School of Molecular and Biomedical Science

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Comprising the disciplines of biochemistry, genetics, physiology, microbiology and immunology.

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13C-sucrose breath test: Novel use of a noninvasive biomarker of environmental gut health
(Inderscience Publishers, 2009) Ritchie, B.; Brewster, D.; Davidson, G.; Tran, C.; McNeil, Y.; Hawkes, J.; Butler, R.
OBJECTIVE: Environmental enteropathy syndrome may compromise growth and predispose to infectious diseases in children in the developing world, including Australian Aboriginal children from remote communities of the Northern Territory. In this study, we described the use of a biomarker 13C-sucrose breath test (SBT) to measure enterocyte sucrase activity as a marker of small intestinal villus integrity and function. METHODS: This was a hospital-based prospective case-control study of Aboriginal and non-Aboriginal children with and without acute diarrheal disease. Using the SBT, we compared 36 Aboriginal case subjects admitted to a hospital (18 diarrheal and 18 nondiarrheal disease), with 7 healthy non-Aboriginal control subjects. Intestinal permeability using the lactulose/rhamnose (L/R) ratio on a timed 90-minute blood test was performed simultaneously with the SBT. The SBT results are expressed as a cumulative percentage of the dose recovered at 90 minutes. RESULTS: Aboriginal children with acute diarrheal disease have a significantly decreased absorptive capacity, as determined by the SBT, with a mean of 1.9% compared with either Aboriginal children without diarrhea (4.1%) or non-Aboriginal (6.1%) control subjects. The mean L/R ratio in the diarrhea group was 31.8 compared with 11.4 in Aboriginal children without diarrhea. There was a significant inverse correlation between the SBT and the L/R ratio. CONCLUSIONS: The SBT was able to discriminate among Aboriginal children with diarrhea, asymptomatic Aboriginal children with an underlying environmental enteropathy, and healthy non-Aboriginal controls. This test provides a noninvasive, easy-to-use, integrated marker of the absorptive capacity and integrity of the small intestine and could be a valuable tool in evaluating the efficacy of interventions aimed at improving gut health.
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13C-Urea breath test: Reproducibility and association with the severity of Helicobacter pylori-associated antral gastritis
(Blackwell Publishing Asia, 2005) Matthews, G.; Cummins, A.; Lawrence, A.; Johnson, B.; Campbell, F.; Butler, R.
Background
The purpose of the present paper was to assess the reproducibility of the (13)C-urea breath test ((13)C-UBT) and its ability to reflect the level of Helicobacter pylori-associated inflammation.
Methods
Asymptomatic H. pylori-positive subjects (n = 21) performed the (13)C-UBT six times. The H. pylori-positive symptomatic subjects (n = 55) performed the (13)C-UBT and had antral biopsies taken for histopathology, culture, urease activity assay and myeloperoxidase activity assay.
Results
No significant intraindividual variation in (13)C-UBT results were observed for the asymptomatic subjects. The (13)C-UBT results were significantly higher in symptomatic subjects with a moderate to severe gastritis compared to a mild gastritis and to no inflammation (34.5 +/- 4.4 vs 17.7 +/- 2.8 vs 1.7 +/- 0.1, respectively, P < 0.01). The (13)C-UBT results significantly correlated with urease (r = 0.55) and myeloperoxidase activity (r = 0.82) but not with bacterial load. conclusion: The (13)C-UBT is a reproducible determinant of H. pylori infection and non-invasively assesses the severity of antral inflammation.
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2-DE with IPGs
(Wiley-V C H Verlag GMBH, 2009) Gorg, A.; Drews, O.; Luck, C.; Weiland, F.; Weiss, W.
