Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris

A stable GS115 Pichia pastoris recombinant strain was constructed to secrete a truncated form of the human alpha(1,3/4) fucosyltransferase (amino acids 45-361). Enzyme production resulted from a secretory pathway based on the pre-pro- alpha mating factor signal sequence of the yeast Saccharomyces cerevisiae . Following its transit through the Golgi apparatus, the enzyme accumulated in the periplasmic space before its release in the culture broth (about 30 mg/l). Cell-enclosed enzyme ( approximately 0.16%) proved to be fairly stable for many freezing and thawing cycles and could be used several times as an immobilized catalyst. Soluble enzyme (>99.8%) representing the main protein of the culture broth (10%) has been characterized by Western-blotting, substrate specificities and kinetic parameters. The two forms (cell-enclosed and soluble) of recombinant enzyme may be used for in vitro synthesis of Lewisadeterminants.     

Computer simulation of expansions of DNA triplet repeats in the fragile X syndrome and Huntington''s disease

The use of high capacity micron-sized non-porous magnetic metal chelator adsorbents for the direct recovery of a recombinant metal-binding protein from crude liquors is described. Selectivity and interaction strength of magnetic chelator particles were assessed using a set of native proteins with known behaviour towards commercially available immobilised metal chelate adsorbents. Particles charged with Cu2+ were highly effective in recovering a recombinant histidine-tailed T4 lysozyme fusion protein directly from crude E. coli extracts in a single step. Levels of recovery and purity were high and compared favourably with those achieved by chromatography of pre-clarified extracts on Cu(2+)-IDA Sepharose. The magnetic approach offers advantages such as the avoidance of clarification to prevent fouling of chromatography columns, steps that become especially significant at large scale. By detailed characterisation of the magnetic chelators the practical use of tailed T4 lysozyme for repeated production of periplasmic products is a realistic prospect.     

Mediators of change in physical activity following an intervention in primary care: PACE

Lysozyme is able to lyse Gram-positive bacteria acting as muramidase on the peptidoglycan polymer. Gram-negative bacteria in vitro are not lysed by lysozyme. It was assumed that the peptido-glycan is protected by the outer membrane and thus that Gram-negative bacteria are not affected by lysozyme without the aid of other factors such as EDTA or complement which enable lysozyme to penetrate the outer membrane. Accidentally, Pellegrini et al. [(1992) J. Appl. Bacteriol., 72:180-187] found that lysozyme per se is able to kill some Gram-negative bacteria. On the basis of morphological and immunocytochemical findings obtained from chemically fixed bacteria, it was concluded that lysozyme does not lyse Gram-negative bacteria but affects the cytoplasm of for example, Escherichia coli, leading to its disintegration, whilst the membranes do not break down. In an attempt to clarify the action of lysozyme on E. coli, we employed cryotechniques including ultrarapid freezing, cryomicroscopy and freeze substitution, and immunolabeling. Bacteria that were immediately frozen after exposure to lysozyme remained morphologically intact. Individual bacteria plated on agar after exposure to lysozyme were mostly intact when frozen within a few seconds. However, inner and outer membranes of 80% of the bacteria were disrupted, whereas the cytoplasm of only a few bacteria showed signs of disintegration when bacteria were frozen with a delay of only 5 min of plating onto pure agar or agar containing growth medium. After a period of time of 15 min between plating onto agar and freezing, about 97% of the bacteria showed changes of disintegration of various extent. Immunolabeling showed that lysozyme binds to the outer cell membrane and may penetrate the membrane, reaching the periplasmic space and possibly the inner cell membrane. The ultrastructural findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E. coli but is not able to induce disintegration. Disintegration is accomplished by changes of the environment starting at the cell membranes. The mechanism by which lysozyme penetrates the membrane, the way it acts to be bactericidal, and the way disintegration is initiated remain to be clarified.     

