bluuebunny/crossref_metadata_2025 huggingface dataset:
Aedes aegypti ferritin
Authors: Geiser Dawn L. , Chavez Carrie A. , Flores‐Munguia Roberto , Winzerling Joy J. , Pham Daphne Q.‐D.
Published: August 2003

Diseases transmitted by hematophagous (blood‐feeding) insects are responsible for millions of human deaths worldwide. In hematophagous insects, the blood meal is important for regulating egg maturation. Although a high concentration of iron is toxic for most organisms, hematophagous insects seem unaffected by the iron load in a blood meal. One means by which hematophagous insects handle this iron load is, perhaps, by the expression of iron‐binding proteins, specifically the iron storage protein ferritin. In vertebrates, ferritin is an oligomer composed of two types of subunits called heavy and light chains, and is part of the constitutive antioxidant response. Previously, we found that the insect midgut, a main site of iron load, is also a primary site of ferritin expression and that, in the yellow fever mosquito, Aedes aegypti, the expression of the ferritin heavy‐chain homologue (HCH) is induced following blood feeding. We now show that the expression of the Aedes ferritin light‐chain homologue (LCH) is also induced with blood‐feeding, and that the genes of the LCH and HCH are tightly clustered. mRNA levels for both LCH‐ and HCH‐genes increase with iron, H2O2 and hemin treatment, and the temporal expression of the genes is very similar. These results confirm that ferritin could serve as the cytotoxic protector in mosquitoes against the oxidative challenge of the bloodmeal. Finally, although the Aedes LCH has no iron responsive element (IRE) at its 5′‐untranslated region (UTR), the 5′‐UTR contains several introns that are alternatively spliced, and this alternative splicing event is different from any ferritin message seen to date.
Kinetics of enzyme acylation and deacylation in the penicillin acylase‐catalyzed synthesis of β‐lactam antibiotics
Authors: Alkema Wynand B. L. , de Vries Erik , Floris René , Janssen Dick B.
Published: August 2003

Penicillin acylase catalyses the hydrolysis and synthesis of semisynthetic β‐lactam antibiotics via formation of a covalent acyl‐enzyme intermediate. The kinetic and mechanistic aspects of these reactions were studied. Stopped‐flow experiments with the penicillin and ampicillin analogues 2‐nitro‐5‐phenylacetoxy‐benzoic acid (NIPAOB) and d‐2‐nitro‐5‐[(phenylglycyl)amino]‐benzoic acid (NIPGB) showed that the rate‐limiting step in the conversion of penicillin G and ampicillin is the formation of the acyl‐enzyme. The phenylacetyl‐ and phenylglycyl‐enzymes are hydrolysed with rate constants of at least 1000 s−1 and 75 s−1, respectively. A normal solvent deuterium kinetic isotope effect (KIE) of 2 on the hydrolysis of 2‐nitro‐5‐[(phenylacetyl)amino]‐benzoic acid (NIPAB), NIPGB and NIPAOB indicated that the formation of the acyl‐enzyme proceeds via a general acid–base mechanism. In agreement with such a mechanism, the proton inventory of the kcat for NIPAB showed that one proton, with a fractionation factor of 0.5, is transferred in the transition state of the rate‐limiting step. The overall KIE of 2 for the kcat of NIPAOB resulted from an inverse isotope effect at low concentrations of D2O, which is overridden by a large normal isotope effect at large molar fractions of D2O. Rate measurements in the presence of glycerol indicated that the inverse isotope effect originated from the higher viscosity of D2O compared to H2O. Deacylation of the acyl‐enzyme was studied by nucleophile competition and inhibition experiments. The β‐lactam compound 7‐aminodesacetoxycephalosporanic acid (7‐ADCA) was a better nucleophile than 6‐aminopenicillanic acid, caused by a higher affinity of the enzyme for 7‐ADCA and complete suppression of hydrolysis of the acyl‐enzyme upon binding of 7‐ADCA. By combining the results of the steady‐state, presteady state and nucleophile binding experiments, values for the relevant kinetic constants for the synthesis and hydrolysis of β‐lactam antibiotics were obtained.
Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1
Authors: Schoch Guillaume A. , Attias Roger , Le Ret Monique , Werck‐Reichhart Danièle
Published: August 2003

CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4‐hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio‐specific catalysis. The functional significance of these residues was assessed using site‐directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole‐2‐carboxylic acid that can be hydroxylated with different regioselectivities, supports the involvement of N302, I371 and K484 in substrate docking and orientation.
Transient increase of the labile iron pool in HepG2 cells by intravenous iron preparations
Authors: Sturm Brigitte , Goldenberg Hans , Scheiber‐Mojdehkar Barbara
Published: August 2003

