Publications by authors named "Marjorie E Dunlop"

8 Publications

  • Page 1 of 1

Kupffer cell function during the erythocytic stage of malaria.

J Gastroenterol Hepatol 2006 Jan;21(1 Pt 2):313-8

Department of Medicine, University of Melbourne, Austin and Repatriation Medical Center, Melbourne, Victoria, Australia.

Background And Aim: Previous studies using isolated perfused rat liver in vivo have suggested that during the erythrocytic phase of malaria infection, overall phagocytosis by Kupffer cells is enhanced. The aim of the present study was to further investigate the individual phagocytic capacity and prostaglandin E(2) (PGE(2)) secretion of isolated Kupffer cells in vitro, and the immunohistochemical characteristics of Kupffer cells in vivo.

Methods: Malaria was induced in male Sprague-Dawley rats (n = 12) by inoculation with parasitized red cells containing Plasmodium berghei. Kupffer cells were isolated by centrifugal elutriation.

Results: A significantly increased yield of Kupffer cells was obtained from malaria-infected livers compared to controls (36.7 +/- 4.5 vs 11.8 +/- 1.1 x10(6) cells, P < 0.0001, n = 12). There was an increased internalization by phagocytosis of [(3)H]-BSA latex microspheres after 60 min in malaria-infected Kupffer cells compared to controls (65.05 +/- 1.5 vs 48.6 +/- 0.7, P < 0.001, n = 12). PGE(2) secretion into the cell culture medium was significantly suppressed in malaria-infected Kupffer cells compared to controls (1167 +/- 88 vs 4537 +/- 383 pg per 10(6) cells, P < 0.001, n = 5). Staining of ED1, ED2 and PCNA was greater in malaria-infected livers compared to control.

Conclusion: The results indicate that the number of Kupffer cells is significantly increased and their phagocytic activity on a cell-by-cell basis is enhanced during the erythrocytic stage of malaria.
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http://dx.doi.org/10.1111/j.1440-1746.2006.04192.xDOI Listing
January 2006

Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line.

Asian J Androl 2004 Mar;6(1):3-13

Department of Obstetrics and Gynaecology, University of Melbourne, 320 Victoria Parade, East Melbourne 3002 VIC, Australia.

Aim: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests.

Methods: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed.

Results: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZP1 was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZP1 was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed.

Conclusion: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZP1 from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.
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March 2004

Elevated SNAP-25 is associated with fatty acid-induced impairment of mouse islet function.

Biochem Biophys Res Commun 2004 Apr;317(2):472-7

Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Parkville, Vic. 3050, Australia.

The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in insulin secretion following chronic exposure to non-esterified fatty acids (NEFAs) has not been extensively investigated. Here, we show that synaptosome-associated protein of 25 kDa (SNAP-25) levels were predominantly elevated in the soluble fraction of mouse islets exposed to palmitate. This coincided with an impairment of insulin secretion to glucose and non-glucose secretagogues, consistent with a defect at a distal regulatory step in exocytosis. Removal of palmitate from the media restored both SNAP-25 protein levels and insulin secretion to control levels. We conclude that increased expression of SNAP-25 is associated with NEFA-induced impairment of insulin secretion in mouse islets.
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http://dx.doi.org/10.1016/j.bbrc.2004.03.067DOI Listing
April 2004

Matrix protein glycation impairs agonist-induced intracellular Ca2+ signaling in endothelial cells.

J Cell Physiol 2002 Oct;193(1):80-92

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.

