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1.
Life (Basel) ; 13(9)2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-37763312

RESUMO

Transcutaneous electrical stimulation of the spinal cord is used to restore locomotion and body weight support in patients with severe motor disorders. We studied the effects of this non-invasive stimulation on postural control in healthy subjects. Stimulation at the L1-L2 vertebrae was performed to activate the extensor muscles of the lower limbs. Because postural regulation depends on the cognitive style, the effects of the stimulation were analyzed separately in field-dependent (FD) and field-independent (FI) participants. During the study, FD and FI participants (N = 16, 25 ± 5 years, all right dominant leg) stood on a force platform in a soundproof chamber with their eyes closed. Stimulation was applied in the midline between the L1 and L2 vertebrae or over the left or right dorsal roots of the spinal cord; under the control condition, there was no stimulation. Stimulation destabilized posture in healthy subjects, whereas patients with movement disorders usually showed an improvement in postural control. In the FD participants, left dorsal root and midline stimulation increased several postural parameters by up to 30%. Dorsal root stimulation on the side of the supporting leg reduced postural control, while stimulation on the side of the dominant leg did not. No significant changes were observed in the FI participants.

2.
Int J Mol Sci ; 23(3)2022 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-35163756

RESUMO

The increasing antibiotic resistance is a clinical problem worldwide. Numerous Gram-negative bacteria have already become resistant to the most widely used class of antibacterial drugs, ß-lactams. One of the main mechanisms is inactivation of ß-lactam antibiotics by bacterial ß-lactamases. Appearance and spread of these enzymes represent a continuous challenge for the clinical treatment of infections and for the design of new antibiotics and inhibitors. Drug repurposing is a prospective approach for finding new targets for drugs already approved for use. We describe here the inhibitory potency of known detoxifying antidote 2,3-dimercaptopropane-1-sulfonate (unithiol) against metallo-ß-lactamases. Unithiol acts as a competitive inhibitor of meropenem hydrolysis by recombinant metallo-ß-lactamase NDM-1 with the KI of 16.7 µM. It is an order of magnitude lower than the KI for l-captopril, the inhibitor of angiotensin-converting enzyme approved as a drug for the treatment of hypertension. Phenotypic methods demonstrate that the unithiol inhibits natural metallo-ß-lactamases NDM-1 and VIM-2 produced by carbapenem-resistant K. pneumoniae and P. aeruginosa bacterial strains. The 3D full atom structures of unithiol complexes with NDM-1 and VIM-2 are obtained using QM/MM modeling. The thiol group is located between zinc cations of the active site occupying the same place as the catalytic hydroxide anion in the enzyme-substrate complex. The sulfate group forms both a coordination bond with a zinc cation and hydrogen bonds with the positively charged residue, lysine or arginine, responsible for proper orientation of antibiotics upon binding to the active site prior to hydrolysis. Thus, we demonstrate both experimentally and theoretically that the unithiol is a prospective competitive inhibitor of metallo-ß-lactamases and it can be utilized in complex therapy together with the known ß-lactam antibiotics.


Assuntos
Klebsiella pneumoniae/enzimologia , Pseudomonas aeruginosa/enzimologia , Unitiol/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Carbapenêmicos/farmacologia , Reposicionamento de Medicamentos , Farmacorresistência Bacteriana/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Modelos Moleculares , Conformação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , beta-Lactamases/química
3.
Diagnostics (Basel) ; 13(1)2022 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-36611414

RESUMO

SARS-CoV-2 infection may cause such complications as post-COVID-19 syndrome, which includes chronic fatigue, myalgia, arthralgia, as well as a variety of neurological manifestations, e.g., neuropathy of small fibers, hearing and vestibular dysfunction, and cognitive impairment. This clinical case describes a 41-year-old patient suffering from post-COVID-19 syndrome and chronic fatigue syndrome. A detailed examination was performed, including an in-depth study of peripheral and central hearing and vestibular functions, as well as small nerve fibers length and density in the skin and cornea of the eye. Contrary to expectations, no peripheral nervous system dysfunction was detected, despite the presence of dizziness and gait instability in the patient. Hearing tests (gap detection test and dichotic test) showed central auditory processing disorders. The evaluated lesion in the processing of temporal and verbal auditory information can be a significant factor contributing to additional overload of the neural activity and leading to chronic fatigue when performing daily activities in patients with CFS and post-COVID-19 complications.

