Point-of-use water filtration reduces healthcare-associated infections in bone marrow transplant recipients.

Outbreaks of infection with gram-negative bacteria (GNB) have been linked to hospital water. We sought to determine whether point-of-use (POU) water filtration might result in decreased risk of infection in hospitalized bone marrow transplant (BMT) recipients in the absence of any recognized outbreak. Unfiltered water was sampled from taps in the BMT unit of a major US teaching hospital, and cultured at a reference laboratory. POU bacterial-retentive filters (0.2 mum) were installed throughout the unit, and replaced every 14 days. Infection rates were tracked over a 9-month period, and compared with rates for a 16-month period before POU filtration. Unfiltered water samples from 50% (2 of 4) outlets sampled grew P. aeruginosa (2 of 4) and Stenotrophomonas maltophilia (1 of 4). Clinical infection rates in the unit were significantly reduced from 1.4 total and 0.4 GNB infections per 100 patient days in the period before POU filtration to 0.18 total and 0.09 GNB infections per 100 patient days (P=0.0068 and 0.0431, respectively) in the 9-month period for which filters were in place. Infections during the POU filtration period were due to non-waterborne organisms. Point-of-use (POU) water filtration may significantly reduce infection rates in BMT recipients in the absence of any recognized outbreak.

Asymptomatic rupture of a rudimentary uterine horn.

Pregnancy in a noncommunicating rudimentary uterine horn frequently results in rupture. Uterine rupture may cause a massive intraperitoneal hemorrhage, necessitating operative intervention to control bleeding. The following is a report of an unusual patient who had an asymptomatic rupture of a rudimentary horn.

Use of bacteriophages as surrogates for mammalian viruses.

The threat of viral contamination is common to all processes using biological products of animal or human origin. Therefore, demonstration of virus clearance (i.e. validation of virus removal and/or inactivation steps) is of utmost importance to the biopharmaceutical industries. Ultimately, virus clearance studies should show that any virus removal/inactivation stage incorporated into the manufacturing process not only removes or inactivates known viruses that may be conceivably present (e.g. from cell banks and source materials), but also other viruses that may be introduced adventitiously (e.g. by addition of supplements downstream of the manufacturing process). In this paper, we outline the shared properties of mammalian viruses and similar sized bacteriophages, and factors that may influence the virus clearance process. We also present test data from filtration studies, showing similar titre reductions for both types of virus. We propose that well-characterised bacteriophage, such as PP7 and PR772 can be used as models for mammalian viruses if the virus removal mechanism is based on size exclusion.

Stability and solubility engineering of the EphB4 tyrosine kinase catalytic domain using a rationally designed synthetic library.

The inability to generate soluble, correctly folded recombinant protein is often a barrier to successful structural and functional studies. Access to affordable synthetic genes has, however, made it possible to design, make and test many more variants of a target protein to identify suitable constructs. We have used rational design and gene synthesis to create a controlled randomised library of the EphB4 receptor tyrosine kinase, with the aim of obtaining soluble, purifiable and active catalytic domain material at multi-milligram levels in Escherichia coli. Three main parameters were tested in designing the library--construct length, functional mutations and stability grafting. These variables were combined to generate a total of 9720 possible variants. The screening of 480 clones generated a 3% hit rate, with a purifiable solubility of up to 15 mg/L for some EphB4 constructs that was largely independent of construct length. Sequencing of the positive clones revealed a pair of hydrophobic core mutations that were key to obtaining soluble material. A minimal kinase domain construct containing these two mutations exhibited a +4.5°C increase in thermal stability over the wild-type protein. These approaches will be broadly applicable for solubility engineering of many different protein target classes. Atomic coordinates and structural factors have been deposited in PDB under the accession 2yn8 (EphB4 HP + staurosporine).

Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis.

