Journal of the New Zealand Medical Association, 17-December-2004, Vol 117 No 1207
Proceedings of the 175th meeting of the Otago Medical School Research Society, 11 November 2004
Investigative studies of dysgalacticin, a novel antimicrobial protein produced by Streptococcus dysgalactiae. H Baird, N Heng, J Tagg, R Jack. Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin.
Dysgalacticin is a novel 21 kDa, heat-labile, acid-stable, anionic antimicrobial protein produced by the gram-positive bacterium Streptococcus dysgalactiae subsp. equisimilis. The inhibitory spectrum of dysgalacticin includes predominantly streptococci belonging to Lancefield serogroups A, C, and G. These three serogroups contain essentially all the major streptococcal pathogens of humans. Dysgalacticin is of interest because of its ability to kill the human pathogen Streptococcus pyogenes by an unknown mechanism that does not involve cell lysis. Studies on how dysgalacticin exerts its inhibitory activity may provide insight into novel methods for the control of S. pyogenes infections. The aim of this study was to determine the structural features of dysgalacticin essential for its inhibitory activity.
Analysis of the primary amino acid sequence of dysgalacticin revealed a putative disulphide bond between Cys132 and Cys186. To ascertain if this disulphide bond is critical for activity, recombinant dysgalacticin was expressed in Escherichia coli. The inhibitory spectrum of recombinant dysgalacticin was indistinguishable from the native protein but its biological activity was lost following reductive alkylation using 2-mercaptoethanol and 4-vinylpyridine. In addition, an engineered dysgalacticin variant (Cys186Ala-dysgalacticin), unable to form a disulphide bond, was also expressed and found to be biologically inactive. Both these independent findings confirm that a disulphide bond is indeed present and essential for biological activity of dysgalacticin.
In conclusion, this study has provided essential information on the structure-function relationship of dysgalacticin. Future studies will focus on elucidating the mode of action of dysgalacticin and will involve (i) determining the effects of dysgalacticin on sensitive S. pyogenes cells as well as (ii) identifying the dysgalacticin target (receptor) using transposon mutagenesis.
Supported by a grant from the Health Research Council of New Zealand and a University of Otago Research Grant
Proliferation and apoptosis in the ovarian surface epithelium, cyst epithelium and rete ovarii of mice of different ages and total lifetime ovulation number. C Beaugié, H J McQuillan, J S Fleming. Department of Anatomy and Structural Biology, Otago School of Medical Sciences, University of Otago, Dunedin.
The risk of developing epithelial ovarian cancer correlates with age and total number of ovulations experienced in a lifetime. Over 90% of ovarian cancers are epithelial and thought to be derived from invagination of ovarian surface epithelium (OSE) within the stroma to form inclusion cysts. Incessant ovulation increases inclusion cyst numbers in mice, as well as increasing the number of cysts derived from rete ovarii (RO) tubules. The present study investigated how proliferation and apoptosis rates within these ovarian epithelial compartments change with age and total lifetime ovulation number.
Incessant ovulation was induced in Swiss Webster mice (n = 10/group) by housing them in screen-divided cages alongside a male, until 3, 6, 9, and 12 months of age. Mice housed away from males, with a lower ovulation number, were used as age-matched controls, as were 6- and 9-month breeding animals. All animals were injected with 3 x 30 μg of bromodeoxyuridine (BrdU) per g body weight i.p. at 2 hour intervals and ovaries collected 2 hours later. Incorporated BrdU was determined by immunohistochemistry, with diaminobenzidine (DAB) visualisation. Apoptosis was determined by active caspase-3 immunohistochemistry with DAB visualisation and with TdT-mediated dUTP nick end labelling. A significant decline in proliferation with age was observed in OSE, but not cysts (negative binomial regression model; p < 0.0001). OSE proliferation rates were 1.16, 1.27, 0.45 and 0.33% for the 3, 6, 9 and 12 month-old animals respectively. Proliferation was highest in cysts (1.42%) and lowest in RO (0.20%). Incessantly ovulated ovaries showed significantly more proliferation than those with lower ovulation number (p < 0.03). Extremely low numbers of apoptotic cells were seen in all epithelial compartments.
We conclude apoptosis occurs infrequently in all ovarian epithelia studied. Unlike OSE cells, cyst cell proliferation does not decline significantly with age, suggesting cell cycle control is different in these cells.
Supported by grants from the NZ Lottery Grants Board (Health) and the HS & JC Anderson Charitable Trust.