4.
RSC Adv ; 11(18): 10727-10737, 2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-35423580

RESUMO

Methoxy-substituted benzenes are the simplest fragments from the lignin separation feedstock. Extensive experimental thermochemical studies of these compounds were carried out, including vapor pressure measurements, combustion and differential scanning calorimetry. These data were evaluated using empirical, semi-empirical and quantum chemical methods. The consistent sets of evaluated thermodynamic data were used to design the method for predicting enthalpies of vaporisation and enthalpies of formation of di- and tri-substituted benzenes. It has been found that the agglomeration of substituents on the benzene ring has dramatic consequences for the energetics of the molecule (in terms of the enthalpy of formation), as well as for the energetics of intermolecular interactions (in terms of the enthalpy of vaporisation). These observations are essential to reliably assess the energetics of the molecules that appear in reaction products of lignin transformations in value-adding chemicals and materials.

5.
Artigo em Inglês | MEDLINE | ID: mdl-32138383

RESUMO

One of the most important components of atmospheric aerosols are microorganisms. Therefore, it is necessary to assess the hazard to humans, both from individual microorganisms which are present in atmospheric bioaerosols as well as from their pool. An approach for determining the hazard of bacteria and yeasts found in atmospheric bioaerosols for humans has previously been proposed. The purpose of this paper is to compare our results for 2006-2008 with the results of studies obtained in 2012-2016 to identify changes in the characteristics of bioaerosols occurring over a decade in the south of Western Siberia. Experimental data on the growth, morphological and biochemical properties of bacteria and yeasts were determined for each isolate found in bioaerosol samples. The integral indices of the hazards of bacteria and yeast for humans were constructed for each isolate based on experimentally determined isolate characteristics according to the approach developed by authors in 2008. Data analysis of two datasets showed that hazard to humans of culturable microorganisms in the atmospheric aerosol in the south of Western Siberia has not changed significantly for 10 years (trends are undistinguishable from zero with a confidence level of more than 95%) despite a noticeable decrease in the average annual number of culturable microorganisms per cubic meter (6-10 times for 10 years).


Assuntos
Aerossóis , Microbiologia do Ar , Bactérias , Saúde Ambiental , Humanos , Medição de Risco , Sibéria
6.
Biochim Biophys Acta Proteins Proteom ; 1868(4): 140365, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31958502

RESUMO

The glycolytic pathway is one of the most important pathways for living organisms, due to its role in energy production and as supplier of precursors for biosynthesis in living cells. This work focuses on determination of the standard Gibbs energy of reaction ΔRg'0 of the enolase reaction, the ninth reaction in the glycolysis pathway. Exact ΔRg'0 values are required to predict the thermodynamic feasibility of single metabolic reactions or even of metabolic reaction sequences under cytosolic conditions. So-called "apparent" standard data from literature are only valid at specific conditions. Nevertheless, such data are often used in pathway analyses, which might lead to misinterpretation of the results. In this work, equilibrium measurements were combined with activity coefficients in order to obtain new standard values ΔRg'0 for the enolase reaction that are independent of the cytosolic conditions. Reaction equilibria were measured at different initial substrate concentrations and temperatures of 298.15 K, 305.15 K and 310.15 K at pH 7. The activity coefficients were predicted using the equation of state electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT). The ePC-SAFT parameters were taken from literature or fitted to new experimentally determined osmotic coefficients and densities. At 298.15 K and pH 7, a ΔRg'0(298.15 K, pH 7) value of -2.8 ± 0.2 kJ mol-1 was obtained. This value differs by up to 5 kJ mol-1 from literature data. Reasons are the poorly defined "standard" conditions and partly undefined reaction conditions of literature works. Finally, using temperature-dependent equilibrium constants and the van 't Hoff equation, the standard enthalpy of reaction of ΔRh'0(298.15 K, pH 7) = 27 ± 10 kJ mol-1 was determined, and a similar value was found by quantum-chemistry calculations.


