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Biomedical subjects

J D Scott

Publications and source records attributed to J D Scott.

At least 127 records · Page 7Linked to original sources
PubMed1994

Oral atovaquone compared with intravenous pentamidine for Pneumocystis carinii pneumonia in patients with AIDS. Atovaquone Study Group.

OBJECTIVE: To test the hypothesis that the therapeutic success rate of oral atovaquone is not worse than that of intravenous pentamidine in the primary treatment of mild and moderate Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome and to detect differences in the toxicity rates of the two treatments. DESIGN: Patients were randomly assigned to receive 21 days of open-label therapy with either atovaquone, 750 mg orally with meals three times daily, or intravenous pentamidine, 3 to 4 mg per kg body weight once daily. SETTING: Multicenter study including university and community treatment facilities. PATIENTS: Patients with human immunodeficiency virus infection and clinical presentations consistent with mild or moderate P. carinii pneumonia were eligible. For efficacy and safety analyses, patients with histologically confirmed P. carinii pneumonia were emphasized. MEASUREMENTS: Patients were monitored by clinical and laboratory evaluations for therapeutic efficacy and adverse events during the acute treatment phase and for 8 weeks after therapy was discontinued. RESULTS: As initial therapy for a histologically confirmed episode of P. carinii pneumonia, 56 patients received atovaquone and 53 received pentamidine. More patients were successfully treated with atovaquone (57%) than with pentamidine (40%), a difference of 17% (95% CI, -3% to 38%; P = 0.085), but more patients failed to respond to atovaquone (29%) than to pentamidine (17%), a difference of 12% (CI, -6% to 29%; P = 0.18). Discontinuation of original therapy because of treatment-limiting adverse events was more frequent in the pentamidine group (36%) than in the atovaquone group (4%) (difference, -32%; CI, -48% to -17%; P < 0.001). Nine patients in each treatment group died during the study. CONCLUSIONS: Oral atovaquone and intravenous pentamidine have similar rates for successful treatment of mild and moderate P. carinii pneumonia, but atovaquone has significantly fewer treatment-limiting adverse events.

AIDS-Related Opportunistic Infections↗
PubMed1994

Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons.

Phosphorylation of molecules involved in synaptic transmission by multifunctional protein kinases modulates both pre- and post-synaptic events in the central nervous system. The positioning of kinases near their substrates may be an important part of the regulatory mechanism. The A-kinase-anchoring proteins (AKAPs; ref. 3) are known to bind the regulatory subunit of cyclic AMP-dependent protein kinase A with nanomolar affinity. Here we show that anchoring of protein kinase A by AKAPs is required for the modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate channels. Intracellular perfusion of cultured hippocampal neurons with peptides derived from the conserved kinase binding region of AKAPs prevented the protein kinase A-mediated regulation of AMPA/kainate currents as well as fast excitatory synaptic currents. This effect could be overcome by adding the purified catalytic subunit of protein kinase. A control peptide lacking kinase-binding activity had no effect. To our knowledge, these results provide the first evidence that anchoring of protein kinase A is crucial in the regulation of synaptic function.

A Kinase Anchor Proteins↗
PubMed1994

Cloning and characterization of AKAP 95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase.

The subcellular location of the type II cAMP-dependent protein kinase is dictated by the interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Using an interaction cloning strategy with RII alpha as a probe, we have isolated cDNAs encoding a novel 761-amino acid protein (named AKAP 95) that contains both RII- and DNA-binding domains. Deletion analysis and peptide studies revealed that the RII-binding domain of AKAP 95 is located between residues 642 and 659 and includes a predicted amphipathic helix. Zinc overlay and DNA binding studies suggest that the DNA-binding domain is composed of two CC/HH-type zinc fingers between residues 464 and 486 and residues 553 and 576. The AKAP was detected in a nuclear matrix fraction, and immunofluorescence using purified anti-AKAP 95 antibodies revealed a distinct nuclear staining in a variety of cell types. Direct overlay of fluorescein isothiocyanate-labeled RII alpha onto fixed rat embryo fibroblasts showed that high-affinity binding sites for RII exist in the nucleus and that these sites are blocked by an anchoring inhibitor peptide. Furthermore, AKAP 95 was detected in preparations of RII that were purified from cellular extracts using cAMP-agarose. The results suggest that AKAP 95 could play a role in targeting type II cAMP-dependent protein kinase for cAMP-responsive nuclear events.