Intravenous iron, used for the treatment of anemia in chronic renal failure and other diseases, represents a possible source of free iron in tissue cells, particularly in the liver. In this study we examined the effect of different sources of intravenous iron (IVI) on the labile iron pool (LIP) which represents the nonferritin‐bound, redox‐active iron that is implicated in oxidative stress and cell injury. Furthermore, we examined the role of the LIP for the synthesis of ferritin. We used HepG2 cells as a well known model for hepatoma cells and monitored the LIP with the metal‐sensitive fluorescent probe, calcein‐AM, the fluorescence of which is quenched on binding to iron. We showed that steady state LIP levels in HepG2 cells were increased transiently, up to three‐fold compared to control cells, as an adaptive response to long‐term IVI exposure. In relation to the amount of iron in the LIP, the ferritin levels increased and the iron content of ferritin decreased. As any fluctuation in the LIP, even when it is only transient (e.g. after exposure to intravenous iron in this study), may result either in impairment of synthesis of iron containing proteins or in cell injury by pro‐oxidants. Such findings in nonreticuloendothelial cells may have important implications in the generation of the adverse effects of chronic iron exposure reported in dialysis patients.
Gelatinase B/MMP‐9 and neutrophil collagenase/MMP‐8 process the chemokines human GCP‐2/CXCL6, ENA‐78/CXCL5 and mouse GCP‐2/LIX and modulate their physiological activities
Authors: Van den Steen Philippe E. , Wuyts Anja , Husson Steven J. , Proost Paul , Van Damme Jo , Opdenakker Ghislain
Published: August 2003

On chemokine stimulation, leucocytes produce and secrete proteolytic enzymes for innate immune defence mechanisms. Some of these proteases modify the biological activity of the chemokines. For instance, neutrophils secrete gelatinase B (matrix metalloproteinase‐9, MMP‐9) and neutrophil collagenase (MMP‐8) after stimulation with interleukin‐8/CXCL8 (IL‐8). Gelatinase B cleaves and potentiates IL‐8, generating a positive feedback. Here, we extend these findings and compare the processing of the CXC chemokines human and mouse granulocyte chemotactic protein‐2/CXCL6 (GCP‐2) and the closely related human epithelial‐cell derived neutrophil activating peptide‐78/CXCL5 (ENA‐78) with that of human IL‐8. Human GCP‐2 and ENA‐78 are cleaved by gelatinase B at similar rates to IL‐8. In addition, GCP‐2 is cleaved by neutrophil collagenase, but at a lower rate. The cleavage of GCP‐2 is exclusively N‐terminal and does not result in any change in biological activity. In contrast, ENA‐78 is cleaved by gelatinase B at eight positions at various rates, finally generating inactive fragments. Physiologically, sequential cleavage of ENA‐78 may result in early potentiation and later in inactivation of the chemokine. Remarkably, in the mouse, which lacks IL‐8 which is replaced by GCP‐2/LIX as the most potent neutrophil activating chemokine, N‐terminal clipping and twofold potentiation by gelatinase B was also observed. In addition to the similarities in the potentiation of IL‐8 in humans and GCP‐2 in mice, the conversion of mouse GCP‐2/LIX by mouse gelatinase B is the fastest for any combination of chemokines and MMPs so far reported. This rapid conversion was also performed by crude neutrophil granule secretion under physiological conditions, extending the relevance of this proteolytic cleavage to the in vivo situation.
Mapping the binding domains of the αIIb subunit
Authors: Biris Nikolaos , Abatzis Morfis , Mitsios John V. , Sakarellos‐Daitsiotis Maria , Sakarellos Constantinos , Tsoukatos Demokritos , Tselepis Alexandros D. , Michalis Lambros , Sideris Dimitrios , Konidou Georgia , Soteriadou Ketty , Tsikaris Vassilios
Published: August 2003

αIIbβ3, a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma‐membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand‐binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the αIIb subunit is presented, using overlapping 20‐peptides, in order to identify the binding sites of αIIb potentially involved in the platelet‐aggregation event. Regions αIIb 313–332, αIIb 265–284 and αIIb 57–64 of αIIbβ3 were identified as putative fibrinogen‐binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC‐1 to the activated form of αIIbβ3. Furthermore, αIIb 313–332 was found to bind to fibrinogen in a solid‐phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in αIIb 313–332, 265–284 and 57–64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.
Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes
Authors: Di Marco Annalise , Yao Dan , Laufer Ralph
Published: August 2003

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)‐mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP‐mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well‐characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 µm and 0.74 nmol·min−1 per mg microsomal protein vs. 0.11 nmol·min−1 per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O‐demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2–3‐fold lower IC50 in hepatocytes than in microsomes. The cell‐associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.
Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis
Authors: Zhao Ming , Antunes Fernando , Eaton John W. , Brunk Ulf T.
Published: August 2003

Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant pro‐apoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O‐methyl‐serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 − but not cathepsins B or D − causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.
In vitro characterization of a plastid terminal oxidase (PTOX)
Authors: Josse Eve‐Marie , Alcaraz Jean‐Pierre , Labouré Anne‐Marie , Kuntz Marcel
Published: August 2003

The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu2+, Zn2+ or Mn2+. This plastoquinol oxidase activity is independent of temperature over the 15–40 °C range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I50 = 0.4 µm) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.
Insulin/protein kinase B signalling pathway upregulates metastasis‐related phenotypes and molecules in H7721 human hepatocarcinoma cell line
Authors: Qi Hui‐Ling , Zhang Ying , Ma Jun , Guo Peng , Zhang Xia‐Ying , Chen Hui‐Li
Published: August 2003

The effect of insulin on cancer metastatic potential was studied in a human hepatocarcinoma cell line, H7721. Cell adhesion to human umbilical vein endothelial cells (HUVECs) and laminin as well as chemotactic cell migration and invasion were selected as the indices of metastasis‐related phenotypes for assessment of metastatic potential ex vivo. The results indicated that insulin enhanced all of these metastasis‐related phenotypes. After the cells were treated with specific inhibitor of PI3K (LY294002) or transfected with antisense cDNA of PKB (AS‐PKB), all of the above phenotypes were attenuated, and they could not be significantly stimulated by insulin, indicating that the insulin effect on metastatic potential was mediated by PI3K and PKB. Only the monoclonal antibody to the sialyl Lewis X (SLex), but not antibodies to other Lewis antigens, significantly blocked the cell adhesion to HUVECs, cell migration and invasion, suggesting that SLex played a crucial role in the metastatic potential of H7721 cells. The upregulation of cell surface SLex and α‐1,3‐fucosyltransferase‐VII (α‐1,3 Fuc T‐VII, enzyme for SLex synthesis) was also mediated by PI3K and PKB, since LY294002 and AS‐PKB also reduced the expressions of SLex and α‐1,3 FucT‐VII, and attenuated the response to insulin. Furthermore, the alterations in the expressions of PKB protein and activity were correlated to the changes of metastatic phenotypes and SLex expression. Taken together, the insulin/PKB signalling pathway participated in the enhancement of metastatic potential of H7721 cells, which was mediated by the upregulation of the expression of SLex and α‐1,3 FucT‐VII.
Membrane orientation of laminin binding protein
Authors: Bandyopadhyay Keya , Karmakar Sudipan , Biswas Aruna , Das Pijush K.
Published: August 2003

Earlier we presented several lines of evidence that a 67‐kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67‐kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti‐LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X‐114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C‐terminal is located in the cytosol. A 34‐kDa transmembrane part of LBP could be identified using the photoactive probe, 3‐(trifluoromethyl)‐3‐(m‐iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin‐released fragment indicated that N‐terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N‐terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C‐terminal cytosolic end.
Regulatory modes of rod outer segment membrane guanylate cyclase differ in catalytic efficiency and Ca2+‐sensitivity
Authors: Hwang Ji‐Young , Lange Christian , Helten Andreas , Höppner‐Heitmann Doris , Duda Teresa , Sharma Rameshwar K. , Koch Karl‐Wilhelm
Published: August 2003

In rod phototransduction, cyclic GMP synthesis by membrane bound guanylate cyclase ROS‐GC1 is under Ca2+‐dependent negative feedback control mediated by guanylate cyclase‐activating proteins, GCAP‐1 and GCAP‐2. The cellular concentration of GCAP‐1 and GCAP‐2 approximately sums to the cellular concentration of a functional ROS‐GC1 dimer. Both GCAPs increase the catalytic efficiency (kcat/Km) of ROS‐GC1. However, the presence of a myristoyl group in GCAP‐1 has a strong impact on the regulation of ROS‐GC1, this is in contrast to GCAP‐2. Catalytic efficiency of ROS‐GC1 increases 25‐fold when it is reconstituted with myristoylated GCAP‐1, but only by a factor of 3.4 with nonmyristoylated GCAP‐1. In contrast to GCAP1, myristoylation of GCAP‐2 has only a minor effect onkcat/Km. The increase with both myristoylated and nonmyristoylated GCAP‐2 is 10 to 13‐fold. GCAPs also confer different Ca2+‐sensitivities to ROS‐GC1. Activation of the cyclase by GCAP‐1 is half‐maximal at 707 nmfree [Ca2+], while that by GCAP‐2 is at 100 nm. The findings show that differences in catalytic efficiency and Ca2+‐sensitivity of ROS‐GC1 are conferred by GCAP‐1 and GCAP‐2. The results further indicate the concerted operation of two ‘GCAP modes’ that would extend the dynamic range of cyclase regulation within the physiological range of free cytoplasmic Ca2+in photoreceptor cells.
Use of lithium and SB‐415286 to explore the role of glycogen synthase kinase‐3 in the regulation of glucose transport and glycogen synthase
Authors: MacAulay Katrina , Hajduch Eric , Blair Anne S. , Coghlan Matthew P. , Smith Stephen A. , Hundal Harinder S.
Published: August 2003

