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Biomedical subjects

C A Phillips

Publications and source records attributed to C A Phillips.

At least 37 records · Page 2Linked to original sources
PubMed1996

Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions.

The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA. It is known that regA protein exists as a dimer in protein crystals. Thus, the stoichiometry may be indicative of a regA dimer bound to two RNAs or a regA monomer bound to one RNA. Gel filtration through Sephadex G-75 revealed that wild-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, consistent with the mass of a dimer. However, wild-type regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at approximately 20 kDa, consistent with a regA monomer-RNA complex. Covalent crosslinking of surface lysines with glutaraldehyde confirmed that wild-type and R91L proteins exist as dimers and higher oligomers in solution. However, the addition of RNA to wild-type regA protein prior to crosslinking inhibited the formation of crosslinked dimers. Thus, the regA protein-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA. Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer.

Chromatography, Gel↗
PubMed1996

Complete analysis of the presenilin 1 gene in early onset Alzheimer's disease.

The presenilin 1 gene has recently been identified as the locus on chromosome 14 which is responsible for a large proportion of early onset, autosomal dominantly inherited Alzheimer's disease (AD). We have elucidated the intron/exon structure of the gene and designed intronic primers to enable direct sequencing of the entire coding region (10 exons) of the presenilin gene in a large number of families. This strategy has enabled us to find a further two novel mutations in the gene. We discuss the distribution of mutations and the proportions of autosomal dominant AD with a mean age of onset below 60 years caused by mutations in this gene.

Alzheimer Disease↗
PubMed1996

CD28-dependent killing by human YT cells requires phosphatidylinositol 3-kinase activation.

CD28/B7 interactions have been demonstrated to provide a co-stimulatory signal for the generation of CD8+ cytotoxic T lymphocytes in the absence of CD4+ T helper cells. The CD28 signals required for induction of cytotoxicity have yet to be described. To investigate further the biochemical signaling pathways associated with CD28-dependent cytotoxicity, we have studied the human thymic leukemia cell line, YT. YT cells kill B7+ targets in a non-major histocompatibility complex (MHC)-restricted, CD28-dependent manner. CD28 ligation on the surface of YT cells caused a rapid increase in the tyrosine phosphorylation of four major cellular substrates with masses estimated to be 110, 95, 85, and 44 kDa. The 110 and 85 kDa substrates were identified as the catalytic and regulatory subunits, respectively, of phosphatidylinositol 3-kinase (PI3-K). Engagement of CD28 caused the rapid receptor association and activation of PI3-K but did not activate phospholipase C gamma. CD28-induced tyrosine phosphorylation and PI3-K activation was independent of p56lck protein tyrosine kinase (PTK) activity (previously reported to be associated with CD28) and was insensitive to inhibition by the PTK inhibitor herbimycin A. Two structurally and mechanistically dissimilar inhibitors of PI3-K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also failed to block CD28-dependent tyrosine phosphorylation events or the association of PI3-K with the CD28 receptor. However, both drugs inhibited CD28-dependent cytotoxicity and CD28 receptor associated PI3-K activity with IC50 values similar to the reported IC50 values for PI3-K inhibition. Although herbimycin A did not significantly block the observed CD28-dependent tyrosine phosphorylation or PI3-K activation, herbimycin did block CD28-dependent cytotoxicity in a dose-dependent manner. These data support a role for PI3-K activation in the CD28-dependent initiation of cytotoxic effector function and suggest that a herbimycin sensitive step(s) is either CD28-independent, resides within a PI3-K-independent CD28 signaling pathway, or is downstream of CD28-dependent PI3-K activation.

Amino Acid Sequence↗
PubMed1995

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Antigenic Variation↗
PubMed1995

Phosphatidylinositol 3-kinase activity is not essential for CD28 costimulatory activity in Jurkat T cells: studies with a selective inhibitor, wortmannin.

