Biomedical subjects
Publications and source records attributed to P M McCready.
Suppression subtractive hybridization, a cost-effective approach for targeting unique DNA, was used to identify a 41.7-kb Yersinia pestis-specific region. One primer pair designed from this region amplified PCR products from natural isolates of Y. pestis and produced no false positives for near neighbors, an important criterion for unambiguous bacterial identification.
PURPOSE: Vertical diplopia is an uncommon but disappointing complication of otherwise successful local anaesthetic cataract surgery. We studied strabismus patterns in a group of such patients to identify the nature and extent of extraocular muscle involvement. METHODS: A retrospective review identified 15 cases of vertical diplopia following local anaesthetic cataract surgery between July 1994 and January 1998. Peribulbar anaesthesia was used in all cases and given by right-handed professionals. RESULTS: All cases had otherwise successful cataract surgery (mean age 80.5 years; median pre-operative VA 6/18; median post-operative VA 6/9). The mean level of vertical diplopia was 7.2 prism dioptres (PD) in the primary position (range 2-25 PD). The left inferior rectus (IR) was paretic in 6 cases and restricted in 5 cases. The left superior rectus (SR) was not affected in any of the cases. The right IR was restricted in a single case. The right SR was paretic in 2 cases and restricted in a single case. None of the cases had clinical involvement of the oblique muscles. Eleven of the cases were managed successfully with prisms. Two of the cases required strabismus surgery. CONCLUSIONS: The incidence of left eye extraocular muscle involvement was greater than right eye involvement, although this did not reach statistical significance (73% vs 27%; p = 0.075). This may be due to the more difficult access of right-handed individuals giving left eye peribulbar injections with the needle tract being directed more closely to the muscle cone. The IR muscle is more commonly affected than the SR (80% vs 20%; p = 0.019). An equal incidence of paretic and restricted rectus muscle pathology was found in this study (53% vs 47%; p = 0.818). The exact aetiology of muscle injury is unknown but could be due to direct muscle or nerve trauma, anaesthetic toxicity, periocular haemorrhage or a combination of these.
We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.
The cis-acting elements located within 15 kb 5' of the murine CD4 gene transcriptional start site and the first intron of the CD4 gene have been investigated using deletion constructs. Our transient transfection data indicate that the expression of the murine CD4 gene is controlled by multiple positive and negative regulatory cis-acting elements. There are at least two cis-acting elements that have a positive effect on the expression of the CD4 gene and at least four regions of DNA that have a negative effect. The positive control elements are located about 13.5 kb 5' of the promoter and within the flanking sequences of the first intron. The DNA between the 5' enhancer and the promoter contains at least two regions that exert a negative effect on CD4 expression. In addition to the positive effect that the first intron has on CD4 expression, there are two regions within the first intron that have a negative effect. These two negative regulatory elements correspond to two T-cell-specific DNase I hypersensitive sites found in the first intron.
A rapid method for the detection of simian immunodeficiency virus (SIV) RNA from peripheral blood mononuclear cells (PBMC) of experimentally infected rhesus macaques by the polymerase chain reaction (PCR) is reported. The PCR was carried out with a complementary DNA (cDNA) template using 3 pairs of primers that were designed to anneal to homologous sequences in conserved regions of 3 molecular clones of SIVmac. The specificity of the primers was confirmed by performing the PCR with template DNA from the 3 molecular clones. SIV-specific RNA was detected from 30 and 50 infected PBMC/6.25 x 10(5) PBMC of two animals.
The Bacillus subtilis gene senS, when present in high copy number, stimulates the expression of several extracellular protein genes during the onset of stationary phase, e.g. aprE. A novel integration vector, pINT, was constructed for transcription expression studies; it employed a unique method of promoter insert production for fusion with the lacZ reporter gene. Deletions were made of the 5' flanking region of the aprE promoter to localize the site responsible for SenS-mediated enhancement activity. pINT was used translationally fuse aprE promoter deletion fragments with the lacZ reporter gene. A site between -177 and -415 with respect to the aprE start site of transcription was found to be required for the maximal SenS-mediated transcription increase from the aprE promoter. A multicopy vector containing the senS coding region without its native negative regulation was highly unstable in B. subtilis; this was due to the expressed senS insert.