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Biomedical subjects

A A Stewart

Publications and source records attributed to A A Stewart.

18 recordsLinked to original sources
PubMed2006

The efficacy of intratumoural 5-fluorouracil for the treatment of equine sarcoids.

OBJECTIVE: To document the efficacy of intratumoural injections of 5-fluorouracil for the treatment of equine sarcoids. DESIGN: A prospective study that included 13 horses and one donkey. PROCEDURE: Sarcoids were confirmed by histological examination and treated with intratumoural 5-fluorouracil every 2 weeks. If the sarcoids did not resolve after seven treatments, treatment was considered a failure. All cases were re-examined 6 months after treatment commenced and owners were telephoned 3 years after commencement of treatment to report on tumour recurrence. Outcome comparisons were performed to determine the effect of previous treatment, tumour size and tumour location on sarcoid resolution. The efficacy of intratumoural 5-fluorouracil was compared with other previously documented treatments of equine sarcoids. RESULTS: Sarcoids smaller than 13.5 cm3 were significantly (P = 0.032) more likely to resolve with treatment than larger sarcoids. Sarcoids that were not responsive to previous therapies were significantly (P = 0.007) more likely to recur after 3 years than sarcoids that had not been treated prior to this study. In this study, there were similar rates of resolution in cases with mutiple tumours (66.6%) when compared to cases with single tumours (60%). The numbers in this study were too small to properly evaluate the effect of tumour location on the success of treatment. Intratumoural 5-fluorouracil appeared to have resolved sarcoids in 9 of 13 cases (61.5%) as determined by follow up conversation with the owners 3 years after the initial treatment. CONCLUSION: The use of intratumoural 5-fluorouracil compares favourably with other treatment modalities for sarcoids, with a long term successful resolution rate of 61.5%. Owners should be warned that resistant sarcoids and sarcoids larger than 13.5 cm3 have a poorer prognosis for resolution and more aggressive therapeutic options should be considered.

Animals↗
PubMed1997

Muscle balance at the knee--moment arms for the normal knee and the ACL-minus knee.

Forces, moments and stresses at the knee are dependent upon external and internal loading factors including muscle forces, segmental position and velocity, load carried, and the moment arms (mechanical advantage) of the muscle-tendon units. Requisite to prediction of forces and moments is a detailed understanding of effective moment arms throughout the knee range-of-motion (ROM). Existing muscle models for the knee are based upon limited static studies of only a few preserved specimens. The objectives of this report are to develop a comprehensive description of muscle-tendon moment arms for the normal knee and the anterior cruciate ligament (ACL)-minus knee during flexion-extension motion. Recent research results describe two nonorthogonal, nonintersecting axes of motion for the knee--one describing flexion-extension (FE) and the other longitudinal rotation (LR, equivalent to internal-external rotation). The effective flexion-extension moment arms of the muscles crossing the knee were developed with respect to the FE axis in 15 fresh, hemi-pelvis cadaver specimens. The normal moment arms for each of 13 muscles plus the patellar tendon exhibited variable, yet repeatable and recognizable patterns throughout the ROM. For most muscles there was no significant difference between the normal and ACL-minus moment arms. The results provide a basis for more accurate predictions of joint reaction forces and moments as well as useful knowledge for practitioners and therapists to assist in the assessment of muscle balance at the knee following injury, repair, and throughout rehabilitation.

Adult↗
PubMed1996

Umbilical cord transforming growth factor-beta 3: isolation, comparison with recombinant TGF-beta 3 and cellular localization.

The transforming growth factor beta (TGF-beta) family of growth modulators play critical roles in tissue development and maintenance. Recent data suggest that individual TGF-beta isoforms (TGF-beta 1, -beta 2 and -beta 3) have overlapping yet distinct biological actions and target cell specificities, both in developing and adult tissues. The TGF-beta 3 isoform was purified to homogeneity from both natural and recombinant sources and characterized by laser desorption mass spectrometry, by protein sequencing, by amino acid analysis and by biological activity. TGF-beta 3 was the major TGF-beta isoform in umbilical cord (230 ng/g), and was physically and biologically indistinguishable from recombinant TGF-beta 3 and from the tumor growth inhibitory (TGI) protein found in umbilical cord. Immunohistochemistry using antipeptide TGF-beta 3 specific antibody showed TGF-beta 3 localization in perivascular smooth muscle.