The interaction of CD28 with its counter-receptor, B7-1 (CD 80), on antigen-presenting cells induces a co-signal in T cells required to promote antigen-dependent interleukin-2 (IL-2) production and to prevent clonal anergy. CD28 stimulation causes both protein-tyrosine kinase and phosphatidylinositol3-kinase (PI3-K) activation, suggesting a possible role for these enzyme activities in CD28 co-signal transduction. Here, we investigate the effect of wortmannin, a selective and irreversible PI3-K inhibitor on CD28 co-signaling events in the Jurkat T cell line. Wortmannin added to cell cultures partially inhibits CD28-induced tyrosine phosphorylation of the putative p110 catalytic subunit of PI3-K, but does not block CD28-induced association of the p85 PI3-K regulatory subunit with the CD28 receptor. Wortmannin inhibits in a dose-dependent manner both total cellular PI3-K activity and CD28-induced receptor-associated PI3-K activity. Wortmannin (1 microM) inhibits cellular PI3-K activity by 90% with complete inhibition achieved at 10 microM. The inhibitory effect of wortmannin on cellular PI3-K activity is prolonged ( > 18 h), suggesting that the drug is not readily metabolized by Jurkat T cells. Wortmannin, at concentrations that blocked PI3-K activity, fails to inhibit the synergistic effect of CD28 on IL-2 secretion in the presence of phorbol 12-myristate 13-acetate and ionomycin. These data demonstrate that CD28-induced signaling events other than the activation of PI3-K catalytic activity contribute to the control of IL-2 secretion.

Androstadienes↗
PubMed1995

Transdermal delivery of drugs with differing lipophilicities using azone analogs as dermal penetration enhancers.

Six model drugs were selected for this study based on their degree of lipophilicity as represented by their log P values (range = -0.95 to 3.51). They included 2,4-dihydroxy-5-fluoropyrimidine (5-fluorouracil); 1,3,7-trimethylxanthine (caffeine); [(2-hydroxybenzoyl)amino]-acetic acid (salicyluric acid); 2-hydroxybenzoic acid (salicylic acid); 9 alpha-fluoro-16 alpha-hydroxyprednisolone 16 alpha, 17 alpha-acetonide (triamcinolone acetonide); and alpha-methyl-4-[2-methylpropyl]benzeneacetic acid (ibuprofen). Six dermal penetration enhancers [Azone or 1-dodecylhexahydro-2H-azepin-2-one (1), N-dodecyl-2-pyrrolidinone (2), N-dodecyl-2-piperidinone (3), N-dodecyl-N-(2-methoxyethyl)acetamide (4), N-(2,2-dihydroxyethyl)dodecylamine (5), and 2-(1-nonyl)-1,3-dioxolane (6)] were tested in vitro across full-thickness hairless mouse skin with each of the drugs. The relationship between lipophilicity (log P) and efficacy (represented by the enhancement ratio of flux) of the drugs when coadministered with the enhancers was examined using linear regression. The three cyclic enhancers (1-3) exhibited linear relationships, indicating that they were more effective at enhancing the penetration of hydrophilic drugs R2 = 0.8997 for 1, 0.8801 for 2, and 0.804 for 3) when evaluating all the model drugs except triamcinolone acetonide (TA). The two acyclic enhancers (4 and 5) showed a similar relationship, but their correlation coefficients were lower at 0.6463 for 4 and 0.6213 for 5. Studies with the dioxolane (6) yielded no relationship between the lipophilicity of the drug and the efficacy of the enhancer, with an R2 of 0.002. Overall, 6 was the least effective enhancer studied. The steroid TA was not included in the linear regression analysis. Of the six model drugs studied, TA exhibited the largest increase in transdermal delivery when enhancers 1-6 were used.

Animals↗
PubMed1995

Irreversible inhibition of human natural killer cell natural cytotoxicity by modification of the extracellular membrane by the adenine nucleotide analog 5'-p-(fluorosulfonyl)benzoyl adenosine.

