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Biomedical subjects

N A Turner

Publications and source records attributed to N A Turner.

At least 19 recordsLinked to original sources
PubMed2004

Resistance, biguanide sorption and biguanide-induced pentose leakage during encystment of Acanthamoeba castellanii.

AIMS: This study investigates the effects of biguanides during encystment of Acanthamoeba castellanii. METHODS AND RESULTS: A non-nutrient encystment system was used to investigate the changes in the levels of sorption (uptake) of three non-cysticidal concentrations (10, 20 and 50 microg ml(-1)) of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) as well as their effects on viability and leakage of pentose sugars during the first 36 h of encystment. Trophozoites treated with CHA or PHMB were more sensitive and generally sorbed more of each biocide than cysts. During encystment, the largest increases in resistance developed between 18 and 36 h for both biguanides with the resistance emerging to biguanide concentrations of 10 or 20 microg ml(-1) between 18 and 24 h. At 50 microg ml(-1) resistance emerged between 24 and 36 h. There was a general decrease in biocide sorption during encystment between 0-24 and 0-21 h for CHA and PHMB, respectively, at a concentration of 50 microg ml(-1). The greatest decline in biguanide-induced pentose leakage was between 0 and 12 h. CONCLUSIONS: The results suggest that during encystment some of the changes in the susceptibility to CHA or PHMB may be related to decreases in the levels of biocide sorption, which is limited by the developing cyst wall. SIGNIFICANCE AND IMPACT OF THE STUDY: During encystation, changes occur in biguanide sensitivity. The physical barrier of the cyst wall may be an important factor in limiting biocide sorption.

Acanthamoeba↗
PubMed2003

A novel flow cytometric analysis for platelet activation on immobilized von Willebrand factor or fibrillar collagen.

Under flow conditions, platelets adhere singly or in small aggregates on von Willebrand factor (VWF)-coated surfaces, but form large aggregates on immobilized fibrillar collagen. We developed a novel flow cytometric analysis to study the mechanisms underlying these distinct platelet deposition patterns. Flow cytometry was used to measure platelet activation after platelet adherence onto microspheres coated with either VWF or collagen fibrils. Two representative indices were calculated to quantify activated GpIIb-IIIa and P-selectin expression on adherent platelets. The signaling pathways responsible for platelet activation after interacting with fibrillar collagen were elucidated using various inhibitors. An in vitro endothelial cell wound model was also used to study the roles of VWF and fibrillar collagen in platelet deposition onto subendothelial matrixes. The adherent platelets on fibrillar collagen express more activated GpIIb-IIIa and P-selectin than those on VWF. Activation of GpIIb-IIIa and expression of P-selectin after platelet interaction with collagen occur via different intracellular signaling pathways; however, Ca2+ released from intracellular pools is common to both phenomena. Platelets were deposited singly or formed small aggregates on the endothelial cell wounded area, and this deposition pattern was dependent on VWF molecules secreted by endothelial cells and the absence of subendothelial collagen fibrils. As less activated GpIIb-IIIa and P-selectin are expressed after platelets interact with immobilized VWF alone, subsequent flowing platelet recruitment is minimal. Collagen fibrils, however, can activate adherent platelets sufficiently to promote the formation of large platelet aggregates.

Calcium Channel Blockers↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed2001

Blockade of adenosine diphosphate receptors P2Y(12) and P2Y(1) is required to inhibit platelet aggregation in whole blood under flow.

Using heparinized whole blood and flow conditions, it was shown that adenosine 5'-diphosphate (ADP) receptors P2Y(12) and P2Y(1) are both important in direct shear-induced platelet aggregation and platelet aggregation subsequent to initial adhesion onto von Willebrand factor (vWf)-collagen. In the viscometer, whole blood was subjected to shear rates of 750, 1500, and 3000 s(-1) for 30 seconds at room temperature. The extent of aggregation was determined by flow cytometry. The P2Y(12) antagonist AR-C69 931MX (ARMX) reduced shear-induced aggregation at these rates by 56%, 54%, and 16%, respectively, compared to control samples. Adenosine 3',5'-diphosphate (A3P5P; P2Y(1) antagonist) inhibited shear-induced aggregation by 40%, 30% and 29%, respectively, compared to control samples. Blockade of both ADP receptors at 3000 s(-1) with ARMX plus A3P5P further reduced the platelet aggregation by 41% compared to the addition of ARMX alone (57% compared to control samples). Using a parallel-plate flow chamber, whole blood was perfused over bovine collagen type 1 at a wall shear rate of 3000 s(-1) for 60 seconds. Platelet deposition was quantified with epifluorescence video microscopy and digital image processing. Blockade of P2Y(12) alone or blockade of P2Y(1) alone did not reduce thrombus formation on vWf-collagen. In contrast, blockade of both P2Y(12) and P2Y(1) reduced platelet deposition by 72%. These results indicate that combinations of antagonists of the ADP receptors P2Y(12) and P2Y(1) are effective inhibitors of direct shear-induced platelet aggregation and of platelet aggregation subsequent to initial adhesion under flow conditions. Inhibitors of these pathways are potentially useful as antiarterial thrombotic agents.

