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A H Ross

Publications and source records attributed to A H Ross.

At least 37 records · Page 2Linked to original sources
PubMed1997

TrkA expression decreases the in vivo aggressiveness of C6 glioma cells.

We stably expressed the nerve growth factor receptor trkA or a truncated trkA lacking the kinase domain (trkA delta) in a highly tumorigenic rat glioma cell line, C6. Survival of rats with large intrastriatal inocula of C6trkA cells was significantly longer than for rats bearing C6 or C6trkA delta cells. Histological studies revealed that C6trkA cells were much less invasive than C6 or C6trkA delta cells and had a greater rate of apoptosis. There was no apparent induction of differentiation of C6 cells by trkA. Therefore, unlike what is observed in neuroblastomas, trkA decreases tumorigenicity by modulating invasiveness and tumor cell death independent of inducing differentiation. This novel mechanism suggests a new therapeutic strategy for malignant gliomas.

Animals↗
PubMed1997

Role of Rho family proteins in phospholipase D activation by growth factors.

Treatment of fibroblasts with growth factors results in activation of phospholipase D (PLD). In order to determine the role of the Rho family of small GTPases in growth factor-mediated PLD activation, we used cells transfected with wild type and mutant Rac1. In response to epidermal growth factor (EGF), PLD activity was greatly increased in Rat1 fibroblasts expressing wild type Rac1 (wtRac1), and completely abrogated in cells expressing dominant negative N17Rac1, consistent with Rac1 mediating the action of this growth factor. In contrast, in cells treated with platelet-derived growth factor (PDGF) or phorbol ester, the wtRac1 cells showed little or no enhancement of PLD activity, and the response was not affected in the N17Rac1 cells, implying that Rac1 played a minimal role in the activation of PLD by PDGF or protein kinase C. Both growth factors produced an attenuated PLD response in cells expressing constitutively active V12Rac1, but these cells showed other changes, including altered morphology, increased basal PLD, and decreased growth factor receptor autophosphorylation. The effects of EGF and PDGF on phosphoinositide phospholipase C activity were not enhanced in cells expressing wtRac1 or inhibited in those expressing N17Rac1. In cells expressing constitutively active V12Rac1, basal phosphoinositide phospholipase C was elevated, but there were no significant effects of EGF or PDGF. We used C3 transferase of Clostridium botulinum, which ADP-ribosylates and inactivates RhoA, to investigate the involvement of RhoA in the activation of PLD by PDGF. Cells expressing wtRac1 and N17Rac1 showed a decreased PLD in response to PDGF when treated with C3 transferase, indicating a role for RhoA. In summary, these data indicate a major role for Rac1 in the activation of PLD by EGF, but not PDGF or protein kinase C.

Animals↗
PubMed1997

Embryonic precursor cells that express Trk receptors: induction of different cell fates by NGF, BDNF, NT-3, and CNTF.

Epidermal growth factor (EGF)-treated neurosphere cultures from embryonal striatum contain multipotential cells capable of neuronal, astrocytic, and oligodendroglial differentiation. In this study, we tested whether these neural precursor cells differentiate in the presence of neurotrophic factors. We first assayed neurosphere cells for expression of neurotrophin receptors. TrkA, TrkB, TrkC, and gp75 were detected by immunofluorescence microscopy in 60-80% of cells. In addition, the ciliary neurotrophic factor receptor alpha was expressed in 50-60% of cells. In the presence of the mitogen, EGF, treatment of stem cells with neurotrophic factors had no apparent effect. Removal of EGF from cells resulted in cessation of cell proliferation and pronounced astrocytic (glial fibrillary acidic protein+) differentiation. Neuronal (neurofilament+) and oligodendroglial (galactocerebroside+) cells appeared in cultures treated with neurotrophic factors. Nerve growth factor (NGF) resulted in bipolar neuronal cells, and brain-derived neurotrophic factor led to multipolar neuronal cells. Treatment with neurotrophin-3 or ciliary neurotrophic factor resulted in bipolar neuronal cells and oligodendrocytes. Neuronal differentiation in the presence of NGF was enhanced by extracellular matrix, and the resulting neuronal cells expressed choline acetyltransferase and, to a lesser degree, tyrosine hydroxylase. These studies demonstrate that neurotrophic factors influence the fates of these multipotential precursor cells. Indeed, the true utility of multipotential precursor cells is the production of different types of cells in different situations. Local cues, such as neurotrophic factors and extracellular matrix, may regulate production of different types of neural cells during development or in response to other stimuli, such as injury.

