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Biomedical subjects

M J Fisher

Publications and source records attributed to M J Fisher.

At least 19 recordsLinked to original sources
PubMed2004

Evaluation of perennial ryegrass straw as a forage source for ruminants.

Two experiments were conducted to evaluate perennial ryegrass straw as a forage source for ruminants. Experiment 1 evaluated digestion and physiological variables in steers offered perennial ryegrass straw containing increasing levels of ergot alkaloid, lolitrem B. Sixteen ruminally cannulated Angus x Hereford steers (231+/-2 kg BW) were blocked by weight and assigned randomly to one of four treatments. Steers were provided perennial ryegrass straw at 120% of the previous 5-d average intake. Before straw feeding, soybean meal was provided (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Low (L) and high (H) lolitrem B straws (<100 and 1,550 ppb, respectively; DM basis) were used to formulate treatment diets: 100% L; 67% L:33% H; 33% L:67% H; 100% H (DM basis). Intake and digestibility of DM and OM, and ruminal pH, total VFA, and NH3-N were not affected by increasing lolitrem B concentration. Ruminal indigestible ADF (IADF) fill increased linearly (P = 0.01) and IADF passage rate decreased linearly (P = 0.04) as lolitrem B increased. Experiment 2 evaluated performance and production by 72 Angus x Hereford cows (539+/-5 kg BW) consuming perennial ryegrass straw containing increasing lolitrem B during the last third of gestation. Cows were blocked by body condition score and randomly assigned to one of three treatments. Cows were provided perennial ryegrass straw ad libitum and supplemented with soybean meal (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Mixtures of a L and H lolitrem B straw (467 and 2,017 ppb, respectively) were used to formulate treatment diets: 100% L, 50% L:50% H, 100% H (DM basis). Thirteen of 24 cows on the 100% H treatment exhibited signs of ryegrass staggers and were removed from the study; nevertheless, lolitrem B concentration did not influence pre- or postcalving weight or body condition score change. These data suggest that feeding perennial ryegrass straw containing up to 1,550 ppb lolitrem B (DM basis) did not adversely affect nutrient digestion or physiological response variables in steers. However, providing straw with a lolitrem B concentration of approximately 2,000 ppb (DM basis) resulted in 54% of cows exhibiting signs of ryegrass staggers. These data suggest that blending straws with a high (>2,000 ppb) and low (<500 ppb) concentration of lolitrem B can be a successful management practice.

Animal Feed↗
PubMed2001

Adenoviral-mediated transfer of tissue plasminogen activator gene into brain capillary endothelial cells in vitro.

Tissue plasminogen activator (tPA) has a critical role in fibrinolysis, converting plasminogen into active protease plasmin. Because intravenous tPA has only limited effectiveness as acute stroke therapy, enhancement of endogenous tPA represents a potential alternative to stroke treatment. Adenoviral-mediated gene transfer was used to enhance production of tPA in bovine brain capillary endothelial cells (BEC). Antigen and activity levels of tPA and plasminogen activator inhibitor-1 (PAI-1) in media from BEC infected with AdCMVtPA were analyzed. Conditioned media were analyzed for thrombomodulin, the integral membrane antithrombotic molecule that co-activates protein C. BEC infected with AdCMVtPA demonstrated enhanced expression of tPA antigen (40.2 +/- 0.4 ng/mL vs 1.1 +/- 1.5 ng/mL [p<0.001] and 0.3 +/- 0.5 ng/mL [p<0.0001], respectively) and increased tPA enzymatic activity (27.4 +/- 5.7 IU/mL vs 8.3 +/- 1.7 IU/mL [p<0.05] and 13.3 +/- 3.2 IU/mL [p<0.05], respectively) compared to BEC infected with the control adenovirus (Adl327) or uninfected BEC. There was a moderate increase in PAI-1 protein 4 days after transfection with AdCMVtPA, and the integral membrane protein thrombomodulin was released into media by transfected BEC. These results demonstrate that adenoviral-mediated delivery in vitro of the human tPA gene resulted in high levels of expression of tPA in BEC. Transient overexpression of tPA by gene transfer might be a useful strategy to protect against thrombotic occlusion during the period of risk of acute stroke.

Adenoviridae↗
PubMed2001

Sequence analysis of the rat phenylalanine hydroxylase gene promoter.

