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Biomedical subjects

P J Casey

Publications and source records attributed to P J Casey.

At least 19 recordsLinked to original sources
PubMed2002

Blood plasma concentrations of insulin-like growth factor-I (IGF-I) in resting standardbred horses.

A survey of standardbred horses was conducted to build up a normal population profile for insulin like growth factor-I (IGF-I) concentrations in racing standardbreds and to ascertain how age, sex and geographic location affect IGF-I. Blood samples were drawn by jugular venepuncture from 202 racing standardbred horses aged one to eight years located in five different geographic regions of New Zealand. IGF-I concentrations were determined by insulin like growth factor-I binding protein (IGFBP)-blocked radioimmunoassay validated for the horse. As described in other species, age played a significant (P<0.05) role in IGF-I concentrations with the highest concentrations occurring in the younger horses. There was a significant (P<0.05) sex effect, intact males having significantly higher IGF-I concentrations compared of mares and/or geldings. Geographic location had a significant (P<0.05) influence on IGF-I. A significant (P<0.05) trainer effect also was noted both within and between geographic locations. We concluded that IGF-I concentrations in racing standardbred horses are affected by age, sex, trainer and geographic location.

Age Factors↗
PubMed2001

Lysine(164)alpha of protein farnesyltransferase is important for both CaaX substrate binding and catalysis.

Protein farnesyltransferase (FTase) catalyses the formation of a thioether linkage between proteins containing a C-terminal CaaX motif and a 15-carbon isoprenoid. The involvement of substrates such as oncogenic Ras proteins in tumour formation has led to intense efforts in targeting this enzyme for development of therapeutics. In an ongoing programme to elucidate the mechanism of catalysis by FTase, specific residues of the enzyme identified in structural studies as potentially important in substrate binding and catalysis are being targeted for mutagenesis. In the present study, the role of the positive charge of Lys(164) of the alpha subunit of FTase in substrate binding and catalysis was investigated. Comparison of the wild-type enzyme with enzymes that have either an arginine or alanine residue substituted at this position revealed unexpected roles for this residue in both substrate binding and catalysis. Removal of the positive charge had a significant effect on the association rate constant and the binding affinity of a CaaX peptide substrate, indicating that the positive charge of Lys(164)alpha is involved in formation of the enzyme (E).farnesyl diphosphate (FPP).peptide ternary complex. Furthermore, mutation of Lys(164)alpha resulted in a substantial decrease in the observed rate constant for product formation without alteration of the chemical mechanism. These and additional studies provide compelling evidence that both the charge on Lys(164)alpha, as well as the positioning of the charge, are important for overall catalysis by FTase.

Alkyl and Aryl Transferases↗
PubMed2001

Distinct regions of the cadherin cytoplasmic domain are essential for functional interaction with Galpha 12 and beta-catenin.

Heterotrimeric G proteins of the G(12) subfamily mediate cellular signals leading to events such as cytoskeletal rearrangements, cell proliferation, and oncogenic transformation. Several recent studies have revealed direct effector proteins through which G(12) subfamily members may transmit signals leading to various cellular responses. Our laboratory recently demonstrated that Galpha(12) and Galpha(13) specifically interact with the cytoplasmic domains of several members of the cadherin family of cell adhesion molecules (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 519-524). This interaction causes beta-catenin to release from cadherin and relocalize to the cytoplasm and nucleus, where it participates in transcriptional activation. Here we report that two distinct regions of the epithelial cadherin (E-cadherin) tail are required for interaction with beta-catenin and Galpha(12), respectively. Deletion of an acidic, 19-amino acid region of E-cadherin abolishes its ability to bind beta-catenin in vitro, to inhibit beta-catenin-mediated transactivation, or to stabilize beta-catenin; causes subcellular mislocalization of beta-catenin; and disrupts cadherin-mediated cell adhesion. On the other hand, deletion of a distinct 11-amino acid region of E-cadherin dramatically attenuates interaction with Galpha(12); furthermore, Galpha(12) is ineffective in stimulating beta-catenin release from an E-cadherin cytoplasmic domain lacking this putative Galpha(12)-binding region. These findings indicate that Galpha(12) and beta-catenin do not compete for the same binding site on cadherin and provide molecular targets for selectively disrupting the interaction of these proteins with cadherin.

