RESUMO
The influence of the impregnation process of pine wood (Pinus sylvestris L.) samples on the electrical resistance changes and the moisture-content measurement accuracy is presented in this paper. In this study, the resistances of impregnated and nonimpregnated green pine timber harvested from northern Poland were compared. An impregnation method based on a vacuum-pressure chamber was used. Copper salts were applied as the impregnated solutions. The obtained results of the electrical resistance comparison showed a dependence of wood resistance on the moisture content. Higher conductivity occurred in impregnated wood samples filled with copper salt compared with wood samples without impregnation. Noticeable differences in the electrical resistance values were observed when the wood moisture content was significantly above the Fibre Saturation Point (FSP).
Assuntos
Pinus sylvestris , Pinus , Polônia , MadeiraRESUMO
Type II toxin-antitoxin (TA) systems are genetic elements usually encoding two proteins: a stable toxin and an antitoxin, which binds the toxin and neutralizes its toxic effect. The disturbance in the intracellular toxin and antitoxin ratio typically leads to inhibition of bacterial growth or bacterial cell death. Despite the fact that TA modules are widespread in bacteria and archaea, the biological role of these systems is ambiguous. Nevertheless, a number of studies suggests that the TA modules are engaged in such important processes as biofilm formation, stress response or virulence and maintenance of mobile genetic elements. The Dickeya dadantii 3937 strain serves as a model for pathogens causing the soft-rot disease in a wide range of angiosperm plants. Until now, several chromosome-encoded type II TA systems were identified in silico in the genome of this economically important bacterium, however so far only one of them was experimentally validated. In this study, we investigated three putative type II TA systems in D. dadantii 3937: ccdAB2Dda, phd-docDda and dhiTA, which represents a novel toxin/antitoxin superfamily. We provide an experimental proof for their functionality in vivo both in D. dadantii and Escherichia coli. Finally, we examined the prevalence of those systems across the Pectobacteriaceae family by a phylogenetic analysis.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Dickeya , Doenças das Plantas/microbiologia , Sistemas Toxina-Antitoxina , Dickeya/genética , Dickeya/metabolismo , Dickeya/patogenicidade , Regulação Bacteriana da Expressão Gênica , VirulênciaRESUMO
Background and objective: Allergy belongs to a group of mast cell-related disorders and is one of the most common diseases of childhood. It was shown that asthma and allergic rhinitis diminish the risk of various cancers, including colon cancer and acute lymphoblastic leukemia. On the other hand, asthma augments the risk of lung cancer and an increased risk of breast cancer in patients with allergy has been observed. Thus, the relation between allergy and cancer is not straightforward and furthermore, its biological mechanism is unknown. The HTRA (high temperature requirement A) proteases promote apoptosis, may function as tumor suppressors and HTRA1 is known to be released by mast cells. Interleukin-12 (Il-12) is an important cytokine that induces antitumor immune responses and is produced mainly by dendritic cells that co-localize with mast cells in superficial organs. Material and methods: In the present study we have assessed with ELISA plasma levels of the HTRA proteins, Il-12, and of the anti-HTRA autoantibodies in children with allergy (40) and in age matched controls (39). Children are a special population, since they usually do not have comorbidities and take not many drugs the processes we want to observe are not influenced by many other factors. Results: We have found a significant increase of HTRA1, 2 and 3, and of the Il-12 levels in the children with atopy (asthma and allergic rhinitis) compared to controls. Conclusion: Our results suggest that the HTRA1-3 and Il-12 levels might be useful in analyzing the pro- and antioncogenic potential in young atopic patients.
