RESUMO
BACKGROUND AND THE PURPOSE OF THE STUDY: Ofloxacin is a quinolone synthetic antibiotic, which acts against resistant mutants of bacteria by inhibiting DNA gyrase. This antibacterial agent is widely used in the treatment of respiratory tract, urinary tract and tissue-based infections, which are caused by Gram-positive and Gram-negative bacteria. In this work, an efficient modified ionic liquid cold-induced aggregation dispersive liquid-liquid microextraction (M-IL-CIA-DLLME) was combined with spectrofluorimetry for trace determination of ofloxacin in real samples. METHODS: In this microextraction method, hydrophobic 1-hexyl-3-methylimidazolium hexafluorophosphate ([Hmim] [PF(6)]) ionic liquid (IL) as a microextraction solvent was dispersed into a heated sample solution containing sodium hexafluorophosphate (NaPF(6)) (as a common ion) and the analyte of interest. Afterwards, the resultant solution was cooled in an ice-water bath and a cloudy condition was formed due to a considerable decrease of IL solubility. After centrifuging, the enriched phase was introduced to the spectrofluorimeter for the determination of ofloxacin. RESULTS AND MAJOR CONCLUSION: In this technique, the performance of the microextraction method was not influenced by variations in the ionic strength of the sample solution (up to 30% w/v). Furthermore, [Hmim][PF(6)] IL was chosen as a green microextraction phase and an alternative to traditional toxic organic solvents. Different parameters affecting the analytical performance were studied and optimized. At optimum conditions, a relatively broad linear dynamic range of 0.15-125 µg l(-1) and a limit of detection (LOD) of 0.029 µg l(-1) were obtained. The relative standard deviation (R.S.D.) obtained for the determination of five replicates of the 10 ml solution containing 50 µg l(-1) ofloxacin was 2.7%. Finally, the combined methodology was successfully applied to ofloxacin determination in actual pharmaceutical formulations and biological samples.
RESUMO
The intestinal guanylyl cyclase-C (GC-C) was originally identified as an Escherichia coli heat-stable enterotoxin (STa) receptor. STa stimulates GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin, causing severe diarrhea. To investigate the interactions of the endogenous and bacterial ligands with GC-C, we designed and characterized a soluble and properly folded fragment of the extracellular ligand-binding domain of GC-C. The membrane-bound guanylyl cyclases exhibit a single transmembrane spanning helix and a globularly folded extracellular ligand-binding domain that comprises about 410 of 1050 residues. Based on the crystal structure of the dimerized-binding domain of the guanylyl cyclase-coupled atrial natriuretic peptide receptor and a secondary structure-guided sequence alignment, we generated a model of the extracellular domain of GC-C comprised of two subdomains. Mapping of mutational and cross-link data onto this structural model restricts the ligand-binding region to the membrane proximal subdomain. We thus designed miniGC-C, a 197 amino acid fragment that mimics the ligand-binding membrane proximal subdomain. Cloning, expression and spectroscopic studies reveal miniGC-C to be a soluble and properly folded protein with a distinct secondary and tertiary structure. MiniGC-C binds STa with nanomolar affinity.
Assuntos
Guanilato Ciclase/química , Guanilato Ciclase/genética , Modelos Moleculares , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Animais , Sítios de Ligação , Escherichia coli/enzimologia , Vetores Genéticos , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Suínos/genéticaRESUMO
A novel and selective procedure for the determination of l-cysteine and l-cystine based on vapour-generation Fourier transform infrared spectrometry is described. Potassium iodate solution was injected into a glass vessel containing l-cysteine and/or l-cystine. The evolved CO was swept by a stream of nitrogen to an infrared gas cell. The vapour phase FTIR spectra were continuously recorded, as a function of time, between 2240 and 2000cm(-1), which includes the CO absorption band. The maximum absorbance at 2170cm(-1) was selected as a measurement criterion. The calibration curve was linear over the range 6-300mgL(-1). The method provided a limit of detection of 2mgL(-1) of l-cysteine, a throughput of 12samples h(-1) and an R.S.D. of 1.76% for five independent analyses of a 75mgL(-1)l-cysteine solution. For the measurement of l-cysteine and l-cystine separately, after measuring total concentration of l-cysteine and l-cystine, l-cysteine was masked with p-benzoquinone at a pH of 3 and individual l-cystine was determined. The amount of l-cysteine was obtained by difference. The method was applied to the determination of l-cysteine and l-cystine in pharmaceutical and urine samples. Results obtained for real samples compared well with those obtained by a reference spectrometric method.
