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Biomedical subjects

A Gilchrist

Publications and source records attributed to A Gilchrist.

At least 37 records · Page 2Linked to original sources
PubMed1998

p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF-alpha or FMLP stimulation.

Mitogen-activated protein (MAP) kinase-mediated signal-transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the roles of MAP kinases in neutrophils are not well understood yet. Protein phosphorylation analysis of cellular MAP kinases indicates that exposure of human neutrophils to chemotactic factor FMLP as well as granulocyte-macrophage CSF, PMA, or ionomycin rapidly induced the activation of p38 and p44/42 MAP kinases, but stimulation with inflammatory cytokine TNF-alpha triggered the activation of p38 MAP kinase only. To study the cellular functions of these MAP kinases, the inhibitor SB20358, which specifically inhibited enzymatic activity of cellular p38 MAP kinase, and the inhibitor PD98059, which specifically blocked the induced protein phosphorylation and activation of p44/42 MAP kinase in intact neutrophils, were utilized. Inhibition of the cellular p38 MAP kinase activation almost completely abolished the TNF-alpha-stimulated IL-8 production and superoxide generation of human neutrophils. In addition, the FMLP-induced neutrophil chemotaxis as well as superoxide generation were suppressed markedly by inhibiting the activation of cellular p38 MAP kinase, but not p44/42 MAP kinase. Moreover, RIA indicates that the activation of cellular p38 MAP kinase was required for the neutrophil IL-8 production stimulated by granulocyte-macrophage CSF or LPS as well as TNF-alpha, but not for that induced by PMA or ionomycin. These results demonstrate that the activation of cellular p38 MAP kinase is indispensable for the TNF-alpha- or FMLP-mediated cellular functions in human neutrophils, and suggest that p38 MAP kinase may play a different role in response to distinct stimulation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

High expression and activation of MAP kinase-activated protein kinase 2 in cardiac muscle cells.

Recently, three mammalian mitogen-activated protein (MAP) kinases, ERK, SAPK/JNK, and p38/HOG-1 have been identified, each with apparently unique signal transduction pathways. The p38 MAP kinase mediates an intracellular stress-activated signaling pathway by regulating down-stream molecules, such as MAP kinase-activated protein (MAPKAP) kinase 2. To study the tissue specificity of MAPKAP kinase 2, mRNA blots containing multiple human tissues were hybridized with a specific oligonucleotide probe corresponding to human MAPKAP kinase 2. The Northern blot analysis revealed that two mRNA species of MAPKAP kinase 2, with sizes of 4.8 and 3.3 kb, were expressed in high levels in both human heart and skeletal muscle tissues. To better understand how MAPKAP kinase 2 is regulated in myocardium, cultured rat cardiac myoblast (H9c2) cells were stimulated with heat shock, H2O2-induced oxidative stress, or phorbol ester (PMA). Enzymatic activity of cellular MAPKAP kinase 2 in the cell lysates was evaluated using an in vitro kinase assay. Exposure of H9c2 cells to heat shock or oxidative stress induced a transient increase of cellular MAPKAP kinase 2 activity, which reached its peak level within 5 min. In contrast, stimulation of H9c2 cells with PMA, a potential myocardial hypertrophic factor, induced a sustained increase of cellular MAPKAP kinase 2 activity that was detectable for over 1 h. In addition, in vitro protein phosphorylation analysis with recombinant MAPKAP kinase 2 showed that small heat shock protein (hsp25) served as a major substrate molecule for the kinase in H9c2 cells and the protein phosphorylation of cellular hsp25 was stimulated by H2O2-induced oxidative stress or PMA treatment in intact H9c2 cells. Moreover, exposure of H9c2 cells to H2O2-induced oxidative stress or PMA rapidly activated cellular p38 MAP kinase as detected by the induced protein phosphorylation of the kinase. Taken together, these results strongly suggest that MAPKAP kinase 2 may be involved in stress-activated signal transduction in myocardium.

Animals↗
PubMed1996

Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation.

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen-activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase-activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP-stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.

Amino Acid Sequence↗
PubMed1996

Protection of the side of the head.

