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Biomedical subjects

M C Willis

Publications and source records attributed to M C Willis.

At least 19 recordsLinked to original sources
PubMed2001

Enantioselective and diastereoselective Mukaiyama-Michael reactions catalyzed by bis(oxazoline) copper(II) complexes.

The scope of highly enantioselective and diastereoselective Michael additions of enolsilanes to unsaturated imide derivatives has been developed with use of [Cu((S,S)-t-Bu-box)](SbF6)2 (1a) as a Lewis acid catalyst. The products of these additions are useful synthons that contain termini capable of differentiation under mild conditions. Michael acceptor pi-facial selectivity is consistent with two-point binding of the imide substrate and can be viewed as an extension of substrate enantioselection in the corresponding Diels-Alder reactions. A model analogous to the one employed to describe the hetero Diels-Alder reaction is proposed to account for the observed relation between enolsilane geometry and product absolute diastereocontrol. Insights into modes of catalyst inactivation are given, including spectroscopic evidence for inhibition of the catalyst by a dihydropyran intermediate that evolves during the course of the reaction. A procedure is disclosed in which an alcohol additive is used to hydrolyze the inhibiting dihydropyran and afford the desilylated Michael adduct in significantly shortened reaction time.

Acrylates↗
PubMed2000

Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers.

High sensitivity and specificity of two modified ssDNA aptamers capable of photocross-linking recombinant human basic fibroblast growth factor (bFGF((155))) were demonstrated. The aptamers were identified through a novel, covalent, in vitro selection methodology called photochemical systematic evolution of ligands by exponential enrichment (PhotoSELEX). The aptamers exhibited high sensitivity for bFGF((155)) comparable with commercially available ELISA monoclonal antibodies with an absolute sensitivity of at least 0.058 ppt bFGF((155)) under prevailing test conditions. The aptamers exquisitely distinguished bFGF((155)) from consanguine proteins, vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). A commercially viable diagnostic system incorporating PhotoSELEX-evolved aptamers capable of simultaneous quantification of a large number of analyte molecules is also described. Such a system benefits from covalent bonding of aptamer to target protein allowing vigorous washing with denaturants to improve signal to noise.

Base Sequence↗
PubMed2000

Computed tomography image-guided surgery in complex acetabular fractures.

Eleven complex acetabular fractures in 10 patients were treated by open reduction with internal fixation incorporating computed tomography image guided software intraoperatively. Each of the implants placed under image guidance was found to be accurate and without penetration of the pelvis or joint space. The setup time for the system was minimal. Accuracy in the range of 1 mm was found when registration was precise (eight cases) and was in the range of 3.5 mm when registration was only approximate (three cases). Added benefits included reduced intraoperative fluoroscopic time, less need for more extensive dissection, and obviation of additional surgical approaches in some cases. Compared with a series of similar fractures treated before this image guided series, the reduction in operative time was significant. For patients with complex anterior and posterior combined fractures, the average operation times with and without application of three-dimensional imaging technique were, respectively, 5 hours 15 minutes and 6 hours 14 minutes, revealing 16% less operative time for those who had surgery using image guidance. In the single column fracture group, the operation time for those with three-dimensional imaging application, was 2 hours 58 minutes and for those with traditional surgery, 3 hours 42 minutes, indicating 20% less operative time for those with imaging modality. Intraoperative computed tomography guided imagery was found to be an accurate and suitable method for use in the operative treatment of complex acetabular fractures with substantial displacement.

Acetabulum↗
PubMed1999

Catalytic enantioselective Michael additions to unsaturated ester derivatives using chiral copper(II) Lewis acid complexes.

[formula: see text] Chiral Cu(II) bisoxazoline (box) Lewis acids have been developed as catalysts of the Michael addition of enolsilanes to unsaturated ester derivatives. While enantioselection is stereoregular, the sense of diastereoselection is directly related to thioester enolsilane geometry: (E) enolsilanes give anti adducts and (Z) enolsilanes afford syn adducts. The size of the enolsilane alkylthio substituent directly impacts the magnitude of diastereoselection.

Acids↗
PubMed1999

The use of aptamers in large arrays for molecular diagnostics.

BACKGROUND: Aptamers are single-stranded oligonucleotides derived from an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment (SELEX). They bind tightly and specifically to target molecules; most aptamers to proteins bind with Kds (equilibrium dissociation constant) in the range of 1 pM to 1 nM. METHODS AND RESULTS: The SELEX protocol has been automated; therefore, hundreds to thousands of aptamers can be made in an economically feasible fashion. Blood and urine can be analyzed on chips that capture and quantitate proteins. SELEX has been adapted to the use of 5-bromo (5-Br) and 5-iodo (5-I) deoxyuridine residues. These halogenated bases can be specifically cross-linked to proteins. Selection pressure during in vitro evolution can be applied for both binding specificity and specific photo-cross-linkability. These are sufficiently independent parameters to allow one reagent, a photo-cross-linkable aptamer, to substitute for two reagents, the capture antibody and the detection antibody, in a typical sandwich array. After a cycle of binding, washing, cross-linking, and detergent washing, proteins will be specifically and covalently linked to their cognate aptamers. CONCLUSIONS: Because no other proteins are present on the chips, protein-specific stain will now show a meaningful array of pixels on the chip. Learning algorithms and retrospective studies should lead to a robust, simple, diagnostic chip.

