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Biomedical subjects

G F Lee

Publications and source records attributed to G F Lee.

At least 19 recordsLinked to original sources
PubMed1999

The effect of O-fucosylation on the first EGF-like domain from human blood coagulation factor VII.

The first epidermal growth factor-like domain (EGF-1) from blood coagulation factor VII (FVII) contains two unusual O-linked glycosylation sites at Ser-52 and Ser-60. We report here a detailed study of the effect of O-fucosylation at Ser-60 on the structure of FVII EGF-1, its Ca2+-binding affinity, and its interaction with tissue factor (TF). The in vitro fucosylation of the nonglycosylated FVII EGF-1 was achieved by using O-fucosyltransferase purified from Chinese hamster ovary cells. Distance and dihedral constraints derived from NMR data were used to determine the solution structures of both nonglycosylated and fucosylated FVII EGF-1 in the presence of CaCl2. The overall structure of fucosylated FVII EGF-1 is very similar to the nonfucosylated form even for the residues near the fucosylation site. The Ca2+ dissociation constants (Kd) for the nonfucosylated and fucosylated FVII EGF-1 were found to be 16.4 +/- 1.8 and 8.6 +/- 1.4 mM, respectively. The FVII EGF-1 domain binds to the extracellular part of TF with a low affinity (Kd approximately 0. 6 mM), and the addition of fucose appears to have no effect on this affinity. These results indicate that the FVII EGF-1 alone cannot form a tight complex with TF and suggest that the high binding affinity of FVIIa for TF requires cooperative interaction among the four domains in FVII with TF. Although the fucose has no significant effect on the interaction between TF and the individual FVII EGF-1 domain, it may affect the interaction of full-length FVIIa with TF by influencing its Ca2+-binding affinity.

Amino Acid Sequence↗
PubMed1998

A novel soluble tissue factor variant with an altered factor VIIa binding interface.

Tissue factor (TF) residues Lys20 and Asp58 form part of a binding epitope previously shown by alanine scanning to be critical for high affinity interactions with factor VIIa (FVIIa). To explore the possibility of enhancing the affinity of a TF-based antagonist for FVIIa, we created libraries in which residues at 20, 58, and adjacent positions were varied in constructs containing the soluble extracellular domain of TF (sTF) fused to the bacteriophage M13 tail coat protein. TF variants monovalently displayed on phage were then sorted on the basis of binding to FVIIa. Sorting of preliminary libraries, in which position 58 and/or 20 and surrounding residues were randomized, led to the selection of TF proteins of essentially wild-type sequence. Therefore, we devised a strategy wherein TF position 20 was held fixed as alanine and 5 specific residues near to, and including, position 58 were randomized to effectively obtain alternative sequences at this interface. The consensus sequence reached with this library included wild-type residues at positions 61, 62, 65, and 66 but exclusively tryptophan at position 58. Analyses of the soluble K20A,D58W (A20W58) TF protein indicated that it binds FVIIa with an affinity comparable with wild-type sTF but is defective as a cofactor for FVIIa-dependent factor X activation. Further experiments designed to elucidate the mechanism of binding suggest that the new binding interactions involve more than the simple addition of hydrophobic surface area.

Anilides↗
PubMed1997

Potent bifunctional anticoagulants: Kunitz domain-tissue factor fusion proteins.

A strategy to design potent antagonists of human coagulation factor VIIa (FVIIa) by linking two proteins that independently inhibit activity and bind at separate, nonoverlapping sites is presented. A bifunctional inhibitor (KDTF5), comprising a Kunitz-type domain engineered to inhibit the FVIIa active site and a soluble tissue factor (TF) variant that is defective as a cofactor for factor X (FX) activation, was developed from structure-based modeling of a ternary FVIIa-Kunitz domain-TF complex. KDTF5 inhibited FVIIa-dependent FX activation with a Ki* of 235 +/- 45 pM, a 193-fold and 398-fold increase in potency compared to the TF variant and Kunitz domain individually. Similarly, KDTF5 was a more potent anticoagulant in vitro compared to either inhibitory domain alone. The results demonstrate the harnessing of a macromolecular chelate effect by fusing two inhibitory ligands that bind a target at spatially distinct sites.