The gene for an enzyme with single or dual specificity on complex carbohydrates has been transferred from its native host (Thermoanaerobium brockii), a thermophilic anaerobe, into Escherichia coli and Bacillus subtilis. Most of the gene coding region is in a 2.2-kilobase PstI fragment that is common to the E. coli and B. subtilis chimeric vectors pCPC902 and pCPC903, respectively. Although the T. brockii debranching enzyme secreted from B. subtilis was unglycosylated and had less thermostability, more enzyme was secreted from B. subtilis (0.80 to 1.0 U/ml) than from T. brockii (0.23 U/ml). E. coli did not export any measurable enzyme. From the fermentation broth of B. subtilis containing pCPC903, three active species of the debranching enzyme were separated; two species are possibly protease digestion products of the larger protein (105,000 molecular weight). Whereas the enzyme can cleave all of the alpha-1----6 glucosidic linkages (and none of the alpha-1----4 bonds) in pullulan, it hydrolyzed mostly alpha-1----4 and very few of the alpha-1----6 linkages in starch. Upon hydrolysis of pullulan by the enzyme, only maltotriose was produced, while starch was digested to various-sized oligomers.

Interactions between neutral phospholipid bilayer membranes.

We have obtained force vs. separation relations between bilayers in 10 different phospholipid preparations: dilauroyl-dimyristoyl-, dipalmitoyl-, distearoyl-, or dioleoylphosphatidylcholine (PC); egg phosphatidylethanolamine; cholesterol-containing bilayers of dipalmitoyl PC and of egg PC. The chemical potential of water in the multilamellar lattice is determined at all water contents and changes continuously with bilayer separation; no discrete classes of water are observed. The interbilayer van der Waals force is estimated from the balance of forces at the bilayer separation where the multilayer lattice is in equilibrium with pure water. Although quantitative differences are evident for different phospholipids, all force curves but one show a clear, exponentially decaying "hydration "repulsion" whose decay distance is 2-3 A . Estimates of forces between bilayer vesicles show great sensitivity to the identity of the phospholipid polar group and to the packing of the hydrocarbon acyl chains. On major implication of this is the likelihood of local structural changes and lipid segregation in the area of closest approach of interacting vesicles of mixed phospholipids.

Measurement of the lateral compressibility of several phospholipid bilayers.

Lateral compressibilities of bilayers in multilayer lattices are given for 10 phospholipid preparation:dilauryl-, dimyristoyl-, dipalmitoyl-, distearoyl-, and dioleoylphosphatidylcholine (PC); egg phosphatidylethanolamine (PE); as well as cholesterol-containing bilayers of dipalmitoyl PC or of egg PC. Bilayer deformability is highly nonlinear and does not permit description in terms of a simple modulus. The presence of cholesterol or C=C bonds (dioleoyl PC) increases deformability, but freezing of acyl chains does not cause dramatic stiffening of the bilayer. Lateral compression of dilauryl PC an dimyristoyl PC causes a transition from "melted" to "frozen" acyl chains above the normal transition temperatures. Our measurements do not correspond in any obvious way to lateral compressibilities in monolayers at the air-water interface.

NMR analysis of the N-terminal SRCR domain of human CD5: engineering of a glycoprotein for superior characteristics in NMR experiments.

CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily for which the three-dimensional polypeptide fold is as yet unknown. Glycosylated CD5 domain 1 (CD5d1) has been obtained by expression by secretion from both Chinese hamster ovary (CHO) cells and Pichia pastoris. Recombinant CD5d1 expressed in this manner was shown to be correctly folded by binding to anti-CD5 L17F12/Leu1 monoclonal antibody. Preliminary nuclear magnetic resonance (NMR) spectra obtained for CD5d1 (residues 1-118) had spectral dispersion typical of a folded protein, but otherwise of such poor quality that NMR structural studies were not feasible. The analysis of glycoproteins by NMR is frustrated by sample heterogeneity and poor spectral quality associated with glycan resonance overlap and the potential for increased line-widths due to the large hydrodynamic volume. In order to pursue NMR structural studies of CD5d1 it was necessary to optimize the quality of NMR spectra of CD5d1. A range of constructs of varying length and carbohydrate content were expressed in CHO cells and in P. pastoris. In addition the P. pastoris CD5d1 proved susceptible to N-glycan cleavage with endoglycosidase H. The protein products were characterised using size exclusion chromatography, NMR measurement of translational self-diffusion coefficients and two-dimensional 1H nuclear Overhauser effect spectroscopy experiments. Removal of an eight residue O-glycosylated C-terminal peptide, in particular, resulted in significant improvements in the quality of the CD5d1 NMR data, while retaining native protein structure. Two-dimensional heteronuclear NMR spectroscopy of nitrogen-15 isotope labelled deglycosylated CD5d1 (residues 1-110) prepared from P. pastoris suggests that this protein product is now amenable to solution structure determination.