Assuntos
Glicólise , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/metabolismo , Concentração de Íons de Hidrogênio , Magnésio/química , Osmose , Saccharomyces cerevisiae/enzimologia , Termodinâmica
7.
FEBS Open Bio ; 8(1): 117-129, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29321962

RESUMO

Highly mutable ß-lactamases are responsible for the ability of Gram-negative bacteria to resist ß-lactam antibiotics. Using site-directed mutagenesis technique, we have produced in vitro a number of recombinant analogs of naturally occurring TEM-type ß-lactamases, bearing the secondary substitution Q39K and key mutations related to the extended-spectrum (E104K, R164S) and inhibitor-resistant (M69V) ß-lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of ß-lactamases. However, in combination with the key mutations, this substitution resulted in decreased KM values towards hydrolysis of a chromogenic substrate, CENTA. The ability of enzymes to restore catalytic activity after exposure to elevated temperature has been examined. All double and triple mutants of ß-lactamase TEM-1 bearing the Q39K substitution showed lower thermal stability compared with the enzyme with Q39 intact. A sharp decrease in the stability was observed when Q39K was combined with E104K and M69V. The key R164S substitution demonstrated unusual ability to resist thermal inactivation. Computer analysis of the structure and molecular dynamics of ß-lactamase TEM-1 revealed a network of hydrogen bonds from the residues Q39 and K32, related to the N-terminal α-helix, towards the residues R244 and G236, located in the vicinity of the enzyme's catalytic site. Replacement of Q39 by lysine in combination with the key drug resistance mutations may be responsible for loss of protein thermal stability and elevated mobility of its secondary structure elements. This effect on the activity of ß-lactamases can be used as a new potential target for inhibiting the enzyme.

8.
Arch Virol ; 162(8): 2489-2492, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28455670

RESUMO

The complete genome of ϕ16, a temperate corynephage from Corynebacterium glutamicum ATCC 21792, was sequenced and annotated (GenBank: KY250482). The electron microscopy study of ϕ16 virion confirmed that it belongs to the family Siphoviridae. The ϕ16 genome consists of a linear double-stranded DNA molecule of 58,200 bp (G+C = 52.2%) with protruding cohesive 3'-ends of 14 nt. Four major structural proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting technique. Using bioinformatics analysis, 101 putative ORFs and 5 tRNA genes were predicted. Only 27 putative gene products could be assigned to known biological functions. The ϕ16 genome was divided into functional modules. Seven putative promoters and eight putative unidirectional intrinsic terminators were predicted. One site of putative «-1¼ programmed ribosomal frameshifting was proposed in the phage tail assembly genome region. C. glutamicum genetic tools could be broadened by exploiting the known integrase gene (gp33) and the newly identified excisionase gene (gp47), participating in site-specific recombination between ϕ16-attP/attB.


Assuntos
Corynebacterium glutamicum/virologia , DNA Viral/genética , Genoma Viral , Siphoviridae/genética , Biologia Computacional , DNA Nucleotidiltransferases/genética , Eletroforese em Gel de Poliacrilamida , Integrases/genética , Anotação de Sequência Molecular , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Recombinação Genética , Análise de Sequência de DNA , Siphoviridae/classificação , Siphoviridae/isolamento & purificação , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
9.
Bioorg Med Chem Lett ; 27(7): 1588-1592, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28237762

RESUMO

The novel classes of acylated phenoxyanilide and thiourea compounds were investigated for their ability to inhibit TEM type ß-lactamase enzyme. Two compounds 4g and 5c reveal the inhibition potency in micromolar range and show their action by non-covalent binding in the vicinity of the TEM-171 active site. The structure activity relationship around carbon chain length and different substituents in ortho- and para-positions of acylated phenoxyanilide as well as molecular modelling study has been performed.


Assuntos
Proteínas de Escherichia coli/antagonistas & inibidores , Tioureia/análogos & derivados , Inibidores de beta-Lactamases/química , beta-Lactamases/química , Anilidas/química , Domínio Catalítico , Proteínas de Escherichia coli/química , Ligação de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Éteres Fenílicos/química , Relação Estrutura-Atividade , Tioureia/química
10.
Biosens Bioelectron ; 88: 283-289, 2017 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-27567265