A Kinase Anchor Proteins↗
PubMed1994

Type II regulatory subunits of cAMP-dependent protein kinase and their binding proteins in the nervous system of Aplysia californica.

Two type II regulatory (R) subunits of cAMP-dependent protein kinase (PKA) of 50 and 47 kDa have been identified in Aplysia neurons by several criteria which include phosphorylation by the catalytic subunit of PKA and nanomolar affinity for a peptide fragment of the human thyroid protein Ht 31, properties that in mammals distinguish type II from type I R subunits. The neuronal type II R subunits are differentially localized within cells. For example, the 50-kDa polypeptide is enriched in taxol-stabilized microtubules. In addition, at least seven high molecular mass neuronal RII-binding proteins ranging in mass from 110 to 420 kDa have been demonstrated by a blot overlay technique, which uses 32P-labeled bovine RII alpha as a probe. The RII-binding proteins also exhibit discrete patterns of subcellular localization. For example, the 420 kDa species is enriched in taxol-stabilized microtubules and therefore may serve to anchor the 50-kDa RII subunit. The localization of PKA through the association of RII subunits with the binding proteins may anchor the multifunctional kinase close to key substrates and thereby contribute to the spatial organization required to mediate the orderly phosphorylation events that underly neuronal modulation.

Animals↗
PubMed1994

Human regulatory subunit RI beta of cAMP-dependent protein kinases: expression, holoenzyme formation and microinjection into living cells.

The human regulatory subunit RI beta of cAMP-dependent protein kinases was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Purification was performed by affinity chromatography on glutathione-agarose beads after cleavage with thrombin. The human recombinant RI beta protein migrated at 55 kDa on SDS-PAGE and displayed immunoreactivity with an anti-human RI beta antiserum. Furthermore, the purified recombinant RI beta protein was shown to exist as a dimer that was able to form holoenzyme with the catalytic subunit C alpha. The rate of RI beta 2C alpha 2 holoenzyme formation was faster in the presence than in the absence of MgATP. The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presence of cAMP resulted in holoenzyme dissociation and release of active C alpha-subunit, due to cAMP binding to RI beta. Compared to a RI alpha 2C alpha 2 holoenzyme, the RI beta 2C alpha 2 holoenzyme exhibited a more than twofold higher sensitivity to cAMP. The subcellular localization of RI beta was analyzed in quiescent REF-52 fibroblasts and Wistar rat thyroid (WRT) cells after microinjection of fluorescently labeled proteins into the cytoplasm. A cytoplasmic distribution was observed when free RI beta was injected, whereas free C alpha injected into the cytoplasm appeared in the nucleus. When holoenzymes with labeled RI beta and unlabeled C alpha, or unlabeled RI beta and labeled C alpha, were injected, unstimulated cells showed fluorescence in the cytoplasm of both cell types. REF-52 cells stimulated with 8-bromo-cAMP (8-Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluorescence mainly in the cytoplasm when RI beta was the labeled subunit of the in vivo dissociated holoenzyme. In contrast, nuclear fluorescence was evident from the release and translocation of labeled C alpha from the holoenzyme complex after stimulation with 8-Br-cAMP or TSH.

Allosteric Regulation↗
PubMed1994

Stickler syndrome: correlation between vitreoretinal phenotypes and linkage to COL 2A1.

