169 results on '"Rodney F. Minchin"'
Search Results
2. Trimodal distribution of arylamine N-acetyltransferase 1 mRNA in breast cancer tumors: association with overall survival and drug resistance
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Rodney F. Minchin and Neville J. Butcher
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Breast cancer ,Arylamine N-acetyltransferase ,Drug resistance ,Overall survival ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Arylamine N-acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that has been associated with cancer cell proliferation in vitro and with survival in vivo. NAT1 expression has been associated with the estrogen receptor and it has been proposed as a prognostic marker for estrogen receptor positive cancers. However, little is known about the distribution of NAT1 mRNA across an entire patient population or its effects on outcomes. To address this, gene expression data from breast cancer patient cohorts were investigated to identify sub-populations based on the level of NAT1 expression. Patient survival and drug response was examined to determine whether NAT1 mRNA levels influenced any of these parameters. Results NAT1 expression showed a trimodal distribution in breast cancer samples (n = 1980) but not in tumor tissue from ovarian, prostate, cervical or colorectal cancers. In breast cancer, NAT1 mRNA in each sub-population correlated with a separate set of genes suggesting different mechanisms of NAT1 gene regulation. Kaplan-Meier plots showed significantly better survival in patients with highest NAT1 mRNA compared to those with intermediate or low expression. While NAT1 expression was elevated in estrogen receptor-positive patients, it did not appear to be dependent on estrogen receptor expression. Overall survival was analyzed in patients receiving no treatment, hormone therapy or chemotherapy. NAT1 expression correlated strongly with survival in the first 5 years in those patients receiving chemotherapy but did not influence survival in the other two groups. This suggests that low NAT1 expression is associated with chemo-resistance. The sensitivity of NAT1 mRNA levels as a single parameter to identify non-responders to chemotherapy was 0.58 at a log(2)
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- 2018
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- View/download PDF
3. Dose-Dependent Production of Anti-PEG IgM after Intramuscular PEGylated-Hydrogenated Soy Phosphatidylcholine Liposomes, but Not Lipid Nanoparticle Formulations of DNA, Correlates with the Plasma Clearance of PEGylated Liposomal Doxorubicin in Rats
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Christopher N. Subasic, Neville J. Butcher, Rodney F. Minchin, and Lisa M. Kaminskas
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Drug Discovery ,Pharmaceutical Science ,Molecular Medicine - Published
- 2023
4. Supplementary Table 1 from Induction of Human Arylamine N-Acetyltransferase Type I by Androgens in Human Prostate Cancer Cells
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Rodney F. Minchin, Gysell M. Broadhurst, Catherine Cheung, Natasha L. Tetlow, and Neville J. Butcher
- Abstract
Supplementary Table 1 from Induction of Human Arylamine N-Acetyltransferase Type I by Androgens in Human Prostate Cancer Cells
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- 2023
5. The pharmacokinetics of PEGylated liposomal doxorubicin are not significantly affected by sex in rats or humans, but may be affected by immune dysfunction
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Gary Cowin, Lisa M. Kaminskas, Rodney F. Minchin, Esther Kuilamu, and Christopher Subasic
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Male ,medicine.medical_specialty ,Pharmaceutical Science ,Polyethylene Glycols ,Immune system ,Pharmacokinetics ,Neoplasms ,Internal medicine ,medicine ,Animals ,Humans ,Doxorubicin ,Body surface area ,Liposome ,Antibiotics, Antineoplastic ,business.industry ,Monocyte ,Rats ,medicine.anatomical_structure ,Endocrinology ,Lymphatic system ,Liposomes ,Female ,Lymph ,business ,medicine.drug - Abstract
PEGylated liposomal doxorubicin (PLD, Caelyx®, Doxil®) has been suggested to show significant sex-based differences in plasma clearance, as well as high inter-individual variability that may be driven by monocyte counts in cancer patients. This study aimed to establish if these differences are similarly observed in rats, which exhibit similar liposome clearance mechanisms to humans, and to use this model to identify sources of inter-individual and sex-based pharmacokinetic variability. The plasma and lymphatic pharmacokinetics of PLD were evaluated in male and female rats by quantifying doxorubicin as well as the 3H-labelled liposome. In general, the pharmacokinetics of doxorubicin and the 3H-liposome did not differ significantly between male and female rats when corrected for body surface area. Female rats did, however, show significantly higher doxorubicin concentrations in lymph compared to male rats. With the exception of serum testosterone concentrations in males, none of the physiological parameters evaluated correlated with plasma clearance. Further, reanalysis of published human data that formerly reported sex-differences in PLD plasma clearance similarly revealed no significant differences in PLD plasma clearance between males and females with solid tumours, but increased plasma clearance in patients with Kaposi's sarcoma (generally HIV+/immunocompromised). These data suggest that with the exception of lymphatic exposure, there are unlikely to be significant sex effects in the pharmacokinetics of liposomes, but immune function may contribute to inter individual variability.
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- 2021
6. An Automatic Image Based Single Dilution Method for End Point Titre Quantitation of Antinuclear Antibodies Tests Using HEp-2 Cells.
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Arnold Wiliem, Peter Hobson, Rodney F. Minchin, and Brian C. Lovell
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- 2011
- Full Text
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7. Automatic classification of Human Epithelial type 2 cell Indirect Immunofluorescence images using Cell Pyramid Matching.
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Arnold Wiliem, Conrad Sanderson, Yongkang Wong, Peter Hobson, Rodney F. Minchin, and Brian C. Lovell
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- 2014
- Full Text
- View/download PDF
8. A chameleonic macrocyclic peptide with drug delivery applications†
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Angela Song, Mark F. Fisher, Jingjing Zhang, Carl Eliasson, Courtney E. McAleese, Grishma Vadlamani, Colton D. Payne, Fatemeh Hajiaghaalipour, Achala S. Jayasena, Richard J. Clark, Bastian Franke, K. Johan Rosengren, Rodney F. Minchin, and Joshua S. Mylne
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0303 health sciences ,biology ,Chemistry ,Dimer ,030302 biochemistry & molecular biology ,Zinnia elegans ,General Chemistry ,biology.organism_classification ,Combinatorial chemistry ,Micelle ,Rhodamine ,03 medical and health sciences ,Macrocyclic peptide ,chemistry.chemical_compound ,Monomer ,Drug delivery ,Efflux ,030304 developmental biology - Abstract
Head-to-tail cyclized peptides are intriguing natural products with unusual properties. The PawS-Derived Peptides (PDPs) are ribosomally synthesized as part of precursors for seed storage albumins in species of the daisy family, and are post-translationally excised and cyclized during proteolytic processing. Here we report a PDP twice the typical size and with two disulfide bonds, identified from seeds of Zinnia elegans. In water, synthetic PDP-23 forms a unique dimeric structure in which two monomers containing two β-hairpins cross-clasp and enclose a hydrophobic core, creating a square prism. This dimer can be split by addition of micelles or organic solvent and in monomeric form PDP-23 adopts open or closed V-shapes, exposing different levels of hydrophobicity dependent on conditions. This chameleonic character is unusual for disulfide-rich peptides and engenders PDP-23 with potential for cell delivery and accessing novel targets. We demonstrate this by conjugating a rhodamine dye to PDP-23, creating a stable, cell-penetrating inhibitor of the P-glycoprotein drug efflux pump., The cyclic peptide PDP-23 adopts a different structure depending on conditions. In water it forms a dimer, but can unfold allowing its hydrophobic core to interact with membranes. PDP-23 shows promise as a cell penetrating scaffold for drug delivery.
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- 2021
9. Arylamine N-acetyltransferase 1 deficiency inhibits drug-induced cell death in breast cancer cells: switch from cytochrome C-dependent apoptosis to necroptosis
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Courtney E. McAleese, Neville J. Butcher, and Rodney F. Minchin
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Isoenzymes ,Cancer Research ,Caspase 8 ,Oncology ,Cell Death ,Arylamine N-Acetyltransferase ,Necroptosis ,Cytochromes c ,Humans ,Antineoplastic Agents ,Apoptosis ,Breast Neoplasms ,Female - Abstract
Purpose Arylamine N-acetyltransferase 1 (NAT1) deficiency has been associated with drug resistance and poor outcomes in breast cancer patients. The current study aimed to investigate drug resistance in vitro using normal breast cancer cell lines and NAT1-deficient cell lines to understand the changes induced by the lack of NAT1 that resulted in poor drug response. Methods The response to seven chemotherapeutic agents was quantified following NAT1 deletion using CRISPR-Cas 9 in MDA-MB-231 and T-47D cells. Apoptosis was monitored by annexin V staining and caspase 3/7 activity. Cytochrome C release and caspase 8 and 9 activities were measured by Western blots. Caspase 8 was inhibited using Z-IETD-FMK and necroptosis was inhibited using necrostatin and necrosulfonamide. Results Compared to parental cells, NAT1 depleted cells were resistant to drug treatment. This could be reversed following NAT1 rescue of the NAT1 deleted cells. Release of cytochrome C in response to treatment was decreased in the NAT1 depleted cells, suggesting suppression of the intrinsic apoptotic pathway. In addition, NAT1 knockout resulted in a decrease in caspase 8 activation. Treatment with necrosulfonamide showed that NAT1 deficient cells switched from intrinsic apoptosis to necroptosis when treated with the anti-cancer drug cisplatin. Conclusions NAT1 deficiency can switch cell death from apoptosis to necroptosis resulting in decreased response to cytotoxic drugs. The absence of NAT1 in patient tumours may be a useful biomarker for selecting alternative treatments in a subset of breast cancer patients.
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- 2022
10. Modulation of Human Arylamine N-Acetyltransferase 1 Activity by Lysine Acetylation: Role of p300/CREB-Binding Protein and Sirtuins 1 and 2
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Neville J. Butcher, Rodney F. Minchin, and Rachel Burow
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0301 basic medicine ,Pharmacology ,biology ,SIRT3 ,Chemistry ,medicine.drug_class ,Allosteric regulation ,Lysine ,Histone deacetylase inhibitor ,Arylamine N-Acetyltransferase 1 ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Biochemistry ,Acetylation ,Sirtuin ,biology.protein ,medicine ,Molecular Medicine ,CREB-binding protein ,030217 neurology & neurosurgery - Abstract
Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.