Determination of pK(a) values of carboxyl groups in the N-terminal domain of rat CD2: anomalous pK(a) of a glutamate on the ligand-binding surface.

The ligand-binding surface of the T-lymphocyte glycoprotein CD2 has an unusually high proportion of charged residues, and ionic interactions are thought to play a significant role in defining the ligand specificity and binding affinity of CD2 with the structurally homologous ligands CD48 (in rodents) and CD58 (in humans). The determination of the electrostatic properties of these proteins can therefore contribute to our understanding of structure-activity relationships for these adhesion complexes that underpin T-cell adhesion to antigen-presenting cells. In this study, we investigated the pH titration behavior of the carboxyl groups of the N-terminal domain of rat CD2 (CD2d1) using the chemical shifts of backbone amide nitrogen-15 ((15)N) and proton NMR resonances, and carboxyl carbon-13 ((13)C) signals. The analysis revealed the presence of a glutamate (Glu41) on the binding surface of rat CD2 with an unusually elevated acidity constant (pK(a) = 6.73) for CD2d1 samples at 1.2 mM concentration. pH titration of CD2d1 at low protein concentration (0.1 mM) resulted in a slight decrease of the measured pK(a) of Glu41 to 6.36. The ionization of Glu41 exhibited reciprocal interactions with a second glutamate (Glu29) in a neighboring location, with both residues demonstrating characteristic biphasic titration behavior of the carboxyl (13)C resonances. Measurements at pH 5.5 of the two-bond deuterium isotope shift for the (13)C carboxyl resonances for Glu41 and Glu29 [(2)DeltaC(delta)(O(epsilon)D) = 0.2 and 0.1 ppm, respectively] were consistent with the assignment of the anomalous pK(a) to Glu41, under the strong influence of Glu29. The characterization of single site mutations of CD2d1 residues Glu41 and Glu29 to glutamine confirmed the anomalous pK(a) for Glu41, and indicated that electrostatic interaction with the Glu29 side chain is a significant contributing influence for this behavior in the wild-type protein. The implications of these observations are discussed with respect to recent structural and functional analyses of the interaction of rat CD2 with CD48. In particular, CD2 Glu41 must be a candidate residue to explain the previously reported strong pH dependence of binding of these two proteins in vitro.

The effect of cholesterol on measured interaction and compressibility of dipalmitoylphosphatidylcholine bilayers.

We have examined the phase diagram of dipalmitoylphosphatidylcholine (DPPC)--cholesterol-water mixtures at low cholesterol content, and report phase separation between 3 and 10 mol% cholesterol. The two lamellar phases at equilibrium in this region appear to be pure DPPC and 11 mol% cholesterol in DPPC. For these two lamellar phases, which are made up of alternating layers of water and bimolecular lipid leaflets, we have measured the forces of interaction between leaflets and the lateral pressure and compressibility of the leaflets. Both bilayers experience a strong repulsive force when forced together only a few ångströms (1 A = 0.1 nm) closer than their maximum separation in excess water. However, the presence of 11 mol% cholesterol causes the bilayers to move apart of 35-A separation from the 19-A characteristic of pure DPPC in excess water. This swelling may result from a decrease in van der Waals attraction between bilayers or from an increase in bilayer repulsion. Differences in bilayer interaction can be a cause for phase separation. More importantly these differences can cause changes in the composition of regions of membranes approaching contact. At 11 mol%, cholesterol substantially increases the lateral compressibility of DPPC bilayers leading to higher lateral density fluctuations and potentially higher bilayer permeability.