Extracellular adenine nucleotides are inhibitors of the human natural killer cell line NK3.3 natural cytotoxicity activity. Natural cytotoxicity was inhibited approximately 26% by 1 mM ATP and 21% by 1 mM ADP. 5'-Adenylyl imidodiphosphate, a nonhydrolyzable ATP analog, inhibited natural cytotoxicity by 41% at a concentration of 1 mM and > 97% at a concentration of 10 mM. In contrast, AMP was not inhibitory. Adenosine was a weak inhibitor of natural cytotoxicity and may represent an alternate regulatory pathway. Removal of the nucleotides resulted in the restoration of control levels of natural cytotoxicity activity. The affinity label 5'-p-(fluorosulfonyl)benzoyladenosine (5'-FSBA) is a synthetic analog of ATP or ADP containing an electrophilic fluorosulfonyl group capable of covalently modifying proteins at adenine di- and triphosphate nucleotide-binding sites. Natural cytotoxicity was irreversibly inhibited by modification of the extracellular membrane of NK3.3 cells by 5'-FSBA. This inhibition was concentration dependent with an I50 approximately 100 microM and complete inhibition at 1 mM. Modification of NK3.3 by 5'-FSBA did not affect the formation of effector-target cell conjugates; however, granule release was inhibited. This targets the site of inhibition by 5'-FSBA modification to a pathway preceding granule release. Irreversible, covalent modification of surface adenine nucleotide-binding proteins by 5'-FSBA provides a probe to study the role of specific adenine nucleotide-binding proteins in the extracellular regulation of natural killer cytolytic activity by adenine nucleotides.

Adenosine↗
PubMed1995

Walking when utilizing a sensory feedback system and an electrical muscle stimulation gait orthosis.

Electrical muscle stimulators (EMS) have been combined with a reciprocating gait orthosis (RGO) to produce an EMS-RGO system for constant velocity ambulation exercise in a spinal cord injured (SCI) individual. The objective of this study is to evaluate an auditory feedback system (AFS) used by the SCI subject when ambulating with the EMS-RGO. Three different types of auditory signals (M = metronome alone, MSL = metronome plus stride length information, and NONE = no auditory feedback) were examined at three different constant walking velocities (WV1 = 0.64 kph, WV2 = 1.22 kph, and WV3 = 1.80 kph). The experimental design was 3 x 3 full factorial with repeated measures, and the dependent variable was the absolute error (AE) of walking distance (absolute value of the distance the subject walked in one minute subtracted from the target distance). A block diagram and circuit schematic of the AFS is provided. The results indicate that at WV1 there is no significant difference of AE among the three auditory conditions (M, MSL and NONE). At WV2, the AE for the NONE auditory condition is significantly greater than the AE for M and MSL (p < 0.05). At WV3, the AE for the NONE auditory condition is significantly less than the AE for M and MSL (p < 0.05). Finally, there was no significant difference in AE between both the M and MSL auditory conditions at any of the three walking velocities. This study indicates that an adequate sensory feedback system for constant velocity control of ambulation in this SCI subject consists of metronome only auditory feedback.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

The revised velocity-strain relationship: phase-plane analysis of left ventricular function and dysfunction.

Cyclical cardiac mechanical performance has been described by plotting mid-wall left ventricular circumferential velocity or strain rate (V) as a function of instantaneous mid-wall strain (epsilon). This results in individual velocity-strain (V-epsilon) parameters. For any given individual, a standard velocity-strain plot is represented by a sinusoidally varying strain and strain rate. Three standard velocity-strain variables are then defined. The peak velocity, or peak strain rate (VM), the cycle strain at VM (epsilon M), and the system time constant (tau M). The individual velocity-strain parameters are then divided by these standard V-epsilon parameters to result in normalized V-epsilon parameters for both cardiac systole and cardiac diastole. Thirty-six patients were evaluated, consisting of four groups: Normals (N = 15 patients), Compensated Volume Overload (CVO = 6 patients), Decompensated Volume Overload (DVO = 9 patients) and Congestive Cardiomyopathy (CCM = 6 patients). Five individual velocity-strain parameters significantly differentiate the CCM group from the N group. However, they do not differentiate CVO group and DVO group from the N group. Two standard-epsilon parameters and one normalized V-epsilon parameter significantly differentiated the CVO group and the DVO group from the N group. Two standard V-epsilon parameters and one normalized V-epsilon parameter significantly differentiated the CCM group from the N group. It is recommended that future phase-plane analyses of non-linear cardiac phenomena include both standardization and normalization techniques.

Adult↗
PubMed1995

Separating population structure from population history: a cladistic analysis of the geographical distribution of mitochondrial DNA haplotypes in the tiger salamander, Ambystoma tigrinum.

