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Biomedical subjects

M A Kennedy

Publications and source records attributed to M A Kennedy.

At least 19 recordsLinked to original sources
PubMed2006

IMPDH1 promoter mutations in a patient exhibiting azathioprine resistance.

Around 9% of inflammatory bowel disease (IBD) patients are resistant to azathioprine. We hypothesized that these patients may carry mutations within inosine-5'-monophosphate dehydrogenase (IMPDH). To test this hypothesis, we screened 20 azathioprine-resistant patients for variations in the two IMPDH genes (IMPDH1 and IMPDH2) using dHPLC and DNA sequencing. A 9 bp insertion within the IMPDH1 P3 promoter was found in a patient exhibiting severe azathioprine resistance. The insertion is predicted to abolish a cAMP-response element (CRE) and was found to significantly reduce IMPDH1 P3 promoter activity in a luciferase reporter gene assay (P-value <0.001). This in vitro assay suggests the variant promoter has altered function in vivo and consequently may have contributed to the thiopurine resistance observed in this patient. The absence of functional variants within the other patients indicates that if IMPDH genetic variability contributes to azathioprine resistance it does so infrequently.

Animals↗
PubMed2004

Glycosylphosphatidylinositol-specific phospholipase D immunoreactivity is present in islet amyloid in type 2 diabetes.

Numerous apolipoproteins associate with amyloid plaques. A minor high-density lipoprotein-associated protein, glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), has recently been described by the authors and others. Since GPI-PLD is synthesized by, and secreted from, pancreatic islet beta cells, the present study examined the hypothesis that GPI-PLD associates with islet amyloid. GPI-PLD immunoreactivity was examined in pancreatic tissues from type 2 diabetic and non-diabetic humans. GPI-PLD binding to heparan sulphate proteoglycan was determined in the absence or presence of heparan sulphate or heparin. Fibril formation from human islet amyloid polypeptide was determined in the absence or presence of GPI-PLD. In non-diabetics, GPI-PLD immunoreactivity was present and co-localized with insulin, as opposed to co-localizing with amyloid in diabetics. No immunoreactivity for apolipoprotein A-I was present in islet cells or islet amyloid. Heparan sulphate proteoglycan, which is commonly present in most amyloid, bound GPI-PLD in vitro. GPI-PLD inhibited the formation of amyloid fibrils from synthetic islet amyloid polypeptide in vitro. GPI-PLD is therefore present in islet amyloid and appears to derive from local production from islets. This localization likely derives from interaction between GPI-PLD and heparan sulphate proteoglycan. Since GPI-PLD also inhibited islet amyloid polypeptide fibril formation in vitro, it is concluded that GPI-PLD may play a role in islet amyloid formation in type 2 diabetes.

Amyloid↗
PubMed2003

Thiopurine S-methyltransferase (TPMT) genotype does not predict adverse drug reactions to thiopurine drugs in patients with inflammatory bowel disease.

BACKGROUND: Azathioprine and mercaptopurine (MP) are well established treatments for inflammatory bowel disease but they have severe adverse effects that prevent their use in some patients. The likelihood and type of adverse effect may relate to thiopurine methyltransferase (TPMT) enzyme activity and genotype. AIM: To compare the TPMT genotype frequencies in patients with inflammatory bowel disease who have had severe adverse effects to those who tolerate azathioprine or MP (controls). METHODS: Patients with inflammatory bowel disease who had been treated with azathioprine or MP in Christchurch between 1996 and 2002 were identified. Patients with adverse effects, and controls, were invited to provide a peripheral blood sample for analysis of TPMT genotype. The genotype frequencies were then compared between the two groups. RESULTS: Fifty-six patients were identified with adverse effects requiring cessation of therapy, of which 50 were genotyped. Reactions included allergic-type (25%), hepatitis (33%), nausea/vomiting (14%), bone marrow suppression (10%), pancreatitis (6%) and other (12%). Five of 50 patients with reactions had TPMT genotype *1/*3, one had *3/*3, and the rest had the wildtype genotype *1/*1. The patient with genotype *3/*3 had severe pancytopenia requiring hospitalization. Three of 50 controls had the *1/*3 genotype and the rest were *1/*1. CONCLUSIONS: The TPMT allele frequency in our population with inflammatory bowel disease is similar to that reported elsewhere. There was a slight trend for more frequent TPMT mutations in the patients with adverse reactions, but this was not statistically significant. Most patients with reactions did not have gene mutations.

Adolescent↗
PubMed2003

Schizophrenia and bipolar affective disorder: perspectives for the development of therapeutics.

