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Biomedical subjects

C L Cooney

Publications and source records attributed to C L Cooney.

At least 19 recordsLinked to original sources
PubMed2000

Measurement of heat evolution and correlation with oxygen consumption during microbial growth. Reprinted from Biotechnology and Bioengineering, Vol. XI, Issue 3, Pages 269-281 (1968).

A procedure for measuring the rate of heat production from a fermentation has been developed. The method is based on measuring the rate of temperature rise of the fermentation broth resulting from metabolism, when the temperature controller is turned off. The heat accumulation measured in this manner is then corrected for heat losses and gains. A sensitive thermistor is used to follow the temperature rise with time. This procedure is shown to be as accurate as previous methods but much simpler in execution. Using this technique, the rate of heat production during metabolism was found to correlate with the rate of oxygen consumption. Experiments were performed using bacteria (E. coli and B. subtilis), a yeast (C. intermedia), and a mold (A. niger). The substrates investigated included glucose, molasses, and soy bean meal. The proportionality constant for the correlation is independent of the growth rate, slightly dependent on the substrate, and possibly dependent on the type of organism growth. This correlation has considerable potential for predicting heat evolution from the metabolism of microorganisms on simple or complex substrates and providing quantitative parameters necessary for heat removal calculations.

Aspergillus niger↗
PubMed1999

Regional heparinization via simultaneous separation and reaction in a novel Taylor-Couette flow device.

The development of a safe and efficient bioreactor design has remained a challenge for the clinical application of immobilized enzymes. Specifically, the use of immobilized heparinase I has been the target of many studies to make heparin anticoagulation therapy safer for the critically ill patient with kidney failure or heart disease. We have investigated the use of Taylor-Couette flow for a novel type of bioreactor. In a previous study, we showed that the fluidization of agarose immobilized heparinase within Taylor vortices in whole blood can lead to extensive blood damage in the form of cell depletion and hemolysis. Based on these findings, we designed and developed a reactor, referred to as vortex-flow plasmapheretic reactor (VFPR), that incorporated plasmapheresis and fluidization of the agarose in the reactive compartment, separate from the whole-blood path. In the present study, immobilized heparinase I was tested as a means of achieving regional heparinization of a closed circuit. This is a method in which heparin is infused into the extracorporeal circuit predialyzer and neutralized postdialyzer. Saline studies were performed with an immobilized heparinase I-packed bed and with the VFPR. An in vitro feasibility study was performed with the VFPR using human blood. The VFPR achieved heparin conversions of 44 +/- 0.5% and 34 +/- 2% in saline and blood, respectively. In addition, the VFPR caused no blood damage. We report a novel method to achieve fluidization which depended on secondary, circumferencial flow, and was independent of the primary flow through the device.

Biocompatible Materials↗
PubMed1999

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.

Acute Kidney Injury↗
PubMed1999

Heparan sulfate mediates bFGF transport through basement membrane by diffusion with rapid reversible binding.

Basic fibroblast growth factor (bFGF) is a pluripotent cytokine with a wide range of target cells. Heparan sulfate binds bFGF, and this interaction has been demonstrated to protect bFGF against physical denaturation and protease degradation. The high concentrations of heparan sulfate in basement membranes have implicated these matrices as storage sites for bFGF in vivo. However, the mechanisms by which basement membranes modulate bFGF storage and release is unknown. To gain insight into these mechanisms, we have developed experimental and mathematical models of extracellular growth factor transport through basement membrane. Intact Descemet's membranes isolated from bovine corneas were mounted within customized diffusion cells and growth factor transport was measured under a variety of conditions that decoupled the diffusion process from the heparan sulfate binding phenomenon. Transport experiments were conducted with bFGF and interleukin 1beta. In addition, bFGF-heparan sulfate binding was disrupted in diffusion studies with high ionic strength buffer and buffers containing protamine sulfate. Transport of bFGF was enhanced dramatically when heparan sulfate binding was inhibited. This process was modeled as a problem of diffusion with fast reversible binding. Experimental parameters were incorporated into a mathematical model and independent simulations were run that showed that the experimental data were accurately predicted by the mathematical model. Thus, this study indicated that basement membranes function as dynamic regulators of growth factor transport, allowing for rapid response to changing environmental conditions. The fundamental principles controlling bFGF transport through basement membrane that have been identified here might have applications in understanding how growth factor distribution is regulated throughout an organism during development and in the adult state.