Glycogen synthase kinase 3 (GSK3) is inactivated by insulin and lithium and, like insulin, Li also activates glycogen synthase (GS) via inhibition of GSK3. Li also mimics insulin's ability to stimulate glucose transport (GT), an observation that has led to the suggestion that GSK3 may coordinate hormonal increases in GT and glycogen synthesis. Here we have used Li and SB‐415286, a selective GSK3 inhibitor, to establish the importance of GSK3 in the hormonal activation of GT in terms of its effect on GS in L6 myotubes and 3T3‐L1 adipocytes. Insulin, Li and SB‐415286 all induced a significant inhibition of GSK3, which was associated with a marked dephosphorylation and activation of GS. In L6 myotubes, SB‐415286 induced a much greater activation of GS (6.8‐fold) compared to that elicited by insulin (4.2‐fold) or Li (4‐fold). In adipocytes, insulin, Li and SB‐415286 all caused a comparable activation of GS despite a substantial differentiation‐linked reduction in GSK3 expression (≈ 85%) indicating that GSK3 remains an important determinant of GS activation in fat cells. Whilst Li and SB‐415286 both inhibit GSK3 in muscle and fat cells, only Li stimulated GT. This increase in GT was not sensitive to inhibitors of PI3‐kinase, MAP kinase or mTOR, but was suppressed by the p38 MAP kinase inhibitor, SB‐203580. Consistent with this, phosphorylation of p38 MAP kinase induced by Li correlated with its stimulatory effect on GT. Our findings support a crucial role for GSK3 in the regulation of GS, but based on the differential effects of Li and SB‐415286, it is unlikely that acute inhibition of GSK3 contributes towards the rapid stimulation of GT by insulin in muscle and fat cells.
Cellular refractoriness to the heat‐stable enterotoxin peptide is associated with alterations in levels of the differentially glycosylated forms of guanylyl cyclase C
Authors: Ghanekar Yashoda , Chandrashaker Akhila , Visweswariah Sandhya S.
Published: August 2003

The heat‐stable enterotoxin peptides (ST) produced by enterotoxigenic Escherichia coli are one of the major causes of transitory diarrhea in the developing world. Toxin binding to its receptor, guanylyl cyclase C (GC‐C), results in receptor activation and the production of high intracellular levels of cGMP. GC‐C is expressed in two differentially glycosylated forms in intestinal epithelial cells. Prolonged exposure of human colonic cell lines to ST peptides induces cellular refractoriness to the ST peptide, in terms of intracellular cGMP accumulation. We have investigated the mechanism of cellular desensitization in human colonic Caco2 cells, and observe that exposure of cells to ST leads to a time and dose‐dependent inability of cells to respond to the peptide in terms of GC‐C stimulation, both in whole cells and membranes prepared from desensitized cells. This is concomitant with a 50% reduction in ST‐binding activity in desensitized cells. Desensitization was correlated with a loss of the plasma membrane‐associated, hyperglycosylated 145 kDa form of GC‐C, while the predominant 130 kDa form, localized both on the plasma membrane and the endoplasmic reticulum, continued to be present in ST‐treated cells. Desensitized cells recovered ST‐responsiveness on removal of the ST peptide, which was correlated with a reappearance of the 145 kDa form on the cell surface, following processing of the endoplasmic reticulum‐associated pool of the 130 kDa form. Selective internalization of the 145 kDa form of the receptor was required for cellular desensitization, as ST‐treatment of cells at 4 °C did not lead to refractoriness. We therefore show a novel means of regulation of cellular responsiveness to the ST peptide, whereby altering cellular levels of the differentially glycosylated forms of GC‐C can lead to differential ligand‐mediated activation of the receptor.
Predictors of Outcome Following Brief Psychodynamic-Interpersonal Therapy for Deliberate Self-Poisoning
Authors: Guthrie Elspeth , Kapur Navneet , Mackway-Jones Kevin , Chew-Graham Carolyn , Moorey James , Mendel Elizabeth , Marino Francis Fredrika , Sanderson Sarah , Turpin Clive , Boddy Gary
Published: September 2003

Background: We found that brief psychodynamic-interpersonal therapy was more helpful than usual care in deliberate self-poisoning patients, and resulted in reduced suicidal ideation and repetition of self-harm in the 6 months post-treatment. Here, we explore which baseline factors predicted outcome following treatment.

