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Biomedical subjects

B Ellis

Publications and source records attributed to B Ellis.

At least 19 recordsLinked to original sources
PubMed1999

A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors.

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.

Amino Acid Sequence↗
PubMed1999

Cytosolic citrate and malonyl-CoA regulation in rat muscle in vivo.

In liver, insulin and glucose acutely increase the concentration of malonyl-CoA by dephosphorylating and activating acetyl-CoA carboxylase (ACC). In contrast, in incubated rat skeletal muscle, they appear to act by increasing the cytosolic concentration of citrate, an allosteric activator of ACC, as reflected by increases in the whole cell concentrations of citrate and malate [Saha, A. K., D. Vavvas, T. G. Kurowski, A. Apazidis, L. A. Witters, E. Shafrir, and N. B. Ruderman. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E641-E648, 1997]. We report here that sustained increases in plasma insulin and glucose may also increase the concentration of malonyl-CoA in rat skeletal muscle in vivo by this mechanism. Thus 70 and 125% increases in malonyl-CoA induced in skeletal muscle by infusions of glucose for 1 and 4 days, respectively, and a twofold increase in its concentration during a 90-min euglycemic-hyperinsulinemic clamp were all associated with significant increases in the sum of whole cell concentrations of citrate and/or malate. Similar correlations were observed in muscle of the hyperinsulinemic fa/fa rat, in denervated muscle, and in muscle of rats infused with insulin for 5 h. In muscle of 48-h-starved rats 3 and 24 h after refeeding, increases in malonyl-CoA were not accompanied by consistent increases in the concentrations of malate or citrate. However, they were associated with a decrease in the whole cell concentration of long-chain fatty acyl-CoA (LCFA-CoA), an allosteric inhibitor of ACC. The results suggest that increases in the concentration of malonyl-CoA, caused in rat muscle in vivo by sustained increases in plasma insulin and glucose or denervation, may be due to increases in the cytosolic concentration of citrate. In contrast, during refeeding after starvation, the increase in malonyl-CoA in muscle is probably due to another mechanism.

Acetyl-CoA Carboxylase↗
PubMed1998

SH2- and SH3-mediated interactions between focal adhesion kinase and Src.

Intramolecular SH2 and SH3 interactions mediate enzymatic repression of the Src kinases. One mechanism of activation is disruption of these interactions by the formation of higher affinity SH2 and SH3 interactions with specific ligands. We show that a consensus Src SH3-binding site residing upstream of the Src SH2-binding site in FAK can function as a ligand for the Src SH3 domain. Surface plasmon resonance experiments indicate that a FAK peptide containing both the Src SH2- and SH3-binding sites exhibits increased affinity for Src. Furthermore, the presence of both sites in vitro more potently activates c-Src. A FAK mutant (FAKPro-2) with substitutions destroying the SH3-binding site shows reduced binding to Src in vivo. This mutation also reduces Src-dependent tyrosine phosphorylation on the mutant itself and downstream substrates, such as paxillin. These observations suggest that an SH3-mediated interaction between Src-like kinases and FAK may be important for complex formation and downstream signaling in vivo.

Amino Acid Sequence↗
PubMed1998

Restorative treatment need assessment by dentally and nondentally trained subjects.

A study was carried out which compared how two groups of people, one with clinical dental experience and one without, assessed restorative dental treatment need. Using a visual analogue scale, a group of final year dental students (n = 50) and nonclinical university students (n = 50) assessed the extent to which they considered common dental imperfections, viz. spacing of the upper anterior teeth and discolouration of upper anterior teeth, warranted restorative correction. The group of dental students judged the necessity for treatment of discolouration to be more urgent than correction of spacing. The nondental group did not differentiate between the degrees of need. Data were non-normal in distribution but the use of appropriate statistical tests showed the differences in mean assessments to be significant.

Adolescent↗
PubMed1998

Monitoring of PPAR alpha protein expression in human tissue by the use of PPAR alpha-specific MAbs.

We report the production and characterization of two PPAR alpha subtype-specific monoclonal antibodies raised against the N-terminal domain of PPAR alpha. P alpha b 11.80A is a Western-reactive antibody, whereas P alpha b 32.51 is useful for immunohistochemistry. Both antibodies exhibited high affinity against the immunogen based on BIAcore analysis, recognized full-length PPAR alpha protein in PPAR alpha-transfected CV-1 cells, and displayed no cross-reactivity against the N-terminal domains of PPAR gamma or PPAR delta proteins as demonstrated by various immunoassays. The application of these antibodies to a panel of normal human tissues revealed that PPAR alpha protein expression is highest in skeletal muscle, liver, and kidney, consistent with previously reported mRNA expression data. These antibodies provide us with valuable tools to further explore the function of PPAR alpha.

