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1.
Electrophoresis ; 2020 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-32335940

RESUMO

Lipid A represents a heterogeneous group of bacterial outer membrane phosphoglycolipids, which play a major role in the pathogenesis of Gram-negative sepsis. The number and position of phosphoryl and acyl groups in lipid A molecules are key structural determinants in their bioactivities. In this study, a NACE-ESI-MS/MS method was developed for the simultaneous analysis of lipid A isomers possessing a different degree of phosphorylation and acylation. Various C4'- and C1-monophosphorylated lipid A isobars, as well as acylation isomers, were baseline separated within 43 min in a separation medium of methanol/dichloromethane/triethylamine/acetic acid 60:40:1.08:0.36 (v/v/v/v). Both normal and reverse CE polarities could be applied for proper detection of the analytes owing to the combination of a suction effect caused by the nebulizer gas at the outlet end of the capillary and external pressure applied on the inlet vial. The separated lipid A species could be identified unequivocally by their characteristic fragmentation patterns through CID performed in both negative- and positive-ionization modes. The uniqueness of the NACE-ESI-MS/MS method lies in its simplicity and reliability for proving the phosphorylation isomerism (C1 or C4') and acylation pattern of native lipid A species or those designed for therapeutic applications.

2.
Electrophoresis ; 40(3): 447-454, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30407655

RESUMO

Perchloric acid (PCA) precipitation is a well-known method for the separation of heavily glycosylated proteins and for reducing the masking effect of major serum proteins. The aim of this study is to characterize PCA-soluble serum proteins in healthy individuals and in patients with systemic inflammatory diseases, such as Crohn's disease and sepsis. A PCA precipitation protocol was prepared and adapted to the analytical methods. After PCA treatment of the serum, the soluble proteins in the supernatant were analyzed by SDS-PAGE and by microchip gel electrophoresis (MGE). Characteristic changes of the electrophoretic patterns of the PCA-soluble fractions were observed. Four characteristic bands (at ∼11, ∼65, ∼85, and ∼120 kDa) with varying intensity were detected by MGE. The proportion of the ∼65, ∼85, and ∼120 kDa bands were significantly higher in systemic inflammatory conditions than in healthy individuals (p < 0.001), and characteristic patterns were observed in patients with acute inflammation. The marked differences in the acid-soluble protein patterns, which were observed in patients with ongoing systemic inflammation, might be a good indicator of inflammation. The MGE analysis is a fast screening and quantification method for the detection of characteristic changes among acid-soluble serum proteins.


Assuntos
Análise Química do Sangue/métodos , Proteínas Sanguíneas/análise , Eletroforese em Gel de Poliacrilamida/métodos , Procedimentos Analíticos em Microchip/métodos , Percloratos/química , Adolescente , Adulto , Proteínas Sanguíneas/química , Doença de Crohn/sangue , Doença de Crohn/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sepse/sangue , Sepse/diagnóstico , Adulto Jovem
3.
Gut Pathog ; 10: 47, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30386437

RESUMO

Background: Shigella spp. are Gram-negative intracellular pathogenic bacteria belonging to the family Enterobacteriaceae and can cause bacterial dysentery, a severe diarrheal disease. The pathophysiological impact of the Gram-negative bacteria is highly related to the composition and structural variability of lipopolysaccharides, the major lipoid components of the outer membrane. Out of the 114 genes involved in the lipopolysaccharide biosynthesis pathway, 47 genes are specific to Shigella spp. Changes in the specific genes can lead to loss of the O polysaccharide side chain, resulting in rough (R) type bacteria with increased sensitivity to temperature, or hydrophobic antibiotics. The formation of various different lipopolysaccharides or lipooligosaccharides has been observed previously in a mutant line showing altered biological properties, but the genetic background has not been investigated in detail. Results: The parental strain of the mutant line, Shigella sonnei 4303, was subjected to whole genome sequencing to gain a better insight into the structure and biosynthesis of lipopolysaccharides. The sequencing revealed a 4,546,505 bp long genome including chromosomal and plasmid DNA, and the lipopolysaccharide biosynthesis genes were also identified. A comparison of the genome was performed with the phylogenetically closely related, wild type, well characterized, highly virulent strain, S. sonnei 53G. Conclusion: Analysis of the lipopolysaccharide biosynthetic genes helped us to get more insight into the pathogenicity and virulence of the bacteria. The genome revealed high similarities with S. sonnei 53G, which can be used as a standard in characterizing the S. sonnei 4303's R-type isogenic derivatives.

