NP's contributions are honored by a colleague.
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Education, Nursing, Continuing↗
Biomedical subjects
S Austin
Publications and source records attributed to S Austin.
Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function.
A mutation, serine 170 to alanine, in the proposed ATP binding site of the activator protein NTRC prevents transcriptional activation at sigma 54-dependent promoters both in vivo and in vitro. The rate of phosphorylation of the mutant protein by NTRB and the stability of mutant NTRC-phosphate were similar to those of wild-type NTRC. The phosphorylated mutant protein shows only a slight decrease in affinity (around 2-fold) for tandem NTRC binding sites in the Klebsiella pneumoniae nifL promoter suggesting that the mutation primarily influences the positive control function of NTRC. Moreover the mutant protein is trans dominant to the wild-type protein with respect to transcriptional activation at both the glnAp2 and nifL promoters. In vitro footprinting experiments reveal that the mutant protein is unable to catalyse isomerisation of closed promoter complexes between sigma 54-RNA polymerase and the nifL promoter to open promoter complexes. However, the mutant protein retains the ability to increase the occupancy of the -24, -12 region by sigma 54-RNA polymerase, forming closed complexes at the nifL promoter, which are not detectable in the absence of NTRC. These data support a model in which the activator influences the formation of closed complexes at the nifL promoter in addition to its role in catalysing open complex formation.
Adenosine Triphosphate↗
Interaction between phorbol dibutyrate and anaesthetics on synaptic responses from olfactory cortex of rat.
Phorbol esters have been shown to enhance the release of transmitters and to potentiate the effect of local anaesthetics in olfactory cortex of the rat. This work examined the interaction between phorbol dibutyrate and a range of substances, which act on axonal conduction and synaptic transmission. Synaptically-evoked field responses were elicited by stimulation of the lateral olfactory tract of slices of olfactory cortex, maintained in vitro. Ketamine (0.2-1.0 mM), benzocaine (0.2-1 mM), atropine (0.1-2 mM) and tetrodotoxin (20-200 nM) depressed synaptic transmission and, in the presence of phorbol dibutyrate (1 microM), these substances were more potent by 3.7 +/- 0.5, 1.5 +/- 4, 2.6 +/- 0.6 and 5.5 +/- 1.8 fold, respectively. Pentobarbitone (0.1-2.0 mM) with or without bicuculline, urethane (10-200 mM), halothane (0.5-5.0 mM) (with bicuculline) and ethanol (50-500 mM) also depressed synaptic transmission but their effectiveness was not potentiated by phorbol dibutyrate. It is thought that the increased potency, produced by phorbol ester, was associated with a presynaptic action of those substances.
Anesthetics↗
Riboflavin deficiency in the rat: effects on iron utilization and loss.
Iron absorption and daily loss of Fe were measured in riboflavin-deficient (B2-) Norwegian hooded rats and controls (B2+). Animals were fed on a test meal extrinsically labelled with 59Fe and whole-body radioactivity measured for 15 d. Riboflavin deficiency led to a reduction in the percentage of the 59Fe dose absorbed and an increased rate of 59Fe loss. All post-absorption 59Fe loss could be accounted for by faecal 59Fe, confirming that the loss was gastrointestinal. Fe concentrations and 59Fe as a percentage of retained whole-body 59Fe were higher in the small intestine of riboflavin-deficient animals than their controls, 14 d after the test meal. A separate experiment demonstrated that riboflavin deficiency was associated with a significant proliferative response of the duodenal crypts of the small intestine. These observations may explain the enhanced Fe loss in riboflavin deficiency.
Absorption↗
Substitutions at a single amino acid residue in the nitrogen-regulated activator protein NTRC differentially influence its activity in response to phosphorylation.
Four substitutions at serine residue 160 which increase the activity of the sigma 54-dependent activator protein NTRC in the absence of NTRB have been analysed in detail. Mutagenesis of the putative phosphoacceptor site of NTRC and analysis of double mutants indicate that the positive control function of the S160W and S160C mutants is phosphorylation-dependent, whereas the activity of the S160Y and S160F mutants is phosphorylation-independent. This was confirmed with two purified mutant proteins in vitro. Occupancy of tandem NTRC-binding sites upstream of the Klebsiella pneumoniae nifL promoter by S160W protein is also phosphorylation-dependent in contrast to occupancy by S160F protein, confirming that both the DNA-binding and activator functions of NTRC are influenced by phosphorylation. The S160W and S160C mutants are apparently more responsive than wild-type protein to 'cross-talk' by other members of the histidine protein kinase family but are less responsive to phosphorylation and dephosphorylation mediated by NTRB.
