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Biomedical subjects

D Reeves

Publications and source records attributed to D Reeves.

At least 37 records · Page 2Linked to original sources

Preliminary study using species-specific oligonucleotide probe for rRNA of Bilophila wadsworthia.

Portions of the 16S RNA from a urease-positive Bilophila wadsworthia strain were sequenced, and a probe was constructed. The probe was end labeled with [32P]ATP and polynucleotide kinase and hybridized on a nylon filter (by dot blot hybridization) to the immobilized rRNA of 12 B. wadsworthia strains and eight other anaerobic isolates. The probe efficiently hybridized only to the Bilophila strains. Cross-reactivity at high RNA levels (2,000 ng) was observed with one strain of Bacteroides thetaiotamicron and one strain of Bacteroides fragilis (with 10x SET buffer [20x SET buffer is 0.5 M NaCl, 0.03 M Tris, and 2 mM EDTA]) but was not seen at lower RNA levels or with 5x SET buffer. When tested against mixed cultures of aerobic and anaerobic isolates representative of appendiceal abscess flora, the probe did not react with mixed cultures containing no Bilophila cells and could detect > or = 10(5) Bilophila CFU/ml when the mixture was seeded with Bilophila cells. This probe is of potential use in the rapid identification of pure isolates and in the direct identification of B. wadsworthia in clinical specimens.

Base Sequence

Expression of human alpha 1 antitrypsin in transgenic sheep.

We have recently described the production of large amounts (< or = 65 grams per litre) of enzymatically active human alpha 1 antitrypsin in the milk of transgenic sheep (Wright et al., 1991). Here, we describe in more detail the expression of the human protein in the milk of these animals throughout the lactation period. Human alpha 1 antitrypsin is also found at much lower levels in the plasma of transgenic ewes before, during and after lactation. It is also detected in male plasma at very low levels. We have previously shown human alpha 1 antitrypsin purified from transgenic sheep milk to be indistinguishable from commercially available human plasma derived alpha 1 antitrypsin in terms of gross sugar content and in vitro activity. Here we extend this comparison to more detailed analyses of glycosylation state, amino-terminal sequence, pI value, and molecular weight determination by mass spectrometry.

Amino Acid Sequence

High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep.

We describe the generation of five sheep transgenic for a fusion of the ovine beta-lactoglobulin gene promotor to the human alpha 1-antitrypsin (h alpha 1AT) genomic sequences. Four of these animals are female and one male. Analysis of the expression of h alpha 1AT in the milk of three of these females shows that all express the human protein at levels greater than 1 gram per liter. In one case initial levels exceeded 60 grams per liter and stabilized at approximately 35 grams per liter as lactation progressed. Human alpha 1AT purified from the milk of these animals appears to be fully N-glycosylated and has a biological activity indistinguishable from human plasma-derived material.

Animals

In-vitro activity of temafloxacin against anaerobic bacteria: a comparative study.

The antimicrobial activity of temafloxacin for 328 anaerobic bacteria was determined and compared to that of cefotetan, cefoxitin, ciprofloxacin and metronidazole. The Wadsworth agar dilution technique using Brucella-lysed sheep blood agar was used throughout. At the recommended breakpoint concentration 4 mg/L, temafloxacin inhibited 60/62 (97%) of the isolates of Bacteroides fragilis and 82/87 (94%) of the isolates of other species of the B. fragilis group. Ninety-six percent of the 24 isolates of Peptostreptococcus, 97% of the 31 isolates of other Bacteroides species and 88% of 66 isolates of Fusobacterium species were also inhibited by 4 mg/L temafloxacin. Metronidazole (breakpoint 16 mg/L) had a broader spectrum of activity than temafloxacin (judged by the percentage of strains tested susceptible at the breakpoints employed) with the exception of non-sporing Gram-positive bacilli. The cephalosporins tested (breakpoint 32 mg/L) had a narrower spectrum of activity. Ciprofloxacin (breakpoint 2 mg/L) was the least effective agent against the majority of the anaerobes tested.

Anti-Infective Agents

Application of phase-induced lateralization to the Stenger test.

The Stenger test was employed to estimate the genuine hearing thresholds in normally hearing volunteer subjects simulating a total unilateral loss. The test was carried out in its standard form and in a modified form in which a phase shift was introduced between the signal delivered to the two ears, set to produce phase-induced lateralization towards the 'poor' ear. The standard test estimated the thresholds at a mean of 13.5 dB above the true thresholds at five frequencies from 250 Hz to 4 kHz. Thresholds at the different frequencies were compared, and although thresholds were lower for the higher frequencies, the apparent effect of frequency was not statistically significant. The modified test, using a 90 degrees phase shift, was found to enhance the test at 250 and 500 Hz (thresholds estimated at about 7 dB above true values), but not significantly at 1 kHz.

Adult

Response of oral leukoplakia to beta-carotene.

Leukoplakia is associated with increased risk of oral cancer and is considered a premalignant lesion. Retinoids, particularly 13-cis retinoic acid, can frequently reverse leukoplakia. However, these drugs have considerable toxicity and are not suitable for large-scale use in the prevention of oral cancer. Beta-carotene is a naturally occurring, nontoxic carotenoid with biologic properties that suggest that it might be efficacious against oral leukoplakia. In 1986, we began a randomized study of 13-cis retinoic acid (1 mg/kg/d) versus beta-carotene (30 mg/d) in leukoplakia. However, owing to the marked differences in toxicity between the two compounds outlined in the consent form, 11 of the initial 16 eligible patients refused to participate unless they were "guaranteed" beta-carotene. Therefore, the study design was changed to a phase II trial of beta-carotene in which the compound was given daily for 3 months. Responding patients were continued for another 3 months of treatment. All lesions were examined histologically at entry. Responses were monitored by bidimensional measurements and photography done at entry, then monthly while on treatment and at study completion. Twenty-four evaluable patients were treated, and 17 had major responses (two complete, 15 partial), a response rate of 71% (95% confidence limits, 53% to 89%). There was no significant toxicity requiring drug discontinuation or dose reduction. These results indicate that beta-carotene has substantial activity in oral premalignancy. Because of its lack of toxicity, it is an excellent candidate for a preventive agent for oral cancer.

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