Characterization of H. parasuis periplasmic nucleotide pyrophosphatase as a potential target enzyme for inhibition of growth

The periplasmic nucleotide pyrophosphatase from Haemophilus parasuis was purified 750-fold to electrophoretic homogeneity through salt fractionation and ion-exchange and affinity chromatography. The purified enzyme was monomeric with an apparent M(r) of 70,000 and catalyzed the hydrolysis of the pyrophosphate bond of NAD to yield NMN and AMP as products. The enzyme exhibited negative cooperativity in the hydrolysis of a number of pyridine dinucleotides and structurally-related pyrophosphate compounds as indicated by biphasic double-reciprocal plots and Hill coefficients of 0.5. The kinetic parameters, K(m) and Vm, determined titrimetrically and analyzed through computer programs, were used to compare the relative effectiveness of dinucleotides containing nitrogen bases other than nicotinamide or adenine to that of NAD. Effective substrate-competitive inhibition of the pyrophosphatase was observed with purine and pyrimidine nucleoside diphosphates in the low micromolar concentration range. Although less effective, N1-alkylnicotinamide chlorides also inhibited competitively with respect to the substrate, NAD. In addition to being an effective inhibitor of the purified enzyme, adenosine diphosphate also inhibited growth of H. parasuis at a low micromolar concentration. This inhibition of growth correlates well with inhibition of the periplasmic pyrophosphatase which is supported by the fact that adenosine diphosphate does not effectively inhibit growth when the pyrophosphatase is by-passed by growth on nicotinamide mononucleotide. These observations are all consistent with the periplasmic nucleotide pyrophosphatase being essential for the growth of the organism on NAD and therefore, a very important enzyme with respect to the pathogenesis of the organism. 3-Aminopyridine mononucleotide, which also inhibited growth of H. parasuis at a low micromolar concentration, did not effectively inhibit the purified pyrophosphatase and a different target enzyme needs to be considered to explain growth inhibition by this derivative.     

Neurodermatitis and psyche

Intracellular lysozyme concentration was measured in neutrophilic granulocytes from 25 patients with multiple myeloma. At diagnosis intraneutrophil lysozyme activity was significantly reduced (mean reduction 50%). During clinical remission after 1-4 months of intensive chemotherapy values were normalized. In 18 cases studied at various stages of the disease from 6 to 70 months after diagnosis there was a significant negative correlation between the duration of the disease and neutrophil lysozyme concentration. The decrease in neutrophil lysozyme concentration was significantly correlated to clinical disease activity and the percentage of plasma cells in bone marrow aspirates, whereas there was no correlation between the concentration of M-protein in serum and the neutrophil lysozyme concentration. Plasma lysozyme concentration was normal. In contrast, neutrophil lysozyme concentration was normal in 18 patients with stage III-IV malignant lymphoma. Plasma lysozyme in this group was significantly higher than normal. The difference in neutrophil lysozyme patterns between multiple myeloma and malignant lymphoma supports the hypothesis that the defect in neutrophil maturation seen in malignant blood disorders is directly related to the infiltration of the bone marrow by pathologic cells.     

Characterization of protein release through glucose-sensitive hydrogel membranes

Glucose-sensitive phase-reversible hydrogels have been prepared based on the specific interaction between polymer-bound glucose and concanavalin A (Con-A). The main goal of this study was to characterize the release of model proteins (insulin and lysozyme) through the hydrogel membrane as the free glucose concentration in the environment was changed. The diffusion of the model proteins through the hydrogel membrane was examined using a diffusion cell. Porous poly(hydroxyethyl methacrylate) (PHEMA) membranes were used to sandwich the mixture of glucose-containing polymers and Con-A in between the donor and receptor chambers. The porous PHEMA membranes allowed diffusion of glucose, insulin and lysozyme, while preventing loss of glucose-containing polymers and Con-A in the sol state. The release rate of model proteins through the glucose-sensitive hydrogel membrane was dependent on the concentration of free glucose. The release rate of the proteins did not remain constant, however, due to the change in free glucose concentration resulting from diffusion of glucose from the receptor chamber to the donor chamber. This study demonstrated the possibility that the glucose-sensitive phase-reversible hydrogels can be used to regulate the insulin release as a function of the free glucose concentration in the environment.     

