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S Austin

Publications and source records attributed to S Austin.

At least 73 records · Page 4Linked to original sources
PubMed1994

An overview of a feasibility study for the production of industrial enzymes in transgenic alfalfa.

The overall aims of the research are to develop genetically engineered alfalfa producing high levels of industrially important enzymes and to develop rapid methods for extracting and purifying these enzymes from alfalfa juice. Using a reporter gene beta-glucuronidase (GUS) as a model system, we were able to demonstrate production of a foreign protein in alfalfa and gain valuable insight into the molecular approaches required for the expression and accumulation of foreign proteins in leaf tissue. GUS activity varied among individual transformants, and GUS was expressed in all plant tissues. GUS activity was shown to segregate in sexual progeny. There was no correlation between copy number of the GUS gene and activity. We have recently demonstrated the production of Mn-dependent lignin peroxidase and alpha-amylase in transgenic alfalfa. Concurrent research in the agricultural engineering aspects of this feasibility study focused on extraction strategies for the recovery of alfalfa juice, and on an evaluation of methods for processing and concentrating the juice. Thus, we are in a position to use plants expressing enzymes that have current or potential industrial importance to complete a feasibility study, and determine whether we can indeed economically recover target enzymes from field-grown transgenic alfalfa plants. The technology developed for these enzymes can be used to extract other value-added products from plants in the future.

Bacillus↗
PubMed1994

DNA distortion and nucleation of local DNA unwinding within sigma-54 (sigma N) holoenzyme closed promoter complexes.

The sigma N (sigma 54) RNA polymerase holoenzyme has the distinctive property of binding to promoters to form a closed promoter complex, which only isomerizes to the open complex when acted upon by an enhancer binding activator protein. We probed promoter complexes that form between sigma N and its holoenzyme with the conformationally sensitive footprinting reagents ortho-copper phenanthroline, potassium permanganate, and diethylpyrocarbonate. Results from these experiments indicate that the contacts sigma N makes at the -12 promoter element are necessary to promote a local DNA distortion immediately adjacent to this promoter element when the holoenzyme but not sigma N alone binds promoter DNA. Complexes in which this local distortion is not detected are not activatable, and the altered DNA conformation is diminished in the activated complex. We propose that a barrier to open complex formation in the sigma N holoenzyme closed complex is at some step or steps after the initial nucleation of DNA strand separation, which is detected as an altered DNA conformation stably maintained within the closed complex. Thus the activator protein may promote a conformational change in the sigma N holoenzyme to allow propagation of the altered DNA conformation, probably local unwinding, which we propose is necessary for formation of the melted DNA state, characteristic of the open promoter complex.

Bacterial Proteins↗
PubMed1994

Gender, work and illness: the influence of a research unit on an agricultural community in The Gambia.

Changes in employment opportunities and medical services are exploited by men and women in different ways. This paper examines gender-based variation in the selective use of employment and health opportunities in a Gambian village which has been the subject of medical and nutritional research by the Medical Research Council (MRC) for 43 years. The seasonal workloads of 105 men and women in Keneba were compared during one calendar year. Women carried a heavier burden of agricultural labour, while men had a higher rate of waged employment. The impact of the MRC field station on the local economy was assessed and evidence of associated male dependence on MRC employment found. Illness reporting patterns and the treatment choices of men and women were examined. Women made greater use of the MRC medical service, while men resorted more frequently to local remedies and healers. Female dependence on the MRC medical services is suggested by the data, and may be linked to the greater attention paid to them by researchers and medical practitioners.

Adult↗
PubMed1994

Identification of a DNA-contacting surface in the transcription factor sigma-54.

The transcription factor sigma-54 (sigma 54) is a sequence-specific DNA-binding protein that directs RNA polymerase to a particular class of promoter. The interaction of sigma 54 with promoter DNA has been analysed by protein-DNA crosslinking and enzymatic and chemical proteolysis. Direct physical evidence for a DNA-contacting surface within the carboxy-terminal one-third of the protein has been obtained. This region of sigma 54 is likely to be close to the surface of the protein, and contacts DNA when either sigma 54 or the sigma 54-holoenzyme bind specifically to promoter DNA. The amino-terminal region of sigma 54 appears to be highly susceptible to proteolysis, and its integrity influences the accessibility towards proteolysis of a second region of sigma 54, which includes the DNA-contacting surface. Thus the amino-terminal region of sigma 54 may have a role in influencing its DNA-binding properties, the major determinants of which appear to reside in the carboxy-terminal one-third of the protein.

Bacterial Proteins↗
PubMed1994

Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL.