Amino Acid Sequence↗
PubMed1995

Physical and biological characterization of a growth-inhibitory activity purified from the neuroepithelioma cell line A673.

Epithelial- and haematopoietic-cell growth-inhibitory activities have been identified in the conditioned medium of the human peripheral neuroepithelioma cell line A673. An A673-cell-derived growth-inhibitory activity was previously fractionated into two distinct components which inhibited the proliferation of human carcinoma and leukaemia cells in culture. One inhibitory activity was shown to comprise interleukin-1 alpha (IL-1 alpha). Here, we have purified to homogeneity a distinct activity which inhibited the growth of the epithelial cells in vitro. Using a combination of protein-sequence analysis and mass spectrometry, we demonstrated that biological activity can be assigned to a dimeric protein with a molecular mass of 25,576 (+/- 4) Da and an N-terminal sequence identical with that of transforming growth factor-beta 1 (TGF-beta 1). Further characterization of the growth inhibitor with TGF-beta-isoform-specific antibodies showed that > 90% of the bioactivity consists of TGF-beta 1 and not TGF-beta 2 or TGF-beta 3. Although A673 cells were growth-inhibited by exogenous TGF-beta 1, we showed that TGF-beta 1 in A673-cell-conditioned media was present in the latent, biologically inactive, form which did not act as an autocrine growth modulator of A673 cells in vitro.

Amino Acid Sequence↗
PubMed1995

Gene expression as a target for new drug discovery.

Over the last 7 years we have carried out a major research effort focused on gene transcription as a novel approach to drug discovery. The goal is to identify small molecular weight compounds that modulate the expression of a target gene in a specific manner, thereby either increasing or decreasing the concentration of the corresponding protein product. Transcriptional modulation not only provides a potential means to replace recombinant proteins as drugs, but also provides a novel approach to manipulate key gene targets in many therapeutic areas. This article describes some of the features and advantages of transcription-based pharmaceuticals and illustrates how this approach can be applied to drug discovery with a program we are pursuing to identify new treatments for sickle cell disease and beta-thalassemia.

Anemia, Sickle Cell↗
PubMed1994

Prevention of chemotherapy-induced ulcerative mucositis by transforming growth factor beta 3.

Mucositis is a common, dose-limiting complication in patients receiving cancer chemotherapy, which appears to be a consequence of the rate of epithelial proliferation. The beta transforming growth factors have been shown to be negative regulators of epithelial cell proliferation. Here we show that transforming growth factor beta 3 administration reduced proliferation of oral epithelium in vitro and in vivo. Topical application of transforming growth factor beta 3 to the oral mucosa of the Syrian golden hamster prior to chemotherapy significantly reduced the incidence, severity, and duration of oral mucositis, reduced chemotherapy-associated weight loss, and increased survival.

Animals↗
PubMed1992

Time-resolved x-ray diffraction study of the troponin-associated reflexions from the frog muscle.

The vertebrate skeletal muscle gives rise to a series of x-ray reflexions indexed as orders (n) of 77 nm, the even orders being meridional whereas the odd orders being near-meridional. The diffraction intensities associated with these reflexions originate from the axial period of 39 nm attributable to the repeat of troponin-tropomyosin on the thin filament. In the present study, the x-ray intensities of the furthest inner reflexions, A2 (n = 2) reflexion at an axial spacing of 1/39 nm-1 and A4 (n = 4) reflexion at 1/19 nm, of this series were measured with a time resolved manner. Upon activation of the frog striated muscle, the two reflexions underwent biphasic time courses of the intensity changes. With A2 reflexion, a rapid intensity increase by 16%, being completed by the time when tension rises to 5%, was followed by a slow intensity decrease down to 50%, which was associated with the tension rise. In both phases, lateral widths remained unchanged. A4 reflexion also behaves in the same way, although the first phase (the intensity increase) was not clear due to unsatisfactory statistics. We interpret phase one as being caused by conformational change of the troponin-tropomyosin complex upon binding of Ca2+ to troponin, whereas phase two being due to direct contribution of the mass of the myosin heads bound to the thin filament, although possible contribution of conformational changes of the regulatory proteins to phase two is not excluded. The results indicated that the calcium activation of the thin filament leads the onset of the actomyosin interaction.

