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B W Murray

Publications and source records attributed to B W Murray.

At least 19 recordsLinked to original sources
PubMed2001

SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase.

Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (K(i) = 0.19 microM). SP600125 is a reversible ATP-competitive inhibitor with >20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-gamma, TNF-alpha, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharide-induced expression of tumor necrosis factor-alpha and inhibited anti-CD3-induced apoptosis of CD4(+) CD8(+) thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.

Adenosine Triphosphate↗
PubMed2001

Mechanistic effects of autophosphorylation on receptor tyrosine kinase catalysis: enzymatic characterization of Tie2 and phospho-Tie2.

Activation of receptor tyrosine kinases by autophosphorylation is one of the most common and critical transformations in signal transduction, yet its role in catalysis remains controversial. Autophosphorylation of the angiogenic receptor tyrosine kinase Tie2 was studied in terms of the autophosphorylation sites, sequence of phosphorylation at these sites, kinetic effects, and mechanistic consequences. Isoelectric focusing electrophoresis and mass spectrometric analysis of a Tie2 autophosphorylation time course showed that Tyr992 on the putative activation loop was phosphorylated first followed by Tyr1108 in the C-terminal tail (previously unidentified autophosphorylation site). Autophosphorylation of Tie2 to produce pTie2 resulted in a 100-fold increase in k(cat) and a 460-fold increase in k(cat)/K(m). Viscosity studies showed that the unphosphorylated Tie2 was partially limited by product diffusion ((k(cat))(eta) = 0.67 +/- 0.06), while product release was more rate-limiting ((k(cat))(eta) = 0.94 +/- 0.08) for autophosphorylated Tie2 (pTie2). Furthermore, autophosphorylation did not significantly affect the phosphoacceptor dissociation constants. There was a significant (k(cat))(H)/(k(cat))(D) solvent isotope effect (SIE) for unphosphorylated Tie2 (2.42 +/- 0.12) and modest SIE (1.28 +/- 0.04) for pTie2, which is consistent with the chemistry step being more rate-limiting for Tie2 as compared to pTie2. The pH-rate profiles of Tie2 and pTie2 revealed a >0.5 unit shift in the pK(a) values of catalytically relevant ionizable residues upon autophosphorylation. The shift in rate-limiting step will result in a different distribution of enzyme pools (e.g., E, E*S, E*P, etc.) which may modulate the susceptibility to inhibition. Tie2 and pTie2 were profiled with a panel of known ATP-competitive kinase inhibitors. Tie2 activation perturbs catalytic residue ionizations, shifts the rate-limiting step to almost exclusive diffusion-control, and transforms the kinase into a more perfect catalyst.

Adenosine Triphosphate↗
PubMed2000

Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping.

In the HLA, H2, and other mammalian MHC:, the class I and II loci are separated by the so-called class III region comprised of approximately 60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the MHC: 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-MHC: that existed before the class I/II genes arose. The pre-MHC: may not have contained the complement and other class III genes involved in immune response.

Animals↗
PubMed2000

A contig map of the Mhc class I genomic region in the zebrafish reveals ancient synteny.

In contrast to the human and mouse Mhc, in which the clusters of class I and class II loci reside in close vicinity to one another, in the zebrafish, Danio rerio, they are found in different linkage groups. Chromosome walking using BAC (bacterial artificial chromosome) and PAC (P1 artificial chromosome) clones reveals the zebrafish class I region to occupy a segment of approximately 450 kb and to encompass at least 19 loci. These include three class I (Dare-UDA, -UEA, -UFA), five proteasome subunit beta (PSMB8, -9A, -9C, -11, -12), two TAPs (TAP2A, TAP2B), and one TAP binding protein (TAPBP). This arrangement contrasts with the arrangements found in human and mouse Mhc, in which the orthologues of the PSMB, TAP, and TAPBP loci reside within the class II region. In addition to this main zebrafish class I contig, a shorter contig of about 150 kb contains two additional class I (UBA, UCA) and at least five other loci. It probably represents a different haplotype of part of the class I region. The previously identified UAA gene shares an identical 5' part with UEA, but the two genes differ in their 3' parts. One of them is probably the result of an unequal crossing over. The described organization has implications for the persistence of syntenic relationships, coevolution of loci, and interpretation of the origin of the human/mouse Mhc organization.