Adenosine Diphosphate↗
PubMed2001

Alteration of lipase chain length specificity in the hydrolysis of esters by random mutagenesis.

The feasibility of altering the chain length specificity of industrially important Rhizomucor miehei lipase was investigated by randomly mutating Phe94 in the protein groove which is responsible for accommodating the acyl chain of the substrate. The recombinant lipase was initially expressed in E. coli. Individual colonies were selected, grown, and the DNA sequence of the lipase gene determined. Fourteen of the 19 possible mutants were identified and each of these was transformed into Pichia pastoris which expresses the enzyme extracellularly. The yeast was grown and the supernatants assessed in several assays with long and short chain substrates. Based on this preliminary screen, one mutant, Phe94Gly, was selected and purified to homogeneity for further analysis. It was found that the substitution of phenylalanine 94 with glycine led to an enzyme which was about six times less active against resorufin ester but displayed 3-4 times higher activity with short chain substrates such as butyric acid esters. The observed alteration to the enzyme specificity was rationalised using the available 3D structure of the lipase.

Amino Acid Substitution↗
PubMed2001

Analysis of conformational states of Candida rugosa lipase in solution: implications for mechanism of interfacial activation and separation of open and closed forms.

In this study, an analysis of the transition between the inactive ("closed") and active ("open") conformations of Candida rugosa lipase in solution is performed using irreversible enzyme inhibitors, cyclic saligenin phosphates. It is shown that >90% inhibition of the enzyme activity toward water-soluble substrates (esterolytic activity) can be achieved with as little as 0.3 mol of the inhibitor per mole of enzyme, whereas activity toward emulsified substrates decreases by approximately 20% under the same conditions. It is also shown that short-term exposure of this inhibited enzyme preparation to an interface leads to a significant increase in esterolytic activity, which even exceeds that of the untreated control. These experimental observations suggest that the inhibitors interact predominantly, if not exclusively, with the open form of the enzyme and that any transitions occurring between the two conformers of the enzyme in solution, in the absence of an interface, are extremely slow. This conclusion is verified by separating the open and closed forms of the enzyme by hydrophobic interaction column chromatography on phenyl-sepharose. Fractions enriched with the respective conformations of the enzyme are further purified using gel-permeation chromatography. On the basis of the elution pattern from this step, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the open (active in the absence of interface) form of the lipase is found to be present in solution as a dimer, whereas the closed form appears to be a monomer. The latter form of the enzyme may be activated by up to 60-fold when exposed to triolein.

Benzyl Alcohols↗
PubMed2001

The mechanism of angiotensin II-induced extracellular signal-regulated kinase-1/2 activation is independent of angiotensin AT(1A) receptor internalisation.

The aim of this study was to determine whether internalisation of the angiotensin II (Ang II) AT(1A) receptor (AT(1A)R) was a prerequisite for Ang II-induced activation of the extracellular signal-regulated kinases, ERK-1/2. The human embryonic kidney (HEK293) cell line stably transfected with either the wild-type rat AT(1A)R or an internalisation-deficient C-terminal truncated mutant of the AT(1A)R (AT(1A)T318R) was used as a model for these studies. Inhibition of AT(1A)R internalisation by treatment with an inhibitor of clathrin-mediated endocytosis, Concanavalin A (Con A), did not inhibit Ang II-induced ERK-1/2 activation. Furthermore, cells transfected with the internalisation-deficient AT(1A)T318R mutant readily activated ERK-1/2 in response to Ang II. Ang II activated ERK-1/2 via two distinct signalling pathways in HEK-AT(1A)R cells. Approximately half of Ang II-induced ERK-1/2 activation was protein kinase C (PKC)-dependent, and the remainder was calcium- and c-Src-dependent and involved transactivation of the epidermal growth factor receptor (EGFR). In summary, Ang II-induced activation of ERK-1/2 occurs via two distinct pathways in HEK293 cells, neither of which requires AT(1A)R internalisation.