Antibodies, Monoclonal↗
PubMed1998

Cytokines and adhesion molecules in patients with polymyalgia rheumatica.

Serum levels of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1ra), tumour necrosis factor alpha (TNF-alpha), IL-6, soluble IL-6 receptor (sIL-6R), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble E-selectin were measured in 15 patients with newly diagnosed polymyalgia rheumatica (PMR) before and after 3 months of corticosteroid therapy. Both IL-6 and IL-1ra were significantly increased in untreated PMR and remained elevated compared with controls during therapy, although significantly only for sIL-1ra. sICAM-1 was raised in 12/15 (87%) patients at diagnosis and remained high in 10/14 (71%) patients; soluble E-selectin levels were initially raised in 6/15 (40%) patients and decreased with therapy in those with the highest levels. IL-6, IL-1ra and sICAM-1 are sensitive indicators of continuing immunological activation in PMR; the advantages of these markers in assessing the response to therapy should be investigated in a longitudinal study.

Aged↗
PubMed1998

Osteocalcin production in primary osteoblast cultures derived from normal and Hyp mice.

Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts. Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17-25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response. Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.

Animals↗
PubMed1998

Dislocation of the mandible during the course of cataract surgery.

Spontaneous dislocation of the mandible is a well-described clinical entity with which the ophthalmologist may not be familiar. However, the widespread use of intravenous sedation places susceptible individuals at risk for the development of dislocation of the mandible. The authors describe a patient who underwent routine cataract surgery and suffered a dislocation of the mandible that initially went unrecognized. Ophthalmic surgeons who use intravenous sedation need to be aware of this complication and need to recognize the symptoms so that patients may receive timely attention.

Aged↗
PubMed1997

Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system.

Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.

Animals↗
PubMed1997

Joint angle measurement: a comparative study of the reliability of goniometry and wire tracing for the hand.

OBJECTIVES: To compare the inter- and intra-rater reliability of goniometry and wire tracing in the assessment of finger joint angles: metacarpo-phalangeal (MCPJ), proximal (PIPJ) and distal interphalangeal joints (DIPJ). DESIGN: Twenty occupational therapists and 20 physiotherapists with a range of clinical experience were recruited from nine different centres. Using a masked goniometer and wire tracing they carried out repeated assessments of the MCPJ, PIPJ and DIPJ of a normal subject fixed in two different positions. RESULTS: The two assessment methods did not produce comparable angle measurements. Goniometry showed greater inter- and intra-rater reliability than wire tracing. Regardless of the assessment tool, the repeatability coefficient indicated that DIPJ measurement was less reliable than the other joints. Clinical and specialist experience did not affect reliability. CONCLUSION: Although both goniometry and wire tracing show limitations as reliable assessment tools, it is recommended that where possible goniometry should be used.

Equipment Design↗
PubMed1997

Personality and depression: a validation study of the depressive experiences questionnaire.

This study investigated the validity of Blatt's model of depression as indicated by his operational measure of its constructs via the Depressive Experiences Questionnaire (DEQ; Blatt, D'Afflitti, & Quinlan, 1976). Hypothesized relations between the two relevant scales of the DEQ and Tellegen's (1982) Multidimensional Personality Questionnaire (MPQ) were examined. Participants consisted of 195 women, including 67 hospitalized unipolar depressives, 77 never-hospitalized unipolar depressives, and 51 nonpsychiatric controls. Overall, the results partially supported the validity of the DEQ even though all participants were women and prior studies have indicated the DEQ's greater discriminative validity for men than for women. However, several of the most strongly predicted relations, such as between DEQ Self-Criticism and MPQ Achievement were not confirmed. Coherent, significant relations between scales of the two measures remained after partialling out the effects of severity of depression.

Adolescent↗
PubMed1996

Correlation of the phosphorylation states of pp60c-src with tyrosine kinase activity: the intramolecular pY530-SH2 complex retains significant activity if Y419 is phosphorylated.