RESUMO

We have demonstrated label-free and real-time detection of prostate specific antigen (PSA) in human serum using silicon nanowire field effect transistors (NW FETs) with Schottky contacts (Si-Ti). The NW FETs were fabricated from SOI material using high-resolution e-beam lithography, thin film vacuum deposition and reactive-ion etching processes eliminating complicated processes of doping and thermal annealing. This allowed substantial simplifying the transistors manufacturing. A new method for covalent immobilization of half-fragments of antibodies on silicon modified by 3-glycidopropyltrimethoxysilane with thiol groups and 5nm gold nanoparticles (GNPs) was established. NW FETs functionalized by GNPs revealed extremely high pH sensitivity of 70mV/pH and enhanced electrical performance in the detection of antigen due to enhanced surface/volume ratio, favorable orientation of antibody active sites and approaching the source of the electric field close to the transistor surface. Si NWFETs were applied for quantitative detection of PSA in a buffer and human serum diluted 1/100. Response time was about 5-10s, and analysis time per sample was 1min. The limit of PSA detection was of 23fg/mL, concentration range of 23fg/mL-500ng/mL (7 orders of magnitude). The PSA concentrations determined by the NW FETs in serum were compared with well-established ELISA method. The results matched well with the correlation coefficient of 0.97.


Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais/métodos , Ouro/química , Nanopartículas Metálicas/química , Nanofios/química , Antígeno Prostático Específico/sangue , Silício/química , Técnicas Biossensoriais/instrumentação , Humanos , Nanopartículas Metálicas/ultraestrutura , Nanofios/ultraestrutura , Transistores Eletrônicos
11.
Microb Drug Resist ; 21(4): 477-90, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25803299

RESUMO

Antimicrobial resistance and plasmid profile of Gram-positive and Gram-negative bacterial strains isolated from the urbanized Eltsovka-1 River (Russia) were investigated. Sequencing of the 16S rRNA of of G+ strains showed 99-100% identity to that of Bacillus aerophilus, Bacillus altitudinis, Bacillus amyloliquefaciens, Bacillus anthrancis, Bacillus barbaricus, Bacillus cereus, Bacillus flexus, Bacillus indriensis, Bacillus stratosphericus, Bacillus subtilis subsp. subtilis, Bacillus thuringiensis, Streptomyces albidoflavus, Streptomyces albus, Streptomyces exfoliatus, Streptomyces odorifer, and Streptomyces sampsonii. Sequencing of the 16S rRNA of G-strains was similar in 99-100% to that of Aeromonas bestiarum, Aeromonas encheleia, Aeromonas hydrophila, A. hydrophila subsp. anaerogenes, A. hydrophila subsp. dhakensis, Aeromonas media, Aeromonas molluscorum, Aeromonas popoffii, Aeromonas salmonicida subsp. masoucida, A. salmonicida subsp. pectinolytica, A. salmonicida subsp. salmonicida, Aeromonas punctata, Aeromonas sobria, and Shewanella putrefaciens. The highest percentage (88.4%) of strains was resistant to polymyxin B followed by 69% to lincomycin, 61.5% to benzilpenicillin, 57.7% to ampicillin, and 50% to carbenicillin. A low level of resistance (4%) was found to kanamycin (8%), to streptomycin (11.5%), to neomycin and tetracycline, and (15%) to erythromycin. No resistance was found to gentamycin, monomycin, and chloroamphenicol. The majority (80.7%) of strains was multidrug-resistant. Ninety-two percent of all strains carried plasmid DNA of various sizes.


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/genética , Farmacorresistência Bacteriana/genética , Plasmídeos/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Testes de Sensibilidade Microbiana , RNA Ribossômico 16S/genética , Federação Russa/epidemiologia , População Urbana
12.
Expert Rev Vaccines ; 13(2): 257-64, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24350587

RESUMO

WHO recommends the inclusion of PCVs in childhood vaccination programs world-wide. Many countries including the Russian Federation are currently planning the inclusion of PCVs in their National Immunization Programs and, therefore, data on the pneumococcal serotype distribution is important to estimate the potential disease impact. Here we review eight recent epidemiological studies on the pneumococcal serotype distribution from Russia. Across all studies, serotypes 6B, 14, 19F and 23F were the most prevalent. Interestingly, serotype 3 was relatively common. Serotype 19A was prevalent among AOM, CAP and nasopharyngeal isolates and among antibiotic resistant isolates in all age groups. The differences in serotype coverage between PCV10 and PCV13 were up to 26%. Based on the current data on serotype distribution, a wide use of PCVs in Russia may lead to a significant reduction of the pneumococcal disease burden.