We have characterized the 5'-end (3218 bp) of the rat phenylalanine hydroxylase (PAH) gene. Within this PAH promoter sequence, we have identified a number of putative regulatory sites analogous to those present in the human and murine PAH promoters. In particular, potential HNF 1 binding sites and a CRE have been identified. These sequences respectively bind HNF1 and CREB transcription factors present in rat nuclear extracts and may be significant in the tissue-specific and hormonal control of PAH expression.

5' Flanking Region↗
PubMed2001

Nucleotide substitution in the ectodomain of trail receptor DR4 is associated with lung cancer and head and neck cancer.

Allelic loss of chromosome 8p21-22 occurs frequently in cancer, including lung and head and neck squamous cell cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors, including proapoptotic DR4 and KILLER/DR5, are located on 8p21-22. TRAIL receptors are candidate tumor suppressor genes, because their inactivation would be expected to result in deficient apoptotic signaling. To investigate the involvement of DR4 in human cancer, we have determined the genomic structure of DR4 and screened 31 lung cancer cell lines [14 small cell lung cancer and 17 non-small cell lung cancer (NSCLC)], many with deletions at 8p21-22, and 21 primary NSCLC samples for mutations in DR4. We found two missense alterations in the ectodomain of DR4. One, at nucleotide 626, changes a cytosine to a guanine (C626G) and results in a substitution of an arginine for threonine. The other, at nucleotide 422, changes a guanine to adenine (G422A) and results in a substitution of a histidine for arginine. Using genomic DNA sequencing and RFLP analysis, we show that these two alterations cosegregated in 96% of all of the samples (n = 243) evaluated (tumor and normal). The frequency of being homozygous for both altered alleles was 35% in the lung cancer cell lines but only 13% in age- and race-matched controls, which was a significant increase (chi(2) = 5.2, P = 0.023). The frequency of homozygosity for both alleles was also significantly increased in the primary NSCLC samples (chi(2) = 9.2, P = 0.002) as compared with the age- and race-matched controls. To determine whether the altered alleles are specific for lung cancer, we evaluated 19 head and neck squamous cell cancer and 25 gastric adenocarcinoma samples. Forty-seven % of the former and 44% of the latter were homozygous for both the C626G and G422A alterations, and this was significantly elevated relative to age- and race-matched controls (chi(2) = 8.6, P = 0.003 and chi(2) = 8.2, P = 0.004). These alterations result in amino acid changes in or near the ligand-binding domain of DR4 and, based on the crystal structure of DR5 and its homology with DR4, have the potential to affect TRAIL binding to DR4. Our results suggest that the altered DR4 alleles may be associated with, and should be investigated additionally as potential markers for, predisposition to common malignancies.

Adenine↗
PubMed2000

Characterization of ecdysteroid 26-hydroxylase: an enzyme involved in molting hormone inactivation.

Insect molting hormone (ecdysteroid) inactivation occurs by several routes, including 26-hydroxylation and further oxidation to the 26-oic acids. Thus, the ecdysteroid 26-hydroxylase is a critical enzyme involved in precise regulation of ecdysteroid titers during insect development. Administration of the ecdysteroid agonist, RH-5849 (1,2-dibenzoyl, 1-tert-butyl hydrazone), or 20-hydroxyecdysone to the tobacco hornworm, Manduca sexta, results in induction of ecdysteroid 26-hydroxylase activity in midgut mitochondria and microsomes. The biochemical and kinetic properties of the ecdysteroid 26-hydroxylase were investigated. The mitochondrial enzyme was found to have optimal activity at a pH of 7. 5 in a Hepes or sodium phosphate buffer at 30-37 degrees C. The apparent K(m) of the microsomal 26-hydroxylase for 20-hydroxyecdysone substrate was lower than that of the mitochondrial enzyme for either 20-hydroxyecdysone or ecdysone substrate. The V(max) of the 26-hydroxylase in both subcellular fractions was slightly higher using 20-hydroxyecdysone as substrate compared to ecdysone. Demonstration that activity of the mitochondrial 26-hydroxylase was inhibited by incubation in a CO (or N(2)) atmosphere, taken together with the requirement for reducing cofactor and the efficacy of the P450 inhibitors, ketoconazole and fenarimol, provided strong evidence that the hydroxylase is cytochrome P450-dependent. Indirect evidence suggested that the mitochondrial and microsomal ecdysteroid 26-hydroxylase(s) could exist in a less active dephosphorylated state or more active phosphorylated state. Using Escherichia coli alkaline phosphatase to remove covalently bound phosphate groups, the activity of the 26-hydroxylase was decreased and, conversely, activity was enhanced using a cAMP-dependent protein kinase with appropriate cofactors. In addition, the protein kinase was shown to reactivate the 26-hydroxylase activity in alkaline phosphatase-treated fractions.

