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Biomedical subjects

F Salinas

Publications and source records attributed to F Salinas.

At least 19 recordsLinked to original sources
PubMed2005

HPLC determination of enoxacin, ciprofloxacin, norfloxacin and ofloxacin with photoinduced fluorimetric (PIF) detection and multiemission scanning: application to urine and serum.

The fluorescence emission of the fluoroquinolones enoxacin (ENO), ciprofloxacin (CIPRO), norfloxacin (NOR) and ofloxacin (OFLO) notably increased by UV irradiation during few minutes, in ethanolic-water medium. An HPLC method has been developed, for the determination of these fluoroquinolones, based in the separation of the formed irradiation photoproducts. Optimization of the analytical wavelengths has been carried out by fast multiemission scanning fluorescence detection. The highest sensitivity has been found when measuring at emission wavelengths of 407 and 490 nm, for ENO and OFLO, respectively, and at 444 nm for both NOR and CIPRO (exciting at 277 nm). According to the criterium of Clayton, using 0.05 as false positive and false negative error assurance probabilities, detection limits of 7.3, 6.0, 6.3 and 14.5 ng/mL, for ENO, NOR, CIPRO and OFLO, respectively, have been found. Urine and serum samples have been successfully analyzed, with recovery values ranging among 99-97% and 98-103%, for urine and serum, respectively.

Chromatography, High Pressure Liquid↗
PubMed2005

Improvements in europium sensitized fluorimetric determination of demeclocycline and methacycline.

Demeclocycline (DM) and methacycline (MT) have been determined by europium-sensitized fluorescence, using EDTA as co-ligand and cetyltrimethylammonium chloride as surfactant. The methods have been developed in slightly alkaline solutions, with the formation of a new chelate where the lanthanide ion is bound to the beta-diketone group. Calibration graphs between 0.01 and 0.1 microg mL(-1) have been obtained for DM and MT determination. Both methods have been applied to the determination of these tetracyclines in serum samples with satisfactory recovery results.

Demeclocycline↗
PubMed2001

Resolution by polarographic techniques of the ternary mixture of captan, captafol and folpet by using PLS calibration and artificial neuronal networks.

The simultaneous polarographic determination of the ternary mixture of captan-captafol and folpet is studied. The polarographic signals of these compounds in their mixture show a high overlapping. For this reason different chemometric methods such as PLS, PCR and artificial neuronal network (ANN) have been utilized for the simultaneous determination of these compounds in mixtures. The calibration model is built from solutions containing river water of known pesticide concentrations and the signals obtained by Sampled DC and DPP (differential pulse polarography) have been used. The analysis of both synthetic and real samples (river water) has been carried out by PLS with satisfactory results in most cases. It is possible to determine 0.25 ppm of each pesticide in river water samples after a preconcentration step by extraction into diethyl ether. ANN has also been applied to improve the results obtained by the PLS tool when the sampled DC current is recorded or when liquid-solid extraction with C18 cartridges is performed.

Calibration↗
PubMed2001

Replisome-mediated DNA replication.

The elaborate process of genomic replication requires a large collection of proteins properly assembled at a DNA replication fork. Several decades of research on the bacterium Escherichia coli and its bacteriophages T4 and T7 have defined the roles of many proteins central to DNA replication. These three different prokaryotic replication systems use the same fundamental components for synthesis at a moving DNA replication fork even though the number and nature of some individual proteins are different and many lack extensive sequence homology. The components of the replication complex can be grouped into functional categories as follows: DNA polymerase, helix destabilizing protein, polymerase accessory factors, and primosome (DNA helicase and DNA primase activities). The replication of DNA derives from a multistep enzymatic pathway that features the assembly of accessory factors and polymerases into a functional holoenzyme; the separation of the double-stranded template DNA by helicase activity and its coupling to the primase synthesis of RNA primers to initiate Okazaki fragment synthesis; and the continuous and discontinuous synthesis of the leading and lagging daughter strands by the polymerases. This review summarizes and compares and contrasts for these three systems the types, timing, and mechanism of reactions and of protein-protein interactions required to initiate, control, and coordinate the synthesis of the leading and lagging strands at a DNA replication fork and comments on their generality.

Bacterial Proteins↗
PubMed2000

Characterization of bacteriophage T4-coordinated leading- and lagging-strand synthesis on a minicircle substrate.

The DNA replication complex of bacteriophage T4 has been assembled as a single unit on a minicircle substrate with a replication fork that permits an independent measurement of the amount of DNA synthesis on both the leading and lagging strands. The assembled replisome consists of the T4 polymerase [gene product 43 (gp43)], clamp protein (gp45), clamp loader (gp44/62), helicase (gp41), helicase accessory factor (gp59), primase (gp61), and single-stranded DNA binding protein (gp32). We demonstrate that on the minicircle the synthesis of the leading and lagging strands are coordinated and that the C-terminal domain of the gp32 protein regulates this coordination. We show that the reconstituted replisome encompasses two coupled holoenzyme complexes and present evidence that this coupling might include a gp43 homodimer interaction.