Protective effects of melatonin against secondary cardiac dysfunction to ischaemic stroke. J A Heaney, A V Tramoudanas, K K Narayan, R M A Rahman, S M Nair, J C Harrison, I A Sammut. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin.
Secondary cardiac dysfunction to ischaemic stroke represents the primary complication in patients with a history of stroke. Inflammation and increased sympathetic activity during the initial ischaemic episode induce oxidative stress in peripheral tissues, including the cardiovascular system. Melatonin has recently been indicated to possess anti-inflammatory, anti-adrenergic and anti-oxidative properties. Furthermore, melatonin has been shown to display efficacy in attenuating pathological processes associated with cardiotoxic drugs. The potential protective effects of melatonin on myocardial function and energetics were investigated in a clinically relevant animal model of ischaemic stroke.
Male Sprague-Dawley rats (265-295 g, n = 32) underwent transient middle cerebral artery occlusion (MCAO) surgery. Melatonin (10 mg/kg, i.p., n = 8) was administered at 1-h post-ischaemic insult and then once every 24 h. At 72 h, animals were sacrificed and hearts perfused with Krebs-Henseleit buffer on a Langendorff system to assess haemodynamic function. Intact ventricular mitochondria were prepared through homogenisation and differential centrifugation. Flavin adenine dinucleotide (FAD)-linked respiratory function and mitochondrial enzyme kinetic analysis was performed to determine bioenergetic capacity. Comparisons amongst treatment groups were performed using One-way analysis of variance, followed by Bonferroni t-tests.
MCAO surgery reduced sinus coronary flow (p < 0.05) and cardiac FAD-linked respiration (p < 0.001), however, ventricular function was not found to be compromised. Mitochondrial enzyme kinetic analysis also showed reduced bioenergetic capacity following MCAO surgery. Melatonin administration led to improved ventricular contractility and sinus coronary flow compared to vehicle treated animals. Melatonin treatment significantly improved FAD-linked respiration (p < 0.001) and led to improved mitochondrial enzyme kinetics.
These results demonstrate for the first time that, in the MCAO model used, cardiac mitochondria and sinus coronary flow are both significantly compromised. Melatonin administration resulted in the maintenance of mitochondrial activity and may oppose the pathophysiological processes resulting in secondary cardiac dysfunction to ischaemic stroke; potentially reducing cardiovascular complications.
The human gene PEG10 utilises backwards ribosomal frameshifting. M Jänicke, W P Tate. Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin.
Ribosomal frameshifting is a rare step in protein synthesis, in which the translating ribosome shifts its reading frame on the messenger RNA and continues synthesis of the protein in a new reading frame. Backwards (-1) frameshifting is widely used by viruses, such as HIV-1, during normal replication and is therefore a target in viral biology that can be exploited for new antiviral therapeutics. It is important to know if there are human genes expressed in adult tissue that utilise -1 frameshifting and must therefore be considered in any anti-viral therapy targeting this mechanism. The human paternally expressed gene 10 (PEG10) has been found to possess two overlapping reading frames with the second in a -1 reading frame. This study investigated whether PEG10 utilises -1 frameshifting for expression in mammalian cells.
Bioinformatic analysis revealed highly conserved homologues of PEG10 in nine other mammalian species (e.g. chimpanzee, cat, dog and mouse), with the seven nucleotide slippery sequence and secondary structure necessary for a -1 frameshift being 100% conserved. To investigate translation of PEG10 in cell culture, the single open reading frames as well as the full-length gene were cloned into a mammalian expression vector. In addition, a control construct expressing both reading frames as a fusion protein was used to determine the size of the full-length protein. Constructs were transfected into monkey kidney (COS-7) cells. Specific antibodies against an N-terminal Flag-tag and a C-terminal His-tag revealed that two proteins are produced, a ~50 kDa protein that is the product of the first open reading frame and a ~100 kDa protein that is the product of both reading frames.
These results indicate that the human gene PEG10 utilises a -1 frameshift mechanism for protein synthesis and expresses two proteins in mammalian cells. This is the first human gene identified that uses -1 frameshifting for its expression.
A role for Pax2 in polycystic kidney disease. C Li1, C Stayner1, A Jeffs1, P Goodyer2, J Zhou3, M R Eccles1. 1Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand; 2Pediatric Nephrology, Montreal Children's Hospital, QC, Canada; 3Brigham and Women's Hospital, Harvard Medical School, Boston, USA.