Molecular adaptation of Drosophila melanogaster lysozymes to a digestive function

A lysozyme (pI 5.5) was purified to homogeneity from heated acid extracts of Drosophila melanogaster larvae, using gel filtration in a Superose column and ion-exchange chromatography in a Mono Q column. The final yield was 67%. The purified lysozyme with Mr 13,700 (determined by SDS-polyacrylamide gel electrophoresis) decreases in activity and has its pH optimum displaced towards acidic values and Km increases as the ionic strength of the medium becomes higher. The lysozyme is resistant to a cathepsin D-like proteinase present in cyclorrhaphous Diptera and displays a chitinase activity which is 11-fold higher than that of chicken lysozyme. Microsequencing of an internal peptide of the purified lysozyme showed that this enzyme is the product of the previously sequenced Lys D gene. The results suggest that the product of the Lys P gene has pI 7.2, a pH optimum around 5 and is not a true digestive enzyme. The most remarkable sequence convergence of D. melanogaster lysozyme D and lysozymes from vertebrate foregut fermenters are serine 104 and a decrease in the number of basic amino acids, suggesting that these features are necessary for digestive function in an acid environment. Adaptive residues putatively conferring stability in an acid proteolytic environment differ between insects and vertebrates, probably because they depend on the overall three-dimensional structure of the lysozymes. A maximum likelihood phylogeny and inferences from insect lysozyme sequences showed that the recruitment of lysozymes as digestive enzymes is an ancestral condition of the flies (Diptera: Cyclorrhapha).     

EXTRACTION OF GOLD AND SILVER FROM TURKISH GOLD ORE BY AMMONIACAL THIOSULPHATE LEACHING

Although cyanide is a widespread leaching reagent for the recovery of precious metals, there still are continuing investigations on alternative processes due to the related environmental problems concerned. Thiosulphate leaching of precious metals is one of the processes that were developed as an alternative and nontoxic technique to conventional cyanidation. The aim of this experimental study was to investigate the possibilities of leaching gold and silver in ammoniacal thiosulphate solutions on a laboratory scale. Samples used in the experiments were taken from Ovac?k Gold Mine, located in the Bergama region of Turkey. The influence of temperature, copper sulphate concentration, ammonia concentration, thiosulphate concentration, sodium sulfite concentration, and solid–liquid ratio on gold and silver leaching recoveries was investigated and optimum leaching conditions were determined. At these conditions, 99.57% Au and 95.87% Ag leaching recoveries were achieved.     

Intracellular accumulation, subcellular distribution, and efflux of tilmicosin in chicken phagocytes

Tilmicosin is a semi-synthetic macrolide antibiotic, currently approved for veterinary use in cattle and swine respiratory disease, and is in development for use in poultry mycoplasma air sacculitis. In order to provide an understanding of clinical efficacy, the in vitro interaction of tilmicosin with three types of chicken phagocytes (MQ-NCSU macrophages, monocyte-macrophages, and heterophils) was evaluated. After incubation with radiolabeled tilmicosin, uptake was determined and expressed as the ratio of the cellular (Cc) to the extracellular (Ce) drug concentration (Cc:Ce). Tilmicosin was avidly accumulated by heterophils (Cc: Ce 138 at 4 h incubation vs 32 and 66, respectively, in MQ-NCSU and monocyte-macrophages) with 61 to 88% localized in the lysosomes. Uptake was dependent on cell viability, temperature, and pH, but was not influenced by metabolic inhibitors. However, phagocytosis of Pasteurella multocida and lipopolysaccharide exposure increased tilmicosin uptake by the chicken phagocytes. Upon removal of extracellular tilmicosin, 50% of the intracellular tilmicosin was effluxed within the first 30 min, but after 4 h of incubation in antibiotic-free medium, 30% remained cell-associated. Opsonized P. multocida significantly enhanced the release of tilmicosin from all three types of chicken phagocytes. Tilmicosin uptake was observed to increase lysosomal enzyme (acid phosphatase, lysozyme, avidin, and beta-glucuronidase) production. Finally, neutrophils were shown to transport and efflux bioactive tilmicosin in a test system measuring both neutrophil chemotaxis under agarose and a bioassay measuring inhibition of bacterial growth in the presence of antibiotic in agar. These in vitro observations of cellular pharmacology suggest a complex interaction between phagocytes and tilmicosin that contribute to clinical efficacy.     