Animals↗
PubMed1991

X-ray diffraction studies on muscle regulation.

Using the intensity of the outer part of the second actin layer line as an indicator of thin filament conformation in vertebrate muscle we were able to identify the four different states of rest, and the three states induced by the presence of Ca2+ ions, rigor bridge attachment and actively cycling bridges, respectively. These findings are in qualitative agreement with a number of biochemical studies by Eisenberg and Greene and others, indicating that activation of the thin filament depends both on Ca2+ ions and crossbridge binding. Yet quantitatively, the biochemical data and our structural data are contradictory. Whereas the biochemical studies suggest a strong coupling between structural changes of the thin filament and the ATPase activity, the structural studies indicate that this is not necessarily the case. Troponin molecules also change their conformation upon activation depending on both Ca2+ ions and crossbridge binding as demonstrated by the early part of the time course of the thin filament meridional reflections in contracting frog muscle. Low ionic strength which has been shown by Brenner and collaborators to increase weakly binding crossbridges in relaxed rabbit psoas muscle does not influence the intensity of the second actin layer line in this muscle. Yet in contracting frog muscle the increase of the second actin layer line increases very rapidly in one step, suggesting that weak binding bridges which are attached to actin prior to force production may indeed influence the thin filament conformation. It therefore appears that weakly bound bridges in the low ionic strength state do not have the same effect on the thin filament conformation as weakly bound bridges in an actively contracting muscle. Arthropod muscles like the thin filament regulated lobster muscles differ from vertebrate muscle in not showing an increase of the second layer line during contraction, which may have to do with differences in crossbridge attachment. The myosin-regulated molluscan muscle ABRM shows a large increase on the second actin layer line upon phasic contraction and a much smaller increase in catch or rigor, indicating that actively cycling bridges influence the thin filament conformation differently than catch or rigor bridges. Several pieces of evidence which we have briefly outlined in this paper suggest that the thin filament conformational changes we have observed do not arise solely from tropomyosin movements and that conformational changes of actin domains should be considered.

Animals↗
PubMed1991

X-ray studies of order-disorder transitions in the myosin heads of skinned rabbit psoas muscles.

Using x-rays from a laboratory source and an area detector, myosin layer lines and the diffuse scattering between them in the moderate angle region have been recorded. At full overlap, incubation of rigor muscles with S-1 greatly reduces the diffuse scattering. Also, three of the four actin-based layer lines lying close to the meridian (Huxley, H. E., and W. Brown, 1967. J. Mol. Biol. 30:384-434; Haselgrove, J. C. 1975. J. Mol. Biol. 92:113-143) increase, suggesting fuller labeling of the actin filaments. These results are consistent with the idea (Poulsen, F. R., and J. Lowy, 1983. Nature [Lond.]. 303:146-152) that some of the diffuse scattering in rigor muscles is due to a random mixture of actin monomers with and without attached myosin heads (substitution disorder). In relaxed muscles, regardless of overlap, lowering the temperature from 24 to 4 degrees C practically abolishes the myosin layer lines (a result first obtained by Wray, J.S. 1987. J. Muscle Res. Cell Motil. 8:62 (a). Abstr.), whilst the diffuse scattering between these layer lines increases appreciably. Similar changes occur in the passage from rest to peak tetanic tension in live frog muscle (Lowy, J., and F.R. Poulsen. 1990. Biophys. J. 57:977-985). Cooling the psoas demonstrates that the intensity relation between the layer lines and the diffuse scattering is of an inverse nature, and that the transition occurs over a narrow temperature range (12-14 degrees C) with a sigmoidal function. From these results it would appear that the helical arrangement of the myosin heads is very temperature sensitive, and that the disordering effect does not depend on the presence of actin. Measurements along the meridian reveal that the intensity of the diffuse scattering increases relatively little and does so in a nearly linear manner: evidently the axial order of the myosin heads is much less temperature sensitive. The combined data support the view (Poulsen, F. R., and J. Lowy. 1983. Nature [Lond.]. 303:146-152) that in relaxed muscles a significant part of the diffuse scattering originates from disordered myosin heads. The observation that the extent of the diffuse scattering is greater in the equatorial than in the meridional direction suggests that the disordered myosin heads have an orientation which is on average more parallel to the filament axis.