Assuntos
Asma/sangue , Serina Peptidase 1 de Requerimento de Alta Temperatura A/análise , Interleucina-12/análise , Rinite Alérgica/sangue , Adolescente , Biomarcadores/análise , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Feminino , Serina Peptidase 1 de Requerimento de Alta Temperatura A/sangue , Humanos , Interleucina-12/sangue , Masculino , Polônia , Estudos ProspectivosRESUMO
Huntington disease (HD) is an inherited neurodegenerative disorder caused by mutations in the huntingtin gene. Involvement of mitochondrial dysfunctions in, and especially influence of the level of mitochondrial DNA (mtDNA) on, development of this disease is unclear. Here, samples of blood from 84 HD patients and 79 controls, and dermal fibroblasts from 10 HD patients and 9 controls were analysed for mtDNA levels. Although the type of mitochondrial haplogroup had no influence on the mtDNA level, and there was no correlation between mtDNA level in leukocytes in HD patients and various parameters of HD severity, some considerable differences between HD patients and controls were identified. The average mtDNA/nDNA relative copy number was significantly higher in leukocytes, but lower in fibroblasts, of symptomatic HD patients relative to the control group. Moreover, HD women displayed higher mtDNA levels in leukocytes than HD men. Because this is the largest population analysed to date, these results might contribute to explanation of discrepancies between previously published studies concerning levels of mtDNA in cells of HD patients. We suggest that the size of the investigated population and type of cells from which DNA is isolated could significantly affect results of mtDNA copy number estimation in HD. Hence, these parameters should be taken into consideration in studies on mtDNA in HD, and perhaps also in other diseases where mitochondrial dysfunction occurs.
Assuntos
DNA Mitocondrial/metabolismo , Fibroblastos/metabolismo , Doença de Huntington/metabolismo , Leucócitos/metabolismo , Adulto , Idoso , DNA Mitocondrial/genética , Feminino , Humanos , Doença de Huntington/genética , Masculino , Pessoa de Meia-Idade , Mutação , Pele/metabolismo , Adulto JovemRESUMO
Recent studies have demonstrated elevated levels of iron (Fe) in brains of patients with Huntington's disease (HD). Striatal cells carrying mutated Huntingtin presented increased sensitivity to cadmium (Cd) toxicity, decreased sensitivity to manganese (Mn) toxicity and deficits in Mn uptake. The hypothesis arose that the observed alterations result from the altered expression and/or activity of proteins engaged in the transport of these metals, that is: transferrin (TF), transferrin receptor (TFR), divalent metal transporter 1 (DMT1) and ZIP8 protein. Here we examined the expression levels of genes encoding these proteins in blood of HD patients and control subjects. A decreasing tendency in the level of TF transcript and increasing tendency of SLC11A2 mRNA encoding DMT1 was observed in the blood of HD patients compared to the control subjects, but neither attained statistical significance. No changes were found in the levels of TFRC coding for TFR and SLC39A8 coding for ZIP8 between HD patients and controls. The results indicate that HD-associated changes in metal homeostasis occur are not related to mechanisms other than the expression level of the here analyzed metal transporters.
Assuntos
Doença de Huntington/sangue , Proteínas de Membrana Transportadoras/genética , Metais/metabolismo , RNA/sangue , Adulto , Idoso , Feminino , Humanos , Doença de Huntington/genética , Masculino , Pessoa de Meia-IdadeRESUMO
The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. In this report, using bacteriophage λ and Shiga toxin-converting bacteriophage Ï24Β, we demonstrate that the presence of this region on a multicopy plasmid results in impaired lysogenization of Escherichia coli and delayed, while more effective, induction of prophages following stimulation by various agents (mitomycin C, hydrogen peroxide, UV irradiation). Spontaneous induction of λ and Ï24Β prophages was also more efficient in bacteria carrying additional copies of the corresponding exo-xis region on plasmids. No significant effects of an increased copy number of genes located between exo and xis on both efficiency of adsorption on the host cells and lytic development inside the host cell of these bacteriophages were found. We conclude that genes from the exo-xis region of lambdoid bacteriophages participate in the regulation of lysogenization and prophage maintenance.