The side of the head is a frequent impact site for pedal cyclists, motorcyclists, and industrial workers. It is vulnerable to impact, yet many helmet standards do not have impact tests at the side of the helmet. Laboratory impact tests should reproduce the phenomena in real impacts, but usually they do not allow the headform to rotate, or ignore the effect of the neck on the motion of the head. The authors designed a linkage, simulating the flexibility of the neck, for use with anthropomorphic dummies in helmet testing. Impacts at the side of the helmet, normal to the helmet surface, were made with flat and hemispherical strikers. Head rotation during impacts caused the impact site to move, in some cases to below the level protected by the helmet. The peak angular head acceleration correlated with the peak linear accelerations. Neck flexibility means that helmets can protect the head from higher kinetic energy impacts than those specified in standards using immovable headforms or impact anvils. The load spreading efficiency of polypropylene foam, used in soft shell bicycle helmets, is better than that of polystyrene foam. The authors conclude that the side of the head can be protected by a suitable helmet design.

Acceleration↗
PubMed1996

Heterotrimeric G proteins.

Over the past year, the thrust of work in the field of heterotrimeric G proteins has been primarily in the following areas: first, resolution of their three-dimensional structures by X-ray crystallography; second, elucidation of the effect of lipid modifications on the Galpha and Ggamma subunits; third, understanding the role of the Gbetagamma dimer in stimulation of a variety of effectors following receptor activation; and fourth, identification of the points of contact among the Galpha, Gbeta, and Ggamma subunits, and between these subunits and their cognate receptor or effector molecules.

Animals↗
PubMed1995

Ectopic expression of human and feline CD9 in a human B cell line confers beta 1 integrin-dependent motility on fibronectin and laminin substrates and enhanced tyrosine phosphorylation.

Few molecules have been shown to confer cell motility. Although the motility-arresting properties of anti-CD9 monoclonal antibody (mAb) suggest the transmembrane 4 superfamily (TM4SF) member CD9 can induce a motorgenic signal, gene transfection studies have failed to confirm this hypothesis. We report here that ectopic expression of human CD9 (CD9h) and feline CD9 (CD9f) in the CD9-negative, poorly motile, human B cell line Raji dramatically enhances migration across fibronectin- and laminin-coated polycarbonate filters. Migration of Raji/CD9h and Raji/CD9f on either substrate was inhibited by the anti-CD9 mAb 50H.19 and by the anti-beta 1 integrin mAb AP-138. Migration of Raji/CD9h on laminin was potently inhibited by the anti-VLA-6 integrin mAb GoH3 and by the anti-VLA-4 integrin mAb 44H6, whereas migration of Raji/CD9h on fibronectin was inhibited only by mAb 44H6. Since CD9h-transfected Raji cells adhered to fibronectin as effectively as mock transfectants, expression of CD9 enhanced motility, but not adhesion. CD9-enhanced migration was inhibited by the protein tyrosine kinase inhibitor herbimycin A suggesting that tyrosine phosphorylation played a role in the generation of a motorgenic signal. Raji/CD9h transfectants adherent to fibronectin expressed 6-fold higher levels of phosphotyrosine than Raji. Raji/CD9f transfectants also phosphorylated proteins on tyrosine more effectively than Raji including a protein of 110 kDa which was phosphorylated on the motility-inducing substrates laminin and fibronectin, but not on bovine serum albumin. Our results support a role for CD9 in the amplification of a motorgenic signal in B cells involving beta 1 integrins and the activation of protein tyrosine kinases.

Animals↗
PubMed1995

The critical role of relative luminance relations in White's effect and grating induction.

It has been proposed that both White's effect and the grating induction effect are examples of brightness induction phenomena modeled in terms of local spatial filters. We have shown that for these illusions to occur it is necessary that the luminance of the gray target elements falls between that of the inducing stripes of the square-wave pattern. This critical role of luminance relationships is not predicted by existing models of these illusions.

Color Perception↗
PubMed1995

Nucleotide sequence of tomato ringspot virus RNA1.

