Maisonneuve fracture equivalent with proximal tibiofibular dislocation. A case report and literature review.
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Adolescent↗
Biomedical subjects
P A Cole
Publications and source records attributed to P A Cole.
Less Invasive Stabilization System (LISS) for fractures of the proximal tibia: indications, surgical technique and preliminary results of the UMC Clinical Trial.
The surgical treatment of proximal tibia fractures, with or without intraarticular involvement, is associated with well-described patterns of failure and significant complication rates. Recent surgical advances allow for a minimally invasive approach to such injuries, which may improve healing times, increase union rates, and decrease complication rates. The Less Invasive Stabilization System (LISS) for proximal tibia fractures employs a laterally based fixed angled implant, allowing for the placement of locking screws proximal and distal to the fracture, which may be placed percutaneously. This manuscript presents the surgical technique and indications of the Tibia LISS. A preview of the early clinical results of the treatment of bicondylar tibial plateau fractures and proximal tibia diaphysis will also be presented.
Adult↗
Compartment pressures after submuscular fixation of proximal tibia fractures.
INTRODUCTION: Tibial fractures are known to be associated with elevated compartment pressures, which may potentially result in a compartment syndrome. The Tibial Less Invasive Stabilization System (LISS PLT) is an internal fixator device that is designed for fixation of proximal tibia fractures. The technique includes submuscular placement of the implant along the lateral border of the tibia. Such placement of the Tibia LISS into the anterior leg compartment could increase compartment pressures, and therefore increase the risk of compartment syndrome. The objective of this prospective study is to determine whether submuscular placement of a tibia LISS fixator increases compartment pressures and what is the incidence of compartment syndrome. MATERIALS AND METHODS: Twenty-eight consecutive proximal tibia fractures were fixed using the Tibia LISS (LISS PLT). Compartment pressures in all four leg compartments were measured pre- and postoperatively. Diastolic pressures were recorded and perfusion pressures calculated. RESULTS: There were no statistically significant differences between pre- and postoperative compartment pressures. One patient out of twenty-eight developed an impending compartment syndrome after placement of the LISS implant. CONCLUSIONS: Submuscular fixation of the tibia with the Less Invasive Stabilization System does not cause a significant rise in leg compartment pressures, nor is it associated with an increased incidence of compartment syndrome.
Bone Plates↗
Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis.
Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.
Amino Acid Sequence↗
Protein tyrosine kinase Csk-catalyzed phosphorylation of Src containing unnatural tyrosine analogues.
Using expressed protein ligation, five unnatural tyrosine analogues (amino-phenylalanine, homotyrosine, 2-methyl-tyrosine, (alphaS,betaR)-beta-methyl-tyrosine, and 2,6-difluoro-tyrosine) were incorporated into Src in place of the natural tail tyrosine residue. These semisynthetic substrates were evaluated as Csk substrates or allosteric activators. It appears that the tyrosine phenol hydroxyl is unlikely to be contributing significantly to Src's ground-state binding affinity for Csk. It has been observed that stabilizing tyrosine conformers can further optimize Src's already high substrate efficiency. These latter findings contrast similar studies with synthetic peptide substrates and highlight the value of investigation of protein kinase substrate selectivity with protein substrates.
CSK Tyrosine-Protein Kinase↗
Bisubstrate ketone analogues as serotonin N-acetyltransferase inhibitors.
Serotonin N-acetyltransferase, also called the melatonin rhythm enzyme, is thought to play an important regulatory role in circadian rhythm in animals and people. A series of analogues were synthesized in which indole and coenzyme A were linked via ketone tethers as designed inhibitors of this enzyme. These compounds were tested against purified serotonin N-acetyltransferase. The parent ketone compound was found to be as potent as an amide linked compound studied previously, suggesting that there are no key hydrogen bonds to the nitrogen atom of the corresponding substrate necessary for tight inhibition. Reduction of the parent ketone afforded the diastereomeric carbinol mixture which showed reduced inhibitory potency, arguing against tetrahedral analogue mimicry as an important inhibitory theme. Several conformationally constrained ketone analogues were synthesized and investigated, and the results indicated that directing the orientation of the two substrates within the bisubstrate system could be used to maximize enzyme inhibition.
