Biomedical subjects

M A Quesada

Publications and source records attributed to M A Quesada.

9 recordsLinked to original sources

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↗

PubMedDate not supplied

Neuroradiologic abnormalities in Marchiafava-Bignami disease of benign evolution.

Marchiafava-Bignami disease has been recognized since 1903, but only recently has it become possible to achieve a probable diagnosis before death occurs. Imaging of the central nervous system with MR and CT have contributed significantly to such a diagnosis. Two cases of the disease are reported in patients, aged 33 and 59 years, with benign evolution of neurologic symptoms the diagnosis was confirmed by neurologic imaging with MR and CT and both were studied with evoked responses. Reversibility of the disease and possible prognostic indicators in these and other patients reported in the literature are discussed. The important role that diagnostic procedures, especially MR imaging, play in the management of this disease is emphasized.

Adult↗

PubMedDate not supplied

[Correlation between magnetic resonance and disability scales (EDSS and ISS) in multiple sclerosis].

In the present study the lesions in MRI imaging were quantified and compared with the clinical and functional abnormalities in 56 patients (27 females and 29 males) with definite multiple sclerosis (MS). The evolution was relapsing in 30, there were relapses followed by progressive evolution in 10, and evolution was progressive from the onset in 16. A good correlation was found between the disability scale EDSS and total lesions in MRI (r = 0.45), and also with the disability scale EDSS and hemispheric (r = 0.45) and centrioval (r = 0.41) involvements and with lesions near the third ventricle (r = 0.32). The clinical parameters predicting a greater surface of involvement in MRI were late onset (beyond 45 years) and progressive evolution. The best correlation between disability scale and MRI lesions was found between EDSS and periventricular (lateral ventricles) lesions (r = 0.46). The linearly correlation between both disability scales was good (r = 0.86), but it improved when an exponential equation was used (r = 0.91). This could allow to use the more simple scale (ISS) with a mathematical transformation. When the patients with a greatest surface of involvement were compared with the remaining MS patients, significant differences were found for nearly all evaluated disability items. Patients with greater involvement surfaces in MRI had greater disability.

Adult↗