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J Boyd

Publications and source records attributed to J Boyd.

At least 199 records · Page 11Linked to original sources
PubMed1988

Proton-NMR studies show that the Thr-102 mutant of yeast iso-1-cytochrome c is a typical member of the eukaryotic cytochrome c family.

The Thr-102 mutant of yeast iso-1-cytochrome c is a useful system for the study of structure-function relationships in this important class of electron transfer proteins, but little is known about its structure. Furthermore, few assignments of individual amino acid residues in yeast iso-1-cytochrome c have been made by proton NMR. Here we report assignments for nearly half of the amino acids in the reduced Thr-102 mutant of yeast iso-1-cytochrome c. We also report assignments for the oxidized Thr-102 mutant. While the crystal structure of the reduced iso-1-cytochrome c (N. B. not the Thr-102 mutant) has been reported, there is currently little structural information concerning its solution structure and none concerning the oxidized protein. There is also no information concerning the structure of either oxidation state of the Thr-102 mutant. Comparison of the chemical shift and NOE data for the reduced Thr-102 mutant and comparison of paramagnetic shifts for analogous residues between this mutant and horse-heart and tuna cytochromes c reveal that both the basic fold of Thr-102 yeast iso-1-cytochrome c and the region around the site of the mutation are the same as those found in the latter two proteins. It is concluded that the results from structure function studies using the Thr-102 mutant will be applicable to eukaryotic cytochrome c in general. This knowledge allows us to proceed to a description of some mutants of yeast iso-1-cytochrome c in the next paper.

Amino Acids↗
PubMed1988

Two-dimensional NMR as a probe of structural similarity applied to mutants of cytochrome c.

Using site-directed mutagenesis, it is possible to prepare many mutants of a protein in a short time, and to uncover differences in function. To understand the changes in function, it is essential to understand the effect(s) of the mutation in terms of structural and dynamic changes. It is particularly important to establish a rapid method for comparing the structure of the mutants with that of the wild-type protein. We propose that a combination of overlayed and difference two-dimensional NOE spectra between the wild-type and mutant protein provide a rapid method for determination of structural similarity. The observation of differences other than those due directly to the field effects of the exchanged side chain allow both local and distant conformational changes to be assessed. Here we compare NOESY spectra from a mutant of yeast iso-1-ferrocytochrome c in which the invariant residue Phe-82 has been changed to a Tyr. We conclude that NMR can show subtle changes in protein structure. Specifically, we show the change must involve the reorientation of the side chain of Leu-85 which is proximal to the mutation. The dynamics of the aromatic side chain at position 82 are shown not to give rise to measurable differences between the wild-type and mutant protein. Structural changes are not propagated to a measurable degree in other parts of the protein.

Binding Sites↗
PubMed1988

Analysis of the diphtheria tox promoter by site-directed mutagenesis.

By oligonucleotide-directed mutagenesis, we introduced alterations in the two putative -10 regions of the diphtheria tox promoter which are positioned at -50 and -56 from the GUG tox initiation signal. The -10 region positioned at -50 is favored in the expression of ADP-ribosyltransferase activity from the wild-type tox promoter in recombinant Escherichia coli; however, the promoter down mutation at position -50 is compensated for by increased activity of the -10 region positioned at -56.

ADP Ribose Transferases↗
PubMed1988

Effects of trichostrongyle larval challenge on the reproductive performance of immune ewes.

Ostertagia circumcincta larvae were administered to adult immune ewes either during the breeding period or around parturition to investigate whether reproductive performance was affected. Two groups of 25 ewes were given water or 12,000 ostertagia larvae three times weekly for five weeks before and five weeks after breeding to synchronised oestrus. Plasma pepsinogen levels were raised in the larval-challenged ewes during the treatment period but no significant effect of treatment was observed on ovulation rate or plasma progesterone concentration. Neither ewe live-weight nor body condition score differed between groups throughout gestation or for four weeks post partum and there was no effect from larval challenge during the breeding period on lambing rate, lamb birthweight or liveweight at four weeks old. A second experiment involved 20 twin-bearing immune ewes which were administered 17,000 ostertagia larvae three times weekly for three weeks before and three weeks after parturition. Plasma pepsinogen concentration was elevated during the larval challenge period and nematode eggs were detected in the faeces after parturition but there was no effect from treatment either on ewe liveweight and body condition score or on lamb birthweight and lamb liveweight at four weeks old. These results indicate that larval challenge with ostertagia larvae at dose rates comparable to larval intake from heavily contaminated pasture did not affect the general well being and reproductive performance of immune Greyface ewes.