Adenine Nucleotides↗
PubMed2000

Diamino benzo[b]thiophene derivatives as a novel class of active site directed thrombin inhibitors. 5. Potency, efficacy, and pharmacokinetic properties of modified C-3 side chain derivatives.

A systematic investigation of the structure-activity relationships of the C-3 side chain of the screening hit 1a led to the identification of the potent thrombin inhibitors 23c, 28c, and 31c. Their activities (1240, 903, and 1271 x 10(6) L/mol, respectively) represent 2200- and 2900-fold increases in potency over the starting lead 1a. This activity enhancement was accomplished with an increase of thrombin selectivity. The in vitro anticoagulant profiles of derivatives 28c and 31c were determined, and they compare favorably with the clinical agent H-R-1-[4aS, 8aS]perhydroisoquinolyl-prolyl-arginyl aldehyde (D-Piq-Pro-Arg-H; 32). The more potent members of this series have been studied in an arterial/venous shunt (AV shunt) model of thrombosis and were found to be efficacious in reducing clot formation. However, their efficacy is currently limited by their rapid and extensive distribution following administration.

Animals↗
PubMed2000

Fused bicyclic Gly-Asp beta-turn mimics with potent affinity for GPIIb-IIIa. Exploration of the arginine isostere.

6-[4-Amidinobenzoyl]amino]-tetralone-2-acetic acid is a potent antagonist of GPIIb-IIIa. Substitution in the meta position of the benzamidine, or replacement with a heteroaryl amidine was tolerated in this series. Use of an acyl-linked 4-alkyl piperidine as an arginine isostere also provided active compounds. Compounds from this series provided substantial systemic exposure in the rat following oral administration.

Acetates↗
PubMed2000

Spatio-logical processes in intracellular signalling.

Classical models of intracellular signalling describe how small changes in a cell's external environment can bring about major changes in cellular activity. Recent findings from experimental biology indicate that many intracellular signalling systems show a high level of spatial organisation. This permits the modification, by protein kinase or protein phosphatase action, of specific subsets of intracellular proteins - an attribute that is not addressed in classical signalling models. Here we use ideas and concepts from computer science to describe the information processing nature of intracellular signalling pathways and the impact of spatial heterogeneity of their components (e.g. protein kinases and protein phosphatases) on signalling activity. We argue that it is useful to view the signalling ecology as a vast parallel distributed processing network of agents operating in heterogeneous microenvironments, and we conclude with an overview of the mathematical and semantic methodologies that might help clarify this analogy between biological and computational systems.

Models, Biological↗
PubMed2000

Stroke precipitated by moderate blood pressure reduction.

Rapid lowering of blood pressure can precipitate or worsen ischemic strokes. This usually has been observed in the setting of profoundly lowered pressure and hypotension. We report on six patients in whom ischemic neurologic injury ensued or worsened after moderate reduction of blood pressure by pharmacological treatment. The 6 patients suffered new or worsened ischemic neurologic deficits after receiving oral or intravenous antihypertensive medications, mostly after relatively small doses. Mean arterial blood pressure in these patients was decreased by 25 +/- 7.7%, or 37 +/- 16 mm Hg (mean +/- SD) without resultant hypotension. These cases emphasize the potential hazards of moderate blood pressure reduction by antihypertensive medications in the setting of an acute ischemic stroke or transient ischemic attack (TIA), as well as rapidly treated hypertension even in those who have not yet manifested ischemic symptoms.

Adult↗
PubMed2000

Retrograde venous perfusion with hypothermic saline and adenosine for protection of the ischemic spinal cord.