Animals↗
PubMed1999

Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks.

Gene therapy is a promising process for the prevention, treatment and cure of diseases such as cancer, acquired immunodeficiency syndrome (AIDS) and cystic fibrosis. One of the methods used to administer therapeutic genes is the direct injection of naked or lipid-coated plasmid DNA, but this requires considerable amounts of plasmid DNA. There are several problems and bottlenecks associated with the design and operation of large-scale processes for the production of pharmaceutical-grade plasmid DNA for gene therapy.

Biopharmaceutics↗
PubMed1999

Immobilization of oligonucleotides on a large pore support for plasmid purification by triplex affinity interaction.

Triplex affinity interaction provides a new process for the purification of plasmid DNA, which is especially suited to meet the demands of a gene therapy use. We developed a method for the functionalization of a large pore affinity support suitable for this application. A 5'-modified DNA oligonucleotide containing an aldehyde group was coupled to adipic acid hydrazide functionalized Sephacryl beads with a yield of 31% (over all immobilization yield 22.6% from starting oligonucleotide). The resulting selective and covalent immobilization of the ligand via a 16 atom, hydrophilic linker arm enables the oligonucleotide bases to freely bind to the target sequence. The proposed method provides affinity supports that might be used in large scale affinity purification of plasmid DNA.

Base Sequence↗
PubMed1998

Purification of plasmids by triplex affinity interaction.

Production of pharmaceutical grade plasmid DNA is an important issue in gene therapy. We developed a method for affinity purification of plasmids by triple helix interaction. This method is based on sequence-specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid. Using design criteria derived from thermodynamic data, we produced a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions. Plasmid DNA was purified from clarified Escherichia coli lysate by incubation with the affinity beads at pH 5.0 and high NaCl concentration. After extensive washing of the beads, purified plasmid DNA was eluted with alkaline buffer. The purified plasmid showed no RNA or cell DNA contamination in HPLC analysis and total protein concentration was reduced considerably. Due to its mechanical stability and porosity this support can be used in a continuous affinity purification process, which has a high potential for scale up.

DNA, Bacterial↗
PubMed1996

Heparinase I from Flavobacterium heparinum. Identification of a critical histidine residue essential for catalysis as probed by chemical modification and site-directed mutagenesis.

We recently identified cysteine-135 as an important amino acid for heparinase I (EC 4.2.2.7) activity. In this study, we have identified a second residue critical for enzymatic activity. We observe concentration-dependent inactivation of heparinase I in the presence of reversible histidine-modifying diethyl pyrocarbonate (DEPC); 0.3 mM DEPC results in 95% of heparinase I inactivation in less than 3 min, and as low as 10 microM DEPC results in a 85% loss of heparinase I activity in 15 min. Heparinase I activity is restored following hydroxylamine treatment. This, along with other experiments, strongly suggests that the inactivation of heparinase I by DEPC is specific for histidine residues. Chemical modification, under nondenaturing conditions, of the histidines using nonradiolabeled and [14C]DEPC indicates that between one and two histidine residues are modified. Chemical modification of the surface-accessible histidines, in the presence and absence of heparin, suggests that the histidine(s) lie(s) in or near the active site of heparinase I. The wild-type heparinase I has four histidine residues; site-directed mutagenesis of H129A, H165A, and H339A did not affect enzyme activity and the kinetic parameters, suggesting that these residues are not essential for heparinase I activity. However, H203A inactivates heparinase I while a H203D mutant has residual activity, indicating a role of this residue in catalysis. We propose that histidine-203, contained in the heparin binding site, is immediately adjacent to cysteine-135, and these residues together form the catalytic domain of heparinase I.

Amino Acid Sequence↗
PubMed1996

Expression in Escherichia coli, purification and characterization of heparinase I from Flavobacterium heparinum.