Assuntos
Bacteriófago lambda/genética , Toxina Shiga/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Escherichia coli Shiga Toxigênica/virologia , Ativação Viral , Sequência de Aminoácidos , Bacteriófago lambda/fisiologia , Dados de Sequência Molecular , Plasmídeos , Prófagos/genética , Prófagos/fisiologia , Alinhamento de Sequência , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/fisiologiaRESUMO
Precise regulation of DNA replication is necessary to ensure the inheritance of genetic features by daughter cells after each cell division. Therefore, determining how the regulatory processes operate to control DNA replication is crucial to our understanding and application to biotechnological processes. Contrary to early concepts of DNA replication, it appears that this process is operated by large, stationary nucleoprotein complexes, called replication factories, rather than by single enzymes trafficking along template molecules. Recent discoveries indicated that in bacterial cells two processes, central carbon metabolism (CCM) and transcription, significantly and specifically influence the control of DNA replication of various replicons. The impact of these discoveries on our understanding of the regulation of DNA synthesis is discussed in this review. It appears that CCM may influence DNA replication by either action of specific metabolites or moonlighting activities of some enzymes involved in this metabolic pathway. The role of transcription in the control of DNA replication may arise from either topological changes in nucleic acids which accompany RNA synthesis or direct interactions between replication and transcription machineries. Due to intriguing similarities between some prokaryotic and eukaryotic regulatory systems, possible implications of studies on regulation of microbial DNA replication on understanding such a process occurring in human cells are discussed.
Assuntos
Carbono/metabolismo , Replicação do DNA , Escherichia coli/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Modelos Biológicos , Ativação TranscricionalRESUMO
The aim of our study was to evaluate the importance of insulin-like growth-factor-binding protein 7 (IGFBP-7) as a potential marker of symptomatic peripheral artery disease (PAD) occurrence. The study group consisted of 145 patients with diagnosed PAD, who qualified for the invasive treatment. The control group consisted of 67 individuals representing the local population and an ischemic heart disease (IHD) group of 88 patients after myocardial infarction or percutaneous coronary intervention. Patients with PAD had significantly higher IGFBP-7 concentrations than control group (1.80 ± 1.62 vs. 1.41 ± 0.45 ng/mL, p = 0.04). No significant differences between PAD patients and IHD patients were found (1.80 ± 1.62 vs. 1.76 ± 1.04 ng/mL, p = 0.783). Patients with multilevel PAD presented significantly higher IGFBP-7 concentrations than patients with aortoiliac PAD-median 1.18 (IQR 0.48-2.23) vs. 1.42 ng/mL (0.71-2.63), p = 0.035. In the group of patients who died or had a major adverse cardiovascular event (MACE) during six months of follow-up, a statistically significant higher IGFBP-7 concentration was found (median 2.66 (IQR 1.80-4.93) vs. 1.36 ng/mL (IQR 0.65-2.34), p = 0.004). It seems that IGFBP-7 is elevated in patients with atherosclerotic lesions-regardless of their locations. Further research should be conducted to verify IGFBP-7 usefulness as a predictor of MACE or death.
Assuntos
Doença Arterial Periférica , Somatomedinas , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Doença Arterial Periférica/diagnóstico , Projetos Piloto , PrognósticoRESUMO
BACKGROUND: Mucopolysaccharidoses (MPS) are inherited metabolic disorders caused by mutations leading to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs). Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs. Substrate reduction therapy is one of potential treatment of these diseases. It was demonstrated previously that genistein (4', 5, 7-trihydroxyisoflavone) inhibits synthesis and reduces levels of GAGs in cultures of fibroblasts of MPS patients. Recent pilot clinical study indicated that such a therapy may be effective in MPS III (Sanfilippo syndrome). METHODS: To learn on details of the molecular mechanism of genistein-mediated inhibition of GAG synthesis, efficiency of this process was studied by measuring of incorporation of labeled sulfate, storage of GAGs in lysosomes was estimated by using electron microscopic techniques, and efficiency of phosphorylation of epidermal growth factor (EGF) receptor was determined by using an ELISA-based assay with fluorogenic substrates. RESULTS: Effects of genistein on inhibition of GAG synthesis and accumulation in fibroblasts from patients suffering from various MPS types were abolished in the presence of an excess of EGF, and were partially reversed by an increased concentration of genistein. No such effects were observed when an excess of 17beta-estradiol was used instead of EGF. Moreover, EGF-mediated stimulation of phsophorylation of the EGF receptor was impaired in the presence of genistein in both wild-type and MPS fibroblasts. CONCLUSION: The results presented in this report indicate that the mechanism of genistein-mediated inhibition of GAG synthesis operates through epidermal growth factor (EGF)-dependent pathway.