A method to measure the interaction of Rac/Cdc42 with their binding partners using fluorescence resonance energy transfer between mutants of green fluorescent protein.

Enhanced blue fluorescent protein (EBFP) and enhanced green fluorescent protein (EGFP) mutants of GFP in close proximity to one another can act as a fluorescence resonance energy transfer (FRET) pair. Unstructured amino acid linkers of varying length were inserted between EBFP and EGFP, revealing that linkers even as long as 50 amino acids can be accommodated and still allow FRET to occur. This led to the development of a novel biosensor for Rac/Cdc42 binding to their effector proteins based on the insertion of amino acids 75-118 of p21-activated kinase (PAK) between the GFP mutants. We demonstrate that this protein construct allows significant FRET between EBFP and EGFP and retains the ability to bind to Rac in its GTP-bound form with a binding affinity similar to the uncomplexed PAK fragment, and furthermore, on binding to Rac or Cdc42 a marked change in FRET takes place. This forms the basis for a simple, sensitive, and rapid method to measure binding of Rac/Cdc42 to their effector proteins. Since the signal is dependent upon the interaction with active GTP-bound forms it acts as a biosensor for the activation of Rac/Cdc42. It has the potential for use in live cells and for identifying localization of Rac/Cdc42 within subcellular compartments.

Delineation of the Cdc42/Rac-binding domain of p21-activated kinase.

p21-activated kinases (PAKs) serve as effector proteins for the GTP-binding proteins Cdc42 and Rac. They are serine/threonine kinases containing the Cdc42/Rac interactive binding (CRIB) motif. The main aim of this study was to define the minimal domain of alphaPAK required for Cdc42/Rac binding. Eight stable PAK fragments of varying lengths, each containing the CRIB motif (residues 75-88), were expressed in Escherichia coli, and their ability to interact with Cdc42 and Rac was assessed using scintillation proximity assays, isothermal titration calorimetry, and fluorescence techniques. The shortest fragments examined (residues 70-94 and 75-94) bound only weakly to either Cdc42 or Rac. A longer fragment starting at residue 75 and ending at residue 105 showed binding to Q61L Rac.GTP with Kd = 1.9 microM. Highest affinity binding (Kd approximately 0.05 microM) was seen with longer fragments ending at residue 118 or 132. A small increase in affinity was seen with those fragments starting at residue 70 rather than residue 75. PAK fragments bound with approximately 3-10-fold higher affinity to Cdc42 than to Rac and bound Q61L variants with 5-10-fold higher affinity than wild type. The dissociation rates of Q61L Rac.mant-GTP and of Q61L Cdc42. mant-GTP from PAK fragment residues 70-132 were measured to be 0.66 and 0.25 min-1, respectively, which are 100-fold lower than dissociation rates for Ras:Ras-effector domains, although their affinities are similar. Calorimetric measurements revealed that binding was associated with a relatively slow heat change. It is suggested that these PAK fragments (in the absence of Cdc42 or Rac) might exist predominantly in an inactive conformation that slowly interconverts with an active conformation and/or a slow conformational change may occur upon binding to Cdc42/Rac. In conclusion, the PAK CRIB motif itself is insufficient for high-affinity binding to Cdc42/Rac, but a 30 amino acid region of PAK (residues 75-105), containing this motif, is sufficient.

Isolation and characterisation of an antifungal antibiotic (GR135402) with protein synthesis inhibition.

A novel antifungal antibiotic GR135402 has been isolated from a fermentation broth of Graphium putredinis which inhibited protein synthesis in Candida albicans but not rabbit reticulocytes. The spectrum of activity included C. albicans and Cryptococcus neoformans but not some other Candida species or Aspergillus species. Therapeutic efficacy in a mouse model of systemic candidosis was attained following parenteral dosing.

Helicobacter pylori porCDAB and oorDABC genes encode distinct pyruvate:flavodoxin and 2-oxoglutarate:acceptor oxidoreductases which mediate electron transport to NADP.

