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Biomedical subjects

R C Robinson

Publications and source records attributed to R C Robinson.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of Arp2/3 complex.

We determined a crystal structure of bovine Arp2/3 complex, an assembly of seven proteins that initiates actin polymerization in eukaryotic cells, at 2.0 angstrom resolution. Actin-related protein 2 (Arp2) and Arp3 are folded like actin, with distinctive surface features. Subunits ARPC2 p34 and ARPC4 p20 in the core of the complex associate through long carboxyl-terminal alpha helices and have similarly folded amino-terminal alpha/beta domains. ARPC1 p40 is a seven-blade beta propeller with an insertion that may associate with the side of an actin filament. ARPC3 p21 and ARPC5 p16 are globular alpha-helical subunits. We predict that WASp/Scar proteins activate Arp2/3 complex by bringing Arp2 into proximity with Arp3 for nucleation of a branch on the side of a preexisting actin filament.

Actin Cytoskeleton↗
PubMed2001

Differences in patterns of activation of MAP kinases induced by oncogenic ras-p21 and insulin in oocytes.

Oncogenic ras (Val 12-containing)-p21 protein induces oocyte maturation by a pathway that is blocked by peptides from effector domains of ras-p21, i.e., residues 35-47 (that block Val 12-p21-activated raf) and 96-110 and 115-126, which do not affect the ability of insulin-activated cellular p21 to induce maturation. Oncogenic p21 binds directly to jun-N-terminal kinase (JNK), which is blocked by the p21 96-110 and 115-126 peptides. This finding predicts that oncogenic p21, but not insulin, induces maturation by early and sustained activation of JNK. We now directly confirm this prediction by showing that oncogenic p21 induces activating phosphorylation of JNK (JNK-P) and of ERK (MAP kinase) (MAPK-P), whose levels correlate with oocyte maturation. p21 peptides 35-47 and 96-110 block formation of JNK-P and MAPK-P, further confirming this correlation and suggesting, unexpectedly, that raf-MEK-MAPK and JNK-jun pathways strongly interact on the oncogenic p21 pathway. In contrast, insulin activates only low levels of JNK-P, and, surprisingly, we find that insulin induces only low levels of MAPK-P, indicating that insulin and activated normal p21 utilize MAP kinase-independent signal transduction pathways.

Animals↗
PubMed2001

Mandibular distraction force: laboratory data and clinical correlation.

PURPOSE: In vitro data were collected to measure torque-force values of an internal distraction device. The measurements were correlated with in vivo torque readings in an attempt to better understand the force required to distract the osteogenic bone callus of the human mandible during distraction osteogenesis. METHODS AND MATERIALS: Five internal craniofacial distraction devices were mounted on an apparatus to test load limits and torque measurements. The apparatus aligned the devices so that weight provided a force opposite and parallel to the vector of distraction. Weights were added in 5-lb increments, and the devices were activated 0.5 mm for each torque reading. Torque readings were obtained from a calibrated torque wrench. Measurements were plotted on a graph and correlated with clinical torque readings obtained from 8 patients undergoing mandibular lengthening. RESULTS: The average torque for distracting the human mandible 0.5 mm twice a day was 4.2 +/- 1.6 Newton-centimeters (N-cm). The average slope of the in vitro data shows that 4.2 N-cm of torque is equivalent to a force of 35.6 N. The average force of device failure was 235.8 N. CONCLUSION: Torque-force diagrams offer an effective means for calibrating safety margins and load capabilities for internal distraction devices. Quantification of axial forces encountered in mandibular lengthening will help contribute to the overall understanding and biomechanics of mandibular distraction osteogenesis.

Adolescent↗
PubMed2001

Screening for problematic prescription opioid use.