RESUMO
The refolding reaction of S54G/P55N ribonuclease T1 is a two-step process, where fast formation of a partly folded intermediate is followed by the slow reaction to the native state, limited by a trans --> cis isomerization of Pro39. The hydrodynamic radius of this kinetic folding intermediate was determined by real-time diffusion NMR spectroscopy. Its folding to the native state was monitored by a series of 128 very fast 2D (15)N-HMQC spectra, to observe the kinetics of 66 individual backbone amide probes. We find that the intermediate is as compact as the native protein with many native chemical shifts. All 66 analyzed amide probes follow the rate-limiting prolyl isomerization, which indicates that this cooperative refolding reaction is fully synchronized. The stability of the folding intermediate was determined from the protection factors of 45 amide protons derived from a competition between refolding and H/D exchange. The intermediate has already gained 40% of the Gibbs free energy of refolding with many protected amides in not-yet-native regions.
Assuntos
Dobramento de Proteína , Guanidina , Cinética , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Desnaturação ProteicaRESUMO
A review of n = 5216 semen analyses performed in our two Clinics from January 1986 to December 1989 allowed to identify n = 35 patients whose sperm had constantly very low motility (less than 5% progressive motile gametes in three subsequent analyses; necrozoospermia cases were excluded from this study). This apparently rare but severe anomaly was found to be associated not only with ultrastructural anomalies (n = 18), but also with positive seminal bacteriology (n = 8) or the presence of antisperm antibodies (n = 2). In eight cases the cause(s) for this constant asthenozoospermia remained obscure. The fertility potential of the men affected was followed-up and is discussed in relation to their anamnesis, physical exam and seminal characteristics.
Assuntos
Infertilidade Masculina/etiologia , Motilidade dos Espermatozoides , Adulto , Autoanticorpos/análise , Bactérias/isolamento & purificação , Feminino , Humanos , Infertilidade Masculina/terapia , Masculino , Microscopia Eletrônica , Gravidez , Prognóstico , Sêmen/microbiologia , Espermatozoides/anormalidades , Espermatozoides/imunologia , Espermatozoides/ultraestruturaRESUMO
The authors wish to stress the importance of the psychosomatic approach in the investigation of infertility. Following the hypothesis that infertile patients may present emotional conflicts with regard to the wish of having a child, psychodynamic interviews were carried out with 116 infertile couples concomitantly with their first consultation at the Sterility Department. The psychological findings show that severe conflicts with regard to wish of parenthood were present in the majority of the female patients and especially in those without clinically defined organic infertility factors. The relationship between clinical diagnosis and psychological outcome was less evident in the male partners. The 11 pregnancies which occurred in the study group are also discussed with regard to the motivational quality of the wish of child of these patients. A recommendation is made that psychological interviews should be included in any infertility investigation as a matter of routine and that therapeutic procedures should take into account both the clinical and psychological diagnosis to prevent unsuccessful situations.
Assuntos
Mecanismos de Defesa , Infertilidade Feminina/psicologia , Transtornos Psicofisiológicos/psicologia , Adulto , Conflito Psicológico , Feminino , Humanos , Infertilidade Masculina/psicologia , Masculino , Motivação , Gravidez , Fatores de RiscoRESUMO
The authors present the problems that have been raised by psychologically interviewing 740 couples who ask for A.I.D. They describe the procedure that they adopted and the features that came up during the interview and particularly in the way the couples experienced their sterility. They conclude by pointing out how important a consultation is, both from the point of view of evaluating the couple psychologically in regard to the treatment and from the point of view of clarifying the needs, the doubts and the expectations that the two partners have when they decide to undertake A.I.D.