Studies have shown diabetes to be associated with alterations in composition of extracellular matrix and that such proteins modulate signal transduction. The present studies examined if non-enzymatic glycation of fibronectin or a mixed matrix preparation (EHS) alters endothelial cell Ca(2+) signaling following agonist stimulation. Endothelial cells were cultured from bovine aorta and rat heart. To glycate proteins, fibronectin (10 microg/ml), or EHS (2.5 mg/ml) were incubated (37 degrees C, 30 days) with 0.5 M glucose-6-phosphate. Matrix proteins were coated onto cover slips after which cells (10(5) cells/ml) were plated and allowed to adhere for 16 h. For measurement of intracellular Ca(2+), cells were loaded with fura 2 (2 microM) and fluorescence intensity monitored. Bovine cells on glycated EHS showed decreased ability for either ATP (10(-6) M) or bradykinin (10(-7) M) to increase Ca(2+) (i). In contrast, glycated fibronectin did not impair agonist-induced increases in Ca(2+) (i). In the absence of extracellular Ca(2+), ATP elicited a transient increase in Ca(2+) (i) consistent with intracellular release. Re-addition of Ca(2+) resulted in a secondary rise in Ca(2+) (i) indicative of store depletion-mediated Ca(2+) entry. Both phases of Ca(2+) mobilization were reduced in cells on glycated mixed matrix; however, as the ratio of the two components was similar in all cells, glycation appeared to selectively impair Ca(2+) release from intracellular stores. Thapsigargin treatment demonstrated an impaired ability of cells on glycated EHS to increase cytoplasmic Ca(2+) consistent with decreased endoplasmic reticulum Ca(2+) stores. Further support for Ca(2+) mobilization was provided by increased baseline IP(3) levels in cells plated on glycated EHS. Impaired ATP-induced Ca(2+) release could be induced by treating native EHS with laminin antibody or exposing cells to H(2)O(2) (20-200 microM). Glycated EHS impaired Ca(2+) signaling was attenuated by treatment with aminoguanidine or the antioxidant alpha-lipoic acid. The results demonstrate that matrix glycation impairs agonist-induced Ca(2+) (i) increases which may impact on regulatory functions of the endothelium and implicate possible involvement of oxidative stress.
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http://dx.doi.org/10.1002/jcp.10153DOI Listing
October 2002

Prevention of diabetes-induced albuminuria in transgenic rats overexpressing human aldose reductase.

Endocrine 2002 Jun;18(1):47-56

University of Melbourne Department of Medicine, Royal Melbourne Hospital, Parkville, VIC, Australia.

Studies using pharmacologic inhibitors have implicated the enzyme aldose reductase in the pathogenesis of albuminuria and diabetic renal disease. However, a clear conclusion is not easily drawn from such studies since these pharmacologic inhibitors have nonspecific properties. To examine further the role of aldose reductase, we have overexpressed the human enzyme in a transgenic rat model. Transgene expression in the kidney was predominantly localized to the outer stripe of the outer medulla, compatible with the histotopography of the straight (S3) proximal tubule. The effect of enzyme overexpression on diabetes-induced renal function and structure was then investigated. Contrary to what may have been anticipated from the previous enzyme inhibition studies, diabetes-induced albuminuria was completely prevented by the overexpression of aldose reductase. No effect of overexpression of aldose reductase on renal structure nor on urinary excretion of beta2-microglobulin and N-acetyl-beta-D-glucosaminidase was observed in this transgenic rat model. In conclusion, our study strongly suggests that multiple roles for aldose reductase may give it a more complex place in diabetic nephropathy than is currently recognized.
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http://dx.doi.org/10.1385/ENDO:18:1:47DOI Listing
June 2002

Phorbol myristate acetate induces ruffling of the acrosome of human sperm.

Fertil Steril 2002 Jul;78(1):128-36

Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Royal Women's Hospital, Victoria, Australia.

Objective: To determine the effect of phorbol myristate acetate (PMA) on human acrosome morphology and the acrosome reaction.

Design: Controlled experiments on sperm and unfertilized oocytes from volunteers.

Setting: Academic research and teaching tertiary hospital.

Patient(s): Sperm samples were from normospermic men and unfertilized oocytes from IVF patients.

Main Outcome Measure(s): Acrosome morphology was assessed by using transmission and scanning electron microscopy. The acrosome reaction was assessed by using fluorescein-labeled Pisum sativum agglutinin.

Result(s): PMA induced acrosome ruffling, indicated by a marked wavy appearance. A significant correlation was found between PMA-induced ruffling and PMA enhancement of the zona pellucida-induced acrosome reaction. Protein kinase C inhibitors bisindolylmalemide I and sangivamycin had no effect on PMA-induced acrosomal ruffling, but actin polymerization inhibitors cytochalasin B and cytochalasin D significantly decreased PMA-induced acrosomal ruffling. In contrast, bisindolylmalemide I, sangivamycin, cytochalasin B, and cytochalasin D significantly decreased both the zona pellucida-induced acrosome reaction and the PMA enhancement of the zona pellucida-induced acrosome reaction.

