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Biomedical subjects

M A Quesada

Publications and source records attributed to M A Quesada.

At least 19 recordsLinked to original sources
PubMed1999

Improved germination under osmotic stress of tobacco plants overexpressing a cell wall peroxidase.

The cell wall is a fundamental component in the response of plants to environmental changes. To directly assess the role of the cell wall we have increased the expression and activity of a cell wall associated peroxidase (TPX2), an enzyme involved in modifying cell wall architecture. Overexpression of TPX2 had no effect on wild-type development, but greatly increased the germination rate under high salt or osmotic stress. Differential scanning calorimetry showed that transgenic seeds were able to retain more water available for germination than wild-type seeds. Thermoporometry calculations indicated that this could be due to a lower mean pore size in the walls of transgenic seeds. Therefore, the higher capacity of transgenic seeds in retaining water could result in higher germination rates in conditions where the availability of water is restricted.

Amino Acid Sequence↗
PubMed1998

Multi-capillary optical waveguides for DNA sequencing.

Cylindrical capillaries can be used as optical elements in a waveguide, where refraction will confine an appropriately focused light beam to pass through the interiors of successive capillaries in a flat parallel array. Such a capillary waveguide allows efficient illumination of samples in multiple capillaries with relatively little laser power. Analytical expressions derived under paraxial and thin-lens approximations provide guidance in selecting the capillary sizes and the refractive indices that will produce the waveguiding effect, but accurate predictions require exact ray tracing. Small reflective losses as the light passes through the capillary surfaces cause cumulative intensity decreases, but the resulting lack of uniformity can be compensated to a considerable extent by illuminating the capillary array from both sides. A 12-capillary waveguide illuminated from both sides in air has a difference of less than 10% from the strongest to the weakest illumination. By increasing the refractive index of both the external medium and the contents of the capillaries, a 96-capillary waveguide for DNA sequencing could be produced that would also provide nearly uniform illumination. A 12-capillary, bi-directionally illuminated waveguide system for DNA sequencing has been constructed. The two focused laser beams are delivered by integrated fiber optic transmitters (IFOTs), and fluorescence is collected by a set of optical fibers whose spacing exactly matches that of the capillaries in the waveguide. The system is easy to align and provides sensitive detection of fluorescence with minimal cross-talk between channels.

Electrophoresis, Capillary↗
PubMed1997

Expression of a highly basic peroxidase gene in NaCl-adapted tomato cell suspensions.

A tomato peroxidase gene, TPX2, that is only weakly expressed in the roots of young tomato seedlings is highly expressed in tomato suspension cells adapted to high external NaCl concentration. The protein encoded by this gene, with an isolectric point value of approximately 9.6, is found in the culture medium of the growing cells. Our data suggest that the expression of TPX2 in the salt-adapted cells is not the result of the elicitation imposed by the in vitro culture or the presence of high NaCl concentration in the medium.

Adaptation, Physiological↗
PubMed1997

Replaceable polymers in DNA sequencing by capillary electrophoresis.

Much progress has been made in the development of replaceable sieving polymers and capillary coatings for high-performance DNA sequencing by capillary electrophoresis. Several studies of parameters that affect resolution, read length and reproducibility have begun to reveal the physical mechanisms acting on single-stranded DNA during electrophoresis through semidilute polymer solutions. Recently developed electro-osmosis-inhibiting matrix polymers have simplified the process of coating capillaries, facilitating the automation of high-throughput parallel systems for large-scale sequencing.

Biotechnology↗
PubMed1996

Polyacrylamide solutions for DNA sequencing by capillary electrophoresis: mesh sizes, separation and dispersion.

Two preparations of linear polyacrylamide with average molecular weights of 0.37 million and 1.14 million Da, and a deuterated preparation with an average molecular weight of 1.71 million Da, were used to study the effects of molecular weight, polydispersity, and concentration on the mesh size of entangled polymers in a DNA sequencing buffer solution and their ability to resolve DNA sequencing reactions by capillary electrophoresis. The polyacrylamide concentrations were above the overlap threshold C*, the concentration above which an entangled polymer network is expected to form. Small angle neutron scattering experiments showed that between 1% and 8% polyacrylamide, the mesh size ( xi ) can be expressed by the relation xi = 2.09C-0.76, where xi is in A and C is the polymer concentration in g/mL. The mesh size depended only on the concentration and was independent of the average molecular weight of the polyacrylamide. Consistent with this result, electrophoretic mobilities of DNA moving through the polymer network depended almost entirely on the polyacrylamide concentration and not on its molecular weight or polydispersity. Although separation was little affected, band sharpness persisted to longer DNAs when the polymer network contained a higher fraction of larger polyacrylamide molecules. We postulate a dispersive effect that depends on the size of the DNA and the resiliency of the polymer network. This interpretation provides a rationale for optimizing the design of polymer solutions to sieve DNA for sequencing by capillary electrophoresis.

