Drowning in antibiotics.

INTRODUCTION: The National Confidential Enquiry into Perioperative Deaths (NCEPOD) report recommended that 'fluid prescribing be given the same value as drug prescribing', yet fluid prescription is commonly delegated to junior doctors despite being a notoriously challenging topic. When antibiotics are given as an infusion they are diluted in 100ml of fluid, which is often unaccounted for when thinking about a patient's fluid requirements. This closed-loop audit aimed to assess first, intravenous (IV) fluid therapy and second, electrolyte prescribing compliance with National Institute for Health and Care Excellence (NICE) guidelines, with and without the additional fluid given with antibiotic administration. METHODS: Two retrospective audits were performed. Total fluid and electrolyte volume received with and without antibiotic fluids was correlated with recommendations in the NICE guidelines. Between cycles 1 and 2, potassium chloride with sodium chloride and glucose (PSG) was introduced as an alternative to IV maintenance fluid, and bolusing of antibiotics was mandatory. RESULTS: When analysing total fluid volume input per day, 10.4% and 7.45% of patients met their fluid requirement accurately in the first and second cycles, respectively. Within cycle 1, the mean total additional fluid that was given over 3 days with antibiotics was 1,572.73ml. In cycle 2, this decreased to 469.44ml when antibiotics were given as a bolus. CONCLUSIONS: In this closed-loop audit we noted that patients receiving IV fluids and IV antibiotics received too much additional fluid when the antibiotic dilution fluid was taken into account. Additional fluid was reduced alongside the proportion of electrolyte complications when bolusing of antibiotics was introduced. We recommend that that all nurses are trained to give antibiotics as a bolus because it can help to reduce fluid-related complications.

Identification of beta-secretase-like activity using a mass spectrometry-based assay system.

We describe an assay system for the identification of site-specific proteases. The assay is based on a protein substrate that is immobilized on ceramic beads. After incubation with cell homogenates, the beads are washed and digested with endoproteinase Lys-C to liberate a defined set of peptides. The peptide fragments are identified by mass spectrometry. The assay was used to screen for beta-secretase, the protease that cleaves amyloid precursor protein (APP) at the beta-site. Cathepsin D was identified as the enzyme responsible for beta-secretase-like activity in two cell lines. Subsequent analysis of the related aspartic protease, cathepsin E, revealed almost identical cleavage specificity. Both enzymes are efficient in cleaving Swedish mutant APP at the beta-site but show almost no reactivity with wild-type APP. Treatment of cell lines with pepstatin inhibited the production of amyloid peptide (Abeta) when they were transfected with a construct bearing the Swedish APP mutant. However, when the cells were transfected with wild-type APP, the generation of Abeta was increased. This suggests that more than one enzyme is capable of generating Abeta in vivo and that an aspartic protease is involved in the processing of Swedish mutant APP.

Controlling polymerization of beta-amyloid and prion-derived peptides with synthetic small molecule ligands.

The Alzheimer beta-amyloid peptide (Abeta) and a fragment of the prion protein have the capacity of forming amyloid-like fibrils when incubated under physiological conditions in vitro. Here we show that a small amyloid ligand, RO-47-1816/001, enhances this process severalfold by binding to amyloid molecules and apparently promote formation of the peptide-to-peptide bonds that join the monomers of the amyloid fibrils. This effect could be antagonized by other ligands, including analogues of RO-47-1816/001, as well as the structurally unrelated ligand Congo red. Analogues of RO-47-1816/001 with low affinity for amyloid did not display any antagonistic effect. In conclusion, these data suggest that synthetic molecules, and possibly also small natural substances present in the brain, may act in a chaperone-like fashion, promoting Abeta polymerization and growth of amyloid fibrils in vitro and possibly also in vivo. Furthermore, we demonstrate that small organic molecules can be used to inhibit the action of amyloid-enhancing compounds.

Expression and characterization of human beta-secretase candidates metalloendopeptidase MP78 and cathepsin D in beta APP-overexpressing cells.