Angiotensin II↗
PubMed2001

Microsomal malonyl-CoA-sensitive carnitine acyltransferase.

Liver microsomes contain two carnitine acyltransferase activities. One of these has properties closely corresponding to those of 88 kDa mitochondrial carnitine palmitoyltransferase-1 (CPT-1). Antisera against CPT-1 cross-react with an 88 kDa microsomal protein, suggesting that CPT-1 may be targeted to both microsomal and mitochondrial membranes. However, no experiments using cDNAs corresponding to CPT-1 involving in vitro translation with microsomes or involving in vivo COS-1 cell transfection provided any evidence to support this hypothesis.

Animals↗
PubMed2001

Enantioselectivity of recombinant Rhizomucor miehei lipase in the ring opening of oxazolin-5(4H)-ones.

Enantioselectivity of enzyme catalysis is often rationalized via active site models. These models are constructed on the basis of comparing the enantiomeric excess of product observed in a series of reactions which are conducted with a range of homologous substrates, typically carrying various side chain substitutions. Surprisingly the practical application of these simple but informative 'pocket size' models has been rarely tested in genetic engineering experiments. In this paper we report the construction, purification and enantioselectivity of two recombinant Rhizomucor miehei lipases which were designed to check the validity of such a model in reactions of ring opening of oxazolin-5(4H)-ones.

Catalytic Domain↗
PubMed2000

Effects of abciximab, ticlopidine, and combined Abciximab/Ticlopidine therapy on platelet and leukocyte function in patients undergoing coronary angioplasty.

BACKGROUND: Abciximab and ticlopidine reduce adverse cardiovascular events after percutaneous transluminal coronary angioplasty (PTCA). The goal of the current study was to determine if combined abciximab/ticlopidine therapy inhibits arterial thrombosis more effectively than either treatment alone. The effect of each therapy on platelet-leukocyte interactions was also investigated. METHODS AND RESULTS: Whole blood samples from 14 patients undergoing PTCA who received abciximab therapy, ticlopidine therapy, or both treatments were evaluated using dynamic experimental systems. Mural thrombus formation under arterial shear conditions (1500 s(-1)) was determined in a parallel plate flow chamber. Shear-induced platelet aggregation was evaluated using a cone-and-plate viscometer at a shear rate of 3000 s(-1). Of the 3 treatments, combined abciximab/ticlopidine therapy produced the most consistent reduction in shear-induced platelet aggregation and the most prolonged inhibition of mural thrombosis. Three days after PTCA, abciximab/ticlopidine treatment decreased mural thrombus formation to approximately 50% of baseline values. Abciximab treatment alone inhibited mural thrombosis for only 1 day after PTCA, whereas ticlopidine treatment alone had no significant effect. Two hours after PTCA, abciximab therapy significantly decreased the number of circulating platelet-neutrophil aggregates but significantly enhanced P-selectin-mediated leukocyte adhesion on the collagen/von Willebrand factor-platelet surface. CONCLUSIONS: Combined therapy with abciximab and ticlopidine has a prolonged inhibitory effect on mural thrombosis formation relative to either treatment alone. Further, we demonstrated an unexpected effect of abciximab in enhancing P-selectin-mediated leukocyte adhesion.

Abciximab↗
PubMed2000

Emergence of resistance to biocides during differentiation of Acanthamoeba castellanii.

A synchronous encystment method was used to study the order of development of resistance of Acanthamoeba castellanii to a range of biocides. The emerging resistance during encystation to short-term exposure to the minimum amoebicidal concentrations of each biocide tested was recorded during the first 36 h of the differentiation process. Hydrochloric acid and moist heat were tested as possible resistance markers. Development of the acid-insoluble, proteincontaining, ectocyst wall and the cellulose endocyst wall was followed by quantification of the acid- and alkali-insoluble residues of cell samples removed from synchronous encystment cultures up to 36 h. Resistance to chemical agents (polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamidine isethionate, hydrogen peroxide) and to moist heat was seen to develop between 14 and 24 h after trophozoites were inoculated into the encystment media. Resistance to hydrochloric acid developed between 0 and 2 h and to chlorhexidine diacetate between 24 and 36 h. Levels of acid-insoluble residues began to increase after 8 h and alkali-insoluble residues (cellulose) were detected after 16 h and coincided with the emergence of resistance to all the agents tested except hydrochloric acid. The results suggest that resistance to the biocides tested probably results largely from the physical barrier of the cyst walls rather than as a consequence of a metabolically dormant cyst.