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Fibroblastos/efeitos dos fármacos , Genisteína/farmacologia , Glicosaminoglicanos/biossíntese , Mucopolissacaridoses/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Receptores ErbB/metabolismo , Estradiol/farmacologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Mucopolissacaridoses/genética , Fosforilação , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Mucopolysaccharidoses (MPSs) are a group of severe metabolic disorders caused by deficiencies in enzymes involved in the degradation of glycosaminoglycans (GAGs)-long chains of sugar carbohydrates in cells that help build bone, cartilage, tendons, corneas, skin, and connective tissue. Although enzyme replacement therapy has become available for the treatment of some types of MPS, effective treatment of neurodegenerative forms of MPS has yet to be determined. Recently, genistein (4',5,7-trihydroxyisoflavone), a specific inhibitor of protein tyrosine kinase, has been found to inhibit GAG synthesis and to reduce GAG concentrations in cultures of fibroblasts of MPS patients. Therefore, a potential substrate reduction therapy has been proposed. OBJECTIVE: The aim of this study was to examine urinary GAG concentration, hair morphology, and cognitive function in patients receiving genistin treatment for Sanfilippo syndrome (MPS type III). METHODS: Patients aged 3 to 14 years with a biochemically confirmed diagnosis of MPS IIIA or MPS IIIB were eligible to enroll in this open-label, pilot study. Genistin-rich soy isoflavone extract 5 mg/kg/d was administered PO for 12 months. Urinary GAG concentration, hair morphology,and cognitive function (measured using a modified version of the Brief Assessment Examination [BAE] and parent observations)were measured at baseline and after 12 months of treatment. RESULTS: Ten patients (6 girls, 4 boys; mean age, 8 years [range,3\2-14 years];mean weight, 28 kg [range, 17\2-43 kg]) were included in the study. All patients had Sanfilippo syndrome; 5 patients had MPS IIIA and 5 had MPS IIIB. After 1 year, statistically significant improvement was found in urinary GAG concentration, hair morphology, and cognitive function. Urinary GAG concentration decreased significantly in all 5 patients with MPS IIIA and in 2 patients with MPS IIIB (P = 0.028). Hair morphology improved significantly in all 5 MPS IIIA patients and in 3 MPS IIIB patients (P = 0.012). A significant increase in the BAE score (by 2-6 points) was noted in 8 patients, while the scores of 2 patients did not change after 12 months of treatment (P = 0.012). No adverse events (AEs) considered related to treatment were reported. Moreover, no AEs not related to the treatment (apart from classical symptoms of MPS III) were noted. CONCLUSIONS: This pilot study found some improvements in GAG concentration, hair morphology, and cognitive function in these pediatric patients with Sanfilippo syndrome treated with genistin-rich soy isoflavone extract for 1 year. Clinical trials are needed to evaluate the efficacy and safety of this potential treatment.
RESUMO
Determination of mtDNA copy number in the cell is crucial to understand many cellular processes. Recently, the number of studies with the use of mitochondrial DNA (mtDNA) content as the determinant of mitochondrial abnormalities increased greatly and is still growing, therefore, optimization of technical conditions for this analysis is crucial. Despite using similar laboratory protocols, some results cannot be compared between research centers, thus causing discrepancies in the assessment of mtDNA content. The aim of this work was to test which conditions of biological sample collection and storage affect estimation of mtDNA level relative to the nuclear DNA (nDNA) in the blood samples and dermal fibroblasts. We found that the time and temperature of sample storage, as well as the type of the blood sample (whole blood or leukocytes) influence the estimate of mtDNA/nDNA ratio in the blood. In the case of dermal fibroblasts collected from healthy control and Huntington disease patients, our data indicate that the passage number of cells is essential to obtain reliable results.
