Ex vivo evaluation of a Taylor-Couette flow, immobilized heparinase I device for clinical application.
Efficient and safe heparin anticoagulation has remained a problem for continuous renal replacement therapies and intermittent hemodialysis for patients with acute renal failure. To make heparin therapy safer for the patient with acute renal failure at high risk of bleeding, we have proposed regional heparinization of the circuit via an immobilized heparinase I filter. This study tested a device based on Taylor-Couette flow and simultaneous separation/reaction for efficacy and safety of heparin removal in a sheep model. Heparinase I was immobilized onto agarose beads via cyanogen bromide activation. The device, referred to as a vortex flow plasmapheretic reactor, consisted of two concentric cylinders, a priming volume of 45 ml, a microporous membrane for plasma separation, and an outer compartment where the immobilized heparinase I was fluidized separately from the blood cells. Manual white cell and platelet counts, hematocrit, total protein, and fibrinogen assays were performed. Heparin levels were indirectly measured via whole-blood recalcification times (WBRTs). The vortex flow plasmapheretic reactor maintained significantly higher heparin levels in the extracorporeal circuit than in the sheep (device inlet WBRTs were 1. 5 times the device outlet WBRTs) with no hemolysis. The reactor treatment did not effect any physiologically significant changes in complete blood cell counts, platelets, and protein levels for up to 2 hr of operation. Furthermore, gross necropsy and histopathology did not show any significant abnormalities in the kidney, liver, heart, brain, and spleen. (+info)
Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1.
As part of a study of protein-carbohydrate interactions, linear N-acetyl-polyllactosamines [Galbeta1,4GlcNAcbeta1,3]nwere synthesized at the 10-100 micromol scale using enzymatic methods. The methods described also provided specifically [1-13C]-galactose-labeled tetra- and hexasaccharides ([1-13C]-Galbeta1,4GlcNAcbeta1,3Galbeta1,4Glc and Galbeta1, 4GlcNAcbeta1,3[1-13C]Galbeta1,4GlcNAcbeta1,3Galbeta 1,4Glc) suitable for NMR studies. Two series of oligosaccharides were produced, with either glucose or N-acetlyglucosamine at the reducing end. In both cases, large amounts of starting primer were available from human milk oligosaccharides (trisaccharide primer GlcNAcbeta1,3Galbeta1, 4Glc) or via transglycosylation from N-acetyllactosamine. Partially purified and immobilized glycosyltransferases, such as bovine milk beta1,4 galactosyltransferase and human serum beta1,3 N- acetylglucosaminyltransferase, were used for the synthesis. All the oligo-saccharide products were characterized by1H and13C NMR spectroscopy and MALDI-TOF mass spectrometry. The target molecules were then used to study their interactions with recombinant galectin-1, and initial1H NMR spectroscopic results are presented to illustrate this approach. These results indicate that, for oligomers containing up to eight sugars, the principal interaction of the binding site of galectin-1 is with the terminal N-acetyllactosamine residues. (+info)
The SH2-containing 5'-inositol phosphatase (SHIP) is tyrosine phosphorylated after Fc gamma receptor clustering in monocytes.
Current models of Fc gamma R signal transduction in monocytes describe a molecular cascade that begins upon clustering of Fc gamma R with the phosphorylation of critical tyrosine residues in the cytoplasmic domains of Fc gamma RIIa or the gamma-chain subunit of Fc gamma RI and Fc gamma RIIIa. The cascade engages several other tyrosine-phosphorylated molecules, either enzymes or adapters, to manifest ultimately an array of biological responses, including phagocytosis, cell killing, secretion of a variety of inflammatory mediators, and activation. Continuing to assess systematically the molecules participating in the cascade, we have found that the SH2-containing 5'-inositol phosphatase (SHIP) is phosphorylated on tyrosine early and transiently after Fc gamma R clustering. This molecule in other systems, such as B cells and mast cells, mediates an inhibitory signal. We find that clustering of either Fc gamma RIIa or Fc gamma RI is effective in inducing SHIP phosphorylation, that SHIP binds in vitro to a phosphorylated immunoreceptor tyrosine-based activation motif, peptide from the cytoplasmic domain of Fc gamma RIIa in activation-independent fashion, although SHIP binding increases upon cell activation, and that Fc gamma RIIb and Fc gamma RIIc are not responsible for the observed SHIP phosphorylation. These findings prompt us to propose that SHIP inhibits Fc gamma R-mediated signal transduction by engaging immunoreceptor tyrosine-based activation motif-containing cytoplasmic domains of Fc gamma RIIa and Fc gamma RI-associated gamma-chain. (+info)
Development of recombinant, immobilised beta-1,4-mannosyltransferase for use as an efficient tool in the chemoenzymatic synthesis of N-linked oligosaccharides.