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- 2020
11. Interaction of the Brain-Selective Sulfotransferase SULT4A1 with Other Cytosolic Sulfotransferases: Effects on Protein Expression and Function
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Neville J. Butcher, Rodney F. Minchin, Deanne J. Mitchell, Richard D. Gordon, Misgana Idris, and Neelima P. Sidharthan
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Sulfotransferase ,Immunoprecipitation ,Dopamine ,Pharmaceutical Science ,Endogeny ,030226 pharmacology & pharmacy ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,medicine ,Humans ,Neurons ,Pharmacology ,biology ,Chemistry ,C-terminus ,Brain ,Gene Expression Regulation, Developmental ,RNA ,Cell Differentiation ,Arylsulfotransferase ,Cell biology ,Cytosol ,Gene Knockdown Techniques ,030220 oncology & carcinogenesis ,Chaperone (protein) ,Mutation ,biology.protein ,Protein Multimerization ,Sulfotransferases ,medicine.drug - Abstract
Sulfotransferase (SULT) 4A1 is a brain-selective sulfotransferase-like protein that has recently been shown to be essential for normal neuronal development in mice. In the present study, SULT4A1 was found to colocalize with SULT1A1/3 in human brain neurons. Using immunoprecipitation, SULT4A1 was shown to interact with both SULT1A1 and SULT1A3 when expressed in human cells. Mutation of the conserved dimerization motif located in the C terminus of the sulfotransferases prevented this interaction. Both ectopically expressed and endogenous SULT4A1 decreased SULT1A1/3 protein levels in neuronal cells, and this was also prevented by mutation of the dimerization motif. During differentiation of neuronal SH-SY5Y cells, there was a loss in SULT1A1/3 protein but an increase in SULT4A1 protein. This resulted in an increase in the toxicity of dopamine, a substrate for SULT1A3. Inhibition of SULT4A1 using small interference RNA abrogated the loss in SULT1A1/3 and reversed dopamine toxicity. These results show a reciprocal relationship between SULT4A1 and the other sulfotransferases, suggesting that it may act as a chaperone to control the expression of SULT1A1/3 in neuronal cells. SIGNIFICANCE STATEMENT: The catalytically inactive sulfotransferase (SULT) 4A1 may regulate the function of other SULTs by interacting with them via a conserved dimerization motif. In neuron-like cells, SULT4A1 is able to modulate dopamine toxicity by interacting with SULT1A3, potentially decreasing the metabolism of dopamine.
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- 2020
12. ArylamineN-Acetyltransferase 1 Regulates Expression of Matrix Metalloproteinase 9 in Breast Cancer Cells: Role of Hypoxia-Inducible Factor 1-α
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Rodney F. Minchin, Neville J. Butcher, and Pengcheng Li
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0301 basic medicine ,Pharmacology ,Messenger RNA ,Metalloproteinase ,Small interfering RNA ,Chemistry ,Arylamine N-Acetyltransferase 1 ,MMP9 ,medicine.disease ,body regions ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Breast cancer ,Transcription (biology) ,Cancer cell ,medicine ,Cancer research ,Molecular Medicine ,030217 neurology & neurosurgery - Abstract
Arylamine N-acetyltransferase 1 (NAT1) is a drug-metabolizing enzyme that influences cancer cell proliferation and survival. However, the mechanism for these effects is unknown. Because of previous observations that NAT1 inhibition decreases invasiveness, we investigated the expression of the metalloproteinase matrix metalloproteinase 9 (MMP9) in human breast cancer samples and in cancer cells. We found a negative correlation between the expression of NAT1 and MMP9 in 1904 breast cancer samples. Moreover, when NAT1 was deleted in highly invasive breast cancer cells, MMP9 mRNA and protein significantly increased, both of which were reversed by reintroducing NAT1 into the knockout cells. After NAT1 deletion, there was an increased association of acetylated histone H3 with the SET and MYND-domain containing 3 (SMYD3) element in the MMP9 promoter, consistent with an increase in MMP9 transcription. NAT1 deletion also up-regulated hypoxia-inducible factor 1-α (HIF1-α). Treatment of the NAT1 knockout cells with small interfering RNA directed toward HIF1-α mRNA inhibited the increased expression of MMP9. Taken together, these results show a direct inverse relationship between NAT1 and MMP9 and suggest that HIF1-α may be essential for the regulation of MMP9 expression by NAT1. SIGNIFICANCE STATEMENT The expression of the enzyme NAT1 was found to be negatively correlated with MMP9 expression in tumor tissue from breast cancer patients. In cells, NAT1 regulated MMP9 expression at a transcriptional level via HIF1-α. This finding is important as it may explain some of the pathological features associated with changes in NAT1 expression in cancer.
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- 2019
13. Drug formulation and nanomedicine approaches to targeting lymphatic cancer metastases
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Lili Wang, Christopher Subasic, Rodney F. Minchin, and Lisa M. Kaminskas
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Drug ,Lymphatic metastasis ,Drug Compounding ,media_common.quotation_subject ,Biomedical Engineering ,Medicine (miscellaneous) ,Cancer metastasis ,Antineoplastic Agents ,Bioengineering ,02 engineering and technology ,Development ,Lymphatic System ,03 medical and health sciences ,medicine ,Animals ,Humans ,General Materials Science ,030304 developmental biology ,media_common ,0303 health sciences ,business.industry ,Cancer ,021001 nanoscience & nanotechnology ,medicine.disease ,Nanomedicine ,Lymphatic system ,Chemotherapy Drugs ,Lymphatic Metastasis ,Drug delivery ,Cancer research ,0210 nano-technology ,business - Abstract
Lymphatic metastasis plays an important role in cancer progression and prognosis. However, conventional small-molecule chemotherapy drugs inefficiently access the lymphatic system, making the effective eradication of lymphatic metastases difficult without dose-limiting toxicity. Various formulation and nanomedicine-based approaches can be used to significantly enhance the trafficking of small-molecule, peptide and protein drugs toward the lymphatic system to enhance drug exposure at sites of lymphatic cancer growth. However, a number of obstacles exist in translating improved lymphatic exposure into improved chemotherapeutic outcomes. This review highlights the opportunities and challenges inherent in employing formulation and nanomedicinal approaches to improve chemotherapeutic drug activity within the lymphatic system and, importantly, at sites of lymphatic cancer metastasis.
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- 2019
14. Polymorphism in the human arylamine N-acetyltransferase 1 gene 3′-untranslated region determines polyadenylation signal usage
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Chandra Choudhury, Neville J. Butcher, and Rodney F. Minchin
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Isoenzymes ,Pharmacology ,MicroRNAs ,Arylamine N-Acetyltransferase ,Untranslated Regions ,Adenine ,Leukocytes, Mononuclear ,Humans ,Polyadenylation ,Biochemistry - Abstract
Human arylamine N-acetyltransferase 1 (NAT1) encodes a drug-metabolising enzyme that plays a role in chemical-associated cancer risk, cancer cell survival and mitochondrial function. Its expression and protein activity are regulated by transcriptional, translational, and post-translational processes, including microRNAs such as miR-1290. Several studies have shown the presence of multiple polyadenylation sites in the NAT1 gene. However, their role in NAT1 expression is poorly understood. Here, we have investigated the genetic sequence of the NAT1 gene in human cell lines, peripheral blood mononuclear cells and breast tumour tissue. We identified five potential polyadenylation signals, two of which carry known single nucleotide polymorphism that affect site usage. Cells that are homozygous for adenine at base 1642, the most distal polyadenylation site, use this site whereas those homozygous for cytosine at base 1642 could not. We also found that the presence of adenine at base 1642 is associated with the NAT1*10 haplotype. Because the putative binding site for miR-1290 is located between the last two polyadenylation sites, we hypothesised that cells that do not use the most distal site will be unaffected by miR-1290. However, this was not the case. NAT1 activity was positively correlated with miR-1290, and induction of miR-1290 in SH-SY5Y cells was associated with induction, not inhibition, of NAT1 activity. The use of PolyA
- Published
- 2022
15. Allosteric regulation of arylamine N-acetyltransferase 1 by adenosine triphosphate
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Neville J. Butcher, Rodney F. Minchin, K. Johan Rosengren, and Rachel Burow
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0301 basic medicine ,Allosteric modulator ,Arginine ,Arylamine N-Acetyltransferase ,Allosteric regulation ,Lysine ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,Allosteric Regulation ,medicine ,Humans ,Pharmacology ,chemistry.chemical_classification ,Dose-Response Relationship, Drug ,Adenosine ,Protein Structure, Tertiary ,Isoenzymes ,030104 developmental biology ,Enzyme ,chemistry ,Acetylation ,Adenosine triphosphate ,HeLa Cells ,medicine.drug - Abstract
In the present study, a screen of adenosine analogs as potential modulators of arylamine-N-acetyltransferase 1 activity identified ATP as an inhibitor within its range of physiological concentrations. Kinetically, ATP was a non-competitive inhibitor with respect to the acetyl acceptor but a competitive inhibitor with respect to the acetyl donor (acetyl-coenzyme A). In silico modelling predicted that ATP bound within the active site cleft arranged with the triphosphate group in close proximity to arginine 127. Since lysine 100 has previously been implicated in the binding of acetyl-coenzyme A to the enzyme, this amino acid was mutated to either an arginine or a glutamine. Both substitutions significantly changed the affinity of ATP for the enzyme, as well as the nature of the interaction to one with a large Hill coefficient (>3). Under these conditions, ATP was a strong allosteric modulator of arylamine-N-acetyltransferase 1 activity. Western blot analysis identified lysine 100 as a site of post-translational modification by acetylation. The results suggest that acetylation of lysine 100 converts arylamine-N-acetyltransferase 1 into a switch modulated by ATP. This observation provides important understanding of the molecular regulation of NAT1 activity and may reveal possible insight into the endogenous role of the enzyme.