Anticoagulants↗
PubMed1997

Analysis of protein structure in intact cells: crosslinking in vivo between introduced cysteines in the transmembrane domain of a bacterial chemoreceptor.

Oxidative crosslinking of cysteines introduced by site-specific mutagenesis is a powerful tool for structural analysis of proteins, but the approach has been limited to studies in vitro. We recently reported that intact cells of Escherichia coli could be treated with Cu(II)-(o-phenanthroline)3 or molecular iodine in a way that left unperturbed flagellar function or general chemotactic response, yet crosslinks were quantitatively formed between select cysteines in adjoining transmembrane helices of chemoreceptor Trg. This suggested that oxidative crosslinking might be utilized for structural analysis in vivo. Thus, we used our comprehensive collection of Trg derivatives, each containing a single cysteine at one of the 54 positions in the two transmembrane segments of the receptor monomer to characterize patterns of crosslinking in vivo and in vitro for this homodimeric protein. We found that in vivo crosslinking compared favorably as a technique for structural analysis with the more conventional in vitro approach. Patterns of crosslinking generated by oxidation treatments of intact cells indicated extensive interaction of transmembrane segment 1 (TM1) with its homologous partner (TM1') in the other subunit and a more distant placement of TM2 and TM2', the same relationships identified by crosslinking in isolated membranes. In addition, the same helical faces for TM1-TM1' interaction and TM2-TM2' orientation were identified in vivo and in vitro. The correspondence of the patterns also indicates that structural features identified by analysis of in vitro crosslinking are relevant to the organization of the chemoreceptor in its native environment, the intact, functional cell. It appears that the different features of the two functionally benign treatments used for in vivo oxidations can provide insights into protein dynamics.

Bacterial Proteins↗
PubMed1996

Identification of a GDP-L-fucose:polypeptide fucosyltransferase and enzymatic addition of O-linked fucose to EGF domains.

An assay of GDP-fucose:polypeptide fucosyltransferase has been established. The enzyme catalyzes the reaction that attaches fucose through an O-glycosidic linkage to a conserved serine or threonine residue in EGF domains. The assay uses recombinant human factor VII EGF-1 domain as acceptor substrate and GDP-fucose as donor substrate. Synthetic peptides with sequences taken from five proteins previously shown to contain O-linked fucose (Harris and Spellman, 1993; Glycobiology, 3, 219-224) did not serve as efficient acceptor substrates. These synthetic peptides did not compromise complete EGF domains and did not contain all six cysteine residues that define the EGF structure. Therefore, the enzyme appears to require more than just a consensus primary sequence and likely requires that the EGF domain disulfide bonds be properly formed. The enzymatic reaction showed linear dependency of its activity on time, amount of enzyme, and substrates. Although the enzyme did not exhibit an absolute requirement for Mn2+, enzymatic activity did increase ten fold in the presence of 50 mM MnCl2. The in vitro glycosylation reaction resulted in complete conversion of the acceptor substrate to glycosylated product, and characterization of the purified product by electrospray mass spectrometry revealed that one fucose was added onto the polypeptide. Most of the enzymatic activity was found to be in the soluble fraction of CHO cell homogenates. However, when enzyme was prepared from rat liver in the presence of protease inhibitors, 37% of the activity was recovered by Triton X-100 extraction of the membrane particles after extensive aqueous washes. The result suggests that the enzyme is probably a membrane protein and, by analogy with other glycosyltransferases, probably has a 'stem' region that is very susceptible to proteolysis.

Amino Acid Sequence↗
PubMed1995

Identification of functionally important helical faces in transmembrane segments by scanning mutagenesis.