The prokaryotic enhancer-binding protein NifA stimulates transcription at a distance by binding to sequences upstream of nitrogen fixation (nif) promoters and catalyzing the formation of open promoter complexes by RNA polymerase containing the alternative sigma factor, sigma 54. The activity of NifA in vivo is modulated by the negative regulatory protein NifL in response to environmental oxygen and fixed nitrogen. To date, a detailed biochemical analysis of these proteins from the model diazotroph Klebsiella pneumoniae has been hindered by their insolubility. We have now purified NifA and NifL from Azotobacter vinelandii in their native form. NifA is competent in specific DNA binding, transcriptional activation, and response to negative regulation by NifL in vitro. In contrast to the conserved mechanism of phosphotransfer demonstrated by other two-component regulatory systems, our results support a model in which NifL regulates the activity of NifA via a protein-protein steric block interaction rather than a catalytic modification of NifA.

Azotobacter vinelandii↗
PubMed1994

Prognosis in cystic fibrosis treated with continuous flucloxacillin from the neonatal period.

All newborn infants in East Anglia are screened for cystic fibrosis by blood immunoreactive trypsin assay at 7 days. Thirty eight infants with cystic fibrosis were randomised to treatment with either continuous oral flucloxacillin 250 mg/day (group P, n = 18) or with episodic antimicrobials as clinically indicated (group E, n = 20). Their progress was monitored from diagnosis to 24 months by a nurse coordinator who visited all infants regularly, at home and in hospital, to collect anthropometric, dietary, clinical, and microbiological data. Mean (range) age of confirmation of diagnosis was 5.7 weeks (1-14 weeks). There was no significant difference in birth weight, genotype, immunoreactive trypsin concentration, neonatal history, symptoms at diagnosis, pancreatic enzyme supplementation, or parental smoking history between the groups. Infants in group E had more frequent cough and a greater number of Staphylococcus aureus isolates than infants in group P. More infants of group E were admitted to hospital, had higher admission rates during the second year (19 v 5), for longer periods (6.4 v 2.2 days), despite receiving more than double the number of courses of antibiotics than group P infants (in addition to flucloxacillin). Continuous prophylactic flucloxacillin from early diagnosis of cystic fibrosis is associated with improved clinical progress during the first two years of life.

Bacterial Infections↗
PubMed1993

Convulsive threshold differences in right unilateral and bilateral ECT.

UNLABELLED: This study examines how the convulsive threshold in electroconvulsive therapy (ECT) varies with electrode placement (bilateral [BL] versus right unilateral [RUL]), age, gender, weight, and the nasion-inion measurements of the head. METHOD: Twenty-eight subjects underwent stimulus dose titration to determine the convulsive threshold. Titration was accomplished for each subject with both electrode placements during the first and second ECT in a balanced design. Head measurements were made prior to the first ECT. Results indicated that the convulsive threshold was higher for BL than RUL, higher for men than women, increased with age, and increased with an increasing nasion-inion measurement in women but not in men. We concluded that the convulsive threshold varies according to the choice of electrode placement, gender, and age as has been reported by others. We additionally report that the convulsive threshold increases with increasing head size as reflected in the nasion-inion distance.

Adult↗
PubMed1993

Effect of foot compression on the velocity and volume of blood flow in the deep veins.

The A-V Impulse System reduces the incidence of deep vein thrombosis by pneumatically compressing the venae comitantes of the lateral plantar artery, causing an increase in the velocity of blood in the proximal axial veins. Using a duplex scanner the effects of altering the pressure, pulse duration and frequency of foot compression on the velocity and volume of blood flow in the superficial femoral and popliteal veins were quantified. In 20 legs, foot compression of 50, 125 and 200 mmHg significantly increased the maximum venous blood flow by 9.0, 13.4 and 15.1 ml/s respectively (P < 0.001). Conversely, reducing the frequency of compression from 6 to 3 cycles per min significantly increased the rise in peak flow from 10.1 to 14.8 ml/s (P < 0.001). Changing the duration of compression from 1 to 3 s had no significant effect on peak flow. Increased blood flow is best achieved with high-pressure low-frequency foot compression. Increasing the duration of compression beyond 1 s has no effect on augmentation of flow in the deep veins.

Adult↗
PubMed1993

A proposed intestinal mechanism for the effect of riboflavin deficiency on iron loss in the rat.

The effect of riboflavin deficiency on gastrointestinal Fe distribution and loss was studied in weanling rats. Riboflavin deficiency was associated with a significant increase in crypt depth in the upper and mid small intestine and a twofold increase in the rate of crypt cell production compared with weight-matched and ad lib.-fed control rats. The rate of loss of endogenous Fe, measured as faecal 59Fe after intraperitoneally administered 59Fe, was twice that from riboflavin-deficient rats compared with weight-matched controls. We suggest that while there may be a contribution from turnover of enterocytes with an enhanced Fe content, enhanced Fe loss associated with riboflavin deficiency is due predominantly to an accelerated rate of small-intestinal epithelial turnover.