4.
Electrophoresis ; 39(13): 1597-1605, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29635703

RESUMO

A fast and simple method with CZE coupled to ESI/QTOF-MS was optimized and validated for quantitative determination of organic acids (lactic acid, succinic acid, malic acid, tartaric acid, shikimic acid, and citric acid) in red wines. The BGE was ammonium acetate and the separation of the analytes was performed in a polybrene-coated capillary in the presence of EOF. The sample preparation included dilution and filtration of the wine. The method showed satisfactory performance characteristics: good linearity for each organic acid, with correlation coefficients ranging from r2  = 0.9902 (shikimic acid) to r2  = 0.9990 (tartaric acid). The limit of quantification was between 0.0034 mM (for shikimic acid) and 0.107 mM (for citric acid), and the recovery data fell between 95.8% (malic acid) and 102.7% (lactic acid); the total run time was less than 4 min. The RSD values for the interday repeatability and intraday reproducibility were between 3.44 and 9.50%, and between 1.75 and 8.29%, respectively. Seventeen Macedonian red Vranec wines were studied demonstrating a wide variation in the organic acids' concentration, which should be most probably due to the variation of the climate conditions in the vine areas.


Assuntos
Ácidos Carboxílicos/análise , Vinho/análise , Acetatos , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Espectrometria de Massas por Ionização por Electrospray
5.
J Mass Spectrom ; 53(2): 146-161, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29144587

RESUMO

In this study, we report the detailed analysis of the fragmentation patterns of positively charged lipid A species based on their tandem mass spectra obtained under low-energy collision-induced dissociation conditions of an electrospray quadrupole time-of-flight mass spectrometer. The tandem mass spectrometry experiments were performed after the separation of the compounds with a reversed-phase high performance liquid chromatography method. We found that both, phosphorylated and nonphosphorylated lipid A molecules can be readily ionized in the positive-ion mode by adduct formation with triethylamine added to the eluent. The tandem mass spectra of the lipid A triethylammonium adduct ions showed several product ions corresponding to inter-ring glycosidic cleavages of the sugar residues, as well as consecutive and competitive eliminations of fatty acids, phosphoric acid, and water following the neutral loss of triethylamine. Characteristic product ions provided direct information on the phosphorylation site(s), also when phosphorylation isomers (ie, containing either a C1 or a C4' phosphate group) were simultaneously present in the sample. Continuous series of high-abundance B-type and low-abundance Y-type inter-ring fragment ions were indicative of the fatty acyl distribution between the nonreducing and reducing ends of the lipid A backbone. The previously reported lipid A structures of Proteus morganii O34 and Escherichia coli O111 bacteria were used as standards. Although, the fragmentation pathways of the differently phosphorylated lipid A species significantly differed in the negative-ion mode, they were very similar in the positive-ion mode. The complementary use of positive-ion and negative-ion mode tandem mass spectrometry was found to be essential for the full structural characterization of the C1-monophosphorylated lipid A species.


Assuntos
Lipídeo A/química , Acilação , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/química , Estrutura Molecular , Fosforilação , Polissacarídeos/química , Proteus/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem
6.
J Sep Sci ; 40(24): 4825-4834, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29064636

RESUMO

Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low-level ampholytes (substances that are migrating within the pH gradient). The appearance of the substances within, or outside the pH gradient depends on, e.g., the composition of the ampholytes (broad or narrow pH range) or on the composition of electrolyte solutions. The experiments performed in coated capillaries (with polyvinyl alcohol or with polyacrylamide) show that the amount and the injection length of the ampholytes influence the length of the pH gradient formed in the capillary.


Assuntos
Eletroforese Capilar , Focalização Isoelétrica , Espectrometria de Massas , Proteínas/isolamento & purificação , Misturas Anfolíticas , Concentração de Íons de Hidrogênio
7.
Methods Mol Biol ; 1600: 151-165, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28478566