Animals↗
PubMed1988

Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients.

Hematopoietic progenitor cell levels were monitored in the peripheral blood and bone marrow of 30 cancer patients receiving recombinant human granulocyte-colony stimulating-factor (rG-CSF) in a phase I/II clinical trial. The absolute number of circulating progenitor cells of granulocyte-macrophage, erythroid, and megakaryocyte lineages showed a dose-related increase up to 100-fold after four days of treatment with rG-CSF and often remained elevated two days after the cessation of therapy. The relative frequency of different types of progenitor cells in peripheral blood remained unchanged. The frequency of progenitor cells in the marrow was variable after rG-CSF treatment but in most patients was slightly decreased. The responsiveness of bone marrow progenitor cells to stimulation in vitro by rG-CSF and granulocyte-macrophage colony-stimulating factor did not change significantly during rG-CSF treatment. In patients nine days after treatment with melphalan and then rG-CSF, progenitor cell levels were very low with doses of rG-CSF at or below 10 micrograms/kg/d, but equaled or exceeded pretreatment values when 30 or 60 micrograms/kg/d of rG-CSF was given.

Bone Marrow↗
PubMed1987

High-resolution 1H NMR study of the solution structure of alamethicin.

A 1H NMR study of the peptide alamethicin, which forms voltage-gated ion channels in membranes, is described. The molecule was studied in methanol as a function of temperature and pH. A complete assignment of the spectra is given, including several stereospecific assignments. Alamethicin was found to have a structure substantially similar to the crystal although, in solution, the C-terminal dipeptide adopts a somewhat extended conformation. The overall conformation was insensitive to the ionization of the side chain of the only ionizable group, Glu-18.

Alamethicin↗
PubMed1987

1H nuclear magnetic resonance studies of an integral membrane protein: subunit c of the F1F0 ATP synthase.

The membrane-traversing subunit c parallel from the F0 part of the ATP synthase molecule has been studied in chloroform/methanol by high-resolution 1H n.m.r. Various one-dimensional and two-dimensional techniques have been used for assignment purposes, some NOE connectivities were established and some 3JHN alpha coupling constants were measured from spin--echo experiments. The effects of varying pH, solvent composition, lanthanide concentration and temperature have been investigated. Evidence is presented that the molecule has extensive alpha-helical segments, and the hairpin structure suggested by other groups is supported by our n.m.r. data. Only one ionizable group, assigned to the C-terminal carboxyl, is observed to titrate in the pH range 2 to 10; so the conserved residue, Asp61, which binds dicyclohexylcarbodiimide, presumably has (at least in this solvent system) an abnormally high pK value.

Amino Acid Sequence↗
PubMed1987

The solution conformations of the subclasses of human IgG deduced from sedimentation and small angle X-ray scattering studies.

The solution conformations of human immunoglobulin G subclass molecules have been investigated by sedimentation and small-angle X-ray scattering techniques. Both methods qualitatively indicate IgG3 to be an extended molecule relative to IgG1. Sedimentation data have been collected for a number of paraproteins of all four subclasses and the hinge-deleted IgG1Dob protein. The known crystal structure of Dob allows the use of this protein as a basis for the proposal of models of the average conformations of IgG subclasses which are consistent with experimental s(0)20,w values. IgG1 is suggested to have a hinge length of 0-15 A and non-coplanar Fab arms; IgG2 to be effectively hingeless with folded-back Fab arms; IgG3 to have an extended hinge of the order of 100 A and IgG4 to be effectively hingeless and T-shaped. The possible correlation of these conformations with subclass function is discussed.

Humans↗
PubMed1987

A method for the rapid assignment of 1H NMR spectra of oligosaccharides using homonuclear Hartmann-Hahn spectroscopy.

An NMR method is described, based on coherence transfer in the rotating frame, which allows for the rapid determination of proton resonance assignments in oligosaccharides. The composite one-dimensional spectrum of the oligosaccharide is decomposed into a subset of spectra that are derived from individual monosaccharide residues. Proton resonance assignments can be obtained from these subspectra by inspection of the multiplicities of the component resonances and by spectral simulation. The method is shown to have particular value in the assignment of proton resonances derived from primary sequence branch-point residues, which often have complex spin-coupling patterns. Other advantages of this method over conventional techniques are discussed.