- Published
- 2018
16. Monocytes Do Not Contribute to Sex Differences Seen in the Pharmacokinetics of Pegylated Liposomal Doxorubicin
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Lili Wang, Rodney F. Minchin, Lisa M. Kaminskas, and Neville J. Butcher
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Male ,medicine.medical_specialty ,Phagocytosis ,Population ,Pharmaceutical Science ,02 engineering and technology ,030226 pharmacology & pharmacy ,Peripheral blood mononuclear cell ,Monocytes ,Polyethylene Glycols ,03 medical and health sciences ,0302 clinical medicine ,Pharmacokinetics ,Internal medicine ,medicine ,Humans ,Doxorubicin ,education ,Testosterone ,education.field_of_study ,Sex Characteristics ,Antibiotics, Antineoplastic ,business.industry ,Monocyte ,021001 nanoscience & nanotechnology ,enzymes and coenzymes (carbohydrates) ,medicine.anatomical_structure ,Endocrinology ,lipids (amino acids, peptides, and proteins) ,Female ,0210 nano-technology ,business ,Hormone ,medicine.drug - Abstract
Pegylated liposomal doxorubicin (PLD) is widely utilised in cancer chemotherapy, but exhibits large inter-individual pharmacokinetic variability and sex differences in plasma clearance. Population pharmacokinetic modelling has suggested PLD plasma clearance correlates with peripheral monocyte counts, while sex hormones are known to affect endocytosis and phagocytosis in mononuclear cells. This study investigated whether sex hormones affect the uptake of PLD by human monocytes and macrophages in vitro. 17β-estradiol, but not testosterone, inhibited the uptake of PLD in a concentration dependent manner in undifferentiated (but not differentiated) THP-1 cells, and primary monocytes obtained from women, but not men. Effects of estradiol were only evident at very high concentrations seen during pregnancy. No differences were observed in monocyte count or monocyte subtypes between males and females. These data show that monocytes do not contribute to sex differences seen in PLD clearance in humans of reproductive age.
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- 2021
17. Cetuximab Exhibits Sex Differences in Lymphatic Exposure after Intravenous Administration in Rats in the Absence of Differences in Plasma Exposure
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Esther Kuilamu, Christopher Subasic, Lisa M. Kaminskas, Fiona Simpson, Rodney F. Minchin, and Gary Cowin
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Male ,medicine.medical_specialty ,Biodistribution ,Metabolic Clearance Rate ,Cetuximab ,Pharmaceutical Science ,Spleen ,02 engineering and technology ,030226 pharmacology & pharmacy ,Lymphatic System ,Rats, Sprague-Dawley ,03 medical and health sciences ,Antineoplastic Agents, Immunological ,Sex Factors ,0302 clinical medicine ,Pharmacokinetics ,Antigen ,Internal medicine ,medicine ,Animals ,Tissue Distribution ,Pharmacology (medical) ,Testosterone ,Pharmacology ,biology ,business.industry ,Organic Chemistry ,021001 nanoscience & nanotechnology ,Endocrinology ,medicine.anatomical_structure ,Lymphatic system ,biology.protein ,Molecular Medicine ,Administration, Intravenous ,Female ,Lymph ,Antibody ,0210 nano-technology ,business ,Biotechnology - Abstract
The aim of this work was to identify whether biochemical and physiological sources of mAb pharmacokinetic sex-effects could be identified in the rat model where target-mediated disposition is avoided. Plasma and lymphatic pharmacokinetics of the humanised anti-EGFR antibody cetuximab, along with potential physiological and biochemical drivers of pharmacokinetic sex differences, were examined in male and female rats. Cetuximab was used as a model mAb since plasma clearance is slower in female patients. When plasma concentrations were normalised to dose, female rats displayed slower plasma clearance than males, but no significant differences were observed in liver and spleen biodistribution. Sex differences in apparent plasma clearance, however, were abolished after normalisation to body weight, surface area or fat-free mass. Significant sex differences were observed in plasma testosterone, endogenous IgG and fat free mass, but did not correlate with apparent clearance. Females did, however, show two-fold higher lymphatic exposure compared to males. These data suggested that mAbs more efficiently access lymph in females, but this does not affect plasma pharmacokinetics or biodistribution. Further, the data suggest that sex differences observed in humans could be a function of antigen density.
- Published
- 2020
18. Hypoxia-mediated drug resistance in breast cancers
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Rodney F. Minchin, Courtney E. McAleese, Chandra Choudhury, and Neville J. Butcher
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0301 basic medicine ,Cancer Research ,Antineoplastic Agents ,Breast Neoplasms ,Drug resistance ,Metastasis ,03 medical and health sciences ,0302 clinical medicine ,Breast cancer ,Medicine ,Humans ,Molecular Targeted Therapy ,Transcription factor ,Tumor hypoxia ,business.industry ,Hypoxia (medical) ,medicine.disease ,Hypoxia-Inducible Factor 1, alpha Subunit ,Oxygen tension ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Oncology ,Hypoxia-inducible factors ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Cancer research ,Tumor Hypoxia ,Female ,medicine.symptom ,business - Abstract
Tissue hypoxia in solid tumors is caused by several pathological changes associated with tumor growth, including altered microvasculature structure, increased diffusional distances, and tumor-associated anemia. As the oxygen tension decreases, tumor cells adapt to the limited oxygen supply. Previous studies have shown that such adaptation leads to an aggressive phenotype that is resistant to many anti-cancer therapies. Induction of hypoxia inducible factors (HIFs) mediates many proteomic and genomic changes associated with tumor hypoxia. In breast cancers, HIFs not only predict poor prognosis, but also promote metastasis and drug resistance. Several studies have proposed HIF-1α as a druggable target in drug-resistant breast cancers, leading to the synthesis and development of small molecule inhibitors. Disappointingly, however, none of these small molecule inhibitors have progressed to clinical use. In this review, we briefly discuss the role of HIF-1α in breast cancer drug resistance and summarize the current and future approaches to targeting this transcription factor in breast cancer treatment.
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- 2020
19. Modulation of Human Arylamine
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Neville J, Butcher, Rachel, Burow, and Rodney F, Minchin
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Models, Molecular ,Niacinamide ,Arylamine N-Acetyltransferase ,Protein Conformation ,Lysine ,Acetylation ,Crystallography, X-Ray ,Hydroxamic Acids ,Transfection ,Benzoates ,CREB-Binding Protein ,Isoenzymes ,Sirtuin 2 ,Sirtuin 1 ,Acetyl Coenzyme A ,Mutagenesis, Site-Directed ,Humans ,Pyrazoles ,Pyrazolones ,E1A-Associated p300 Protein ,Nitrobenzenes ,HeLa Cells - Abstract
Arylamine
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- 2019
20. Role for human arylamine N-acetyltransferase 1 in the methionine salvage pathway
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Rodney F. Minchin, Katey Witham, and Neville J. Butcher
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0301 basic medicine ,Isomerase activity ,Homocysteine ,Arylamine N-Acetyltransferase ,Arylamine N-Acetyltransferase 1 ,Biology ,Biochemistry ,HeLa ,03 medical and health sciences ,chemistry.chemical_compound ,Methionine ,Cell Line, Tumor ,Humans ,Methionine synthase ,Pharmacology ,Gene knockdown ,biology.organism_classification ,Isoenzymes ,030104 developmental biology ,chemistry ,Gene Knockdown Techniques ,Cancer cell ,biology.protein ,Cell Division - Abstract
The Phase II drug metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) has been implicated in the growth and survival of cancer cells, although the mechanisms that underlies these effects are unknown. Here, a focused metabolomics approach was used to identify changes in folate catabolism as well as the S-adenosylmethionine (SAM) cycle following NAT1 knockdown with shRNA. Although acetylation of the folate catabolite p-aminobenzoylglutamate (pABG) was significantly decreased, there were no changes in intracellular pABG or the various components of the SAM cycle. By contrast, the flux of homocysteine in the medium was different following NAT1 knockdown after the methionine content was exhausted suggesting a need for this metabolite in methionine synthesis. Analysis of the growth of various cancer cells in methylthioadenosine-supplemented medium showed that NAT1 knockdown inhibited the methionine salvage pathway in HT-29 cells but not in HeLa or MDA-MB-436 cells. The cause of this was a low level of expression of the isomerase MRI-1 in the HT-29 cells. Knocking down both NAT1 and MRI-1 in HeLa cells with siRNA further demonstrated a redundancy between these 2 enzymes, although direct isomerase activity by NAT1 could not be demonstrated. The present study has identified a novel endogenous role for human NAT1 that might explain some of its effects in cancer cell growth and survival.
- Published
- 2017
21. Arylamine
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Pengcheng, Li, Neville J, Butcher, and Rodney F, Minchin
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Gene Expression Regulation, Neoplastic ,Isoenzymes ,Gene Knockout Techniques ,Matrix Metalloproteinase 9 ,Arylamine N-Acetyltransferase ,Cell Line, Tumor ,Humans ,Breast Neoplasms ,Female ,Hypoxia-Inducible Factor 1, alpha Subunit ,HT29 Cells ,Gene Expression Regulation, Enzymologic ,HeLa Cells - Abstract
Arylamine
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- 2019
22. Loss of human arylamine N-acetyltransferase I regulates mitochondrial function by inhibition of the pyruvate dehydrogenase complex
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Patricia Essebier, Rodney F. Minchin, Lili Wang, and Neville J. Butcher
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0301 basic medicine ,Pyruvate dehydrogenase kinase ,Arylamine N-Acetyltransferase ,Pyruvate Dehydrogenase Complex ,Oxidative phosphorylation ,Mitochondrion ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Glycolysis ,Arylamine N-acetyltransferase ,Chemistry ,Biological Transport ,Cell Biology ,Pyruvate dehydrogenase complex ,Cell biology ,Mitochondria ,Gene Expression Regulation, Neoplastic ,Isoenzymes ,030104 developmental biology ,Glucose ,030220 oncology & carcinogenesis ,Cancer cell ,Phosphorylation ,HT29 Cells ,Gene Deletion - Abstract
Human arylamine N-acetyltransferase 1 (NAT1) has been widely reported to affect cancer cell growth and survival and recent studies suggest it may alter cell metabolism. In this study, the effects of NAT1 deletion on mitochondrial function was examined in 2 human cell lines, breast carcinoma MDA-MB-231 and colon carcinoma HT-29 cells. Using a Seahorse XFe96 Flux Analyzer, NAT1 deletion was shown to decrease oxidative phosphorylation with a significant loss in respiratory reserve capacity in both cell lines. There also was a decrease in glycolysis without a change in glucose uptake. The changes in mitochondrial function was due to a decrease in pyruvate dehydrogenase activity, which could be reversed with the pyruvate dehydrogenase kinase inhibitor dichloroacetate. In the MDA-MB-231 and HT-29 cells, pyruvate dehydrogenase activity was attenuated either by an increase in phosphorylation or a decrease in total protein expression. These results may help explain some of the cellular events that have been reported recently in cell and animal models of NAT1 deficiency.
