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C W Gray

Publications and source records attributed to C W Gray.

At least 37 records · Page 2Linked to original sources
PubMed1994

PA28 activator protein forms regulatory caps on proteasome stacked rings.

The proteasome is a 700,000 dalton proteolytic complex found in eukaryotes and, in a simpler form, in archaebacteria. Its distinctive architecture consists of a stack of four rings, each containing approximately six to eight 21,000 to 35,000 dalton subunits. In this report, we describe the use of electron microscopy of negatively stained specimens, including alignment and averaging of multiple digitized images, to investigate the complexes formed between eukaryotic proteasomes and a recently-isolated 28,000 dalton eukaryotic proteasome activator, PA28. We find that purified PA28, which was previously shown to have a native molecular mass consistent with oligomerization of the PA28 polypeptide, occurs as a ring of variably-positioned protein subunits. In one set of images, these subunits appear to be tethered to a central hub. When incubated with proteasomes, PA28 forms oligomeric regulatory caps on both ends of eukaryotic proteasomes, with the proteasome outer rings serving as scaffolds to which the bases of the regulatory caps are attached. The base of each cap consists of a thickened protein mass, so that the base is wider than the tip of the cap. A stain-filled channel penetrates into each cap from its tip, indicating that the protein subunits tend to separate somewhat at the tip. The caps are about 10 to 11 nm wide at the base and 7 to 8 nm long from base to tip. This is the first direct visualization of the interaction between proteasomes and a purified, functionally characterized protein modulator of proteasome activity, and it gives the first insight as to the particle geometry through which activation by PA28 must occur. The capping at both ends implies the existence of an underlying 2-fold symmetry (or pseudosymmetry) in the eukaryotic proteasome, suggesting that proteasomes may consist of two identical or quasi-identical structural units which interact at a central interface.

Animals↗
PubMed1993

Regulation of beta-amyloid precursor protein isoform mRNAs by transforming growth factor-beta 1 and interleukin-1 beta in astrocytes.

In cultured astrocytes, all three major transcripts of beta-amyloid precursor protein (APP) were expressed with the ratio for APP695, APP751 and APP770 isoform mRNAs being 1:4:2. In comparison with controls, treatment of astrocytes with transforming growth factor-beta 1 (TGF-beta 1) produced about 6 fold increase in total APP mRNA, while elevation in the interleukin-1 beta (IL-1 beta) treated group was small and may relate to the mitogenic effect of IL-1 beta on astrocytes. Treatment of astrocytes with cytokines also produced marked changes in the upregulation in expression of different APP isoforms. The net increase in mRNAs of KPI-containing isoforms APP751 and APP770 was relatively more than for the APP695 isoform. This phenomenon was mainly related to the differences in the expression of KPI-containing APP isoforms and APP695 isoform in the controls. The present findings provide further evidence for the involvement of astrocytes in a cascade of events leading to the development of senile plaques in Alzheimer's disease and Down's syndrome.

Alzheimer Disease↗
PubMed1993

Induction of beta-amyloid precursor protein isoform mRNAs by bFGF in astrocytes.

All three major transcripts of beta-amyloid precursor protein (APP) were expressed in astrocytes. The ratio for APP695, APP751 and APP770 isoform mRNAs being 1:4:2. Treatment with bFGF and EGF produced upregulation of APP mRNAs in cultured astrocytes in varying proportions. In comparison with controls, increases in APP mRNAs in bFGF and EGF treated groups were about 4.5 and 2 fold, respectively. The elevation in mRNAs of KPI-containing isoforms APP751 and APP770 was relatively more than the elevation in mRNA of APP695 isoform. Our findings provide further evidence for the involvement of astrocytes in a cascade of events leading to the development of senile plaques in Alzheimer's disease and Down's syndrome.

Amyloid beta-Protein Precursor↗
PubMed1992

Characterization of a neurotrophic factor produced by cultured astrocytes involved in the regulation of subcortical cholinergic neurons.