Actins↗
PubMed1983

The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle.

Protein phosphatase-2B was purified from extracts of rabbit skeletal muscle by a procedure that involved fractionation with ammonium sulphate, chromatography on DEAE-Sepharose, fractionation with poly(ethylene glycol), gel filtration on Sephadex G-200 (Mr = 98000 +/- 4000), chromatography on Affi-Gel Blue and affinity chromatography on calmodulin-Sepharose. The enzyme was purified 3500-fold in seven days with an overall yield of 0.5%. The alpha-subunit of phosphorylase kinase, protein phosphatase inhibitor-1 and the myosin P-light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase-2B with similar kinetic constants. The alpha-subunit of phosphorylase kinase was dephosphorylated at least 100-fold more rapidly than the beta-subunit, while glycogen phosphorylase, glycogen synthase, histones H1 and H2B, ATP-citrate lyase, acetyl-CoA carboxylase, L-pyruvate kinase and protein synthesis initiation factor eIF-2 were not dephosphorylated at significant rates. Protein phosphatase-2B became activated 10-fold by calmodulin (A0.5 = 6 nM) after chromatography on DEAE-Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the Vmax of the reaction without altering the Km for inhibitor-1. The activity of protein phosphatase-2B was completely dependent on Ca2+ in the presence or absence of calmodulin. Half-maximal activation was observed at 1.0 microM Ca2+ in the absence, and at 0.5 microM Ca2+ in the presence, of 0.03 microM calmodulin. Protein phosphatase-2B was inhibited completely by trifluoperazine; half-maximal inhibition occurred at 45 microM in the absence and 35 microM in the presence of 0.03 microM calmodulin. The metabolic role of protein phosphatase-2B in vivo is discussed in the light of the observation that this enzyme is probably identical to a major calmodulin-binding protein of neural tissue termed calcineurin or CaM-BP80 [Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Lett. 137, 80-84].

Animals↗
PubMed1983

The protein phosphatases involved in cellular regulation. 6. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles.

Methods were developed for quantifying protein phosphatases-1, 2A, 2B and 2C in cell extracts, and these procedures were exploited to determine their tissue and subcellular distributions. In addition, the contribution of each enzyme to the total protein phosphatase activity in skeletal muscle and liver extracts towards nine proteins involved in the control of glycogen metabolism, glycolysis/gluconeogenesis, fatty acid synthesis and cholesterol synthesis was assessed. Each protein phosphatase was present at significant concentrations in skeletal muscle, heart muscle, liver, brain and adipose tissue, although the relative amounts differed considerably. In skeletal muscle, protein phosphatase-1 was the major enzyme acting on phosphorylase, glycogen synthase and phosphorylase kinase (beta-subunit), and thus was the major protein phosphatase responsible for the inactivation of glycogenolysis and stimulation of glycogen synthesis. This idea was reinforced by the observation that 50% of the protein phosphatase-1 activity was associated with the protein-glycogen complex. In the liver, protein phosphatases-1, 2A and 2C each appear to play a role in the regulation of glycogen metabolism. Protein phosphatase-1 accounted for a significant fraction of the total potential activity towards phosphorylase and glycogen synthase, and was the major phosphorylase kinase (beta-subunit) phosphatase of this tissue. In addition, it was the only protein phosphatase present in the protein-glycogen complex. Protein phosphatase 2A was also a major phosphorylase phosphatase and glycogen synthase phosphatase in this tissue. Protein phosphatase 2C was a significant glycogen synthase phosphatase in the liver, but had negligible activity toward phosphorylase or phosphorylase kinase (beta-subunit). In the absence of Ca2+, protein phosphatase 2A was the major phosphorylase kinase (alpha-subunit) phosphatase and the only inhibitor-1 phosphatase, in skeletal muscle or liver. In the presence of Ca2+, protein phosphatase 2B accounted for most of the activity towards these substrates. Protein phosphatase 2A was the major enzyme acting on L-pyruvate kinase, ATP-citrate lyase and acetyl-CoA carboxylase in rat liver, suggesting an important role in the regulation of glycolysis/gluconeogenesis and fatty acid synthesis. Protein phosphatase 2C was the major enzyme acting on hydroxymethylglutaryl-CoA (HMG-CoA) reductase and HMG-CoA reductase kinase, suggesting an important role in the regulation of cholesterol synthesis. However, the observation that 20% of the protein phosphatase-1 in liver was associated with the microsomal fraction suggests that this enzyme may also be involved in regulating HMG-CoA reductase, which is tightly associated with microsomes. The activity of protein phosphatase-1 in dilute skeletal muscle and liver extracts was just as sensitive to inhibitor-1 and inhibitor-2 as the purified enzyme. In concentrated extracts, higher concentrations of the inhibitor proteins were required and the inhibition was time-dependent...

