A study to assess the feasibility of using glucometer measurement of blood glucose on fresh blood sampled from the surgical field
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Journal Article↗
Biomedical subjects
S Austin
Publications and source records attributed to S Austin.
A plasmid partition system of the P1-P7par family from the pMT1 virulence plasmid of Yersinia pestis.
The complete sequence of the virulence plasmid pMT1 of Yersinia pestis KIM5 revealed a region homologous to the plasmid partition (par) region of the P7 plasmid prophage of Escherichia coli. The essential genes parA and parB and the downstream partition site gene, parS, are highly conserved in sequence and organization. The pMT1parS site and the parA-parB operon were separately inserted into vectors that could be maintained in E. coli. A mini-P1 vector containing pMT1parS was stably maintained when the pMT1 ParA and ParB proteins were supplied in trans, showing that the pMT1par system is fully functional for plasmid partition in E. coli. The pMT1par system exerted a plasmid silencing activity similar to, but weaker than those of P7par and P1par. In spite of the high degree of similarity, especially to P7par, it showed unique specificities with respect to the interactions of key components. Neither the P7 nor P1 Par proteins could support partition via the pMT1parS site, and the pMT1 Par proteins failed to support partition with P1parS or P7parS. Typical of other partition sites, supernumerary copies of pMT1parS exerted incompatibility toward plasmids supported by pMT1par. However, no interspecies incompatibility effect was observed between pMT1par, P7par, and P1par.
Bacterial Proteins↗
The stability region of the large virulence plasmid of Shigella flexneri encodes an efficient postsegregational killing system.
The large virulence plasmid pMYSH6000 of Shigella flexneri contains a determinant that is highly effective in stabilizing otherwise unstable plasmids in Escherichia coli. Expression of two small contiguous genes, mvpA and mvpT (formerly termed STBORF1 and STBORF2), was shown to be sufficient for stability. Mutations in mvpT abolished plasmid stability, and plasmids expressing only mvpT killed the cells unless mvpA was supplied from a separate plasmid or from the host chromosome. When replication of a plasmid carrying the minimal mvp region was blocked, growth of the culture stopped after a short lag and virtually all of the surviving cells retained the plasmid. Thus, the mvp system stabilizes by a highly efficient postsegregational killing (PSK) mechanism, with mvpT encoding a cell toxin and mvpA encoding an antidote. The regions that surround the mvp genes in their original context have an inhibitory effect that attenuates plasmid stabilization and PSK. The region encompassing the mvp genes also appears to contain an additional element that can aid propagation of a pSC101-based plasmid under conditions where replication initiation is marginal. However, this appears to be a relatively nonspecific effect of DNA insertion into the plasmid vector.
DNA, Bacterial↗
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Accreditation↗
Cyclo-oxygenase products and atherothrombosis.
The advent of selective inhibitors of the cyclo-oxygenase (COX)-2 enzyme has afforded the opportunity to reduce the incidence of gastrointestinal complications of traditional nonsteroidal anti-inflammatory drugs (NSAIDs). The widespread use of these drugs has increased interest in their role in the cardiovascular system. Although deletion of the prostacyclin receptor (the IP) accelerates atherogenesis in the mouse, retention of one copy of the IP is atheroprotective. This is consistent with the failure of biochemically defined, selective doses of a COX-2 inhibitor to accelerate atherogenesis in the mouse, despite suppressing prostacyclin biosynthesis by roughly 60%. Inhibition of both COX isozymes, by contrast, markedly retards atherogenesis. Consistent with these observations, antagonism of the thromboxane receptor (the TP) retards atherogenesis and diminishes the proliferative response to vascular injury in the mouse. Even partial suppression of prostacyclin (without coincident inhibition of platelet COX-1-dependent thromboxane formation) by COX-2 inhibitors may be undesirable in acute vascular occlusive syndromes. However, these drugs are unlikely to accelerate progression of the underlying vascular disease. By contrast, the effects of TP antagonists, aspirin, and even traditional NSAIDs on atherosclerotic plaque progression merit further evaluation in humans.
Animals↗
Spectroscopic studies of the structure and stability of glycoconjugate vaccines.
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Animals↗
The upstream region of the nodD3 gene of Sinorhizobium meliloti carries enhancer sequences for the transcriptional activator NtrC.
In Sinorhizobium meliloti the expression of the nodulation genes nodABC is regulated in response to the level of fixed nitrogen (ammonia). Previous results suggested that the response to the nitrogen status is mediated by the two-component NtrB/NtrC system which controls transcription of the nodD3 gene, encoding a positive regulatory protein for the activation of nodABC transcription. Here we confirm by DNase I footprinting and gel shift assays that NtrC, when phosphorylated by NtrB, is able to interact with the enhancer sequences present upstream of nodD3. A model is proposed whereby NtrC functions to control the transcription from the two promoters in the upstream region of nodD3 in response to nitrogen status.
Bacterial Proteins↗
Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences.