Acetylglucosamine↗
PubMed1987

Genetic assembly and selective toxicity of diphtheria-toxin-related polypeptide hormone fusion proteins.

The reports of Miyanohara et al. (1986) and Murphy et al. (1986) were the first to describe the genetic construction, expression, and receptor-specific selective toxicity of a chimaeric toxin. In the present report, we have extended these earlier observations and have shown that the fusion of a modified gene encoding IL-2 to a truncated diphtheria toxin gene also results in the expression of a biologically active chimaeric IL-2 toxin. In both instances we have used receptor-binding-domain substitution and have genetically coupled those portions of the diphtheria toxin structural gene that encode the ADP-ribosyl transferase activity of fragment A and lipid-associating domains of fragment B to modified genes which encode either the polypeptide hormone alpha-MSH or the T-cell growth factor IL-2. The chimaeric toxins expressed from these gene fusions have been shown to be selectively targeted to those eukaryotic cells that carry specific surface receptors for the ligand compounds of the hybrid. For example, in the case of the IL-2 toxin, it is clear that the selective action of this hybrid protein is based upon both its diphtheria-toxin and IL-2-related components. Following binding to the IL-2R on activated and/or malignant T-cell, IL-2 toxin is internalized by receptor-mediated endocytosis. Upon acidification of the endosome, diphtheria toxin fragment B portions of the chimaeric toxin facilitate the delivery of fragment A to the cytosol where it catalyses the ADP ribosylation of EF-2. The assembly of chimaeric toxins at the level of the gene offers several advantages over chemical linkage. Since chemical linkage of the toxophore and ligand components of the conjugate toxins requires activation of the epsilon-amino moiety of lysine residues with reagents that will allow for subsequent disulphide linkage, the precise site of coupling is generally not known. In addition, there has been considerable concern over the lability of the disulphide bond between the toxophore and ligand components in vivo due to the action of disulphide reductases. The assembly of chimaeric toxins at the level of the gene allows for precise linkage of the toxophore and ligand components. Since the linkage between the toxophore and ligand is a peptide bond, the chimaeric toxin should be stable in vivo. In addition, the genetic construction of chimaeric toxins also allows for further protein engineering through site-directed mutagenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1986

Conformational transitions in N-linked oligosaccharides.

An assignment strategy involving 1H-1H correlated spectroscopy (COSY), relayed correlation spectroscopy (RECSY), nuclear Overhauser effect spectroscopy (NOESY), and triple quantum filtered correlated spectroscopy (TQCOSY) is described for six related N-linked oligosaccharides. These are of three "types", i.e., complex, bisected complex, and oligomannose. Using spin-spin coupling constant data derived from these assignments, together with semiempirical quantum mechanical energy calculations, we have examined the rotamer distributions at the Man alpha 1-6Man beta-linkage in each structure, and additionally at the Man alpha 1-6Man alpha-linkage in oligomannose oligosaccharides. We show that while several primary sequence differences are "passive", certain key residues modulate the orientation of the alpha 1-6 arms. These residues may be proximal or distal to the site of the conformational change. There is no direct correlation between these perturbations and the oligosaccharide type. These data are discussed in terms of the proposed recognition function of oligosaccharides in biological systems.

Animals↗
PubMed1986

Monoclonal antibodies to human hypoglossal nucleus which stain neurons and astrocytes in normal brains and brains from cases of Alzheimer-type dementia.

Monoclonal antibodies were raised to membranes of hypoglossal nuclei from normal human post-mortem brain. Two of these clones were recloned to yield antibodies ES.18 and ES.19. Antibody ES.18 stained some, but not all, neuronal perikarya in the medulla oblongata and other brain areas. Neurons stained by this antibody did not have a common neurotransmitter or physiological function, although they tended to be large. Perikarya in the basal forebrain nucleus from a case of Alzheimer-type dementia were stained much more intensely by ES.18 than were these perikarya in a control brain. Antibody ES.19 did not stain neuronal perikarya but stained glial fibrillary acidic protein-positive processes below the pia, in the subependymal layer and in the molecular layer of the cerebellum of control and Alzheimer brains. This antibody also stained the numerous glial fibrillary acidic protein-positive astrocytes in Alzheimer cerebral cortex, but did not stain glial fibrillary acidic protein-positive astrocytes in the white matter of brains from controls or cases of Alzheimer-type dementia. The staining pattern of ES.19 suggests that fibrous astrocytes in Alzheimer cerebral cortex are antigenically different from fibrous astrocytes in white matter.

