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Biomedical subjects

G Perkins

Publications and source records attributed to G Perkins.

At least 19 recordsLinked to original sources
PubMed1999

NH2-Terminal targeting motifs direct dual specificity A-kinase-anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum.

Subcellular localization directed by specific targeting motifs is an emerging theme for regulating signal transduction pathways. For cAMP-dependent protein kinase (PKA), this is achieved primarily by its association with A-kinase-anchoring proteins (AKAPs). Dual specificity AKAP1, (D-AKAP1) binds to both type I and type II regulatory subunits and has two NH2-terminal (N0 and N1) and two COOH-terminal (C1 and C2) splice variants (. J. Biol. Chem. 272:8057). Here we report that the splice variants of D-AKAP1 are expressed in a tissue-specific manner with the NH2-terminal motifs serving as switches to localize D-AKAP1 at different sites. Northern blots showed that the N1 splice is expressed primarily in liver, while the C1 splice is predominant in testis. The C2 splice shows a general expression pattern. Microinjecting expression constructs of D-AKAP1(N0) epitope-tagged at either the NH2 or the COOH terminus showed their localization to the mitochondria based on immunocytochemistry. Deletion of N0(1-30) abolished mitochondrial targeting while N0(1-30)-GFP localized to mitochondria. Residues 1-30 of N0 are therefore necessary and sufficient for mitochondria targeting. Addition of the 33 residues of N1 targets D-AKAP1 to the ER and residues 1-63 fused to GFP are necessary and sufficient for ER targeting. Residues 14-33 of N1 are especially important for targeting to ER; however, residues 1-33 alone fused to GFP gave a diffuse distribution. N1(14-33) thus serves two functions: (a) it suppresses the mitochondrial-targeting motif located within residues 1-30 of N0 and (b) it exposes an ER-targeting motif that is at least partially contained within the N0(1-30) motif. This represents the first example of a differentially targeted AKAP and adds an additional level of complexity to the PKA signaling network.

A Kinase Anchor Proteins↗
PubMed1999

Clinical, haematological and biochemical findings in foals with neonatal Equine herpesvirus-1 infection compared with septic and premature foals.

A retrospective multicentre study comparing historical, clinical, haematological, acid-base and biochemical findings of foals with Equine herpesvirus-1 (EHV-1) infection, septicaemia or prematurity was performed to determine if early diagnosis of EHV-1 foals was possible. Fifty-three foals were studied and were assigned to one of 2 groups: herpes positive (n = 14) or herpes negative (n = 39). The latter group included 20 septic, 11 premature, and 8 premature and septic foals. The presence of herpes antigen was confirmed by immunoperoxidase histochemical staining of tissues from necropsied foals. A nonparametric statistical analysis followed by a backwards elimination logistic regression was performed to establish a model at a P value of <0.05. All herpes positive foals died, while 47% (9/19) of the septic foals survived. Based upon our analysis, herpes positive foals were more likely to have total white blood cell counts less than 3 x 10(9)/l and to be icteric as compared to the septic and premature foals. Despite profound hepatic necrosis in the herpes positive foals, liver enzymes were not elevated and were not significantly different from the controls.

Acid-Base Equilibrium↗
PubMed1998

Evidence for a Cdc6p-independent mitotic resetting event involving DNA polymerase alpha.

Eukaryotic DNA replication is limited to once per cell cycle because cyclin-dependent kinases (cdks), which are required to fire origins, also prevent re-replication. Components of the replication apparatus, therefore, are 'reset' by cdk inactivation at the end of mitosis. In budding yeast, assembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) at origins can only occur during G1 because it is blocked by cdk1 (Cdc28) together with B cyclins (Clbs). Here we describe a second, separate process which is also blocked by Cdc28/Clb kinase and, therefore, can only occur during G1; the recruitment of DNA polymerase alpha-primase (pol alpha) to chromatin. The recruitment of pol alpha to chromatin during G1 is independent of pre-RC formation since it can occur in the absence of Cdc6 protein. Paradoxically, overproduction of Cdc6p can drive both dephosphorylation and chromatin association of pol alpha. Overproduction of a mutant in which the N-terminus of Cdc6 has been deleted is unable to drive pol alpha chromatin binding. Since this mutant is still competent for pre-RC formation and DNA replication, we suggest that Cdc6p overproduction resets pol alpha chromatin binding by a mechanism which is independent of that used in pre-RC assembly.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1998

Nucleotide-dependent prereplicative complex assembly by Cdc6p, a homolog of eukaryotic and prokaryotic clamp-loaders.