The nucleotide sequence of tomato ringspot nepovirus (TomRSV) RNA1 has been determined. TomRSV RNA1 is 8214 nucleotides in length, excluding the 3' poly(A) tail, and contains a single long open reading frame (ORF) of 6591 nucleotides beginning at the first AUG codon at nucleotide position 78. This ORF accounts for 80% of the RNA1 sequence and would give rise to a polyprotein with a predicted molecular mass of 244 kDa. Amino acid sequence comparisons between portions of the TomRSV RNA1-encoded polyprotein and proteins encoded by several members of the picornavirus superfamily have provided information concerning the genomic organization and putative functions of TomRSV-encoded proteins. The putative TomRSV protease retains a conserved histidine residue present in the proteases encoded by members of the como-, poty- and poliovirus groups which is thought to be involved in dipeptide cleavage site recognition. Interestingly, this histidine residue is replaced by a leucine in the proteases of other sequenced nepoviruses. This suggests that the TomRSV protease shares dipeptide cleavage site specificity with that of como-, poty- and picornaviruses rather than the other nepoviruses.

Amino Acid Sequence↗
PubMed1995

Local and global processes in surface lightness perception.

Various demonstrations show that a target of constant luminance can be made to appear darker in perceived lightness merely by introducing an adjacent region of higher luminance. This has often been interpreted as a manifestation of contrast effects produced by lateral inhibition, a relatively local process. An alternative interpretation holds that the highest luminance in such a display serves as an anchor that defines the white level. This interpretation is global in the sense that the anchor need not be located near any particular target in order to serve as its standard. Edge integration processes have been postulated that would enable such remote comparisons, but there is controversy about the strength of these processes. We report a series of experiments in which local and global processes were assessed. Specifically, we tested whether the introduction of a higher luminance has a greater darkening effect on an adjacent target than on a remote target. We found no difference, suggesting that the darkening effect is a matter of anchoring, not contrast, and that edge integration processes required by anchoring are relatively strong.

Attention↗
PubMed1994

The primary structure of a human MAP kinase activated protein kinase 2.

Mitogen-activated protein (MAP) kinase is of central importance in mediating intracellular actions in response to a variety of extracellular stimuli. MAP kinase activated protein (MAPKAP) kinase 2 is one of the two known protein kinases that can be phosphorylated and activated by MAP kinase. Here we present the first complete primary structure of MAPKAP kinase 2 elucidated from a human cDNA sequence. Sequence analysis reveals that MAPKAP kinase 2 is a 370 amino acid protein containing a proline-rich N-terminal region and a well conserved catalytic domain. Northern blot analysis of MAPKAP kinase 2 showed a 4.8 kb mRNA species in HL-60 cells. In addition, we also show the first evidence that recombinant MAPKAP kinase 2 is phosphorylated and activated by MAP kinase in vitro.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein.

The entire nucleotide sequence of the rsaA gene, encoding the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A, was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98,132. Protease cleavage of mature RsaA protein and amino acid sequencing of retrievable peptides yielded two peptides: one aligned with a region approximately two-thirds the way into the predicted amino acid sequence and the second peptide corresponded to the predicted carboxy terminus. Thus, no cleavage processing of the carboxy portion of the RsaA protein occurred during export, and with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted RsaA amino acid profile was unusual, with small neutral residues predominating. Excepting aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein, with a predicted pI of 3.46. As with most other sequenced S-layer proteins, RsaA contained no cysteine residues. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted RsaA sequence. However, RsaA protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the RsaA protein that was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in the RsaA protein may perform a similar function, since S-layer assembly and surface attachment requires calcium. RsaA protein also shared some homology with 10 other S-layer proteins, with the Campylobacter fetus S-layer protein scoring highest.

Amino Acid Sequence↗
PubMed1991

Transformation of freshwater and marine caulobacters by electroporation.

We performed plasmid electrotransformation of Caulobacter crescentus strains and obtained up to 3 x 10(8) transformants per micrograms of pKT230. The presence and integrity of the paracrystalline protein surface (S) layer influenced electroporation; caulobacters lacking the S layer were electrotransformed 10 times more efficiently than caulobacters possessing the S layers. A procedure yielding 1,500 transformants per micrograms of pKT230 was developed for a marine caulobacter. Electroporation was used in combination with several genetic techniques, including introduction of ligation mixtures, suicide transposon mutagenesis, gene replacement, and plasmid electrotransfer from Escherichia coli to caulobacters.

Bacteria↗