Body Fluids↗
PubMed1999

Mass spectral characterization of a protein-nucleic acid photocrosslink.

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.

Alkaline Phosphatase↗
PubMed1999

Rank order analysis of tibial plafond fractures: does injury or reduction predict outcome?

We investigated the effects of severity of initial injury pattern and the quality of the articular reduction on outcome of displaced intra-articular distal tibial fractures, using a series of 25 patients who were treated with articulated external fixation and limited internal fixation, which provided a spectrum of reduction quality. Outcome was assessed by clinical ankle scores and radiographic arthrosis. The results demonstrate the rank order method to be a reliable means of stratifying severity of injury and quality of reduction. Neither injury nor reduction correlated with clinical ankle score. Reduction had a significant correlation with radiographic arthrosis. We conclude that the rank order method is useful in stratification of fracture patients, and that factors other than injury pattern and quality of articular reduction are important in determining outcome of patients with this severe articular injury.

Adolescent↗
PubMed1997

Post-SELEX combinatorial optimization of aptamers.

In vitro selection techniques provide a means of isolating nucleic acid ligands for binding to particular protein targets. Although most aptamers have quite high affinities for their target proteins, it has been shown that post-SELEX modification can result in further enhancement of binding affinity, as well as other desired properties. This has led to the current development of a more systematic approach to aptamer optimization using a combinatorial screening methodology.

Base Sequence↗
PubMed1996

DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.

Animals↗
PubMed1995

Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands.

We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection. Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.

Base Sequence↗
PubMed1995

The effects of the interosseous membrane and partial fibulectomy on loading of the tibia: a biomechanical study.

The biomechanical basis for the treatment of delayed union of tibial fractures by partial fibulectomy has yet to be fully evaluated. To gain further insight into this problem, nine intact cadaveric lower extremities were instrumented with strain gauges on the surfaces of the tibia and fibula. The limbs were then subjected to axial loading with the ankle and subtalar joints placed in multiple positions. The specimens were loaded either through the distal femur or by direct loading of the tibial plateau. All specimens were first tested intact then after sectioning of the interosseous membrane and finally after partial fibulectomy. It was shown that during loading of the leg, the primary effects of the interosseous membrane were to stabilize the fibula and constrain its posterolateral bending. The fibular strains were not reduced to zero following sectioning of the interosseous membrane. Tibial strains measured on the anteromedial and anterolateral surfaces were consistently in relative tension, indicating a posterior bending force (anterior bowing) of the tibia. After partial fibulectomy, strains on these surfaces became relatively more compressive. With the ankle and subtalar joints in neutral position (0 degree flexion, 0 degree inversion/eversion) the strains on the anterior surface averaged approximately 10% more compressive relative to the intact condition. Tibial strains were observed to vary with the position of the ankle and subtalar joints. The fact that the anteromedial and anterolateral tibia surfaces were always in tension may explain why partial fibulectomy has not proved to be a uniformly successful treatment method for delayed union of the tibia. Furthermore, it points to the important role of "fracture personality" in the selection of treatment.

Aged↗
PubMed1994

An RNA-protein contact determined by 5-bromouridine substitution, photocrosslinking and sequencing.

An analogue of the replicase translational operator of bacteriophage R17, that contains a 5-bromouridine at position -5 (RNA 1), complexes with a dimer of the coat protein and photocrosslinks to the coat protein in high yield upon excitation at 308 nm with a xenon chloride excimer laser. Tryptic digestion of the crosslinked nucleoprotein complex followed by Edman degradation of the tryptic fragment bearing the RNA indicates crosslinking to tyrosine 85 of the coat protein. A control experiment with a Tyr 85 to Ser 85 variant coat protein showed binding but no photocrosslinking at saturating protein concentration. This is consistent with the observation from model compound studies of preferential photocrosslinking of BrU to the electron rich aromatic amino acids tryptophan, tyrosine, and histidine with 308 nm excitation.

Amino Acid Sequence↗
PubMed1994

Telomeric protein-DNA point contacts identified by photo-cross-linking using 5-bromodeoxyuridine.