Amino Acid Sequence↗
Phorbol dibutyrate enhances local anaesthetic action.
1. Synaptically-evoked field responses were elicited by stimulation of the lateral olfactory tract of rat olfactory cortex slices maintained in vitro. 2. Various concentrations of lignocaine (5-500 microM) were applied to the solution bathing the slices. These produced dose-dependent depressions of the synaptically-evoked potential over the concentration range 20-500 microM. The responses completely recovered on washing out the lignocaine. Similar depressions were also noted for procaine (100-1000 microM). 3. In the 47 slices tested, application of beta-phorbol 12,13-dibutyrate (1 microM) increased the amplitude of the synaptic response (from 0.99 +/- 0.05 to 1.36 +/- 0.06 mV). beta-Phorbol 13-monbutyrate (1 microM) had no effect. 4. In the presence of phorbol dibutyrate the depressant effect of lignocaine was increased: the EC50 changed from 91 +/- 10 to 24 +/- 2 microM (a mean potency increase of 3.47 +/- 0.14). A similar increase in potency for procaine was observed with phorbol dibutyrate (from 264 +/- 23 to 49 +/- 9 microM: a 5.49 +/- 0.82 increase in potency). If the tissue was pre-equilibrated in a concentration of lignocaine which produced a 60-80% depression, addition of phorbol ester caused a complete abolition of the evoked potential. 5. beta-Phorbol 13-monobutyrate (1 microM) had no effect on the potency of lignocaine. 6. The Na and K currents generating the action potential in the presynaptic nerve terminals were unaffected by phorbol dibutyrate. The depressant effect of lignocaine on these currents was not modified by phorbol dibutyrate. The depressant effect of lignocaine on these currents was not modified by phorbol dibutyrate. 7. The potentiation of lignocaine could not be accounted for by membrane depolarization or by nonspecific actions of phorbol dibutyrate, and was distinct from the action on transmitter release. Therefore, it seems likely that protein kinase C activation was responsible for the modified action of lignocaine, although the mechanism for this is unclear.
Anesthetics, Local↗
Fine-structure analysis of the P1 plasmid partition site.
P1 plasmid partition requires two plasmid-encoded Par proteins and a cis-acting site. The site, parS, lies in a region consisting of a 13-bp palindrome and an adjacent AT-rich sequence. A series of point mutations were analyzed for their effects on partition site activity. The results indicated that only the left arm of the palindrome and some adjacent bases were needed. The limits of the functional site were further refined to a maximum of 22 bp, which includes binding sites for the P1 ParB protein. Mutations in the 22-bp site cause concomitant defects in partition and the ability to exert partition-mediated incompatibility. Like the region immediately to the left of the 22-bp region, the right arm of the palindrome is not essential for partition but does contain information that affects the specificity of incompatibility.
Bacterial Proteins↗
Critical sequences in the core of the P1 plasmid replication origin.
The core of the P1 plasmid replication origin consists of a series of 7-bp repeats and a G+C-rich stretch. Methylation of the GATC sequences in the repeats is essential. Forty different single-base mutations in the region were isolated and assayed for origin function. A single-base change within any 7-bp repeat could block the origin, irrespective of whether GATC bases were affected. The repeats themselves were critical, but the short intervals between them were not. Mutations in the G+C-rich region showed it to be a spacer whose exact length is important but whose sequence can vary considerably. It maintains a precise distance between the 7-bp repeats and binding sites for the P1 RepA initiator protein. It may also serve as a clamp to limit strand separation during initiation.
Bacterial Proteins↗
Small intestinal length: a factor essential for gut adaptation.
Changes in small intestinal structure, cytokinetics, and function are dynamic ways in which the gut adapts to diet, disease, and damage. Adequate length provides a static 'reserve' permitting an immediate response to pathophysiological changes. The length of the small intestine from conception to adulthood using data taken from eight published reports of necropsy measurement of 1010 guts is described. Mean length at 20 weeks' gestation was 125 cm, at 30 weeks' 200 cm, at term 275 cm, at 1 year 380 cm, at 5 years 450 cm, at 10 years 500 cm, and at 20 years 575 cm. Prenatal small intestinal growth exceeded that of body length according to the law: small intestinal length alpha body length to the power 4/3. After birth there was a noticeable deceleration: small intestinal length alpha body length to the power 1/2. The coefficient of variation of small intestinal length postnatally was 24%, sixfold greater than for body length. The rapid prenatal small intestinal growth rate ensures that the mature newborn has adequate small intestine to meet postnatal nutritional demands, but handicaps the preterm infant who undergoes intestinal resection. The wide variation in lengths suggests a 'surplus' surface area that is immediately available to respond, independent of dynamic mucosal changes, to fluctuations in food availability, local intestinal disease, damage, rapid transit, and resection.