Bacteriophage T4↗
PubMed2000

Using an AraC-based three-hybrid system to detect biocatalysts in vivo.

Recent methods to create large libraries of proteins have greatly advanced the discovery of proteins with novel functions. However, one limitation in the discovery of new biocatalysts is the screening or selection methods employed to find enzymes from these libraries. We have developed a potentially general method termed QUEST (QUerying for EnzymeS using the Three-hybrid system), which allows the construction of an easily screened or selected phenotype for, in theory, any type of enzymatic reaction. The method couples the in vivo concentration of an enzyme's substrate to changes in the transcriptional level of a reporter operon. Using the arabinose operon activator AraC, we constructed a system capable of detecting the fungal enzyme scytalone dehydratase (SD) in bacteria, and demonstrated its sensitivity and usefulness in library screening.

AraC Transcription Factor↗
PubMed1999

Use of a stopped-flow pneumatic mixing module to analyze dinitrophenol pesticides. Simultaneous determination of dinoseb and dinobuton.

A kinetic study of the degradation of dinobuton in an alkaline medium has been undertaken by using a stopped-flow pneumatic system. A semiautomatic method for determining dinobuton is proposed for the first time. A detection limit of 0.40 microg mL(-)(1) was calculated. Simultaneous determination of dinobuton and dinoseb can be made by combination of equilibrium and kinetic measurements. The proposed method has been applied to analyze binary mixtures of dinobuton and dinoseb samples with the amount of both components simulating the composition of one undegraded and several degraded dinobuton samples. Also, the procedure has been tested in the analysis of a commercial formulation of dinobuton and the results validated with high-performance liquid chromatography (HPLC).

2,4-Dinitrophenol↗
PubMed1998

High-performance liquid chromatographic-fluorometric determination of glyoxal, methylglyoxal, and diacetyl in urine by prederivatization to pteridinic rings.

A sensitive and simple liquid chromatographic method to determine glyoxal, methylglyoxal, and diacetyl is reported. The method is based on the conversion to the corresponding pteridin derivatives (pterin, 6-methylpterin, and 6,7-dimethylpterin). The proposed method using fluorometric detection has been applied to the determination of the three alpha-dicarbonyl compounds in human urine. Linearity (peak area vs concentration of alpha-dicarbonyl) was observed at least up to 43 microM. Detection limits of 32 pmol for glyoxal, 11 pmol for methylglyoxal, and 99 pmol for diacetyl were calculated (20 microliters was injected). Levels of 132 microM for glyoxal and 15 microM for methylglyoxal were determined in normal urine samples, while diacetyl was not detected.

Chemical Phenomena↗
PubMed1998

Simultaneous determination of pteridines in multicomponent mixtures using derivative spectrophotometry and partial least-squares calibration.

Simple binary mixtures composed of very similar pteridines (neopterin and pterin) have been resolved by derivative spectrophotometry. Detection limits of 0.30 microgram ml-1 and 0.12 microgram ml-1 for pterin and neopterin, respectively, have been calculated. Also, different mixtures of pteridines considered as disease markers, such as pterin, neopterin, xanthopterin and isoxanthopterin, have been determined by using a partial least-squares (PLS-2) model. Calibration set containing 0-7 micrograms ml-1 for each component was used. The resolution of several mixtures and single determination was tested in artificial samples.

Drug Combinations↗
PubMed1997

Determination of nitrofurantoin, furazolidone and furaltadone in milk by high-performance liquid chromatography with electrochemical detection.

A HPLC method with coulometric detection has been established to carry out the separation of the three nitrofuran derivatives, nitrofurantoin, furazolidone and furaltadone. A Nova-Pak C18 column (150 x 3.9 mm) and a Coulochem II detector from ESA have been used. After obtaining the hydrodynamic curves of the three compounds in the porous graphite electrode a potential of -600 mV was selected as the working potential. The influence of other variables such as mobile phase composition and flow-rate were studied. The mobile phase considered as an optimum was acetonitrile-0.1 M aqueous solution of sodium perchlorate (28:72), with 0.5% glacial acetic acid. The oxygen of the mobile phase was removed with a vacuum system on-line and a nitrogen stream was used to remove the oxygen of the samples. The calibration graphs and the detection limits were established. The method proposed was used, with good results, for the determination of the three compounds in milk.

Animals↗
PubMed1997

The gene 32 single-stranded DNA-binding protein is not bound stably to the phage T4 presynaptic filament.

A central reaction in homologous recombination is synapsis, which involves invasion of duplex DNA by a homologous single strand. A key intermediate in this process is the presynaptic filament, a protein-DNA complex composed of a "strand transferase" polymerized along the invading single strand. In this report, the organization and mechanism of assembly of the bacteriophage T4 presynaptic filament are explored. Three T4 proteins, encoded by the uvsX, uvsY and 32 genes, are involved in this process. It is demonstrated that a well-defined series of events involving multiple protein-DNA and protein-protein interactions is required to mediate a transition from an initial gene 32-DNA complex to a mature presynaptic filament in which the UvsX and UvsY proteins are in contact with the DNA and each other, while most or all of the gene 32 protein is removed from the complex.