The Oxytricha telomere protein specifically recognizes single-stranded telomeric DNA, forming an extremely salt resistant and kinetically stable nucleoprotein complex. The absence of information on how this heterodimeric protein binds to DNA prompted this photo-cross-linking study. Multiple protein-DNA photo-cross-links are formed upon UV irradiation of Oxytricha telomeres reconstituted with a synthetic oligonucleotide terminating in 5'-T16T15T14T13G12G11G10G9T8T7T6T5G4G3G2G1-3'. Site-specific substitution of certain nucleotides with 5-bromodeoxyuridine (BrdU) greatly increased the photo-cross-linking yield, each substitution favoring a specific protein-DNA cross-link. For example, substitution of BrdU for T7 resulted in 25% cross-linking of the bound DNA, a 10-fold increase over the unsubstituted DNA. Both subunits of the telomere protein cross-link to, and are therefore near, the DNA. Three point contacts within this nucleoprotein complex, involving the alpha subunit, were established using BrdU substitution: Tyr239, Tyr142, and His292 cross-link to G3, T15, and T7, respectively. One photo-cross-link, Tyr239-G3, occurs amid a short acidic stretch of the alpha subunit, counter to expectations for amino acids that approach the polyanionic DNA. The two remaining cross-links are to amino acids in hydrophobic regions of the primary polypeptide sequence, consistent with the hypothesis that hydrophobic interactions account for the salt resistance (> 2 M NaCl) of this protein-DNA complex. These two photo-cross-links suggest that the telomere protein may bind telomeric single-stranded DNA by intercalation of aromatic residues into a nucleotide lattice.

Amino Acid Sequence↗
PubMed1993

Photocrosslinking of 5-iodouracil-substituted RNA and DNA to proteins.

5-Iodouracil-substituted RNA and DNA were crosslinked regiospecifically to associated proteins in yields of 70 to 94% of bound nucleic acid. Irradiation of the iodouracil chromophore with monochromatic, long-wavelength ultraviolet radiation (325 nanometers) eliminates excitation of other nucleic acid and protein chromophores. The combination of high crosslinking yields, excellent specificity, and elimination of photodamage to other chromophores represents an important advance toward the precise identification of contacts in nucleoprotein complexes.

Animals↗
PubMed1991

A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA.

The well-characterized RNA binding site of the bacteriophage R17 coat protein has been used to investigate the cross-linking of protein to 5-bromouridine (BrU)-substituted RNA using medium-wavelength UV light. We have demonstrated a specific RNA-protein cross-link and identified the site on the RNA of protein attachment. Formation of the covalent complex is dependent upon the presence of BrU at position -5 of the RNA and specific binding of the RNA by coat protein. The amount of cross-linking increases with time and depends on the light source and conditions used. Irradiations using a broad-spectrum UV transilluminator (peak at 312 nm) or monochromatic XeCl excimer laser (308 nm) gave levels of cross-linking exceeding 20 and 50%, respectively. The quantum yield of photo-cross-linking, determined with 308-nm excitation, was 0.003. While little strand breakage or debromination of the RNA occurred, significant protein photodamage was observed.

Base Sequence↗
PubMed1985

Evaluation of a mini-column chromatographic procedure for the measurement of hemoglobin A1c.

An ion-exchange chromatographic procedure is described which facilitates the determination of beta-chain aminoterminal modified glycated hemoglobin. The procedure includes an erythrocyte lysis reagent which eliminates the labile aldimine component (pre-A1c) and a two-stage elution step which separates HbA, 1a + b from HbA1c. This procedure also includes calibrator material which aids in correcting for temperature fluctuations during the analysis. Within-run CV's for samples with HbA1c levels between 4.0% and 13.7% were 1.4 to 3.2%. The between-run CV for an HbA1c control was 5.5%. A comparison of the present test to an ion-exchange HPLC method yielded the equation: HPLC = 0.96 (present method) -0.2% (n = 101 and r = 0.984). Two separate reference range studies yielded comparable results (n = 220/65, mean = 4.77/4.78%, S.D. = 0.68/0.55). Studies with pooled erythrocytes and various lipemic plasmas did not reveal any assay interferences. Various abnormal hemoglobins were studied for their effect on the assay.

Chromatography, Ion Exchange↗
PubMedDate not supplied

Liposome-anchored vascular endothelial growth factor aptamers.

Nuclease-resistant aptamers identified from randomized nucleic acid libraries represent a novel class of drug candidates. Aptamers are synthesized chemically and therefore can be readily modified with functional groups that modulate their properties. We report here on the preparation, initial characterization, and functional properties of a nuclease-resistant vascular endothelial growth factor (VEGF) aptamer anchored in liposome bilayers through a lipid group on the aptamer. While the high-affinity binding to VEGF is maintained, the plasma residence time of the liposome-anchored aptamer is considerably improved compared with that of the free aptamer. The lipid group attachment and/or liposome anchoring leads to a dramatic improvement in inhibitory activity of the aptamer toward VEGF-induced endothelial cell proliferation in vitro and vascular permeability increase and angiogenesis in vivo.

Animals↗