Evidence that spontaneous situs inversus in cultured neural plate staged rat embryos is additive with and not mediated through adrenergic mechanisms

Immunoadsorbents comprising Fv fragments specific for hen egg lysozyme were used to recover the enzyme from a 20-fold excess of bovine albumin. We designed automatic equipment to run this model purification system for 100 cycles non-stop and monitored the deterioration of the immunoadsorbents during the cycling procedure. Only minor losses (approximately 25%) in the immunoadsorbents' capacity were detected; this correlated well with ligand loss (measured by enzyme-linked immunosorbent assay) which was approximately 0.2% per cycle. A surprising finding was that the use of "single-chain" Fv fragments conferred only a minor advantage with respect to stability of the immunoadsorbents.     

Noncovalent polycationic coatings for capillaries in capillary electrophoresis of proteins

The adsorption of proteins with net positive charges (pI > pH) on the walls of fused-silica capillaries is a common problem in the analysis of proteins by capillary electrophoresis. This paper explores the use of polycationic polymers as noncovalent coatings to limit this problem. The behavior of three sets of proteins was compared using uncoated and coated capillaries: (i) a protein charge ladder obtained by acetylation of lysozyme (EC 3.2.1.17); (ii) a protein charge ladder obtained by acetylation of carbonic anhydrase II (EC 4.2.1.1); (iii) a test panel of proteins with a range of values of molecular weight and pI. Four polycationic polymers were examined: polyethylenimine (PEI; MWav = 15000), Polybrene (MWav = 25000), poly(methoxyethoxyethyl)ethylenimine (MWav = 64000), and poly(diallyldimethylammonium chloride) (MWav = 10000). Detection of proteins with high pI was readily achieved using the first three of these polycationic polymer coatings but not with the poly(diallyldimethyl-ammonium chloride). Examination of the stability of these coatings indicates that they are robust: the change in electroosmotic flow was less than 10% for 25 replications of the same separations, using capillaries coated with PEI or Polybrene. This study demonstrates that the charge ladder obtained by acetylation of lysozyme is a good model with which to test the efficiency of polycationic coatings. A study of the electrophoretic mobilities of the members of this charge ladder at pH 8.3 determined the effective charge of lysozyme (Zp(0) = +7.6 +/- 0.1) and established the acidity of the alpha-ammonium group of lysozyme (pKa = 7.8 +/- 0.1). Results from the test panel of proteins suggest that protein adsorption is mainly driven by electrostatic interactions.     

The effect of mammary gland expression of human lysozyme on the properties of milk from transgenic mice

Transgenic mice were used as model systems to evaluate the impact of human lysozyme expression in the mammary gland. We previously generated two lines of transgenic mice that express human lysozyme mRNA in the mammary gland under the tissue-specific and developmentally correct control of the bovine gene promoter for alpha s1-casein. Concentrations of human lysozyme protein in milk of transgenic mice varied from .25 to .71 micrograms/microliters of milk. Human lysozyme secreted into mouse milk retained its antimicrobial activity, as determined by a denaturing polyacrylamide gel activity assay. The physical and functional properties of the milk were also altered, because mouse milk containing human lysozyme had a 35% decrease in rennet clotting time, a smaller median micelle size (157 nm vs. 172 nm), and a 2.5- to 3-fold greater gel strength than control milk. From these results, we conclude that the use of transgenic animals producing lysozyme in the milk is feasible and potentially useful to the dairy industry.     

Biogenesis and topology of integral membrane proteins: characterization of lactose permease-chloramphenicol acetyltransferase hybrids

Use of beta-lactamase in gene fusions to study membrane protein topology permits exploitation of its biological activity to select for positive (external) hybrids on ampicillin agar plates. When the enzyme is attached to cytoplasmic loops of a membrane protein, it is not secreted and is therefore unable to confer ampicillin resistance. In this study, we examine the use of the cytoplasmic enzyme chloramphenicol acetyltransferase (Cat) as a complement to the use of periplasmic beta-lactamase, in gene fusion studies. This enzyme is responsible for chloramphenicol resistance in Escherichia coli. We show that Cat confers substantial antibiotic resistance when fused to cytoplasmic loops of lactose permease. As expected, periplasmically exposed Cat is enzymatically active in vitro but unable to confer significant chloramphenicol resistance, presumably because of the absence of acetylcoenzyme A in the periplasm. Therefore, Cat may serve as a topogenic sensor in gene fusion studies. The new Cat fusion approach is discussed with regard to its potential use for selecting E. coli mutants which are defective in the assembly of membrane proteins.     

