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Biomedical subjects

L M Smith

Publications and source records attributed to L M Smith.

At least 109 records · Page 6Linked to original sources
PubMed1996

Plasma nitrate concentration in infective gastroenteritis and inflammatory bowel disease.

BACKGROUND: In subjects on a low nitrate diet, plasma nitrate concentration and urinary nitrate excretion are thought to reflect endogenous nitric oxide (NO) production, and have been reported to increase during infective and inflammatory bowel disease. AIMS: To compare the extent of NO production in patients with infective versus non-infective forms of bowel dysfunction. SUBJECTS: Four groups: 20 healthy, volunteer clerical and laboratory staff, 12 patients with irritable bowel syndrome, 19 patients with inflammatory bowel disease, and 20 patients with infective gastroenteritis. METHODS: The plasma nitrate concentration was determined with a copper coated cadmium column and spectrophotometry. Mean and median plasma nitrate concentrations were calculated and compared within the four groups. Mann-Whitney distribution free rank testing was used to compare the median values. RESULTS: Median plasma nitrate concentrations in the four groups were: controls 32.7 mumol/l; irritable bowel syndrome 35.5 mumol/l; inflammatory bowel disease 35.1 mumol/l; and gastroenteritis 117.9 mumol/l (p < 0.001 gastroenteritis v all other groups). CONCLUSIONS: Plasma nitrate concentration could serve as a discriminant between infective and inflammatory or functional bowel disease in patients presenting with diarrhoea. It is not clear why there is considerable difference in endogenous nitrate synthesis in these two conditions, which are both characterised by severe gut inflammation.

Adolescent↗
PubMed1996

Use of transcription factors as agents and targets for drug development.

Cells respond to external signals by either activating or inhibiting key regulatory proteins of gene expression called transcription factors (TFs). Abnormal expression of these factors plays a critical role in many human cancers. Recently, many TFs have been identified and their structure-function relationship has been characterized. Such new information has been useful in designing new chemotherapeutic drugs targeting these regulatory proteins. This review discusses the use of dominant-negative (DN) mutants of TFs as inhibitors of cell growth, as well as possible strategies for screening potential inhibitors of TF activity.

Animals↗
PubMed1995

A human chromosome 7 yeast artificial chromosome (YAC) resource: construction, characterization, and screening.

The paradigm of sequence-tagged site (STS)-content mapping involves the systematic assignment of STSs to individual cloned DNA segments. To date, yeast artificial chromosomes (YACs) represent the most commonly employed cloning system for constructing STS maps of large genomic intervals, such as whole human chromosomes. For developing a complete YAC-based STS-content map of human chromosome 7, we wished to utilize a limited set of YAC clones that were highly enriched for chromosome 7 DNA. Toward that end, we have assembled a human chromosome 7 YAC resource that consists of three major components: (1) a newly constructed library derived from a human-hamster hybrid cell line containing chromosome 7 as its only human DNA; (2) a chromosome 7-enriched sublibrary derived from the CEPH mega-YAC collection by Alu-polymerase chain reaction (Alu-PCR)-based hybridization; and (3) a set of YACs isolated from several total genomic libraries by screening for > 125 chromosome 7 STSs. In particular, the hybrid cell line-derived YACs, which comprise the majority of the clones in the resource, have a relatively low chimera frequency (10-20%) based on mapping isolated insert ends to panels of human-hamster hybrid cell lines and analyzing individual clones by fluorescence in situ hybridization. An efficient strategy for polymerase chain reaction (PCR)-based screening of this YAC resource, which totals 4190 clones, has been developed and utilized to identify corresponding YACs for > 600 STSs. The results of this initial screening effort indicate that the human chromosome 7 YAC resource provides an average of 6.9 positive clones per STS, a level of redundancy that should support the assembly of large YAC contigs and the construction of a high-resolution STS-content map of the chromosome.

Animals↗
PubMed1995

Histaminase activity in patients with vernal keratoconjunctivitis.

