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Biomedical subjects

J Feeney

Publications and source records attributed to J Feeney.

At least 73 records · Page 4Linked to original sources
PubMed1992

Reassessment of the acceptor specificity and general properties of the Lewis blood-group gene associated alpha-3/4-fucosyltransferase purified from human milk.

The acceptor specificity and general properties of a Lewis blood-group gene associated alpha-3/4-L-fucosyltransferase isolated from human milk have been examined at the penultimate purification stage involving affinity chromatography on GDP-hexanolamine Sepharose, and after a subsequent gel filtration step on Sephacryl S-200. Both preparations transferred fucose to the O-4 position of N-acetylglucosamine in Type 1 (Gal beta 1-3GlcNAc-R) acceptors and the O-3 position of glucose in lactose-based (Gal beta 1-4Glc) oligosaccharides, and both used Type 1 sialylated compounds when the terminal N-acetylneuraminic acid was present in alpha-2,3 linkage. The striking difference between the two preparations was in their reactivity with Type 2 (Gal beta 1-4GlcNAc-R) chains; after Sephacryl S-200 chromatography the apparent KM values for the alpha-3/4- preparation with unsubstituted low-molecular-weight Type 2 oligosaccharides were considerably increased. Substitution of the terminal galactose with sialic acid in alpha-2,3 linkage decreased the KM values for low-molecular-weight oligosaccharides but no detectable incorporation of fucose was observed into N-acetyllactosamine end-groups of glycoproteins with N-linked oligosaccharide chains, irrespective of the presence of sialic acid in the terminal sequences.

Carbohydrate Sequence↗
PubMed1991

Characterisation of minor tetra- to hepta-saccharides O-linked to human meconium glycoproteins by t.l.c.-m.s. microsequencing of neoglycolipid derivatives in conjunction with conventional m.s. and 1H-n.m.r. spectroscopy.

As part of the establishment of a data base for core and backbone sequences of O-linked oligosaccharides of human meconium glycoproteins, the minor tetra- to hepta-saccharides released from mild-acid treated blood group [corrected] H-active glycoproteins have been studied. These oligosaccharides are heterogeneous and difficult to isolate, and a t.l.c.-m.s. microsequencing procedure has been applied to the neoglycolipid derivatives, in conjunction with 1H-n.m.r. spectroscopy, methylation analysis, and mass spectrometry (m.s.) of native and methylated oligosaccharides. Among an array of oligosaccharides characterised are those having the branched beta-GlcNAc-(1----6)[beta-Gal- (1----3)]-GalNAcol core, and others with the following linear sequences not characterised previously from this source: beta-Gal-(1----3)-beta-GlcNAc-(1----3)-beta-Gal-(1----3)-GalNAcol, beta-Gal-(1----4)-beta-GlcNAc-(1----3)-beta-Gal-(1----3)-GalNAcol, alpha-GalNAc- (1----3)-beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)-GalNAcol, beta-GlcNAc- (1----3)-beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)- GalNAcol, beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)-beta-Gal-(1----3/4)-beta- GlcNAc-(1----3)-beta-Gal-(1----3)-GalNAcol, and beta-GlcNAc-(1----3)-beta-Gal-(1----3/4)-beta-GlcNAc-(1----3)-beta-Gal- (1----3/4)-beta-GlcNAc-(1----3)-GalNAcol.

Anthocyanins↗
PubMed1991

Dihydrofolate reductase: sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution.

Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential 1H and 15N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D 15N/1H nuclear Overhauser-heteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 1H-1H through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows NOE cross peaks to be detected between NH protons even when their 1H chemical shifts are degenerate as long as the amide 15N chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+1 NOEs define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR.methotrexate.NADPH complex [Bolin et al. (1982) J. Biol. Chem. 257, 13650-13662], while the NMR-determined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-110 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of beta-sheet structure were characterized in the binary complex by strong alpha CHi-NHi+1 NOEs and by slowly exchanging amide protons. In addition, several long-range NOEs were identified linking together these stretches to form a beta-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR.methotrexate.NADPH complex, which contains an eight-stranded beta-sheet, indicating that the main body of the beta-sheet is preserved in the binary complex in solution.

Amino Acid Sequence↗
PubMed1991

19F NMR evidence for interactions between the c-AMP binding sites on the c-AMP receptor protein from E. coli.