Rapid digestion of pp60c-src tyrosine kinase (src TK) in combination with electrospray ionization mass spectrometry enabled the determination of the time course for autophosphorylation of three tyrosine sites (Y338, Y419, and Y530) and a correlation with src TK activity. A form of src TK was purified from baculovirus-infected cells which contains only Y338 partially phosphorylated. Incubation with MgATP increases the phosphorylation of all three sites. The autophosphorylation and dephosphorylation of Y419 are directly correlated with the level of src TK activity. The role of Y338 phosphorylation is unknown. Conditions resulting in complete autophosphorylation of Y530 were identified by electrospray ionization mass spectrometry. Surface plasmon resonance detection and size exclusion chromatography provide direct evidence for an intramolecular pY530-SH2 complex, supporting previous models [Matsuda, M., Mayer, B.J., Fukui, Y., & Hanafusa, H. (1990) Science 248, 1537-1539]. Contrary to these models, when the enzyme is fully phosphorylated on Y530, phosphorylated on Y419, and present only as the intramolecular pY530-SH2 complex, 20% of the kinase activity is retained. In addition, the k(m)'s for substrates are unaffected. Disruption of the pY530-SH2 interaction and activation of kinase activity by a high-affinity SH2 ligand yield a Kactivation which is 200-fold larger than the Kd for ligand binding to the uncomplexed src SH2 domain. These data suggest a Keq of 200 (unitless) for the intramolecular association of pY530 with the SH2 domain. We propose that the pY530-SH2 interaction modulates signal transduction by down-regulating src TK activity 5-fold, and perhaps more importantly by inhibiting protein-protein interactions with the SH2 domain. These results have significant implications relative to the development of SH2 ligands as therapeutics to control aberrant signal transduction. These ligands will be 200-fold less effective at inhibiting protein-protein interactions versus down-regulated src TK than versus activated src TK. This should minimize activation of src TK activity in normal cells and lead to an increased therapeutic index.

Adenosine Triphosphate↗
PubMed1995

Media calcium attenuates mitochondrial 1,25(OH)2D production in phosphorus or vitamin D-deprived rats.

Although PTH and hypophosphatemia are the best known stimulators of 25-hydroxyvitamin D-1 alpha-hydroxylase, 1,25(OH)2D3 production in rats and humans can be modulated by circulating calcium, independent of PTH. To test whether calcium modulates this function directly in mitochondria, we examined effects of calcium on 1 alpha-hydroxylase in isolated mitochondrial preparations under basal and stimulated conditions. Rats were fed a low phosphorus (or matched control) diet for 4 or 7 d or a vitamin D-deficient (or matched control) diet for 2, 4, or 7 wk. Renal mitochondria were isolated and assayed for 1 alpha-hydroxylase activity in the presence or absence of added calcium. Calcium did not alter 1 alpha-hydroxylase in rats on control diets. After 4 d of low phosphorus diet, 1 alpha-hydroxylase was increased 2-fold over basal activity; media calcium prevented this stimulatory response. By 7 d the calcium effect was not evident. After 4 wk of vitamin D deprivation, activity was approximately 30-fold greater than controls; calcium reduced this response significantly (15-fold). A significant, but less marked inhibition of activity by calcium was present in rats subjected to 7 wk of vitamin D deprivation. Extramitochondrial calcium can directly modulate 1,25(OH)2D3 production, but this effect appears to be secondary to the primary physiologic regulators of this function. The calcium effect can be overcome after longer term exposure to phosphorus deprivation, but is sustained in the presence of long term vitamin D deprivation.

Animals↗
PubMed1995

Effects of extreme pH on the physiology of the Australian 'yabby' Cherax destructor: acute and chronic changes in haemolymph carbon dioxide, acid-base and ionic status

Freshwater habitats throughout the world are becoming increasingly threatened by the likelihood of acidification, but little consideration has been given to the importance of severe alkalization. Acute and chronic fluctuations in haemolymph acid­base status (PCO2, CCO2 and pH), [Na+] and [Ca2+] were monitored for up to 504 h (21 days) in the Australian freshwater crayfish Cherax destructor exposed to low- and high-pH water. The importance of carapace [Ca2+] during acid exposure was assessed. Crayfish were exposed to pH 7.1, pH 4.5 and pH 8.0 water containing calcium at 500 µmol l-1 while the effect of a lower calcium concentration (50 µmol l-1) was assessed in pH 4.5 water. Cherax in acid water containing 50 µmol l-1 Ca2+ exhibited a significant decrease in CO2 content after 2 h (mean decrease 1.13 mmol l-1, venous; 1.57 mmol l-1, arterial) and large ranges in PCO2 throughout the treatment (2.4­7.3 mmHg). The overall acid­base response was a metabolic acidosis compensated by a respiratory alkalosis. The haemolymph Na+ concentration in both control (pH 7.1, 50 µmol l-1) and acid-exposed animals in lower-Ca2+ water was up to 50 % reduced compared with that in animals in pH 7.1, 500 µmol l-1 Ca2+ water. Ion regulatory mechanisms, causing a subsequent increase in haemolymph [Na+] after 288 h, were implicated as an important component in acid­base homeostasis. Crayfish in acid, low-Ca2+ water also exhibited a 3.2 mmol l-1 increase in haemolymph [Ca2+] and showed a haemolymph alkalosis compared with animals in acid water with higher [Ca2+]. At higher water [Ca2+] in pH 4.5 water (500 µmol l-1 Ca2+), the haemolymph pH of Cherax was only 0.1 unit lower than that of animals in 50 µmol l-1 Ca2+ acid water after 96 h, and both CaCO2 and CvCO2 were unchanged compared with the initial condition. As with low-Ca2+ acid-exposure, the potential haemolymph acidosis appeared largely to be compensated by respiratory alkalosis. There was a transient 31 % reduction in haemolymph [Na+], although osmolality was unchanged (control 411±7.29 mosmol kg-1). Acid­base equilibrium recovered rapidly, probably in association with changes in ion flux and the re-establishment of normal haemolymph Na+ concentration. Alkaline-exposed Cherax destructor exhibited a mixed respiratory alkalosis and metabolic acidosis. Whereas haemolymph [Ca2+] increased by 1.8 mmol l-1 after only 1 h, haemolymph Na+ levels increased by 36 % after 2 h, possibly as part of a net H+ loss from the haemolymph. Increased HCO3-/Cl- exchange could contribute to the 4.3 mmol l-1 decrease in haemolymph CO2 level after 0.5 h of alkaline exposure. The responses of Cherax to extreme pH are different from those of the European and North American crayfish species studied to date.