PURPOSE: To investigate the activity of histamine-degradating enzymes in tears and plasma of patients with vernal keratoconjunctivitis (VKC). METHOD: Tear and plasma samples were collected from patients with VKC and from age-matched control subjects. Histamine was measured by enzyme-linked immunosorbent assay in acid samples treated with perchloric to deactivate histaminase and in untreated samples. Tear cytology, skin test reactivity to histamine, and the sum clinical score of allergic signs and symptoms in patients with VKC also were evaluated. Nineteen patients with active VKC and six age-matched control subjects participated in this study. RESULTS: In untreated samples, tear histamine (mean +/- standard error of the mean) was 11.15 +/- 2.16 ng/ml in patients with VKC and 0.855 +/- 0.225 ng/ml in control tears (P < 0.001). In treated samples, mean tear histamine was 22.25 +/- 4.17 ng/ml in patients with VKC versus 10.64 +/- 2.85 ng/ml in control subjects (not statistically different). The ratio of histamine in treated to untreated samples (indicating histaminase activity) was significantly lower in patients with VKC (2.30 +/- 0.263) than in control subjects (17.57 +/- 5.97; P = 0.0001). Plasma histamine levels in untreated and treated samples were significantly higher in patients with VKC (untreated, 2.23 +/- 0.334 ng/ml; treated, 4.37 +/- 0.357 ng/ml) than in control subjects (untreated, 0.254 +/- 0.068, P = 0.0002; treated, 2.96 +/- 0.171 ng/ml, P = 0.0082). The enzymatic breakdown of histamine (treated/ untreated) in plasma was significantly decreased in patients with VKC (2.54 +/- 0.447) compared with control subjects (14.78 +/- 4.86; P = 0.0012). Skin reactivity to histamine was not increased in VKC. Tear histamine levels were significantly correlated to tear lymphocyte content in the general population and to tear basophils in the patients with tarsal-vernal VKC only. An increased number of tear eosinophils were correlated with elevated enzyme activity only in patients with tarsal-vernal VKC and to the clinical score only in limbal-vernal patients. CONCLUSION: The enzymatic degradation of histamine was significantly decreased in patients with VKC compared with control subjects in both tears and plasma, suggesting that this dysfunction may be a primary factor in the pathophysiology of VKC.

Adolescent↗
PubMed1995

Evaluation of 0.05% levocabastine versus 4% sodium cromolyn in the allergen challenge model.

PURPOSE: This study was conducted to evaluate the efficacy of 0.05% levocabastine compared with 4% cromolyn for treating allergic conjunctivitis induced by ocular allergen challenge. METHODS: Subjects who met all entry criteria and reacted positively to ocular allergen challenge at two previous visits (n = 50) received placebo in one eye and cromolyn in the fellow eye, four times daily for 2 weeks. On day 18, subjects received the final dose of cromolyn in the pretreated eye and one drop of levocabastine in the fellow eye. Subjects were challenged and evaluated after 3, 5, and 10 minutes. Four hours after drug administration, subjects were rechallenged and evaluated after 3, 5, and 10 minutes. RESULTS: Levocabastine was significantly more effective than cromolyn in inhibiting itching, hyperemia, eyelid swelling, chemosis, and tearing after the initial challenge and 4-hour rechallenge (P < 0.05). CONCLUSION: These results suggest that levocabastine is superior to cromolyn for treating allergen-induced conjunctivitis and has a duration of action of at least 4 hours.

Allergens↗
PubMed1995

Comparative effectiveness of an integrated pest management system and an insecticidal perimeter spray for control of smokybrown cockroaches (Dictyoptera: Blattidae).

Two treatments were compared for effectiveness in reducing abundance of smokybrown cockroaches, Periplaneta fuliginosa (Serville), around houses; an integrated pest management strategy consisting of sanitation, landscape management, and reduced insecticide usage, and a perimeter insecticide treatment applied according to label directions. Regression of means through time indicated that the integrated pest management treatment lasted approximately 60 d, whereas the perimeter treatment lasted only 30 d. The greater duration of control in the integrated pest management treatment was likely the result of decreased abundance of cockroaches outside the spray zone. In the perimeter treatment, cockroach abundance was unaffected outside the spray zone with no reduction in adult abundance in the spray zone except in the week following treatment application. The difference between the integrated pest management and perimeter treatments suggests that reinvasion of cockroaches from peripheral habitats decreases the duration of cockroach suppression. Abundance of 1st instars and small and medium nymphs was reduced for the duration of the experiment by both methods. Sampling procedures did not influence cockroach abundance. Reduction in abundance of cockroaches outside of houses reduced abundance of cockroaches inside houses.

Animals↗
PubMed1995

Mass spectrometry of nucleic acids: the promise of matrix-assisted laser desorption-ionization (MALDI) mass spectrometry.

In the past several years, significant progress has been made in the application of matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to the analysis of large biopolymers, including nucleic acids. By isolating analyte molecules in an appropriate matrix and irradiating the sample with a high-intensity, pulsed laser beam, MALDI can generate intact, gas-phase ions of these analytes. Primarily used with time-of-flight mass spectrometers, this relatively new, soft ionization technique has allowed for the routine analysis of oligonucleotides up to 60 or so nucleotides in length. Recent results have also shown that base specific, matrix-dependent fragmentation is an important factor in the MALDI analysis of oligonucleotides. Further extension of the technique to longer oligonucleotides will rely on both the continued search for new matrix materials and an increased understanding of the desorption and ionization process in MALDI.