RESUMO

Endotoxins (lipopolysaccharides, LPS; lipooligosaccharides, LOS) are components of the envelope of Gram-negative bacteria. These molecules, responsible for both advantageous and harmful biological activity of these microorganisms, are highly immunogenic and directly involved in numerous bacterial diseases in humans, such as Gram-negative sepsis. The characterization of endotoxins is of importance, since their physiological and pathophysiological effects depend on their chemical structure. The differences among the LPS from different bacterial serotypes and their mutants include variations mainly within the composition and length or missing of their O-polysaccharide chains. Microchip electrophoretic methodology enables the structural characterization of LPS molecules from several bacteria and the quantitative evaluation of components of endotoxin extracts. The improved microchip electrophoretic method is based on the direct labeling of endotoxins by covalent binding of a fluorescent dye. The classification of the S-type LPSs can be done according to their electrophoretic profiles, which are characteristics of the respective bacterial strains. According to the number, distribution, and the relative amounts of components in an endotoxin extract, it is possible to differentiate between the S-type endotoxins from different Gram-negative bacterial strains. The microchip electrophoresis affords high-resolution separation of pure and partially purified (e.g., obtained from whole-cell lysate) S and R endotoxins. This microchip technique provides a new, standardizable, fast, and sensitive method for the detection of endotoxins and for the quantitative evaluation of components of an endotoxin extract.


Assuntos
Eletroforese Capilar/métodos , Endotoxinas/análise , Lipopolissacarídeos/análise
8.
Methods Mol Biol ; 1600: 187-198, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28478568

RESUMO

Lipopolysaccharides (LPSs, endotoxins) are components of the outer cell membrane of most Gram-negative bacteria and can play an important role in a number of diseases of bacteria, including Gram-negative sepsis. The hydrophilic carbohydrate part of LPSs consists of a core oligosaccharide (in the case of an R-type LPS or lipooligosaccharide, LOS) linked to an O-polysaccharide chain (in the case of an S-type LPS), which is responsible for O-specific immunogenicity. The hydrophobic lipid A anchor is composed of a phosphorylated diglucosamine backbone to which varying numbers of ester- and amide-linked fatty acids are attached and this part of the LPSs is associated with endotoxicity. The detailed chemical characterization of endotoxins requires long-lasting large-scale isolation procedures, by which high-purity LPSs can be obtained. However, when a large number of bacterial samples and their LPS content are to be compared prompt, small-scale isolation methods are used for the preparation of endotoxins directly from bacterial cell cultures. The purity of the endotoxins extracted by these methods may not be high, but it is sufficient for analysis.Here, we describe a fast and easy micromethod suitable for extracting small quantities of LOS and a slightly modified micromethod for the detection of the lipid A constituents of the LPSs from bacteria grown in different culture media and evaluate the structures with mass spectrometry. The cellular LOS and lipid A were obtained from crude isolates of heat-killed cells, which were then subjected to matrix-assisted laser desorption/ionization mass spectrometry analysis. The observed ions in the 10-colony samples were similar to those detected for purified samples. The total time for the sample preparation and the MS analysis is less than 3 h.


Assuntos
Lipídeo A/química , Lipopolissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Oligossacarídeos/química
9.
Electrophoresis ; 38(5): 677-688, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27699824

RESUMO

This paper comprises a continuation of computer simulation studies dealing with carrier ampholyte based CIEF in presence of narrow pH gradients. With this technique, amphoteric sample components with pI values outside the pH gradient are migrating isotachophoretically toward the cathode or anode whereas components with pI values within the gradient become focused. In order to understand the processes occurring in presence of the electric field, the behavior of both carrier ampholytes and amphoteric sample components is investigated by computer modeling. Characteristics of two pH unit gradients with end components having pI values at or around 7.00 and conditions that lead to the formation of a water zone at neutrality were investigated. Data obtained reveal that a zone of water is formed in focusing with carrier ampholytes when the applied pH range does not cover the neutral region, ends at pH 7.00 or begins at pH 7.00. The presence of additional amphoteric components that cover the neutrality region prevent water zone formation under current flow. This situation is met in experiments with narrow pH gradients that end or begin around neutrality. Simulation data reveal that no water zone evolves when atmospheric carbon dioxide dissolved in the catholyte causes the migration of carbonic acid (in the form of carbonate and/or hydrogen carbonate ions) from the catholyte through the focusing structure. An electrolyte change in the electrode solution does not have an impact on the focusing part but does change the isotachophoretic pattern migrating behind the leading ion.