When dissociated subcortical cells were cultured in the presence of conditioned medium of relatively differentiated astrocytes (ACM), a marked increase was observed in the expression of choline acetyltransferase (ChAT), an enzyme required for the synthesis of the neurotransmitter acetylcholine. Astrocytes from the target regions of subcortical neurons, the hippocampus and the cerebral cortex, produced neurotrophic factor consistently more than those derived from the nontarget region, the cerebellum. The production of cholinergic trophic activity was increased with the maturation of astrocytes. Even though, nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF) are known cholinergic trophic compounds produced by astrocytes in vitro, a large part of the neurotrophic activity in our ACM was not related to either of these 2 factors. This is because (i) ACM and NGF produced an additive effect on ChAT activity, (ii) only a small proportion of the cholinergic trophic activity in ACM was abolished by anti-NGF antibody, and (iii) treatment with CNTF had no effect on ChAT activity of basal forebrain cholinergic neurons. On the other hand, when cholinergic neurons are cultured on a preformed layer of astrocytes, addition of basal fibroblast growth factor (bFGF) failed to increase further the ChAT activity. Similarly the effects of ACM and bFGF were not additive. A large proportion of the cholinergic trophic activity in ACM was neutralized by anti-bFGF antibody. These findings would suggest that the trophic activity on septal cholinergic neurons in our ACM was due to bFGF or a bFGF-like compound.

Acetylcholine↗
PubMed1992

Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate.

A factor of low M(r) with growth-promoting effects on rabbit embryos was extracted and purified from commercial bovine serum albumin (BSA). This embryotrophic factor was extracted from BSA dissolved in formic acid by membrane filtration (membrane cutoff of M(r) 10,000) and then freeze-drying of the filtrate. The extract was purified successively by chromatography on G-10 Sephadex, QAE-Sephadex A-25 anion exchange and high-performance liquid chromatography (HPLC) reverse-phase columns. Mass spectrometry of the active reverse-phase material indicated that the major component in this material had an M(r) of 192. The embryotrophic factor in the low M(r) extract of BSA was shown to be citrate, because: (i) the mass spectra of the active reverse-phase material and citrate were identical, (ii) the activity was eluted at the identical position to citrate on an analytical HPLC anion-exchange column, (iii) the original BSA sample was shown by enzyme assay to be heavily contaminated by citrate and (iv) citrate stimulated cell proliferation and expansion of blastocysts.

Animals↗
PubMed1989

13C NMR of methylated lysines of fd gene 5 protein: evidence for a conformational change involving lysine 24 upon binding of a negatively charged lanthanide chelate.

Helical complexes formed between fd DNA and reductively methylated fd gene 5 protein were indistinguishable by electron microscopy from complexes formed with the nonmethylated protein. 13C NMR spectroscopy of 13C-enriched N epsilon, N epsilon-dimethyllsyl residues of the protein showed that three of these residues (Lys-24, Lys-46, and Lys-69) were selectively perturbed by binding of the oligomer d(pA)7. These were the same lysyl residues that we previously found to be most protected from methylation by binding of the protein to poly[r(U)] [Dick, L. R., Sherry, A. D., Newkirk, M. M., & Gray D. M. (1988) J. Biol. Chem. 263, 18864-18872]. Thus, these lysines are probably directly involved in the nucleic acid binding function of the protein. Negatively charged chelates of lanthanide ions were used to perturb the 13C NMR resonances of labeled lysyl and amino-terminal residues of the gene 5 protein. The terbium chelate was found to bind tightly (Ka approximately 10(5) M-1) to the protein with a stoichiometry of 1 chelate molecule per protein dimer. 13C resonances of Lys-24, Lys-46, and Lys-69 were maximally shifted by the terbium chelate and were maximally relaxed by the gadolinium chelate. Also, the terbium chelate was excluded by the oligomer d(pA)7. Computer fits of the induced chemical shifts of 13C resonances with those expected for various positions of the terbium chelate failed to yield a possible chelate binding site unless the chemical shift for Lys-24 was excluded from the fitting process.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophages↗
PubMed1989

Three-dimensional structure of complexes of single-stranded DNA-binding proteins with DNA. IKe and fd gene 5 proteins form left-handed helices with single-stranded DNA.

Specimen-tilting in an electron microscope was used to determine the three-dimensional architecture of the helical complexes formed with DNA by the closely related single-stranded DNA binding proteins of fd and IKe filamentous viruses. The fd gene 5 protein is the only member of the DNA-helix-destabilizing class of proteins whose structure has been determined crystallographically, and yet a parameter essential to molecular modeling of the co-operative interaction of this protein with DNA, the helix handedness, has not been available prior to this work. We find that complexes formed by titrating fd viral DNA with either the fd or IKe gene 5 protein have a left-handed helical sense. Complexes isolated from Escherichia coli infected by fd virus are also found to be left-handed helical; hence, the left-handed fd helices are not an artefact of reconstitution in vitro. Because the proteins and nucleic acid of the complexes are composed of asymmetric units which cannot be fitted equivalently to right-handed and left-handed helices, these results rule out a previous computer graphics atomic model for the helical fd complexes: a right-handed helix had been assumed for the model. Our work provides a defined three-dimensional structural framework within which to model the protein-DNA and protein-protein interactions of two structurally related proteins that bind contiguously and co-operatively on single-stranded DNAs.