- Published
- 2019
23. The MBNL/CELF Splicing Factors Regulate Cytosolic Sulfotransferase 4A1 Protein Expression during Cell Differentiation
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Rodney F. Minchin, Misgana Idris, and Neville J. Butcher
- Subjects
Sulfotransferase ,Cellular differentiation ,Induced Pluripotent Stem Cells ,Pharmaceutical Science ,Biology ,030226 pharmacology & pharmacy ,03 medical and health sciences ,Exon ,Mice ,0302 clinical medicine ,Cell Line, Tumor ,Animals ,Humans ,RNA, Messenger ,Pharmacology ,Neurons ,Messenger RNA ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Exons ,Embryo, Mammalian ,Phenotype ,Cell biology ,Alternative Splicing ,030220 oncology & carcinogenesis ,RNA splicing ,Ectopic expression ,Female ,RNA Splicing Factors ,Sulfotransferases ,Minigene - Abstract
Sulfotransferase 4A1 (SULT4A1) is a sulfotransferase-like protein that is highly conserved between species. In human tissues, there are two transcripts, one that produces a full-length protein and one that produces an unstable truncated protein. The second transcript, which includes a pseudo-exon between exons 6 and 7 (6p), is widely expressed, whereas the first is more restricted. Differentiation of neuronal cells results in the removal of the pseudo-exon and subsequent SULT4A1 protein expression. Recent studies with SULT4A1 knockout mice showed that the protein is essential for normal development and that its absence leads to a severe neurologic phenotype. Here, the regulation of SULT4A1 6p splicing was investigated during neuronal differentiation using SH-SY5Y cells, human induced pluripotent stem cells, and mouse embryonic tissue. In all three models, pseudo-exon 6p was removed during differentiation, resulting in stable SULT4A1 protein expression. Using a minigene splicing assay, a region upstream of pseudo-exon 6p was identified that is essential for correct splicing of SULT4A1 mRNA. Within this region, there were binding motifs for four RNA processing factors (MBNL-1, MBNL-2, CELF-1, and CELF-2). Time-dependent changes in SULT4A1 protein and MBNL/CELF protein during differentiation supported their role in correctly splicing the SULT4A1 mRNA. Furthermore, ectopic expression of each factor produced efficient splicing in the minigene assay as well as correct splicing of the endogenous SULT4A1 mRNA. These results show that SULT4A1 mRNA is a target for MBNL/CELF-dependent splicing, which may be essential in producing stable, functional SULT4A1.
- Published
- 2018
24. Release of bioactive peptides from polyurethane films in vitro and in vivo: Effect of polymer composition
- Author
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Jing Zhang, Rodney F. Minchin, Trent M. Woodruff, Darren J. Martin, and Richard J. Clark
- Subjects
Male ,0301 basic medicine ,Materials science ,Biocompatibility ,Polyurethanes ,Melanoma, Experimental ,Biomedical Engineering ,Biocompatible Materials ,Peptide ,02 engineering and technology ,Peptides, Cyclic ,Biochemistry ,Biomaterials ,03 medical and health sciences ,chemistry.chemical_compound ,In vivo ,Animals ,Organic chemistry ,Amino Acid Sequence ,Molecular Biology ,Cell Proliferation ,Polyurethane ,chemistry.chemical_classification ,Temperature ,General Medicine ,Polymer ,021001 nanoscience & nanotechnology ,Small molecule ,Cyclic peptide ,Mice, Inbred C57BL ,Drug Liberation ,030104 developmental biology ,chemistry ,Drug delivery ,Biophysics ,Peptides ,0210 nano-technology ,Biotechnology - Abstract
Thermoplastic polyurethanes (TPUs) are widely used in biomedical applications due to their excellent biocompatibility. Their role as matrices for the delivery of small molecule therapeutics has been widely reported. However, very little is known about the release of bioactive peptides from this class of polymers. Here, we report the release of linear and cyclic peptides from TPUs with different hard and soft segments. Solvent casting of the TPU at room temperature mixed with the different peptides resulted in reproducible efflux profiles with no evidence of drug degradation. Peptide release was dependent on the size as well as the composition of the TPU. Tecoflex 80A (T80A) showed more extensive release than ElastEon 5-325, which correlated with a degree of hydration. It was also shown that the composition of the medium influenced the rate and extent of peptide efflux. Blending the different TPUs allowed for better control of peptide efflux, especially the initial burst effect. Peptide-loaded TPU prolonged the plasma levels of the anti-inflammatory cyclic peptide PMX53, which normally has a plasma half-life of less than 30 min. Using a blend of T80A and E5-325, therapeutic plasma levels of PMX53 were observed up to 9 days following a single intraperitoneal implantation of the drug-loaded film. PMX53 released from the blended TPUs significantly inhibited B16-F10 melanoma tumor growth in mice demonstrating its bioactivity in vivo . This study provides important findings for TPU-based therapeutic peptide delivery that could improve the pharmacological utility of peptides as therapeutics. Statement of Significance Therapeutic peptides can be highly specific and potent pharmacological agents, but are poorly absorbed and rapidly degraded in the body. This can be overcome by using a matrix that protects the peptide in vivo and promotes its slow release so that a therapeutic effect can be achieved over days or weeks. Thermoplastic polyurethanes are a versatile family of polymers that are biocompatible and used for medical implants. Here, the release of several peptides from a range of polyurethanes was shown to depend on the type of polymer used in the polyurethane. This is the first study to examine polyurethane blends for peptide delivery and shows that the rate and extent of peptide release can be fine-tuned using different hard and soft segment mixtures in the polymer.
- Published
- 2016
25. Stable non-covalent labeling of layered silicate nanoparticles for biological imaging
- Author
-
Gysell M. Mortimer, Anthony W. Musumeci, Darren J. Martin, Kevin S. Jack, and Rodney F. Minchin
- Subjects
Materials science ,Nanoparticle ,Bioengineering ,Nanotechnology ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Cell Line ,Nanomaterials ,law.invention ,Biomaterials ,chemistry.chemical_compound ,Confocal microscopy ,law ,Humans ,Cyanine ,Fluorescent Dyes ,Benzoxazoles ,Microscopy, Confocal ,Quinolinium Compounds ,Silicates ,Cationic polymerization ,021001 nanoscience & nanotechnology ,Fluorescence ,0104 chemical sciences ,chemistry ,Mechanics of Materials ,Drug delivery ,Nanoparticles ,0210 nano-technology ,Biological imaging ,HeLa Cells - Abstract
Layered silicate nanoparticles (LSN) are widely used in industrial applications and consumer products. They also have potential benefits in biomedical applications such as implantable devices and for drug delivery. To study how nanomaterials interact with cells and tissues, techniques to track and quantify their movement through different biological compartments are essential. While radiolabels can be very sensitive, particularly for in vivo studies, fluorescent labeling has been preferred in recent years because of the array of methods available to image and quantify fluorescent nanoparticles. However, labeling can be problematic, especially if it alters the physical properties of the nanomaterial. Herein is described a novel non-covalent labeling technique for LSN using readily available fluorescent dimeric cyanine dyes without the need to use excess amounts of dye to achieve labeling, or the need for removal of unbound dye. The approach utilizes the cationic binding properties of layered silicate clays and the multiple quaternary nitrogens associated with the dyes. Preparation of YOYO-1 labeled LSN with optimal dispersion in aqueous media is presented. The utilization of the labeled particles is then demonstrated in cell binding and uptake studies using flow cytometry and confocal microscopy. The labeled LSN are highly fluorescent, stable and exhibit identical physical properties with respect to the unlabeled nanoparticles. The general approach described here is applicable to other cyanine dyes and may be utilized more widely for labeling nanoparticles that comprise a crystalline plate structure with a high binding capacity.
- Published
- 2016
26. Trimodal distribution of arylamine N-acetyltransferase 1 mRNA in breast cancer tumors: association with overall survival and drug resistance
- Author
-
Neville J. Butcher and Rodney F. Minchin
- Subjects
0301 basic medicine ,lcsh:QH426-470 ,Antineoplastic Agents, Hormonal ,medicine.drug_class ,Arylamine N-Acetyltransferase ,lcsh:Biotechnology ,Estrogen receptor ,Antineoplastic Agents ,Breast Neoplasms ,Drug resistance ,Kaplan-Meier Estimate ,Biology ,03 medical and health sciences ,0302 clinical medicine ,Breast cancer ,Prostate ,lcsh:TP248.13-248.65 ,Gene expression ,Genetics ,medicine ,Humans ,Overall survival ,RNA, Messenger ,Proportional Hazards Models ,Regulation of gene expression ,Arylamine N-acetyltransferase ,medicine.disease ,3. Good health ,Isoenzymes ,lcsh:Genetics ,030104 developmental biology ,medicine.anatomical_structure ,Receptors, Estrogen ,Estrogen ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Cancer research ,Female ,Biotechnology ,Research Article - Abstract
Background Arylamine N-acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that has been associated with cancer cell proliferation in vitro and with survival in vivo. NAT1 expression has been associated with the estrogen receptor and it has been proposed as a prognostic marker for estrogen receptor positive cancers. However, little is known about the distribution of NAT1 mRNA across an entire patient population or its effects on outcomes. To address this, gene expression data from breast cancer patient cohorts were investigated to identify sub-populations based on the level of NAT1 expression. Patient survival and drug response was examined to determine whether NAT1 mRNA levels influenced any of these parameters. Results NAT1 expression showed a trimodal distribution in breast cancer samples (n = 1980) but not in tumor tissue from ovarian, prostate, cervical or colorectal cancers. In breast cancer, NAT1 mRNA in each sub-population correlated with a separate set of genes suggesting different mechanisms of NAT1 gene regulation. Kaplan-Meier plots showed significantly better survival in patients with highest NAT1 mRNA compared to those with intermediate or low expression. While NAT1 expression was elevated in estrogen receptor-positive patients, it did not appear to be dependent on estrogen receptor expression. Overall survival was analyzed in patients receiving no treatment, hormone therapy or chemotherapy. NAT1 expression correlated strongly with survival in the first 5 years in those patients receiving chemotherapy but did not influence survival in the other two groups. This suggests that low NAT1 expression is associated with chemo-resistance. The sensitivity of NAT1 mRNA levels as a single parameter to identify non-responders to chemotherapy was 0.58 at a log(2)
- Published
- 2018
27. Human Arylamine N-Acetyltransferase Type 1
- Author
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Pengcheng Li, Lili Wang, Rodney F. Minchin, and Neville J. Butcher
- Subjects
Arylamine N-acetyltransferase ,Cell growth ,Cell ,Biology ,medicine.disease ,Metastasis ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Acetylation ,Cancer cell ,medicine ,Cancer research ,Epigenetics ,Xenobiotic - Abstract
Human arylamine N-acetyltransferase 1 (NAT1) is one of the two functional NATs in humans that acetylate primary arylamine and hydrazine xenobiotics. There is a growing body of evidence that suggests NAT1 may have an important physiological role in the cell in addition to its seemingly secondary role in xenobiotic metabolism. Specifically, roles in folate homeostasis, epigenetic regulation of gene expression, cell growth and survival, and cancer cell invasion and metastasis have been proposed. This chapter discusses the current evidence linking NAT1 to physiological processes and cancer cell biology, as well as its potential as a diagnostic/prognostic biomarker and therapeutic target.