Animals↗
PubMed1993

The function of the upstream region of the sigma 54-dependent Klebsiella pneumoniae nifL promoter is sensitive to DNA supercoiling.

The positive control protein NTRC activates transcription from the sigma 54-dependent nifL and glnAp2 promoters of Klebsiella pneumoniae by binding to upstream enhancer-like sequences and contacting downstream bound sigma 54-RNA polymerase via looping of the intervening DNA. In contrast to the glnAp2 promoter, the activity of the nifL promoter is very sensitive to changes in DNA supercoiling both in vivo and in vitro. We have shown previously that the downstream elements of the nifL promoter are involved in the supercoiling response. In this study we find that the upstream region of nifL influences the supercoiling response of a hybrid nifL-glnAp2 promoter both in vivo and in vitro, demonstrating that the nifL upstream region also confers supercoiling sensitivity. DNA supercoiling did not appear to influence binding of NTRC to its sites in the nifL upstream region, suggesting that another function of this region, most probably DNA loop formation, is sensitive to changes in DNA topology.

Bacterial Proteins↗
PubMed1993

Core RNA polymerase assists binding of the transcription factor sigma 54 to promoter DNA.

The sigma subunit of bacterial RNA polymerase is necessary for the specific binding of RNA polymerase holoenzyme to promoter DNA. Promoter complexes which form with holoenzyme containing sigma 54 remain as closed complexes unless they are activated by one class of enhancer binding protein. The sigma 54 transcription factor can bind specifically to certain promoter sites in the absence of the core RNA polymerase subunits. This property has allowed demonstration of a new role for core polymerase in transcription, namely that it assists the binding of sigma 54 to promoter DNA. An altered form of sigma 54 with a deletion within the amino-terminal region showed increased affinity for specific DNA-binding sites. Although able to complex with core RNA polymerase the mutant sigma 54 failed to respond to core polymerase in the manner characteristic of the wild-type sigma 54 by altering its footprint. This result indicates that sigma 54 has a latent DNA-binding activity which is revealed by core RNA polymerase, and possibly involves a change in sigma 54 conformation. Promoter complexes which formed with sigma 54-holoenzyme appeared to be qualitatively different, depending upon the target promoter sequence, suggesting that different activatable complexes form at different promoter sequences.

Bacterial Proteins↗
PubMed1993

Evidence of two levels of control of P1 oriR and host oriC replication origins by DNA adenine methylation.

A mutant mini-P1 plasmid with increased copy number can be established in Dam- strains of Escherichia coli, where mini-P1 plasmid replication is normally blocked. Comparison of this plasmid and a plasmid driven by the host oriC replication origin showed that both origins are subject to control by methylation at two different levels. First, both origins appear to be subject to negative regulation acting at the level of hemimethylation. This probably involves the sequestration of the hemimethylated DNA produced by replication, as has been previously described for oriC. Second, both origins show a positive requirement for adenine methylation for efficient function in vivo. This conclusion is supported by the behavior of the P1 origin in an improved in vitro replication system. In vitro, where sequestration of hemimethylated DNA is not expected to occur, the hemimethylated P1 origin DNA was fully functional as a template. However, the activity of fully unmethylated DNA was severely restricted in comparison with that of either of the methylated forms. This in vitro uncoupling of the two effects of origin methylation suggests that two separate mechanisms are involved.

Adenine↗
PubMed1993

Cystic fibrosis identified by neonatal screening: incidence, genotype, and early natural history.

The incidence of cystic fibrosis over the last 10 years in East Anglia (a region of the United Kingdom with a population of 2.1 million) has halved. This has happened during the establishment of a neonatal screening programme, which has enabled early diagnosis, genetic counselling, and lately the option of prenatal diagnosis in subsequent pregnancies. One hundred and seven children were born with cystic fibrosis between 1981 and 1990, eight of whom were siblings. The Guthrie blood spots of 82 infants detected by neonatal immunoreactive trypsin screening between 1981 and 1990 were examined for the presence of the most common cystic fibrosis gene mutation (delta F508). It was present in 135 (82%) of the 164 cystic fibrosis genes analysed with 54 (66%) cases being homozygous and 27 (33%) heterozygous. Sixty nine per cent of infants were symptomatic at the time of diagnosis regardless of genotype. No association was found between the early clinical or biochemical features of the disease and homozygosity or heterozygosity for this mutation. Screening for cystic fibrosis using the blood immunoreactive trypsin assay alone remains an effective method of identifying infants with the disease soon after birth, thereby allowing early therapeutic intervention. Genetic counselling and prenatal diagnosis have contributed to a reduction in the number of children born with cystic fibrosis, but may not entirely explain the decreasing incidence of the disease.