The proper medicinal use of opioids, in light of their notorious history and current relation to social ills, continues to be debated and remains unclear in several areas of medicine. This article will review several areas and points of controversy related to screening for potential problematic opioid behavior in chronic nonmalignant pain patients. Controversy over the prescription of opioids for chronic nonmalignant pain continues, despite the growing acceptance of this practice. Indeed, past research supports the beneficial use of opioids for noncancer pain. Unfortunately, traditional definitions of abuse and dependence, with their emphasis on tolerance and withdrawal, are inappropriate for chronic pain patients prescribed opioids. The component of traditional definitions of abuse and dependence that appears most applicable to chronic pain patients centers on the criterion that the patient continue to take the drug (in this case, the opioid) despite negative and harmful effects or despite any decrease in pain level. Although clinical observations exist about risk factors for opioid misuse in chronic pain patients, there is limited research. Further, the area of prescreening for problematic drug behavior is in its infancy. However, researchers have begun to delve into this challenging area and the application of rigorous empirical research will bring us closer to identifying those patients at risk so that their pain is managed without destructive outcomes in other areas of their life.

Chronic Disease↗
PubMed2000

Conformational modulation of human cytochrome P450 2E1 by ethanol and other substrates: a CO flash photolysis study.

The alcohol-inducible cytochrome P450 2E1 is a major human hepatic P450 which metabolizes a broad array of endogenous and exogenous compounds, including ethanol, low-molecular weight toxins, and fatty acids. Several substrates are known to stabilize this P450 and inhibit its cellular degradation. Furthermore, ethanol is a known modulator of P450 2E1 substrate metabolism. We examined the CO binding kinetics of P450 2E1 after laser flash photolysis of the heme-CO bond, to probe the effects of ethanol and other substrates on protein conformation and dynamics. Ethanol had an effect on the two kinetic parameters that describe CO binding: it decreased the rate of CO binding, suggesting a decrease in the protein's conformational flexibility, and increased the photosensitivity, which indicates a local effect in the active site region such as strengthening of the heme-CO bond. Other substrates decreased the CO binding rate to varying degrees. Of particular interest is the effect of arachidonic acid, which abolished photodissociation in the absence of ethanol but had no effect in the presence of ethanol. These results are consistent with a model of P450 2E1 whereby arachidonic acid binds along a long hydrophobic binding pocket and blocks exit of CO from the heme region.

Arachidonic Acid↗
PubMed2000

Phosphorylation of Acanthamoeba actophorin (ADF/cofilin) blocks interaction with actin without a change in atomic structure.

LIM-kinase activated by GST-Pak1 phosphorylates Acanthamoeba actophorin stoichiometrically and specifically on serine 1. The atomic structure of phosphorylated actophorin determined by X-ray crystallography is essentially identical with the structure of unphosphorylated actophorin. We compared biochemical properties of phosphorylated actophorin, unphosphorylated actophorin and mutants of actophorin with serine 1 replaced by aspartic acid or alanine. Phosphorylation strongly inhibits interaction of actophorin with Mg-ADP- or Mg-ATP-actin monomers and Mg-ADP-actin filaments, so Ser1 phosphorylation directly blocks interaction of actin-depolymerizing factor (ADF)/cofilin proteins with actin. About 30 % of actophorin is phosphorylated in live amoebas grown in suspension culture. Phosphorylation of ADF/cofilin proteins by LIM-kinase or other enzymes will tend to stabilize actin filaments by inhibiting the ability of these proteins to sever and depolymerize older actin filaments that have hydrolyzed their bound ATP and dissociated the phosphate.

Acanthamoeba↗
PubMed2000

Induction of oocyte maturation by jun-N-terminal kinase (JNK) on the oncogenic ras-p21 pathway is dependent on the raf-MEK signal transduction pathway.

PURPOSE: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. METHODS: We microinjected raf dominant negative mutant mRNA (DN-raf) and the MEK-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. RESULTS: We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. CONCLUSION: We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MEK inhibitors. The results imply that blockade of both MEK and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras-induced tumors.