We applied mutational analysis to a protein domain that functions in neither catalysis nor binding but, rather, in transmembrane signaling. The domain is part of chemoreceptor Trg from Escherichia coli. It contains four transmembrane segments, two from each subunit of the homodimer. We used cysteine scanning to investigate the functional importance of each of 54 residues in the two transmembrane segments. Cysteines at some positions resulted in subtle but significant reductions in tactic response. Those positions defined a specific helical face on each segment, implying that the segments function as helices. The functionally important faces corresponded to structural, helical packing faces identified independently by biochemical studies. All functionally impaired receptors exhibited altered signaling properties, either reduced signaling upon stimulation or induced signaling in the absence of stimulation. The distribution of substitutions creating these two phenotypes implied that conformational signaling involves movement between the two transmembrane helices within a subunit and that signaling is optimal when stable interactions are maintained across the interface between subunits.

Cell Membrane↗
PubMed1995

Transmembrane signaling characterized in bacterial chemoreceptors by using sulfhydryl cross-linking in vivo.

Transmembrane signaling by bacterial chemoreceptors is thought to involve conformational changes within a stable homodimer. We investigated the functional consequences of constraining movement between pairs of helices in the four-helix structure of the transmembrane domain of chemoreceptor Trg. Using a family of cysteine-containing receptors, we identified oxidation treatments for intact cells that catalyzed essentially complete sulfhydryl cross-linking at selected positions and yet left flagellar and sensory functions largely unperturbed. Constraining movement by cross-links between subunits had little effect on tactic response, but constraining movement between transmembrane segments of the monomer drastically reduced function. We deduce that transmembrane signaling requires substantial movement between transmembrane helices of a monomer but not between interacting helices across the interface between subunits.

Bacterial Proteins↗
PubMed1995

Quantitative approaches to utilizing mutational analysis and disulfide crosslinking for modeling a transmembrane domain.

The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit. We had previously used mutational analysis and sulfhydryl cross-linking between introduced cysteines to obtain data relevant to the three-dimensional organization of this domain. In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the sequences of the segments. The analyses provided a strong indication of alpha-helical periodicity in the first transmembrane segment and a substantial indication of that periodicity for the second segment. On this basis, we considered both segments as idealized alpha-helices and proceeded to model the transmembrane domain as a unit of four helices. For this modeling, we calculated helical crosslinking moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslinking data. Crosslinking moments were used to define the relative separation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg. Utilization of Fourier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with helical crosslinking moments to position helical pairs should be useful in modeling other transmembrane domains.

Amino Acid Sequence↗
PubMed1994

Deducing the organization of a transmembrane domain by disulfide cross-linking. The bacterial chemoreceptor Trg.

The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four segments, two from each subunit of the homodimer. We used site-specific mutagenesis to introduce cysteines into those segments and oxidative cross-linking of cysteine pairs to identify residues that are near each other in space. Propensity for cross-linking was determined for pairs of homologously placed cysteines in the two subunits of the dimer at all 54 possible positions. Also, combinations of cysteines were identified that readily oxidized to join heterologous segments within or between monomers. These patterns of cross-linking were used to develop a model for the three-dimensional structure of the transmembrane domain in which the four transmembrane segments are helices associated in a bundle, with stronger interactions near the periplasm and weaker interactions near the cytoplasm. The striking similarity of this model to a model for the transmembrane domain of chemoreceptor Tar, derived using the same experimental strategy, strengthens the notion that a combination of comprehensive cysteine substitutions and analysis of patterns of disulfide cross-linking is sufficient to deduce a detailed three-dimensional structure for a transmembrane domain.

Bacterial Proteins↗
PubMed1991

Alternative liability insurance: a physician-owned captive insurance company.

The physician-owned captive insurance company is a lesser known but dynamic alternative to commercial insurance. The Physicians Reimbursement Fund, Ltd., was founded in 1975 in response to the malpractice crisis of that year. The company insures about 100 physicians in high-risk specialties. Approximately one half are obstetrician-gynecologists. Innovative management has enabled this company to operate successfully at a fraction of the premium charged by typical insurance companies. Fourteen years of experience have demonstrated the ability of this company to successfully serve the needs of the community.