Acetyl Coenzyme A↗
Probing the mechanism of enzymatic phosphoryl transfer with a chemical trick.
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Adenosine Triphosphatases↗
Transcriptional coactivator protein p300. Kinetic characterization of its histone acetyltransferase activity.
The p300/cAMP response element-binding protein-binding protein (CBP) family members include human p300 and cAMP response element-binding protein-binding protein, which are both important transcriptional coactivators and histone acetyltransferases. Although the role of these enzymes in transcriptional regulation has been extensively documented, the molecular mechanisms of p300 and CBP histone acetyltransferase catalysis are poorly understood. Herein, we describe the first detailed kinetic characterization of p300 using full-length purified recombinant enzyme. These studies have employed peptide substrates to systematically examine the substrate specificity requirements and the kinetic mechanism of this enzyme. The importance of nearby positively charged residues in lysine targeting was demonstrated. The strict structural requirement of the lysine side chain was shown. The catalytic mechanism of p300 was shown to follow a ping-pong kinetic pathway and viscosity experiments revealed that product release and/or a conformational change were likely rate-limiting in catalysis. Detailed analysis of the p300 selective inhibitor Lys-CoA showed that it exhibited slow, tight-binding kinetics.
Acetyltransferases↗
Molecular determinants for Csk-catalyzed tyrosine phosphorylation of the Src tail.
Phosphorylation of a critical tail tyrosine residue in Src modulates its three-dimensional structure and protein tyrosine kinase activity. The protein tyrosine kinase Csk is responsible for catalyzing the phosphorylation of this key Src tyrosine residue, but the detailed molecular basis for Src recognition and catalysis is poorly understood. In this study, we investigate this phosphorylation event using purified recombinant Csk and Src proteins and mutants. It was shown that the apparent k(cat) and K(m) values for Csk phosphorylation of catalytically impaired Src (dSrc) are similar to the parameters for Csk-catalyzed phosphorylation of the Src family member Lck. The SH3 (Src homology 3) and SH2 (Src homology 2) domains of dSrc were fully dispensable with respect to rapid phosphorylation, indicating that the catalytic domain and tail of dSrc are sufficient for the high efficiency of dSrc as a substrate. Of the eight Src tail residues examined, only the fully conserved Glu (Y-3 position) and Gln (Y-1 position) investigated by alanine scanning mutagenesis caused large reductions (10--40-fold) in dSrc substrate efficiency. The Y-3 Glu requirement was stringent as conservative replacements with Asp or Gln were no better than Ala whereas replacement of the Y-1 Gln with Ile was readily tolerated. Interestingly, en bloc replacement of the tail with a seven amino acid consensus sequence derived from a peptide library analysis was no better than the wild-type sequence. Surprisingly, the dSrc Y527F protein, although not a Csk substrate, enhanced Csk-catalyzed phosphorylation of dSrc. These results and other data suggest that Src dimerization (or higher order oligomerization) is important for high-efficiency Csk-catalyzed phosphorylation of the Src tail.
Amino Acid Substitution↗
Distal femoral fracture fixation utilizing the Less Invasive Stabilization System (L.I.S.S.): the technique and early results.
The treatment of supracondylar femoral fractures in the past three decades has evolved from non-operative to operative treatment. While operative fixation utilizing either plate fixation or rigid intramedullary nail fixation has improved patient outcomes, the problems of malunion, nonunion, need for bone grafting, joint stiffness, and infection persist. An emphasis on maintenance of the soft tissue envelope around fractures has improved efficacy in increasing osseous healing and decreasing infection. Out of this movement grew the concept of submuscular plating for distal femoral fractures, and subsequently L.I.S.S. fixation (Less Invasive Stabilization System) for distal femoral fractures. The technique and early results utilizing the L.I.S.S. for distal femoral fractures is described. The technique of L.I.S.S. fixation first begins with traditional direct visualization and internal fixation of the articular surface. Closed reduction is then performed on the metaphyseal / diaphyseal component of the fracture, followed by submuscular fixation utilizing the L.I.S.S. fixation. The L.I.S.S. can best be thought as an "internal" external fixator.