Journal Article↗
PubMed1995

Effects of extreme pH on the physiology of the Australian 'yabby' Cherax destructor: acute and chronic changes in haemolymph oxygen levels, oxygen consumption and metabolic levels

Respiration and metabolism of the freshwater crayfish Cherax destructor were investigated with respect to the acidification and alkalization of its environment. Crayfish were exposed for up to 504 h (21 days) to pH 4.5, pH 7.1 (control) or pH 8.0 water and oxygen consumption rate, haemolymph oxygen transport and haemolymph glucose and lactate concentrations were determined. The effect of reducing environmental [Ca2+] in acid water from 500 to 50 µmol l-1 was also examined. In acid water (500 µmol l-1 Ca2+), oxygen uptake by Cherax was reduced by 79 % after 504 h (21 days) compared with 'control' animals (pH 7.1, 500 µmol l-1 Ca2+). Haemolymph lactate concentration (mean 0.6 mmol l-1) remained constant, indicating that anaerobiosis was not important, while glucose concentrations were regulated within the range of control values (0.32±0.01 mmol l-1). The arterial-venous CO2 difference of Cherax haemolymph decreased after 288 h and PaO2 increased from 11.1±0.5 mmHg to 42.4±1.0 mmHg between 96 h and 288 h. Decreased oxygen uptake and delivery without compensatory increases in anaerobiosis or glucose levels describe a hypometabolic response to low pH. The hypometabolic response of Cherax was greater in alkaline water as shown by a 53 % reduction in O2 uptake rate compared with a 44 % reduction in acid-exposed (500 µmol l-1 Ca2+) animals after 96 h. This decrease in M(dot)O2 of alkaline-exposed animals was correlated with decreased haemolymph glucose levels (from 0.32±0.01 at 0 h to 0.06±0.01 mmol l-1 at 96 h). Lowering the [Ca2+] of the water both increased the magnitude of the effects of acid exposure and elicited further changes in haemolymph oxygen transport. The maintenance of high haemolymph PO2 during pH stress appears to reduce the involvement of haemocyanin, since this promotes decreased a­v CO2. Hypometabolism probably permits Cherax to conserve resources that might otherwise be used, however, for growth and reproduction. The implications for the fitness of the animal are discussed.

Journal Article↗
PubMed1994

Trial of labor following cesarean delivery.

OBJECTIVE: To examine several variables that may affect the success rate for a trial of labor after previous cesarean delivery, as well as those affecting the rate of uterine rupture. METHODS: Between June 1, 1990 and December 31, 1991, we performed a consecutive, prospective study of 593 pregnant women who had had at least one abdominal delivery in the past, and attempted a trial of labor in each. Particular attention was given to the success rate of vaginal delivery, the type of previous uterine incision, use of oxytocin, estimated maternal blood loss, 5-minute Apgar scores, and reason for the previous cesarean operation. RESULTS: Four hundred seventy-eight patients (81%) had a successful vaginal delivery. Oxytocin induction or augmentation was successful in 46 of 67 (69%) and 117 of 167 cases (70%), respectively. Estimated maternal blood loss was less than 500 mL in 453 cases (95%). Five patients (0.8%) experienced true uterine rupture, resulting in severe neurologic sequelae in one infant. The only consistent indication of uterine rupture was an abrupt and prolonged fetal bradycardia. The majority (463; 97%) of infants who were delivered vaginally had 5-minute Apgar scores of 8 or greater. CONCLUSION: Our success rate of 81% suggests that a trial of labor after previous cesarean delivery is a safe and desirable option, but only after thorough patient counseling. An abrupt and persistent fetal bradycardia may be the only indication that uterine rupture has occurred.

Apgar Score↗