Animals↗
PubMed2000

Major histocompatibility complex class II A genes in cichlid fishes: identification, expression, linkage relationships, and haplotype variation.

Two cichlid species, the haplochromine Aulonocara hansbaenschi and the tilapiine Oreochromis niloticus, were used to study the major histocompatibility complex (Mhc) class II A variation within this group. Multiple class II A sequences were recovered from A. hansbaenschi and O. niloticus cDNA libraries and three sequence families, DAA, DBA, and DCA, were identified. Sets of O. niloticus haploid embryo families were used to determine the linkage relationships of these genes. Two independently assorting linkage groups were detected, DAA and DBA/DCA, neither of which is linked to the previously described Mhc class I gene cluster. Three DCA genes and up to four DBA genes were found to segregate in different haplotypes, whereas DAA occurred as a single locus. Four DBA haplotypes, DBA*H1-H4, were identified and shown to co-segregate with the previously described class II B haplotypes. Four DCA haplotypes, DCA*H1-H4, were found at a distance of 37 cM from the DBA/class II B cluster; in one DCA haplotype, DCA*H5, the genes were tightly linked to the DBA/class II B clusters. Transcripts of DAA and DBA genes were found in O. niloticus hepatopancreas and spleen; transcripts of DCA genes were detected in the A. hansbaenschi cDNA library, but not in O. niloticus. These findings provide a basis for using class II haplotypes as markers in the study of adaptive radiation in the cichlid species flocks of the East African Great Lakes.

Amino Acid Sequence↗
PubMed2000

Nonlinkage of major histocompatibility complex class I and class II loci in bony fishes.

In tetrapods, the functional (classical) class I and class II B loci of the major histocompatibility complex (Mhc) are tightly linked in a single chromosomal region. In an earlier study, we demonstrated that in the zebrafish, Danio rerio, order Cypriniformes, the two classes are present on different chromosomes. Here, we show that the situation is similar in the stickleback, Gasterosteus aculeatus, order Gasterosteiformes, the common guppy, Poecilia reticulata, order Cyprinodontiformes, and the cichlid fish Oreochromis niloticus, order Perciformes. These data, together with unpublished results from other laboratories suggest that in all Euteleostei, the classical class I and class II B loci are in separate linkage groups, and that in at least some of these taxa, the class II loci are in two different groups. Since Euteleostei are at least as numerous as tetrapods, in approximately one-half of jawed vertebrates, the class I and class II regions are not linked.

Animals↗
PubMed2000

Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic probes for glycosyltransferases.

An effective procedure for the synthesis of 2-deoxy-2-fluoro-sugar nucleotides via Select fluor-mediated electrophilic fluorination of glycals with concurrent nucleophilic addition or chemo-enzymatic transformation has been developed, and the fluorinated sugar nucleotides have been used as probes for glycosyltransferases, including fucosyltransferase III, V, VI, and VII, and sialyl transferases. In general, these fluorinated sugar nucleotides act as competitive inhibitors versus sugar nucleotide substrates and form a tight complex with the glycosyltransferase.

Animals↗
PubMed2000

Identification and linkage of the proteasome activator complex PA28 subunit genes in zebrafish.

PA28 is an activator of the latent 20S proteasome, a large multisubunit complex involved in intracellular proteolysis. Two forms of hexameric PA28 have been identified, PA28-(alphabeta)3 and PA28-(gamma)6, of which the former is of immunological importance. Both the PA28-alpha and PA28-beta subunits are inducible by interferon-gamma (IFN-gamma) and the PA28-(alphabeta)3 complex enhances the ability of the 20S proteasome to produce peptides suited for binding to major histocompatibility complex (Mhc) class I molecules. To identify the homologues of the PA28 subunits in zebrafish we screened a cDNA library and obtained full-length cDNA sequences of the genes PSME1, PSME2 and PSME3 coding for the PA28-alpha, PA28-beta and PA28-gamma subunits, respectively. Phylogenetic analysis indicates the existence of the ancestors of all three genes prior to the divergence of tetrapods and bony fishes. The IFN-gamma-inducible subunits, PA28-alpha and PA28-beta, evolve faster than the presumably older PA28-gamma subunit. Using zebrafish radiation hybrid panels, the genes PSME2 and PSME3 were mapped to linkage group 12 and shown to be separated by a distance of less than 2.4 cM. This observation suggests that an intrachromosomal duplication event created the precursor of the IFN-gamma-inducible genes from a PA28-gamma-like ancestor prior to their recruitment into the Mhc class I peptide presentation pathway.