The SeqA protein binds to the post-replicative forms of the origins of replication of the Escherichia coli chromosome (oriC) and the P1 plasmid (P1oriR) at hemimethylated GATC adenine methylation sites. It appears to regulate replication by preventing premature reinitiation. However, SeqA binding is not exclusive to replication origins: different fragments with hemimethylated GATC sites can bind SeqA in vitro when certain rules apply. Most notably, more than one such site must be present on a bound fragment. The protein appears to recognize individual hemimethylated sites, but must undergo an obligate cooperative interaction with a nearby bound protein for stable binding. SeqA contacts both DNA strands in a discrete patch at each hemimethylated GATC sequence. All four GATC bases are contacted and are essential for binding. Although the recognized sequence is symmetrical, the footprint on the methylated strand is always broader, suggesting that the bound protein is positioned asymmetrically with its orientation dictated by the position of the unique methyl group. Studies of alternative spacings and relative orientations of adjacent sites suggest that each site may be recognized by a symmetrical dimer with an induced asymmetry in one of the subunits similar to that seen with certain type II restriction endonucleases.
Bacterial Outer Membrane Proteins↗
Expression of testicular genes in haematological malignancies.
The gene expression of a new group of tumour antigens known as cancer/testis (CT) antigens is now well-recognized in some solid tumours. However, their expression in haematological malignancies remained unclear. In this study, we have used reverse transcription polymerase chain reaction and Southern blot analysis to examine the presence of transcripts for the three CT antigens, NY-ESO-1, SSX2 and SCP1 in haematological malignant cells. We found that transcripts for SCP1 could be detected in 10% of myeloma, 5.7% of acute myeloid leukaemia and 23% of chronic myeloid leukaemia. In contrast, NY-ESO-1 and SSX2 were not detected in any of the 107 tumour samples.
Antigens, Neoplasm↗
Isolation and properties of the complex between the enhancer binding protein NIFA and the sensor NIFL.
In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation.
Adenosine Diphosphate↗
Not a mouse stirring: deletion of the EP2 and love's labor's lost.
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Animals↗
New methods for the characterisation of biopharmaceuticals: conjugate vaccines against Haemophilus influenzae type b.
Modern physicochemical methods allow biological pharmaceuticals, particularly those arising from recombinant DNA technology, to be characterised with a degree of precision not previously possible. These techniques, which tell us what a material is (rather than what it does) provide an approach complementary to traditional bioassays for the control of biological pharmaceuticals. As we come to understand the mechanisms by which structural variation modulates the various biological activities of a product, structure-based assays will be able to replace biological identity and potency assays, although replacement of safety tests to find trace impurities (such as endotoxin) may be more difficult.
Animal Testing Alternatives↗
Properties of a mutant form of the prokaryotic enhancer binding protein, NTRC, which hydrolyses ATP in the absence of effectors.
The mutation S170A in the proposed nucleotide binding site of the transcriptional activator protein NTRC abolishes its ability to catalyse open promoter complex formation by the sigma(N)-RNA polymerase holoenzyme. NTRC(S170A) has significant ATPase activity, which, in contrast to the wild-type protein, is unaffected by phosphorylation or binding to enhancer sites on DNA. The mutant protein appears to oligomerise normally on DNA in response to phosphorylation but the ATPase activity is apparently not responsive to changes in oligomerisation state. The defect in transcriptional activation is discussed in relation to mutations in other sigma(N)-dependent activators.
Adenosine Triphosphatases↗
Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems.
The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells. It encodes two DNA sites and two proteins, ParA and ParB. Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific. Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules. The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition. Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts. We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA. Similarly, ParA acts in partition by making a specific contact with ParB bound at parS. Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB. As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition. The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules.
Amino Acid Sequence↗
Electron donation to the flavoprotein NifL, a redox-sensing transcriptional regulator.
Transcriptional control of the nitrogen fixation (nif) genes in response to oxygen in Azotobacter vinelandii is mediated by nitrogen fixation regulatory protein L (NifL), a regulatory flavoprotein that modulates the activity of the transcriptional activator nitrogen fixation regulatory protein A (NifA). CD spectra of purified NifL indicate that FAD is bound to NifL in an asymmetric environment and the protein is predominantly alpha-helical. The redox potential of NifL is -226 mV at pH 8 as determined by the enzymic reduction of NifL by xanthine oxidase/xanthine in the presence of appropriate mediators. The reduction of NifL by xanthine oxidase prevented NifL from acting as an inhibitor of NifA. In the absence of electron mediators NifL could also be reduced by Escherichia coli flavohaemoprotein (Hmp) with NADH as reductant. Hmp contains a globin-like domain with haem B as prosthetic group and an FAD-containing oxidoreductase module. The carboxyferrohaem form of Hmp was competent to reduce NifL, suggesting that electron donation to NifL originates from the flavin in Hmp rather than by direct electron transfer from the haem. Spinach ferredoxin:NAD(P) oxidoreductase, which adopts a folding similar to the FAD- and NAD-binding domains of Hmp, also reduced NifL with NADH as reductant. Re-oxidation of NifL occurs rapidly in the presence of air, raising the possibility that NifL might sense intracellular oxygen. We propose a physiological redox cycle in which the oxidation of NifL by oxygen and hence the activation of its inhibitory properties occurs rapidly, in contrast with the switch from the active to the reduced form of NifL, which occurs more slowly.