Assuntos
Coleta de Amostras Sanguíneas/métodos , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Reação em Cadeia da Polimerase/métodos , Células Cultivadas , DNA Mitocondrial/sangue , Fibroblastos/fisiologia , Humanos , Reprodutibilidade dos TestesRESUMO
Mucopolysaccharidoses (MPS) are inherited, severe, progressive, metabolic disorders caused by deficiencies in different enzymes involved in degradation of glycosaminoglycans (GAGs). Although enzyme replacement therapy (ERT) has recently been available for MPS type I, and clinical trials have been performed in ERT for MPS II and MPS VI, there is little chance that this kind of treatment may be effective for neurodegenerative forms of MPS (due to inefficient delivery of enzymes to central nervous system through the blood-brain barrier), hence currently there is no effective therapy available for them. Therefore, we aim to develop an alternative therapy for these diseases. We found that genistein (4',5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) inhibits synthesis of GAGs considerably in cultures of fibroblasts of MPS patients (types I, II, IIIA and IIIB were tested). Prolonged cultivation of these cells in the presence of genistein resulted in reduction of GAG accumulation and normalization of cells as estimated by biochemical tests and electron microscopic analysis, respectively. As genistein inhibits kinase activity of epidermal growth factor receptor, which is required for full expression of genes coding for enzymes involved in GAG production, we propose to consider a substrate reduction therapy for MPS, which is referred to as 'gene expression-targeted isoflavone therapy'.
Assuntos
Fibroblastos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Glicosaminoglicanos/biossíntese , Mucopolissacaridoses/tratamento farmacológico , Mucopolissacaridoses/metabolismo , Biópsia , Linhagem Celular , Receptores ErbB/antagonistas & inibidores , Fibroblastos/química , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Mucopolissacaridoses/patologia , Pele/microbiologiaRESUMO
There are two 'pathways' of replication of lambda plasmids in Escherichia coli. One pathway requires the assembly of a new replication complex before replication and the second pathway is based on the activity of the replication complex inherited by one of two daughter plasmid copies after a preceding replication round. Such a phenomenon was postulated to occur also in other replicons, including Saccharomyces cerevisiae autonomously replicating sequences. Here we investigated directionality of lambda plasmid replication carried out by the heritable and newly assembled replication complexes. Using two-dimensional agarose gel electrophoresis and electron microscopy we demonstrated that in both normal growth conditions and during the relaxed response to amino acid starvation (when only replication carried out by the heritable complex is possible), bidirectionally and undirectionally replicating plasmid molecules occurred in host cells in roughly equal proportions. The results are compatible with the hypothesis that both complexes (heritable and newly assembled) are equivalent.
Assuntos
Bacteriófago lambda/genética , Plasmídeos/genética , Replicon , Replicação Viral , Bacteriófago lambda/ultraestrutura , DNA Viral/genética , DNA Viral/ultraestrutura , Eletroforese em Gel Bidimensional , Escherichia coli/genética , Escherichia coli/virologia , Ligases/genética , Substâncias Macromoleculares , Mutação , Plasmídeos/ultraestruturaRESUMO
DnaA and SeqA proteins are main regulators (positive and negative, respectively) of the chromosome replication in Escherichia coli. Nevertheless, both these replication regulators were found recently to be also transcription factors. Interestingly, both DnaA and SeqA control activity of the bacteriophage lambdap(R) promoter by binding downstream of the transcription start site, which is unusual among prokaryotic systems. Here we asked what are functional relationships between these two transcription regulators at one promoter region. Both in vivo and in vitro studies revealed that DnaA and SeqA can activate the p(R) promoter independently and separately rather than in co-operation, however, increased concentrations of one of these proteins negatively influenced the transcription stimulation mediated by the second regulator. This may suggest a competition between DnaA and SeqA for binding to the p(R) regulatory region. The physiological significance of this DnaA and SeqA-mediated regulation of p(R) is demonstrated by studies on lambda plasmid DNA replication in vivo.
Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/genética , Proteínas da Membrana Bacteriana Externa , Bacteriófagos/fisiologia , Primers do DNA/química , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Técnicas In Vitro , Óperon Lac/fisiologia , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da Polimerase , Transcrição Gênica , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , beta-Galactosidase/metabolismoRESUMO
The outbreak of an infectious disease in captive-bred Lepidoptera can cause death of all the caterpillars within days. A mixed baculoviral-bacterial infection observed among Actias selene (Hubner 1807), the Indian moon moth (Insecta: Lepidoptera: Saturniidae), larvae was characterized and followed by a photographic documentation of the disease progression. The etiological agents were determined using mass spectrometry and polymerase chain reaction (PCR). It appeared that the disease was caused by a mixed infection of larvae with a baculovirus and Morganella morganii. A molecular phylogenetic analysis of the virus and microbiological description of the pathogenic bacterium are presented.
Assuntos
Baculoviridae/isolamento & purificação , Morganella morganii/isolamento & purificação , Mariposas/microbiologia , Mariposas/virologia , Animais , Baculoviridae/genética , Sequência de Bases , DNA Bacteriano/genética , DNA Viral/genética , Larva/microbiologia , Larva/virologia , Morganella morganii/genética , Análise de Sequência de DNARESUMO
An Escherichia coli CM2555 strain, sensitive to chloramphenicol when expressing the cat gene and producing active chloramphenicol acetyltransferase (CAT), was described recently. It was proposed that this sensitivity is due to decreased levels of acetyl coenzyme A (Acetyl CoA) in cat-expressing CM2555 cells in the presence of chloramphenicol. CAT catalyzes transfer of the acetyl moiety from Acetyl CoA to a chloramphenicol molecule. Thus, a very efficient acetylation of chloramphenicol may cause deprivation of Acetyl CoA and cell death. A specific mutation causing the chloramphenicol sensitivity phenotype of CM2555 was not reported to date. Therefore, we aimed to identify a genetic defect causing this phenotype. Here, we found that overexpression of the acrEF genes, encoding a transmembrane pump, or the acrE gene alone, results in restoration of chloramphenicol-resistance of cat-expressing CM2555 strain. Although no mutation exists in the CM2555 acrE locus, a nonsense mutation in the 67th codon of the acrA gene, which encodes a component of another transmembrane pump, has been found. Although introduction of the deltaacrAB allele into CM732, a parental strain of CM2555, and into some other commonly used E. coli strains led to their chloramphenicol sensitivity in the presence of CAT, the same genetic manipulation did not result in such a phenotype in other genetic backgrounds, including "wild-type" E. coli MG1655. These results suggest that the acrA dysfunction is one of more mutations responsible for chloramphenicol sensitivity of cat-expressing CM2555 strain.
Assuntos
Proteínas de Bactérias/genética , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol/farmacologia , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli , Escherichia coli/efeitos dos fármacos , Genes Bacterianos , Lipoproteínas/genética , Sequência de Bases , Western Blotting , Códon sem Sentido , Primers do DNA , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Membrana Transportadoras , Testes de Sensibilidade MicrobianaRESUMO
The marine environment is estimated to be one of the most significant sources of biological activity in the world. In the last few decades an increase in the research intensity conducted on marine microorganisms has been observed, which confirms the great potential of these organisms in the field of bioactive compounds' production. In order to efficiently use the natural resources of the marine environment, metagenomics can be applied. This powerful technique allows for efficient screening of microbial biodiversity for bioactive compounds. The primary aim of this review is to present some aspects of the construction of metagenomic libraries, and strategies of screening for novel bioactives in the marine surrounding. This paper also illustrates several examples of the application of metagenomic methods in the discovery of novel enzymes and drugs in various marine environments.