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cystic growths in the kidneys, pancreas, and liver. Over 85% of ADPKD cases are caused by disruptions of PKD1, encoding polycystin-1. Over-expression of the transcription factor, Pax2, is also associated with kidney cyst formation. To assess a role for Pax2 in an animal model of ADPKD, mice carrying a disrupted Pkd1 gene were crossed with Pax2 heterozygous mutant mice. At embryonic day 18.5 (E18.5), Pkd1 homozygous mutant (Pkd1-/-) embryos had a kidney mass (12.6 ± 0.93 mg) more than twice that of wild-type litter mates (5.9 ± 0.53 mg). The presence of a Pax2 mutation in Pkd1-/- mice resulted in a significant reduction in kidney mass (4.2 ± 0.61 mg, p < 0.001) when compared to Pkd1-/- mice, and attenuation of cyst development. To identify genes that may participate in cystogenesis, the expression levels of eight candidate genes, in the polycystin-1 signaling pathway, including c-Myc, Egf and β-catenin, were quantitated in mRNA from wild-type and mutant E18.5 kidneys, using realtime PCR.
There was no significant change in the mRNA levels of these genes, suggesting that (1) there is no role for these genes during cystogenesis in this mouse model of ADPKD, or (2) changes in gene expression levels may occur at an earlier developmental stage, or in specific kidney structures, or (3) post-transcriptional processing may alter the function of these genes during cystogenesis. In immunohistochemical studies β-catenin was retained at the lateral membrane of cystic cells in the Pkd1-/- kidneys, but cytoplasmic β-catenin staining was lost. The cytoplasmic localization of β-catenin was restored in kidneys of Pkd1-/- mice with a Pax2 mutation, coincident with attenuation in cyst formation.
These observations suggest that persistent Pax2 expression in ADPKD may be linked to changes in the localization of β-catenin, which has previously been suggested to form a complex with polycystin-1.
Efficacy of a micronutrient-fortified seasoning powder on biochemical and functional outcomes in Thai primary school children. MS Manger1, P Winichagoon3, T Pongcharoen3, S Gorwachirapan3, A Boonpraderm3, KB Bailey1, RA Cook1, J McKenzie2, Andrew Gray2, BA Ryan4, RS Gibson1. 1Department of Human Nutrition and 2Preventive and Social Medicine, University of Otago, New Zealand; 3Institute of Nutrition, Mahidol University, Thailand; 4Department of Family Relations and Applied Nutrition, College of Social and Applied Human Sciences, University of Guelph, Canada.
Deficiencies of iron, iodine, vitamin A, and zinc co-exist in children in northeast Thailand and may contribute to impairments in growth, immune competence, and cognitive function.
The aim was to determine the efficacy of a seasoning powder fortified with iron, iodine, vitamin A and zinc served with noodles or rice consumed for school lunch on biochemical status, morbidity, cognition, and growth in rural northeast Thai children.
A double blind, randomised controlled trial of children (n = 566) aged 6-12 years recruited from ten rural schools in Ubon Ratchanthani province was done. Children were stratified by age and gender, and then randomly assigned to receive either an unfortified or a fortified seasoning powder containing iron (5 mg), iodine (50 µg), zinc (5 mg) and vitamin A (270 µg) per serve on each school day for 31 weeks. Baseline and final micronutrient status, and haemoglobinopathies were assessed from blood and urine. Symptoms of diarrhoea-related and respiratory-related disease were recorded daily. Cognitive function was assessed at follow-up by visual memory and digit span tests. Anthropometrical measurements were carried out at baseline and follow-up.
At follow-up, the odds of zinc and iodine deficiencies in the fortified group were 0.63 (95% CI 0.42, 0.95) and 0.52 (0.38, 0.71) times those in the unfortified group, respectively, and haemoglobin was 1.70 g/L (0.45, 2.96) higher. Treatment had no effect on serum retinol or growth, but resulted in a significantly lower incidence of respiratory-related symptoms [ratio 0.82 (0.70, 0.96)], and a significantly higher number of items visually recalled [difference 0.56 (0.15, 0.99)], compared with children in the unfortified group.
In conclusion, a micronutrient-fortified seasoning powder reduced the incidence of zinc and iodine deficiency, increased haemoglobin concentration, reduced morbidity, and improved cognitive function after 31 weeks. It is therefore a promising vehicle for improving the micronutrient status of NE Thai school children.