Human beta-secretase candidates, MP78 (h-MP78, EC 3.4.24.15) and cathepsin D (Cat D, EC 3.4.23.5), were evaluated for their ability to enhance amyloid-beta-protein (A beta) secretion when overexpressed in beta APP-containing cells. HEK-293 cells stably co-expressing h-MP78 or Cat D and h-beta APP695 were metabolically labeled with [35S]methionine and A beta secretion was quantified in the conditioned media by immunoprecipitation and ELISA without showing any significant increase in A beta production. Because Cat D is known to have a higher affinity for APP-substrate containing the Swedish familial Alzheimer's disease double mutation (SFAD, K595N and M596L substitutions in beta APP695) than for the wild type substrate [Dreyer et al., Eur. J. Biochem., 224 (1994) 265-271], the effect of Cat D overexpression was tested in a HEK293/beta APPSFAD stable cell line. ELISA analysis of the conditioned media from these cells did also not reveal any increase in A beta generation. In addition, recombinant h-MP78 purified from E. coli cleaved an APP-derived substrate spanning the beta-secretase site (ISEVKMD1AEFRHDS) at multiple sites, but the beta-site cleavage was only a minor one; cleavage occurred predominantly at K-M and E-F bonds. Human liver Cat D also cleaved the same substrate at multiple sites, yet the major cleavage at pH 4.0 occurred at the amyloidogenic D1 site. These findings indicate that h-MP78 does not have the cleavage specificity required for a beta-secretase protease and although Cat D fulfilled the amyloidogenic cleavage specificity, the results of the co-expression experiments make both enzymes less likely candidates as relevant beta-secretases.

Expression and characterisation of plasmepsin I from Plasmodium falciparum.

Two aspartic proteinases, plasmepsins I and II, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin I. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of Lys110P to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH-dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin I. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin II. Plasmepsin I had 5-10-fold lower k(cat)/Km values than plasmepsin II for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P. falciparum growth, were found to have up to 80-fold lower inhibition constants for plasmepsin I compared to plasmepsin II. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of P. falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin II gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest they are not functionally redundant but play distinct roles in the parasite.

Crystal structure of phytase from Aspergillus ficuum at 2.5 A resolution.

Phytase is a high molecular weight acid phosphatase. The structure has an alpha/beta-domain similar to that of rat acid phosphatase and an alpha-domain with a new fold.

Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases.

Obtaining high quality protein crystals remains a rate-limiting step in the determination of three-dimensional X-ray structures. A frequently encountered problem in this respect is the high or heterogeneous carbohydrate content of many eukaryotic proteins. A number of reports have demonstrated the use of enzymatic deglycosylation in the crystallization of certain glycoproteins. Although this is an attractive tool, there are some problems that hinder the more widespread use of glycosidases in crystallization. First, commercially available glycosidases are relatively expensive, which virtually prohibits their use on a large scale. Second, the glycosidase must be removed from the glycoprotein of interest following deglycosylation, which is not always straightforward. To circumvent these problems we have cloned the two most generally useful glycosidases, peptide-N-glycosidase F and endoglycosidase F1 from Flavobacterium meningosepticum, as fusion proteins with glutathione S-transferase. The fusion not only allows rapid purification of these enzymes from Escherichia coli cell extracts, but also permits rapid removal from target proteins following deglycosylation. We have used these enzymes to obtain crystals of phytase from Aspergillus ficuum and acid phosphatase from Aspergillus niger and to obtain a new crystal form of recombinant human renin.

Identification of a human immunodeficiency virus-1 protease cleavage site within the 66,000 Dalton subunit of reverse transcriptase.

The human immunodeficiency virus-1 reverse transcriptase is a heterodimer of related 51 and 66 kDa subunits. The smaller subunit arises by viral protease-catalyzed cleavage of the carboxy-terminal domain of the 66 kDa species. Comparison of the amino acid composition analyses of the isolated 51 kDa and 66 kDa subunits indicates that the carboxyl terminus of 51 kDa is Phe440. This site was confirmed in vitro using purified recombinant protease and a peptide spanning the postulated cleavage area. The sequence surrounding this site does not show significant homology to other protease cleavage sites in the viral gag and pol precursors; thus, this new information may contribute to our understanding of the sequence specificity of the viral protease.

Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography.

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.

HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA.

The virion cores of the replication competent type 1 human immunodeficiency virus (HIV-1), a retrovirus, contain and RNA genome associated with nucleocapsid (NC) and reverse transcriptase (RT p66/p51) molecules. In vitro reconstructions of these complexes with purified components show that NC is required for efficient annealing of the primer tRNALys,3. In the absence of NC, HIV-1 RT is unable to retrotranscribe the viral RNA template from the tRNA primer. We demonstrate that the HIV-1 RT p66/p51 specifically binds to its cognate primer tRNALys,3 even in the presence of a 100-fold molar excess of other tRNAs. Cross-linking analysis of this interaction locates the contact site to a region within the heavily modified anti-codon domain of tRNALys,3.