The 19F NMR spectra of 3-fluorotyrosine containing c-AMP receptor protein (CRP) from E. coli have been recorded in the presence of increasing amounts of c-AMP. One of the signals (from Tyr B) shifts upfield by 0.6 ppm in the presence of excess c-AMP and shows both slow and fast exchange behaviour during the titration. This is evidence for interactions between the two c-AMP binding sites on the CRP dimer leading to different dissociation rate constants (less than or equal to 75 s-1; greater than or equal to 350 s-1) for complexes containing one and two c-AMP molecules.

Binding Sites↗
PubMed1991

Critical care nurse perceptions of family needs.

Family needs research has for the most part focused on the families' perceptions when a significant other is admitted to the intensive care unit. We examined critical care nurse perceptions of family needs. The questionnaire "Needs of Families of Critically Ill Patients" was given to 126 intensive care unit nurses. The tool was an adaptation of Molter's questionnaire "Needs of Relatives of Critically Ill Patients." The revised tool examined nurse perception of family needs, perception of time available to meet the needs in daily practice, and the best professional to meet the family need if the need was identified as best met by someone other than the nurse. The majority of the nurses perceived family needs as important or very important, and 85% of the nurses indicated that they were able to meet family needs and had the time to do so. Cognitive family were ranked higher than psychologic or personal and physical needs. Nurses from the four intensive care units ranked family needs significantly differently, a result that may be influenced by differing patient acuity and patient length of stay on individual units. Nurses' perceptions of family needs were influenced by units worked, length of time practicing in critical care, educational preparation, and length of time in nursing.

Analysis of Variance↗
PubMed1990

NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine.

1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1990

A novel method of preparing totally alpha-deuterated amino acids for selective incorporation into proteins. Application to assignment of 1H resonances of valine residues in dihydrofolate reductase.

The pyridoxal/2H2O exchange reaction of the alpha-CH of amino acids is known to be accompanied by racemisation: Thus by using a D-amino acid as the starting material any L-amino acid formed in the reaction will be essentially fully deuterated at its alpha-position. We have used this method to prepare alpha-deuterated L-valine and incorporated this biosynthetically into L. casei dihydrofolate reductase. A comparison of the alpha CH-NH fingerprint regions of COSY spectra of deuterated and normal DHFR complexes allows one to identify cross-peaks from 15 of the 16 valine residues.

Amino Acids↗
PubMed1990

The 1H-NMR assignments of the aromatic resonances in complexes of Lactobacillus casei dihydrofolate reductase and the origins of their chemical shifts.

All the aromatic proton resonances in the 500-MHz NMR spectra of Lactobacillus casei dihydrofolate reductase have been assigned for several of its complexes with inhibitors. For the complexes with methotrexate and trimethoprim this was achieved by using a combination of NMR techniques in conjunction with a selectively deuterated protein designed to simplify the spectra such that nuclear Overhauser effect (NOE) connections could be detected with greater ease and certainty. By correlating these NOE data with crystal structure data on related complexes it was possible to assign all the aromatic resonances and to extend these assignments to spectra of other complexes of dihydrofolate reductase. The conformation-dependent chemical shifts observed for many of the resonances could be explained qualitatively, but not quantitatively, in terms of ring-current shifts. The discrepancies between calculated ring-current shifts and the observed conformation-dependent shifts could not in general be accounted for satisfactorily in terms of carbonyl-group anisotropic shielding contributions calculated using presently available models. In the case of the H delta 1, delta 2 protons of Phe30 some of the discrepancy probably results from a difference in the conformation of the Phe ring between the solution and crystal states.

Chemical Phenomena↗
PubMed1990

NMR studies of interactions of ligands with dihydrofolate reductase.

NMR spectroscopy is a useful technique for studying interactions, conformations and dynamic processes within ligand-protein complexes. Several examples of the application of the method to studies of complexes of anti-folate drugs with their target enzyme, dihydrofolate reductase, are discussed.

Drug Interactions↗
PubMed1990

19F-n.m.r. studies of ligand binding to 5-fluorotryptophan- and 3-fluorotyrosine-containing cyclic AMP receptor protein from Escherichia coli.