Adaptation, Physiological↗
Partition-mediated incompatibility of bacterial plasmids.
Explore the source record for details and available documents.
Bacteria↗
Characterisation of the Klebsiella pneumoniae nitrogen-fixation regulatory proteins NIFA and NIFL in vitro.
Transcriptional activation by the Klebsiella pneumoniae nitrogen-fixation-specific positive control protein, NIFA, (nifA gene product) has been demonstrated in vitro in S30 extracts from cells which overproduce this protein. The activity of NIFA was dramatically reduced in vitro in the presence of the negative regulatory protein NIFL (nifL gene product) but was not inhibited by the presence of a mutant NIFL protein, NIFL2404. Transcriptional activation from the nifH promoter by NIFA was dependent on the alternative sigma factor, sigma 54, and also required the presence of an upstream activator sequence. NIFA activity was temperature-sensitive in vitro (as it is in vivo) which is due, at least in part, to the intrinsic lability of the protein itself. The majority of overproduced NIFA and NIFL was insoluble after low-speed centrifugation and was inactive in vitro. A low level of less aggregated NIFA protein present in cell extracts was responsible for in vitro activity and this fraction was partially purified.
Bacterial Proteins↗
Transcriptional activation of the Klebsiella pneumoniae nifLA promoter by NTRC is face-of-the-helix dependent and the activator stabilizes the interaction of sigma 54-RNA polymerase with the promoter.
Activation of transcription at the Klebsiella pneumoniae nifLA promoter requires the phosphorylated form of the positive control protein NTRC, together with RNA polymerase modified by the alternative sigma factor sigma 54. Dimethylsulphate and potassium permanganate were used as probes to analyse the interaction of NTRC and sigma 54-RNA polymerase with supercoiled nifLA promoter DNA in vitro. In contrast to the glnAp2 promoter, sigma 54 holoenzyme did not protect guanine residues in the nifLA promoter from methylation in the absence of the activator. We propose that NTRC stabilizes the interaction of sigma 54-RNA polymerase with the -24, -12 region, in addition to its role in catalysing open complex formation. Phosphorylated NTRC binds to two sites located greater than 100 nucleotides upstream of the -24, -12 region; it also induces hyper-methylation of a G residue at -23. Enhanced methylation at -23 is not co-operative with the binding of activator to the upstream sites and may account for the ability of NTRC, when present at high concentration, to activate transcription in the absence of the upstream binding sites. The insertion of spacer mutations at -86 indicates that transcriptional activation of the nifLA promoter at low NTRC concentrations is face-of-the-helix dependent, both in vivo and in vitro. We propose that correct positioning of activator molecules at the upstream binding sites stabilizes the interaction of sigma 54-RNA polymerase with the downstream region via the formation of a DNA loop.
Bacterial Proteins↗
Amino acid metabolism in muscle and in the whole body of man before and after ingestion of a single mixed meal.
Whole-body energy expenditure, protein oxidation, and exchange of amino acids, glutathione, and other metabolites across forearm muscle were assessed before and for 4 h after ingestion of a single meal containing 3274 kJ (783 kcal) and 25.5 g protein (4.08 g nitrogen). The study 1) demonstrates the dominance of glutamine over all other amino acids in carrying N out of skeletal muscle (1.33 mumol.L muscle-1.min-1 between 0 and 4 h--equivalent to 71% of the amino acid release or 82% of the amino acid N release); 2) suggests that glucose-alanine recycling between muscle and liver is not increased in the postprandial period despite the large uptake of glucose by muscle; 3) provides no support for the hypothesis that a substantial amount of N is exchanged between blood and muscle in the form of glutathione; and 4) considers the overall quantitative importance of amino acid oxidation in the whole body and the accompanying exchange of amino acids in skeletal muscle.
Adult↗
DNA supercoiling and aerobic regulation of transcription from the Klebsiella pneumoniae nifLA promoter.