Method: Patients presenting to an emergency department with deliberate self-poisoning were randomly assigned to brief psychodynamic-interpersonal therapy (PIT) or usual care. Severity of suicidal ideation 6 months post-treatment was used as the main outcome measure. Sociodemographic features and baseline psychological measures were used as predictor variables. Univariate and regression analyses were used to identify predictors of outcome for the whole group and for those who received psychotherapy.

Results: Principal predictors for the psychotherapy group were baseline severity of depression and a prior history of self-harm. For the group as a whole predictors were severity of suicidal ideation, anxiety and prior history of self-harm.

Conclusions: Four session PIT for deliberate self-poisoning is effective in reducing suicidal ideation in patients with less severe depression, no prior history of self-harm, and who have not consumed alcohol with the overdose. Extended therapy may be indicated for those with more severe depression.
Clinical and Service Implications of a Cognitive Analytic Therapy Model of Psychosis
Authors: Kerr Ian B. , Birkett Paul B. L. , Chanen Andrew
Published: September 2003

Cognitive analytic therapy (CAT) is an integrative, interpersonal model of therapy predicated on a radically social concept of self, developed over recent years in the UK by Anthony Ryle. A CAT-based model of psychotic disorder has been developed much more recently based on encouraging early experience in this area. The model describes and accounts for many psychotic experiences and symptoms in terms of distorted, amplified or muddled enactments of normal or ‘neurotic’ reciprocal role procedures (RRPs) and of damage at a meta-procedural level to the structures of the self.

Reciprocal role procedures are understood in CAT to represent the outcome of the process of internalization of early, sign-mediated, interpersonal experience and to constitute the basis for all mental activity, normal or otherwise. Enactments of maladaptive RRPs generated by early interpersonal stress are seen in this model to constitute a form of ‘internal expressed emotion’. Joint description of these RRPs and their enactments (both internally and externally) and their subsequent revision is central to the practice of CAT during which they are mapped out through written and diagrammatic reformulations.

This model may usefully complement and extend existing approaches, notably recent CBTbased interventions, particularly with ‘difficult’ patients, and generate meaningful and helpful understandings of these disorders for both patients and their treating teams. We suggest that use of a coherent and robust model such as CAT could have important clinical and service implications in terms of developing and researching models of these disorders as well as for the training of multidisciplinary teams in their effective treatment.
Quality of Life of Community-Based Chronic Schizophrenia Patients in Penang, Malaysia
Authors: Mubarak A. R. , Baba Ismail , Heng Chin Low , Soon Hoe Quah
Published: September 2003

Objective: This is a study of the quality of life (QOL) of 174 community-based chronic schizophrenia patients in Penang, Malaysia.

Method: The study samples were selected from the Out Patient Department, Department of Psychiatry, Penang General Hospital, Malaysia. The data was collected through personal interviews with the respondents. A questionnaire prepared by the research team was used to collect data on background characteristics. Lehman's (1988) Quality of Life Interview was used to collect data on patients’ QOL.

Result: Equal number of males and females participated in the study. The interviews on QOL indicated problems in the areas of life in general, place of living, daily activities, social relations, finance, work and general health. The results also revealed that community-based schizophrenia patients had acute poverty and experienced social isolation, discrimination and exploitation in the workplace.

Conclusion: Implications of these results on the implementation process of National Mental Health Policy in Malaysia are discussed. The research paper also discusses the negative impacts of limited rehabilitation facilities available in the community and its implications on the QOL of severely mentally ill patients. The need for immediate research attention on QOL of such patients in the South-east Asian region has been highlighted.
Delusions: The Continuum Versus Category Debate
Authors: Mullen Richard
Published: September 2003

Objective: In this paper I critically review the recently developed idea that delusions are best considered as part of a continuum along with more ordinary human beliefs.

Method: A literature review of the area was guided by a Medline search, and supplemented with material already known to the author. Claims that recent research supports the continuum hypothesis is critiqued.

Results: The argument and evidence advanced for the continuum approach to delusions depends largely on: (i) inadequacies of prevailing definitions of delusion, particularly in the light of evidence that delusional conviction is often not absolute; and (ii) the high prevalence of unusual beliefs in community populations. To the extent that the delusion construct contains much in addition to conviction and unusual or false belief content, the continuity approach is weak. The category of delusions continues to have some validity.

