measured with a photon-counting TV camera. The light intensity measured on seven different bacteria correlated with initial viable counts; the correlation coefficient was calculated to be 0.89. The number of light emission points measured on Escherichia coli also correlated with the initial viable counts (r = 0.81) in a range from 1 to 100 CFU. Presumptive bacterial counts by the present bioluminescence assay determined on 79 samples of vegetables and 122 samples of environmental water correlated well with the viable counts obtained by the conventional plating method, with correlation coefficients of 0.87 and 0.82, respectively.
Assuntos
Contagem de Colônia Microbiana/métodos , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Verduras/microbiologia , Microbiologia da Água , Meios de Cultura , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Água Doce/microbiologia , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/crescimento & desenvolvimento , Medições Luminescentes , Filtros Microporos , Fótons , Água do Mar/microbiologia , TelevisãoRESUMO
VH (heavy-chain variable region) and VL (light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and MAb-18 which inhibited Japanese radish acid phosphatase. Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar VH and identical VL domains. Initially, the VH and VL genes were expressed in Escherichia coli as single-chain Fv (ScFv) fragments. Fragments ScFv-11 and ScFv-18, named for MAb-11 and MAb-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources. ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine. The PCR-amplified VH and VL genes were subsequently expressed separately in Escherichia coli as fusion products with glutathione S-transferase. The fusion proteins had little effect on Japanese radish acid phosphatase. Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none. Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18.
Assuntos
Fosfatase Ácida/imunologia , Inibidores Enzimáticos/imunologia , Fragmentos de Imunoglobulinas , Raízes de Plantas/enzimologia , Verduras/enzimologia , Fosfatase Ácida/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Bacteriófagos , Clonagem Molecular , Escherichia coli , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência de Aminoácidos , Baço/metabolismoRESUMO
The random amplified polymorphic DNA (RAPD) band patterns from 23 Salmonella spp. produced by use of an oligonucleotide primer (called du primer) designed on the basis of the N-terminal sequence of dulcitol 1-phosphate dehydrogenase (5'-GTGGTGACCCAGGATGGCCAGGTG-3') were different from those from 16 non-Salmonella spp. The bands at 460 and 700 bp were produced in all Salmonella strains tested. These RAPD fragments obtained from Salmonella typhimurium strongly hybridized with the corresponding RAPD bands from the other strains of Salmonella, but not with those from non-Salmonella spp. in Southern blot analysis. The RAPD bands were detected by ethidium bromide staining even when genomic DNA prepared from as few as 2.8 x 10(3) cells was used. The minimum detectable cell number in the initial inoculum of S. typhimurium was 4 x 10(-1) CFU/25 g of raw beef after the preenrichment in Enterobacteriaceae enrichment mannitol (EEM) broth for 6 h and the selective enrichment in dulcitol-magnesium chloride-pyridinesulfonic acid-brilliant green-novobiocin (DMPBN) medium for 18 h at 42 degrees C. Seven raw foods inoculated with S. typhimurium at numbers from 4 x 10(-1) to 2.6 x 10(2) CFU/25 g of food were positive in both the RAPD analysis and the conventional culture method.
Assuntos
DNA Bacteriano/análise , Microbiologia de Alimentos , Técnica de Amplificação ao Acaso de DNA Polimórfico , Salmonella/isolamento & purificação , Sequência de Bases , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
IMP dehydrogenase was purified from a crude extract of B, cereus cells. The molecular mass of the purified enzyme was estimated to be 56 kDa by SDS-PAGE and 225 kDa by gel filtration. The optimum pH of the enzyme was about 9.5. The first seven residues at N-terminus of the enzyme was determined to be Met-Trp-Glu-Ser-Lys-Phe-Val. The enzyme showed a significant specificity for inosine nucleotides among 15 purines and pyrimidines tested, but not acted on other purines and pyrimidines including inosine. Among 11 metal ions and 3 enzyme inhibitors tested, Al3+ activated the IMP dehydrogenase. The enzyme activity was strongly inhibited by Zn2+ and Fe3+.
Assuntos
Bacillus cereus/enzimologia , IMP Desidrogenase/isolamento & purificação , Sulfato de Amônio , Bacillus cereus/crescimento & desenvolvimento , Fracionamento Químico , Cromatografia em Agarose , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , IMP Desidrogenase/química , IMP Desidrogenase/metabolismo , Metais/farmacologia , Esporos Bacterianos , Especificidade por SubstratoRESUMO
Cephalexin, cefaclor, cefadroxil, and cefotaxime strongly inhibited sporulation of Bacillus cereus ts-4 at 1 microgram/ml. Cephalexin was most inhibitory on the sporulation of B. cereus when the antibiotic was added at 3 h after induction of sporulation by nutrient downshift technique. Examination of 4',6-diamidino-2-phenylindole-stained cells by fluorescence-phase contrast microscopy showed that cephalexin inhibited the formation of asymmetric septum. By using [3H]penicillin, eight penicillin-binding proteins (PBPs) were detected from the cells of B. cereus ts-4. Among them, four PBPs were also detected in sporulating cells. Affinity of cephalexin to PBPs were measured indirectly by competition for subsequent binding of radioactive penicillin G. Cephalexin strongly bound to PBP 4 with molecular weight of 72,000 in sporulating cells.