Helicobacter pylori, a major cause of human gastric disease, is a microaerophilic bacterium that contains neither pyruvate nor 2-oxoglutarate dehydrogenase activity. Previous studies (N. J. Hughes, P. A. Chalk, C. L. Clayton, and D. J. Kelly, J. Bacteriol. 177:3953-3959, 1995) have indicated that the major routes for the generation of acetyl coenzyme A (acetyl-CoA) and succinyl-CoA are via pyruvate:flavodoxin oxidoreductase (POR) and 2-oxoglutarate:acceptor oxidoreductase (OOR), respectively. The purified POR is a heterotetrameric protein, with subunits of 48 (PorA), 36 (PorB), 24 (PorC), and 14 (PorD) kDa. In this study OOR has been purified, and it is similarly composed of polypeptides of 43 (OorA), 33 (OorB), 24 (OorC), and 10 (OorD) kDa. Both POR and OOR are oxygen labile and are likely to be major contributors to the microaerophilic phenotype of H. pylori. Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor. Although the purified enzymes were unable to reduce NAD(P), electrons from both pyruvate and 2-oxoglutarate could reduce NADP in cell extracts, consistent with a role for these oxidoreductases in the provision of NADPH as a respiratory electron donor. The H. pylori por, oor, and fldA genes were cloned and sequenced. The deduced por gene products showed significant sequence similarity to archaeal four-subunit 2-oxoacid:acceptor oxidoreductases. However, the amino acid sequences of OorA and -B were more closely related to that of the two-subunit POR of the aerobic halophile Halobacterium halobium. Both porD and oorD encode integral ferredoxin-like subunits. POR and OOR are probably essential enzymes in H. pylori, as insertion inactivation of porB and oorA appeared to be lethal.

Use of NMR to Study H. pylori Metabolism.

Until recently, the culture of Helicobacter pylori in vitro has only been achievable with complex undefined media supplemented with blood products, such as Brain-heart infusion (BHI) medium, blood agar, chocolate agar, or lysed blood agar (1). Substrate utilization and metabolic activity during growth on these media is difficult to define; this has led to the use of techniques such as nuclear magnetic resonance (NMR) spectroscopy to investigate the metabolic pathways active in H. pylori (e.g., refs. 2-5). Defined media for the growth of H. pylori have now been described (6,7), however, and may facilitate future metabolic studies in this organism by more conventional techniques.

Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori.

A heat-stable dehydroquinase was purified to near homogeneity from a plate-grown suspension of the Gram-negative stomach pathogen Helicobacter pylori, and shown from both its subunit and native molecular masses to be a member of the type II family of dehydroquinases. This was confirmed by N-terminal amino acid sequence data. The gene encoding this activity was isolated following initial identification, by random sequencing of the H. pylori genome, of a 96 bp fragment, the translated sequence of which showed strong identity to a C-terminal region of other type II enzymes. Southern blot analysis of a cosmid library identified several potential clones, one of which complemented an Escherichia coli aroD point mutant strain deficient in host dehydroquinase. The gene encoding the H. pylori type II dehydroquinase (designated aroQ) was sequenced. The translated sequence was identical to the N-terminal sequence obtained directly from the purified protein, and showed strong identity to other members of the type II family of dehydroquinases. The enzyme was readily expressed in E. coli from a plasmid construct from which several milligrams of protein could be isolated, and the molecular mass of the protein was confirmed by electrospray MS. The aroQ gene in H. pylori may function in the central biosynthetic shikimate pathway of this bacterium, thus opening the way for the construction of attenuated strains as potential vaccines as well as offering a new target for selective enzyme inhibition.

The respiratory chain of Helicobacter pylori: identification of cytochromes and the effects of oxygen on cytochrome and menaquinone levels.