Supported by the Micronutrient Initiative Fund and the University of Otago
Interleukin-6 stimulates both STAT3 and ERK1/2 phosphorylation in adrenal chromaffin cells. B Milne, S Bunn. Department of Anatomy and Structural Biology, Otago School of Medical Sciences, University of Otago, Dunedin.
Chromaffin cells of the adrenal medulla function as part of the stress response by releasing catecholamines. Stress can come in many forms including infection and tissue damage. It is therefore possible that activation of the immune system could be involved in regulation of the adrenal medulla. This project investigated whether adrenal chromaffin cells respond to stimulation by an immune signal, interleukin-6 (IL-6). IL-6 is known to play a major role in the inflammatory response.
Chromaffin cell cultures from bovine adrenal medullae were incubated with or without IL-6 (3 separate cultures in each case). For immunofluorescence microscopy and for Western blotting, antibodies were employed for phospho-STAT3 and phospho-ERK1/2. These phosphoproteins belong to two independent intracellular signalling cascades that are activated by IL-6 in other cells. An antibody for tyrosine hydoxylase (TH), the rate-limiting enzyme in catecholamine production, was employed for dual-labelled immunofluorescence.
IL-6 caused time- and concentration-dependent responses in phosphorylation of the STAT3 proteins. At 1 nM, IL-6 induced a 147 ± 8% (mean ± SEM) change from basal levels, with the maximal response after 15 min. The immunofluorescence supported this by showing increased levels of phospho-STAT3 in TH-positive cells, plus evidence of translocation to the nucleus, another indicator of STAT activation. Western blotting also showed increased levels of phospho-ERK1/2, with a maximal response by 1 nM IL-6 after 5 min.
This work shows for the first time that chromaffin cells respond to stimulation by IL-6. IL-6 activation of both the STAT3 and ERK1/2 proteins may have a role in catecholamine release, by altering the activity of TH. STAT3 may induce gene transcription, potentially including the transcription of TH. ERK1/2 causes phosphorylation of TH in other cells, and may play a similar role here. Therefore, through the actions of IL-6, the immune system may have a role in the stress response.
Supported by a grant from the Lotteries Board.
Cardio-mitochondrial protection by haem oxygenase products: CO and bilirubin. K K Narayan, A V Tramoundanas, P Zhu, J C Harrison, I A Sammut. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin.
Inducible haem oxygenase (HO) has been suggested to function as an effective antioxidant system in various types of cells thus conferring protection against oxidative stress/ischaemia-reperfusion injury both in vivo and in vitro. The byproducts of HO catalysed haem degradation, biliverdin and its metabolite bilirubin, demonstrate antioxidant properties whilst carbon monoxide (CO) possesses potent vasodilatory/cytoprotective effects. Therefore, this study investigated the individual effects of bilirubin and a CO-releasing molecule, tricarbonyldichlororuthenium (TCDR) on cardiac haemodynamic and mitochondrial function in an isolated, perfused rat heart model subjected to ischaemia-reperfusion injury.
Male Lewis rats (250-300 g) were anaesthetised with diethyl ether, and sodium heparin was injected intravenously. Hearts were excised, rapidly cannulated and Langendorff perfused with Krebs-Henseleit buffer containing either vehicle or drug; TCDR (20 μM) or bilirubin (0.5 and 1 μM). Hearts were then subjected to a 30-min period of warm global ischaemia followed by 1-h reperfusion. Cardiac haemodynamic parameters including left ventricular developed pressure (LVDP), heart rate (HR) and sinus coronary flow rates were measured at various time points (pre-ischaemia, 20, 40, 60 minutes into reperfusion). Intact cardiac ventricular mitochondria were isolated and purified through homogenisation and differential centrifugation. Mitochondrial respiratory function was measured in intact organelles, using a water-jacketed Clark-type oxygen electrode.
Significant recovery in LVDP at 10 mmHg and coronary flow rates were observed for all the treatment groups in comparison to the corresponding vehicle controls (n = 10, p < 0.05). Mitochondrial state 3-respiration rate (measure of oxygen consumption during phosphorylation) and FAD-linked respiration (marker of oxygen consumption) were also markedly improved in all treated groups versus vehicle controls (n = 10, p < 0.05 as analysed by one-way ANOVA for repeated measures followed by Dunn’s post hoc test).
The byproducts, TCDR and bilirubin, protect cardiac haemodynamic and mitochondrial function against ischaemia-reperfusion injury, therefore replicating the protection obtained following HO-1 upregulation.