Animals↗
PubMed1996

A comparison of the effects of oral conjugated equine estrogen and transdermal estradiol-17 beta combined with an oral progestin on quality of life in postmenopausal women.

OBJECTIVE: To compare the effect of transdermal estradiol-17 beta and oral conjugated equine estrogen when combined with an oral progestin on quality of life in post-menopausal women. DESIGN: Randomized controlled double-blind trial. A randomization error lead to the exclusion of six subjects but the soundness of the remaining randomization was confirmed. SETTING: Large urban community. PATIENTS: Women 2-7 years after menopause with a uterus and ovaries, and not currently using hormone replacement therapy. Seventy-four women completed the trial. INTERVENTIONS: After baseline measures of quality of life, subjects were randomly assigned to either continuous oral conjugated equine estrogen 0.625 mg daily or continuous transdermal estradiol-17 beta 50 mcg twice weekly, for four 4-week cycles. Medroxyprogesterone acetate 10 mg oral tablets was administered to both groups for the last 12 days of each cycle. OUTCOMES MEASURED: Quality of life was determined using the Menopause-Specific Quality of Life Questionnaire. Tolerability was determined by a specifically designed list of adverse effects. Both measures were recorded at base-line and in mid-cycle during the second, third and fourth cycles of treatment. RESULTS: There were no statistically significant differences in any of the domains at baseline between the oral and transdermal treatment groups. In the vasomotor domain-scores for the oral and transdermal groups improved from baseline levels of 3.14 and 3.09, respectively, to 1.32 and 1.23; physical domain scores improved from 2.45 and 2.73 to 2.04 and 1.78; psychosocial domain scores improved from 2.72 and 3.04 to 2.21 and 1.94; sexual domain scores improved from 2.32 and 2.16 to 1.64 and 1.30. There were no statistically significant group differences or time/group interactions. Both forms of therapy were equally well tolerated. CONCLUSIONS: Improvement in all domains, measured by the Menopause-Specific Quality of Life Questionnaire, was observed in both the oral and transdermal groups. In the absence of a placebo control group, the improvements observed cannot be attributed solely to the therapy. Neither form of therapy offered an advantage over the other in respect to improvement in quality of life.

Administration, Cutaneous↗
PubMed1996

The neurotrophin receptor, gp75, forms a complex with the receptor tyrosine kinase TrkA.

The high-affinity NGF receptor is thought to be a complex of two receptors , gp75 and the tyrosine kinase TrkA, but direct biochemical evidence for such an association had been lacking. In this report, we demonstrate the existence of such a gp75-TrkA complex by a copatching technique. Gp75 on the surface of intact cells is patched with an anti-gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with and anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression of wild-type and mutated NGF receptors. TrkA and gp75 copatch in both the absence and presence of NGF. The association is specific, since gp75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor-beta, and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains show partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copatching. A point mutation which inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with gp75. Hence, although interactions between the gp75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role.

Animals↗
PubMed1996

The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells.

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.

Aphidicolin↗
PubMed1996

Caliber estimation from cranial entrance defect measurements.