In order to overcome the limitations of carrier ampholyte generated pH gradients, IPGs were developed in the late 1970s. However, the 2-DE pattern we included in the first publication on IEF with IPGs [Bjellqvist et al., J. Biochem. Biophys. Methods 1982, 6, 317–339] was far from being competitive to O'Farrell's high-resolution 2-DE with carrier ampholytes. Our 2-DE pattern in this article was, more or less, only a proof of principle. It was, however, the beginning of a long journey of stepwise improved 2-DE protocols we developed in our laboratory and summarized in the reviews published in Electrophoresis 1988, 9, 531–546 and in Electrophoresis 2000, 21, 1037–1053. Milestones were the design of the IPG strip, and the “reduction-alkylation equilibration protocol” of IPG strips after IEF for the efficient transfer of proteins from first to second dimension. The protocol of 2-DE with IPGs has been constantly refined, e.g. by the generation of tailor-made IPGs with different pH intervals from the acidic to the basic extremes (pH 2.5–12), and extended separation distances for improved resolution. In the present review, a historical outline from the technical difficulties encountered during the development of 2-DE with IPGs, to the establishment of the actual “standard protocol” will be given, as well as the modified procedures for the separation of very acidic, very alkaline, low-abundance and hydrophobic proteins, followed by a brief discussion of the advantages and technical challenges of gel-based proteomic technologies.
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The 25-Hydroxyvitamin D 24-Hydroxylase
(Academic Press, 1997) Omdahl, John L.; May, B.; School of Molecular and Biomedical Science : Biochemistry
This chapter reviews the role of 25-hydroxyvitamin D 24-hydroxylase in vitamin D metabolism. The 24-hydroxylase enzyme is a widely distributed component of the vitamin D pathway. It is regulated by a spectrum of hormones and functions to synthesise 24-hydroxylated metabolites, with preferential action in promoting bone mineralization and possibly other undisclosed cellular functions that may become evident through the use of gene knockout models. It also plays a central role in directing the metabolic turnover of several 25-hydroxylated vitamin D metabolites.
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25-Hydroxyvitamin D requirement for maintaining skeletal health utilising a Sprague-Dawley rat model
(Pergamon-Elsevier Science Ltd, 2007) Anderson, P.; Sawyer, A.; May, B.; O'Loughlin, P.; Morris, H.
To study the role of vitamin D to optimise bone architecture, we have developed an animal model to investigate the effects of frank vitamin D-deficiency as well as graded depletion of circulating 25-hydroxyvitamin D₃ (25D) levels on the skeleton. Rats fed on dietary vitamin D levels from 0 to 500 ng/day achieved diet-dependent circulating levels of 25D ranging from 11 to 115 nmol/L. Levels of serum 1,25-dihydroxyvitamin D₃ (1,25D) increased as dietary vitamin D increased between 0 and 200 ng/day at which point a maximum level was achieved and retained with higher vitamin D intakes. The renal levels of 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) mRNA were highest in animal groups fed on vitamin D between 0 and 300 ng/day. In contrast, renal 25-hydroxyvitamin D 24-hydroxylase (CYP24) mRNA levels increased as dietary vitamin D increased achieving maximum levels in animals receiving 500 ng vitamin D/day. This animal model of vitamin D depletion is suitable to provide invaluable information on the serum levels of 25D and dietary calcium intake necessary for optimal bone structure. Such information is essential for developing nutritional recommendations to reduce the incidence of osteoporotic hip fractures.
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-308 Nco I polymorphism of tumour necrosis factor a in overweight caucasians
(Elsevier Sci Ireland Ltd, 2003) Coates, A.; Heilbronn, L.; Noakes, M.; Kind, K.; Clifton, P.
