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PubMed2005

Probing the interface between biomolecules and inorganic materials using yeast surface display and genetic engineering.

Although promising for biomimetic materials applications, polypeptides binding inorganic material surfaces and the mechanism of their function have been difficult to characterize. This paper reports sequence-activity relationships of peptides interfacing with semiconductor CdS, and presents methodologies broadly applicable to studying peptide-solid surface interactions. We first employed yeast surface display with a human repertoire antibody library and identified rarely-occurring scFv fragments as CdS-binding polypeptides. Using our semi-quantitative cell-surface binding assay, site-directed mutational analysis, and genetic engineering we defined short distal regions of the displayed polypeptides necessary and sufficient for CdS binding. Alanine scanning mutagenesis in combination with a series of engineered polyhistidine peptides elucidated a direct relationship between histidine number and binding strength, which appeared to be further modulated by arginine and basic residues. The minimum strength of interaction was established by competition studies using soluble synthetic peptide analogs, which showed half-maximal inhibition of yeast binding to CdS at approximately 2 microM peptide. We then showed the ability of cells displaying material-specific polypeptides to form self-healing biofilms and discriminate between materials of fabricated heterostructure surfaces. Furthermore, we demonstrated the synthetic potential of the selected soluble CdS peptide in mediating aqueous synthesis of fluorescent CdS nanoparticles at room temperature. This platform may be further applied to elucidate mechanisms governing interfacial interactions and to generate material-specific reagents useful in medicine, biosensors, and bioproduction of high value inorganic materials.

Amino Acid Sequence↗
PubMed2005

Endothelin-1 and isoprenaline co-stimulation causes contractile failure which is partially reversed by MEK inhibition.

OBJECTIVE: The mitogen-activated kinase kinases (MEK)-extracellular signal-regulated kinases (ERK) signaling pathway is activated by agonists like catecholamines or endothelin-1 (ET-1) and has been implicated in cardiac pathology, such as the progression from cardiac hypertrophy to failure. The purpose of the present study, performed in an in vitro model of contractile failure, was to evaluate whether MEK inhibition prevents functional deterioration. METHODS AND RESULTS: Contractile dysfunction was induced in reconstituted rat heart tissue by concomitant treatment with ET-1 (10 nmol/l) and isoprenaline (ISO, 10 nmol/l) for 5 days. While basal force of contraction was unchanged, contractile responsiveness to beta-adrenoceptor agonists was markedly impaired (active force declined to 51% of controls) and was associated with decreased lusitropy. Moreover, in ET-1+ISO-treated heart tissues, reprogramming of gene expression was observed with an increased ratio of beta-myosin heavy chain (MHC) to alpha-MHC mRNA and increased transcript levels of ANF and skeletal/smooth muscle alpha-actin isoforms. The MEK inhibitor U0126 (10 micromol/l) almost completely prevented the reduction in beta-adrenergic responsiveness and the negative lusitropic effect of ET-1+ISO co-stimulation. In addition, U0126 completely normalized ANF gene expression, but did not affect or only marginally affected expression of MHC and alpha-actin isoforms. CONCLUSIONS: These results suggest that interruption of the MEK-ERK signaling pathway with a specific MEK inhibitor prevents, in part, the occurrence of a pathologic phenotype secondary to excessive stimulation with neurohumoral factors. The MEK-ERK pathway seems to be an important but not exclusive regulatory pathway responsible for the development of contractile dysfunction.

Actins↗
PubMed2004

Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica.

The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering. In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene. We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures. Batch culture led to a lipase production of 26450 U ml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources. Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158246 U ml(-1) after 80 h of cultivation. In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain.

Bioreactors↗
PubMed2006

Assembly of bacteriophage Qbeta virus-like particles in yeast Saccharomyces cerevisiae and Pichia pastoris.

Recombinant bacteriophage Qbeta coat protein (CP), which has been proposed as a promising carrier of foreign epitopes via their incorporation either by gene engineering techniques or by chemical coupling, efficiently self-assembles into virus-like particles (VLPs) when expressed in Escherichia coli. Here, we demonstrate expression and self-assembly of Qbeta CP in yeast Saccharomyces cerevisiae and Pichia pastoris. Production reached 3-4 mg/1g of wet cells for S. cerevisiae and 4-6 mg for P. pastoris, which was about 15-20% and 20-30% of the E. coli expression level, respectively. Qbeta VLPs were easily purified by size-exclusion chromatography in both cases and contained nucleic acid, shown by native agarose gel electrophoresis. The obtained particles were highly immunogenic in mice and the resulting sera recognized both E. coli- and yeast-derived Qbeta VLPs equally well.

Allolevivirus↗
PubMed2006

Microscale Marangoni actuation: all-optical and all-electrical methods.