Two fluorine-containing analogues of the cyclic AMP receptor protein (CRP) from Escherichia coli were prepared by biosynthetic incorporation of 5-fluorotryptophan (5-F-Trp) and 3-fluorotyrosine (3-F-Tyr). The 19F-n.m.r. spectrum of the [5-F-Trp]CRP showed two signals corresponding to the two tryptophan residues, and that of the [3-F-Tyr]CRP showed six signals (two overlapping) corresponding to the six tyrosine residues: these results are as expected for a symmetrical dimer. A comparison of the 19F-n.m.r. spectra of the CRP analogues in the presence and in the absence of cyclic AMP reveals that the chemical shifts of both tryptophan residues and of two of the six tyrosine residues show differences. Since none of these residues is in direct contact with the bound nucleotide (although Trp-85 is fairly close), these shift changes must arise from induced conformational effects. The 19F-n.m.r. spectra of complexes with cyclic GMP showed chemical-shift perturbations different from those caused by cyclic AMP, indicating that different conformational changes are induced by the binding of cyclic GMP. The 19F-n.m.r. spectrum of the complex of [3-F-Tyr]CRP with tubercidin 3',5'-(cyclic)monophosphate (which can activate transcription) showed essentially the same chemical-shift changes as seen for the cyclic AMP complex, indicating that similar conformational changes have been induced by the nucleotide binding. [3-F-Tyr]CRP in the presence of an equimolar amount of the 20 bp self-complementary DNA oligomer 5'-AATGTGAGTTAACTCACATT-3' and excess cyclic AMP gave an 19F-n.m.r. spectrum that was almost identical with that for the [3-F-Tyr]CRP-cyclic AMP complex, indicating that the binding of DNA does not induce significant conformational changes involving the tyrosine residues. Proteolysis of [3-F-Tyr]CRP with chymotrypsin produced a 31 kDa fragment that is a dimer containing the cyclic AMP-binding domain. This fragment contains five of the six tyrosine residues, and its 19F-n.m.r. chemical shifts were essentially the same as those of the intact protein except for one missing signal (signal F): this signal could be assigned to Tyr-206 and shown to be unperturbed by the binding of cyclic nucleotide to the intact [3-F-Tyr]CRP. The similarity of the 19F-n.m.r. chemical shifts in the alpha-fragment and the intact CRP indicates that the alpha-fragment retains the same structure as found in the intact protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclic AMP↗
PubMed1990

Cardiac surgery in patients over the age of 80 years.

The risk-benefit relationship of open heart surgery in octogenarians is not well established. Eighty consecutive patients over the age of 80 who underwent cardiac operations under cardiopulmonary bypass were evaluated. Twenty-five patients were in functional class IV, 42 in class III, and 13 in class II. Forty-four patients had only coronary artery bypass grafts (CABG), 12 only aortic valve replacement (AVR), 6 only mitral valve replacement (MVR), 12 CABG and AVR, 4 CABG and MVR, 1 CABG and aneurysmectomy, and 1 had resection of left atrial myxoma. Operative mortality (within 30 days) was 12.5% for the group. Mortality was related to bleeding, left ventricular failure, primary ventricular fibrillation, pulmonary failure, and renal failure. Mortality was higher in patients with (1) advanced functional class, (2) mitral valve replacement, (3) postoperative hemorrhage, and (4) associated pulmonary disease. While a generally conservative approach is recommended for octogenarian patients, many with life-threatening cardiac disease, especially those free of major multisystem illnesses, should not be denied the benefit of surgical treatment.

Aged↗
PubMed1990

Cephalocele involving the oral cavity.

A cephalocele is an uncommon neural tube defect that rarely involves the oral cavity. We present a case of an oral cephalocele associated with polyhydramnios discovered on antenatal ultrasound. The differential diagnosis includes epignathus and epulis. The prognosis is dependent not only on the extent of brain involvement and associated anomalies, but also on the exact location of the herniated mass. If the oral cavity is extensively involved, airway management at birth may be difficult. Therefore, preparations for delivery may include maternal tertiary site referral, antenatal neurosurgical and pediatric consultation, and anticipation of complications such as rupture of the sac, dystocia, or the need for immediate tracheostomy if intubation is not possible.

Abnormalities, Multiple↗
PubMed1989

Characterisation by mass spectrometry and 500-MHz proton nuclear magnetic resonance spectroscopy of penta- and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins).

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500-MHz 1H NMR are described of the major penta- and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied after depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio-Gel P4 chromatography and HPLC. Eleven penta- and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J. 256, 397-401] and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features: (table; see text) Sequences in these oligosaccharides not commonly found in mucins so far studied are chain-terminating GlcNAc alpha 1-4Gal, repeating-type-I (Gal beta 1-3GlcNAc) backbones, the backbone branch GlcNAc beta 1-6(GlcNAc beta 1-3)Gal and the backbone sequence GlcNAc beta 1-6Gal beta 1- in the absence of a substituent at C3 of galactose.