- Published
- 2018
28. Arylamine N-acetyltransferase 1 protects against reactive oxygen species during glucose starvation: Role in the regulation of p53 stability
- Author
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Lili Wang, Rodney F. Minchin, and Neville J. Butcher
- Subjects
0301 basic medicine ,Arylamine N-Acetyltransferase ,Cultured tumor cells ,lcsh:Medicine ,Apoptosis ,Biochemistry ,Metastasis ,HeLa ,Gene Knockout Techniques ,Oxidative Damage ,Basic Cancer Research ,Medicine and Health Sciences ,Small interfering RNAs ,RNA, Small Interfering ,lcsh:Science ,Multidisciplinary ,biology ,Cell Death ,Chemistry ,Organic Compounds ,Monosaccharides ,Cell biology ,Isoenzymes ,Nucleic acids ,Oncology ,Cell Processes ,Physical Sciences ,Cell lines ,Biological cultures ,HT29 Cells ,Intracellular ,Research Article ,Programmed cell death ,Blotting, Western ,Carbohydrates ,Cell Growth ,03 medical and health sciences ,DNA-binding proteins ,Genetics ,Humans ,HeLa cells ,Non-coding RNA ,Cell Proliferation ,Cell growth ,lcsh:R ,Organic Chemistry ,Chemical Compounds ,Contact inhibition ,Biology and Life Sciences ,Proteins ,Cell Biology ,biology.organism_classification ,Cell cultures ,Acetylcysteine ,Gene regulation ,Research and analysis methods ,030104 developmental biology ,Glucose ,Cancer cell ,RNA ,lcsh:Q ,Gene expression ,CRISPR-Cas Systems ,Tumor Suppressor Protein p53 ,Reactive Oxygen Species - Abstract
Human arylamine N-acetyltransferase 1 (NAT1) has been associated with cancer cell growth and invasion, but the underlying molecular mechanisms remain unknown. NAT1 is located on the short arm of chromosome 8 (8p21), a region that is commonly deleted in colon cancer. Previously, it was reported that HT-29 colon cancer cells, which have a large deletion at 8p21-22, show marked morphological changes, increased E-cadherin expression and altered cell-cell contact inhibition following down-regulation of NAT1 with shRNA. By contrast, no effects on growth were observed in HeLa cells. In the present study, cellular changes following knockout of NAT1 with CRISPR/Cas9 in HT-29 and HeLa cells were compared in the presence and absence of glucose. Cell growth decreased in both cell-lines during glucose starvation, but it was enhanced in HT-29 cells following NAT1 deletion. This was due to an increase in ROS production that induced cell apoptosis. Both ROS production and cell death were prevented by the glutathione precursor N-acetylcysteine. NAT1 knockout also resulted in a loss of the gain-of-function p53 protein in HT-29 cells. When p53 expression was inhibited with siRNA in parental HT-29 cells, ROS production and apoptosis increased to levels seen in the NAT1 knockout cells. The loss of p53 may explain the decreased colony formation and increased contact inhibition previously reported following NAT1 down-regulation in these cells. In conclusion, NAT1 is important in maintaining intracellular ROS, especially during glucose starvation, by stabilizing gain-of-function p53 in HT-29 cells. These results suggest that NAT1 may be a novel target to decrease intracellular gain-of -function p53.
- Published
- 2017
29. Fluoromica nanoparticle cytotoxicity in macrophages decreases with size and extent of uptake
- Author
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Gysell M. Mortimer, Darren J. Martin, Rodney F. Minchin, Yingdong Zhu, and Nicolin Tee
- Subjects
Materials science ,Biocompatibility ,media_common.quotation_subject ,Biophysics ,Pharmaceutical Science ,Nanoparticle ,Bioengineering ,Nanotechnology ,Protein Corona ,Apoptosis ,Biomaterials ,high energy milling ,Mice ,In vivo ,International Journal of Nanomedicine ,Cell Line, Tumor ,Drug Discovery ,Macrophage ,Animals ,Humans ,Scavenger receptor ,Cytotoxicity ,Internalization ,media_common ,Original Research ,Fluorescent Dyes ,Macrophages ,Organic Chemistry ,phagocytosis ,General Medicine ,RAW 264.7 Cells ,layered silicates ,Nanoparticles ,accumulation - Abstract
Nicolin Tee,1 Yingdong Zhu,2 GysellM Mortimer,1 Darren J Martin,2 Rodney F Minchin11School of Biomedical Science, University of Queensland, Brisbane, QLD, Australia; 2Australian Institute ofBioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, AustraliaAbstract: Polyurethanes are widely used in biomedical devices such as heart valves, pacemaker leads, catheters, vascular devices, and surgical dressings because of their excellent mechanical properties and good biocompatibility. Layered silicate nanoparticles can significantly increase tensile strength and breaking strain of polyurethanes potentially increasing the life span of biomedical devices that suffer from wear in vivo. However, very little is known about how these nanoparticles interact with proteins and cells and how they might exert unwanted effects. A series of fluoromica nanoparticles ranging in platelet size from 90 to over 600 nm in diameter were generated from the same base material ME100 by high energy milling and differential centrifugation. The cytotoxicity of the resulting particles was dependent on platelet size but in a manner that is opposite to many other types of nanomaterials. For the fluoromicas, the smaller the platelet size, the less toxicity was observed. The small fluoromica nanoparticles (
- Published
- 2015
30. Effects of human arylamine N ‐acetyltransferase I knockdown in triple‐negative breast cancer cell lines
- Author
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Rodney F. Minchin, Jacky M. Tiang, and Neville J. Butcher
- Subjects
Cancer Research ,Lung Neoplasms ,Arylamine N-Acetyltransferase ,MDA-MB-231 ,Mice, Nude ,Triple Negative Breast Neoplasms ,Vimentin ,Biology ,Metastasis ,Small hairpin RNA ,Transduction, Genetic ,Cell Line, Tumor ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,metastasis ,Neoplasm Invasiveness ,Radiology, Nuclear Medicine and imaging ,Pseudopodia ,skin and connective tissue diseases ,beta Catenin ,Triple-negative breast cancer ,Cancer Biology ,Original Research ,Filopodia ,Mice, Inbred BALB C ,Cell growth ,Lentivirus ,Cancer ,Cadherins ,invasion ,medicine.disease ,Molecular biology ,3. Good health ,Isoenzymes ,Oncology ,N-acetyltransferase ,Cell culture ,Gene Knockdown Techniques ,Cancer research ,biology.protein ,Female ,Snail Family Transcription Factors ,Neoplasm Transplantation ,Transcription Factors - Abstract
Expression of human arylamine N-acetyltransferase I (NAT1) has been associated with various cancer subtypes and inhibition of this enzyme with small molecule inhibitors or siRNA affects cell growth and survival. Here, we have investigated the role of NAT1 in the invasiveness of breast cancer cells both in vitro and in vivo. We knocked down NAT1 using a lentivirus-based shRNA approach and observed marked changes in cell morphology in the triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-436, and BT-549. Most notable was a reduction in the number and size of the filopodia protrusions on the surface of the cells. The loss of filopodia could be rescued by the reintroduction of NAT1 into the knockdown cells. NAT1 expression was localized to the lamellipodia and extended into the filopodia protrusions. In vitro invasion through Geltrex was significantly inhibited in both the MDA cell lines but not in the BT-549 cells. The expression of Snail increased when NAT1 was knocked down, while other genes associated with mesenchymal to epithelial transition (vimentin, cytokeratin-18, and Twist) did not show any changes. By contrast, both N-cadherin and β-catenin were significantly reduced. When MDA-MB-231 cells expressing shRNA were injected in vivo into BALB/c nu/nu nude mice, a significant reduction in the number of colonies that formed in the lungs was observed. Taken together, the results show that NAT1 can alter the invasion and metastatic properties of some triple-negative breast cancer cells but not all. The study suggests that NAT1 may be a novel therapeutic target in a subset of breast cancers.
- Published
- 2015
31. A bag of cells approach for antinuclear antibodies HEp-2 image classification
- Author
-
Arnold Wiliem, Peter Hobson, Rodney F. Minchin, and Brian C. Lovell
- Subjects
Histology ,Contextual image classification ,Computer science ,business.industry ,Inference ,Pattern recognition ,CAD ,Cell Biology ,Pathology and Forensic Medicine ,Set (abstract data type) ,Bag-of-words model in computer vision ,Bag-of-words model ,Computer-aided diagnosis ,Histogram ,Artificial intelligence ,business - Abstract
The antinuclear antibody (ANA) test via indirect immunofluorescence applied on Human Epithelial type 2 (HEp-2) cells is a pathology test commonly used to identify connective tissue diseases (CTDs). Despite its effectiveness, the test is still considered labor intensive and time consuming. Applying image-based computer aided diagnosis (CAD) systems is one of the possible ways to address these issues. Ideally, a CAD system should be able to classify ANA HEp-2 images taken by a camera fitted to a fluorescence microscope. Unfortunately, most prior works have primarily focused on the HEp-2 cell image classification problem which is one of the early essential steps in the system pipeline. In this work we directly tackle the specimen image classification problem. We aim to develop a system that can be easily scaled and has competitive accuracy. ANA HEp-2 images or ANA images are generally comprised of a number of cells. Patterns exhibiting in the cells are then used to make inference on the ANA image pattern. To that end, we adapted a popular approach for general image classification problems, namely a bag of visual words approach. Each specimen is considered as a visual document containing visual vocabularies represented by its cells. A specimen image is then represented by a histogram of visual vocabulary occurrences. We name this approach as the Bag of Cells approach. We studied the performance of the proposed approach on a set of images taken from 262 ANA positive patient sera. The results show the proposed approach has competitive performance compared to the recent state-of-the-art approaches. Our proposal can also be expanded to other tests involving examining patterns of human cells to make inferences.