We present experimental results from an all-optical microfluidic platform that may be complimented by a thin film all-electrical network. Using these configurations we have studied the microfluidic convective flow systems of silicone oil, glycerol, and 1,3,5-trinitrotoluene on open surfaces through the production of surface tension gradients derived from thermal gradients. We show that sufficient localized thermal variation can be created utilizing surface plasmons and/or engaging individually addressable resistive thermal elements. Both studies manipulate fluids via Marangoni forces, each having their unique exploitable advantages. Surface plasmon excitation in metal foils are the driving engine of many physical-, chemical-, and bio-sensing applications. Incorporating, for the first time, the plasmon concept in microfluidics, our results thus demonstrate great potential for simultaneous fluid actuation and sensing.

Glycerol↗
PubMed2005

Absorption of odourous substances using selective gas-liquid separation processes.

Absorption is one of the most widely used techniques for treating odourous waste gases. An improvement of the effectiveness of gas scrubbers can be achieved by using adapted washing liquids. A screening test is proposed as a low cost tool for testing washing liquids on industrial waste gases. The odour index is used to identify the main cause of the specific smell and to simplify the analysis of absorptive performances of washing liquids. The method is verified by experiments at a chocolate factory and a fat and oil refinery.

Absorption↗
PubMed2006

Chlorine decay in drinking-water transmission and distribution systems: pipe service age effect.

Water quality can deteriorate in the transmission and distribution system beyond the treatment plant. Minimizing the potential for biological regrowth can be attained by chlorinating the finished water. While flowing through pipes, the chlorine concentration decreases for different reasons. Reaction with the pipe material itself and the reaction with both the biofilm and tubercles formed on the pipe wall are known as pipe wall demand, which may vary with pipe parameters. The aim of this paper was to assess the impact of the service age of pipes on the effective chlorine wall decay constant. Three hundred and two pipe sections of different sizes and eight different pipe materials were collected and tested for their chlorine first-order wall decay constants. The results showed that pipe service age was an important factor that must not be ignored in some pipes such as cast iron, steel, cement-lined ductile iron (CLDI), and cement-lined cast iron (CLCI) pipes especially when the bulk decay is not significant relative to the wall decay. For the range of the 55 years of pipe service age used in this study, effective wall decay constants ranged from a decrease by -92% to an increase by +431% from the corresponding values in the recently installed pipes. The effect of service age on the effective wall decay constants was most evident in cast iron pipes, whereas steel pipes were less affected. Effective chlorine wall decay for CLCI and CLDI pipes was less affected by service age as compared to steel and cast iron pipes. Chlorine wall decay constants for PVC, uPVC, and polyethylene pipes were affected negatively by pipe service age and such effect was relatively small.

Chlorine↗
PubMed1997

Ag/AgCl/Cl- coated silver-stripe reference electrode.

This paper introduces a new type of miniaturized coated silver-stripe reference electrode without internal solution for single use and intends to give in detail the method used to prepare a Ag/AgCl/Cl- miniaturized coated silver-stripe reference electrode of a second kind which acts as a half-cell of a sensor system measuring Ca2+ ion activity in human blood as well as the difficulties encountered. The technique described is a cost-effective and time saving method for biomedical measurement.

Biomedical Engineering↗
PubMed2002

Lipases as practical biocatalysts.

Lipases are the most used enzymes in synthetic organic chemistry, catalyzing the hydrolysis of carboxylic acid esters in aqueous medium or the reverse reaction in organic solvents. Recent methodological advancements regarding practical factors affecting lipase activity and enantioselectivity are reviewed. Select practical examples concerning the use of lipases in the production of chiral intermediates are also highlighted.

Catalysis↗
PubMed1988

Carbonyl 13C NMR spectrum of basic pancreatic trypsin inhibitor: resonance assignments by selective amide hydrogen isotope labeling and detection of isotope effects on 13C nuclear shielding.

The carbonyl region of the natural abundance 13C nuclear magnetic resonance (NMR) spectrum of basic pancreatic trypsin inhibitor is examined, and 65 of the 66 expected signals are characterized at varying pH and temperature. Assignments are reported for over two-thirds of the signals, including those of all buried backbone amide groups with slow proton exchange and all side-chain carbonyl groups. This is the first extensively assigned carbonyl spectrum for any protein. A method for carbonyl resonance assignments utilizing amide proton exchange and isotope effects on nuclear shielding is described in detail. The assignments are made by establishing kinetic correlation between effects of amide proton exchange observed in the carbonyl 13C region with development of isotope effects and in the amide proton region with disappearance of preassigned resonances. Several aspects of protein structure and dynamics in solution may be investigated by carbonyl 13C NMR spectroscopy. Some effects of side-chain primary amide group hydrolysis are described. The main interest is on information about intramolecular hydrogen-bond energies and changes in the protein due to amino acid replacements by chemical modification or genetic engineering.

Amides↗
PubMedDate not supplied

Metabolic control analysis of eucaryotic pyruvate dehydrogenase multienzyme complex.