Bone Screws↗
Fixation of distal femoral fractures above total knee arthroplasty utilizing the Less Invasive Stabilization System (L.I.S.S.).
Supracondylar femoral fractures above total knee arthroplasty remain a treatment challenge. Complication rates as high as 30% are associated with both nonoperative and operative treatment. Conventional plate fixation and rigid intramedullary nail fixation has improved the treatment of these fractures. However, problems still exist in the setting of a short distal femoral block and/or significant osteoporosis. Less Invasive Stabilization System (L.I.S.S.) fixation has been utilized for the treatment of supracondylar femoral fractures above total knee arthroplasty. Multiple fixed angle screws give optimal fixation around the femoral component. Advantages appear to include maintenance of distal femoral fixation, low infection, and low need for bone grafting.
Bone Plates↗
Kinase chips hit the proteomics era.
Protein kinase chips, in which kinases are tested for their ability to phosphorylate immobilized substrates, have been developed and used to evaluate the protein kinases encoded by the yeast genome. This new technology promises to be a valuable addition to the biochemists' and cell biologists' arsenal for evaluating the substrate selectivity and function of protein kinases in cell signaling.
Binding Sites↗
Mechanistic studies on the alkyltransferase activity of serotonin N-acetyltransferase.
BACKGROUND: Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) catalyzes the first, rate-limiting step in the biosynthesis of the circadian hormone melatonin (5-methoxy-N-acetyltryptamine) from serotonin. Our recent discovery that, in addition to catalyzing the acetyl transfer from acetyl-coenzyme A (acetyl-CoASH) to serotonin, AANAT is also a robust catalyst for the alkyl transfer reaction between CoASH and N-bromoacetyltryptamine has not only opened up a new way to develop cell-permeable AANAT acetyltransferase inhibitors that are valuable in vivo tools in helping elucidate melatonin's (patho)physiological roles, but has also raised a question - how does AANAT accelerate the alkyl transfer reaction? In this study, mechanistic aspects of the AANAT-catalyzed alkyl transfer reaction were explored by employing CoASH and a series of N-haloacetyltryptamines that were also evaluated for their AANAT acetyltransferase inhibitory activities. RESULTS: Investigation of various N-haloacetyltryptamine analogs showed a similar leaving group effect on the enzymatic and non-enzymatic reaction rates. Steady-state kinetic analyses demonstrated that AANAT alkyltransferase obeys a sequential, ternary complex mechanism, with random substrate binding. Rate versus pH profiles revealed the catalytic importance of an ionizable group with pK(a) of approximately 7. All those N-haloacetyltryptamines that serve as substrates of AANAT alkyltransferase are also potent (low micromolar) in vitro inhibitors against AANAT acetyltransferase activity. In particular, N-chloroacetyltryptamine was also shown to be a potent inhibitor of intracellular melatonin production in a pineal cell culture assay. CONCLUSIONS: This is the first detailed investigation of the alkyltransferase activity associated with an acetyltransferase. Our results indicate that AANAT does not accelerate the alkyl transfer reaction by simple approximation effect as previously proposed for the similar alkyl transfer reaction catalyzed by other acyltransferases. This study has general implications for developing novel inhibitors by taking advantage of the promiscuous alkyltransferase activity associated with several acyltransferases.
Alkyl and Aryl Transferases↗
Site-specific incorporation of a phosphotyrosine mimetic reveals a role for tyrosine phosphorylation of SHP-2 in cell signaling.