Assuntos
Misturas Anfolíticas/química , Eletroforese Capilar/métodos , Focalização Isoelétrica/métodos , Ácido Carbônico/química , Simulação por Computador , Concentração de Íons de Hidrogênio , Água/química
10.
Int J Clin Pharm ; 39(1): 78-87, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27888454

RESUMO

Background Growth hormones are widely available on the Internet for those who want to enhance their physical performance and improve body satisfaction. Illegitimate websites market somatropin injections without medical prescription and encourage misuse. Customers potentially put their health at risk when purchasing parenteral medications online. Objective The objective of our study was to evaluate the online market of no-prescription somatropin products and to analyse and document Internet pharmacy characteristics, distribution and pharmaceutical quality. Setting Websites indexed in Google promoting somatropin for sale direct to patients. Method Websites promoting the sale of growth hormone products were identified and analysed from June to August 2014. Internet vendor sites were evaluated to identify possible patient and medication safety concerns. Website characteristics, delivery time, storage conditions, packaging and attached product information were assessed. Investigation of the somatropin content was achieved using capillary electrophoresis with UV detection and electrospray ionization mass spectrometry. Main outcome measure Accessibility and quality of somatropin injections. Results Seventeen individual Internet vendor websites distributed somatropin products directly to patients, majority (94%) did not require a valid medical prescription before dispensing the products. Majority (70%) of Internet pharmacies displayed no medical information and none (0%) of the vendors displayed any regulatory body logo. All online samples had significantly (p < 0.001) lower somatropin concentration than labelled. Conclusion Our results clearly illustrate that prescription only biologic drugs are widely available online and can be easily accessed by anyone. Unprofessional distribution and handling is likely to cause degradation and possible patient safety concerns.


Assuntos
Medicamentos Falsificados , Hormônio do Crescimento Humano/normas , Internet/normas , Disponibilidade de Medicamentos Via Internet/normas , Medicamentos sob Prescrição/normas , Humanos , Internet/legislação & jurisprudência , Disponibilidade de Medicamentos Via Internet/legislação & jurisprudência
11.
J Mass Spectrom ; 51(11): 1043-1063, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27506631

RESUMO

We established a new reversed phase-high performance liquid chromatography method combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross-reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2' or the C3' sites. The fragmentation of the various, C4' monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri-acylated, tetra-acylated, penta-acylated, hexa-acylated and hepta-acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa-acylated ion at m/z 1716 with the E. coli-type acyl distribution and the hepta-acylated ion at m/z 1954 with the Salmonella-type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative-ion mode MS permitted efficient detection for non-phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Lipídeo A/química , Acilação , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/química , Hidrólise , Fosforilação , Proteólise , Proteus/química , Salmonella/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
12.
J Chromatogr A ; 1456: 152-61, 2016 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-27317005

RESUMO

Cavitands are cavity-shaped cyclic oligomers and they can create host-guest interactions with various analytes, therefore they have applications in supramolecular chemistry, nanoscale reactions, chromatographic separations, drug encapsulation and delivery, biochemistry. The investigation of the chromatographic behavior of large molecules, such as resorcinarenes and cavitands is meager up to now. To understand the retention of resorcinarenes and cavitands in liquid chromatography, we studied their retention mechanism by the thermodynamic parameters calculated from the van't Hoff equation and by generation of an adsorption isotherm, which can describe the adsorption of the solute on the stationary phase surface. We compared the thermodynamics of the retention for cyclic oligomers in acetonitrile:water and methanol:water mobile phases. Furthermore, we determined the equilibrium adsorption isotherm by inverse method and we made an error analysis of the estimation obtained with the inverse method to ascertain the validity of the obtained isotherm parameters over a broader concentration range.


Assuntos
Calixarenos/química , Cromatografia Líquida/métodos , Éteres Cíclicos/química , Fenilalanina/análogos & derivados , Resorcinóis/química , Acetonitrilos , Adsorção , Algoritmos , Metanol , Fenilalanina/química , Solventes , Termodinâmica , Água
13.
J Mass Spectrom ; 51(8): 615-628, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28239963

RESUMO

Non-phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non-phosphorylated lipid A species under low-energy collision-induced dissociation conditions of an electrospray ionization quadrupole time-of-flight instrument. Charge-promoted consecutive and competitive eliminations of the acyl chains and cross-ring cleavages of the sugar residues were observed. The A-type fragment ion series and the complementary X-type fragment(s) with corresponding deprotonated carboxamide(s) were diagnostic for the distribution of the primary and secondary acyl residues on the non-reducing and the reducing ends, respectively, of the non-phosphorylated lipid A backbone. Reversed-phase liquid chromatography in combination with negative-ion electrospray ionization quadrupole time-of-flight tandem mass spectrometry could provide sufficient information on the primary and secondary acyl residues of a non-phosphorylated lipid A. As a standard, the hexa-acylated ion at m/z 1636 with the Escherichia coli-type acyl distribution (from E. coli O111) was used. The method was tested and refined with the analysis of other non-phosphorylated hexa- and several hepta-, penta-, and tetra-acylated lipid A species detected in crude lipid A fractions from E. coli O111 and Proteus morganii O34 bacteria. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lipídeo A/análise , Lipídeo A/química , Espectrometria de Massas em Tandem/métodos , Escherichia coli/química , Modelos Moleculares , Fosforilação , Proteus/química
14.
J Sep Sci ; 38(17): 2975-82, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26084870