Caliber estimation from entrance defects has long been rejected by forensic scientists. This appears to be a consequence of soft tissue perspective of forensic pathologists. This study examined the relation between caliber and cranial entrance defects and maximum cranial thickness. The calibers considered in this inquiry were .22, .25, .32, and .38. The sample consisted of 73 specimens obtained at autopsy (thirty-seven of .22 caliber, five of .25, six of .32, and twenty-five of .38). To test the strength of the relation between caliber, minimum diameter, and maximum thickness Pearson correlation coefficients were conducted. The strongest relationship was observed between caliber and minimum diameter. A relationship between minimum diameter and maximum thickness was also observed. To test the null hypothesis that the mean minimum diameter is not significantly different between calibers an analysis of variance procedure was performed. The ANOVA yielded a strong relationship between dependent variable minimum diameter and caliber. Multiple regression analysis measuring the association between minimum diameter, caliber, and maximum thickness was also conducted. The Pr > F .0001 suggests that the overall model is significant. Discriminant functions and canonical variables were obtained. Classification was first performed by using two values small and large calibers. The large caliber group consisted of .38, while the small caliber group included .22, .25, and .32. The correct classification rate using crossvalidation for large caliber is 86.96%, and 93.33% for the group small caliber. A narrower classification was also performed by using three values, .23 caliber (.22 and .25 calibers grouped), .32, and .38 as the criterion variable groups also using minimum diameter and maximum thickness as predictors. The correct classification rate using crossvalidation is 82.02% for .23 caliber, 73.94% for .38 caliber, and 16.67% for .32 caliber defects. The discriminant functions can be used with appropriate caution to classify observations into groups defined by caliber using minimum diameter and maximum thickness as the predictors. Caution is suggested when attempting to estimate caliber from defects that are not produced from the perpendicular entrance of a bullet.

Analysis of Variance↗
PubMed1996

Distal mucus fistula following resection for perforated sigmoid diverticular disease.

A retrospective study of 62 consecutive patients undergoing resection of perforated diverticular disease was undertaken to assess influence of distal stump closure (DSC) or creating a distal mucus fistula (DMF) on subsequent restoration of bowel continuity. Forty-one patients underwent DSC and 21 DMF. Mean operating time and hospital stay were 164 min and 30 days for patients undergoing DSC compared with 142 min and 18 days for those undergoing DMF. Of the 54 survivors, 44 underwent reversal (25/35; DSC, 19/19; DMF). Mean operative time and hospital stay was 208 min and 17 days for DSC compared to 143 min and 10 days for DMF (P = 0.0003). Overall mortality and morbidity of either groups were comparable. Distal mucus fistula can often be safely created following resection of perforated sigmoid diverticulitis. This may result in higher reversal rates and significantly reduces the operating time and hospital stay without compromising the outcome.

Aged↗
PubMed1995

Regulation of phospholipase D in HL60 cells. Evidence for a cytosolic phospholipase D.

Phospholipase D (PLD) activity that was stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was detected in cytosol and membranes of HL60 cells. GTP gamma S-stimulated PLD activity was detected in the membranes when exogenous labeled phosphatidylcholine was used in the presence of phosphatidylethanolamine and phosphatidylinositol 4,5-bisphosphate, but not when [3H]myristic acid-labeled endogenous substrate was used. Cytosolic PLD co-chromatographed with small GTP-binding proteins on anion-exchange columns, but subsequent chromatography separated these. Reconstitution studies demonstrated ADP ribosylation factor (ARF) as a regulator of cytosolic PLD, whereas the Rho proteins RhoA and CDC42Hs were ineffective. The cytosolic enzyme showed very little activity in the absence of GTP gamma S and was stimulated by 2 mM Ca2+, whereas the membrane enzyme had significant basal activity and was inhibited by Ca2+. Rho-specific GDP dissociation inhibitor inhibited GTP gamma S stimulation of membrane PLD activity in the presence and absence of cytosol. The stimulation in GDP dissociation inhibitor-treated membranes could be partially recovered by the addition of recombinant Rho proteins (RhoA, Rac1, CDC42Hs). RhoA and Rac1 were also stimulatory in untreated membranes. However, Western blot analysis of membranes showed the presence of RhoA, but not Rac1 or CDC42Hs, suggesting that RhoA was the endogenous small GTP-binding protein involved in GTP-dependent PLD activity in membranes in the absence of cytosol. ARF also stimulated the membrane PLD in the presence of GTP gamma S, and the combination of RhoA and ARF showed a synergistic effect. These results show the presence of ARF-dependent PLD activity in both cytosol and membranes. The membranes contain another PLD activity for which the endogenous regulator appears to be RhoA. The data suggest the existence of at least two different PLD isozymes in HL60 cells.

Cell Line↗
PubMed1995

Interaction with TrkA immobilizes gp75 in the high affinity nerve growth factor receptor complex.