Carbohydrate Conformation↗
PubMed1989

A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei.

A kinetic scheme is presented for Lactobacillus casei dihydrofolate reductase that predicts steady-state kinetic parameters. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. Two major features of this kinetic scheme are the following: (i) product dissociation is the rate-limiting step for steady-state turnover at low pH and follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex; (ii) the rate constant for hydride transfer from NADPH to dihydrofolate (H2F) is rapid (khyd = 430 s-1), favorable (Keq = 290), and pH dependent (pKa = 6.0), reflecting ionization of a single group. Not only is this scheme identical in form with the Escherichia coli kinetic scheme [Fierke et al. (1987) Biochemistry 26, 4085] but moreover none of the rate constants vary by more than 40-fold despite there being less than 30% amino acid homology between the two enzymes. This similarity is consistent with their overall structural congruence. The role of Trp-21 of L. casei dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Trp-21, a strictly conserved residue near both the folate and coenzyme binding sites, was replaced by leucine. Two major effects of this substitution are on (i) the rate constant for hydride transfer which decreases 100-fold, becoming the rate-limiting step in steady-state turnover, and (ii) the affinities for NADPH and NADP+ which decrease by approximately 3.5 and approximately 0.5 kcal mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1989

Photolabile precursors of inositol phosphates. Preparation and properties of 1-(2-nitrophenyl)ethyl esters of myo-inositol 1,4,5-trisphosphate.

1-(2-Nitrophenyl)ethyl esters of D-myo-inositol 1,4,5-trisphosphate (InsP3) have been synthesized and shown to have suitable properties for use as photolabile precursors of InsP3. Synthesis was accomplished by treatment of InsP3 with 1-(2-nitrophenyl)diazoethane in a CHCl3/water mixture. This resulted in esterification of each of the three phosphate residues in InsP3, the 1-phosphate being more reactive than the 4- or 5-phosphate. Singly esterified P-1, P-4, and P-5 esters, termed P-1, P-4, and P-5 caged InsP3, were isolated from the reaction mixture by anion-exchange HPLC and characterized by 500-MHz 1H NMR spectroscopy. Each of these caged InsP3 esters exists as a pair of diastereoisomers and was identified by examining the effects of pH and nitrophenyl ring current shielding on the chemical shifts of nonexchangeable inositol protons. 1H NMR spectra of InsP3 were analyzed for comparison. On photolysis the compounds released InsP3 with rate constants of 175 (P-1), 225 (P-4), and 280 s-1 (P-5) as determined by monitoring the aci-nitro decay reaction at pH 7.1, 0.2 M ionic strength, 21 degrees C. Quantum yields determined by steady-state near-UV photolysis were 0.65 +/- 0.08 for each compound. P-4 and P-5 caged InsP3 were the most promising biologically inactive InsP3 precursors since at concentrations up to 50 microM they did not release Ca2+ from smooth muscle sarcoplasmic reticulum (SR) and were not metabolized by vascular smooth muscle InsP3 5-phosphatase or bovine brain InsP3 3-kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Dihydrofolate reductase: multiple conformations and alternative modes of substrate binding.

The complex of Lactobacillus casei dihydrofolate reductase with the substrate folate and the coenzyme NADP+ has been shown to exist in solution as a mixture of three slowly interconverting conformations whose proportions are pH-dependent [Birdsall, B., Gronenborn, A. M., Hyde, E. I., Clore, G. M., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1982) Biochemistry 21, 5831]. The assignment of the resonances of all the aromatic protons of the ligand molecules in all three conformational states of the complex has now been completed by using a variety of NMR methods, particularly two-dimensional exchange experiments. The resonances of the nicotinamide protons of the coenzyme and the pteridine 7-proton of the folate have different chemical shifts in the three conformations, in some cases differing by more than 1 ppm. Comparison of the COSY spectra of the complex at low pH (conformation I) and high pH (conformations IIa and IIb) with that of the enzyme-methotrexate-NADP+ complex shows only slight differences in the conformation of the protein. The pattern of chemical shift changes in the ligand and the protein indicates that the structural differences are localized within the active site of the enzyme. Nuclear Overhauser effects (NOEs) are observed between the nicotinamide 5- and 6-protons and the methyl resonance of Thr 45 at both low and high pH, indicating that there is no major movement of the nicotinamide ring. By contrast, NOEs are observed between the pteridine 7-proton and the methyl protons of Leu 19 and Leu 27 in conformations I and IIa but not in conformation IIb.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