RESUMO

The understanding of the retention behavior of large molecules is an area of interest in liquid chromatography. Resorcinarene-based cavitands are cavity-shaped cyclic oligomers that can create host-guest interactions. We have investigated the chromatographic behavior of two types of cyclic tetramers as analytes in high-performance liquid chromatography. The experiments were performed at four different temperatures (15, 25, 35, 45°C) on two types of reversed stationary phases (C8 and C18 ) from two different manufacturers. We have found a huge difference between the retention of resorcinarenes and cavitands. In some cases, the retention factor of cavitands was even a hundred times larger than the retention factor of resorcinarenes. The retention of methylated derivates was two to four times larger compared to that of demethylated compounds on every column. The opposite retention behavior of the resorcinarenes and cavitands on the two types of stationary phases showed well the difference of the selectivity of the XTerra and BDS Hypersil columns. The retention mechanism was studied by the thermodynamic parameters calculated from the van't Hoff equation.


Assuntos
Calixarenos/química , Éteres Cíclicos/química , Fenilalanina/análogos & derivados , Resorcinóis/química , Calixarenos/análise , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida/métodos , Fenilalanina/análise , Fenilalanina/química , Temperatura , Termodinâmica
15.
Electrophoresis ; 36(11-12): 1336-43, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25630395

RESUMO

The structure of the oligosaccharide repeating units of endotoxins from Gram-negative bacteria is characteristic for the different serogroups and serotypes of bacteria. Detailed examination of the cross-reactions of three enterobacterial serotypes, Proteus morganii O34, Escherichia coli O111, and Salmonella enterica sv. Adelaide O35, was performed using sensitive tests (ELISA, immunoblotting). Fine differences between the endotoxins of the bacteria were detected using silver staining of SDS-PAGE gels and chip-technology for the intact lipopolysaccharides (LPSs). The compositions of the O-specific polysaccharides of LPSs extracted from the bacteria were studied, and it was proven that the three cross-reacting bacteria contain O-antigens built from the same monosaccharides, namely colitoses linked to glucose, galactose, and N-acetyl-galactosamine. The NMR and GC-MS studies revealed that the most probable component for the cross-reaction is the rare sugar, colitose.


Assuntos
Reações Cruzadas , Enterobacteriaceae/imunologia , Configuração de Carboidratos , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Lipopolissacarídeos/química , Ressonância Magnética Nuclear Biomolecular
16.
Neuropeptides ; 48(6): 371-8, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25455106

RESUMO

The aim of the present study was to evaluate the therapeutic potential of local capsaicinoid (EMSPOMA(®) cream) treatment on chronic low back pain in patients with degenerative spine diseases and to investigate the possible mechanism of action of the therapy. The qualitative and quantitative analyses of capsaicinoids in EMSPOMA(®) cream were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the clinical study 20 patients with degenerative spine diseases were involved in a self-controlled examination. During the 21 day therapy they received 30 min daily treatment with capsaicinoid (EMSPOMA(®)) cream to the lumbar region of the back. The pain (VASs, Oswestry Disability Index) and the mobility of the lumbar region of the spine (Schober's, Domján's L and R test) were detected at baseline and at the end of the 1st, 2nd and 3rd weeks. The plasma level of somatostatin-like immunoreactivity (SST-LI) was measured by radioimmunoassay (RIA) before and after the treatment on the first and the last day of the therapy. Nonivamide (0.01%) was identified as the only capsaicinoid molecule in the cream. In the clinical study the 21 day local nonivamide treatment reduced the pain sensation. Oswestry Disability Index decreased from 39 ± 3.9% to 32.5 ± 4.4%. VASs showed 37.29%-59.51% improvement. In the plasma level of SST-LI threefold elevation was observed after the first nonivamide treatment. We conclude that nonivamide treatment exerts analgesic action in chronic low back pain and causes the release of the antinociceptive and anti-inflammatory neuropeptide somatostatin which may play pivotal role in the pain-relieving effect.