Conclusion(s): PMA-induced acrosomal ruffling involves actin polymerization, possibly independent of conventional protein kinase C. Acrosomal ruffling is involved in the PMA augmentation of the zona pellucida-induced acrosome reaction.
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http://dx.doi.org/10.1016/s0015-0282(02)03166-7DOI Listing
July 2002

Comparison of insulin secretory function in two mouse models with different susceptibility to beta-cell failure.

Endocrinology 2002 Jun;143(6):2085-92

University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia.

Type 2 diabetes is characterized by a susceptibility to beta-cell failure. However, subjects at risk of developing type 2 diabetes, such as those with obesity or a family history of diabetes, have been shown to display hyperinsulinemia. Although this hyperinsulinemia may be an adaptive response to insulin resistance, the possibility that insulin hypersecretion may be a primary defect has not been thoroughly investigated. The DBA/2 mouse is a model of pancreatic islet susceptibility. Unlike the resistant C57BL/6 mouse strain, the DBA/2 mouse islet fails when stressed with insulin resistance or when exposed to chronic high glucose concentrations. The aim of this study was to compare insulin secretory function in the DBA/2 and C57BL/6 strains in the absence of insulin resistance or high glucose. Insulin secretion was assessed in vivo using the iv glucose tolerance test and in vitro using isolated islets in static incubations. It was shown that DBA/2 mice hypersecreted insulin in vivo, compared with C57BL/6 mice, at 1 d and at 4 and 10 wk of age. This hypersecretion was not attributable to insulin resistance (as assessed by the insulin tolerance test) or increased parasympathetic nervous system outflow. Insulin hypersecretion was also demonstrated in vitro. This was associated with higher glycolysis and glucose oxidation, and elevated activity (but not protein levels) of islet glucokinase and hexokinase. Furthermore, GLUT2 protein levels were higher, which may explain an increase in glucokinase activity in DBA/2 mouse islets. In summary, the DBA/2 mouse, a model of islet failure, has increased glucose-mediated insulin secretion from a very early age, which is associated with an increase in glucose utilization. Further studies will determine whether there is a link between insulin hypersecretion and subsequent beta-cell failure.
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http://dx.doi.org/10.1210/endo.143.6.8859DOI Listing
June 2002

Hyaluronan increases glomerular cyclooxygenase-2 protein expression in a p38 MAP-kinase-dependent process.

Kidney Int 2002 May;61(5):1729-38

Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria, Australia.

Background: Accumulation of the matrix glycosaminoglycan hyaluronan occurs in many types of renal injury but could follow any provision of hyaluronan substrate to the kidney, for example, through widespread use of supplementary glucosamine in osteoarthritic conditions. Hyaluronan can increase cyclooxygenase-2 (COX-2) protein and prostaglandin production. This effect was characterized in rat renal glomeruli to determine the cellular mechanism of activation.

Methods: Isolated glomeruli were treated with purified hyaluronan (molecular mass 2 x 105 D) for up to 24 hours.

Results: An increase in cyclooxygenase capacity and COX-2 protein was shown to follow the activation of p38-mitogen-activated protein (MAP) kinase and to be inhibited by a specific pyridinyl imadazole inhibitor (SB 202190). Hyaluronan-induced activation of cytosolic phospholipase A2 also was shown to be a p38 MAP kinase effect in these preparations. Prostaglandin production was inhibited by COX-2-specific non-steroidal anti-inflammatory compounds (NS-398 and celecoxib) but, as shown for many non-steroidal anti-inflammatory drugs (NSAIDs), an increase in COX-2 protein accompanied this inhibition.

Conclusions: We propose that these findings have clinical relevance. Prostaglandins have a number of important intrarenal regulatory effects leading to some debate over renal function with the use of NSAIDs. Where hyaluronan is increased, p38 MAP-kinase-dependent provision of prostaglandin substrate, via activation of cytosolic phospholipase A2, and a concomitant increase in cyclooxygenase-2 protein would raise renal prostaglandin levels. While NSAID treatment can prevent a rise in prostaglandin levels, it needs to be maintained to avoid possible exacerbation of pro-inflammatory conditions due to increased COX-2 protein levels.
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http://dx.doi.org/10.1046/j.1523-1755.2002.00334.xDOI Listing
May 2002