- Published
- 2014
32. Nanotoxicology and Nanovaccines
- Author
-
Gysell M. Mortimer and Rodney F. Minchin
- Subjects
0301 basic medicine ,business.industry ,Pseudoallergy ,02 engineering and technology ,Pharmacology ,021001 nanoscience & nanotechnology ,medicine.disease ,Bioinformatics ,03 medical and health sciences ,030104 developmental biology ,Immune system ,Human use ,Nanotoxicology ,Toxicity ,Medicine ,0210 nano-technology ,business ,Adverse effect - Abstract
Toxicity elicited by nanoparticles has been extensively studied, especially for those nanomaterials designed for human use. Safety concerns with nanomedicines have led to a better understanding of the molecular and cellular events that occur when nanoparticles enter biological systems. The key adverse effects include oxidative stress, inflammatory reactions, and genotoxicity. There are now a number of clinical trials using various nanoparticle formulations that show trends in the type and severity of unwanted effects that might be expected with nanomedicines. One of the major concern is the propensity for nanoparticles to activate the immune system, resulting in minor side effects such as local inflammation and more serious side effects including complement activation-related pseudoallergy to life-threatening side effects such as hypercytokinemia. Many adverse effects are observed in only a subgroup of patients, which raises the important question of how these individuals may be identified before treatment. For nanovaccines there is the potential of guiding the development of new treatments by understanding how adverse effects are elicited and how they may be avoided.
- Published
- 2017
33. Cryptic Epitopes of Albumin Determine Mononuclear Phagocyte System Clearance of Nanomaterials
- Author
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Neville J. Butcher, Rodney F. Minchin, Darren J. Martin, Zhou J. Deng, Gysell M. Mortimer, and Anthony W. Musumeci
- Subjects
Models, Molecular ,biology ,Protein Conformation ,Chemistry ,Macrophages ,Silicates ,General Engineering ,Albumin ,Serum albumin ,General Physics and Astronomy ,Biological Transport ,Protein Corona ,Mononuclear phagocyte system ,Blood proteins ,Epitope ,Cell Line ,Epitopes ,Biochemistry ,biology.protein ,Humans ,Nanoparticles ,General Materials Science ,Scavenger receptor ,Receptor ,Serum Albumin - Abstract
While plasma proteins can influence the physicochemical properties of nanoparticles, the adsorption of protein to the surface of nanomaterials can also alter the structure and function of the protein. Here, we show that plasma proteins form a hard corona around synthetic layered silicate nanoparticles (LSN) and that one of the principle proteins is serum albumin. The protein corona was required for recognition of the nanoparticles by scavenger receptors, a major receptor family associated with the mononuclear phagocyte system (MPS). Albumin alone could direct nanoparticle uptake by human macrophages, which involved class A but not class B scavenger receptors. Upon binding to LSN, albumin unfolded to reveal a cryptic epitope that could also be exposed by heat denaturation. This work provides an understanding of how albumin, and possibly other proteins, can promote nanomaterial recognition by the MPS without albumin requiring chemical modification for scavenger receptor recognition. These findings also demonstrate an additional function for albumin in vivo.
- Published
- 2014
34. Implementation of a Virtual Laboratory Practical Class (VLPC) module in pharmacology education
- Author
-
Steven Chen, Mary-Louise Manchadi, Matthew J. Cheesman, Teague Jacob, Peter A. Tregloan, and Rodney F. Minchin
- Subjects
Class (computer programming) ,Resource (project management) ,education ,Virtual Laboratory ,Minor (academic) ,Pharmacology ,Completion time ,Laboratory experiment ,Psychology ,Session (web analytics) ,Task (project management) - Abstract
The Virtual Laboratory Practical Class (VLPC) computer program is a resource aimed at improving practical laboratory performance and understanding in a second-year pharmacology student cohort. In our evaluation of the program, half of the 233 students were provided access to the VLPC before their laboratory session and half after the completion of their experiment. Surveys were conducted to gauge student perception and understanding of the laboratory experiment. In addition, data measuring student completion times for the laboratory task were collected. Students who used the VLPC before the class reported an increase in their confidence in successfully completing the live practical experiment. They also showed a significant decrease in the mean completion time for the real laboratory session. No effect of the VLPC on assignment scores was observed, however this is likely due to the content of the program, which comprised only a very minor portion of these assessable laboratory reports. While some operational issues affected students’ use and engagement in the early stages of the project, students reported benefits to their practical skills and understanding, and perceived the VLPC as a realistic and useful depiction of the laboratory environment.
- Published
- 2014
35. Regulation of MMP9 expression by human arylamine N-acetyltransferase (NAT1) via Hif-1a
- Author
-
Pengcheng Li, Neville J. Butcher, and Rodney F. Minchin
- Subjects
Oncology ,Arylamine N-acetyltransferase ,business.industry ,Medicine ,Hematology ,MMP9 ,business ,Molecular biology - Published
- 2019
36. Interaction of Human Arylamine N-Acetyltransferase 1 with Different Nanomaterials
- Author
-
Zhongfan Jia, Neville J. Butcher, Zhou J. Deng, Michael J. Monteiro, Rodney F. Minchin, Gysell M. Mortimer, and Darren J. Martin
- Subjects
Models, Molecular ,Protein Denaturation ,Hot Temperature ,Arylamine N-Acetyltransferase ,Polymers ,Surface Properties ,Silicon dioxide ,Pharmaceutical Science ,Nanoparticle ,Arylamine N-Acetyltransferase 1 ,Nanotechnology ,Protein Corona ,Nanomaterials ,chemistry.chemical_compound ,Microscopy, Electron, Transmission ,Humans ,Particle Size ,Titanium ,Pharmacology ,Binding Sites ,biology ,Chemistry ,Silicon Dioxide ,Enzyme assay ,Isoenzymes ,Titanium dioxide ,biology.protein ,Biophysics ,Nanoparticles ,Hydroxide ,Zinc Oxide - Abstract
Humans are exposed to nanoparticles in the environment as well as those in nanomaterials developed for biomedical applications. However, the safety and biologic effects of many nanoparticles remain to be elucidated. Over the past decade, our understanding of the interaction of proteins with various nanomaterials has grown. The protein corona can determine not only how nanoparticles interact with cells but also their biologic effects and toxicity. In this study, we describe the effects that several different classes of nanoparticles exert on the enzymatic activity of the cytosolic protein human arylamine N-acetyltransferase 1 (NAT1), a drug-metabolizing enzyme widely distributed in the body that is also responsible for the activation and detoxification of known carcinogens. We investigated three metal oxides (zinc oxide, titanium dioxide, and silicon dioxide), two synthetic clay nanoparticles (layered double hydroxide and layered silicate nanoparticles), and a self-assembling thermo-responsive polymeric nanoparticle that differ in size and surface characteristics. We found that the different nanoparticles induced very different responses, ranging from inhibition to marked enhancement of enzyme activity. The layered silicates did not directly inactivate NAT1, but was found to enhance substrate-dependent inhibition. These differing effects demonstrate the multiplicity of nanoparticle-protein interactions and suggest that enzyme activity may be compromised in organs exposed to nanoparticles, such as the lungs or reticulo-endothelial system.
- Published
- 2013
37. Effect of Supercritical Carbon Dioxide on the Loading and Release of Model Drugs from Polyurethane Films: Comparison with Solvent Casting
- Author
-
Jing Zhang, Darren J. Martin, Kristofer J. Thurecht, Elena Taran, and Rodney F. Minchin
- Subjects
chemistry.chemical_classification ,Materials science ,Thermoplastic ,Supercritical carbon dioxide ,Polymers and Plastics ,Biocompatibility ,Organic Chemistry ,Condensed Matter Physics ,Elastomer ,Supercritical fluid ,Solvent ,chemistry.chemical_compound ,chemistry ,Chemical engineering ,Polymer chemistry ,Materials Chemistry ,Rhodamine B ,Organic chemistry ,Physical and Theoretical Chemistry ,Polyurethane - Abstract
Thermoplastic polyurethanes (TPUs) are widely used in biomedical applications because of their excellent mechanical properties and biocompatibility. However, their role as possible matrices for drug delivery has received little attention. In this study, the loading and release of model drugs from different TPUs using supercritical CO 2 (scCO 2 ) as an inert and convenient solvent are investigated. The effects of scCO 2 treatment on the physical properties of ElastEon TPU films are assessed. The results indicate subtle effects on the microphase morphology of the TPU without significant changes in its mechanical properties. Model drugs (rhodamine B, fluorescein, and 7-hydroxycoumain) are readily loaded into the films and show homogeneous impregnation across the thickness of the film. The loading capacity under the conditions used in the present study varies from less than 1% for rhodamine B to approximately 4% for 7-hydroxycoumarin. The release kinetics not only vary between the different drugs, but are also altered by changes in the composition of the hard and soft segments of the TPUs. These results demonstrate the potential of scCO 2 treatment in preparing drug-loaded polyurethanes used in medical devices. This information may be helpful in designing TPUs with different drug-release profiles for a variety of disease treatments.