Acrylic Resins↗
PubMed1996

Multiple capillary DNA sequencer that uses fiber-optic illumination and detection.

An 8-capillary prototype electrophoresis system for DNA sequencing has been constructed. The sequence of 400-450 bases can be obtained from each capillary in less than an hour from sequencing reactions generated with four-color fluorescent terminators. Illumination of each capillary and collection of fluorescence is through individual optical fibers. Resolution of the DNA ladder is through a replaceable sieving matrix of linear polyacrylamide in reusable coated capillaries. Light from an argon ion laser is introduced into a fused biconically tapered fiber-optic splitter, and individual fibers deliver approximately 10 mW of 514 nm light to each of the eight electrophoresis capillaries. Illumination and collection are by fibers normal to the surface of the electrophoresis capillary and at right angle to each other. Illumination by a fiber with low numerical aperture and collection by a fiber with high numerical aperture provides good sensitivity and signal-to-noise ratios without the need for microlenses (limit of detection: 1.5 x 10(-11)M fluorescein analog dye with a signal-to-noise ratio of 2). The eight collection fibers are passed in parallel through holographic filters for Rayleigh rejection and into an imaging spectrograph, which simultaneously displays the full fluorescence spectrum (475-648 nm) from the eight capillaries in parallel on the surface of an intensified charge-coupled device (CCD). The CCD is read out at a rate of 3.4 complete images per second.

DNA↗
PubMed1994

Induction of a tomato peroxidase gene in vascular tissue.

Expression of a tomato peroxidase gene that is constitutively expressed only in roots was induced in stems and leaves as a result of mechanical wounding. However, wound-induction of TPX1 transcript accumulation in leaves was limited to the mid-rib. No TPX1 transcript was detected in the lamina of the leaf after wounding. Peroxidase isozyme studies indicated the presence of a unique basic isoform in stems after wounding.

Enzyme Induction↗
PubMed1994

Characterization and in situ localization of a salt-induced tomato peroxidase mRNA.

NaCl treatment of tomato plants in hydroponic culture at concentrations as low as 50 mM resulted in enhanced accumulation of transcripts of TPX1, a full-length cDNA clone that we had isolated from a library of NaCl-treated tomato plants using a peroxidase-specific oligonucleotide probe. Although the overall amino acid sequence identity of TPX1 to other peroxidase genes was less than 45%, there was a very high degree of identity in all of the conserved domains. The deduced amino acid sequence included the presence of a N-terminal signal peptide but not the C-terminal extension present in peroxidases targeted to the vacuole. The mature protein has a theoretical pI value of 7.5. Transcripts that hybridized to TPX1 were detected only in the roots with higher levels of mRNA in epidermal and subepidermal cell layers. Isoelectric focusing of root extracts showed two major bands of peroxidase activity at pI 5.9 and 6.2. Both activities increased with salt treatment. Southern analysis indicated the presence of only a single TPX1 gene in tomato.

Amino Acid Sequence↗
PubMed1993

Fluorometric assay using dimeric dyes for double- and single-stranded DNA and RNA with picogram sensitivity.

Thiazole orange homodimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methy l-2,3- dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow homodimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) bind with very high affinity to nucleic acids with more than a 1000-fold fluorescence enhancement upon binding. A linear dependence of fluorescence intensity on DNA concentration over a range from 0.5 to 100 ng/ml in the presence of 2 x 10(-7) M TOTO or YOYO in 4 mM Tris-acetate/0.1 mM EDTA/50 mM NaCl, pH 8.2 allows sensitive quantitation of double-stranded DNA in a conventional fluorometer. With nucleic acid-dye mixtures in an array of 25-microliters wells in a block of low autofluorescence plastic and detection with a laser-excited confocal fluorescence scanner, as little as 20 pg of double-stranded DNA can be detected per well. The array scanning method is rapid, has high throughput, and requires small amounts of sample. It also allows quantitation of single-stranded DNA and RNA.

Animals↗
PubMed1993

Picogram detection of stable dye-DNA intercalation complexes with two-color laser-excited confocal fluorescence gel scanner.