We investigated whether a polymorphism in the promoter region of the TNFα gene (−308 A/G) is associated with reduced weight loss in obese Australian subjects on an energy restricted diet. 189 healthy subjects and 91 subjects with type II diabetes were genotyped for the −308 Nco I polymorphism using PCR-RFLP techniques. A subset of these subjects (211 females and 45 males), were placed on a 30% energy restricted diet (6200 kJ) for 12 weeks. Subjects were assessed every 2 weeks and changes in body weight, waist circumference and BMI were used as determinants of weight loss. Fasting plasma was analysed for glucose, insulin, lipids and free fatty acids. 64% of subjects were GG homozygotes, 31% were AG heterozygotes and 5% were AA homozygotes. There was no significant difference between the allele frequency in healthy subjects (0.21) and type 2 diabetic patients (0.24). The presence of the −308 A/G polymorphism did not significantly influence initial BMI, the amount of weight lost (GG, 8.1±0.65 kg, AG, 6.9±0.77 kg, AA, 7.6±0.12 kg), waist circumference or any metabolic variable. The AA variant at position −308 in the promoter region of the TNFα gene does not influence the amount of weight lost in overweight and obese men and women on a 30% energy restricted diet.
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360His polymorphism of the apolipoproteinA-IV gene and plasma lipid response to energy restricted diets in overweight subjects
(Elsevier Sci Ireland Ltd, 2000) Heilbronn, L.; Noakes, M.; Coates, A.; Kind, K.; Clifton, P.
Obesity is commonly associated with high rates of cardiovascular disease (CVD). Weight loss in obese subjects reduces risk factors for CVD but this response is not uniform. Genetic factors could be involved in this variability. The 360His polymorphism of apolipoproteinA-IV (apoA-IV) influences the lipid response to fat intake, but it is unclear whether this polymorphism could contribute to lipid variability during weight loss. Therefore, we assessed the effects of an energy restricted diet (6.3 MJ) for 12 weeks on weight loss and plasma lipids according to apoA-IV genotype in 186 overweight/obese subjects (BMI mean 33+/-4.3, range 25.0-48.0 kg/m(2)). The frequency of the 360His allele was 0.083. Energy restriction for 12 weeks resulted in an average weight loss of 8. 25+/-0.28 kg. HDL-C increased 5.4% in subjects with the apoA-IV-1/1 genotype with weight loss compared to a 2.6% decrease in apoA-IV-1/2 subjects (P=0.035). This was more apparent when only the subjects with type 2 diabetes (n=57) were analyzed (P=0.003). ApoA-IV genotype was not related to change in total cholesterol, LDL-C or triglyceride concentrations. Therefore, weight loss as a treatment to reduce CVD risk factors may be more effective in subjects with the apoA-IV-1/1 variant as compared to those with the apoA-IV-1/2 variant, especially in subjects with type 2 diabetes.
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5-Benzylidenerhodanine and 5-benzylidene-2-4-thiazolidinedione based antibacterials
(Pergamon-Elsevier Science Ltd, 2012) Zvarec, O.; Polyak, S.; Tieu, W.; Kuan, K.; Dai, H.; Pedersen, D.; Morona, R.; Zhang, L.; Booker, G.; Abell, A.
Herein we outline the antibacterial activity of amino acid containing thiazolidinediones and rhodanines against Gram-positive bacteria Staphylococcus aureus ATCC 31890, Staphylococcus epidermidis and Bacillus subtilis ATCC 6633. The rhodanine derivatives were generally more active than the analogous thiazolidinediones. Compounds of series 5 showed some selectivity for Bacillus subtilis ATCC 6633, the extent of which is enhanced by the inclusion of a non-polar amino acid at the 5-position of the core thiazolidinediones and rhodanines scaffolds. SAR data of series 8 demonstrated improved activity against the clinically more significant Staphylococci with selectivity over Bacillus subtilis ATCC 6633 induced by introduction of a bulky aryl substituent at the 5-position of the core scaffolds.
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5-Fluorouracil Induces an Acute Reduction in Neurogenesis and Persistent Neuroinflammation in a Mouse Model of the Neuropsychological Complications of Chemotherapy
(Springer Nature, 2022) Subramaniam, C.B.; Wardill, H.R.; Davies, M.R.; Heng, V.; Gladman, M.A.; Bowen, J.M.