It has been proposed that the high affinity nerve growth factor (NGF) receptor required for NGF response is a complex of two receptor proteins, gp75 and the tyrosine kinase TrkA, but direct biochemical or biophysical evidence has been lacking. We have previously shown using fluorescence recovery after photobleaching that gp75 is highly mobile on NGF-nonresponsive cells, but relatively immobile on NGF-responsive cells. In this report, we show that a physical interaction with TrkA causes gp75 immobilization. We found that gp75 is relatively mobile on TrkA negative nnr5 cells, a PC12 variant which is nonresponsive to NGF. In contrast, on T14 nnr5 cells (which bear a TrkA expression vector) gp75 is relatively immobile. Similarly, using baculoviruses to express gp75 and TrkA on Sf9 insect cells, we found that TrkA immobilizes gp75 molecules. The related receptor, TrkB, caused a more modest immobilization of gp75. Immobilization was found to require intact TrkA kinase and gp75 cytoplasmic domains, paralleling the requirements of high affinity binding of NGF. Analysis of gp75 diffusion coefficients indicates that mutated gp75 and TrkA molecules may form a complex, even in the absence of the ability to bind NGF with high affinity.

Animals↗
PubMed1995

TrkA neurogenic receptor regulates differentiation of neuroblastoma cells.

We examined events associated with neuronal differentiation of neuroblastoma cell line SH-SY5Y. Treatment with nerve growth factor (NGF) and aphidicolin, an inhibitor of DNA polymerases alpha and delta, induces terminal differentiation of SH-SY5Y cells. Following 3-4 days of treatment with NGF + aphidicolin, the cells irreversibly ceased proliferation and differentiated. There was a succession of events preceding differentiation. Down-regulation of c-myc was an early event occurring after less than 1 day of treatment with NGF + aphidicolin. Upregulation of the trkA and low-affinity NGF receptors (LNGFR) occurred after 3 days of NGF + aphidicolin treatment and required treatment with both NGF and aphidicolin. To test the role of TrkA in neuroblastic differentiation, we transfected SH-SY5Y cells with a TrkA-expression plasmid. In response to NGF in the absence of aphidicolin, the TrkA-transformant line ceased proliferation and irreversibly differentiated. SH-SY5Y cells bearing a control plasmid displayed only modest, reversible differentiation and did not cease cell proliferation in response to NGF. Hence, expression of NGF receptors is upregulated during differentiation of SH-SY5Y cells, and overexpression of TrkA enhances NGF-induced differentiation.

Aphidicolin↗
PubMed1995

Differential biological effects of K252 kinase inhibitors are related to membrane solubility but not to permeability.

K252a and K252b are related protein kinase inhibitors that, dependent on conditions, can either inhibit or potentiate the effects of neurotrophic factors. K252a, an ester, is more potent and more cytotoxic on intact cells than K252b, a carboxylic acid. To understand better why these drugs elicit different degrees of biological responses, we analyzed their hydrophobicity, cell permeability, and subcellular distribution. As judged by partitioning between organic and aqueous phases, both compounds are hydrophobic. The partition coefficients were 15.6:1 (organic/aqueous phases) for K252a and 4.4:1 for K252b. The ratio of fluorescence excitation at 352 nm to that at 340 nm for the K252 compounds in the organic alcohol 1-decanol versus water provides a simple assay of binding of these compounds to phospholipid membranes. This ratio shifted for K252a, but not K252b, in the presence of phospholipid vesicles, indicating that K252a dissolved in the hydrophobic interior of the membrane. Using quantitative video fluorescence microscopy, we found that K252a strongly labeled both Sf9 insect cells and PC12 rat pheochromocytoma cells, probably staining intracellular membranes. The uptake of K252a was rapid and apparently irreversible. K252b also quickly entered Sf9 and PC12 cells, but staining was much weaker. Hence, K252a and K252b are similar in that they both rapidly enter cells but greatly differ in their membrane solubility.

Animals↗
PubMed1994

Activation of rat liver phospholipase D by the small GTP-binding protein RhoA.