The stable complexes between highly fluorescent, polyfunctional intercalators and dsDNA can be used to detect dsDNA in agarose gels at picogram levels and for multicolor detection of multiplexed dsDNA fragments. Development of additional DNA-binding fluorophores with appropriate spectroscopic properties will expand the range of applications. In principle, the DNA-dye intercalation complexes represent a more sensitive alternative to an established approach to fluorescent labeling and detection of restriction fragments by ligation to single-stranded short oligonucleotides labeled with different fluorochromes, followed by separation on denaturing polyacrylamide gels. The latter technique gives near single-base resolution up to 400 bases and the ability to quantitate fragment size up to 2000 bases, and has been successfully applied to cosmid mapping. Detection of DNA fragments as intercalation complexes requires that the separations be performed on agarose gels under nondenaturing conditions. Such conditions have been used for extensive mapping of yeast cosmids with postelectrophoresis staining with ethidium bromide. For the patterns on agarose gels, the magnitude of the "error window," which specifies how similar two fragments must be before the corresponding fragments in different digests are paired, was reported to be strongly size dependent. The error window was expanded by a factor of 1.3 for fragments from 400 to 600 bp, 1.2 for fragments from 600 to 800 bp, and 1.1 for fragments from 800 to 1000 bp. Moreover, it was necessary to introduce corrections for systematic differences between size estimates taken from two different gels. For the multiplexing procedure described here, the size estimates for fragments from 600 bp to over 23 kbp were in close agreement with actual sizes as determined from DNA sequence (Table I), and certainly within the error windows given above. The multiplexing procedure should also minimize errors introduced by gel-to-gel variations in mobility, because the standard and unknowns are always run in the same lanes. Kohara et al. established a physical map of almost the entire Escherichia coli chromosome by analysis of a large genomic library. In this case, partial restriction digests were used to generate patterns of fragments and the mapping was performed by agarose gel electrophoresis. The disadvantage of this approach is that fewer fragments are generated. However, this is compensated for by the fact that partial digests reveal the order of the fragments produced and thus greatly increase the amount of information relevant to the question of overlap between different DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzothiazoles↗
PubMed1992

DNA sequencing using capillary array electrophoresis.

A DNA sequencing method is presented that utilizes capillary array electrophoresis, two-color fluorescence detection, and a two-dye labeling protocol. Sanger DNA sequencing fragments are separated on an array of capillaries and detected on-column using a two-color, laser-excited, confocal-fluorescence scanner. The four sets of DNA sequencing fragments are separated in a single capillary and then distinguished by using a binary coding scheme where each fragment set is labeled with a characteristic ratio of two dye-labeled primers. Since only two dye-labeled primers are required, it is possible to select dyes that have identical mobility shifts. It is also shown that the ratio of the signal in the two detection channels provides a reliable identification of the sequencing fragment. DNA sequencing results on a 25-capillary array are presented.

Base Sequence↗
PubMed1992

Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications.

The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7- diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiaz ole)-2- methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620-750 nm (dsDNA-EthD emission) and 500-565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laser-excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.

Benzoxazoles↗
PubMed1992

[Positive anti-Borrelia antibodies in patients with clinical manifestations compatible with neuroborreliosis].

The clinical features of 19 patients with neurological manifestations unexplained by another disease and positive serology for Borrelia burgdorferi were studied. ECM was present in only 11% of the cases and 32% referred tick bite. The characteristic features for suspicion of NB according to our series was the presence of polyneuritis in 84% of the cases specially in the form of multiple mononeuritis and involvement of the facial nerve (79%) leading to even greater suspicion with the association of V pair involvement. Seizures, sleep disorders, and higher mental dysfunction may be found in association with other more characteristic neurological features. The typical triad of NB (aseptic meningitis, facial paralysis and polyradiculoneuritis) was found in 21% of the patients and in the absence of another disease to justify the same neuroborreliosis (NB) seemed evident. In all the cases components of this triad were found. Headache, arthralgia, fever and, less frequently, arthritis are other symptoms often past with the presence of anti-BB antibodies. Patients with the shortest evolution most frequently presented antecedents of facial paralysis, sensory alterations and Romberg's sign than patients of longer evolution. CSF demonstrated the presence of pleocytosis in 24% of the cases and in only one patient a slight increase in the intrathecal activity of IgG was observed which may be of use in differential diagnosis with MS. MR showed alterations in 61% of the patients and, while not specific, the lesions present subcortical predominance.

Adult↗
PubMed1991

High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange.