The neuropsychological symptoms associated with chemotherapy treatment remain a major challenge with their prevention hampered by insufcient understanding of pathophysiology. While long-term neuroimmune changes have been identifed as a hallmark feature shared by neurological symptoms, the exact timeline of mechanistic events preceding neuroinfammation, and the relationship between the glial cells driving this neuroinfammatory response, remain unclear. We therefore aimed to longitudinally characterize the neuroimmunological changes following systemic 5-fuorouracil (5-FU) treatment to gain insight into the timeline of events preceding the well-documented chronic neuroinfammation seen following chemotherapy. Eighteen female C57Bl/6 mice received a single intraperitoneal dose of 5-FU and groups were killed at days 1 and 2 (acute timepoint), days 4 and 8 (subacute timepoint), and days 16 and 32 (chronic timepoint). A further six mice were administered with vehicle control with tissues collected from three mice on day 1 and day 32 of the study. The expression of key genes of interest, BCL2, BDNF, TIMP1, MMP-9, MMP-2, TNFα, IL-1β, and IL-6R were assessed using real time polymerase chain reaction. Levels of neurogenesis were determined through immunofuorescent staining of doublecortin (DCX). The density of microglia and astrocytes were assessed using immunofuorescence staining of Iba1 and GFAP respectively. 5-FU treatment caused signifcant decreases to DCX staining at acute timepoints (p=0.0030) which was positively correlated with BCL2 expression levels. An increase to microglial density was observed in the prefrontal cortex (p=0.0256), CA3 region (p=0.0283), and dentate gyrus (p=0.0052) of the hippocampus at acute timepoints. 5-FU caused increases to astrocyte density, across multiple brains regions, at subacute and chronic timepoints which were positively correlated with TNFα, TIMP-1, MMP-2, and IL-6R expression. This study has identified acute objective neuroinflammatory changes suggesting that the role of early intervention should be explored to prevent the development of neuropsychological deficits in the longer term following chemotherapy.
Open Access
A 'bright zone' in male hoverfly (Eristalis tenax) eyes and associated faster motion detection and increased contrast sensitivity
(Company of Biologists Ltd, 2006) Straw, A.; Warrant, E.; O'Carroll, D.
Eyes of the hoverfly Eristalis tenax are sexually dimorphic such that males have a fronto-dorsal region of large facets. In contrast to other large flies in which large facets are associated with a decreased interommatidial angle to form a dorsal `acute zone' of increased spatial resolution, we show that a dorsal region of large facets in males appears to form a `bright zone' of increased light capture without substantially increased spatial resolution. Theoretically, more light allows for increased performance in tasks such as motion detection. To determine the effect of the bright zone on motion detection, local properties of wide field motion detecting neurons were investigated using localized sinusoidal gratings. The pattern of local preferred directions of one class of these cells, the HS cells, in Eristalis is similar to that reported for the blowfly Calliphora. The bright zone seems to contribute to local contrast sensitivity; high contrast sensitivity exists in portions of the receptive field served by large diameter facet lenses of males and is not observed in females. Finally, temporal frequency tuning is also significantly faster in this frontal portion of the world, particularly in males, where it overcompensates for the higher spatial-frequency tuning and shifts the predicted local velocity optimum to higher speeds. These results indicate that increased retinal illuminance due to the bright zone of males is used to enhance contrast sensitivity and speed motion detector responses. Additionally, local neural properties vary across the visual world in a way not expected if HS cells serve purely as matched filters to measure yaw-induced visual motion.
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A 1.4-Mb interval RH map of horse chromosome 17 provides detailed comparison with human and mouse homologues
(Academic Press Inc Elsevier Science, 2004) Lee, E.; Raudsepp, T.; Kata, S.; Adelson, D.; Womack, J.; Skow, L.; Chowdhary, B.