Assuntos
Bacillus cereus/efeitos dos fármacos , Proteínas de Bactérias , Proteínas de Transporte/efeitos dos fármacos , Cefalexina/farmacologia , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/efeitos dos fármacos , Peptidil Transferases , Bacillus cereus/química , Bacillus cereus/fisiologia , Proteínas de Transporte/análise , Cefalexina/metabolismo , Peso Molecular , Muramilpentapeptídeo Carboxipeptidase/análise , Proteínas de Ligação às Penicilinas , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/fisiologiaRESUMO
IMP dehydrogenase activity of B. cereus increased parallel to cell growth in YE-EMM, where B. cereus did not sporulate. When B. cereus was cultured in a modified G medium, a sporulation medium, the activity reached the highest level at 6 hr and decreased thereafter. After induction of sporulation by nutritional shift down in 1/100 G medium, the enzyme activity decreased to about 5% compared with exponentially growing cells at 1 hr of resuspension. The sporulation rate of B. cereus was over 90% in the modified G medium and 1/100 G medium. Sporulation was strongly inhibited by mycophenolic acid at 1 mM, when the drug was added at 0 and 1 hr of resuspension in 1/100 G medium. Intracellular GTP concentration of B. cereus decreased to the lowest level about 1 hr of resuspension. Although GTP increased to about 50% of the exponentially growing cells at 2 hr of resuspension in control cells, the concentration did not increase in the presence of 1 mM mycophenolic acid.
Assuntos
Bacillus cereus/fisiologia , IMP Desidrogenase/fisiologia , Formicinas/farmacologia , Guanosina Trifosfato/análise , Ácido Micofenólico/farmacologia , Esporos Bacterianos/fisiologiaRESUMO
After frozen storage for 7 d, the viability and CO2 productivity of a conventional baker's yeast strain D greatly decreased. The viability of a freeze-tolerant strain, DFT, used for the frozen dough method slightly decreased after the same storage period, while the CO2 productivity greatly decreased. The CO2 productivity and DNase I inhibitory activity of actin of the cell-free extracts prepared immediately after thawing from 7-d frozen-stored cells markedly decreased in both strains. In DFT, however, the productivity and the inhibitory activity of the cell-free extract increased when the extract was prepared after incubation of the frozen-thawed cells at 30 degrees C. The increase in the inhibitory activity first occurred and then the increase in the CO2 productivity. Gel filtration patterns of actin and glycolytic enzymes were compared between cell-free extracts of both strains. Peaks of actin and activity peaks of hexokinase and pyruvate kinase decreased in the strain D after frozen storage, but only slightly in the strain DFT. After frozen storage, phosphofructokinase activity peak shifted to a lower molecular weight in strain D.
Assuntos
Actinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Dióxido de Carbono/metabolismo , Contagem de Células , Sobrevivência Celular , Cromatografia em Gel , Meios de Cultura , Congelamento , Glicólise , Hexoquinase/metabolismo , Peso Molecular , Piruvato Quinase/metabolismo , Saccharomyces cerevisiae/citologiaRESUMO
Hardening-induced soluble proteins of Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27) were isolated and purified by two-dimensional high-performance liquid chromatography (2D-HPLC) on an anion-exchange column, with subsequent reversed-phase chromatography. Some of the proteins were resolved by SDS-PAGE, characterized by amino-terminal sequencing and identified by searching for homologies in databases. Separation of the soluble proteins during the hardening of Chlorella by a combination of 2D-HPLC and SDS-PAGE revealed that at least 31 proteins were induced or increased in abundance. Of particular interest was the induction after 12 h of a 10-kDa protein with the amino-terminal amino acid sequence AGNKPITEQISDAVGAAGQKVG and the induction after 6 h of a 14-kDa protein with the amino-terminal sequence ALGEESLGDKAKNAFEDAKDAVKDAAGNVKEAV. The amino-terminal sequences of these proteins indicated that they were homologous to late embryogenesis abundant (LEA) proteins. Furthermore, the level of a 22-kDa protein also increased after 12 h. The amino-terminal sequence of this protein, AAPLVGGPAPDFTAAAVFD, indicated that it was homologous to thioredoxin peroxidase.
Assuntos
Chlorella/química , Proteínas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Proteínas de Plantas/química , SolubilidadeRESUMO
To investigate the effects of hardening on gene expression in Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27), a frost-hardy strain, 17 cDNA clones corresponding to hardening-induced Chlorella (hiC) genes were isolated by differential screening of a cDNA library from 6-h hardened cells. Northern blot analysis of transcripts of hiC genes showed that these genes are specifically induced by hardening and that their patterns of induction vary. Southern blots of genomic DNAs from two strains (Chlorella ellipsoidea Gerneck IAM C-102, chilling-sensitive; and C. vulgaris C-27, frost-hardy) of Chlorella indicated that ten hiC clones out of 17 hybridized only with DNA of strain C-27 and the other seven clones hybridized with DNA of both strains. However, of these seven clones, transcripts corresponding to six clones did not accumulate in strain C-102 at low temperatures. The sequence of a deduced protein encoded by the most abundant clone, hiC6, exhibited homology to sequences of Group III LEA (late embryogenesis abundant) proteins and had an amino-terminal amino acid sequence that was similar to the sequences of chloroplast transit peptides.
Assuntos
Proteínas de Algas , Chlorella/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Aclimatação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar , DNA de Plantas , Congelamento , Genes de Plantas , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Gd-DTPA enhanced dynamic MR imaging was performed in 11 normal uteri using a 1.5-T MR unit in order to analyze normal dynamic patterns. After intravenous bolus administration of Gd-DTPA, dynamic study was performed with serial imaging-gradient echo (FLASH). In most cases of normal uteri, early enhancement was shown in peripheral zone that was similar to be junctional zone, forming inner region of muscle layer.