Ethidium homodimer (EthD; lambda Fmax 620 nm) at EthD:DNA ratios up to 1 dye:4-5 bp forms stable fluorescent complexes with double-stranded DNA (dsDNA) which can be detected with high sensitivity using a confocal fluorescence gel scanner (Glazer, A.N., Peck, K. & Mathies, R.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3851-3855). However, on incubation with unlabeled DNA partial migration of EthD takes place from its complex with dsDNA to the unlabeled DNA. It is shown here that this migration is dependent on the fractional occupancy of intercalating sites in the original dsDNA-EthD complex and that there is no detectable transfer from dsDNA-EthD complexes formed at 50 bp: 1 dye. The monointercalator thiazole orange (TO; lambda Fmax 530 nm) forms readily dissociable complexes with dsDNA with a large fluorescence enhancement on binding (Lee, L.G., Chen, C. & Liu, L.A. (1986) Cytometry 7, 508-517). However, a large molar excess of TO does not displace EthD from its complex with dsDNA. When TO and EthD are bound to the same dsDNA molecule, excitation of TO leads to efficient energy transfer from TO to EthD. This observation shows the practicability of 'sensitizing' EthD fluorescence with a second intercalating dye having a very high absorption coefficient and efficient energy transfer characteristics. Electrophoresis on agarose gels, with TO in the buffer, of preformed linearized M13mp18 DNA-EthD complex together with unlabeled linearized pBR322 permits sensitive fluorescence detection in the same lane of pBR322 DNA-TO complex at 530 nm and of M13mp18 DNA-EthD complex at 620 nm. These observations lay the groundwork for the use of stable DNA-dye intercalation complexes carrying hundreds of chromophores in two-color applications such as the physical mapping of chromosomes.

Benzothiazoles↗
PubMed1991

[Progression of functional disability in 70 patients with multiple sclerosis].

We have carried out a follow-up study in 70 patients that were included in a follow-up protocol when they met the criteria of MS, so as to evaluate the variations in the disability scale per unit of time of follow-up (index of progression) in relation to the several risk factors. There were 31 females and 39 men followed up for 19.6 +/- 12 months. One half of the patients (35) had their first symptoms between 25 and 45 years of age, in 27 the onset was after age 25 and in 8 after age 45. At the end of follow-up 42 patients were classified as having remitting forms, 12 as progressive forms after a remitting phase and 16 as progressive forms from the onset. Significant differences in the evolutive forms were only found in remitting-progressive forms, which had a quicker progression than the purely remitting and chronic progressive forms. The patients who initially had cerebellar symptoms had a quicker progression than those with any other type of presentation. Whereas patients with late onset had a quicker progression than the group with intermediate onset, the patients with early onset had a slower progression.

Adolescent↗
PubMed1991

High-sensitivity DNA detection with a laser-excited confocal fluorescence gel scanner.

A high-sensitivity, laser-excited confocal fluorescence gel scanner has been developed and applied to the detection of fluorescently labeled DNA. An argon ion laser (1-10 mW at 488 nm) is focused in the gel with a high-numerical aperture microscope objective. The laser-excited fluorescence is gathered by the objective and focused on a confocal spatial filter, followed by a spectral filter and photodetector. The gel is placed on a computer-controlled scan stage, and the scanned image of the gel fluorescence is stored and analyzed in a computer. This scanner has been used to detect DNA separated on sequencing gels, agarose mapping gels and pulsed field gels. Sanger sequencing gels were run on M13mp18 DNA using a fluoresceinated primer. The 400-microns-thick gels, loaded with 30 fmol of DNA fragments in 3-mm lanes, were scanned at 78-microns resolution. The high resolution of our scanner coupled with image processing allows us to read up to approximately 300 bases in four adjacent sequencing lanes. The minimum band size that could be detected and read was approximately 200 microns. This instrument has a limiting detection sensitivity of approximately 10 amol of fluorescein-labeled DNA in a 1 x 3-mm band. In applications to agarose mapping gels, we have exploited the fact that DNA can be prestained with ethidium homodimer, followed by electrophoresis and fluorescence detection to achieve picogram sensitivity. We have also developed methods using both ethidium homodimer and thiazole orange staining which permit two-color detection of DNA in one lane.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

[Intrathecal secretion of antiviral antibodies in multiple sclerosis in patients in Seville].

To assess the presence of antiviral antibody synthesis in the cerebrospinal fluid in patients with multiple sclerosis, concurrent plasma and spinal fluid determination of antibody titers against measles, varicella-zoster, rubella, mumps, cytomegalovirus, and herpes viruses were performed in 29 samples and were compared with a control group. The study revealed an increased titre of antiviral antibodies in the spinal fluid in patients with multiple sclerosis. This increased activity was markedly significant for the varicella-zoster and cytomegalovirus in patients with clinical symptoms of multiple sclerosis. There was also and increased antibody titre against cytomegalovirus and varicella-zoster in patients with well defined illness. No antibody reaction was observed in the control group. The study of the antiviral antibody activity in the spinal fluid in patients with multiple sclerosis is useful in the follow-up control and in the diagnosis of the disorder specially in our community, where the investigation of antibodies anti cytomegalovirus appears to be the most appropriate method due to its high sensitivity and absence of false positive tests.

Antibodies, Viral↗