Animals↗
PubMed2001

Phosphorylation and nuclear translocation of a regulator of G protein signaling (RGS10).

Heterotrimeric G proteins are involved in the transduction of hormonal and sensory signals across plasma membranes of eukaryotic cells. Hence, they are a critical point of control for a variety of agents that modulate cellular function. Activation of these proteins is dependent on GTP binding to their alpha (Galpha) subunits. Regulators of G protein signaling (RGS) bind specifically to activated Galpha proteins, potentiating the intrinsic GTPase activity of the Galpha proteins and thus expediting the termination of Galpha signaling. Although there are several points in most G protein controlled signaling pathways that are affected by reversible covalent modification, little evidence has been shown addressing whether or not the functions of RGS proteins are themselves regulated by such modifications. We report in this study the acute functional regulation of RGS10 thru the specific and inducible phosphorylation of RGS10 protein at serine 168 by cAMP-dependent kinase A. This phosphorylation nullifies the RGS10 activity at the plasma membrane, which controls the G protein-dependent activation of the inwardly rectifying potassium channel. Surprisingly, the phosphorylation-mediated attenuation of RGS10 activity was not manifested in an alteration of its ability to accelerate GTPase activity of Galpha. Rather, the phosphorylation event correlates with translocation of RGS10 from the plasma membrane and cytosol into the nucleus.

Amino Acid Sequence↗
PubMed2001

Farnesylation of nonpeptidic thiol compounds by protein farnesyltransferase.

Protein farnesyltransferase catalyzes the modification of protein substrates containing specific carboxyl-terminal Ca(1)a(2)X motifs with a 15-carbon farnesyl group. The thioether linkage is formed between the cysteine of the Ca(1)a(2)X motif and C1 of the farnesyl group. Protein substrate specificity is essential to the function of the enzyme and has been exploited to find enzyme-specific inhibitors for antitumor therapies. In this work, we investigate the thiol substrate specificity of protein farnesyltransferase by demonstrating that a variety of nonpeptidic thiol compounds, including glutathione and dithiothreitol, are substrates. However, the binding energy of these thiols is decreased 4-6 kcal/mol compared to a peptide derived from the carboxyl terminus of H-Ras. Furthermore, for these thiol substrates, both the farnesylation rate constant and the apparent magnesium affinity decrease significantly. Surprisingly, no correlation is observed between the pH-independent log(k(max)) and the thiol pK(a); model nucleophilic reactions of thiols display a Brønsted correlation of approximately 0.4. These data demonstrate that zinc-sulfur coordination is a primary criterion for classification as a FTase substrate, but other interactions between the peptide and the FTase.isoprenoid complex provide significant enhancement of binding and catalysis. Finally, these results suggest that the mechanism of FTase provides in vivo selectivity for the farnesylation of protein substrates even in the presence of high concentrations of intracellular thiols.

Alkyl and Aryl Transferases↗
PubMed2001

Interaction of Galpha 12 and Galpha 13 with the cytoplasmic domain of cadherin provides a mechanism for beta -catenin release.

The G12 subfamily of heterotrimeric G proteins, comprised of the alpha-subunits Galpha12 and Galpha13, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to cell growth and oncogenesis. In an attempt to elucidate specific roles of this subfamily in cell regulation, we sought to identify molecular targets of Galpha12. Here we show a specific interaction between the G12 subfamily and the cytoplasmic tails of several members of the cadherin family of cell-surface adhesion proteins. Galpha12 or Galpha13 binding causes dissociation of the transcriptional activator beta-catenin from cadherins. Furthermore, in cells lacking the adenomatous polyposis coli protein required for beta-catenin degradation, expression of mutationally activated Galpha12 or Galpha13 causes an increase in beta-catenin-mediated transcriptional activation. These findings provide a potential molecular mechanism for the previously reported cellular transforming ability of the G12 subfamily and reveal a link between heterotrimeric G proteins and cellular processes controlling growth and differentiation.

Adenocarcinoma↗
PubMed2001

Vascular anatomy of the brachioradialis rotational musculocutaneous flap.