PURPOSE: Spinal cord injury and the resultant postoperative paraplegia are devastating complications of thoracic aortic surgery, for which no widely accepted protective interventions exist. We hypothesized that retrograde venous perfusion-cooling of the spinal cord with a hypothermic saline and adenosine solution would protect it from ischemic injury caused by thoracic aortic occlusion. METHODS: Adult domestic swine of either sex (weight range, 20 to 30 kg) were intubated and ventilated. A left thoracotomy was performed. The accessory hemiazygous vein was divided, and a catheter was inserted distally. The aorta was clamped at the left subclavian artery. The venous catheter was not used in the animals in the control group (n = 7); in the animals in the experimental group (n = 7), a cold (4 degrees C) saline and adenosine solution was infused into the accessory hemiazygous vein. After 30 minutes, the clamp and catheter were removed, and the chest was closed. A blinded observer evaluated the animals' hind-leg motor activity 24 hours later. The Tarlov scale was used: 0, complete paralysis; 1, minimal movement; 2, stands with assistance; 3, stands alone; 4, weak walk; 5, normal gait. The animals' rectal temperatures were measured at the end of the experiment, and blood pressure was measured throughout. Two other groups were studied to assess the effect of the intervention on spinal cord temperature. RESULTS: The animals in the control group had a mean Tarlov score of 1.7 +/- 0.6; the animals in the experimental group had a mean Tarlov score of 4.9 +/- 0.1 (P <.01). The animals in the experimental group had a significantly greater drop in spinal cord temperature than those in the control group (4. 05 +/- 0.6 degrees C vs 0.58 +/- 0.12 degrees C; P <.01). No significant difference in rectal temperatures was found, nor did any arrhythmias or hypotensive episodes occur in either group. Perfusion of the spinal cord was confirmed with angiography by using this approach. CONCLUSION: Retrograde venous perfusion-cooling of the spinal cord with a hypothermic saline and adenosine solution protects the cord from ischemic injury caused by clamping of the thoracic aorta.

Adenosine↗
PubMed2000

Homozygous deletion of the death receptor DR4 gene in a nasopharyngeal cancer cell line is associated with TRAIL resistance.

The family of tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptors, including the pro-apoptotic DR4 and p53-regulated KILLER/DR5, as well as the decoys TRID and TRUNDD, are all located on human chromosome 8p21-22. This region of the genome is frequently altered in head and neck cancer. We previously reported that KILLER/DR5 can be mutationally inactivated in head and neck cancer. Here, we report that the FaDu nasopharyngeal cancer cell line contains an abnormal chromosome 8p21-22 region. In addition, there appears to be a homozygous deletion involving DR4 but not KILLER/DR5 in FaDu cells. The homozygous loss within the DR4 gene encompasses its death domain, which is required for apoptotic signaling. The deletion of DR4 in FaDu cells is associated with resistance to the cytotoxic effects of TRAIL. Re-introduction of wild-type DR4 leads to apoptosis and restores TRAIL sensitivity of FaDu cells. These observations suggest that the death inducing DR4 receptor gene may be a rare target for inactivation in human cancer and that DR4 loss may contribute to resistance to TRAIL therapy.

Apoptosis↗
PubMed2000

Molecular determinants of response to TRAIL in killing of normal and cancer cells.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a potent inducer of death of cancer but not normal cells, which suggests its potential use as a tumor-specific antineoplastic agent. TRAIL binds to the proapoptotic death receptors DR4 and the p53-regulated proapoptotic KILLER/DR5 as well as to the decoy receptors TRID and TRUNDD. In the present studies, we identified a subgroup of TRAIL-resistant cancer cell lines characterized by low or absent basal DR4 or high expression of the caspase activation inhibitor FLIP. Four of five TRAIL-sensitive cell lines expressed high levels of DR4 mRNA and protein, whereas six of six TRAIL-resistant cell lines expressed low or undetectable levels of DR4 (chi 2; P < 0.01). FLIP expression appeared elevated in five of six (83%) TRAIL-resistant cell lines and only one of five (20%) TRAIL-sensitive cells (chi 2; P < 0.05). Two TRAIL-resistant lines that expressed DR4 contained an A-to-G alteration in the death domain encoding arginine instead of lysine at codon 441. The K441R polymorphism is present in 20% of the normal population and can inhibit DR4-mediated cell killing in a dominant-negative fashion. The expression level of KILLER/DR5, TRID, TRUNDD or TRID, and TRUNDD did not correlate with TRAIL sensitivity (P > 0.05). These results suggest that the major determinants for TRAIL sensitivity may be the expression level of DR4 and FLIP. TRAIL-resistant cells became susceptible to TRAIL-mediated apoptosis in the presence of doxorubicin. In TRAIL-sensitive cells, caspases 8, 9, and 3 were activated after TRAIL treatment, but in TRAIL-resistant cells, they were activated only by the combination of TRAIL and doxorubicin. Our results suggest: (a) evaluation of tumor DR4 and FLIP expression and host DR4 codon 441 status could be potentially useful predictors of TRAIL sensitivity, and (b) doxorubicin, in combination with TRAIL, may effectively promote caspase activation in TRAIL-resistant tumors.