Structural insights into the hydrolysis of cellular nitric oxide synthase inhibitors by dimethylarginine dimethylaminohydrolase.

Nitric oxide synthase is inhibited by asymmetric NG-methylated derivatives of arginine whose cellular levels are controlled in part by dimethylarginine dimethylaminohydrolase (DDAH, EC 3.5.3.18). Levels of asymmetric NG,NG-dimethylarginine (ADMA) are known to correlate with certain disease states. Here, the first structure of a DDAH shows an unexpected similarity to arginine:glycine amidinotransferase (EC 2.1.4.1) and arginine deiminase (EC 3.5.3.6), thus defining a superfamily of arginine-modifying enzymes. The identification of a Cys-His-Glu catalytic triad and the structures of a Cys to Ser point mutant bound to both substrate and product suggest a reaction mechanism. Comparison of the ADMA-DDAH and arginine-amidinotransferase complexes reveals a dramatic rotation of the substrate that effectively maintains the orientation of the scissile bond of the substrate with respect to the catalytic residues. The DDAH structure will form a basis for the rational design of selective inhibitors, which are of potential use in modulating NO synthase activity in pathological settings.

Survival and nutritional requirements of three bacteria isolated from ultrapure water.

Bacteria isolated previously from ultrapure water (UPW) systems were examined for their ability to survive in UPW, with the ultimate goal of elucidating potential carbon and energy sources for the bacteria. Two strains of Ralstonia pickettii isolated from different areas within the UPW system (pretreatment and polishing loop, and referred to as strains 3A1 and MF254A, respectively) and a strain of Bradyrhizobium sp. were compared to increase our understanding of the fundamental behavior of bacteria contaminating UPW. R. pickettii (3A1) grew significantly slower in R2A medium, with a final cell yield much lower than the isolate from the polishing loop. In addition, R. pickettii MF254A showed a broader substrate range than either strain 3A1 or Bradyrhizobium sp. In UPW, there appears to be a threshold cell concentration (approximately 10(6) colony-forming units/ml), whereby the cell numbers remain constant for a prolonged period of 6 months or more. Below this concentration, rapid proliferation is observed until the threshold concentration is attained. Preliminary experiments suggested that nitrogen gas (frequently added to UPW storage tanks) may contribute to growth of Bradyrhizobium sp. Above the threshold concentration, the strain of Ralstonia sp. isolated from the polishing loop was capable of cryptic growth with heat-killed cells in UPW. However, cryptic growth was not observed when the cells supplied as nutrients were killed using UV254 light. Furthermore, cryptic growth did not appear to contribute significantly to proliferation of Bradyrhizobium sp. or Ralstonia sp. 3A1 (isolated from the pretreatment loop). We believe that cryptic growth may aid survival of the bacteria in UPW, but further experiments are warranted to prove this phenomenon conclusively.

Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2.

Resolution of Holliday junctions into separate DNA duplexes requires enzymatic cleavage of an equivalent strand from each contributing duplex at or close to the point of strand exchange. Diverse Holliday junction-resolving enzymes have been identified in bacteria, bacteriophages, archaea and pox viruses, but the only eukaryotic examples identified so far are those from fungal mitochondria. We have now determined the crystal structure of Ydc2 (also known as SpCce1), a Holliday junction resolvase from the fission yeast Schizosaccharomyces pombe that is involved in the maintenance of mitochondrial DNA. This first structure of a eukaryotic Holliday junction resolvase confirms a distant evolutionary relationship to the bacterial RuvC family, but reveals structural features which are unique to the eukaryotic enzymes. Detailed analysis of the dimeric structure suggests mechanisms for junction isomerization and communication between the two active sites, and together with site-directed mutagenesis identifies residues involved in catalysis.