The purpose of this 2-part vascular injection study was to (1) determine the sources of blood supply to the brachioradialis muscle and the distance around which the brachioradialis muscle flap may be rotated for local soft tissue reconstruction and (2) determine the fasciocutaneous vascular perfusion territory associated with the vascular pedicle of the brachioradialis muscle flap. Lead oxide injections were administered in 16 fresh frozen human upper extremity amputation specimens to determine the contribution of the isolated radial recurrent artery (RRA) and subsequent 3- and 6-cm segments of radial artery (RA) to a rotational brachioradialis muscle flap. The RRA perfused an average of 41% (range, 20% to 60%) of the brachioradialis muscle length. Selective injection of the RRA and the proximal 3-cm segment of the RA perfused 80% (range, 59% to 100%) of the muscle length, corresponding to more than 90% of muscle volume. Flap rotation consistently provided adequate tissue coverage to the antecubital fossa, the lateral elbow, and the proximal one-third volar forearm. Based on these findings, the fasciocutaneous perfusion territory of the isolated vascular pedicle was quantified by selective India ink injection studies in 10 fresh frozen cadaveric specimens. Consistent fasciocutaneous perfusion occurred directly over the muscle belly. No specimen, however, was perfused greater than approximately 1 cm distal to the musculocutaneous junction. This 2-part study defines the vascular anatomy and local utility of the brachioradialis rotational musculocutaneous flap.

Aged↗
PubMed2001

The effect of combined arterial hemodynamics on saphenous venous endothelial nitric oxide production.

INTRODUCTION: Evidence exists that an ideal bypass conduit should have a functional endothelial cell surface combined with mechanical properties similar to those of native arteries. We hypothesized that the effect of combined arterial levels of pulsatile shear stress, flow, and cyclic strain would enhance saphenous venous endothelial cell nitric oxide (NO) production, and that variations in these "ideal" conditions could impair this function. We studied NO production as a measure of endothelial function in response to different hemodynamic conditions. METHODS: Human adult saphenous venous endothelial cells were cultured in 10-cm silicone tubes, similar in diameter (5 mm) and compliance (6%) to a medium-caliber peripheral artery (eg, popliteal). Tube cultures were exposed to arterial conditions: a combined pressure (120/80 mm/Hg; mean, 100 mm/Hg), flow (mean, 115 mL/min) and cyclic strain (2%), with a resultant pulsatile shear stress of 4.8 to 9.4 dyne/cm2 (mean, 7.1). Identical tube cultures were used to study variations in these conditions. Modifications of the system included a noncompliant system, a model with nonpulsatile flow, and a final group exposed to pulsatile pressure with no flow. NO levels were measured with a fluorometric nitrite assay of conditioned media collected at 0, 0.25, 0.5, 1, 2, and 4 hours. Experimental groups were compared with cells exposed to nonpulsatile, nonpressurized low flow (shear stress 0.1 dyne/cm2) and static cultures. RESULTS: All experimental groups had greater rates of NO production than cells under static conditions (P <.05). Cells exposed to ideal conditions produced the greatest levels of NO. Independent decreases in compliance, flow, and pulsatility resulted in significantly lower rates of NO production than those in the group with these conditions intact (vs noncompliant P <.05, vs nonflow P <.05, and vs nonpulsatile P <.05). CONCLUSIONS: Our results show that in the absence of physiologically normal pulsatility, cyclic strain, and volume flow, endothelial NO production does not reach the levels seen under ideal conditions. Pulsatile flow and compliance (producing flow with cyclic stretch) play a key role in NO production by vascular endothelium in a three-dimensional hemodynamically active model. This correlates biologically with clinical experience linking graft inflow and runoff and the mechanical properties of the conduit to long-term patency.

Adult↗
PubMed2000

The C-terminal polylysine region and methylation of K-Ras are critical for the interaction between K-Ras and microtubules.

After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease Rce1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking Rce1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.

Amino Acid Sequence↗
PubMed2000

Isoprenylcysteine carboxyl methyltransferase deficiency in mice.