Bacteriophage T4↗
PubMed1995

Phage T4 homologous strand exchange: a DNA helicase, not the strand transferase, drives polar branch migration.

Homologous strand exchange is a central step in general genetic recombination. A multiprotein complex composed of five purified bacteriophage T4 proteins (the products of the uvsX, uvsY, 32, 41, and 59 genes) that mediates strand exchange under physiologically relevant conditions has been reconstituted. One of these proteins, the product of the uvsY gene, is required for homologous pairing but strongly inhibits branch migration catalyzed by UvsX protein, the phage RecA analog. Branch migration is completely dependent on the gene 41 protein, a DNA helicase that also functions in phage replication. The helicase is delivered to the strand exchange complex by the gene 59 accessory protein in a strand-specific fashion through direct interactions between the gene 59 and gene 32 proteins. These data suggest that strand transferases such as UvsX protein are essential for homologous pairing in vivo, but that a DNA helicase drives polar branch migration.

Bacteriophage T4↗
PubMed1995

Homology dependence of UvsX protein-catalyzed joint molecule formation.

The bacteriophage UvsX protein is a "strand transferase" that promotes the pairing of homologous single and double-stranded DNAs. The efficiency of UvsX protein-mediated joint molecule formation between supercoiled duplex DNA and oligonucleotides is shown to have a sharp dependence on the degree of homology. The reaction proceeded efficiently with oligonucleotides containing 32 homologous positions but not with oligonucleotides containing only 24 homologous bases. This was shown to reflect an intrinsic homology requirement for the formation of stable joint molecules and was not caused by poor binding of the protein to short single-stranded DNAs. Even a single mismatch located in the middle of a region of 40 homologous nucleotides had a detectable effect on the efficiency of pairing. An in vitro recombinationally initiated DNA synthesis reaction that mimics the "secondary mode" of phage T4 DNA replication exhibited the same homology dependence.

Adenosine Triphosphatases↗
PubMed1995

Simultaneous fluorimetric determination of acetylsalicylic acid metabolites in urine by partial least squares multivariate calibration.

A method is described for the simultaneous determination of the main urinary acetylsalicylic acid (aspirin) metabolites, salicyclic, salicyluric and gentisic acids, based on their native fluorescence. The urine was extracted into diethyl ether in acid medium, and back-extracted with glycine/sodium hydroxide buffer solution at pH 9.4. A comparative study of the results found using the excitation, the emission and the combination of the excitation plus the emission spectral data, as analytical signals, was performed. The data set, composed of the excitation plus the emission spectra, was selected as the analytical signal. The optimum wavelengths to record the excitation (lambda(em)=444 nm) and the emission spectra (lambda(ex)=323 nm) were selected to maximize the contribution from gentisic acid, which is the minor urinary metabolite. Partial least squares (PLS-1) multivariate calibration was then applied for the determination. Recovery values from urine samples spiked with salicyclic, salicyluric and gentisic acids varied from 90.1 to 97.6% (mean 93.6%), from 90.0 to 110% (mean 97.9%) and from 89.9 to 104.7% (mean 98.5%), respectively.

Journal Article↗
PubMed1995

Analysis of sulfamethazine in the presence of sulfamerazine or sulfadiazine by first-derivative photochemically induced fluorescence.

A first-derivative photochemically-induced fluorescence method is proposed for the determination of sulfamethazine (SMTZ) in the presence of sulfamerazine (SMRZ) or sulfadiazine (SDZ). Linear calibration plots were obtained for SMTZ in SMTZ-SMRZ or SMTZ-SDZ binary mixtures with correlation coefficients larger than 0.999. For SMTZ the lower limits of detection were 7-13 ppb. Recoveries of 91-114% were obtained in the analysis of SMTZ in pharmaceutical formulations.

Anti-Infective Agents↗
PubMed1994

Strand exchange through a DNA-protein complex requires a DNA helicase.

The phage T4 uvsX and gene 32 proteins are capable of mediating homologous strand exchange, a central reaction in general genetic recombination, in vitro using naked DNA substrates. However, strand exchange is blocked by a sequence specific DNA-protein complex. Since protein-complexed substrates must be employed in vivo, this suggests that another factor(s) is required for strand exchange with protein-complexed DNAs. We show here that a DNA helicase, the T4 dda protein, allows the phage recombination machinery to drive branch migration through a RNA polymerase-promoter complex. This is the first observation of in vitro strand exchange using protein-bound substrates. These results suggest that a DNA helicase is a necessary component of the "protein machine" that mediates recombination in vivo.

Adenosine Triphosphatases↗