Animals↗
PubMed1981

The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities.

The MgATP-dependent phosphorylase phosphatase was found to have a broad substrate specificity. Its activity against all phosphoproteins tested was dependent upon preincubation with the activating factor FA and MgATP. The enzyme dephosphorylated and inactivated phosphorylase kinase and inhibitor 1, and dephosphorylated and activated glycogen synthase and acetyl-CoA carboxylase. Glycogen synthase was dephosphorylated at similar rates whether it had been phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase or glycogen synthase kinase 3. The enzyme also catalysed the dephosphorylation of ATP citrate lyase, initiation factor eIF-2, and troponin I. The properties of the MgATP-dependent protein phosphatase from either dog liver or rabbit skeletal muscle showed a remarkable similarity to highly purified preparations of protein phosphatase 1 from rabbit skeletal muscle. The relative activities of the two enzymes against all phosphoproteins tested was very similar. Both enzymes dephosphorylated the beta-subunit of phosphorylase kinase 40-fold faster than the alpha-subunit, and both enzymes were inhibited by identical concentrations of the two proteins termed inhibitor 1 and inhibitor 2, which inhibit protein phosphatase 1 specifically. These results demonstrate that the MgATP-dependent protein phosphatase is a type-1 protein phosphatase, and is distinct from type-2 protein phosphatases which dephosphorylate the alpha-subunit of phosphorylase kinase and are unaffected by inhibitor 1 and inhibitor 2. The possibility that the MgATP-dependent protein phosphatase is an inactive form of protein phosphatase 1 and that both proteins share the same catalytic subunit is discussed.

Adenosine Triphosphate↗
PubMed1978

Phosphoglycerate kinase B from ram testis. Purification, characterisation and comparison with the muscle isoenzyme.

1. The testis-specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from 'normal' phosphoglycerate kinase which is also present in testis tissue. The purification procedure is described. 2. The best preparations had no detectable impurity on electrophoresis, and had specific activities comparable with the same enzyme from other sources. 3. Kinetic studies indicated that the two isoenzymes have identical properties, within experimental error, for substrate affinity (for MgATP, 3-phosphoglycerate and MgADP), energy of activation and thermal denaturation. 4. The molecular weights of both isoenzymes were not distinguishably different from those previously reported, as measured by polyacrylamide/dodecylsulphate electrophoresis. The amino acid compositions showed only slight differences, and tryptic peptide maps showed that there was close homology of sequence. Starch gel electrophoresis at pH 6.5 indicates that the B isoenzyme has 1--2 more positive charges than the A. 5. Phosphoglycerate kinase A isolated from sheep muscle was shown, within experimental error, to be identical to the phosphoglycerate kinase A isolated from testis. 6. The results further substantiate the suggestion that the B isoenzyme is coded by a gene which was duplicated from the phosphoglycerate kinase A gene.

Amino Acids↗