After isoprenylation, Ras and other CAAX proteins undergo endoproteolytic processing by Rce1 and methylation of the isoprenylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). We reported previously that Rce1-deficient mice died during late gestation or soon after birth. We hypothesized that Icmt deficiency might cause a milder phenotype, in part because of reports suggesting the existence of more than one activity for methylating isoprenylated proteins. To address this hypothesis and also to address the issue of other methyltransferase activities, we generated Icmt-deficient mice. Contrary to our expectation, Icmt deficiency caused a more severe phenotype than Rce1 deficiency, with virtually all of the knockout embryos (Icmt-/-) dying by mid-gestation. An analysis of chimeric mice produced from Icmt-/- embryonic stem cells showed that the Icmt-/- cells retained the capacity to contribute to some tissues (e.g. skeletal muscle) but not to others (e.g. brain). Lysates from Icmt-/- embryos lacked the ability to methylate either recombinant K-Ras or small molecule substrates (e.g. N-acetyl-S-geranylgeranyl-l-cysteine). In addition, Icmt-/- cells lacked the ability to methylate Rab proteins. Thus, Icmt appears to be the only enzyme participating in the carboxyl methylation of isoprenylated proteins.

Animals↗
PubMed2000

Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition.

Protein farnesyltransferase catalyzes the lipid modification of protein substrates containing Met, Ser, Gln, or Ala at their C-terminus. A closely related enzyme, protein geranylgeranyltransferase type I, carries out a similar modification of protein substrates containing a C-terminal Leu residue. Analysis of a mutant of protein farnesyltransferase containing a Tyr-to-Leu substitution at position 361 in the beta subunit led to the conclusion that the side chain of this Tyr residue played a major role in recognition of the protein substrates. However, no interactions have been observed between this Tyr residue and peptide substrates in the crystal structures of protein farnesyltransferase. In an attempt to reconcile these apparently conflicting data, a thorough kinetic characterization of the Y361L variant of mammalian protein farnesyltransferase was performed. Direct binding measurements for the Y361L variant yielded peptide substrate binding that was actually some 40-fold tighter than that with the wild-type enzyme. In contrast, binding of the peptide substrate for protein geranylgeranyltransferase type I was very weak. The basis for the discrepancy was uncovered in a pre-steady-state kinetic analysis, which revealed that the Y361L variant catalyzed farnesylation of a normal peptide substrate at a rate similar to that of the wild-type enzyme in a single turnover, but that subsequent turnover was prevented. These and additional studies revealed that the Y361L variant does not "switch" protein substrate specificity as concluded from steady-state parameters; rather, this variant exhibits severely impaired product dissociation with its normal substrate, a situation resulting in a greatly compromised steady-state activity.

Alkyl and Aryl Transferases↗
PubMed2000

Lysosomal prenylcysteine lyase is a FAD-dependent thioether oxidase.

Prenylated proteins contain either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached via a thioether bond to a cysteine residue at or near their C terminus. As prenylated proteins comprise up to 2% of the total protein in eukaryotic cells, and the thioether bond is a stable modification, their degradation raises a metabolic challenge to cells. A lysosomal enzyme termed prenylcysteine lyase has been identified that cleaves prenylcysteines to cysteine and an unidentified isoprenoid product. Here we show that the isoprenoid product of prenylcysteine lyase is the C-1 aldehyde of the isoprenoid moiety (farnesal in the case of C-15). The enzyme requires molecular oxygen as a cosubstrate and utilizes a noncovalently bound flavin cofactor in an NAD(P)H-independent manner. Additionally, a stoichiometric amount of hydrogen peroxide is produced during the reaction. These surprising findings indicate that prenylcysteine lyase utilizes a novel oxidative mechanism to cleave thioether bonds and provide insight into the unique role this enzyme plays in the cellular metabolism of prenylcysteines.

Carbon-Sulfur Lyases↗
PubMed2000

Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.

After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt-/-). Lysates from Icmt-/- cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such as N-acetyl-S-geranylgeranyl-L-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt-/- cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt-/- cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt-/- cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells.

Animals↗
PubMed2000

The basis for K-Ras4B binding specificity to protein farnesyltransferase revealed by 2 A resolution ternary complex structures.