Assuntos
Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/isolamento & purificação , Adolescente , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Criança , Pré-Escolar , Política de Saúde , Humanos , Programas de Imunização , Lactente , Recém-Nascido , Vacinas Pneumocócicas/administração & dosagem , Prevalência , Federação Russa/epidemiologia , Sorotipagem , Vacinas Conjugadas/administração & dosagem
13.
Appl Microbiol Biotechnol ; 93(1): 179-89, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21870047

RESUMO

A test system is described and expanded upon for mass field immunochromatography assay on porous membrane carriers for rapid diagnostics of potato virus X (PVX) in potato leaf tissue and sprout extracts using colloidal gold nanoparticles as a marker. Sensitivity of the assay developed for PVX identification is found to be comparable to the sensitivity of solid-phase sandwich-ELISA. Complete assay time does not exceed 15 min, and the lower limit of the PVX detection in non-clarified leaf extract is 2 ng/ml. A single measurement requires 0.1-0.2 ml (3-5 drops) of tested solution only (extracted from 10-20 mg of potato leaf tissue or sprouts). The simplicity and reliability of the method makes it especially efficient in direct rapid monitoring of many infected potato specimens in the field, as verified by field trials of 360 clones of 28 domestic and foreign cultivars of potato. A diagnostic kit for routine analyses of potato viral infections both in the laboratory and in the field is described and expanded upon.


Assuntos
Doenças das Plantas/virologia , Potexvirus/isolamento & purificação , Solanum tuberosum/virologia , Virologia/métodos , Cromatografia de Afinidade/métodos , Potexvirus/imunologia , Sensibilidade e Especificidade , Fatores de Tempo
14.
Appl Microbiol Biotechnol ; 93(1): 331-41, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22159605

RESUMO

Pantoea ananatis AJ13355 is a newly identified member of the Enterobacteriaceae family with promising biotechnological applications. This bacterium is able to grow at an acidic pH and is resistant to saturating concentrations of L-glutamic acid, making this organism a suitable host for the production of L-glutamate. In the current study, the complete genomic sequence of P. ananatis AJ13355 was determined. The genome was found to consist of a single circular chromosome consisting of 4,555,536 bp [DDBJ: AP012032] and a circular plasmid, pEA320, of 321,744 bp [DDBJ: AP012033]. After automated annotation, 4,071 protein-coding sequences were identified in the P. ananatis AJ13355 genome. For 4,025 of these genes, functions were assigned based on homologies to known proteins. A high level of nucleotide sequence identity (99%) was revealed between the genome of P. ananatis AJ13355 and the previously published genome of P. ananatis LMG 20103. Short colinear regions, which are identical to DNA sequences in the Escherichia coli MG1655 chromosome, were found to be widely dispersed along the P. ananatis AJ13355 genome. Conjugal gene transfer from E. coli to P. ananatis, mediated by homologous recombination between short identical sequences, was also experimentally demonstrated. The determination of the genome sequence has paved the way for the directed metabolic engineering of P. ananatis to produce biotechnologically relevant compounds.


Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Pantoea/genética , Cromossomos Bacterianos , Conjugação Genética , DNA Circular/química , DNA Circular/genética , Escherichia coli/genética , Transferência Genética Horizontal , Dados de Sequência Molecular , Plasmídeos , Recombinação Genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico
15.
FEMS Microbiol Lett ; 318(1): 55-60, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21306430

RESUMO

Pantoea ananatis accumulates gluconate during aerobic growth in the presence of glucose. Computer analysis of the P. ananatis SC17(0) sequenced genome revealed an ORF encoding a homologue (named gcd) of the mGDH (EC 1.1.99.17) apoenzyme from Escherichia coli and a putative pyrroloquinoline quinone (PQQ) biosynthetic operon homologous to pqqABCDEF from Klebsiella pneumoniae. Construction of Δgcd and Δpqq mutants of P. ananatis confirmed the proposed functions of these genetic elements. The P. ananatis pqqABCDEF was cloned in vivo and integrated into the chromosomes of P. ananatis and E. coli according to the Dual In/Out strategy. Introduction of a second copy of pqqABCDEF to P. ananatis SC17(0) doubled the accumulation of PQQ. Integration of the operon into E. coli MG1655ΔptsGΔmanXY restored the growth of bacteria on glucose. The obtained data show the essential role of pqqABCDEF in PQQ biosynthesis in P. ananatis and E. coli. We propose that the cloned operon could be useful for an efficient phosphoenolpyruvate-independent glucose consumption pathway due to glucose oxidation and construction of E. coli strains with the advantage of phosphoenolpyruvate-derived metabolite production.