Stickler syndrome is an autosomal dominantly inherited condition characterised by ocular, articular, facial, auditory and oral features. There is locus heterogeneity with about two thirds of families showing linkage to the gene encoding type II procollagen (COL 2A1). Clinical overlap with Marshall's, Wagner's and other syndromes has caused considerable confusion but the importance of the congenital vitreous anomaly, as first described by Scott, has not previously been emphasised. This study examines the linkage of two vitreo-retinal phenotype subgroups of Stickler syndrome to COL 2A1. A total of 97 affected patients from 24 pedigrees were examined. This is the largest published series of Stickler syndrome patients to date and all have undergone full clinical and ophthalmological examination by a single investigator. A clinical classification is proposed based on vitreoretinal phenotype. All patients demonstrating the congenital vitreous anomaly have been designated Stickler syndrome type 1 and those without the congenital vitreous anomaly as Stickler syndrome type 2 patients. There were 69 affected patients from 20 unrelated type 1 pedigrees and 28 affected patients from 4 unrelated type 2 pedigrees. Using two markers at the COL 2A1 locus, Stickler syndrome type 1 pedigrees showed complete linkage to COL 2A1 with a maximum lod score of 12.33 at zero recombination. Linkage to COL 2A1 was excluded in the two type 2 pedigrees that were informative. From these data it appears that this clinical classification is a useful first step in resolving the genetic heterogeneity in this condition.

Base Sequence↗
PubMed1994

Vitreous detachment and the posterior hyaloid membrane: a clinicopathological study.

This study examines the prevalence and clinicopathological features of posterior hyaloid membrane separation, or 'true' vitreous detachment, in a randomly selected population of general medical in-patients. One hundred and seven eyes of 60 patients were examined clinically using slit lamp biomicroscopy; in 57% the posterior hyaloid membrane was detached and in 43% the posterior hyaloid membrane was attached. Eighteen eyes have subsequently been examined immunohistochemically, demonstrating that the posterior hyaloid membrane, including the Weiss ring, is made up of type IV collagen. The implications of these findings are discussed.

Age Factors↗
PubMed1994

Cyclocryotherapy for chronic glaucoma after vitreoretinal surgery.

The case records were reviewed of 27 patients with chronic glaucoma after vitreoretinal surgery who underwent 28 cyclocryotherapy procedures between March 1987 and March 1992. The average intraocular pressure after 3 months was between 11.0 and 13.3 mmHg with an average fall of 24-26 mmHg. More than 85% had intraocular pressures of less than 21 mmHg after 3 months; 28% were hypotonic (IOP < 6 mmHg). Six months postoperatively, 68% maintained or had improved vision. The hypotonic eyes were found to have deterioration in vision more frequently than those with an intraocular pressure > 5 mmHg (57% compared with 24%). The odds of a hypotonic eye losing vision were 4.27 times greater than for a non-hypotonic eye. Cyclocryotherapy was successful in relief of pain in all 4 eyes which were painful pre-operatively.

Adolescent↗
PubMed1993

Follicle-stimulating hormone regulation of A-kinase anchoring proteins in granulosa cells.

It has been well established that the biochemical and morphological changes during maturation of granulosa cells that are induced by follicle-stimulating hormone (FSH) occur through the elevation of intracellular cAMP and consequent activation of the cAMP-dependent protein kinase (PKA). In this report we show that FSH action alters the expression of A-Kinase Anchoring Proteins (AKAPs), which function to target the subcellular distribution of the type II PKA. Exposure of granulosa cells grown in primary culture with FSH and estradiol for 72 h resulted in the up-regulation of an 80-kDa AKAP and the RII beta subunit of PKA, whereas cells grown in control medium containing only estradiol produced a time-dependent increase of a 140-kDa AKAP. RII overlays performed with [32P]RII alpha preferentially detected RII-binding bands of 80 and 95 kDa compared to blots probed with [32P]RII beta, suggesting that FSH may alter the subcellular location of PKA in an isoform-specific manner. FSH treatment causes a translocation of RII alpha from the particulate to the cytosolic fraction coincident with the induction of the 80-kDa AKAP, which is also predominately cytosolic. These data suggest that FSH promotes a redistribution of the type II PKA holoenzyme through the selective induction of an RII isoform-specific AKAP.

A Kinase Anchor Proteins↗
PubMed1993

A-kinase anchoring proteins: a key to selective activation of cAMP-responsive events?

The cAMP-dependent protein kinase (PKA) regulates a variety of diverse biochemical events through the phosphorylation of target proteins. Because PKA is a multifunctional enzyme with a broad substrate specificity, its compartmentalization may be a key regulatory event in controlling which particular target substrates are phosphorylated. In recent years it has been demonstrated that differential localization of the type II holoenzyme is directed through interaction of the regulatory subunit (RII) with a family of A-Kinase Anchoring Proteins (AKAPs). In this report, we review evidence for PKA compartmentalization and discuss the structural and functional properties of AKAPs.

