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Biomedical subjects

R J White

Publications and source records attributed to R J White.

At least 19 recordsLinked to original sources
PubMed1992

Mechanism of TATA-binding protein recruitment to a TATA-less class III promoter.

The TATA-binding protein (TBP) is required for transcription by RNA polymerase III (pol III), even though many pol III templates, such as the adenovirus VA1 gene, lack a consensus TATA box. We show that TBP alone does not form a stable, productive interaction with VA1 DNA. However, it can be incorporated into an initiation complex if the other class III basal factors, TFIIIB and TFIIIC, are also present. TFIIIB can associate with the evolutionarily conserved C-terminal domain of TBP in the absence of DNA or TFIIIC, suggesting that TFIIIB exists in solution as a complex with TBP. The stable association of TBP with an essential component of the pol III transcription apparatus may account for the ability of TATA-less class III genes to recruit TBP.

Adenoviridae↗
PubMed1992

Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation. Application to studies of human link protein cleavage by stromelysin.

Monoclonal antibodies were raised that specifically recognize the NH2-terminal neoepitope sequence present in link protein cleavage products derived from stromelysin-degraded proteoglycan aggregate. Competitive enzyme-linked immunosorbent assay, using synthetic peptides as inhibitors, showed that one of these antibodies (CH-3) required, for antibody recognition, the free NH2-terminal amino acid isoleucine (residue 17 of the intact protein) in the sequence NH2-IQAENG at the stromelysin cleavage site of link protein 3. Human proteoglycan aggregate was digested with recombinant human stromelysin, bovine chymotrypsin, bovine trypsin, and porcine elastase, and their respective link protein degradation products were tested for immunoreactivity with antibody CH-3. Only stromelysin- and chymotrypsin-generated link protein 3 were recognized by antibody CH-3. Both of these enzymes generate link protein NH2 termini with the sequence 17IQAENG. . .; hence these studies indicated that monoclonal antibody CH-3 recognized this neoepitope sequence in only specific proteolytically modified link protein molecules. Since the occurrence of link protein 3 increases with aging, the incidence of CH-3 epitope in proteoglycans isolated from human knee articular cartilage of individuals of different ages was investigated. The prevalence of CH-3 epitope was found to be highest in newborn and adolescent articular cartilage samples. However, little CH-3 epitope was detected in older adult cartilage, although considerably more link protein 3 was present in these samples. These results suggest that additional proteolytic agents are responsible for the increased occurrence of link protein degradation products with aging.

Adult↗
PubMed1992

A role for the TATA-box-binding protein component of the transcription factor IID complex as a general RNA polymerase III transcription factor.

The major class of vertebrate genes transcribed by RNA polymerase (EC 2.7.7.6) III, which includes 5S rRNA genes, tRNA genes, and the adenovirus VA genes, is characterized by split internal promoters and no absolute dependence upon specific upstream sequences. Fractionation experiments have shown that transcription of such genes requires two general RNA polymerase III-specific factors, TFIIIB and TFIIIC. We now demonstrate that a third general factor is also employed by these genes. This is the TATA-box-binding protein originally identified as being a component of the general RNA polymerase II transcription factor TFIID. This protein is involved in the transcription by RNA polymerase III of every template tested, even though the promoters of VA and most vertebrate tRNA and 5S rRNA genes do not contain recognizable TATA elements.

Base Sequence↗
PubMed1992

The dermatan sulfate proteoglycans of the adult human meniscus.

The dermatan sulfate proteoglycans decorin and biglycan were extracted from pooled adult human menisci with 4 M guanidinium chloride and purified by successive cesium chloride density gradient centrifugation, ion exchange chromatography, and gel filtration. A final yield of about 2 mg of dermatan sulfate proteoglycan per gram of wet tissue was obtained. The proteoglycan is predominantly decorin with some biglycan, and the dermatan sulfate chains contain about 70% of their uronic acid residues as iduronate and possess about three times as much 4-sulfation as 6-sulfation of their N-acetylgalactosamine residues. On gel filtration under associative conditions, about half of the proteoglycan exhibits self-association. This includes most of the biglycan but also a substantial proportion of decorin. The molecules that show self-association appear to have longer dermatan sulfate chains, though there is no apparent difference in their overall composition. The predominance of decorin in the adult meniscus and its ability to interact both with itself and collagen fibrils is compatible with a role in maintaining tissue integrity and strength.