Amino Acid Substitution↗
PubMed1999

Fused bicyclic Gly-Asp beta-turn mimics with specific affinity for GPIIb-IIIa.

Disubstituted isoquinolones 2 and 3 have affinity for GPIIb-IIIa and represent leads for further structural evaluation. Structure-activity studies centered on the bicyclic beta-turn mimic contained in these molecules indicated that this moiety could accommodate a variety of modifications. Specifically, monocyclic, 6, 5-bicyclic, and 6,7-bicyclic structures provide compounds with affinity for GPIIb-IIIa. Within the 6,6-series, isoquinoline, tetralin, tetralone, and benzopyran nuclei yield potent antagonists that are specific for GPIIb-IIIa. Attachment of the arginine isostere (benzamidine) to the supporting nucleus can be accomplished with an ether or amide linkage, although the latter enhances activity. Several compounds in this series provided measurable blood levels after oral dosing. Conversion of the acid moiety in these molecules to an ester generally provided compounds which gave greater systemic exposure after oral administration. Absolute bioavailabilities in the rat for the ethyl ester prodrug derivatives of the tetralin, tetralone, and benzopyran analogues of 3 were 28%, 23%, and 24%, respectively.

Administration, Oral↗
PubMed1999

Diadenosine polyphosphates and the control of cyclic AMP concentrations in isolated rat liver cells.

Extracellular diadenosine polyphosphates (Ap(n)A), through their interactions with appropriate P(2) receptors, influence a diverse range of intracellular activities. In particular, Ap(4)A stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that, like ATP, Ap(4)A and other naturally occurring diadenosine polyphosphates attenuate glucagon-stimulated accumulation of cyclic AMP in isolated rat liver cells. The characteristics of Ap(4)A- and ATP-dependent modulation of glucagon-stimulated cyclic AMP accumulation are similar. These results are discussed in the context of the repertoire of intracellular signalling processes modulated by extracellular nucleotides.

Adenosine Triphosphate↗
PubMed1999

Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.

Alternative Splicing↗
PubMed1999

Spinal cord protection during aortic cross-clamping using retrograde venous perfusion.

BACKGROUND: Paraplegia remains a devastating complication following thoracic aortic operation. We hypothesized that retrograde perfusion of the spinal cord with a hypothermic, adenosine-enhanced solution would provide protection during periods of ischemia due to temporary aortic occlusion. METHODS: In a rabbit model, a 45-minute period of spinal cord ischemia was produced by clamping the abdominal aorta and vena cava just below the left renal vessels and at their bifurcations. Four groups (n = 8/group) were studied: control, warm saline, cold saline, and cold saline with adenosine infusion. In the experimental groups, saline or saline plus adenosine was infused into the isolated cavae throughout the ischemic period. Clamps were removed and the animals to recovered for 24 hours before blinded neurological evaluation. RESULTS: Tarlov scores (0 = paraplegia, 1 = slight movement, 2 = sits with assistance, 3 = sits alone, 4 = weak hop, 5 = normal hop) were (mean +/- standard error of the mean): control, 0.50 +/- 0.50; warm saline, 1.63 +/- 0.56; cold saline, 3.38 +/- 0.26; and cold saline plus adenosine, 4.25 +/- 0.16 (analysis of variance for all four groups, p < 0.00001). Post-hoc contrast analysis showed that cold saline plus adenosine was superior to the other three groups (p < 0.0001). CONCLUSION: Retrograde venous perfusion of the spinal cord with hypothermic saline and adenosine provides functional protection against surgical ischemia and reperfusion.

Animals↗