Assuntos
Proteínas de Bactérias/genética , Glucose Desidrogenase/genética , Óperon , Cofator PQQ/biossíntese , Pantoea/enzimologia , Pantoea/genética , Proteínas de Bactérias/metabolismo , Gluconatos/metabolismo , Glucose Desidrogenase/metabolismo , Mutação , Pantoea/metabolismo
16.
BMC Mol Biol ; 10: 34, 2009 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-19389224

RESUMO

BACKGROUND: Pantoea ananatis, a member of the Enterobacteriacea family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of Pantoea ananatis have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The lambda Red-recombineering technique previously developed in E. coli and used for gene inactivation in several other bacteria is a high-performance tool for rapid construction of precise genome modifications. RESULTS: In this study, the expression of lambda Red genes in P. ananatis was found to be highly toxic. A screening was performed to select mutants of P. ananatis that were resistant to the toxic affects of lambda Red. A mutant strain, SC17(0) was identified that grew well under conditions of simultaneous expression of lambda gam, bet, and exo genes. Using this strain, procedures for fast introduction of multiple rearrangements to the Pantoea ananatis genome based on the lambda Red-dependent integration of the PCR-generated DNA fragments with as short as 40 bp flanking homologies have been demonstrated. CONCLUSION: The lambda Red-recombineering technology was successfully used for rapid generation of chromosomal modifications in the specially selected P. ananatis recipient strain. The procedure of electro-transformation with chromosomal DNA has been developed for transfer of the marked mutation between different P. ananatis strains. Combination of these techniques with lambda Int/Xis-dependent excision of selective markers significantly accelerates basic research and construction of producing strains.


Assuntos
Bacteriófago lambda/genética , Engenharia Genética/métodos , Pantoea/genética , Recombinação Genética , Biotecnologia/métodos , Cromossomos Bacterianos/genética , Eletroporação/métodos , Mutação , Plasmídeos/genética , Seleção Genética
17.
Lasers Surg Med ; 40(6): 422-32, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18649381

RESUMO

BACKGROUND AND OBJECTIVE: The number of in vitro experimental studies was carried out with the use of intact tissues to establish a mechanism of laser-tissue interaction. However, in the process of degeneration, both biochemical composition and behavior of the disc were altered drastically. The objective of this study was to evaluate the role of the main matrix components in laser modification of annulus fibrosus (AF) under IR laser irradiation. STUDY DESIGNS/MATERIALS AND METHODS: The samples of AF in a motion segment after hyaluronidase treatment, trypsin digestion and glycation by glyceraldehyde were heated in hydrothermal bath (95 degrees C, 2 min) or irradiated by laser at 1.56 microm. Specimens were imaged by cross-polarization optical coherence tomography (CP-OCT), and then analyzed by differential scanning calorimery (DSC). RESULTS AND DISCUSSION: According to CP-OCT and DSC data non-significant alteration was revealed in AF after hyaluronidase treatment, glycation led to stabilization of annulus collagen and trypsin digestion resulted in a noticeable impairment of collagen fibrils. Laser treatment induced subsequent damages of AF matrix but these damages cannot be explained by laser heating only. The specificity of chemical modification of AF matrix has an influence on a character of collagen network alteration due to IR laser effect. Minimal and maximal alterations are observed for hyaluronidase and trypsin treated samples respectively. Glyceraldehyde fixed samples showed failure of the collagen structure after moderate laser treatment; at the same time thermal denaturation of collagen macromolecules was negligible. We assume that a mechanical effect of laser irradiation plays an important role in laser-induced annulus collagen modification and propose the scheme of physico-chemical process occurring under non-uniform IR laser treatment in AF tissue. CONCLUSION: CP-OCT and DSC techniques allow us to record the alteration of collagen network organization as a result of chemical modification. There were detected significant and specific effects of the biochemical composition and material properties on the response of AF collagen network on laser irradiation. The results go in accordance with our hypothesis that the primary effect of laser influence on collagen network under tension is the mechanical damage of collagen fiber.