Conclusion: Both categorical and continuous approaches to delusions have validity that depends at any time on our immediate clinical or scientific needs. No definitive resolution of the category versus continuum debate is likely to emerge.
Dexamethasone‐induced exophthalmos in a group of Holstein calves
Authors: Townsend Wendy M. , Renninger Matthew , Stiles Jean , Townsend Jonathan R. , Krohne Sheryl G.
Published: August 2003

Six Holstein calves were evaluated for progressive exophthalmos. The affected calves were receiving daily injections of dexamethasone (30 µg/kg subcutaneously twice daily) as part of a metabolic study. The control calves did not exhibit exophthalmos. The ocular examinations were normal except for the exophthalmos, which ranged from mild to marked in severity. Upon postmortem examination, marked deposition of retrobulbar adipose tissue was noted in the affected calves. Dexamethasone administration appears to increase deposition of retrobulbar adipose tissue resulting in a progressive exophthalmos.
Nonulcerative keratouveitis as a manifestation of Leptospiral infection in a horse
Authors: Wada Shinya , Yoshinari Masanobu , Katayama Yoshinari , Anzai Toru , Wada Ryuichi , Akuzawa Masao
Published: August 2003

A 2‐year‐old Thoroughbred filly presented with ocular pain and epiphora of the left eye. The pupil was miotic and the cornea edematous near the ventro‐temporal limbus, but did not retain any fluorescein. The topical antibiotics and atropine and diclofenac, and systemic flunixin meglumine and antibiotic therapy did not resolve the condition. A pink and fleshy infiltrate developed near the limbus indicating nonulcerative keratouveitis. The anterior uveitis deteriorated as manifested by the presence of dyscoria, hypopyon, and organized fibrin in the anterior chamber. Ocular signs were improved by topical and subconjunctival corticosteroids, but repeatedly deteriorated as the frequency of medication was reduced. The horse was seropositive to three serovars ofLeptospira interrogans. The animal was diagnosed as blind on day 91 by the absence of pupillary light and menace reflexes, and donated for histopathologic diagnosis. The corneal opacity was histologically fibrotic and infiltrated predominantly by lymphocytes with Descemet's membrane partially disrupted by macrophages. The choroid was infiltrated by lymphocytes, eosinophils and basophils, and was positive to IgG and C3. There were filamentous or spiral structures positive to Warthin‐Starry stain in the renal cortex. There was also polymerase chain reaction amplification of the leptospiral gene in the kidney. From these findings nonulcerative keratouveitis was believed to be caused by systemic infection with Leptospira.
Myxosarcoma in the eye and brain in a dog
Authors: Richter Marianne , Stankeova Simona , Hauser Beat , Scharf Gernot , Spiess Bernhard M.
Published: August 2003

A male mixed breed dog of unknown age was presented with sudden onset of blindness and a slightly exophthalmic right eye. Ophthalmoscopically, a spherical, pinkish mass protruding from the region of the optic disc into the vitreous was seen in the right eye. Ultrasound and computer tomography demonstrated an extension of the mass into the right retrobulbar space, continuing intracranially to the optic chiasm. Cytologic findings, obtained by ultrasound‐guided fine needle aspiration from the retrobulbar space, were consistent with myxosarcoma. On the basis of these findings of a well‐delineated mass in the brain stem area, irradiation was planned as the therapy of choice. The dog was treated under general anesthesia using a proton beam at the Paul Scherrer Institute (PSI, Villigen, Switzerland). A curative protocol (56 Gy in 16 fractions over 4 weeks, 3.5 Gy/fraction) was used. Twenty‐seven days post radiation therapy the dog was euthanized, as clinical symptoms progressed. Enlargement of the intracranial tumor was seen on a CT study repeated on the day of euthanasia. A gross pathologic and histologic examination were performed and confirmed the diagnosis of a myxosarcoma involving the eye, retrobulbar space of the right side, and the optic chiasm. Post‐mortem examination failed to identify any abnormalities or metastases elsewhere in the body.
Effects of transcorneal iridal photocoagulation on the canine corneal endothelium using a diode laser
Authors: Chandler Matthew J. , Moore Phillip A. , Dietrich Ursula M. , Martin Charles L. , Vidyashankar Anand , Chen Guorong
Published: August 2003

Objective To investigate the potential damage to the canine corneal endothelium following transcorneal iridal laser photocoagulation using a semiconductor diode laser.

Animals studied  Sixteen young mongrel dogs.

Procedures Baseline corneal endothelial cell counts and corneal thickness were measured in the central and temporal quadrants using a noncontact specular microscope under general anesthesia. Transcorneal iridal photocoagulation was applied using a semiconductor diode laser in a continuous mode with the use of an operating microscope. Fifteen dogs were treated, and the sixteenth dog served as a control. Fifteen different treatment combinations were randomly assigned to the 30 eyes; the fellow eye was treated differently. Three treatment factors were investigated: (1) laser energy intensity, (2) target tissue to endothelial distance, and (3) laser application duration. After 3 weeks the dogs were euthanized, specular microscopy was repeated, and the cornea was examined by scanning electron microscopy.