Insurance, Liability↗
PubMed1989

Developmentally regulated proteolytic processing of a yolk glycoprotein complex in embryos of the sea urchin, Strongylocentrotus purpuratus.

We have isolated a yolk glycoprotein complex from eggs and early embryos of the sea urchin, Strongylocentrotus purpuratus. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of these complexes and peptide mapping of their individual glycoprotein components indicate that developmental stage-specific changes in molecular composition of the complex are due to proteolytic processing events. Our data revealed that a 180 kDa glycoprotein of the egg complex is separated by a single proteolytic cleavage into intermediate glycoproteins of 115 and 76 kDa early in development. By the hatched blastula stage, each of these intermediate glycoproteins has been further processed to lower molecular weight forms: the 115 kDa protein is proteolytically clipped to a 84 kDa form, perhaps through 110 and 105 kDa intermediaries, while the 76 kDa molecule is directly processed to a 65 kDa form.

Animals↗
PubMed1987

Fine-needle aspiration of the breast: the outpatient management of breast lesions.

Fine-needle aspiration of the breast has become a well-accepted diagnostic tool for the management of breast lesions. When employed on an ambulatory basis it is both accurate and cost-effective. This study of a series of patients managed over a 2-year period demonstrates the use of this technique in an ambulatory gynecologic practice. Results demonstrate this to be a very rapidly accomplished, effective procedure that is very well accepted by the patients. Results are generally available within 24 hours and permit both the physician and the patient to accurately predict the course of management. In addition, it allows the primary physician to accurately determine which patients will require in-hospital management and which may safely undergo ambulatory, office-based biopsy of solid lesions. Experience shows this to be a valuable diagnostic tool that can be safely utilized by the practicing gynecologist.

Adult↗
PubMed1984

Developing a hospital pharmacy purchasing system.

Suggestions for developing or modifying a pharmacy purchasing system are presented and discussed. To achieve an optimal purchasing system, pharmacy managers must understand the development and operation of their current systems and enlist the participation of other hospital departments in planning the new or modified system. A pharmacy purchasing system should ensure that drug products and supplies of the highest quality are obtained at the lowest price, that these items are available when needed, and that the financial investment in drug and supply inventory is appropriate. The job requirements, activities, and responsibilities of pharmacy purchasing personnel are described, and the recording and retrieving of purchasing data are discussed. Various order-placement alternatives are compared, and order-shipment alternatives are described. Equipment and supply requirements for the purchasing function are also presented. An efficient, productive purchasing system can produce cost savings for the pharmacy department and the institution.

Inventories, Hospital↗
PubMed1982

Alternative to the traditional discount method of wholesaler purchasing.

A program of purchasing drugs from wholesalers at the wholesaler's exact invoice cost plus a percentage is described and compared with the traditional method of average wholesale price (AWP) less a discount. The comparison was conducted by the pharmacy department of a 310-bed, teaching hospital that awarded a one-year contract to a wholesaler offering its items at the exact cost plus a pre-established percentage. Data collected from monthly wholesaler computer printouts gave the following information on each product: (1) list price per item, (2) actual cost to pharmacy per item, (3) percentage discount from AWP, and (4) quantity ordered. The net percentage discount from AWP for 12 months was calculated and compared to the former (traditional) discount rate. The net discount from AWP was 15.6% for purchases made by the hospital during the first 12 months of the program. When compared with the smaller discount the hospital traditionally received, the new program saved the hospital $5758 on annual purchases of $136,419. The actual dollar savings to an institution that changes from a traditional discount program to a cost-plus-percentage program depends on: (1) the negotiated percentage added to wholesaler cost, (2) the discount from AWP that the institution was previously receiving, and (3) the volume of wholesale purchases.

Pharmacy Service, Hospital↗