Alzheimer Disease↗
PubMed1986

Genetic construction, expression, and melanoma-selective cytotoxicity of a diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion protein.

The structural gene for diphtheria toxin, tox, has been modified at its Sph I site by the introduction of an oligonucleotide linker encoding a unique Pst I restriction endonuclease site and a synthetic oligonucleotide encoding alpha-melanocyte-stimulating hormone (alpha-MSH). The resulting fusion gene directs the expression of a diphtheria toxin-related alpha-MSH hybrid protein in which the diphtheria toxin receptor-binding domain has been replaced with alpha-MSH sequences. The chimeric toxin has been partially purified from periplasmic extracts of recombinant Escherichia coli K-12 and has been found to be selectively toxic for alpha-MSH receptor-positive human malignant melanoma NEL-M1 cells in vitro.

ADP Ribose Transferases↗
PubMed1986

In vitro activity of difloxacin hydrochloride (A-56619), A-56620, and cefixime (CL 284,635; FK 027) against selected genital pathogens.

Management of sexually transmitted diseases is facilitated by having antimicrobial agents with activity against all of the major genital pathogens. Newer quinolones show promise of being active against Neisseria gonorrhoeae and Chlamydia trachomatis. Two quinolones, difloxacin (A-56619) and A-56620, and an oral cephalosporin, cefixime (CL 284,635; FK 027), were evaluated in vitro. All three were highly active against 400 isolates of N. gonorrhoeae, including penicillinase-producing N. gonorrhoeae, N. gonorrhoeae with chromosomally mediated resistance, and isolates with penicillin MICs of less than 1 microgram/ml. Susceptibilities to one antimicrobial agent were usually strongly correlated with susceptibilities to the other antimicrobial agents evaluated, but isolates with increasing resistance to beta-lactams were least likely to show increasing resistance to quinolones. Difloxacin and, to a lesser extent, A-56620 were active against all 10 strains of C. trachomatis, and both had moderate activity against over 200 strains of Gardnerella vaginalis. Based on in vitro activity, difloxacin and A-56620 merit in vivo assessment for management of both C. trachomatis and N. gonorrhoeae infections, and cefixime shows considerable promise for treatment of N. gonorrhoeae infections.

Anti-Bacterial Agents↗
PubMed1986

Intracranial tumors in patients with facial pain.

Over the past 10 years, 2000 patients with facial pain have been evaluated at the Mayfield Neurologic Institute. Sixteen of these patients were found to harbor intracranial tumors. The presenting features of this group of patients are analyzed, and the literature reviewed. Peripherally placed tumors tend to cause atypical facial pain associated with sensory loss. Middle fossa tumors may present as trigeminal neuralgia, but usually cause severe pain of an atypical nature and a progressive neurological deficit. Posterior fossa tumors are most likely to cause trigeminal neuralgia; these neoplasms are usually accompanied by subtle neurological deficits and are readily detected by current diagnostic studies.

Adult↗
PubMed1985

Assignment of resonances in the 1H NMR spectrum of human lysozyme.

Assignments in the 1H NMR spectrum for more than 120 resonances arising from 38 of the 130 amino acid residues of human lysozyme are presented. Assignments have been achieved using a combination of one and two-dimensional NMR techniques. Two-dimensional double-quantum correlated spectroscopy and relayed coherence transfer spectroscopy were found to be particularly useful for the identification of spin systems in the aromatic and methyl regions of the spectrum. These spin systems were assigned to specific residues in human lysozyme with reference to the X-ray crystal structure using one-dimensional nuclear Overhauser enhancement (NOE) data and a computer-based search procedure. Unique assignments were found for resonances of 27 amino acid residues even when a distance constraint on NOE effects of 0.7 nm was used in the search procedure; for the remaining residues closer constraints or additional information were required. The assignments include all but one of the resonances in the aromatic region of the spectrum and all the methyl group resonances in the region upfield of 0.6 ppm. The assignments presented here provide a basis for a comparison of the NMR spectra of human lysozyme and the more widely studied hen lysozyme.

Amino Acids↗