Azotobacter vinelandii↗
The redox- and fixed nitrogen-responsive regulatory protein NIFL from Azotobacter vinelandii comprises discrete flavin and nucleotide-binding domains.
Azotobacter vinelandii NIFL is a nitrogen fixation-specific regulatory flavoprotein that modulates the activity of the transcriptional activator NIFA in response to oxygen and fixed nitrogen in vivo. NIFL is also responsive to ADP in vitro. Limited proteolysis of NIFL indicates that it comprises a relatively stable N-terminal domain and a C-terminal domain that is protected from trypsin digestion in the presence of adenosine nucleotides. ATP protects the protein from cleavage in the vicinity of potential nucleotide-binding sites in the C-terminus, whereas ADP protects the entire C-terminal domain. NIFL has an apparent Kd of 130 microM for ATP and 16 microM for ADP. The purified N-terminal domain has an identical UV/visible absorption spectrum to the wild-type protein and is reduced by sodium dithionite, demonstrating that it is a flavin-binding domain. The isolated N-terminal domain does not inhibit NIFA activity. A subdomain fragment containing 160 residues of the C-terminal region, including the nucleotide-binding sites, is also not competent to inhibit NIFA. Removal of the first 146 residues of NIFL, which includes a conserved S-motif (PAS-like domain), found in a large family of sensory proteins from eubacteria, archea and eukarya eliminates the redox response. However, this truncated protein remains competent to inhibit NIFA activity in response to ADP in vitro and to the level of fixed nitrogen in vivo. The redox and nitrogen-sensing functions of A. vinelandii NIFL are therefore separable and are discrete functions of the protein.
Adenosine Diphosphate↗
Bile salt-stimulated lipase and digestion of non-breast milk fat.
BACKGROUND: 13 Carbon (13C)-lipid breath tests are an effective, noninvasive way of repeatedly measuring fat digestion. The purpose of this study was to assess the contribution of bile salt-stimulated lipase (BSSL) in human milk to the digestion of non-breast-milk fat in Gambian infants. METHODS: Twelve Gambian infants (aged 3-8 months) were studied on 4 days. 13C-Trioctanoin (7.5 mg/kg, digested by BSSL preduodenal and pancreatic lipases) and 13C-cholesteryl octanoate (25 mg/kg, digested by BSSL and pancreatic lipases) were used as substrates. The percentage dose recovery (PDR) of 13C in breath during 5 hours was compared after ingestion of each substrate with fresh, expressed breast milk (FBM) or heated, expressed breast milk (HBM). Gas isotope ratio-mass spectrometry was used to measure 13C enrichment, and breast milk samples were analysed for esterase activity. RESULTS: Heating breast milk significantly decreased esterase activity (mean +/- SD values: FBM = 12.2 +/- 2.9 IU/ml; HBM = 0.5 +/- 0.3 IU/ml), and there was no difference in the volumes of milk ingested on each test day (approximately 50 ml). The PDR of 13C was comparable to that previously described in healthy English infants and was not increased by BSSL. The mean +/- SD PDR of 13C from trioctanoin was 36.3 +/- 8.4% for FBM and 34.6 +/- 6.3% for HBM (NS). From cholesteryl octanoate, the mean +/- SD PDR of 13C was 24.3 +/- 8.7% for FBM and 27.1 +/- 7.5% for HBM (NS). CONCLUSIONS: Bile salt-stimulated lipase may enhance fat digestion in younger or malnourished infants who have a greater degree of pancreatic enzyme deficiency. However, this study suggests that it does not increase the digestion of non-breast-milk fat in healthy, well-nourished infants aged 3 to 8 months from an underprivileged background, who typically ingest frequent small quantities of breast milk.
Caprylates↗
Effects of protein restriction in early life on growth and function of the gastrointestinal tract of the rat.
BACKGROUND: Undernutrition during early life may have both immediate and later consequences. This study was undertaken to measure the long-term effects of perinatal undernutrition on the growth and function of the gastrointestinal tract. METHODS: Pregnant rats were assigned to one of four groups that received isocaloric diets restricted in protein during pregnancy or lactation and during both or neither. Thereafter, their pups were followed until aged 1 year. RESULTS: At 21 days the body weights of the young of those born of dams with postnatal protein restriction were halved, with comparable reductions in the weights of the stomach and caecum, compared with those of control animals. The lengths of the small and large intestines and mucosal weights of the foregut were also significantly reduced. Lactase activities were significantly increased and sucrase and maltase activities significantly reduced. By 42 days all the effects were less marked, and at 1 year the dimensions of the organs of the gastrointestinal tract and the composition and enzyme levels of the mucosa were all insignificantly different relative to body weight. CONCLUSIONS: Prenatal protein restriction alone had no significant long-term negative effects on body weight, growth, or mucosal hydrolase activity of the gastrointestinal tract. Postnatal protein restriction had a marked effect on these indices in early life and delayed the changes in mucosal hydrolases usually seen at weaning. In contrast with other organs and their functions, long-term growth of the gut and activity of small intestinal hydrolases are preserved in the face of perinatal protein restriction.
Animals↗