Expression of the Cdc6 protein (Cdc6p) is essential for formation of prereplicative complexes at budding yeast replication origins. Analysis of mutations in the conserved nucleoside triphosphate (NTP)-binding site of Cdc6p described here suggests that NTPs are required both for the productive interaction of Cdc6p with replication origins during G1 and the quantitative loading of the Mcm2-7 family of proteins onto chromatin. We show that Cdc6p exhibits significant sequence similarity to subunits of eukaryotic and prokaryotic clamp-loaders, which load ring-shaped DNA polymerase processivity factors onto DNA in an analogous reaction. Similarities in both sequence and mechanism suggest that Cdc6p and the clamp-loaders are members of a superfamily of nucleotide-dependent loading factors.

Alleles↗
PubMed1998

Energy deprivation and a deficiency in downstream metabolic target genes during the onset of embryonic heart failure in RXRalpha-/- embryos.

RXRalpha null mutant mice display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To dissect the molecular basis of the RXRalpha-associated cardiomyopathy, we performed subtractive hybridization and systematically characterized putative downstream target genes that were selectively lacking in the mutant embryos, both at early (E10.5) and late (E13.5) stages of mouse embryonic development. Approximately 50% of the subtracted clones (61/115) encoded proteins involved in intermediary metabolism and electron transport, suggesting an energy deficiency in the RXRalpha-/- embryos. In particular, clone G1, which encodes subunit 14.5b of the NADH-ubiquinone dehydrogenase complex, displayed a dose-dependent expression in the wild-type, heterozygous and RXRalpha mutant mice. This gene was also downregulated in a retinoid-deficient rat embryo model. ATP content and medium Acyl-CoA dehydrogenase mRNA were lower in RXRalpha mutant hearts compared to wild-type mice. Ultrastructural studies showed that the density of mitochondria per myocyte was higher in the RXRalpha mutant compared to wild-type littermates. We propose a model whereby defects in intermediary metabolism may be a causative factor of the RXRalpha-/- phenotype and resembles an embryonic form of dilated cardiomyopathy.

Acyl-CoA Dehydrogenase↗
PubMed1997

The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.

The budding yeast Cdc6 protein (Cdc6p) is essential for formation of pre-replicative complexes (pre-RCs) at origins of DNA replication. Regulation of pre-RC assembly plays a key role in making initiation of DNA synthesis dependent upon passage through mitosis and in limiting DNA replication to once per cell cycle. Cdc6p is normally only present at high levels during the G1 phase of the cell cycle. This is partly because the CDC6 gene is only transcribed during G1. In this article we show that rapid degradation of Cdc6p also contributes to this periodicity. Cdc6p degradation rates are regulated during the cell cycle, reaching a peak during late G1/early S phase. Removal of a 47-amino-acid domain near the N-terminus of Cdc6p prevents degradation of Cdc6p. Likewise, mutations in the Cdc4/34/53 pathway involved in ubiquitin-mediated degradation block proteolysis and genetic evidence is presented indicating that the N-terminus of Cdc6p interacts with the Cdc4/34/53 pathway, probably through Cdc4p. A stable Cdc6p mutant which is no longer degraded by the Cdc4/34/53 pathway is, none the less, fully functional. Constitutive overexpression of either wild-type or stable Cdc6p does not induce re-replication and does not induce assembly of pre-replicative complexes after DNA replication is complete.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1997

Electron tomography of neuronal mitochondria: three-dimensional structure and organization of cristae and membrane contacts.

The structure of neuronal mitochondria from chick and rat was examined using electron microscope tomography of chemically fixed tissue embedded in plastic and sliced in approximately 500 nm-thick sections. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with an intermediate voltage electron microscope (400 kV). The tilt increment was either 1 degree or 2 degrees ranging from -60 degrees to +60 degrees. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each approximately 7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of approximately 22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, approximately 14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes approximately 14 nm). These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. However, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion.

Animals↗
PubMed1997

Three-dimensional structure of the gap junction connexon.

The gap junction membrane channel is composed of macular aggregations of intercellular channels permitting the direct intercellular transfer of ions and small molecules. Each intercellular channel is formed by the apposition of two hexameric transmembrane channels (connexons), one from each cell. The interlocking of the two channels occurs extracellularly in a narrow 2.5-nm "gap" separating the junctional membranes. The channel-channel interaction is known to be selective between members of the family of proteins, called connexins, which oligomerize into the connexons. In addition to selectivity, the molecular interfaces involved in the extracellular interactions between connexons must be very congruent, since the intercellular channel must provide high resistances to the leakage of small ions between the channel lumen and the extracellular space. By using a recently developed biochemical procedure for obtaining ordered arrays of connexons from gap junctions split in the extracellular gap, (Ghoshroy, S., D. A. Goodenough, and G. E. Sosinsky. 1994. Preparation, characterization, and structure of half gap junctional layers spit with urea and EGTA. J. Membr. Biol. 146:15-28) a three-dimensional reconstruction of a connexon has been obtained by electron crystallographic methods. This reconstruction emphasizes the structural asymmetry between the extracellular and cytoplasmic domains and assigns lobed structural features to the extracellular domains of the connexon. The implication of our hemichannel structure is discussed in relation to the in vivo state of unpaired connexons, which have been shown to exist in the plasma membrane.

