Biomedical subjects

R Craig

Publications and source records attributed to R Craig.

At least 19 recordsLinked to original sources

PubMed2000

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments.

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.

Actin Cytoskeleton↗

PubMed2000

p38 MAPK and NF-kappa B collaborate to induce interleukin-6 gene expression and release. Evidence for a cytoprotective autocrine signaling pathway in a cardiac myocyte model system.

In cardiac myocytes, the stimulation of p38 MAPK by the MAPKK, MKK6, activates the transcription factor, NF-kappaB, and protects cells from apoptosis. In the present study in primary neonatal rat cardiac myocytes, constitutively active MKK6, MKK6(Glu), bound to IkappaB kinase (IKK)-beta and stimulated its abilities to phosphorylate IkappaB and to activate NF-kappaB. MKK6(Glu) induced NF-kappaB-dependent interleukin (IL)-6 transcription and IL-6 release in a p38-dependent manner. IL-6 protected myocardial cells against apoptosis. Like IL-6, TNF-alpha, which activates both NF-kappaB and p38, also induced p38-dependent IL-6 expression and release and protected myocytes from apoptotis. While TNF-alpha was relatively ineffective, IL-6 activated myocardial cell STAT3 by about 8-fold, indicating a probable role for this transcription factor in IL-6-mediated protection from apoptosis. TNF-alpha-mediated IL-6 induction was inhibited by a kinase-inactive form of the MAPKKK, TGF-beta activated protein kinase (Tak1), which is known to activate p38 and NF-kappaB in other cell types. Thus, by stimulating both p38 and NF-kappaB, Tak1-activating cytokines, like TNF-alpha, can induce IL-6 expression and release. Moreover, the myocyte-derived IL-6 may then function in an autocrine and/or paracrine fashion to augment myocardial cell survival during stresses that activate p38.

Animals↗

PubMed2000

An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin.

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.

Actin Cytoskeleton↗

PubMed2000

Three-dimensional reconstruction of thin filaments containing mutant tropomyosin.

Interactions of the components of reconstituted thin filaments were investigated using a tropomyosin internal deletion mutant, D234, in which actin-binding pseudo-repeats 2, 3, and 4 are missing. D234 retains regions of tropomyosin that bind troponin and form end-to-end tropomyosin bonds, but has a length to span only four instead of seven actin monomers. It inhibits acto-myosin subfragment 1 ATPase (acto-S-1 ATPase) and filament sliding in vitro in both the presence and absence of Ca(2+) (, J. Biol. Chem. 272:14051-14056) and lowers the affinity of S-1.ADP for actin while increasing its cooperative binding. Electron microscopy and three-dimensional reconstruction of reconstituted thin filaments containing actin, troponin, and wild-type or D234 tropomyosin were carried out to determine if Ca(2+)-induced movement of D234 occurred in the filaments. In the presence and absence of Ca(2+), the D234 position was indistinguishable from that of the wild-type tropomyosin, demonstrating that the mutation did not affect normal tropomyosin movement induced by Ca(2+) and troponin. These results suggested that, in the presence of Ca(2+) and troponin, D234 tropomyosin was trapped on filaments in the Ca(2+)-induced position and was unable to undergo a transition to a completely activated position. By adding small amounts of rigor-bonded N-ethyl-maleimide-treated S-1 to mutant thin filaments, thus mimicking the myosin-induced "open" state, inhibition could be overcome and full activation restored. This myosin requirement for full activation provides support for the existence of three functionally distinct thin filament states (off, Ca(2+)-induced, myosin-induced; cf.;, J. Mol. Biol. 266:8-14). We propose a further refinement of the three-state model in which the binding of myosin to actin causes allosteric changes in actin that promote the binding of tropomyosin in an otherwise energetically unfavorable "open" state.

Actin Cytoskeleton↗

PubMed2000

Children's acceptance of xylitol-based foods.