Nonrandom associations of alleles or haplotypes with geographical location can arise from restricted gene flow, historical events (fragmentation, range expansion, colonization), or any mixture of these factors. In this paper, we show how a nested cladistic analysis of geographical distances can be used to test the null hypothesis of no geographical association of haplotypes, test the hypothesis that significant associations are due to restricted gene flow, and identify patterns of significant association that are due to historical events. In this last case, criteria are given to discriminate among contiguous range expansion, long-distance colonization, and population fragmentation. The ability to make these discriminations depends critically upon an adequate geographical sampling design. These points are illustrated with a worked example: mitochondrial DNA haplotypes in the salamander Ambystoma tigrinum. For this example, prior information exists about restricted gene flow and likely historical events, and the nested cladistic analyses were completely concordant with this prior information. This concordance establishes the plausibility of this nested cladistic approach, but much future work will be necessary to demonstrate robustness and to explore the power and accuracy of this procedure.

Ambystoma↗
PubMed1995

Performance study involving a force-reflecting joystick for spastic individuals performing two types of tracking tasks.

10 upper-extremity spastic subjects and 10 normal subjects were studied with a force-reflecting joystick in the performance of a continuous time-tracking task as well as an acquisition task termed, "Fitts' Law." Certain force-reflection paradigms, in a spatial sense, allowed the spastic subjects to obtain performance proficiency near levels of the normal subjects as measured by a capacity metric.

Hand Strength↗
PubMed1994

CD28 signal transduction: tyrosine phosphorylation and receptor association of phosphoinositide-3 kinase correlate with Ca(2+)-independent costimulatory activity.

The interaction of CD28 with its counter-receptor, B7, induces a cosignal in T cells required to prevent clonal anergy and to promote antigen-dependent interleukin-2 production. The molecular basis of the CD28 cosignal is not well understood but involves the activation of protein tyrosine kinase(s) (PTK). In this report we demonstrate that CD28 cross-linking on Jurkat T leukemic cells causes the activation of at least two PTK pathways. A CD28-induced, p56lck kinase-independent pathway causes tyrosine-phosphorylation of a 110-kDa substrate while recruitment of p56lck kinase activity is apparently required for CD28-induced tyrosine-phosphorylation of 97- and 68-kDa substrates as well as CD28-induced increases in intracellular calcium. The tyrosine phosphorylation of p110, but not p97 or p68, correlated with CD28 calcium-independent costimulatory activity. The pp110 molecule was tentatively identified as the catalytic subunit of phosphoinositide (PI)-3 kinase based upon its coimmunoprecipitation with the p85 regulatory subunit of PI-3 kinase. PI-3 kinase protein and catalytic activity were found complexed with the CD28 receptor if the receptor was "activated" by cross-linking on the surface of intact cells prior to detergent solubilization. The kinetics of association of PI-3 kinase with the "activated" CD28 receptor was rapid, occurring within 30 s of receptor cross-linking and was stable for at least 30 min. Analysis of the CD28 cytoplasmic peptide sequence revealed a putative PI-3 kinase src homology 2 binding motif and CD28 tyrosine phosphorylation site, DYMNM. Tyrosine phosphorylation of CD28 was detected in pervanadate-treated Jurkat B2.7 cells, but not untreated cells. Pervanadate-induced tyrosine phosphorylation of CD28 correlated with receptor association of PI-3 kinase in the absence of CD28 cross-linking, suggesting that CD28 association with PI-3 kinase uses a tyrosine phosphorylation-dependent mechanism. These data provide a model for CD28 signal transduction and support a role for PI-3 kinase in mediating the CD28 calcium-independent, cyclosporin A-insensitive costimulatory signal.

Amino Acid Sequence↗
PubMed1994

Management of the third stage of labor: a survey of practice among Texas obstetricians.