Base Sequence↗
PubMed1995

A small and lightweight three-channel signal-conditioning unit for strain-gage transducers: a technical note.

A small and lightweight signal conditioning unit for use with strain-gage transducers is described. The unit provides bridge-balancing, amplification, and filtering for three channels of Wheatstone bridge circuits. The electronics are housed with a 4.5 cm x 3.0 cm x 2.5 cm box that allows connection to a transducer and to a cable that extends to power supplies and a data storage facility. Evaluation tests showed the signal conditioning unit had low noise (0.356 microV, peak-to-peak, referenced to input) with crosstalk between channels of less than 0.02% of the peak-to-peak input signal. The signal conditioning unit has application in biomechanics where a small and lightweight unit is needed; for example, skin/support interface stress measurements in prosthetics and orthotics research.

Biomechanical Phenomena↗
PubMed1994

Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports.

A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.

Alleles↗
PubMed1994

DNA sequencing with a hexamer string primer and dye-labeled terminators.

A method for fluorescence-based DNA sequencing using a hexamer string primer and dye-labeled terminators is described. Sequencing protocols have been determined that provide an average read length of 393 bases with 99.6% base-calling accuracy for single-stranded M13mp18 DNA, and an average read length of 367 bases with 99.4% base-calling accuracy for double-stranded M13mp19 DNA. The signal intensity is stronger than that obtained with a standard 18-mer primer, while the resolution and length of readable sequence are comparable.

DNA Primers↗
PubMed1994

A direct selection strategy for shotgun cloning and sequencing in the bacteriophage M13.

A new cloning strategy is described which utilizes direct selection of recombinants for shotgun sequencing in the filamentous bacteriophage M13. Direct selection is accomplished by insertional inactivation of the M13 gene X protein, a powerful inhibitor of phage-specific DNA synthesis when overproduced. An extra copy of gene X was inserted into the intergenic region of M13 and placed under the control of the bacteriophage T7 gene 10 promoter and RBS. Random fragments are cloned into the EcoRV cloning site of the new gene X cistron and recombinants are selected in an E. coli male strain producing T7 RNA polymerase. Cloning efficiencies obtained with M13-100 or phosphatase-treated M13mp19 vector are comparable. The direct selection capability of M13-100 was demonstrated to have the following advantages: (a) consistently achieved high ratios of recombinants to religated vector in the libraries, averaging about 500:1 (0.2% background), and (b) the elimination of the need for phosphatase treatment of the vector to reduce background. The direct selection strategy significantly improves the efficiency of shotgun library construction in M13, and should therefore facilitate the cloning aspects of large scale sequencing projects.

Bacteriophage M13↗
PubMed1994

Evaluation of the new ophthalmic antihistamine, 0.05% levocabastine, in the clinical allergen challenge model of allergic conjunctivitis.

The objective of this study was to evaluate the efficacy of 0.05% levocabastine, a new antihistamine formulated for ophthalmic use, compared with the placebo vehicle for the treatment of allergic conjunctivitis induced by ocular allergen challenge. Subjects who reacted. positively in both eyes on two separate occasions to ocular allergen challenge with grass, ragweed, or cat dander (N = 47) received one dose of 1 to 2 drops of 0.05% levocabastine in one eye and its vehicle in the other eye. After 10 minutes, the predetermined dose of allergen was instilled in both eyes. Signs and symptoms of allergic conjunctivitis were evaluated with biomicroscopy and subjective evaluation of itching after 3, 5, and 10 minutes. Four hours after drug administration, subjects were rechallenged and reevaluated to determine levocabastine's duration of action. Results showed that levocabastine was significantly more effective than placebo in inhibiting itching, hyperemia, eyelid swelling, chemosis, and tearing after the initial challenge and in inhibiting all parameters except eyelid swelling after the rechallenge 4 hours later (p < 0.05). These results demonstrate that levocabastine, currently the only ophthalmic antihistamine available that is not combined with a vasoconstrictor, is efficacious in the inhibition of itching, as well as all of the allergic signs of a vascular origin, with a duration of action of at least 4 hours. Because of its strong effects on itching and hyperemia, chemosis, lid swelling, and tearing, levocabastine would be a valuable therapeutic agent to add to the heterogeneous family of antiallergic compounds presently available for the treatment of seasonal allergic conjunctivitis.

Adolescent↗
PubMed1994

Rapid isolation of cosmid insert DNA by triple-helix-mediated affinity capture.