Assuntos
Organismos Aquáticos , Metagenômica , Água do Mar/microbiologia , Organismos Aquáticos/enzimologia , Organismos Aquáticos/genética , Organismos Aquáticos/microbiologia , Biodiversidade , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Biblioteca Gênica , Biologia MarinhaRESUMO
The outbreak of an infectious disease in captive-bred Lepidoptera can cause death of all the caterpillars within days. A mixed baculoviral–bacterial infection observed among Actias selene (Hübner 1807), the Indian moon moth (Insecta: Lepidoptera: Saturniidae), larvae was characterized and followed by a photographic documentation of the disease progression. The etiological agents were determined using mass spectrometry and polymerase chain reaction (PCR). It appeared that the disease was caused by a mixed infection of larvae with a baculovirus and Morganella morganii. A molecular phylogenetic analysis of the virus and microbiological description of the pathogenic bacterium are presented.
RESUMO
Bacteriophage lambda genome is one of the classical model replicons in studies on the regulation of DNA replication. Moreover, since genes coding for Shiga toxins are located in genomes of lambdoid phages, understanding of mechanisms controlling lambda DNA replication may be of bio-medical importance. During lytic development of bacteriophage lambda, its genome is replicated according to the theta (circle-to-circle) mode early after infection, and then it is switched to the sigma (rolling circle) mode. Two mechanisms of regulation of this switch were proposed recently and both suggested a crucial role for directionality of lambda DNA replication. Whereas one hypothesis assumed transient impairment of ClpP/ClpX-mediated proteolysis of the lambdaO initiator protein, another suggested a crucial role for transcriptional activation of the orilambda region and factors involved in the control of the p (R) promoter activity. Here we demonstrate that mutations in clpP and clpX genes had little influence on both directionality of lambda DNA replication and appearance of sigma replication intermediates. On the other hand, regulators affecting activity of the p (R) promoter (responsible for initiation of transcription, which activates orilambda) directly or indirectly influenced directionality of lambda DNA replication to various extents. Therefore, we conclude that regulation of the efficiency of transcriptional activation of orilambda, rather than transient impairment of the lambdaO proteolysis, is responsible for the control of the switch from theta to sigma replication, and propose a model for this control.
Assuntos
Bacteriófago lambda/genética , Replicação do DNA , DNA Viral/genética , Regulação Viral da Expressão Gênica , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Eletroforese em Gel de Ágar , Endopeptidase Clp/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Modelos Genéticos , Chaperonas Moleculares/genética , Mutação , Plasmídeos/metabolismo , Origem de Replicação , Transcrição Gênica , Ativação TranscricionalRESUMO
SeqA protein, a main negative regulator of the replication initiation of the Escherichia coli chromosome, also has several other functions which are still poorly understood. It was demonstrated previously that in seqA mutants the copy number of another replicon, the lambda plasmid, is decreased, and that the activity of the lambda p(R) promoter (whose function is required for stimulation of ori lambda) is lower than that in the wild-type host. Here, SeqA-mediated regulation of lambda phage and plasmid replicons was investigated in more detail. No significant influence of SeqA on ori lambda-dependent DNA replication in vitro was observed, indicating that a direct regulation of lambda DNA replication by this protein is unlikely. On the other hand, density-shift experiments, in which the fate of labelled lambda DNA was monitored after phage infection of host cells, strongly suggested the early appearance of sigma replication intermediates and preferential rolling-circle replication of phage DNA in seqA mutants. The directionality of lambda plasmid replication in such mutants was, however, only slightly affected. The stability of the heritable lambda replication complex was decreased in the seqA mutant relative to the wild-type host, but a stable fraction of the lambda O protein was easily detectable, indicating that such a heritable complex can function in the mutant. To investigate the influence of seqA gene function on heritable complex- and transcription-dependent lambda DNA replication, the efficiency of lambda plasmid replication in amino acid-starved relA seqA mutants was measured. Under these conditions, seqA dysfunction resulted in impairment of lambda plasmid replication. These results indicate that unlike oriC, SeqA modulates lambda DNA replication indirectly, most probably by influencing the stability of the lambda replication complex and the transcriptional activation of ori lambda.