Biophysical characterisation of lysozyme binding to LPS Re and lipid A

The binding of lysozyme to bacterial deep rough mutant lipopolysaccharide (LPS) Re and to its lipid moiety lipid A, the 'endotoxic principle' of LPS, was investigated using biophysical techniques. The beta<-->alpha gel to liquid crystalline phase transition, the nature of the functional groups of the endotoxins, the secondary structure of lysozyme, and competition with polymyxin B were studied by Fourier-transform infrared spectroscopy (FTIR); the supramolecular aggregate structure of the endotoxins was determined with synchrotron radiation X-ray diffraction and the binding stoichiometry with microcalorimetry. The results were compared with those found with zwitterionic and negatively charged phospholipids. It can clearly be shown that lysozyme binds electrostatically to charged groups of the endotoxin molecules with the consequence of acyl-chain rigidification and an initiation of a transition from inverted cubic to multilamellar structures. The binding stoichiometry of endotoxin and lysozyme is a 3:1 molar ratio for both LPS Re and lipid A, indicating a dominant binding of lysozyme to the lipid A-phosphates. This could be confirmed by the analysis of a phosphate vibration and by the use of a dephospho LPS. Parallel to lysozyme binding to endotoxin, a conformational change of the secondary structure in the protein from mainly alpha helix to more unordered structures takes place, while the residual beta-sheet substructure does not exhibit a clear concentration dependence. Binding is found to be specific for the endotoxins since, for the zwitterionic phosphatidylcholine, no binding is observed and, for the negatively charged phosphatidylglycerol, only very weak binding is found. The results are discussed in the context of the ability of lysozyme to reduce endotoxicity.     

Towards a molecular level understanding of protein stabilization: the interaction between lysozyme and sorbitol

The paper is investigating the mechanism of stabilization of proteins by polyols at the molecular level. It is addressing the interactions of sorbitol, a polyol commonly used as a protein stabilizing agent, with hen egg white lysozyme, a well studied protein. Differential scanning calorimetry shows an increase in denaturation temperature of lysozyme upon addition of sorbitol at a concentration of 250 mM and above. Increasing sorbitol concentration also caused an increase in signal intensity of the CD spectrum of lysozyme in the wavelength region of 280-300 nm. Two-dimensional nuclear magnetic resonance spectroscopy was used to examine interactions between lysozyme and sorbitol. Most significant changes are manifest in the anomalous relaxation properties of Ala and Thr methyl groups indicating modifications of local motions and possibly compression of the entire structure. This is further corroborated by new intra-protein nuclear Overhauser effects in the presence of sorbitol. There is also evidence that water is displaced from the enzyme surface close to Ile-88 upon addition of sorbitol. In combination these results reveal a complex interplay of different interactions. Comparison to NMR-spectra of lysozyme with a bound inhibitor (tri-N-acetyl-glucosamine) shows that the interaction with sorbitol affects spatially disparate regions of the protein.     

Effect of Vernonia cinerea Less flower extract in adjuvant-induced arthritis

Denatured lysozyme was refolded by a dilution method. The refolding yield depended greatly on the lysozyme concentration in the refolding mixture. When the concentration of denatured lysozyme was 0.02 g/L, the refolding yield was as high as 60%. However, when the concentration of denatured lysozyme was 0.2 g/L, the refolding yield was as low as 10% due to the formation of aggregates. To prevent the formation of aggregates and to increase the refolding yield at a low cost, inexpensive additives were screened. The addition of acetone, acetoamide, or urea derivatives was very effective for improving the refolding yield. To clarify why the addition of acetoamide in the refolding mixture improved the refolding yield at the high lysozyme concentration, the time courses of the concentration and the average diameter of the aggregates in the refolding mixture were monitored by the dynamic light scattering method. The experimental results showed that acetoamide played a role in preventing the formation and growth of aggregates and secondary aggregation between the lysozyme aggregates.     