Available evidence shows that the benefits of routine oxytocic administration in the third stage of labor far outweigh the potential risks. In 1990, T.F. Baskett, MD, reviewed the routine use of oxytocic drugs by Canadian obstetricians in the management of the third stage of labor. With Dr Baskett's permission, we sent a similar questionnaire to 1500 obstetricians in Texas: 1000 practiced in urban areas and 500 practiced in rural areas of the state. Ninety-four percent of Texas obstetricians answering the survey used oxytocics routinely in managing the third stage of labor. However, only 14.9% administered the oxytocics before delivery of the placenta, in contrast to 92.1% who gave oxytocics after the delivery of the placenta. Oxytocin was the chosen oxytocic drug for routine third-stage management (95%) as well as for postpartum hemorrhage (73.3%). Of the physicians surveyed, 55.9% used 15-methyl prostaglandin F2a (Hemabate) to treat postpartum hemorrhage refractory to other drugs within the past year. Of the respondents, 14.3% had managed acute uterine inversion during the past year. Thus, Texas obstetricians use Oxytocin routinely in the management of the third stage of labor, but few are converted to active management.

Attitude of Health Personnel↗
PubMed1993

Identification and partial characterization of an ectoATPase expressed by human natural killer cells.

An extracellular membrane-associated ectoATPase has been identified on the human natural killer cell line NK3.3. The enzyme is distinct from other classes of ATPases, kinases, and phosphatases. NK3.3 ectoATPase demonstrated a Km for ATP of 41 microM and a Vmax of 0.2 mumol/min and required both Ca2+ and Mg2+ for maximal activity. Purine and pyrimidine nucleotides were competitive inhibitors of the catalytic reaction. Inhibition increased with the addition of increasing negative charge of the phosphate side chain and was also dependent on contributions from the nucleoside. NK3.3 ectoATPase activity was inhibited by reaction with the affinity label [p-(fluorosulfonyl)benzoyl]-5'-adenosine (5'-FSBA), which is shown to modify the enzyme at or near the ATP-binding domain. Photoaffinity labeling of intact NK3.3 cells with [alpha-32P]-8-azidoATP demonstrated an ATP-binding protein of 68-80 kDa unique to NK3.3 cells. A positive correlation was observed between the ability of the various nucleotides to block photoincorporation into the 68-80-kDa protein and their ability to inhibit ectoATPase activity. NK3.3 cells which were made ectoATPase-deficient by reaction with 5'-FSBA demonstrated that this enzyme does not have a major role in the protection of this cytolytic effector cell from the possible lytic effects of extracellular ATP.

Adenosine Triphosphatases↗
PubMed1993

Association of the p56lck protein tyrosine kinase with the Fc gamma RIIIA/CD16 complex in human natural killer cells.

The multimeric Fc gamma RIIIA (CD16) complex is expressed on the surface of natural killer (NK) cells and is composed of a 50-70-kDa transmembrane glycoprotein Fc gamma receptor (CD16), the T cell receptor (TCR)-zeta chain, and the Fc epsilon RI gamma chain. Cross-linking Fc gamma RIIIA initiates the rapid tyrosine phosphorylation of multiple substrates including the zeta subunit and causes subsequent cell activation and antibody-dependent cellular cytotoxicity (ADCC). The subunits of the Fc gamma RIIIA complex lack intrinsic protein tyrosine kinase (PTK) activity, suggesting that receptor-induced tyrosine phosphorylation events are mediated by a nonreceptor PTK. We report here that the human Fc gamma RIIIA is complexed with p56lck, a src-family PTK previously found associated with the CD4 and CD8 receptors on T cells. Upon engagement of the CD16 receptor, p56lck is rapidly (within 30 s) and transiently phosphorylated on tyrosine residues. Several Fc gamma RIIIA-associated proteins are identified in immune complex kinase assays including the TCR-zeta subunit, a p70-90 zeta-associated protein (ZAP), p50a (acidic) and p50b (basic), and p56lck. We demonstrate that the src-family protein tyrosine kinase inhibitor, herbimycin A, blocks increased intracellular calcium levels and ADCC caused by CD16 cross-linking on NK3.3 cells. Likewise cross-linking CD16 with the protein tyrosine phosphatase CD45, abrogates CD16-induced calcium mobilization. These data suggest that p56lck is physically associated with Fc gamma RIIIA (CD16) and functions to mediate signaling events related to the control of NK cellular cytotoxicity.

Antibody-Dependent Cell Cytotoxicity↗