Amino Acid Sequence↗
PubMed2000

Identification of seven genes in the major histocompatibility complex class I region of the zebrafish.

Physical linkage of genes whose products are involved in similar physiological pathways may have functional significance. The identification of conserved gene linkage in distantly related organisms can therefore strengthen the hypothesis of selection acting towards keeping genes on a chromosome. We used the cDNA selection technique and the polymerase chain reaction (PCR) with generic primers for the identification of new genes on the genomic clones bearing the major histocompatibility complex (Mhc) class I genes of the zebrafish (Danio rerio). We found six new genes (BING1, DAXX, TAPBP, KNSL2, TAP2B and KE6) whose orthologues are known to be linked to the Mhc class II region in humans and mice. In addition, a new zebrafish Mhc class I gene, termed Dare-UFA, was detected. By contrast, a search for the human leucocyte antigen (HLA)-linked BING3, KE3 and SACM2L genes revealed that these loci are not located on the class I clones of the zebrafish. The zebrafish class I region contains repetitive elements with similarity to the DANA, SATA and LINE repeats, as well as Tc1 transposable elements. Our findings indicate a high degree of linkage conservation between the zebrafish class I and the mammalian class II regions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Analysis of a 26-kb region linked to the Mhc in zebrafish: genomic organization of the proteasome component beta/transporter associated with antigen processing-2 gene cluster and identification of five new proteasome beta subunit genes.

Sequencing of zebrafish (Danio rerio) bacterial artificial chromosome and P1 artificial chromosome genomic clone fragments and of cDNA clones has led to the identification of five new loci coding for beta subunits of proteasomes (PSMB). Together with the four genes identified previously, nine PSMB genes have now been defined in the zebrafish. Six of the nine genes reside in the zebrafish MHC (Mhc) class I region, four of them reside in a single cluster closely associated with TAP2 on a 26-kb long genomic fragment, and two reside at some distance from the fragment. In addition to homologues of the human genes PSMB5 through PSMB9, two new genes, PSMB11 and PSMB12, have been found for which there are no known corresponding genes in humans. The new genes reside in the PSMB cluster in the Mhc. Homology and promoter region analysis suggest that the Mhc-associated genes might be inducible by IFN-gamma. The zebrafish class I region contains representatives of three phylogenetically distinguishable groups of PSMB genes, X, Y, and Z. It is proposed that these genes were present in the ancestral PSMB region before Mhc class I genes became associated with it.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.

Activation of the transcription factor NF-kappaB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IkappaB. We recently purified a large multiprotein complex, the IkappaB kinase (IKK) signalsome, which contains two regulated IkappaB kinases, IKK1 and IKK2, that can each phosphorylate IkappaBalpha and IkappaBbeta. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappaB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IkappaB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappaB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappaB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappaB activation.

Amino Acid Sequence↗
PubMed1998

Isolation of mhc class II DMA and DMB cDNA sequences in a marsupial: the gray short-tailed opossum (Monodelphis domestica).

We report the cDNA sequences for the DMA and DMB family of Mhc genes of the gray short-tailed opossum. Until now DM sequences were available only in eutherian mammals. The marsupial sequences indicate that both members of the family are old and probably diverged from other classical class II families about the time of the radiation of jawed vertebrates some 450 million years ago. We examine the evolutionary rates of equivalent sets of classical and nonclassical genes to check for rate heterogeneity. We find the alpha-1 domain of the DR genes to be untypically conservative in its evolutionary mode. The DM genes appear to evolve at rates typical of other class II genes, indicating that their placement at the root of class II gene evolutionary trees may be justified.

Amino Acid Sequence↗
PubMed1998

Sequence variation at the major histocompatibility complex DRB loci in beluga (Delphinapterus leucas) and narwhal (Monodon monoceros).