UNLABELLED: Daily consumption of xylitol (5-10 g/day) added to chewing gum and confectionary foods has been previously shown to prevent dental caries in children. METHODS: Snack foods containing xylitol were developed and tested for acceptability in a convenience sample of 31 children ages 3 to 6 years. In order to mimic an after-meal snack, all children were tested during mid-morning, approximately 1 h after eating. Preference testing was based on the methodology of Birch et al. (J Nutr Educ, 1979; 11: 77-80). In the first phase, each child was presented with a tray of six xylitol-based foods (popsicles, pudding, gum drops, gelatin dessert, cookies, popcorn) and asked to taste each item in any desired order. Immediately after tasting a food, the child was asked to place it in front of one of three cartoon faces (smile, frown or neutral) representing the child's response to the taste of that particular food. In the second phase, the child was asked to rank order the foods in each face category (smile, frown or neutral). Ranks within categories were then combined to obtain a rank ordering for all of the foods. RESULTS: Non-parametric data analysis indicated significant differences in ranking between the foods when they were compared to each other (Friedman ANOVA by ranks, P<0.01). Pudding was significantly less preferred than the other foods (sign tests, P<0.04). At least 84% of the children found five of the six foods very good or satisfactory, when considered individually. CONCLUSIONS: These results suggest that snack foods developed with xylitol are generally well accepted by children.

Analysis of Variance↗

PubMed2000

Measure how ACNPs impact outcomes.

Nurse leaders need a way to measure acute care nurse practitioners' (ACNPs') performance and justify their positions. An objective documentation tool can help to evaluate ACNPs' work activities and measure performance and outcomes.

Acute Disease↗

PubMed1999

Low bioavailability of cyclosporine microemulsion and tacrolimus in a small bowel transplant recipient: possible relationship to intestinal P-glycoprotein activity.

With intestine transplants the allograft is dependent on itself for maintenance of adequate immunosuppression. We evaluated an intestinal transplant recipient who required very large doses of either tacrolimus or cyclosporine emulsion to achieve acceptable blood concentrations. Pharmacokinetic studies revealed bioavailabilities of 2% and 6% respectively, while D-xylose and B12 absorption were found to be within normal limits and fecal fat was only slightly increased, suggesting that there was a selective absorptive defect for these drugs. Biopsies of the allograft ileum revealed a high P-glycoprotein activity compared to the jejunum or to intestinal biopsies from other normal subjects. This may be a contributing factor to poor immunosuppressive drug absorption in this patient and others.

Biological Availability↗

PubMed1999

Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy.

Past attempts to detect tropomyosin in electron micrograph images of frozen-hydrated troponin-regulated thin filaments under relaxing conditions have not been successful. This raised the possibility that tropomyosin may be disordered on filaments in the off-state, a possibility at odds with the steric blocking model of muscle regulation. By using cryoelectron microscopy and helical image reconstruction we have now resolved the location of tropomyosin in both relaxing and activating conditions. In the off-state, tropomyosin adopts a position on the outer domain of actin with a binding site virtually identical to that determined previously by negative staining, although at a radius of 3.8 nm, slightly higher than found in stained filaments. Molecular fitting to the atomic model of F-actin shows that tropomyosin is localized over sites on actin subdomain 1 required for myosin binding. Restricting access to these sites would inhibit the myosin-cross-bridge cycle, and hence contraction. Under high Ca(2+) activating conditions, tropomyosin moved azimuthally, away from its blocking position to the same site on the inner domain of actin previously determined by negative staining, also at 3.8 nm radius. These results provide strong support for operation of the steric mechanism of muscle regulation under near-native solution conditions and also validate the use of negative staining in investigations of muscle thin filament structure.

Actins↗

PubMed1999

On scene thoracotomy: a case report.

We report a case of on scene resuscitative thoracotomy performed by an anaesthetist on a patient in cardiac arrest following a stab wound to the chest. The patient made a good recovery and was discharged from hospital within 2 weeks. The rationale for performing resuscitative thoracotomy and who should perform this procedure are discussed.

Adult↗

PubMed1998

Functional analysis of Tpr: identification of nuclear pore complex association and nuclear localization domains and a role in mRNA export.

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.

Animals↗

PubMed1998

The novel murine calmodulin-binding protein Sha1 disrupts mitotic spindle and replication checkpoint functions in fission yeast.

Entry into mitosis is normally blocked in eukaryotic cells that have not completed replicative DNA synthesis; this 'S-M' checkpoint control is fundamental to the maintenance of genomic integrity. Mutants of the fission yeast Schizosaccharomyces pombe defective in the S-M checkpoint fail to arrest the cell cycle when DNA replication is inhibited and hence attempt mitosis and cell division with unreplicated chromosomes, resulting in the 'cut' phenotype. In an attempt to identify conserved molecules involved in the S-M checkpoint we have screened a regulatable murine cDNA library in S. pombe and have identified cDNAs that induce the cut phenotype in cells arrested in S phase by hydroxyurea. One such cDNA encodes a novel protein with multiple calmodulin-binding motifs that, in addition to its effects on the S-M checkpoint, perturbed mitotic spindle functions, although spindle pole duplication was apparently normal. Both aspects of the phenotype induced by this cDNA product, which we term Sha1 (for spindle and hydroxyurea checkpoint abnormal), were suppressed by simultaneous overexpression of calmodulin. Sha1 is structurally related to the product of the Drosophila gene abnormal spindle (asp). These data suggest that calmodulin-binding protein(s) are important in the co-ordination of mitotic spindle functions with mitotic entry in fission yeast, and probably also in multicellular eukaryotes.