The preparation of the conserved core structure of asparagine-linked oligosaccharides found in eukaryotic glycoproteins is an important step towards the synthesis of homogeneous neoglycoproteins. So far, however, the convenient generation of the Manbeta4GlcNAcbeta4GlcNAc (Gn2M) core trisaccharide has proved to be a major obstacle because of the inherent difficulties associated with the synthesis of beta-mannosides. Here we report the overproduction in Escherichia coli of full-length and transmembrane-deleted yeast beta-1, 4-mannosyltransferases as novel N-terminal fusions bearing a decahistidinyl sequence and the minimal human Myc epitope. The recombinant enzymes were highly active and were amenable to immobilisation by nickel(II) chelation and to immunodetection with an anti-Myc monoclonal antibody. The immobilised, transmembrane-deleted enzyme exhibited an apparent Km of 14 microM for the synthetic acceptor substrate analogue, phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside (PPGn2), under saturating donor conditions. This figure is comparable to those previously reported for native and recombinant yeast beta-1, 4-mannosyltransferases with, respectively, the natural dolichyl-linked acceptor and PPGn2. The validity of the reaction product was confirmed by chromatographic and spectroscopic analysis. (+info)
Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli.
The penicillin amidase (PA) from Escherichia coli belongs to a group of proteolytically processed bacterial enzymes. The mechanism of the maturation of the single polypeptide proenzyme has been studied for the PA from E. coli using a slowly processing mutant proenzyme. The mutant proenzyme was constructed by replacing Thr with Gly in the Thr(263)-Ser(264) bond that must be hydrolysed in active PA. The mutant proenzyme was purified by biospecific affinity chromatography using an immobilized monoclonal antibody against PA. The maturation of the free and covalently immobilized purified proenzyme was studied in vitro. For the free proenzyme the same products with PA activity as observed in homogenates of wild-type PA-producing E. coli cells were found to be formed during this process. A kinetic analysis of the possible inter- and intramolecular processes involved in the maturation demonstrated that unambiguous evidence for the existence of intramolecular processes can only be obtained in systems where intermolecular processes are excluded. The Gly(263)-Ser(264) bond was found to be hydrolysed first in the free and immobilized mutant proenzyme, based on determinations of mass spectra, N-terminal sequences and active site concentrations. In the system with immobilized proenzyme intermolecular processes are excluded, demonstrating that this bond is hydrolysed by intramolecular autoproteolysis. Based on the known three-dimensional structure of the PA from E. coli the same maturation mechanism should apply for the wild-type proenzyme. (+info)
Enzymatic synthesis of beta-lactam antibiotics using penicillin-G acylase in frozen media.
Penicillin-G acylase (EC 126.96.36.199) from Escherichia coli catalyzed the synthesis of various beta-lactam antibiotics in ice at -20 degrees C with higher yields than obtained in solution at 20 degrees C. The initial ratio between aminolysis and hydrolysis of the acyl-enzyme complex in the synthesis of cephalexin increased from 1.3 at 20 degrees C to 25 at -20 degrees C. The effect on the other antibiotics studied was less, leading us to conclude that freezing of the reaction medium influences the hydrolysis of each nucleophile-acyl-enzyme complex to a different extent. Only free penicillin-G acylase could perform transformations in frozen media: immobilized preparations showed a low, predominantly hydrolytic activity under these conditions. (+info)
Inhibition of phosphodiesterase/pyrophosphatase activity of PC-1 by its association with glycosaminoglycans.
PC-1 is a type II membrane-bound glycoprotein consisting of a short N-terminal cytoplasmic domain and a large C-terminal extracellular domain, which contains phosphodiesterase/pyrophosphatase activity. When Jurkat T cells were cultured with dibutyryl cAMP, the membrane-bound PC-1 and its soluble form were induced. They were purified as a homodimer of a 130 kDa peptide and a 120 kDa monomer, respectively, and the same two forms could also be obtained from COS-7 cells that had been transfected with PC-1 cDNA. The membrane-bound and soluble forms of PC-1 were indistinguishable from each other in terms of their enzyme kinetics and N-glycosylated moieties. Thus, the enzymatically active and fully glycosylated form of soluble PC-1 was utilized to search for its interacting molecules. The phosphodiesterase/pyrophosphatase activity of PC-1 was competitively inhibited by glycosaminoglycans, such as heparin and heparan sulfate, which are the major components of the extracellular matrix. PC-1 was capable of binding to heparin-Sepharose and the binding was inhibited in the presence of the enzyme substrate, ATP or its nonhydrolyzable analog. The enzyme activity of PC-1 itself, however, was not required for the binding to heparin-Sepharose. These results suggest that PC-1 might function as an adhesion molecule independent of its enzyme activity to associate with glycosaminoglycans in the extracellular matrix. (+info)
Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation.
F0F1, found in mitochondria or bacterial membranes, synthesizes adenosine 5'-triphosphate (ATP) coupling with an electrochemical proton gradient and also reversibly hydrolyzes ATP to form the gradient. An actin filament connected to a c subunit oligomer of F0 was able to rotate by using the energy of ATP hydrolysis. The rotary torque produced by the c subunit oligomer reached about 40 piconewton-nanometers, which is similar to that generated by the gamma subunit in the F1 motor. These results suggest that the gamma and c subunits rotate together during ATP hydrolysis and synthesis. Thus, coupled rotation may be essential for energy coupling between proton transport through F0 and ATP hydrolysis or synthesis in F1. (+info)