Animals↗
PubMed2000

Synthetic peptide mimics of a predicted topographical interaction surface: the cytochrome P450 2B1 recognition domain for NADPH-cytochrome P450 reductase.

In order to identify the cytochrome P450-binding domain for NADPH-cytochrome P450 reductase, synthetic peptide mimics of predicted surface regions of rat cytochrome P450 2B 1 were constructed and evaluated for inhibition of the P450-reductase interaction. A peptide corresponding to residues 116-134, which includes the C helix, completely inhibited reductase-mediated benzphetamine demethylation by purified P450 2B1. Replacement of Arg-125 by Glu yielded a noninhibitory peptide, suggesting that this residue significantly contributes to the reductase-P450 interaction. Additional P450 peptides were prepared which correspond to combinations of regions distant in primary sequence, but predicted to be spatially proximate. A peptide derived from segments of the C and L helices was a more potent inhibitor than peptides derived from either segment alone. This topographically designed peptide not only inhibited P450 2B1 in its purified form, but also when membrane-bound in rat liver microsomes. The peptide also inhibited microsomal aryl hydrocarbon hydroxylase, aniline hydroxylase, and erythromycin demethylase activities derived from other P450s. These results indicate that the C and L helices contribute to a reductase-binding site common to multiple P450s, and present a peptide mimic for this region that is useful for inhibition of P450-mediated microsomal activities.

Amino Acid Sequence↗
PubMed1999

Domain movement in gelsolin: a calcium-activated switch.

The actin-binding protein gelsolin is involved in remodeling the actin cytoskeleton during growth-factor signaling, apoptosis, cytokinesis, and cell movement. Calcium-activated gelsolin severs and caps actin filaments. The 3.4 angstrom x-ray structure of the carboxyl-terminal half of gelsolin (G4-G6) in complex with actin reveals the basis for gelsolin activation. Calcium binding induces a conformational rearrangement in which domain G6 is flipped over and translated by about 40 angstroms relative to G4 and G5. The structural reorganization tears apart the continuous beta sheet core of G4 and G6. This exposes the actin-binding site on G4, enabling severing and capping of actin filaments to proceed.

Actins↗
PubMed1999

The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site.

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.

Binding Sites↗
PubMed1998

Crystal structure of the N-terminal domain of sialoadhesin in complex with 3' sialyllactose at 1.85 A resolution.

The structure of the functional N-terminal domain from the extracellular region of the cell surface receptor sialoadhesin has been determined in complex with the oligosaccharide 3' sialyllactose. This provides structural information for the siglec family of proteins. The structure conforms to the V-set immunoglobulin-like fold but contains several distinctive features, including an intra-beta sheet disulphide and a splitting of the standard beta strand G into two shorter strands. These novel features appear important in adapting the V-set fold for sialic acid-mediated recognition. Analysis of the complex with 3'sialyllactose highlights three residues, conserved throughout the siglec family, as key features of the sialic acid-binding template. The complex is representative of the functional recognition interaction with carbohydrate and as such provides detailed information for a heterotypic cell adhesion interaction.

Animals↗
PubMed1997

The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation.

The structure of gelsolin has been determined by crystallography and comprises six structurally related domains that, in a Ca2+-free environment, pack together to form a compact globular structure in which the putative actin-binding sequences are not sufficiently exposed to enable binding to occur. We propose that binding Ca2+ can release the connections that join the N- and C-terminal halves of gelsolin, enabling each half to bind actin relatively independently. Domain shifts are proposed in response to Ca2+ as bases for models of how gelsolin acts to sever, cap, or nucleate F-actin filaments. The structure also invites discussion of polyphosphoinositide binding to segment 2 and suggests how mutation at Asp-187 could initiate a series of events that lead to deposition of amyloid plaques, as observed in victims of familial amyloidosis (Finnish type).

Actins↗
PubMed1997

Differential interaction of erythromycin with cytochromes P450 3A1/2 in the endoplasmic reticulum: a CO flash photolysis study.