The regulation of protein tyrosine phosphatase (PTPase) SHP-2 is proposed to involve tyrosine phosphorylation on two tail tyrosine residues. Using "expressed protein ligation", nonhydrolyzable phosphotyrosine analogs were introduced at known phosphorylation sites in SHP-2. Biochemical analysis suggests that a phosphonate at Tyr542 interacts intramolecularly with the N-terminal SH2 domain to relieve basal inhibition of the PTPase, whereas a phosphonate at Tyr-580 stimulates the PTPase activity by interaction with the C-terminal SH2 domain. Microinjection experiments indicate that a single phosphorylation of Tyr-542 of SHP-2 is sufficient to activate the MAP kinase pathway in living cells. These studies support a novel mechanism explaining how tyrosine phosphorylation of a PTPase is important in signal transduction.
Adaptor Proteins, Signal Transducing↗
Mechanism-based design of a protein kinase inhibitor.
Protein kinase inhibitors have applications as anticancer therapeutic agents and biological tools in cell signaling. Based on a phosphoryl transfer mechanism involving a dissociative transition state, a potent and selective bisubstrate inhibitor for the insulin receptor tyrosine kinase was synthesized by linking ATPgammaS to a peptide substrate analog via a two-carbon spacer. The compound was a high affinity competitive inhibitor against both nucleotide and peptide substrates and showed a slow off-rate. A crystal structure of this inhibitor bound to the tyrosine kinase domain of the insulin receptor confirmed the key design features inspired by a dissociative transition state, and revealed that the linker takes part in the octahedral coordination of an active site Mg2+. These studies suggest a general strategy for the development of selective protein kinase inhibitors.
Adenosine Triphosphate↗
Chromatin-dependent cooperativity between constitutive and inducible activation domains in CREB.
The cyclic AMP (cAMP)-responsive factor CREB induces target gene expression via constitutive (Q2) and inducible (KID, for kinase-inducible domain) activation domains that function synergistically in response to cellular signals. KID stimulates transcription via a phospho (Ser133)-dependent interaction with the coactivator paralogs CREB binding protein and p300, whereas Q2 recruits the TFIID complex via a direct association with hTAF(II)130. Here we investigate the mechanism underlying cooperativity between the Q2 domain and KID in CREB by in vitro transcription assay with naked DNA and chromatin templates containing the cAMP-responsive somatostatin promoter. The Q2 domain was highly active on a naked DNA template, and Ser133 phosphorylation had no additional effect on transcriptional initiation in crude extracts. Q2 activity was repressed on a chromatin template, however, and this repression was relieved by the phospho (Ser133) KID-dependent recruitment of p300 histone acetyltransferase activity to the promoter. In chromatin immunoprecipitation assays of NIH 3T3 cells, cAMP-dependent recruitment of p300 to the somatostatin promoter stimulated acetylation of histone H4. Correspondingly, overexpression of hTAFII130 potentiated CREB activity in cells exposed to cAMP, but had no effect on reporter gene expression in unstimulated cells. We propose that cooperativity between the KID and Q2 domains proceeds via a chromatin-dependent mechanism in which recruitment of p300 facilitates subsequent interaction of CREB with TFIID.
Animals↗
Chemical rescue of a mutant protein-tyrosine kinase.
Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed k(cat) for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is approximately 3000-fold smaller compared with that of wild type Csk, whereas the K(m) values for ATP and poly(Glu,Tyr) are only mildly affected. The k(cat) value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the k(cat) value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.
Amino Acid Sequence↗
Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate.
The interaction of a synthetic tetrafluorotyrosyl peptide substrate with the activated tyrosine kinase domain of the insulin receptor was studied by steady-state kinetics and x-ray crystallography. The pH-rate profiles indicate that the neutral phenol, rather than the chemically more reactive phenoxide ion, is required for enzyme-catalyzed phosphorylation. The pK(a) of the tetrafluorotyrosyl hydroxyl is elevated 2 pH units on the enzyme compared with solution, whereas the phenoxide anion species behaves as a weak competitive inhibitor of the tyrosine kinase. A structure of the binary enzyme-substrate complex shows the tetrafluorotyrosyl OH group at hydrogen bonding distances from the side chains of Asp(1132) and Arg(1136), consistent with elevation of the pK(a). These findings strongly support a reaction mechanism favoring a dissociative transition state.
Catalytic Domain↗