Point mutations in conserved amino acid residues within the C-terminal domain of HIV-1 reverse transcriptase specifically repress RNase H function.

Two single site substitutions (E478----Q and H539----F) were introduced into the C-terminal RNase H domain of HIV-1 reverse transcriptase. These mutant proteins were expressed in Escherichia coli and purified by Ni2+-nitrilotriacetic acid affinity chromatography. Both enzymes are clearly defective in RNase H function, but exhibit wild type reverse transcriptase activity.

Complexity in the redox titration of the dihaem cytochrome c4.

Redox titration of the dihaem, two domain cytochromes c4 from Pseudomonas aeruginosa, Pseudomonas stutzeri and Azotobacter vinelandii showed complex behaviour indicative of the presence of two redox components. In the case of the P. stutzeri cytochrome c4, two spectroscopically distinct components were present during the redox titration. In contrast, cytochrome c-554(548) from a halophilic Paracoccus species is a stable dimer of a monohaem cytochrome which shows close homology to cytochrome c4, but does not show complexity in its redox titration. The presence of chemically distinct haem environments or anti-cooperative interactions between identical haem groups are two possible explanations for the redox complexity of cytochrome c4. The simple redox titration of cytochrome c-554(548) shows that haems situated relatively close together need not interact, but direct cleavage, separation and study of the domains will be necessary to decide whether they do or do not interact in the case of cytochrome c4.

Structural basis for the variation of pH-dependent redox potentials of Pseudomonas cytochromes c-551.

The redox potentials of many c-type cytochromes vary with pH over the physiological pH range. We have investigated the pH dependence of redox potential for the four homologous cytochromes c-551 from Pseudomonas aeruginosa, Pseudomonas stutzeri strain 221, Pseudomonas stutzeri strain 224, and Pseudomonas mendocina . The pH dependence is due to an ionizable group that ionizes with pKox in ferricytochrome c-551 but with a higher pK, pKred , in ferrocytochrome c-551. For P. aeruginosa cytochrome c-551 it has been shown that this ionizable group is one of the heme propionic acid substituents [Moore, G. R., Pettigrew , G. W., Pitt , R. C., & Williams, R. J. P. (1980) Biochim. Biophys. Acta 590, 261-271]but the values of pKox and pKred are significantly lower in this protein than in the other three cytochromes. NMR and chemical modification studies show that for the two P. stutzeri cytochromes c-551 and P. mendocina cytochrome c-551, this propionic acid substituent is again important for the pH dependence of the redox potential. However, a histidine occurring at position 47 in their sequences hydrogen bonds to the propionic acid and thereby raises its pK. In P. aeruginosa cytochrome c-551, His-47 is substituted by Arg-47. Hydrogen-bonding schemes involving His-47 and the propionic acid are proposed.

The effect of iron-hexacyanide binding on the determination of redox potentials of cytochromes and copper proteins.

The midpoint redox potentials of Pseudomonas aeruginosa cytochrome c-551 and Rhodopseudomonas viridis cytochrome c2 were measured as a function of pH in the presence of Euglena cytochrome c-558 and the results compared with those obtained in the presence of ferro-ferricyanide. The pattern of pH dependence observed for the two bacterial cytochromes was the same whether it was measured by equilibrium with another redox protein or with the inorganic redox couple. Thus, the pH dependence of redox potential is not a consequence of pH-dependent ligand binding. The midpoint potential of Ps. aeruginosa azurin was measured as a function of pH using both ferro-ferricyanide mixtures and redox equilibrium with horse cytochrome c or Rhodopseudomonas capsulata cytochrome c2. In this case also the pattern of pH dependence obtained did not vary with the redox system used and it closely resembled that of Ps. aeruginosa cytochrome c-551. This is consistent with the observation that the equilibrium between cytochrome c-551 and azurin is relatively independent of pH. An equation was derived which described ph-dependent ligand binding and which can produce theoretical curves to fit the experimental pH dependence of redox potential for both cytochrome and azurin. However, the pronounced effect on such curves produced by varying the ligand association constants, and the insensitivity of the experimental data to changes in ionic strength, suggest that ligand binding effects do not account for the pH dependence of redox potential.