Acanthamoeba↗
PubMed1998

The synthesis of sub-micron magnetic particles and their use for preparative purification of proteins.

A novel one-step protocol for the preparation of sub-micron magnetic particles as small as 30 nm in diameter has been developed. The surface of the particles was functionalized with carboxyl groups ( approximately 1 COOH per 15A2) to facilitate the attachment of affinity ligands. The high surface area of the resulting beads, combined with the high density of functional groups on the surface, makes them ideally suited for the preparative purification of proteins as was demonstrated by the efficient isolation of trypsin (36 mg per gram of particles) from pancreatic extract.

Animals↗
PubMed1998

Increased von Willebrand factor binding to platelets in single episode and recurrent types of thrombotic thrombocytopenic purpura.

Extensive microvascular platelet aggregation is characteristic of thrombotic thrombocytopenic purpura (TTP). Previous studies have indicated that abnormalities of von Willebrand factor (vWf) are often present in TTP patient plasma. There has not been previously any direct evidence linking these abnormalities to the process of intravascular platelet aggregation in TTP. We used flow cytometry to analyze the binding of vWf to single platelets, and the presence of platelet aggregates, in the blood of 4 children with chronic relapsing (CR) TTP and 5 adults with single episode or recurrent TTP. vWf on the single platelets of CRTTP patients at all time points studied was significantly increased compared to controls, and was increased further as platelet counts decreased to levels below 40,000/microl. The single episode and recurrent adult TTP patients had platelet aggregates in the blood, as well as increased vWf on single platelets, before therapy commenced and thereafter until recovery was in process. In the one unresponsive single episode TTP patient, vWf on single platelets remained elevated, and platelet aggregates persisted, until her death. The platelet alpha-granular protein, P-selectin, was not increased on the single platelets of most TTP blood samples, suggesting that it is vWf from plasma (rather than from alpha-granules) that attaches to platelet surfaces in association with platelet aggregation. These results suggest that vWf-platelet interactions are involved in the platelet clumping process that characterizes TTP.

Adenosine Diphosphate↗
PubMed1998

Oxidative stress induces DNA fragmentation and caspase activation via the c-Jun NH2-terminal kinase pathway in H9c2 cardiac muscle cells.

The aim of this study was to test the hypothesis that oxidative stress induces apoptosis in the H9c2 cardiac muscle cell line, and that signaling via mitogen-activated protein kinase (MAPK) pathways is involved. Three forms of oxidative stress were utilized: the superoxide generator menadione; hydrogen peroxide; or simulated ischemia followed by reperfusion. Relatively low concentrations of menadione (10 micrometer) or H2O2 (250 micrometer) caused maximal DNA fragmentation and caspase activation, both markers for apoptotic cell death, and preferential activation of the c-Jun NH 2-terminal kinase (JNK) and p38 MAPK pathways. In contrast, higher concentrations of menadione or H 2O2 caused less DNA fragmentation, more necrotic cell death and preferential activation of the extracellular signal-regulated kinase (ERK) pathway. Simulated ischemia alone did not induce DNA fragmentation or caspase activation and activated only the p38 MAPK pathway. However, ischemia plus reperfusion resulted in DNA fragmentation, caspase activation, necrotic cell death and activation of all three MAPK pathways. Selective inhibition of the ERK or p38 MAPK pathways (by PD98059 or SB-203580, respectively) had no effect on the extent of oxidative stress-induced DNA fragmentation or caspase activation. In contrast, inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK markedly reduced the extent of DNA fragmentation and caspase activation induced by oxidative stress. In conclusion, these data suggest that the JNK pathway plays an important role in signaling oxidative stress-induced apoptosis of H9c2 cardiac muscle cells.

Animals↗
PubMed1997

Activation of protein kinase C-alpha and translocation of the myristoylated alanine-rich C-kinase substrate correlate with phorbol ester-enhanced noradrenaline release from SH-SY5Y human neuroblastoma cells.

The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the alpha-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-epsilon from cytosolic and membrane-associated to cytoskeleton- and membrane-associated. TPA had no effect on the cytosolic location of PKC-zeta. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 microM). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 nM) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-alpha (but not -epsilon or -zeta). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-alpha.

Alanine↗