Metabolic control analysis (MCA) of pyruvate dehydrogenase multienzyme (PDH) complex of eucaryotic cells has been carried out using both in vitro and in vivo mechanistic models. Flux control coefficients (FCC) for the sensitivity of pyruvate decarboxylation rate to activities of various PDH complex reactions are determined. FCCs are shown to be strong functions of both pyruvate levels and various components of PDH complex. With the in vitro model, FCCs are shown to be sensitive to only the E1 component of the PDH complex at low pyruvate concentrations. At high pyruvate concentrations, the control is shared by all of the components, with E1 having a negative influence while the other three components, E2, X, and K, exert a positive control over the pyruvate decarboxylation rate. An unusual behavior of deactivation of the E1 component leading to higher net PDH activity is shown to be linked to the combined effect of protein X acylation and E1 deactivation. The steady-state analysis of the in vivo model reveals multiple steady state behavior of pyruvate metabolism with two stable and one unstable steady-states branches. FCCs also display multiplicity, showing completely different control distribution exerted by pyruvate and PDH components on three branches. At low pyruvate concentrations, pyruvate supply dominates the decarboxylation rate and PDH components do not exert any significant control. Reverse control distribution is observed at high pyruvate concentration. The effect of dilution due to cell growth on pyruvate metabolism is investigated in detail. While pyruvate dilution effects are shown to be negligible under all conditions, significant PDH complex dilution effects are observed under certain conditions. Comparison of in vitro and in vivo models shows that PDH components exert different degrees of control outside and inside the cells. At high pyruvate levels, PDH components are shown to exert a higher degree of control when reactions are taking place inside the cells as compared to the in vitro situation.

Cell Division↗
PubMedDate not supplied

Inexpensive and generic affinity purification of recombinant proteins using a family 2a CBM fusion tag.

The selective binding of the family 2a carbohydrate binding module (CBM2a) of xylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosic substrates is shown to provide a new, cost-effective affinity chromatography system for purification of recombinant protein. Genetic linkage of CBM2a to a target protein, in this case protein A from Staphylococcus aureus, results in a fusion protein that binds strongly to the particulate-cellulose resin Avicel PH101 and retains the biological activity of the fusion partner. Affinity purification of protein A-CBM2a from the supernatant of a recombinant E. coli JM101 culture results in a product purity of greater than 95% and a product concentration factor of 34 +/- 3. Measured column parameters are combined with one-dimensional equations governing continuity and intraparticle diffusion to predict product breakthrough curves with good accuracy over the range of realistic operating conditions. Peak spreading within the column is controlled by intraparticle diffusion for CBM2a and by a combination of film mass transfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein.

Canada↗
PubMed2005

Thermochemical and kinetic analyses on oxidation of isobutenyl radical and 2-hydroperoxymethyl-2-propenyl radical.

In recognition of the importance of the isobutene oxidation reaction in the preignition chemistry associated with engine knock, the thermochemistry, chemical reaction pathways, and reaction kinetics of the isobutenyl radical oxidation at low to intermediate temperature range were computationally studied, focusing on both the first and the second O2 addition to the isobutenyl radical. The geometries of reactants, important intermediates, transition states, and products in the isobutenyl radical oxidation system were optimized at the B3LYP/6-311G(d,p) and MP2(full)/6-31G(d) levels, and the thermochemical properties were determined on the basis of ab initio, density functional theory, and statistical mechanics. Enthalpies of formation for several important intermediates were calculated using isodesmic reactions at the DFT and the CBS-QB3 levels. The kinetic analysis of the first O2 addition to the isobutenyl radical was performed using enthalpies at the CBS-QB3 and G3(MP2) levels. The reaction forms a chemically activated isobutenyl peroxy adduct which can be stabilized, dissociate back to reactants, cyclize to cyclic peroxide-alkyl radicals, and isomerize to the 2-hydroperoxymethyl-2-propenyl radical that further undergoes another O2 addition. The reaction channels for isomerization and cyclization and further dissociation on this second O2 addition were analyzed using enthalpies at the DFT level with energy corrections based on similar reaction channels for the first O2 addition. The high-pressure limit rate constants for each reaction channel were determined as functions of temperature by the canonical transition state theory for further kinetic model development.

Journal Article↗
PubMed1999

Folding alphabets.

A new computational approach optimizes searches for reduced protein folding alphabets that use fewer than 20 types of amino acids. The predicted optimal five-letter alphabet happens to be in agreement with the suggestive results of a recent experiment, but whether highly reduced alphabets are sufficient for truly protein-like properties remains an open experimental question.

Algorithms↗
PubMed2004

Oxygen therapeutics: can we tame haemoglobin?

Chemically modified or genetically engineered haemoglobins (Hbs) developed as oxygen therapeutics (often termed 'blood substitutes') are designed to correct oxygen deficit due to ischaemia in a variety of clinical settings. These modifications are intended to stabilize Hb outside its natural environment--red blood cells--in a functional tetrameric and/or polymeric form. Uncontrolled haem-mediated oxidative reactions of cell-free Hb and its reactions with various oxidant/antioxidant and cell signalling systems have emerged as an important pathway of toxicity. Current protective strategies designed to produce safe Hb-based products are focused on controlling or suppressing the 'radical' nature of Hb while retaining its oxygen-carrying function.

Animals↗