Stimulation of phospholipase D by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in rat liver plasma membranes indicates the involvement of GTP-binding proteins. We used RhoGDI, an inhibitor of GDP dissociation from small GTP-binding proteins of the Rho family, to determine the involvement of these proteins. Incubation, and subsequent washing, of plasma membranes with RhoGDI dose-dependently diminished GTP gamma S-stimulated phospholipase D activity, as determined by accumulation of phosphatidylethanol in the presence of ethanol. Incubation with RhoGDI also caused a rapid and dose-dependent appearance of RhoA in the wash, which was associated with the inhibition of phospholipase D. RhoGDI also rapidly extracted Cdc42 from membranes, but Rac1 was not extracted. Full reconstitution of GTP gamma S-stimulated phospholipase D in RhoGDI-washed membranes was achieved with recombinant RhoA. There was partial reconstitution with Rac1 and no enhancement with Cdc42 or ADP-ribosylation factor. The response to RhoA was dose-dependent (EC50 = 0.5 microM). ADP-ribosylation of RhoA by Clostridium botulinum C3 exoenzyme did not affect its ability to recover GTP gamma S-stimulated phospholipase D activity in RhoGDI-washed membranes. These findings support a role for GTP-binding proteins of the Rho family in the activation of membrane-associated phospholipase D and implicate RhoA as the major protein involved.

Adenosine Diphosphate Ribose↗
PubMed1994

Purification and properties of phospholipase A1 from bovine brain.

Phospholipase A1 (PLA1) was isolated from a soluble fraction of bovine brain. The purification included sequential DEAE-Sephacel, phenyl-Sepharose FF, and heparin-Sepharose CL-6B column chromatography. Mono Q, Sephacryl S-300, and Mono S high resolution column chromatography in the presence of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (10 mM) and glycerol (10%, v/v) was required to further separate the enzyme from contaminating material. The purified PLA1 eluted from the Sephacryl S-300HR column in a volume corresponding to a molecular mass of 365 kDa and migrated as two bands (M(r) = 112,000 and 95,000) when separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Chromatofocusing, hydroxylapatite, and lectin affinity column chromatography and nondenaturing polyacrylamide gel electrophoresis were unsuccessful in separating the two electrophoretic bands, implying a close association or similarity. The purified enzyme was stable in solutions containing detergent and glycerol and was insensitive to metal chelators, dithiothreitol, phenylmethylsulfonyl fluoride, and diisopropyl fluorophosphate, but was inactivated by heat (60 degrees C) and ZnCl2. At pH 7.5, the purified enzyme showed highest specific activity, 23.8 mumol/min-mg, when 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylethanolamine was the substrate. The rate of catalysis was optimal at a pH of 9.0 and could be enhanced 2-fold by Ca2+, Mg2+, and Sr2+, but not Mn2+. The enzyme catalyzed the specific hydrolysis of acyl groups from the sn-1 position of a broad range of phospholipid substrates, including lysophospholipids, and accounts for most of the soluble phospholipase A1 activity of bovine brain.

Animals↗
PubMed1994

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low-affinity neurotrophin receptor.

Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neuotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.

Binding, Competitive↗
PubMed1994

Retrograde axonal transport and lesion-induced upregulation of the TrkA high-affinity NGF receptor.

Long-term physiological responses of nerve growth factor (NGF) and other neurotrophins require gene regulation and likely depend on retrograde axonal transport of NGF or a signaling molecule activated by ligand-receptor interaction. The low-affinity neurotrophin receptor p75LANR is retrogradely transported, but this receptor is not sufficient for NGF-dependent cell survival or differentiation. In this study we examined the distribution and transport of the TrkA NGF receptor using two anti-peptide polyclonal antibodies and a monoclonal antibody, all of which are TrkA specific. We find that (1) in the adult rat brain TrkA-like immunoreactivity is similar with all antibodies in striatal and basal forebrain neurons, (2) TrkA is upregulated in neuronal and nonneuronal cells near the sites of injury, and (3) TrkA immunoreactivity builds up within the proximal and distal segments of transected fimbrial axons, which is consistent with its transport in the anterograde and retrograde directions. Thus, TrkA may itself be, or be a component of, the neurotrophic intraaxonal messenger by which NGF regulates gene expression in sensitive neurons.

Animals↗