Comparative genomics has served as a backbone for the rapid development of gene maps in domesticated animals. The integration of this approach with radiation hybrid (RH) analysis provides one of the most direct ways to obtain physically ordered comparative maps across evolutionarily diverged species. We herein report the development of a detailed RH and comparative map for horse chromosome 17 (ECA17). With markers distributed at an average interval of every 1.4 Mb, the map is currently the most informative among the equine chromosomes. It comprises 75 markers (56 genes and 19 microsatellites), of which 50 gene specific and 5 microsatellite markers were generated in this study and typed to our 5000-rad horse × hamster whole genome RH panel. The markers are dispersed over six RH linkage groups and span 825 cR₅₀₀₀. The map is among the most comprehensive whole chromosome comparative maps currently available for domesticated animals. It finely aligns ECA17 to human and mouse homologues (HSA13 and MMU1, 3, 5, 8, and 14, respectively) and homologues in other domesticated animals. Comparisons provide insight into their relative organization and help to identify evolutionarily conserved segments. The new ECA17 map will serve as a template for the development of clusters of BAC contigs in regions containing genes of interest. Sequencing of these regions will help to initiate studies aimed at understanding the molecular mechanisms for various diseases and inherited disorders in horse as well as human.
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A 2.5-Mb contig constructed from Angus Longhorn and horned Hereford DNA spanning the polled interval on bovine chromosome 1
(Blackwell Publishing Ltd, 2006) Wunderlich, K.; Abbey, C.; Clayton, D.; Song, Y.; Schein, J.; Georges, M.; Coppieters, W.; Adelson, D.; Taylor, J.; Davis, S.; Gill, C.
The polled locus has been mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. As an intermediate step in our efforts to identify the polled locus and the underlying causative mutation for the polled phenotype, we have constructed a BACbased physical map of the interval containing the polled locus. Clones containing genes and markers in the critical interval were isolated from the TAMBT (constructed from Angus and Longhorn genomic DNA) and CHORI-240 (constructed from horned Hereford genomic DNA) BAC libraries and ordered based on fingerprinting and the presence or absence of 80 STS markers. A single contig spanning 2.5 Mb was assembled. Comparison of the physical order of STSs to the corresponding region of human chromosome 21 revealed the same order of genes within the polled critical interval. This contig of overlapping BAC clones from horned and polled breeds is a useful resource for SNP discovery and characterization of positional candidate genes.
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A behavioural, neurochemical and proteomic analysis after treatment with MDMA and methamphetamine
(Karger, 2009) Jaehne, E.; Colella, A.; Hoffmann, P.; Irvine, R.J.; MDMA Conference (11 Sep 2008 - 13 Sep 2008 : Melbourne, Australia)
Open Access
A biolistic method for high-throughput production of transgenic wheat plants with single gene insertions
(BioMed Central, 2018) Ismagul, A.; Yang, N.; Maltseva, E.; Iskakova, G.; Mazonka, I.; Skiba, Y.; Bi, H.; Eliby, S.; Jatayev, S.; Shavrukov, Y.; Borisjuk, N.; Langridge, P.
Background: The relatively low efficiency of biolistic transformation and subsequent integration of multiple copies of the introduced gene/s significantly complicate the genetic modification of wheat (Triticum aestivum) and other plant species. One of the key factors contributing to the reproducibility of this method is the uniformity of the DNA/gold suspension, which is dependent on the coating procedure employed. It was also shown recently that the relative frequency of single copy transgene inserts could be increased through the use of nanogram quantities of the DNA during coating. Results: A simplified DNA/gold coating method was developed to produce fertile transgenic plants, via microprojectile bombardment of callus cultures induced from immature embryos. In this method, polyethyleneglycol (PEG) and magnesium salt solutions were utilized in place of the spermidine and calcium chloride of the standard coating method, to precipitate the DNA onto gold microparticles. The prepared microparticles were used to generate transgenics from callus cultures of commercial bread wheat cv. Gladius resulting in an average transformation frequency of 9.9%. To increase the occurrence of low transgene copy number events, nanogram amounts of the minimal expression cassettes containing the gene of interest and the hpt gene were used for co-transformation. A total of 1538 transgenic wheat events were generated from 15,496 embryos across 19 independent experiments. The variation of single copy insert frequencies ranged from 16.1 to 73.5% in the transgenic wheat plants, which compares favourably to published results. Conclusions: The DNA/gold coating procedure presented here allows efficient, large scale transformation of wheat. The use of nanogram amounts of vector DNA improves the frequency of single copy transgene inserts in transgenic wheat plants.