Cystic Fibrosis↗
PubMed1993

Dual marker one day pancreolauryl test.

There is a pressing need for a simple non-invasive test of exocrine pancreatic function for use in children. The pancreolauryl test has been modified by the addition of a second marker (mannitol) to achieve a single day test without the need for two timed urine collections. Six healthy subjects and nine patients with cystic fibrosis were studied. Fluorescein, fluorescein dilaurate, and mannitol were taken by mouth, alone or in combinations, followed by 10 hour urine collections in two hourly aliquots to study the comparative pharmacokinetics of these markers. Urinary fluorescein was determined spectrophotometrically and urinary mannitol enzymatically. When fluorescein dilaurate and mannitol were taken together and the results expressed as ratios of percentage fluorescein to percentage mannitol recovery (F:M ratio) (mean (SD)) there was clear discrimination between healthy subjects and those with cystic fibrosis regardless of enzyme replacement treatment (57.3 (18.2) v 3.4 (1.4) v 3.2 (1.6) respectively). The differences in F:M ratios reached statistical significance in urinary aliquots collected between two and eight hours after marker ingestion. This single day tubeless test will greatly simplify the investigation of the child with suspected exocrine pancreatic dysfunction.

Administration, Oral↗
PubMed1993

Association of radiographic changes of osteoarthritis, symptoms, and synovial fluid particles in 300 knees.

Associations between compartmental distribution of radiographic changes of osteoarthritis (OA), individual features of OA (joint space loss, sclerosis, cyst, osteophyte; each scored 0-3), and presence of synovial fluid calcium particles (calcium pyrophosphate dihydrate (CPPD) crystals identified by polarised light microscopy and other calcium particles by alizarin red positivity (ARP) were sought in 300 osteoarthritic knees (178 patients; mean age 72, range 33-96 years). Patients whose knees were symptom free as well as those with symptoms were included. Osteoarthritis of two or three compartments but not unicompartmental OA was associated with the presence of CPPD or ARP. Involvement of any compartment (not just patellofemoral), and higher mean scores for both total and individual osteoarthritis changes (except cysts) was associated with CPPD and ARP; CPPD, but not ARP, was associated with symptoms: knees reported as having symptoms had higher mean total OA scores. Femoral cortical erosion, found more often in women, was associated with higher mean total OA score at the patellofemoral compartment but not with the presence of particles. Attrition, remodelling, and chondrocalcinosis (each scored as present or absent) occurred more often in knees with CPPD. Age did not correlate with any aspect of the OA score. This study confirms the association of calcium particles with the process of OA. Unlike previous studies confined to symptomatic knees, a radiographic pattern specific to CPPD ('pyrophosphate arthropathy') did not emerge.

Adult↗
PubMed1992

DNA supercoiling response of the sigma 54-dependent Klebsiella pneumoniae nifL promoter in vitro.

Transcription from the sigma 54-dependent Klebsiella pneumoniae nifL and glnAp2 promoters is activated by the general nitrogen regulatory protein NTRC. Unlike the glnAp2 promoter, which is relatively insensitive to changes in DNA supercoiling, transcription from nifL in vitro in a chloride-based buffer is supercoiling-dependent at physiological salt concentrations. The replacement of chloride with an acetate-based buffer decreases the stringency of the nifL supercoiling response, but open complexes formed on linear nifL promoter DNA under these conditions are unstable and less extensive than those found on supercoiled (form I) DNA. We have introduced mutations in particular elements of the nifL promoter that increase its homology to glnAp2. At the wild-type nifL promoter, sigma 54-RNA polymerase makes only limited contacts with the promoter in the absence of NTRC. However, a G to T change at -26 (nifL74) allows the formation of a stable closed complex with sigma 54-holoenzyme on both linear and form I templates in the absence of the activator. The combination of C to T mutations at -3 and -1 (nifL18) increases the A+T rich nature of the melted region and stabilizes open complexes formed on linear DNA. Open complex formation as a function of superhelical density was assessed at each promoter. Formation of open complexes at glnAp2 peaks at -0.024 and declines at higher superhelical densities, whereas at the wild-type nifL promoter, open complex formation peaks at -0.067 and is not detectable at superhelical densities less than -0.032. Both the nifL74 and nifL18 mutations altered the supercoiling response, increasing the ability to form open complexes at low superhelical densities. The presence of the nifL74 and nifL18 mutations in combination further altered the response of the promoter to DNA supercoiling. These observations suggest that the promoter as a whole, and not any one promoter element, mediates the transcriptional response to DNA supercoiling.

Base Sequence↗
PubMed1992

The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent.

The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent. We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites.

Adenosine Triphosphatases↗