Results  Dyscoria and focal iris darkening were noted in all eyes immediately following laser treatment. Focal corneal edema (n = 2) and an incipient anterior capsular cataract (n = 1) were also noted. Baseline mean corneal endothelial cell densities were 2530 cells/mm2centrally and 2607 cells/mm2temporally. Postlaser corneal endothelial cell densities were 2499 cells/mm2centrally and 2523 cells/mm2temporally. Mean prelaser corneal thickness measurements were 0.555 mm centrally and 0.549 mm temporally. Postlaser corneal thickness measurements were 0.580 mm centrally and 0.554 mm temporally. Statistical analyzes revealed no significant changes in endothelial cell densities (P > 0.05) or corneal thickness (P > 0.05) induced by any treatment combination. Aside from tissue handling and processing artifacts, scanning electron microscopy revealed no endothelial cell damage.

Conclusions  Our study demonstrated by specular and scanning electron microscopy that diode laser iridal photocoagulation had no significant effect on the canine corneal endothelium within the parameters described. However, one must take into consideration the young age of the dogs and the potential for corneal endothelial cell regeneration in young dogs, and the relatively short period of postoperative study.
Localization of smooth muscle actin in the iridocorneal angle of normal and spontaneous glaucomatous beagle dogs
Authors: Ryland T. R. , Lewis P. A. , Chisholm M. , Gelatt K. N. , Samuelson D. A.
Published: August 2003

Purpose  To date, our knowledge of the canine trabecular meshwork (TM) with regard to contractility is incomplete. It is important to understand the potential contractile capability within the TM and possible changes associated with spontaneous hypertensive glaucoma. To that end we have examined the presence of actin, including smooth muscle (SM) actin, in the normal and glaucomatous canine iridocorneal angle (ICA) morphologically and immunohistochemically.

Methods  Sections from the ICAs of 12 Beagles with inherited glaucoma (3 months to 6 years old) and age‐matched normal Beagles were treated with target retrieval, protein and power blocked and sequentially incubated with the primary antibody (rat anticanine SM actin) and the secondary antibody (rabbit antirat immunoglobulin), followed by peroxidase labeled streptavidin and incubation with substrate‐chromogen solution (AEC). Smooth muscle fibers that lined an artery within canine heart tissue were used as positive controls. Separate specimens were prepared for ultrastructual observation.

Results  Ultrastructurally, cells within the inner, posterior region of the corneoscleral TM and outer, posterior region of the uveal TM contained many microfilaments, 6 nm in diameter (i.e. actin). Immunohistochemistry demonstrated that cells within these regions possessed SM actin, having been greatest posteriorly, but extended anteriorly to a lesser extent. In the preglaucomatous affected dog the localization pattern for SM actin was identical to that seen in the normal dogs. With the progression of the disease the pattern disappeared.

Conclusions  The interior presence of myofibroblastic cells within the canine ICA suggests that these cells and the smooth muscle cells of the ciliary body along the same plane of orientation function to facilitate the removal of aqueous humor and are likely to be influenced by vascular mediators. The contractile apparatus for the ICA in the dog with inherited glaucoma appeared identical to that of the normal dog prior to expression of the disease, but weakened as the disease progressed.
Effects of growth factors (EGF, PDGF‐BB and TGF‐β1) on cultured equine epithelial cells and keratocytes: implications for wound healing
Authors: Haber Marion , Cao Zhiyi , Panjwani Noorjahan , Bedenice Daniela , Li William W. , Provost Patricia J.
Published: August 2003

Objective  The physiologic mechanisms involving growth factors, including PDGF‐BB, EGF, and TGF‐β1, as potent mediators of fibroblasts and epithelial cells in corneal wound healing remain unknown. The goal of this study was to determine culture methods for equine epithelial cells and keratocytes and to investigate how exogenous growth factors influence proliferation of both cell types.

Procedures  Cell cultures were established from healthy corneas harvested from horses immediately following euthanasia and maintained using standard tissue culture protocols. To determine the effects of PDGF‐BB, EGF, TGF‐β1, keratocytes (1 × 105/well) and epithelial cells (2 × 105/well) were each cultured in 12 well plates and exposed separately to the growth factors. The cells were exposed to concentrations of EGF between 0 and 50 ng/mL; PDGF‐BB between 0 and 75 ng/mL; and TGF‐β1 between 0 and 10 ng/mL. Cell proliferation was measured using 3H‐thymidine assay and differences in growth determined using anova and Tukey's HSD test (P < 0.05).