Assuntos
Analgésicos/administração & dosagem , Capsaicina/análogos & derivados , Dor Lombar/tratamento farmacológico , Somatostatina/sangue , Administração Cutânea , Idoso , Capsaicina/administração & dosagem , Feminino , Humanos , Dor Lombar/etiologia , Masculino , Pessoa de Meia-Idade , Medição da Dor , Doenças da Medula Espinal/complicações
17.
Nat Prod Commun ; 8(9): 1247-50, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24273858

RESUMO

n-Hexane, chloroform, ethyl acetate and 50% ethanol in water extracts prepared from the air-dried flowering parts of Lythrum salicaria L. were tested for in vitro pharmacological properties on Guinea-pig ileum, which is suitable for detecting a whole range of neuronal and smooth muscle effects. UHPLC-MS was used to evaluate polyphenol components of the extracts. In the ileum, the most prominent response (46.4% related to 0.5 microM histamine) of the extracts causing smooth muscle contractions were triggered by the 50% ethanol in water extract in a concentration-dependent manner. Atropine, indomethacin and PPADS plus suramin significantly reduced the contractile response caused by this extract. The strongest inhibition was due to atropine. The results suggest that L. salicaria extracts have a moderate muscarinic receptor agonist effect in Guinea-pig ileum and that prostanoids and purinoceptor mechanisms are involved to some extent. Therefore diluted extracts of L. salicaria p.o. could be used as a mild stimulant of gastrointestinal motility. The 50% ethanol in water extract was rich in polyphenols. n-Hexane, chloroform and ethyl acetate extracts failed to contain catechin, caffeic acid, quercetin-3-D-galactoside and rutin, but they all showed spasmogenic effects, and, therefore we do not think that these compounds could be involved in the spasmogenic activity.


Assuntos
Fármacos Gastrointestinais/análise , Motilidade Gastrointestinal/efeitos dos fármacos , Íleo/efeitos dos fármacos , Lythrum/química , Extratos Vegetais/farmacologia , Animais , Feminino , Cobaias , Masculino , Extratos Vegetais/química , Plantas Medicinais/química
18.
PLoS One ; 8(4): e62142, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23646117

RESUMO

We investigated the antifungal activity of fused Mannich ketone (FMK) congeners and two of their aminoalcohol derivatives. In particular, FMKs with five-membered saturated rings were shown to have minimum inhibitory concentration (MIC90s) ranging from 0.8 to 6 µg/mL toward C. albicans and the closely related C. parapsilosis and C. krusei while having reduced efficacy toward C. glabrata and almost no efficacy against Aspergillus sp. Transcript profiling of C. albicans cells exposed for 30 or 60 min to 2-(morpholinomethyl)-1-indanone, a representative FMK with a five-membered saturated ring, revealed a transcriptional response typical of oxidative stress and similar to that of a C. albicans Cap1 transcriptional activator. Consistently, C. albicans lacking the CAP1 gene was hypersensitive to this FMK, while C. albicans strains overexpressing CAP1 had decreased sensitivity to 2-(morpholinomethyl)-1-indanone. Quantitative structure-activity relationship studies revealed a correlation of antifungal potency and the energy of the lowest unoccupied molecular orbital of FMKs and unsaturated Mannich ketones thereby implicating redox cycling-mediated oxidative stress as a mechanism of action. This conclusion was further supported by the loss of antifungal activity upon conversion of representative FMKs to aminoalcohols that were unable to participate in redox cycles.


Assuntos
Antifúngicos/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Bases de Mannich/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Antifúngicos/química , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Bases de Mannich/química , Mutação , Relação Quantitativa Estrutura-Atividade , Leveduras/efeitos dos fármacos , Leveduras/metabolismo
20.
Methods Mol Biol ; 968: 149-54, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23296892

RESUMO

Chip-based flow cytometry is a rather new method that offers an easy, fast opportunity for examination of yeasts, such as Candida cells. In our study cell-chip technology was tested with ATCC Candida strains to determine their viability and susceptibility against antifungal agents, amphotericin B and fluconazole. We found this technology to be suitable for the detection of Candida cells, for the differentiation between dead and living cells, and for the determination of amphotericin B and fluconazole susceptibility of different Candida strains (Bouquet et al., Mycoses 55:e90-e96, 2012).


Assuntos
Anfotericina B/farmacologia , Candida/efeitos dos fármacos , Citometria de Fluxo/métodos , Fluconazol/farmacologia , Procedimentos Analíticos em Microchip/métodos , Farmacorresistência Fúngica , Testes de Sensibilidade Microbiana
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