The use of heparin for extracorporeal therapies has been problematical due to haemorrhagic complications; as a consequence, heparinase I from Flavobacterium heparinum is used for the determination of plasma heparin and for elimination of heparin from circulation. Here we report the expression of recombinant heparinase I in Escherichia coli, purification to homogeneity and characterization of the purified enzyme. Heparinase I was expressed with an N-terminal histidine tag. The enzyme was insoluble and inactive, but could be refolded, and was purified to homogeneity by nickel-chelate chromatography. The cumulative yield was 43%, and the recovery of purified heparinase I was 14.4 mg/l of culture. The N-terminal sequence and the molecular mass as analysed by matrix-assisted laser desorption MS were consistent with predictions from the heparinase I gene structure. The reverse-phase HPLC profile of the tryptic digest, the Michaelis-Menten constant Km (47 micrograms/ml) and the specific activity (117 units/mg) of purified recombinant heparinase I were similar to those of the native enzyme. Degradation of heparin by heparinase I results in a characteristic product distribution, which is different from those obtained by degradation with heparinase II or III from F. heparinum. We developed a rapid anion-exchange HPLC method to separate the products of enzymic heparin degradation, using POROS perfusion chromatography media. Separation of characteristic di-, tetra- and hexa-saccharide products is performed in 10 min. These methods for the expression, purification and analysis of recombinant heparinase I may facilitate further development of heparinase I-based medical therapies as well as further investigation of the structures of heparin and heparan sulphate and their role in the extracellular matrix.

Amino Acid Sequence↗
PubMed1996

Heparinase I from Flavobacterium heparinum. Mapping and characterization of the heparin binding domain.

In this study we have identified the primary heparin binding site of heparinase I (EC 4.2.2.7). Chemical and proteolytic digests of heparinase I were used in direct binding and competition assays, to map the regions of heparinase I that interact specifically with heparin. We find the heparin binding site contains two Cardin-Weintraub heparin binding consensus sequences and a calcium co-ordination consensus motif. We show that heparin binding to heparinase I is independent of calcium (Kd of 60 nm) and that calcium is able to activate heparinase I catalytically. We find that sulfhydryl selective labeling of cysteine 135 of heparinase I protects the lysines of the heparin binding sequence from proteolytic cleavage, suggesting the close proximity of the heparin binding site to the active site. Site-directed mutagenesis of H203A (contained in the heparin binding site) inactivated heparinase I; however, a H203D mutant retained marginal activity, indicating a role for this residue in catalysis. The above results taken together suggest that histidine 203 (hence the heparin binding site) is immediately adjacent to the scissile bond. We propose that the heparin binding site and active site are in close proximity to each other and that the calcium coordination motif, contained in the heparin binding site, may bridge heparin to heparinase I through calcium in a ternary complex during catalysis.

Amino Acid Sequence↗
PubMed1996

Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.

Apoproteins↗
PubMed1995

Heparinase I from Flavobacterium heparinum: the role of the cysteine residue in catalysis as probed by chemical modification and site-directed mutagenesis.

Heparinase I (heparin lyase I, EC 4.2.2.7), a heparin-degrading enzyme produced by Flavobacterium heparinum, is used to deheparinize blood following extracorporeal procedures in surgery and in other applications. The present study of mapping and characterization of the cysteines of heparinase I represents the first structural characterization of a heparinase. [3H]Iodoacetic acid labeling demonstrated that heparinase I has two free cysteines. One of the two cysteines is surface accessible and lies in a hydrophilic environment while the other is in a hydrophobic environment. Chemical modification of the cysteines, both in the presence and in the absence of heparin, suggests that the surface-accessible cysteine lies in or near the active site of heparinase I. Preferential reactivity of this cysteine with negatively charged sulfhydryl-modifying reagents and the cysteines' high reactivity to iodoacetic acid at pH 6.5 indicate that the surface-accessible cysteine is in a positively charged region. The surface-accessible cysteine (cysteine-135) was mapped as the active-site cysteine by radiolabeling with [3H]iodoacetic acid and by tryptic digestion and peptide sequencing. Site-directed mutagenesis of cysteine-135 to a serine or an alanine in r-heparinase I demonstrates that this cysteine is essential for enzymatic activity. However, replacement of the surface-inaccessible cysteine by a serine or alanine has no effect.

Base Sequence↗
PubMed1995

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Amino Acid Sequence↗
PubMed1995

Enzymatic degradation of glycosaminoglycans.