A simple and rapid method for the isolation of cosmid insert DNA was developed based on triple-helix-mediated affinity capture (TAC). A modified cosmid was constructed from the SuperCos 1 cosmid vector by flanking the cloning site with two homopurine-homopyrimidine triple-helix-forming sequences. The cosmid DNA is digested with NotI restriction enzyme to release the insert DNA. The NotI-digested cosmid DNA is then combined with a biotinylated homopyrimidine oligonucleotide in an acidic buffer solution to form a triple-helix complex. The triple-helix complex is captured with streptavidin-coated magnetic beads. Insert DNA is eluted by adding a pH 9 buffered solution to the captured complex. The purified insert DNA is recovered with a yield of up to 95% and a purity of at least 95%. The isolated insert DNA was directly digested with CviJI restriction endonuclease to generate random fragments for shotgun sequencing.

Base Sequence↗
PubMed1994

A high throughput system for the preparation of single stranded templates grown in microculture.

A high throughput system for the preparation of single stranded M13 sequencing templates is described. Supernatants from clones grown in 48-well plates are treated with a chaotropic agent to dissociate the phage coat protein. Using a semi-automated cell harvester, the free nucleic acid is bound to a glass fiber filter in the presence of chaotrope and then washed with ethanol by aspiration. Individual glass fiber discs are punched out on the cell harvester and dried briefly. The DNA samples are then eluted in water by centrifugation. The processing time from 96 microcultures to sequence quality templates is approximately 1 hr. Assuming the ability to sequence 400 bases per clone, a 0.5 megabase per day genome sequencing facility will require 6250 purified templates a week. Toward accomplishing this goal we have developed a procedure which is a modification of a method that uses a chaotropic agent and glass fiber filter (Kristensen et al., 1987). By exploiting the ability of a cell harvester to uniformly aspirate and wash 96 samples, a rapid system for high quality template preparation has been developed. Other semi-automated systems for template preparation have been developed using commercially available robotic workstations like the Biomek (Mardis and Roe, 1989). Although minimal human intervention is required, processing time is at least twice as long. Custom systems based on paramagnetic beads (Hawkins et al., 1992) produce DNA in insufficient quantity for direct sequencing and therefore require cycle sequencing. These systems require custom programing, have a fairly high initial cost and have not proven to be as fast as the method reported here.

Base Sequence↗
PubMed1993

Basic matrices for the matrix-assisted laser desorption/ionization mass spectrometry of proteins and oligonucleotides.

In order to examine the importance of pH in the matrix-assisted laser desorption/ionization (MALDI) analysis of proteins and oligonucleotides, 37 highly substituted pyrimidine, pyridine, and benzene derivatives containing basic amino groups were screened as potential matrices. Several of these compounds were useful for the preparation and analysis of samples under neutral and basic pH conditions. The matrix, 2-amino-4-methyl-5-nitropyridine, gave good results with small proteins (up to approximately 12,000 Da). The best results with oligonucleotides were obtained with 2-amino-5-nitropyridine, which was a useful matrix for the analysis of mixed-base oligonucleotides smaller than 20 nucleotides in length and homopolymers of thymidine. In general, oligonucleotide analysis using basic matrices was found to be subject to similar constraints of size and base composition that hold when acidic matrices are used. A major advantage of these newly discovered matrix materials over the more commonly used acidic matrices is that they extend the utility of MALDI to the analysis of acid-sensitive species.

Base Sequence↗
PubMed1993

Optimization of electric field strength for DNA sequencing in capillary gel electrophoresis.

Capillary gel electrophoresis (CGE) has demonstrated the ability to separate DNA sequencing reactions at speeds up to 25 times as great as conventional slab gel electrophoresis. These increased speeds are made possible by the efficient heat dissipation of capillaries, which permits higher electric fields to be employed without deleterious thermal effects. The high electric fields, however, also lead to a reduction in the spacing between bands with a concomitant loss of resolution. The resulting tradeoff between speed and resolution is a very important practical aspect of these high-field separations. This work addresses this question by investigating the band broadening and resolution of DNA fragments as they are separated through a fixed distance of gel at field strengths ranging from 50 to 400 V/cm. It is found that the bandwidths of DNA fragments do decrease with the higher field strengths due to a reduction in the diffusional broadening of bands. However, at sufficiently high electric field strengths, the bands begin to broaden again due to the thermal gradient across the gel. This behavior causes the optimum electric field strength for maximum fragment resolution to depend upon the length of fragments being separated. The relative contributions of diffusion and thermal gradients are discussed and used to predict the ultimate performance of constant field capillary gel electrophoresis.

Diffusion↗