Controlled delivery of antibacterial proteins from biodegradable matrices

Prosthetic valve endocarditis is an infrequent, but serious complication of cardiac valve replacement. The infection is caused by the adherence of bacteria to the prosthetic valve or to tissue at the site of implantation. Recently it was shown that antibacterial peptides from blood platelets are involved in clearance and killing of bacteria adhering to vegetations induced in a model for prosthetic valve endocarditis using rabbits. The application of these antibacterial proteins in a release system, incorporated in the Dacron sewing ring of the prosthetic heart valve would diminish the incidence of endocarditis. In this study a release system for small cationic proteins based on cross-linked gelatin was developed and characterised. Furthermore, the system was evaluated with respect to the uptake and in vitro release of lysozyme, a small cationic protein that was chosen as a model protein for small cationic antibacterial proteins. Variation of gelatin type (A and B), and cross-link density resulted in differences in swelling, thermal behaviour, and number of charged groups. Lysozyme uptake was proportional to swelling, but was governed by the number of anionic groups. The latter was also observed for the release profiles: when the amount of free carboxylic acids is higher (gelatin B compared to gelatin A), the lysozyme release lasts for a longer time period. The release into solidified agarose medium, as a model for heart muscle tissue, was measured. After 50 h, 40-100% of the lysozyme was released, which is in accordance with the aimed release period of 24-48 h. The adsorption experiments in vitro suggest an influence of the electrostatic interactions between lysozyme and gelatin. This hypothesis was validated with a mathematical model which takes both diffusion and adsorption interactions into account.     

Insect cell stimulation by LPS requires the activity of cell-released proteases

The hemocyte line BTI-EA-1174-A from the lepidopteran insect Estigmene acraea responds to bacterial lipopolysaccharide (LPS) by an enhanced phagocytic reaction and a dose-dependent increase of lysozyme release [Wittwer et al., Dev Comp Immunol 21:323 (1997)]. This paper provides evidence for a strong proteolytic activity in cell culture supernatants occurring after addition of LPS (1 mg/ml). The proteolysis is caused by cell-released proteases and seems to be necessary for cell activation. Its inhibition by alpha 2-macroglobulin results in a dose-dependent reduction in cellular response strength. Phagocytic reactions, as well as lysozyme release, are lowered to about half in the presence of 0.0001 mg/ml alpha 2-macroglobulin. A nearly complete abolishment of activation was achieved with final concentrations of 1.0 mg/ml alpha 2-macroglobulin. The data presented allow us to conclude that the LPS-triggered proteolytic activity is an important part of the activation process; it occurs outside of the cells and delivers immune response activating factors.     

Oxidoreductase activities of chromaffin granule ghosts isolated from the bovine adrenal medulla

1. Based on estimated s-values of subpopulations of bovine adrenal chromaffin granules (B?dtker-Naess, V., Slinde, E., Terland, O. and Flatmark, T. (1978) Biochim. Biophys. Acta 541, 124--134) a new large-scale procedure is described for the isolation of the total population of chromaffin granules by differential centrifugation in 0.25 M sucrose. 2. Using the total population of chromaffin granules obtained by differential centrifugation, final purification was achieved by density-gradient centrifugation in either sucrose or Percoll-sucrose. In either case, the isolated granule fractions were contaminated with mitochondria to about the same degree. 3. Chromaffin granule ghosts, obtained by hypoosmotic lysis of granules isolated by sucrose density-gradient, centrifugation, were subjected to centrifugation on a discontinuous density gradient (buffer/0.9 M sucrose). By this procedure a substantial purification of the ghosts was achieved as determined from measurements of protein and various marker enzymes. 4. In contrast to preparations of chromaffin granule ghosts prepared by previous standard procedures, those purified by gradient centrifugation (on 0.9 M sucrose) did not reveal any NADH-linked cytochrome b-561 reductase activity. However, experimental evidence is presented for the existence of an intrinsic NADH-oxidizing enzyme system in the granule membrane. 5. No significant difference was observed in the specific content of cytochrome b.561 and NADH:(acceptor) oxidoreductase activities between ghost preparations obtained from populations of heavy and light chromaffin granules. 6. The functional significance of cytochrome b-561 and the NADH:(acceptor) oxidoreductase activities of the granule membrane remains to be determined.