Aged↗
PubMed1992

Postoperative chest infection after upper abdominal surgery: an important problem for smokers.

The incidence and risk factors for postoperative chest infection have been studied in 127 patients undergoing elective upper abdominal surgery. The overall incidence of postoperative chest infection was 20.5%. Mean length of postoperative stay was extended from 7.8 to 10.7 days in those developing infection (P less than 0.05). Patients who smoked cigarettes and those with smoking related diseases had a considerably higher risk of infection. Patients with chronic bronchitis and airflow obstruction had an incidence of infection of 85.7%, those with chronic bronchitis alone 83.3% and those with airflow obstruction and no chronic bronchitis 50%. Patients with a smoking history of greater than or equal to 20 pack years but no chronic bronchitis or airflow obstruction had an incidence of 20.8%. All three were independently significant risk factors for infection. Patients without respiratory disease and who were non-smokers had an incidence of infection of only 7.1%. Smoking and its sequelae are the principal risk factors for postoperative chest infection. This study shows that the high risk patient can readily be identified by enquiry into respiratory symptoms, smoking history and by spirometry. This is the group in whom prophylactic measures are especially important, and they require close supervision to enable early identification and therapy of infection.

Abdomen↗
PubMed1992

Microbial flora of the trachea during intubation of patients undergoing upper abdominal surgery.

BACKGROUND: The presence of Haemophilus influenzae in the oropharynx is correlated with the subsequent development of chest infection. The importance of colonisation of the trachea by bacteria at the time of surgery is uncertain. This study investigated the tracheal flora at the time of intubation in 24 patients undergoing elective upper abdominal surgery. METHODS: The bacterial flora of the trachea was sampled in all 24 patients immediately after intubation and immediately before extubation. Patients were assessed postoperatively for the development of chest infection. RESULTS: Bacteria, including H influenzae in five cases, were isolated from the post-intubation brushings of the trachea of 15 patients. The pre-extubation brushings from only four patients yielded growth. Three of five patients developing a chest infection were colonised by H influenzae according to the postintubation brush, compared with two of 19 without chest infections. Before extubation two of five developing chest infections had H influenzae in the trachea but none of 19 without infection. All but one of the patients from whom H influenzae was isolated were smokers. CONCLUSIONS: These results suggest that the increased risk of postoperative chest infection in cigarette smokers may be due in part to colonisation of the trachea by H influenzae at the time of operation.

Adult↗
PubMed1991

Oropharyngeal flora and chest infection after upper abdominal surgery.

The oropharyngeal flora was determined before and after operation in 127 patients undergoing upper abdominal surgery. Swabs of the oropharynx were obtained on the day before operation and on the first, third, and fifth postoperative days. Isolation of Haemophilus influenzae, Streptococcus pneumoniae, and coliforms was noted. In the 108 patients with the full series of throat swabs the incidence of oropharyngeal colonisation by H influenzae was 16% and was unchanged after operation. S pneumoniae was present in only 5.6 (six patients) before operation and the incidence fell to 1.9% (two patients). There was a transient rise in coliform colonisation postoperatively. Twenty four patients developed a chest infection. In eight a bacterial cause was established, in six H influenzae and in two S pneumoniae. There was a significant relation between the carriage of H influenzae before operation and development of a chest infection. H influenzae was also found more often in cigarette smokers. The presence of S pneumoniae or coliform organisms before surgery was not related to the development of infection. The high incidence of postoperative chest infection in cigarette smokers appears to be due in part to preoperative colonisation of the oropharynx by H influenzae.

Abdomen↗
PubMed1990

Monte-Carlo simulation of multisite echinomycin-DNA interactions detected by in vitro transcription analysis.