Amino Acid Sequence↗
PubMed1993

FG7142 causes opposite changes in [3H]GABA release from nigrocollicular regions.

The activity of GABAergic neurons projecting from the striatum to the substantia nigra (SN) and from the SN to the superior colliculus (SC) may be involved in regulating seizure sensitivity such that striatonigral transmission is decreased and nigrocollicular transmission is increased in proconvulsant states. To test whether these changes occur in FG7142-treated rats, GABA transmission was assessed by measuring [3H]GABA release from superfused slices of the SN and SC and measuring [35S]TBPS binding to GABAA receptors throughout the brain. Nine daily injections of FG7142 (30 mg/kg IP) greatly increased myoclonic seizures in about one half of the animals. These animals exhibited a decrease in stimulated [3H]GABA release from the SN and an increase in both basal and stimulated release from the SC. Animals that were less sensitive to FG7142 treatment also had increased collicular release but not decreased nigral release. [35S]TBPS binding was unchanged by FG7142 treatment. Thus, decreased nigral GABA release may contribute to decreased striatonigral transmission after seizure occurrence whereas increased collicular GABA release may contribute to increased nigrocollicular transmission preceding multiple-seizure occurrence.

Animals↗
PubMed1992

Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79.

Postsynaptic densities (PSD) are a network of proteins located on the internal surface of excitatory synapses just inside the postsynaptic membrane. Enzymes associated with the PSD are optimally positioned to respond to signals transduced across the postsynaptic membrane resulting from excitatory synaptic transmission or neurotransmitter release. We present evidence suggesting that type II cAMP-dependent protein kinase (PKA) is anchored to the PSD through interaction of its regulatory subunit (RII) with an A-Kinase Anchor Protein (AKAPs). A cDNA for the human RII-anchoring protein, AKAP 79, was isolated by screening an expression library with radiolabeled RII. This cDNA (2621 base pairs) encodes a protein of 427 amino acids with 76% identity to bovine brain AKAP 75 and 93% identity to a carboxyl-terminal RII-binding fragment of murine brain AKAP 150. A bacterially expressed 92-amino acid fragment, AKAP 79 (335-427) was able to bind RII alpha. Disruption of secondary structure by site-directed mutagenesis at selected residues within a putative acidic amphipathic helix located between residues 392 and 408 prevented RII binding. Immunological studies demonstrate that AKAP 79 is predominantly expressed in the cerebral cortex and is a component of fractions enriched for postsynaptic densities. AKAP antisera strongly cross-react with a 150-kDa protein in murine PSD believed to be AKAP 150. Co-localization of the type II PKA in purified PSD fractions was confirmed immunologically by detection of RII and enzymologically by measuring cAMP-stimulated phosphorylation of the heptapeptide substrate Kemptide. Approximately 30% of the PSD kinase activity was specifically inhibited by PKI 5-24 peptide, a highly specific inhibitor of PKA. We propose that AKAP 79 and AKAP 150 function to anchor the type II PKA to the PSD, presumably for a role in the regulation of postsynaptic events.

A Kinase Anchor Proteins↗
PubMed1992

Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.

A Kinase Anchor Proteins↗
PubMed1992

A single step purification for recombinant proteins. Characterization of a microtubule associated protein (MAP 2) fragment which associates with the type II cAMP-dependent protein kinase.

A 167 base pair DNA cassette has been constructed to facilitate the detection and purification of recombinant proteins. This cassette, kfc, encodes three distinct peptide units: a phosphorylation site for the cAMP-dependent protein kinase (PKA), called kemptide, a factor Xa cleavage site, and a calmodulin-binding peptide. Expressed kfc fusion proteins can be purified from bacterial lysates in one step by affinity chromatography on calmodulin-agarose using EGTA as eluant. As a test of this system, we describe the expression, purification and characterization of the PKA binding domain of the microtubule associated protein (MAP 2).

Amino Acid Sequence↗