Results  Epithelial cell and keratocyte cultures were successfully established. EGF maximally stimulated keratocyte and epithelial cells at 25 ng/mL and 5 ng/mL, respectively. PDGF‐BB maximally stimulated keratocytes and epithelial cells at 50 ng/mL and 5 ng/mL, respectively. TGF‐β1 inhibited keratocytes at 5 ng/mL and 10 ng/mL, and epithelial cells at 1 ng/mL and 2 ng/mL.

Conclusions  Methods were established to maintain epithelial cells and keratocytes in vitro. PDGF‐BB and EGF stimulate, while TGF‐β1 inhibits the proliferation of epithelial cells and keratocytes. These growth factors may play a role in maintenance and repair of the equine cornea.
A study of the morphology of canine eyes enucleated or eviscerated due to complications following phacoemulsification
Authors: Moore Denise L. , McLellan Gillian J. , Dubielzig Richard R.
Published: August 2003

This study was undertaken to describe the histopathologic abnormalities observed in canine eyes enucleated or eviscerated due to complications following phacoemulsification; to correlate these findings with the clinical abnormalities reported, and to suggest, if apparent, likely causes and effects of these abnormalities. Sixty‐six canine globes or evisceration samples received for histopathologic interpretation over a 10‐year period (1990–2000) were studied. All globes and evisceration samples were obtained from clinical patients examined by board‐certified ophthalmologists. Hematoxylin and eosin (H&E) and alcian blue/periodic acid Schiff (PAS) stained sections were examined by light microscopy in every case. A tissue Gram stain was used when indicated. Clinical information obtained from the pathology submission form was reviewed in all cases, and obtained from questionnaires completed and returned by an ophthalmologist for 51 cases (77%). The most frequent histopathologic diagnoses were glaucoma (76%) and retinal detachment (64%). The most frequently reported clinical abnormalities in this series were glaucoma (86%) and uveitis (82%). Five problem areas were identified that appear to make a significant contribution to the failure of canine cataract surgery and merit further investigation: pre‐iridal fibrovascular membranes; lens fiber regrowth; lens epithelial membranes; endophthalmitis, and the health of the corneal surgical incision.
Intravascular papillary endothelial hyperplasia of the conjunctiva in a horse
Authors: Herrera Héctor Daniel , Duchene Adriana Graciela , Croxatto J. Oscar , Burgués Nora , Amusquibar Hugo
Published: August 2003

Objective Vascular tumors of the conjunctiva in the horse are rare. We present a unique case of an intravascular papillary endothelial hyperplasia of the conjunctiva.

Animal studied Horse.

Procedures Case report. A 6‐year‐old‐mare presented with a red mass in the conjunctiva of the left eye. After complete ophthalmologic examination the lesion was excised. The tissue was processed for light microscopy and studied histopathologically.

Results Pathologic examination revealed a nonencapsulated vascular lesion composed of confluent vascular spaces filled by multiple papillary structures composed of a central collagenous core lined by hyperplastic endothelial cells. There was neither atypical endothelial cell nor mitotic activity.

Conclusions Intravascular papillary endothelial hyperplasia is a benign proliferative lesion that should be differentiated from malignant vascular tumors.
Alginate as a new biomaterial for the growth of porcine retinal pigment epithelium
Authors: Eurell Thomas E. , Brown Daniel R. , Gerding Paul A. , Hamor Ralph E.
Published: August 2003

Objective  Determine the effect of a 3‐dimensional alginate matrix on the growth and differentiation of cells isolated from porcine retinal pigment epithelium (RPE).

Procedures  Porcine RPE cells were harvested from enucleated eyecups, isolated by differential gravity sedimentation and cultured in either alginate alone (Group 1) or on plastic tissue culture plates followed by alginate (Group 2). Group 1 cells were cultured in alginate to evaluate the efficacy of the matrix as a culture medium. Group 2 cells were initially cultured on plastic to induce dedifferentiation. The cells were then harvested, suspended in alginate beads, and incubated for a second culture period to determine if the induced dedifferentiation was reversible.

Results  The number of Group 1 cells was significantly greater (P ≤ 0.01) at the end of the culture period. The amount of pigment and cell morphology of Group 1 cells at the end of the culture period was similar to that seen at initial cell isolation. The initial culture of Group 2 cells on plastic showed characteristic features of dedifferentiation marked by the loss of pigment and alterations in microscopic appearance. Secondary culture of dedifferentiated Group 2 cells in alginate beads resulted in a return to pigmentation and characteristic morphology for a majority of the cultured cells.

Conclusions  Porcine RPE cells can be propagated in alginate culture with a significant increase in cell numbers while maintaining normal morphology. Under the conditions described in the present study, the dedifferentiation of porcine RPE induced by standard in vitro culture methods is reversible.