Animals↗
PubMed1996

Immunocytochemical staining of cytologic specimens. How helpful is it?

The contribution of immunocytochemical (ICC) staining to the diagnosis of a range of cytologic specimens reported over a 20-month period was retrospectively assessed. A total of 194 cases (1.6% of total workload) were stained for diagnostic purposes in this period. Immunocytochemical staining was determined to be helpful in 75.8% of cases. In body fluid cases (105), staining was most often performed to assist with the discrimination of mesothelial cells and metastatic malignancy. Immunocytochemical staining was helpful in 82% of fluid specimens, with the preliminary diagnosis confirmed in 64%, refined in 8%, and revised in 10% of cases. In fine-needle aspiration (FNA) specimens, staining was helpful in 69%, resulting in a refinement of diagnosis in 55%, and confirming the preliminary diagnosis in 14%. The preliminary diagnosis was revised in a single FNA case. Immunocytochemical staining was particularly valuable in assisting the subclassification of poorly differentiated malignancies. A more precise diagnosis was possible following consideration of ICC results in 68% of cases with a preliminary diagnosis of poorly differentiated malignancy. Immunocytochemical staining was also helpful in identifying the primary site of metastatic carcinoma in six FNA cases. It is concluded that the selective use of well-chosen panels of antibodies can be very helpful in resolving diagnostic difficulties in cytologic specimens. In particular, ICC may be an invaluable aid to the diagnosis of difficult serous effusion specimens and in the sub-typing of poorly differentiated malignancy.

Antibodies↗
PubMed1996

Use of a virulence-associated protein based enzyme-linked immunosorbent assay for Rhodococcus equi serology in horses.

An enzyme-linked immunosorbent assay (ELISA) was developed against Rhodococcus equi using Triton X-114 detergent extracted whole cell material, in which the virulence associated protein (VapA) predominated. Enzymelinked immunosorbent assay titres corresponded to antibody reacting with VapA on Western blots. There was considerable variation in antibody titres of nonimmunised mares and in the time when the colostrally derived antibody of their foals had declined to low or undetectable titres. In general, antibodies in foals declined to their lowest levels at age 4-8 weeks. Seroconversion occurred in foals age 8-10 weeks, but the precise time depended on maternal titre and the month in which the foal was born. Foals reaching age 8 weeks in late summer showed more marked seroconversion than foals born earlier. The ELISA was used to follow the response to immunisation with the same Triton X-114 extracted material. Six mares immunised before parturition with the antigen in aluminium hydroxide adjuvant developed high titres, up to > 102,400 and transferred them to their foals through colostrum. Their foals responded to immunisation with 0.5-1.0 mg antigen 3, 5, 7 and 9 weeks after birth. Antibody titres following immunisation with similar dosage reached up to > 102,400 in a separate group of foals of nonimmunised mares. Nonvaccinated control foals seroconverted at age 6-8 weeks. The VapA based ELISA is useful to follow the course of natural infection with R. equi or immunisation with VapA based antigen.

Actinomycetales Infections↗
PubMed1992

The addition of pethidine to epidural bupivacaine in labour--effect of changing bupivacaine strength.

The effects of varying the strength of bupivacaine used in epidurals for the relief of labour pain was examined. The trial randomly allocated sixty women in the first stage of labour to one of three groups. All women were of ASA status 1 or 2 and had uncomplicated pregnancies. Subjects in each group received pethidine 25 mg in 10 ml of either 0.125%, 0.1875%, or 0.25% bupivacaine. Pain scores for each patient were then assessed over the following thirty minutes. Duration of analgesia and subsequent dose requirements were examined. No difference in pain scores between groups at thirty minutes after injection of the test solutions was found. The 0.25% solution group did however have a more rapid onset of analgesia with the majority of patients in this group achieving their maximum effect between ten and twenty minutes after injection. Duration of analgesia was not prolonged by using the stronger solutions. This study suggests that when epidural pethidine 25 mg is added to local anaesthetic solutions of bupivacaine, adequate analgesia for the first stage of labour is achieved with the 0.125% bupivacaine solution. The use of stronger solutions of bupivacaine achieves no greater degree of analgesia nor longer duration of action, although the onset of analgesia may be faster with the stronger solutions. Further investigations are needed to determine if 25 mg of pethidine is the best choice of dose to use under these circumstances.

Adult↗