DNA, Bacterial↗
PubMed1986

Circular dichroism of two conformations of poly[d(G-C)] induced by low pH.

Circular dichroism (CD) and UV absorption data showed that poly[d(G-C)] (at 0.09M NaCl, 0.01M Na+ (phosphate), 20 degrees C) underwent two conformational transitions upon lowering of the pH by the addition of HCl. The first transition was complete at about pH 3.0. The second transition was complete upon lowering the pH to 2.6 or upon raising the temperature, at pH 3.0, to about 40 degrees C. There was no indication of denaturation during either transition. The CD spectrum for the second acid conformation had large CD bands including a positive one at 288nm, a characteristic associated with C X C+ base-pairs. Electron microscopy showed no significant formation of condensed supramolecular aggregates corresponding to the first or second acid forms of poly[d(G-C)]. On the basis of spectral data, electron microscopy, and proton-uptake measurements, we propose models for the secondary structures that poly[d(G-C)] adopts in its two acid conformations.

Base Composition↗
PubMed1984

Complex of fd gene 5 protein and double-stranded RNA.

We report the formation of complexes of the single-stranded DNA binding protein encoded by gene 5 of fd virus, with natural double-stranded RNAs. In the first direct visualization of a complex of the fd gene 5 protein with a double-stranded nucleic acid, we show by electron microscopy that the double-stranded RNA complex has a structure which is distinct from that of complexes with single-stranded DNA and is consistent with uniform coating of the exterior of the double-stranded RNA helix by the protein. Circular dichroism spectral data demonstrate that the RNA double helix in the complex is undisrupted, and that perturbation of the 228-nm circular dichroism assigned to protein tyrosines can occur in the absence of intercalation of nucleotide bases with protein aromatic residues. Our findings emphasize the potential importance of interaction with the sugar-phosphate polynucleotide backbone in binding of the fd gene 5 protein to nucleic acids.

Circular Dichroism↗
PubMed1982

Neutron scattering data on reconstituted complexes of fd deoxyribonucleic acid and gene 5 protein show that the deoxyribonucleic acid is near the center.

We have performed low-angle neutron scattering studies on reconstituted complexes of fd DNA and the gene 5 protein that is produced during infection of Escherichia coli by filamentous fd phage. Essentially identical helical complexes have been made with normal protonated DNA or DNA in which at least 87% of the nonexchangeable protons are replaced by deuterium. From neutron scattering profiles of both complexes over a range of D2O/H2O solvent mixtures, the DNA deuteration is shown to have a dramatic influence on the measured cross-sectional radius of gyration. Most importantly, data for the complex containing deuterated DNA lead to a more negative slope in a plot of the square of the cross-sectional radius of gyration vs. the inverse of the solute-solvent contrast, compared with the slope of a plot of data for the complex containing protonated DNA. This means that, in a cross-sectional view of the complex, the DNA is near the center of the structure. By our analysis, the DNA has a cross-sectional radius of gyration of 17.6 +/- 3 A, while the protein has a cross-sectional radius of gyration of about 33.5 A. Therefore, the model for the structure of the helical complex that has been proposed from X-ray diffraction studies on gene 5 protein crystallized with oligodeoxynucleotides [McPherson, A., Jurnak, F., Wang, A., Kolpak, F., Rich, A., Molineux, I., & Fitzgerald, P. (1980) Biophys. J. 32, 155-170] is not valid for the complex in solution. From our neutron diffraction data we have also obtained values for the solvent-excluded volume and mass per unit length. The relation of our findings to the solution structure of the complex is discussed.

Bacteriophages↗
PubMed1976

Surgical repair of tusk injury (pulpectomy) in an adult, male forest elephant (Loxodonta cyclotis).

A 15-year-old male forest elephant housed in a zoo sustained a fracture of the right tusk that was 10 cm inside the cheek pouch, thus exposing the tusk canal. Treatment of the cavity by packing, topical application of antibiotics, and administration of various antiseptic preparations failed; however, the tusk grew. To treat the infected, growing tusks's root canal or pulp, surgery--comparable to a pulpectomy in man--was performed with successful results.

Animals↗