Expression from the K. pneumoniae nifLA promoter is oxygen sensitive and is also inhibited by the DNA gyrase inhibitor coumermycin A1 under anaerobic growth conditions. The activity of this promoter was found to be highly sensitive to changes in DNA topology in vitro. Transcription was completely dependent on negative supercoiling at physiological salt concentrations although transcription from linear or fully relaxed closed circular templates was detectable at KCl concentrations lower than 50 mM. These observations suggest that aerobic regulation of nif transcription may be mediated through the level of DNA supercoiling.
Aerobiosis↗
Carbohydrate, fat, and protein metabolism in muscle and in the whole body after mixed meal ingestion.
The effect of ingesting a meal containing 3,275 kJ (47.3% carbohydrate, two-thirds of which was in the form of simple sugars, 39.4% fat, and 13.2% protein) on the oxidation of carbohydrate fat and protein (or amino acids) was assessed by indirect calorimetry and measurement of the rate of excretion of nitrogenous end products in urine and changes in the plasma urea concentration. Simultaneously, an assessment was made of substrate metabolism in forearm muscle by measuring forearm blood flow and concentration of metabolites in arterialized and deep venous blood. The mean resting energy expenditure during the first four hours after food ingestion was 15% higher than in the preprandial period (P less than .01). The extra energy dissipated during this time is equivalent to 5.3% of the energy provided in the meal. Carbohydrate oxidation increased by 111% (P less than .01), protein oxidation increased by 40% (P less than .05), and fat oxidation decreased by 21% (P less than .05). The concentration of glucose and amino acids and their uptake by muscle increased after food ingestion, while the reverse occurred with nonesterified fatty acids (NEFA). In the early postprandial period there was a marked suppression in the uptake of NEFA by muscle and a tendency toward decreased rather than increased release of glycolytic products (lactate + pyruvate + alanine), despite an up to sevenfold increase in the uptake of glucose. Fructose, which accounted for about 30% of the carbohydrate in the diet, was not taken up by muscle to any significant extent. It is estimated that during the first four hours after the meal muscle accounted for the uptake of 20% to 25% of the carbohydrate provided in the meal. This was associated with a small and nonsignificant change in the oxygen uptake by muscle. The data suggest that: (1) the increased uptake of glucose by muscle in the postprandial period does not necessarily increase the release of glycolytic products or increase the activity of the glucose-alanine and Cori cycles between muscle and liver; (2) triglyceride may become a more important energy source for muscle than circulating NEFA, especially in the early postprandial period; (3) muscle is not a major site of dietary-induced thermogenesis (zero to four hours); and (4) it is likely that nonmuscular tissues took up more carbohydrate (including fructose) than skeletal muscle during the first four hours after ingestion of this meal (even if it is assumed that as little as 50% of the dietary carbohydrate had been absorbed by four hours).
Adult↗
Idiopathic thenar muscle hypertrophy.
Two patients are described with idiopathic hypertrophy of the first dorsal interosseous and thenar muscles. CT scans have proved helpful in defining the abnormality.
Adult↗
The role of activator binding sites in transcriptional control of the divergently transcribed nifF and nifLA promoters from Klebsiella pneumoniae.
The regulatory region spanning the divergently transcribed nifF and nifLA promoters contains a NIFA-specific upstream activator sequence (UAS) located around +59, and two NTRC binding sites centred at -142 and -163 with respect to the nifLA transcription start site. We have constructed mutations in each of these binding sites and examined their role in transcriptional activation of the divergently transcribed promoters. Analysis of a mutation at +60 confirms that the UAS is required for efficient NIFA-mediated activation of nifF transcription. This sequence is also required for maximal activation of the nifLA promoter. Mutations at -169 and -148, within the two NTRC binding sites, reduce activation of the nifLA promoter by NTRC in vivo and lower the affinity of the activator for these sites in vitro. Phosphorylation of NTRC by NTRB is required for efficient binding of NTRC to these sites.
Adenosine Triphosphate↗
Requirements for transcriptional activation in vitro of the nitrogen-regulated glnA and nifLA promoters from Klebsiella pneumoniae: dependence on activator concentration.
Three proteins involved in nitrogen regulation in Klebsiella pneumoniae, NTRA, NTRB and NTRC, have been purified. In a defined in vitro system all three NTR proteins are required for initiation of transcription at the ntr activatable glnA and nifLA promoters. However, in crude S-30 extracts, transcription from the glnA promoter, but not the nifLA promoter, can be activated in the absence of NTRB. A higher concentration of NTRC is required for activation of nifLA transcription than for glnA transcription. Sequences located between -227 and -158 with respect to the nifL transcription start site are required for efficient activation of the nifLA promoter in vitro.
Base Sequence↗