NMR analysis of interacting soluble forms of the cell-cell recognition molecules CD2 and CD48.

The T cell glycoprotein, CD2, is one of the best characterized molecules mediating recognition at the cell surface. The ligands of murine and human CD2 are CD48 and CD58, respectively, and interactions between these molecules have been shown to influence antigen recognition and T cell activation. The CD58 binding site of human CD2 has been characterized in mutational studies, and here we use heteronuclear NMR spectroscopy to identify the rat CD48 binding site of the N-terminal domain of rat CD2 (CD2d1). The NMR spectrum of bacterially expressed CD2d1, assigned initially at pH 4.3 in the course of determining the three-dimensional solution structure of this domain [Driscoll, P.C., et al. (1991) Nature 353, 762-765], has been reassigned as a two-dimensional 15N-1H heteronuclear single-quantum coherence (HSQC) spectrum at neutral pH. The CD48 binding surface was identified by monitoring perturbations in the line widths and chemical shifts of cross peaks in the HSQC spectrum of CD2d1 during titrations with a soluble form of CD48 expressed in Chinese hamster ovary cells. This first solution NMR analysis of interacting cell surface molecules shows that the ligand binding site extends across an area of ca. 700-800 A2 of the GFCC'C" face corresponding almost exactly to lattice contacts in crystals of soluble CD2 first proposed as a model of the interaction of CD2 with its ligands. The analysis finds no evidence for any large-scale structural changes in domain 1 of CD2 to accompany CD48 binding. Comparisons of the human and rat CD2 ligand binding sites suggest that species- and ligand-specific binding may be determined by as few as three amino acid residues, corresponding to Thr37, Leu38, and Glu41 in rat CD2 (Lys42, Lys43, and Gln46 in human CD2).

Structural analysis of the CD5 antigen--expression, disulphide bond analysis and physical characterisation of CD5 scavenger receptor superfamily domain 1.

CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily, for which no three-dimensional structure has been obtained. Recombinant soluble CD5 domain 1 (CD5d1), the N-terminal SRCR domain, has been expressed in both chinese hamster ovary (CHO) cells and Pichia pastoris. CD5d1 was shown to be correctly folded by binding to the CD5 monoclonal antibody Leul. Circular dichroism and NMR analyses indicate that CD5d1 has a high beta-sheet content. CD5d1 from both CHO cells and P. pastoris have very similar properties. The disulphide bonding pattern was determined and is consistent with that found for the group-A SRCR domain of type-1 macrophage scavenger receptor and MARCO, the macrophage receptor with collagenous structure. Observations have been made of the role of glycosylation of CD5. P. pastoris expression provides large quantities of correctly folded recombinant CD5d1 for multidimensional NMR and for X-ray crystallographic studies. The whole extracellular region of CD5, expressed as a chimaera with rat CD4 domains 3 and 4 (cCD5d1-3-CD4d3+4), was studied by electron microscopy and carbohydrate analysis to gain an overview of the structure of the extracellular portion of intact CD5. Carbohydrate analysis identified N-linked glycans on CD5 domains 1 and 2, and sialylated O-linked glycans on the linker peptide between domains 1 and 2. Electron microscopy and carbohydrate analysis together suggest that the extracellular region of CD5 forms a rod-like structure with domain 1 distal from the cell surface and separated from domains 2 and 3 by an O-glycosylated peptide linker region.

Oligosaccharide analysis and molecular modeling of soluble forms of glycoproteins belonging to the Ly-6, scavenger receptor, and immunoglobulin superfamilies expressed in Chinese hamster ovary cells.

Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specific glycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached.

Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis.

cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/L-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and D-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4x10(6) M(-1) x s(-1)) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1x10(4) M(-1) x s(-1)) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of L-dopachrome compared with non-esterified L-dopachrome (>87000-fold) and a high kcat (approximately 4x10(4) s(-1). The crystal structure, determined to 1.65 A (1 A=0.1 nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5 ng/ml-5 pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500 ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6 ng/ml (55 ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is partially orthologous to mammalian MIF.