The kinetics of CO binding to cytochromes P450, measured by the flash photolysis technique, were used to probe the interaction of erythromycin with cytochromes P450 in rat liver microsomes. Addition of erythromycin generates substrate difference spectra using microsomes from rats treated with phenobarbital or dexamethasone but not from untreated rats, showing that it binds to P450s induced by these agents. In contrast, erythromycin and/or a monoclonal antibody to P450 3A1/2 accelerated CO binding to microsomes from rats treated with phenobarbital but had no effect on microsomes from untreated or dexamethasone-treated rats. Based on the differential amounts and inducibilities of the P450 3A1 and 3A2 forms in these microsomal samples, these results indicate that erythromycin increased the rate for P450 3A2 but not P450 3A1. The divergent effects of erythromycin on these P450s, which exhibit 89% sequence similarity, were consistent with a model of the P450 substrate binding site in which erythromycin forms a more rigid complex with P450 3A1 than P450 3A2. These results demonstrate the sensitivity of P450 conformation/dynamics to substrate binding, and show that CO binding kinetics can distinguish among closely related P450s in a microsomal environment.

Animals↗
PubMed1997

Conformation-dependent phosphorylation of p53.

Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 ((His)p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and beta subunit of casein kinase II (CKIIbeta) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length (His)p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53wt) undergoes better phosphorylation by CKIIbeta and by WCE kinases than mutant forms of p53 at amino acid 249 (p53(249)) or 273 (p53(273)). Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C reduces the phosphorylation of mutant p53 to a greater extent than of p53wt. Comparing truncated forms of p53 revealed that the ability of CKIIbeta, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97-155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97-117 that increase p53 phosphorylation by CKIIbeta while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53(249) exhibits significant changes to the conformation of p53 within amino acids 97-117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase.

Adenosine Triphosphate↗
PubMed1997

Drug-drug interactions: effect of quinidine on nifedipine binding to human cytochrome P450 3A4.

Quinidine is a known inhibitor of cytochrome P450-mediated nifedipine metabolism. The interactions of nifedipine and quinidine with human cytochrome P450 3A4, which metabolizes these drugs, were examined using the kinetics of CO binding to this P450 as a rapid kinetic probe of protein conformation and dynamics. This approach showed that nifedipine and quinidine bind to different P450 3A4 species, respectively termed species I and II, with distinct conformations. When both drugs were present simultaneously, nifedipine interacted with the quinidine-bound P450 species II, but not species I. These findings indicate that quinidine acts as an allosteric inhibitor by switching nifedipine binding from nifedipine-metabolizing species I to the nonmetabolizing species II.

Anti-Arrhythmia Agents↗
PubMed1997

Differential mechanisms of cytochrome P450 inhibition and activation by alpha-naphthoflavone.

The anticarcinogenicity of some flavonoids has been attributed to modulation of the cytochrome P450 enzymes, which metabolize procarcinogens to their activated forms. However, the mechanism by which flavonoids inhibit some P450-mediated activities while activating others is a longstanding, intriguing question. We employed flash photolysis to measure carbon monoxide binding to P450 as a rapid kinetic technique to probe the interaction of the prototype flavonoid alpha-naphthoflavone with human cytochrome P450s 1A1 and 3A4, whose benzo[a]pyrene hydroxylation activities are respectively inhibited and stimulated by this compound. This flavonoid inhibited P450 1A1 binding to benzo[a]pyrene via a classical competitive mechanism. In contrast, alpha-naphthoflavone stimulated P450 3A4 by selectively binding and activating an otherwise inactive subpopulation of this P450 and promoting benzo[a]pyrene binding to the latter. These data indicate that flavonoids enhance activity by increasing the pool of active P450 molecules within this P450 macrosystem. Activators in other biological systems may similarly exert their effect by expanding the population of active receptor molecules.

Benzo(a)pyrene↗