- Published
- 2013
38. Cytosolic Sulfotransferase 1A3 Is Induced by Dopamine and Protects Neuronal Cells from Dopamine Toxicity
- Author
-
Neelima P. Sidharthan, Rodney F. Minchin, and Neville J. Butcher
- Subjects
Sulfotransferase ,Dopaminergic ,Neurotoxicity ,Cell Biology ,Biology ,medicine.disease ,Biochemistry ,Cell biology ,Dopamine receptor D1 ,Dopamine receptor ,Dopamine receptor D3 ,Dopamine ,Dopamine receptor D2 ,medicine ,Molecular Biology ,medicine.drug - Abstract
Dopamine neurotoxicity is associated with several neurodegenerative diseases, and neurons utilize several mechanisms, including uptake and metabolism, to protect them from injury. Metabolism of dopamine involves three enzymes: monoamine oxidase, catechol O-methyltransferase, and sulfotransferase. In primates but not lower order animals, a sulfotransferase (SULT1A3) is present that can rapidly metabolize dopamine to dopamine sulfate. Here, we show that SULT1A3 and a closely related protein SULT1A1 are highly inducible by dopamine. This involves activation of the D1 and NMDA receptors. Both ERK1/2 phosphorylation and calcineurin activation are required for induction. Pharmacological agents that inhibited induction or siRNA targeting SULT1A3 significantly increased the susceptibility of cells to dopamine toxicity. Taken together, these results show that dopamine can induce its own metabolism and protect neuron-like cells from damage, suggesting that SULT1A3 activity may be a risk factor for dopamine-dependent neurodegenerative diseases.
- Published
- 2013
39. Cryptic epitopes and functional diversity in extracellular proteins
- Author
-
Gysell M. Mortimer and Rodney F. Minchin
- Subjects
0301 basic medicine ,Extracellular Matrix Proteins ,030102 biochemistry & molecular biology ,Extracellular proteins ,Proteins ,Cell Biology ,Biology ,Cleavage (embryo) ,Biochemistry ,Epitope ,Cell biology ,Evolution, Molecular ,03 medical and health sciences ,Functional diversity ,Epitopes ,030104 developmental biology ,Protein structure ,Unfolded protein response ,Animals ,Humans ,Denaturation (biochemistry) ,Extracellular Space ,Conserved Sequence - Abstract
The functional diversity of proteins is a major factor determining the complexity of cells and tissues. Both translational and post-translational modifications contribute to this diversity. Recently, protein unfolding and refolding has been recognised as another mechanism for diversity by unmasking buried or cryptic sequences (epitopes) that possess physiological functions. In the current review, we focus on extracellular proteins where folding dynamics can be influenced by mechanical forces, protein-protein interactions and denaturation. Many cryptic epitopes in these proteins are exposed following proteolytic cleavage, but recent data indicate that unfolding/refolding play an important role in regulating the physiological behaviour of extracellular proteins. By understanding how and when hidden sequences are exposed, novel techniques for manipulating the function of these proteins may be uncovered.
- Published
- 2016
40. Sulfotransferase 1A3/4 copy number variation is associated with neurodegenerative disease
- Author
-
Neville J. Butcher, George D. Mellick, Malcolm K. Horne, Christopher Fowler, Colin L. Masters, and Rodney F. Minchin
- Subjects
0301 basic medicine ,Adult ,Male ,DNA Copy Number Variations ,Disease ,Biology ,Bioinformatics ,03 medical and health sciences ,0302 clinical medicine ,Alzheimer Disease ,Chromosome instability ,Genetics ,medicine ,Humans ,Copy-number variation ,Gene ,Genetic Association Studies ,Aged ,Pharmacology ,Aged, 80 and over ,Aryl sulfotransferase ,Chromosome ,Neurodegenerative Diseases ,Parkinson Disease ,Middle Aged ,medicine.disease ,Arylsulfotransferase ,030104 developmental biology ,Molecular Medicine ,Female ,Alzheimer's disease ,030217 neurology & neurosurgery ,Pharmacogenetics - Abstract
The cytosolic aryl sulfotransferase genes SULT1A3 and SULT1A4 are located on chromosome 16p11.2 in a region of chromosomal instability. SULT1A3/4 are important enzymes in the metabolism of catecholamines linked to neurodegenerative diseases such as Parkinson’s and Alzheimer’s. In the present study, copy number variation of the SULT1A3/4 genes in healthy individuals, as well as a cohort of Parkinson’s disease and Alzheimer’s disease patients was examined. In all subjects, SULT1A3/4 copy number varied from 1 to 10. In Alzheimer’s disease patients, there was a significantly lower copy number compared to controls, and a positive correlation between copy number and age of disease onset. By contrast, there were no differences in Parkinson’s disease patients. However, when early-onset Parkinson’s disease was evaluated separately, there appeared to be an association with gene copy number and risk. The current study shows that these neurodegenerative diseases may be related to SULT1A3/4 copy number.
- Published
- 2016
41. Protein corona formation in bronchoalveolar fluid enhances diesel exhaust nanoparticle uptake and pro-inflammatory responses in macrophages
- Author
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Shea P. Connell, Zhou J. Deng, David E. Newby, Gysell M. Mortimer, Rodger Duffin, Rodney F. Minchin, Mark R. Miller, Catherine A Shaw, Edwin Carter, and Patrick W. F. Hadoke
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,endocrine system ,Materials science ,Surface Properties ,Biomedical Engineering ,Nanoparticle ,Protein Corona ,02 engineering and technology ,Plasma protein binding ,Toxicology ,Cell Line ,03 medical and health sciences ,Pulmonary surfactant ,In vivo ,medicine ,Humans ,Particle Size ,Lung ,Vehicle Emissions ,Macrophages ,Interleukin-8 ,Albumin ,Biological activity ,Blood Proteins ,021001 nanoscience & nanotechnology ,030104 developmental biology ,Nanotoxicology ,Leukocytes, Mononuclear ,Biophysics ,Nanoparticles ,0210 nano-technology ,Bronchoalveolar Lavage Fluid ,Protein Binding - Abstract
In biological fluids nanoparticles bind a range of molecules, particularly proteins, on their surface. The resulting protein corona influences biological activity and fate of nanoparticle in vivo. Corona composition is often determined by the biological milieu encountered at the entry portal into the body, and, can therefore, depend on the route of exposure to the nanoparticle. For environmental nanoparticles where exposure is by inhalation, this will be lung lining fluid. This study examined plasma and bronchoalveolar fluid (BALF) protein binding to engineered and environmental nanoparticles. We hypothesized that protein corona on nanoparticles would influence nanoparticle uptake and subsequent pro-inflammatory biological response in macrophages. All nanoparticles bound plasma and BALF proteins, but the profile of bound proteins varied between nanoparticles. Focusing on diesel exhaust nanoparticles (DENP), we identified proteins bound from plasma to include fibrinogen, and those bound from BALF to include albumin and surfactant proteins A and D. The presence on DENP of a plasma-derived corona or one of purified fibrinogen failed to evoke an inflammatory response in macrophages. However, coronae formed in BALF increased DENP uptake into macrophages two fold, and increased nanoparticulate carbon black (NanoCB) uptake fivefold. Furthermore, a BALF-derived corona increased IL-8 release from macrophages in response to DENP from 1720 ± 850 pg/mL to 5560 ± 1380 pg/mL (p = 0.014). These results demonstrate that the unique protein corona formed on nanoparticles plays an important role in determining biological reactivity and fate of nanoparticle in vivo. Importantly, these findings have implications for the mechanism of detrimental properties of environmental nanoparticles since the principle route of exposure to such particles is via the lung.
- Published
- 2016
42. Drug delivery: Unravelling the stealth effect
- Author
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Neville J, Butcher, Gysell M, Mortimer, and Rodney F, Minchin
- Subjects
Drug Carriers ,Drug Delivery Systems ,Liposomes ,Humans - Published
- 2016
43. Plasma protein binding of positively and negatively charged polymer-coated gold nanoparticles elicits different biological responses
- Author
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Michael J. Monteiro, Rodney F. Minchin, Mingtao Liang, Istvan Toth, and Zhou J. Deng
- Subjects
Materials science ,Polymers ,Static Electricity ,Biomedical Engineering ,Fibrinogen ,Metal Nanoparticles ,Nanoparticle ,Protein Corona ,Blood Proteins ,Plasma protein binding ,Hydrogen-Ion Concentration ,Toxicology ,Receptor–ligand kinetics ,Electrophoresis ,Biochemistry ,Colloidal gold ,Cell Line, Tumor ,Static electricity ,Biophysics ,Humans ,Electrophoresis, Gel, Two-Dimensional ,Gold ,Surface charge ,Particle Size ,Protein Binding - Abstract
The binding of proteins to nanoparticles is an important event that can determine the biological effect of nanoparticles in the body. We examined plasma protein binding to gold nanoparticles (5-20 nm) with different surface charge. Positively and negatively charged nanoparticles bound a range of proteins whereas neutral nanoparticle bound very little. As little as 25% neutral polymer on the surface of the charged nanoparticles inhibited protein binding, with only slight change in surface charge. Fibrinogen bound with high affinity to both of the charged nanoparticles. However, binding kinetics and protease digestion suggested that the binding orientation for each nanoparticle was different. Only the negatively charged nanoparticles induced cytokine release from THP-1 cells. While common proteins can bind to different nanoparticles, the biological outcome may not be the same. Consequently, knowledge about the composition of the protein corona is not sufficient to predict biological effects of nanoparticles.
- Published
- 2012
44. ArylamineN-Acetyltransferase 1: A Novel Drug Target in Cancer Development
- Author
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Rodney F. Minchin and Neville J. Butcher
- Subjects
Arylamine N-Acetyltransferase ,Protein Conformation ,Down-Regulation ,Antineoplastic Agents ,Arylamine N-Acetyltransferase 1 ,Biology ,Gene Expression Regulation, Enzymologic ,Epigenesis, Genetic ,Substrate Specificity ,Small Molecule Libraries ,Folic Acid ,Neoplasms ,Drug Discovery ,Genotype ,medicine ,Animals ,Humans ,Gene ,Pharmacology ,Genetics ,Molecular Structure ,Arylamine N-acetyltransferase ,Drug discovery ,Cancer ,medicine.disease ,Isoenzymes ,Drug development ,Cancer cell ,Molecular Medicine - Abstract
The human arylamine N-acetyltransferases first attracted attention because of their role in drug metabolism. However, much of the current literature has focused on their role in the activation and detoxification of environmental carcinogens and how genetic polymorphisms in the genes create predispositions to increased or decreased cancer risk. There are two closely related genes on chromosome 8 that encode the two human arylamine N-acetyltransferases--NAT1 and NAT2. Although NAT2 has restricted tissue expression, NAT1 is found in almost all tissues of the body. There are several single-nucleotide polymorphisms in the protein coding and 3'-untranslated regions of the gene that affect enzyme activity. However, NAT1 is also regulated by post-translational and environmental factors, which may be of greater importance than genotype in determining tissue NAT1 activities. Recent studies have suggested a novel role for this enzyme in cancer cell growth. NAT1 is up-regulated in several cancer types, and overexpression can lead to increased survival and resistance to chemotherapy. Although a link to folate homeostasis has been suggested, many of the effects attributed to NAT1 and cancer cell growth remain to be explained. Nevertheless, the enzyme has emerged as a viable candidate for drug development, which should lead to small molecule inhibitors for preclinical and clinical evaluation.