BACKGROUND: The protein farnesyltransferase (FTase) catalyzes addition of the hydrophobic farnesyl isoprenoid to a cysteine residue fourth from the C terminus of several protein acceptors that are essential for cellular signal transduction such as Ras and Rho. This addition is necessary for the biological function of the modified proteins. The majority of Ras-related human cancers are associated with oncogenic variants of K-RasB, which is the highest affinity natural substrate of FTase. Inhibition of FTase causes regression of Ras-mediated tumors in animal models. RESULTS: We present four ternary complexes of rat FTase co-crystallized with farnesyl diphosphate analogs and K-Ras4B peptide substrates. The Ca(1)a(2)X portion of the peptide substrate binds in an extended conformation in the hydrophobic cavity of FTase and coordinates the active site zinc ion. These complexes offer the first view of the polybasic region of the K-Ras4B peptide substrate, which confers the major enhancement of affinity of this substrate. The polybasic region forms a type I beta turn and binds along the rim of the hydrophobic cavity. Removal of the catalytically essential zinc ion results in a dramatically different peptide conformation in which the Ca(1)a(2)X motif adopts a beta turn. A manganese ion binds to the diphosphate mimic of the farnesyl diphosphate analog. CONCLUSIONS: These ternary complexes provide new insight into the molecular basis of peptide substrate specificity, and further define the roles of zinc and magnesium in the prenyltransferase reaction. Zinc is essential for productive Ca(1)a(2)X peptide binding, suggesting that the beta-turn conformation identified in previous nuclear magnetic resonance (NMR) studies reflects a state in which the cysteine is not coordinated to the zinc ion. The structural information presented here should facilitate structure-based design and optimization of inhibitors of Ca(1)a(2)X protein prenyltransferases.

Alkyl and Aryl Transferases↗
PubMed2000

Thrombosed giant coronary artery aneurysm presenting as an intracardiac mass.

Giant coronary artery aneurysms are rare in adults and are usually found in association with Kawasaki's disease arising in childhood. We report a case of a thrombosed giant right coronary artery aneurysm presenting as an intracardiac mass detected after inferior wall myocardial infarction. Histologic analysis indicated that fibromuscular dysplasia was the underlying cause of the aneurysm.

Aged↗
PubMed2000

The effects of bovine recombinant growth hormone administration on insulin-like growth factor-I and the haemopoietic system in thoroughbred geldings.

The effect of intramuscularly administered recombinant bovine growth hormone (rbGH) on insulin-like growth factor-I (IGF-I) and white and red blood cell indices was studied in Thoroughbred geldings. An insulin-like growth factor binding protein (IGFBP)-blocked radioimmunoassay was modified and validated for the measurement of IGF-I in equine blood plasma. Baseline values of IGF-I and blood indices were determined over a 48 h period and then a single dose of 5 microg/kg, 10 microg/kg or 50 microg/kg of rbGH was administered. Insulin-like growth factor-I levels increased in a dose-dependent manner, with the highest values between 12 h and 24 h. The highest dose (50 microg/kg) yielded the greatest IGF-I response with a 90.2+/-10.8% increase at 24 h. White blood cell count increased following the three doses of rbGH with the highest white blood cell count at 12 h after the 50 microg/kg dose. Haemoglobin was significantly increased at 24 h (P< 0.05), when values following doses of 10 microg/kg and 50 microg/kg were significantly greater than after the vehicle or the dose of 5 microg/kg. Red blood cell count was not affected by any of the rbGH doses. These results indicated that rbGH is biologically active in the horse and that rbGH at a dose rate of 10 microg/kg or more could be used therapeutically.

Animals↗
PubMed2000

Compartment syndromes of the upper extremity.

Upper extremity compartment syndromes are difficult conditions that are manifest in a myriad of ways. Careful attention to the details of the history and physical examination can assist in the development of a useful working diagnosis. Testing ITPs is the best method available to help confirm the diagnosis. Prompt fasciotomy is effective for diminishing compartment pressure and limiting additional tissue necrosis due to pressure elevation. Unfortunately, a number of characteristics specific to compartment syndrome affect ultimate outcome. Other variables, such as the amount of muscle damage from the initial injury, problems originating from any associated fracture, and soft tissue injury, also significantly affect overall patient outcome.

Compartment Syndromes↗