The quinone and cytochrome components of the respiratory chain of the microaerophilic bacterium Helicobacter pylori have been investigated. The major isoprenoid quinone was menaquinone-6, with traces of menaquinone-4; no methyl-substituted or unusual menaquinone species were found. Cell yield was highest after growth at 10% (v/v) oxygen and menaquinone levels (per dry cell mass) were maximal at 5-10% (v/v) oxygen. Helicobacter pylori cells and membranes contained b- and c-type cytochromes, but not terminal oxidases of the a- or d-types, as judged by reduced minus oxidised difference spectra. Spectra consistent with the presence of a CO-binding terminal oxidase of the cytochrome b- or o-type were obtained. The soluble fraction from disrupted cells also contained cytochrome c. There were no significant qualitative differences in the cytochrome complements of cells grown at oxygen concentrations in the range 2-15% (v/v) but putative oxidases were highest in cells grown at 5-10% (v/v) oxygen.

Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori.

The enzyme activities responsible for carboxylation reactions in cell extracts of the gastric pathogen Helicobacter pylori have been studied by H14CO3- fixation and spectrophotometric assays. Acetyl coenzyme A carboxylase (EC 6.4.1.2) and malic enzyme (EC 1.1.1.40) activities were detected, whereas pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.3.1) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) activities were absent. However, a pyruvate-dependent, ATP-independent, and avidin-insensitive H14CO3- fixation activity, which was shown to be due to the isotope exchange reaction of pyruvate:flavodoxin oxidoreductase (EC 1.2.7.1), was present. The purified enzyme is composed of four subunits of 47, 36, 24, and 14 kDa. N-terminal sequence analysis showed that this enzyme is related to a recently recognized group of four-subunit pyruvate:ferredoxin oxidoreductases previously known only from hyperthermophiles. This enzyme from H. pylori was found to mediate the reduction of a number of artificial electron acceptors in addition to a flavodoxin isolated from H. pylori extracts, which is likely to be the in vivo electron acceptor. Indirect evidence that the enzyme is capable of in vitro reduction of the anti-H. pylori drug metronidazole was also obtained.

Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori.

Oxygen uptake by Helicobacter pylori cells and membranes was determined. Cells from stirred broth cultures or agar plates, suspended in buffer, possessed a variable and apparently endogenous respiration which could be sustained for several hours. In contrast, oxygen consumption by cells from statically incubated broth cultures, in the absence of added substrate, was transient or undetectable. These latter cells, however, oxidised ethanol, fumarate, glucose, D-lactate, pyruvate and succinate, though glucose-oxidising ability declined rapidly. The Kms for D-lactate, pyruvate and succinate metabolism were low (< or = 20 microM) and oxygen uptake was approximately 1.5, 2 and 2 mol per mol substrate respectively, indicating metabolism beyond acetate plus CO2 and implying the presence of tricarboxylic acid cycle activity. Cell membranes oxidised fumarate, D-lactate, NADH, NADPH and succinate. NADPH oxidation was six times more rapid than that of NADH. Rates of oxygen uptake by cells suspended in buffer with metabolisable substrate were < 20% of those for cells suspended in a brain heart infusion medium. Uninoculated medium consumed significant quantities of oxygen.

Metabolism of pyruvate and glucose by intact cells of Helicobacter pylori studied by 13C NMR spectroscopy.

The metabolic routes of substrate catabolism by intact cells of H. pylori have been investigated by 13C NMR. Real time analyses of metabolic transformations under anaerobic conditions have been obtained with dense cell suspensions incubated with 13C-labelled pyruvate and glucose. In addition, time point studies have been carried out with cells incubated under aerobic conditions. Anaerobically, pyruvate was rapidly metabolized to lactate, ethanol and acetate. In addition, alanine was produced in significant quantities by cells provided with a nitrogen source and the metabolic incorporation of nitrogen from urea was demonstrated. Under aerobic conditions acetate was the major oxidation product from pyruvate; no evidence was obtained for tricarboxylic acid cycle activity. Glucose was metabolized more slowly than pyruvate. Anaerobically, two major products were observed and identified as sorbitol and gluconate by gas chromatography/mass spectrometry. Evidence was obtained for the oxidation of glucose to acetate under aerobic conditions. The fate of the 13C label with glucose substrates labelled in different positions showed that this oxidation takes place via the Entner-Doudoroff pathway.