3T3 Cells↗

PubMed1998

Dimerization of P-selectin glycoprotein ligand-1 (PSGL-1) required for optimal recognition of P-selectin.

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with alpha1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.

Alanine↗

PubMed1998

An sLex-deficient variant of HL60 cells exhibits high levels of adhesion to vascular selectins: further evidence that HECA-452 and CSLEX1 monoclonal antibody epitopes are not essential for high avidity binding to vascular selectins.

Selectins are carbohydrate-binding cell adhesion molecules that play a key role in the initiation of inflammatory responses. Several studies have suggested that the sialylated, fucosylated tetrasaccharide sialyl Lewis X (sLex) is an important component of leukocyte ligands for E- and P-selectin. We have identified a stable variant of the HL60 cell line, HL60var, which displays a nearly complete absence of staining with several mAb directed against sLex and/or sLex-related structures. HL60var also exhibits a concomitant increase in reactivity with mAb directed against the unsialylated Lewis X (Lex/CD15) structure. Despite this sLex deficiency, HL60var binds well to both E- and P-selectin. No significant differences in expression of alpha1,3-fucosyltransferases, C2GnT (Core2 transferase), or P-selectin glycoprotein ligand-1 between HL60var and typical sLex(high) HL60 cells were detected. Although the precise molecular basis for the sLex(-/low) phenotype of HL60var remains uncertain, flow cytometric analysis with the sialic acid-specific Limax flavus lectin revealed a sharp reduction in HL60var surface sialylation. Thus, the loss in mAb reactivity may result from a loss of sialic acid residues from the mAb carbohydrate epitope. However, binding of HL60var to E- and P-selectin remains sensitive to neuraminidase treatment. Taken together, these data indicate that high levels of surface sLex and/or related epitopes are not essential for interactions with vascular selectins, implying that as yet unidentified sialylated, fucosylated structures serve as physiologically relevant ligands for E- and P-selectin.

Animals↗

PubMed1998

MKK6 activates myocardial cell NF-kappaB and inhibits apoptosis in a p38 mitogen-activated protein kinase-dependent manner.

In cardiac myocytes the stimulation of p38 mitogen-activated protein kinase activates a hypertrophic growth program and the induction of the cardiac-specific genes associated with this program. This study focused on determining whether these novel growth-promoting effects are accompanied by the p38-mediated inhibition of apoptosis, and if so, what signaling pathways might be responsible. Primary neonatal rat ventricular myocytes were driven into apoptosis by treatments known to induce apoptosis in other cell types, e.g. incubation with anisomycin or overexpression constitutively active MEKK-1 (MEKK-1COOH), a protein that strongly activates extracellular signal-regulated kinase and N-terminal c-Jun kinase, but not p38. Overexpression of constitutively active MKK6, MKK6 (Glu), which selectively activates p38 in cardiac myocytes, protected cells from either anisomycin- or MEKK-1COOH-induced apoptosis. This protection was blocked by SB 203580, a selective p38 inhibitor. MKK6 (Glu) also activated transcription mediated by NF-kappaB, a factor which protects other cell types from apoptosis. The activation of NF-kappaB and the protection from apoptosis mediated by MKK6 (Glu) were both blocked by SB 203580. Interestingly, overexpression of a mutant form of I-kappaBalpha, which inhibits nuclear translocation of NF-kappaB, completely blocked MKK6 (Glu)-activated NF-kappaB but had little effect on MKK6s anti-apoptotic effects. These findings suggest that, in part, the overexpression of MKK6 (Glu) may foster growth and survival of cardiac myocytes by protecting them from apoptosis in a p38-dependent manner. Additionally, while NF-kappaB is activated in myocardial cells by p38, this does not appear to be the major mechanism by which MKK6 (Glu) exerts its anti-apoptotic effects in this cell type, suggesting a novel pathway for p38-mediated protection from apoptosis.

Animals↗

PubMed1998

Towards an atomic model of the thick filaments of muscle.