- Published
- 2011
45. Phosphorylation/dephosphorylation of human SULT4A1: Role of Erk1 and PP2A
- Author
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Rodney F. Minchin, Deanne J. Mitchell, and Neville J. Butcher
- Subjects
Threonine ,Sulfotransferase ,Immunoprecipitation ,Protein subunit ,Blotting, Western ,Biology ,PC12 Cells ,ERK1 ,Dephosphorylation ,03 medical and health sciences ,0302 clinical medicine ,Two-Hybrid System Techniques ,Animals ,Humans ,Protein Phosphatase 2 ,Phosphorylation ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Mitogen-Activated Protein Kinase 3 ,Kinase ,Protein phosphatase 2 ,Cell Biology ,Peptidylprolyl Isomerase ,PP2A ,Rats ,NIMA-Interacting Peptidylprolyl Isomerase ,Biochemistry ,PIN1 ,Sulfotransferases ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery ,HeLa Cells ,Protein Binding - Abstract
SULT4A1 is a cytosolic sulfotransferase that shares little homology with other human sulfotransferases but is highly conserved between species. It is found in neurons located in several regions of the brain. Recently, the stability of SULT4A1 was shown to be regulated by Pin1, a peptidyl-prolyl cis-trans isomerase implicated in several neurodegenerative diseases. Since Pin1 binds preferentially to phosphoproteins, these findings suggested that SULT4A1 is post-translationally modified. In this study, we show that the Thr11 residue of SULT4A1, which is involved in Pin1 binding is phosphorylated. MEK inhibition was shown to abolish Pin1 mediated degradation of SULT4A1 while in vitro phosphorylation assays using alanine substitution mutants of SULT4A1 demonstrated phosphorylation of Thr11 by ERK1. We also show that dephosphorylation was catalyzed by the protein phosphatase 2A. The PP2A regulatory subunit, Bβ was identified from a yeast-2-hybrid screen of human brain cDNA as a SULT4A1 interacting protein. This was further confirmed by GST pull-downs and immunoprecipitation. Other members of the B subunit (αδγ) did not interact with SULT4A1. Taken together, these studies indicate that SULT4A1 stability is regulated by post-translational modification that involves the ERK pathway and PP2A. The phosphorylation of SULT4A1 allows interaction with Pin1, which then promotes degradation of the sulfotransferase.
- Published
- 2011
- Full Text
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46. Histone Deacetylase Inhibitors Increase Human Arylamine N-Acetyltransferase-1 Expression in Human Tumor Cells
- Author
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Kok L Sin, Neville J. Butcher, Jacky M. Tiang, Rodney F. Minchin, and Scott Paterson
- Subjects
Transcription, Genetic ,Arylamine N-Acetyltransferase ,Sp1 Transcription Factor ,medicine.drug_class ,Pharmaceutical Science ,Hydroxamic Acids ,Gene Expression Regulation, Enzymologic ,Histones ,Tumor Cells, Cultured ,medicine ,Humans ,Promoter Regions, Genetic ,Pharmacology ,Histone deacetylase 5 ,biology ,Histone deacetylase 2 ,Histone deacetylase inhibitor ,Acetylation ,HDAC4 ,Chromatin ,Up-Regulation ,Cell biology ,Histone Deacetylase Inhibitors ,Isoenzymes ,Histone ,Trichostatin A ,Cancer research ,biology.protein ,Histone deacetylase ,HeLa Cells ,Transcription Factors ,medicine.drug - Abstract
Arylamine N-acetyltransferase-1 (NAT1) has been associated with disorders involving folate metabolism, such as spina bifida, as well as numerous human cancers. As a result, the transcriptional and post-transcriptional regulation of NAT1 activity has been extensively studied. However, little work has been reported on the epigenetic control of NAT1 expression. Here, we demonstrate that the histone deacetylase inhibitor trichostatin A (TSA) increases NAT1 activity in human cancer cells by increasing transcription from the proximal promoter NATb. A specific Sp1 binding site was identified as essential for optimal induction of NAT1 by TSA. However, TSA did not increase the expression of Sp1 in HeLa cells. Instead, TSA increased the acetylation of histones associated with the NATb promoter. This allowed recruitment of Sp1 to the promoter along with acetylated histones. We propose that NAT1 transcription is partially repressed by the local chromatin condensation in the vicinity of NATb and that histone deacetylase inhibition leads to up-regulation of NAT1 expression via a direct change in chromatin conformation.
- Published
- 2010
47. Endosomal disruptors in non-viral gene delivery
- Author
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Rodney F. Minchin and Shu Yang
- Subjects
Genetics ,Endosome ,Genetic enhancement ,Transgene ,Genetic Vectors ,Gene Expression ,Pharmaceutical Science ,Apoptosis ,Endosomes ,Genetic Therapy ,Gene delivery ,Biology ,Endocytosis ,Cell biology ,Gene expression ,Humans ,Transgenes ,Enhancer ,Gene ,HeLa Cells - Abstract
Non-viral gene delivery for the treatment of genetic and non-genetic diseases has been under investigation for several decades, but there has been very little application in patients because of poor gene expression and toxicity.As gene delivery almost invariably involves endocytosis, many of its limitations are related to compartmentalisation of the transgene within the endosomes. Gene expression enhancers have become an essential part of manipulating endosomal release, as well as protecting transgene from intracellular degradation. However, disruption of the endosomes can also release proteases that have been shown to activate apoptotic pathways.An understanding of the role that endosomal release plays in the toxicity of gene delivery vehicles will help identify new approaches to minimise adverse effects while enhancing non-viral gene expression.The future of non-viral gene therapy needs to identify new approaches that limit endosome-induced toxicity while enhancing expression so that a pharmacological response can be reliably observed in vivo.
- Published
- 2010
48. Fluorescent layered double hydroxide nanoparticles for biological studies
- Author
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Anthony W. Musumeci, Zhi Ping Xu, Darren J. Martin, Margaret K. Butler, Gysell M. Mortimer, and Rodney F. Minchin
- Subjects
Inorganic chemistry ,Size-exclusion chromatography ,Nanoparticle ,Geology ,Fluorescence ,Nanomaterials ,chemistry.chemical_compound ,chemistry ,Chemical engineering ,Geochemistry and Petrology ,Hydroxide ,Molecule ,Fluorescein ,Fluorescein isothiocyanate - Abstract
Layered double hydroxide (LDH) nanoparticles have the potential to benefit a myriad of medical, consumer and industrial products if intricate control of the nanoscale architecture and understanding of the biological interactions and potential toxicity of the LDH nanomaterials are achieved. Here, we report the synthesis and fractionation of LDH nanomaterials of well defined spatial characteristics through the careful control of synthesis conditions and the use of ultracentrifugation. The appropriateness of a range of fluorescent dyes, for labelling LDH nanoparticles to enable biological tracking, has also been investigated. These dyes include fluorescein, fluorescein isothiocyanate (FITC), 8-aminonaphthelene-1,3,6-trisulfonic acid (ANTS) and 8-aminopyrene-1,3,6-trisulfonic acid (APTS). Thermal degradation of the popular FITC dye during nanoparticle synthesis was revealed and suitable alternative fluorescent molecules established. Furthermore, we report for the first time the application of size exclusion chromatography on LDH nanoparticles as a fast, reliable and efficient method to achieve purification without causing nanoparticle agglomeration.
- Published
- 2010
49. Minireview: Nanoparticles for Molecular Imaging—An Overview
- Author
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Rodney F. Minchin and Darren J. Martin
- Subjects
Endocrinology ,Human disease ,Medical imaging ,Nanoparticle ,Nanotechnology ,Molecular imaging ,Biology ,Imaging modalities - Abstract
Molecular imaging is a technique for quantifying physiological changes in vivo using imaging probes, or beacons, which can be detected noninvasively. This field of study has advanced rapidly in recent years, in part due to the application of nanotechnology. The versatility of different imaging modalities has been significantly enhanced by innovative nanoparticle development. These nanoprobes can be used to image specific cells and tissues within a whole organism. Some of the nanoparticles under development may be useful to measure biological processes associated with human disease and help monitor how these change with treatment. This review highlights some of the recent advances in nanoparticles for molecular imaging. It also addresses issues that arise with the use of nanoparticles. Whereas much of the technology remains at an experimental stage, the potential for enhancing disease diagnosis and treatment is considerable.
- Published
- 2010
50. Synthesis and Characterization of Dual Radiolabeled Layered Double Hydroxide Nanoparticles for Use in In Vitro and In Vivo Nanotoxicology Studies
- Author
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Anthony W. Musumeci, Zhi Ping Xu, Rodney F. Minchin, Suzanne V. Smith, Tara L. Schiller, and Darren J. Martin
- Subjects
Hydrotalcite ,Chemistry ,Nanoparticle ,Nanotechnology ,Gene delivery ,Combinatorial chemistry ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Nanomaterials ,chemistry.chemical_compound ,General Energy ,Nanotoxicology ,Isomorphous substitution ,Hydroxide ,Radioisotopic Labeling ,Physical and Theoretical Chemistry - Abstract
Layered double hydroxide (LDH) nanomaterials are currently the focus of intense scientific interest due to their potential application in drug and gene delivery research. However, the emerging field of nanotoxicology requires the development of new and more sensitive methodologies to follow the in vivo delivery kinetics as well as the persistence and bioaccumulation of the LDH carriers subsequent to delivery of the payload to the target area. Radioisotopic labeling offers very high detection sensitivity (
- Published
- 2009
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