The variation at loci with similarity to DRB class II major histocompatibility complex loci was assessed in 313 beluga collected from 13 sampling locations across North America, and 11 narwhal collected in the Canadian high Arctic. Variation was assessed by amplification of exon 2, which codes for the peptide binding region, via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Two DRB loci were identified in beluga: DRB1, a polymorphic locus, and, DRB2, a monomorphic locus. Eight alleles representing five distinct lineages (based on sequence similarity) were found at the beluga DRB1 locus. Although the relative number of alleles is low when compared with terrestrial mammals, the amino acid variation found among the lineages is moderate. At the DRB1 locus, the average number of nonsynonymous substitutions per site is greater than the average number of synonymous substitutions per site (0.0806 : 0.0207, respectively; P<0.01). Most of the 31 amino acid substitutions do not conserve the physiochemical properties of the residue, and 21 of these are located at positions implicated as forming pockets responsible for the selective binding of foreign peptide side chains. Only DRB1 variation was examined in 11 narwhal, revealing a low amount of variation. These data are consistent with an important role for the DRB1 locus in the cellular immune response of beluga. In addition, the ratio of nonsynonymous to synonymous substitutions is similar to that among primate alleles, arguing against a reduction in the balancing selection pressure in the marine environment. Two hypotheses may explain the modest amount of Mhc variation when compared with terrestrial mammals: small population sizes at speciation or a reduced neutral substitution rate in cetaceans.

Alleles↗
PubMed1997

IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

Cell Cycle Proteins↗
PubMed1997

p38-2, a novel mitogen-activated protein kinase with distinct properties.

Mitogen-activated protein (MAP) kinases are involved in many cellular processes. Here we describe the cloning and characterization of a new MAP kinase, p38-2. p38-2 belongs to the p38 subfamily of MAP kinases and shares with it the TGY phosphorylation motif. The complete p38-2 cDNA was isolated by polymerase chain reaction. It encodes a 364-amino acid protein with 73% identity to p38. Two shorter isoforms missing the phosphorylation motif were identified. Analysis of various tissues demonstrated that p38-2 is differently expressed from p38. Highest expression levels were found in heart and skeletal muscle. Like p38, p38-2 is activated by stress-inducing signals and proinflammatory cytokines. The preferred upstream kinase is MEK6. Although p38-2 and p38 phosphorylate the same substrates, the site specificity of phosphorylation can differ as shown by two-dimensional phosphopeptide analysis of Sap-1a. Additionally, kinetic studies showed that p38-2 appears to be about 180 times more active than p38 on certain substrates such as ATF2. Both kinases are inhibited by a class of pyridinyl imidazoles. p38-2 phosphorylation of ATF2 and Sap-1a but not Elk1 results in increased transcriptional activity of these factors. A sequential kinetic mechanism of p38-2 is suggested by steady state kinetic analysis. In conclusion, p38-2 may be an important component of the stress response required for the homeostasis of a cell.

Activating Transcription Factor 2↗
PubMed1997

Why is CMP-ketodeoxyoctonate highly unstable?

CMP-ketodeoxyoctonate (CMP-KDO) and analogs, including CMP-5-deoxy-5-fluoro-KDO, CMP-5-deoxy-KDO, and CMP-5-epi-KDO, were prepared from CTP and the corresponding KDO sugars catalyzed by CMP-KDO synthetase. These analogs were found to be much more stable than CMP-KDO (t1/2 = 0.57 h) yet less stable than CMP-sialic acid (t1/2 = 151 h). Fluorination at the 5-position of CMP-KDO has a 200-fold enhanced stability compared to the 156-fold enhancement for the 3R-fluoro analog, probably due to the loss of H-bonding interactions (for the 5-F derivative) and the cause of remote inductive effect (for the 3- and the 5-F analogs) on the glycosidic cleavage. Hydrolysis of CMP-KDO is perhaps facilitated by an intramolecular hydrogen bond from the 5-OH group with the phosphate oxygen as demonstrated by the 3-5-fold enhanced stability of CMP-5-epi-KDO and CMP-5-deoxy-KDO compared to CMP-KDO and by molecular modeling studies of water-solvated CMP-KDO. Hydrolysis of CMP-KDO also was found to be subject to a substantial solvent isotope effect (kH/kD = 2.7), which is significantly different from the reported solvent isotope effect for the hydrolysis of sialyglycosides (kH/kD = 0.86) and dependent on both buffer and magnesium ion concentrations. Considering these results and molecular modeling studies, it is proposed that the hydrolysis of CMP-KDO under neutral conditions proceeds through a glycosidic cleavage which occurs at the electronically favorable twist-boat conformation, facilitated by intramolecular H-bonding interaction of the 4-, 5- and 7- (or 8-) OH groups and the phosphate oxygen and by the leaving group magnesium ion complexation.

Buffers↗