The thick filaments of muscle and non-muscle cells are polymers of myosin molecules whose energy-transducing heads lie on the filament surface, where they interact with actin to generate force. A key structural question is how the myosin heads are arranged in the relaxed state, and how this arrangement changes on activation of contraction. We have fitted the atomic structure of the myosin head to the three-dimensional structure of myosin filaments of tarantula muscle determined by electron microscopy to produce a near-atomic model of the head arrangement. A good fit is obtained only when the two heads from a myosin molecule run along the helical tracks antiparallel to each other. Oppositely oriented heads from axially adjacent molecules in a helix interact with each other, with their nucleotide-binding pockets opposed. This arrangement, supported also by crosslinking evidence, suggests a simple mechanism for the stabilization of myosin head helices in relaxed muscle via the formation of intermolecular "dimers" of heads from axially adjacent myosin molecules.

Actin Cytoskeleton↗

PubMed1998

An atomic model of fimbrin binding to F-actin and its implications for filament crosslinking and regulation.

Using a new procedure that combines electron-density correlation with biochemical information, we have fitted the crystal structure of the N-terminal actin-binding domain of human T-fimbrin to helical reconstructions of fimbrin-decorated actin filaments. The map locates the N-terminal calcium-binding domain and identifies actin-binding site residues on the two calponin-homology domains of fimbrin. Based on this map, we propose a model of a fimbrin crosslink in an actin bundle and its regulation by calcium.

Actins↗

PubMed1998

Inhibition of Tat-mediated transactivation and HIV replication with Tat mutant and repressor domain fusion proteins.

Strategies to inhibit the spread of HIV infection consist of a number of specific molecular approaches. Since viral production is dependent upon Tat-mediated transactivation of the HIV promoter through the Tat activating region (TAR), tat antisense RNA, anti-tat ribozymes, TAR decoys and dominant negative Tat mutant proteins have been suggested as therapeutic inhibitors. We produced and tested several Tat mutant proteins, including a newly generated form Tat delta 58, for the ability to inhibit Tat-mediated transactivation and HIV production. In addition, we generated a new Tat fusion mutant between a C-terminus truncated form of Tat (Tat delta 53) and the Drosophila Engrailed (Eng) transcription repressor domain to test the hypothesis that transcriptional repression can be targeted to the HIV promoter. This fusion mutant was also examined for its capacity to block both Tat-mediated transactivation and HIV replication. We show that three mutants Tat delta 53. Tat delta 58 and Tat delta 53/Eng result in a transdominant phenotype inhibiting wild-type Tat-mediated transactivation, and that the inhibiting potential is increased by the presence of the entire basic domain or the fusion of a repressor domain. However, only the transdominant mutants Tat delta 58 and Tat delta 53/Eng significantly inhibit HIV-1 replication after infection of transfected T cell lines. These results demonstrate the potent inhibiting activity of Tat mutants on HIV replication, and suggest a synergistic effect of Tat transdominant mutant fusion with the Drosophila Engrailed transcription repressor domain.

Cell Line↗

PubMed1998

High level inhibition of HIV replication with combination RNA decoys expressed from an HIV-Tat inducible vector.

Intracellular immunization, an antiviral gene therapy approach based on the introduction of DNA into cells to stably express molecules for the inhibition of viral gene expression and replication, has been suggested for inhibition of HIV infection. Since the Tat and Rev proteins play a critical role in HIV regulation, RNA decoys and ribozymes of these sequences have potential as therapeutic molecular inhibitors. In the present study, we have generated several anti-HIV molecules; a tat-ribozyme, RRE, RWZ6 and TAR decoys and combinations of decoys, and tested them for inhibition of HIV-1 replication in vitro. We used T cell specific CD2 gene elements and regulatory the HIV inducible promoter to direct high level expression and a 3' UTR sequence for mRNA stabilization. We show that HIV replication was most strongly inhibited with the combination TAR + RRE decoy when compared with the single decoys or the tat-ribozyme. We also show that the Tat-inducible HIV promoter directs a higher level of steady-state transcription of decoys and inhibitors and that higher levels of expression directly relate to increased levels of inhibition of HIV infection. Furthermore, a stabilization of the 3' end of TAR + RRE inhibitor transcripts using a beta-globin 3' UTR sequence leads to an additional 15-fold increase in steady-state RNA levels. This cassette when used to express the best combination decoy inhibitor TAR + RRE, yields high level HIV inhibition for greater than 3 weeks. Taken together, both optimization for high level expression of molecular inhibitors and use of combinations of inhibitors suggest better therapeutic application in limiting the spread of HIV.

Cell Line↗