Glycosaminoglycans (GAGs) play an intricate role in the extracellular matrix (ECM), not only as soluble components and polyelectrolytes, but also by specific interactions with growth factors and other transient components of the ECM. Modifications of GAG chains, such as isomerization, sulfation, and acetylation, generate the chemical specificity of GAGs. GAGs can be depolymerized enzymatically either by eliminative cleavage with lyases (EC 4.2.2.-) or by hydrolytic cleavage with hydrolases (EC 3.2.1.-). Often, these enzymes are specific for residues in the polysaccharide chain with certain modifications. As such, the enzymes can serve as tools for studying the physiological effect of residue modifications and as models at the molecular level of protein-GAG recognition. This review examines the structure of the substrates, the properties of enzymatic degradation, and the enzyme substrate-interactions at a molecular level. The primary structure of several GAGs is organized macroscopically by segregation into alternating blocks of specific sulfation patterns and microscopically by formation of oligosaccharide sequences with specific binding functions. Among GAGs, considerable dermatan sulfate, heparin and heparan sulfate show conformational flexibility in solution. They elicit sequence-specific interactions with enzymes that degrade them, as well as with other proteins, however, the effect of conformational flexibility on protein-GAG interactions is not clear. Recent findings have established empirical rules of substrate specificity and elucidated molecular mechanisms of enzyme-substrate interactions for enzymes that degrade GAGs. Here we propose that local formation of polysaccharide secondary structure is determined by the immediate sequence environment within the GAG polymer, and that this secondary structure, in turn, governs the binding and catalytic interactions between proteins and GAGs.

Animals↗
PubMed1994

A stereochemical approach to pyranose ring flexibility: its implications for the conformation of dermatan sulfate.

Glycosaminoglycans, such as heparin, heparan sulfate, and dermatan sulfate, are characterized by a disaccharide repeating unit of a uronate and a hexosamine and are increasingly understood to be important physiologically as soluble components of the extracellular matrix. The secondary structure of this class of acidic polysaccharides is believed to play a key role in determining the wide range of biological specificities. Central to the structural diversity of the glycosaminoglycans is the experimentally documented conformational flexibility of the iduronate residue. Here, we outline an approach to explore the iduronate conformational flexibility by imposing stereochemical criteria of nonbonded contact distances. By performing a complete search of all possible torsions that define the iduronate ring geometry, we eliminate any prior bias with regard to minimum energy conformers. The approach led to alternative feasible conformers for the iduronate ring that are stereochemically satisfactory and are consistent with the available physico-chemical data.

Carbohydrate Conformation↗
PubMed1994

Heparinase inhibits neovascularization.

Neovascularization is associated with the regulation of tissue development, wound healing, and tumor metastasis. A number of studies have focused on the role of heparin-like molecules in neovascularization; however, little is known about the role of heparin-degrading enzymes in neovascularization. We report here that the heparin-degrading enzymes, heparinases I and III, but not heparinase II, inhibited both neovascularization in vivo and proliferation of capillary endothelial cells mediated by basic fibroblast growth factor in vitro. We suggest that the role of heparinases in inhibition of neovascularization is through depletion of heparan sulfate receptors that are critical for growth factor-mediated endothelial cell proliferation and hence neovascularization. The differences in the effects of the three heparinases on neovascularization could be due to different substrate specificities for the enzymes, influencing the availability of specific heparin fragments that modulate heparin-binding cytokines involved in angiogenesis.

Allantois↗
PubMed1993

Cloning and expression of heparinase I gene from Flavobacterium heparinum.

Heparinases, enzymes that cleave heparin and heparin sulfate, are implicated in physiological and pathological functions ranging from wound healing to tumor metastasis and are useful in deheparinization therapies. We report the cloning of the heparinase I (EC 4.2.2.7) gene from Flavobacterium heparinum using PCR. Two degenerate oligonucleotides, based on the amino acid sequences derived from tryptic peptides of purified heparinase, were used to generate a 600-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. This probe was used to screen a Flavobacterium genomic DNA library in pUC18. The open reading frame of heparinase I is 1152 bp in length, encoding a precursor protein of 43.8 kDa. Eleven of the tryptic peptides (approximately 35% of the total amino acids) mapped onto the open reading frame. The amino acid sequence reveals a consensus heparin binding domain and a 21-residue leader peptide with a characteristic Ala-(Xaa)-Ala cleavage site. Recombinant heparinase was expressed in Escherichia coli as a soluble protein, using the T7 polymerase pET expression system. The recombinant heparinase cleavage of heparin was identical to that of native heparinase.

Amino Acid Sequence↗