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A chemokine-like viral protein enhances alpha interferon production by plasmacytoid dendritic cells but delays CD8⁺ T cell activation and impairs viral clearance
(Amer Soc Microbiology, 2013) Wikstrom, M.; Fleming, P.; Comerford, I.; McColl, S.; Andoniou, C.; Degli-Esposti, M.
Murine cytomegalovirus encodes numerous proteins that act on a variety of pathways to modulate the innate and adaptive immune responses. Here, we demonstrate that a chemokine-like protein encoded by murine cytomegalovirus activates the early innate immune response and delays adaptive immunity, thereby impairing viral clearance. The protein, m131/129 (also known as MCK-2), is not required to establish infection in the spleen; however, a mutant virus lacking m131/129 was cleared more rapidly from this organ. In the absence of m131/129 expression, there was enhanced activation of dendritic cells (DC), and virus-specific CD8+ T cells were recruited into the immune response earlier. Viral mutants lacking m131/129 elicited weaker production of alpha interferon (IFN-α) at 40 h postinfection, indicating that this protein exerts its effects during early rounds of viral replication in the spleen. Furthermore, while wild-type and mutant viruses activated plasmacytoid dendritic cells (pDC) equally at this time, as measured by the upregulation of costimulatory molecules, the presence of m131/129 stimulated more pDC to secrete IFN-α, accounting for the stronger IFN-α response than from the wild-type virus. These data provide evidence for a novel immunomodulatory function of a viral chemokine and expose the multifunctionality of immune evasion proteins. In addition, these results broaden our understanding of the interplay between innate and adaptive immunity.
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A childhood epilepsy mutation reveals a role for developmentally regulated splicing of a sodium channel
(Academic Press Inc Elsevier Science, 2007) Xu, R.; Thomas, E.; Jenkins, M.; Gazina, E.; Chiu, C.; Heron, S.; Mulley, J.; Scheffer, I.; Berkovic, S.; Petrou, S.
Seizure susceptibility is high in human infants compared to adults, presumably because of developmentally regulated changes in neural excitability. Benign familial neonatal-infantile seizures (BFNIS), characterized by both early onset and remission, are caused by mutations in the gene encoding a human sodium channel (NaV1.2). We analyzed neonatal and adult splice forms of NaV1.2 with a BFNIS mutation (L1563V) in human embryonic kidney cells. Computer modeling revealed that neonatal channels are less excitable than adult channels. Introduction of the mutation increased excitability in the neonatal channels to a level similar to adult channels. By contrast, the mutation did not affect the adult channel variant. This "adult-like" increased excitability is likely to be the mechanism underlying BFNIS in infants with this mutation. More generally, developmentally regulated NaV1.2 splicing may be one mechanism that counters the normally high excitability of neonatal neurons and helps to reduce seizure susceptibility in normal human infants.
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A chromosome inversion near the KIT gene and the Tobiano spotting pattern in horses
(Karger, 2007) Brooks, S.; Lear, T.; Adelson, D.; Bailey, E.