Oral administration of EGCG modulates CYP450 enzymes in the female BALB/c mouse. M Scandlyn, M Goodin, R Rosengren. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin.
Epigallocatechin gallate (EGCG) is the predominant polyphenolic catechin found in a variety of foods, including green tea, chocolate and fruits. It has demonstrated chemo-preventative activity both in vitro and in vivo, however the mechanism underlying this activity has not been conclusively proven. We have previously demonstrated that EGCG (12.5 and 25 mg/kg, i.p.) can alter the activity of key cytochrome P450 (CYP450) enzymes involved in the synthesis and metabolism of estrogens in the female Swiss Webster mouse. However, 50 mg/kg i.p. EGCG induced considerable mortality and hepatotoxicity. The aim of this study was to examine the ability of EGCG, when administered orally, to modulate CYP450 activity.
Female BALB/c mice (10 mice/group) were administered EGCG (25 and 50 mg/kg) or saline (5 ml/kg) by oral gavage for 7 days. On day 8, mice were euthanased by carbon dioxide inhalation, and ovarian and hepatic microsomes were prepared. Aromatase (CYP19) catalytic activity was determined in ovarian microsomes, and CYP1A, CYP3A and CYP2E1 catalytic activities were determined in hepatic microsomes.
Hepatotoxicity testing, assessed by plasma alanine aminotransferase (ALT) activities, demonstrated that EGCG was well tolerated. Ovarian aromatase activity, determined by the release of [3H]-H2O from [3H]-androstenedione, was reduced by 31% after 25 mg/kg and 46% after 50 mg/kg EGCG administration (15 ± 2, 10 ± 1, 8 ± 1 pmol/mg/h for saline, 25 mg/kg and 50 mg/kg, respectively). Aniline hydroxylation was used to determine CYP2E1 activity, which increased 2-fold following 50 mg/kg EGCG (0.45 ± 0.05 and 0.91 ± 0.06 nmol/mg/min for saline and 50 mg/kg EGCG, respectively). CYP1A and CYP3A activities, however, were not altered by treatment with EGCG.
These results demonstrate that oral administration of EGCG is capable of modulating aromatase and CYP2E1 activities without accompanying toxicity. This suggests that orally administered EGCG may have a potential role as an aromatase inhibitor for the treatment of hormone-sensitive cancers.
Is the signal produced by the arterial baroreceptors in the rabbit affected when arterial pulse pressure increases as occurs, for example, during exercise? M Turner, C Bolter. Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin.
Mean arterial pressure (MAP) is the primary signal encoded in the nerve activity sent to the brainstem from the arterial baroreceptors. During exercise MAP is regulated at a higher value than at rest (baroreflex resetting). Arterial pulse pressure (PP) also increases considerably from ~40 to ≤100 mmHg during exercise in humans. This study investigated whether an increase in PP could contribute to the resetting of MAP during exercise.
Whole nerve activity was recorded from either the aortic depressor nerve (ADN) or carotid sinus nerve (CSN) in rabbits (2.5-4.5 kg) anaesthetised with sodium pentobarbitone. Arterial pressure was recorded from either the aortic arch or a branch of the external carotid artery. Heart rate was held constant throughout the experiment by atrial pacing. Controlled inflation and deflation of cuffs secured around the vena cava and aorta produced ramps of arterial pressure (40-130 mmHg). To increase PP a mixture of isoprenaline (5 μg/ml) and adrenaline (5 μg/ml) was infused (0.4 ml/min) intravenously. For each arterial pressure ramp, the relationship between mean nerve activity and MAP (activity-pressure curve) was fitted with a third order polynomial expression.
For both the ADN and CSN recordings, an increase in PP was associated with a right shift of the activity-pressure curve. To quantify this shift we have expressed the response in terms of change from the control values at a MAP of 90 mmHg. Data are presented as the mean ± SD. Statistical comparisons were performed using the paired t-test. In recordings from the ADN (n = 7), PP increased 11.9 ± 6.8 (p = 0.004) and the response curve shifted by 5.9 ± 2.2 mmHg (p = 0.0004). In recordings from the CSN (n = 6) PP increased 16.8 ± 3.5 (p < 0.0001), and the response curve shifted by 7.7 ± 4.0 mmHg (p = 0.01).
Our results indicate that, at typical values of MAP, mean baroreceptor activity decreases when pulse pressure increases. A right shift in the activity-pressure curve when pulse pressure is increased may contribute to the upward resetting of mean arterial pressure that occurs during exercise.
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