The interaction of echinomycin with DNA was analyzed at 37 degrees C by in vitro transcription analysis using a 497 bp fragment of DNA containing the lac UV5 promoter. Sixteen discrete drug binding sites were detected. The mole fraction of blocked transcript at each site was monitored over 4 h, and the kinetic profile was analyzed by Monte-Carlo simulation. The time course for all 16 sites was fully described by this process. For each drug site, three parameters were resolved with the following variation between sites: relative drug occupancy (1-26), dissociation rate constant (0.06-0.70 min-1), and probability of termination of transcription (0-48%). Eight low-occupancy binding sites were at 5'-CA sequences (relative occupancy of 1.0-2.9). The eight major sites were all at 5'-CG sequences (relative occupancy of 6.3-26) and exhibited an average occupancy some 13-fold greater than the CA sites, corresponding to an average additional stability of approximately 1.6 kcal. The dissociation rates from apparent high-affinity sites were only partially correlated with relative occupancy. Ten binding sites exhibited a 3-48% probability of termination of transcription immediately adjacent to the 5'-CG central sequence. Termination probably arises from distortion of the DNA helix in regions flanking the binding site and was most dramatic (48% probability) where two adjacent CG sites were separated by only 1 bp. This termination phenomenon may well account for the observed effects of echinomycin in vivo.

Base Sequence↗
PubMed1990

Regulation of RNA polymerase III transcription in response to Simian virus 40 transformation.

Transcription by RNA polymerase III of the B2 family of middle-repetitive elements is activated in response to transformation by a variety of agents, including DNA tumour viruses, RNA tumour viruses and chemical carcinogens. We have investigated the mechanism of activation in SV40-transformed cells and we find that the effect is due to an increase in the activity of the general class III transcription factor TFIIIC, achieved both by an increase in factor abundance and by a change in its phosphorylation state. SV40 transformation also stimulates transcription of other genes by RNA polymerase III but the effect may be balanced by compensatory post-transcriptional changes. TFIIIC may mediate the stimulation of polymerase III transcription by a range of transforming viruses.

Animals↗
PubMed1990

Cough threshold after upper abdominal surgery.

The effect of upper abdominal surgery under general anaesthesia on the cough threshold was studied in 26 patients, on the basis of the concentrations of capsaicin and citric acid causing cough. Cough threshold was determined after administering doubling doses of nebulised aerosols of capsaicin and citric acid before operation and on the first and fourth postoperative days. There was an increase in cough threshold (decrease in cough sensitivity) in response to both inhaled irritants on the first postoperative day from the preoperative day and a return towards preoperative values by the fourth day after surgery. The increase in cough threshold on the first postoperative day correlated with the time since opiate administration (r = 0.7 for capsaicin, 0.52 for citric acid). These results show that the threshold concentration of chemical irritants causing cough is increased on the first postoperative day after upper abdominal surgery.

Abdomen↗
PubMed1990

New insight into drug-DNA interactions at individual drug binding sites probed by RNA polymerase during active transcription of the DNA.

An in vitro transcription assay has been used to probe drug-DNA interactions during active transcription of the DNA. The method relies upon the formation of a stable, synchronized population of initiated transcripts comprising a short length of RNA, achieved by the absence of one nucleotide in the initiation mixture. Subsequent equilibration of the transcription complex with drug, followed by elongation of the initiated transcripts, yields lengths of RNA determined by transcription up to each drug-occupied site. A variety of site-specific phenomena have been observed, including delayed termination of transcription 5-10 bp downstream of actinomycin binding sites; 10-20% probability of termination at most echinomycin sites; drug residence-time-dependent termination of bacteriophage polymerases; enhanced residence time compared to physicochemical measurement of drug-DNA dissociation rates. The use of two counter-directed promoters separated by 100 bp results in a sensitive bidirectional transcription footprinting procedure able to resolve adjacent drug sites separated by only 1 bp. The significance of the method of in vitro transcription is the ability to quantitate a range of parameters describing individual drug sites in situations where multiple drug-DNA interactions exist.

Base Sequence↗