Pathways to acetyl-CoA formation in Candida albicans.

The supply of acetyl units from the mitochondrion to the cytosol of Candida albicans appears to be dependent only upon the activity of carnitine acetyltransferase (CAT). The enzyme ATP:citrate lyase (ACL), the major source of acetyl units in oleaginous yeasts, is absent from C. albicans in both the mycelial and yeast forms. There appears to be no other active translocation of acetate or acetyl groups except via the action of carnitine acetyltransferase.

Treatment of cervical intraepithelial neoplasia (CIN) by radical electrocoagulation diathermy: 5 years' experience.

During the first 5 years of a colposcopy clinic established in a London teaching hospital 653 new patients were seen, usually referred because they had had an abnormal cervical smear report suggesting cervical intraepithelial neoplasia (CIN). Of 490 women treated within the district for CIN, 361 (73.7%) underwent radical electrocoagulation diathermy, 91 (18.6%) were treated by cone biopsy, 34 (6.9%) underwent hysterectomy and the other four patients were treated by cryocautery. Of the 245 women treated by radical electrocoagulation diathermy who have been followed for at least 12 months, persistent or recurrent disease has been found in only 15, a first time treatment success rate of 93.9%.

Activities of enzymes of rat hepatic mitochondrial fatty acid oxidation during increased fatty acid degradation.

The activities of the enzymes of hepatic mitochondrial fatty acid oxidation have been compared in normal and fat-fed, streptozotocin diabetic, starved and hyperthyroid adult female rats. The various experimental states resulted in hyperketonaemia and marked increases in the activities of palmitoyl-CoA synthetase and carnitine palmitoyltransferase I, but had little effect on the other enzymes involved.

Hepatic mitochondrial fatty acid oxidation during the perinatal period in the rat.

1. The activity of hepatic mitochondrial carnitine acyltransferase I increases rapidly after birth, is high during the suckling period and falls after weaning. In contrast, carnitine acyltransferase II and acyl-CpA dehydrogenase exhibit few developmental changes. 2. These and previous studies indicate that outer mitochondrial membrane acyl-CoA synthetase and inner membrane carnitine acyltransferase I increase in activity after birth much more rapidly than to any other enzymes of fatty acid oxidation. 3. Studies of the 18 hr after caesarian delivery indicate that whereas the major increase in the activity of acyl-CoA synthetase occurs within 3 hr of birth the change in carnitine acyltransferase I activity is less rapid. 4. Prolonged pregnancy, starvation of the mother or feeding the mother a high polyunsaturated fat content diet resulted in increased activities of acyl-CoA synthetase and carnitine acyltransferase I in the fetal liver.

Changes in the yield, and carbohydrate, lipid and protein content of milk during lactation in the rat.

The milk yield and composition was studied during the first three lactations of a group of rats. Milk yield increased steadily throughout the three lactations, but was somewhat lower during the first than subsequent lactations. Protein concentration was similar during all three lactations and varied little with stage of lactation. In contrast the lactose concentration, which was reasonably constant for the first 8 days post partum, increased thereafter two-fold by the end of the period studied in all three lactations. However, the N-acetyl-neuraminyl lactose concentration showed somewhat reciprocal changes. Considerable variations in the triacylglycerol concentration was found during the first lactation but few changes were observed during subsequent lactations. The free fatty acid concentration was at all times low and showed no significant changes during or between lactations. At most stages of lactation in raw milk, the major fatty acids are palmitate, oleate and linoleate. However, as lactation progresses there is an increase in the proportion of medium-chain saturated fatty acids and a corresponding decrease in the proportion of long chain unsaturated fatty acids in milk fat. Clearly the composition of milk is not invariable but changes both during and between lactations. Such changes may be expected to have some influence on the metabolism of the offspring.