Tobiano is a white spotting pattern in horses caused by a dominant gene, Tobiano(TO). Here, we report TO associated with a large paracentric chromosome inversion on horse chromosome 3. DNA sequences flanking the inversion were identified and a PCR test was developed to detect the inversion. The inversion was only found in horses with the tobiano pattern, including horses with diverse genetic backgrounds, which indicated a common genetic origin thousands of years ago. The inversion does not interrupt any annotated genes, but begins approximately 100 kb downstream of the KIT gene. This inversion may disrupt regulatory sequences for the KIT gene and cause the white spotting pattern.
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A Combination of Local Inflammation and Central Memory T Cells Potentiates Immunotherapy in the Skin
(Amer Assoc Immunologists, 2012) Fiorenza, S.; Kenna, T.; Comerford, I.; McColl, S.; Steptoe, R.; Leggatt, G.; Frazer, I.
Adoptive T cell therapy uses the specificity of the adaptive immune system to target cancer and virally infected cells. Yet the mechanism and means by which to enhance T cell function are incompletely described, especially in the skin. In this study, we use a murine model of immunotherapy to optimize cell-mediated immunity in the skin. We show that in vitro–derived central but not effector memory-like T cells bring about rapid regression of skin-expressing cognate Ag as a transgene in keratinocytes. Local inflammation induced by the TLR7 receptor agonist imiquimod subtly yet reproducibly decreases time to skin graft rejection elicited by central but not effector memory T cells in an immunodeficient mouse model. Local CCL4, a chemokine liberated by TLR7 agonism, similarly enhances central memory T cell function. In this model, IL-2 facilitates the development in vivo of effector function from central memory but not effector memory T cells. In a model of T cell tolerogenesis, we further show that adoptively transferred central but not effector memory T cells can give rise to successful cutaneous immunity, which is dependent on a local inflammatory cue in the target tissue at the time of adoptive T cell transfer. Thus, adoptive T cell therapy efficacy can be enhanced if CD8+ T cells with a central memory T cell phenotype are transferred, and IL-2 is present with contemporaneous local inflammation.
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A combined free flow electrophoresis and DIGE approach to compare proteins in complex biological samples
(Humana Press, 2012) Fung, K.Y.C.; Cursaro, C.; Lewanowitsch, T.; Cosgrove, L.; Hoffmann, P.; Kurien, B.J.; Scofield, R.H.
Free flow electrophoresis (FFE) has been applied in numerous studies as a protein separation technique due to its multiple advantages such as fast and efficient sample recovery, high resolving power, high reproducibility, and wide applicability to protein classes. As a stand-alone platform however, its utility in comparative proteomic analysis is limited as protein samples must be run sequentially rather than simultaneously which introduces inherent variability when attempting to perform quantitative analysis. Here we describe an approach combining fluorescent CyDye technology (DIGE) with FFE to simultaneously separate and identify differentially expressed proteins in a model cell system.
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A combined free-flow electrophoresis and DIGE approach to identify proteins regulated by butyrate in HT29 cells
(Wiley - V C H Verlag GmbH & Co. KGaA, 2011) Fung, K.; Cursaro, V.; Lewanowitsch, T.; Brierley, G.; McColl, S.; Lockett, T.; Head, R.; Hoffmann, P.; Cosgrove, L.
Many biologically active agents exert a pleiotropic response in cells and tissues. This presents challenges in descriptive and comparative analysis of the proteome in response to these agents. Although free-flow electrophoresis has been applied in a number of proteomic studies as a protein separation technique, the combination of free-flow electrophoresis and DIGE has not yet been investigated for comparative proteomic analysis. In this study, we have compared the effects of butyrate on HT29 colorectal cancer cells with a particular focus on apoptosis and describe the utility of a novel approach combining free-flow electrophoresis with DIGE to identify differentially expressed proteins. We verify the results obtained by the combined free-flow electrophoresis and DIGE approach with Western blot analysis of selected proteins. We also report for the first time the regulation of a number of proteins by butyrate in HT29 colorectal cells including peptidyl-prolyl cis-trans isomerase A (cyclophilin A) and profilin-1.

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