Assuntos
Colágenos Fibrilares/efeitos da radiação , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/efeitos da radiação , Terapia com Luz de Baixa Intensidade/métodos , Doenças da Coluna Vertebral/terapia , Animais , Modelos Animais de Doenças , Feminino , Colágenos Fibrilares/metabolismo , Hialuronoglucosaminidase/farmacologia , Quimiólise do Disco Intervertebral/métodos , Vértebras Lombares , Masculino , Probabilidade , Coelhos , Distribuição Aleatória , Sensibilidade e Especificidade , Tomografia de Coerência Óptica , Tripsina/farmacologia
18.
BMC Biotechnol ; 7: 80, 2007 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-18028554

RESUMO

BACKGROUND: RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet. RESULTS: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of lambdaRed-driven recombination between the plasmid and a constructed in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing PlacUV5-->lacI. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range. CONCLUSION: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production.


Assuntos
Escherichia coli/genética , Engenharia Genética/métodos , Pantoea/genética , Plasmídeos/genética , Proteínas Recombinantes/metabolismo , Transfecção/métodos
19.
Span J Psychol ; 10(2): 266-76, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17992953

RESUMO

Auditory perception of the depth of space is based mainly on spectral and amplitude changes of sound waves originating from the sound source and reaching the listener. The perceptive illusion of movement of an auditory image caused by changes in amplitude and/or frequency of the signal tone emanating from an immobile loudspeaker was studied. Analysis of data obtained from the participants revealed the diapason of combinations of amplitude and frequency changes for which the movement direction was perceived similarly by all participants, despite significantly different movement assessment criteria. Additional auditory and visual information of the conditions of radial movement (near or far fields) determined listeners' interpretation of changes in the signal parameters. The data obtained about the perception of approach and withdrawal models are evidence of the fact that the principal cues of the perception of the distance of immobile sound sources manifests similarly to that of an auditory image moving along a radial axis.


Assuntos
Percepção Auditiva , Percepção de Profundidade , Meio Ambiente , Ilusões , Percepção Espacial , Percepção Visual , Adolescente , Adulto , Sinais (Psicologia) , Feminino , Humanos , Masculino , Psicofisiologia/métodos
20.
Span. j. psychol ; 10(2): 266-276, nov. 2007. ilus, tab
Artigo em Inglês | IBECS | ID: ibc-77115

RESUMO

Auditory perception of the depth of space is based mainly on spectral and amplitude changes of sound waves originating from the sound source and reaching the listener. The perceptive illusion of movement of an auditory image caused by changes in amplitude and/or frequency of the signal tone emanating from an immobile loudspeaker was studied. Analysis of data obtained from the participants revealed the diapason of combinations of amplitude and frequency changes for which the movement direction was perceived similarly by all participants, despite significantly different movement assessment criteria. Additional auditory and visual information of the conditions of radial movement (near or far fields) determined listeners’ interpretation of changes in the signal parameters. The data obtained about the perception of approach and withdrawal models are evidence of the fact that the principal cues of the perception of the distance of immobile sound sources manifests similarly to that of an auditory image moving along a radial axis (AU)


La percepción auditiva de la profundidad del espacio se basa principalmente en los cambios de espectro y amplitud de las ondas de sonido que originan desde la fuente del sonido y alcanzan al escuchador. Se estudió la ilusión perceptiva del movimiento de una imagen auditiva causada por los cambios en la amplitud y/o la frecuencia del tono de señal que emana desde un altavoz inmóvil. Análisis de los datos obtenidos de los participantes reveló el diapasón de combinaciones de las modulaciones de amplitud y frecuencia para las cuales el vector de movimiento se percibió de la misma forma por todos los participantes, a pesar de criterios de evaluación del movimiento significativamente diferentes. Información adicional auditiva y visual de las condiciones de movimiento radial (campos cercanos o lejanos) determinó la interpretación de los cambios en los parámetros de la señal por parte de los escuchadores. Los datos obtenidos acerca de la percepción de los modelos de acercamiento y retirada son evidencia del hecho de que los rasgos principales de la percepción de la distancia de fuentes de sonido inmóviles se manifiesta de forma similar a la de una imagen auditiva que se mueve a lo largo de un eje radia (AU)


Assuntos
Humanos , Percepção Auditiva , Ilusões Ópticas , Percepção Visual , Ilusões/psicologia , Percepção do Tempo , Percepção Espacial , Percepção de Distância
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