Schizophrenia and bipolar disorder remain two of the most severe and difficult to treat psychotic disorders hampered by our poor understanding of their pathologies. The development of typical antipsychotic drugs opened an avenue of investigation through the dopamine D2 receptor in schizophrenia. With the reintroduction of the atypical antipsychotic clozapine came the development of a new generation of atypical agents and hypotheses challenging the centrality of this receptor in explaining antipsychotic effects. Evaluation of these competing theories does not provide sufficient evidence to displace the importance of the dopamine D2 receptor in antipsychotic efficacy, but does raise limitations of it as an explanatory hypothesis. Further, the treatment of other symptom domains in schizophrenia remains relatively neglected and open for the development of novel therapies. Similar to schizophrenia, bipolar disorder presents a diversity of clinical states but unlike schizophrenia, its mainstay of treatment, lithium, has not had a clear receptor target impeding understanding of the disorder's pathology and treatment. This has pushed investigation into other domains emphasising a number of intracellular signalling pathways and glial-neuronal interactions. The heavy genetic loading of bipolar disorder has allowed linkage analyses to identify a number of putative regions, however, the diversity of phenotypes complicates such studies. Polymorphisms of candidate genes have yielded potential leads such as dopamine beta hydroxylase in mood disorder and the serotonin transporter for treatment response. It is anticipated that combiningthe above approaches may hold promise for the development of more effective treatments.

Antipsychotic Agents↗
PubMed2002

A common P-glycoprotein polymorphism is associated with nortriptyline-induced postural hypotension in patients treated for major depression.

The multi-drug resistance gene ABCB1 (or MDR1) encodes a P-glycoprotein (P-gp) that regulates passage of many substances across the blood-brain barrier. The antidepressant amitriptyline and its metabolites (including nortriptyline) are substrates for P-gp, and in mice lacking P-gp, penetration of amitriptyline, but not fluoxetine, into the brain is enhanced. We reasoned that polymorphic variation of P-gp may contribute to differing responses of patients to antidepressant drugs. A single nucleotide polymorphism (SNP) of ABCB1 (3435C>T) was recently correlated with expression levels and in vivo function of P-gp. We examined this SNP in patients with major depression enrolled in a randomized antidepressant treatment trial of nortriptyline and fluoxetine, and observed a significant association between nortriptyline-induced postural hypotension and 3435C>T (chi(2) = 6.78, df = 2, P = 0.034). Our results suggest that homozygosity for 3435T alleles of ABCB1 is a risk factor for occurrence of nortriptyline-induced postural hypotension (OR = 1.37, P = 0.042, 95% CI 1.01-1.86).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Characterization of the human ABCG1 gene: liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein.

The human ABCG1 gene encodes a member of the ATP-binding cassette (ABC) superfamily of transporter proteins and is highly induced when macrophages are incubated with oxysterols. Using mRNA from oxysterol-treated human THP-1 cells together with 5'-rapid amplification of cDNA ends and polymerase chain reaction, we identified a novel ABCG1 transcript that encodes a putative protein of 786 residues containing a new amino terminus of 203 amino acids. Characterization of the genomic organization and structure of the human ABCG1 gene demonstrates that: (i) the gene consists of 23 exons spanning 98 kilobase pairs (kb) on chromosome 21q22.3, (ii) the 203 amino acids are encoded on three previously unidentified exons, 8-10, and (iii) a promoter, containing a TATA box and two liver X receptor (LXR) alpha response elements (LXREs), is located upstream of exon 8. Northern analysis using exon-specific probes confirms that oxysterol treatment results in >10-fold induction of ABCG1 transcripts that are derived from either exons 8-23 or exons 5, 7, and 11-23. Electromobility shift assays demonstrate that LXRalpha and retinoid X receptor alpha bind to the two LXREs in intron 7. Cells were transiently transfected with reporter luciferase constructs under the control of either (i) 9 kb of genomic DNA corresponding to intron 7 and part of exon 8 and containing either wild-type or mutant LXREs or (ii) two copies of the wild-type or mutant LXRE. In all cases, the wild-type construct was regulated in an LXR- and oxysterol-dependent manner, and this regulation was attenuated when the LXREs were mutated. In conclusion, the human ABCG1 gene contains multiple promoters, spans more than 98 kb and comprises 23 exons that give rise to alternative transcripts encoding proteins with different amino-terminal sequences. Elucidation of the various roles of different ABCG1 isoforms will be important for our understanding of mammalian cholesterol homeostasis.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2001

DNA-XPA interactions: a (31)P NMR and molecular modeling study of dCCAATAACC association with the minimal DNA-binding domain (M98-F219) of the nucleotide excision repair protein XPA.

Recent NMR-based, chemical shift mapping experiments with the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) suggest that a basic cleft located in the loop-rich subdomain plays a role in DNA-binding. Here, XPA-DNA interactions are further characterized by NMR spectroscopy from the vantage point of the DNA using a single-stranded DNA nonamer, dCCAATAACC (d9). Up to 2.5 molar equivalents of XPA-MBD was titrated into a solution of d9. A subset of (31)P resonances of d9 were observed to broaden and/or shift providing direct evidence that XPA-MBD binds d9 by a mechanism that perturbs the phosphodiester backbone of d9. The interior five residues of d9 broadened and/or shifted before (31)P resonances of phosphate groups at the termini, suggesting that when d9 is bound to XPA-MBD the internal residues assume a correlation time that is characteristic of the molecular weight of the complex while the residues at the termini undergo a fraying motion away from the surface of the protein on a timescale such that the line widths are more characteristic of the molecular weight of ssDNA. A molecular model of the XPA-MBD complex with d9 was calculated based on the (15)N (XPA-MBD) and (31)P (d9) chemical shift mapping studies and on the assumption that electrostatic interactions drive the complex formation. The model shows that a nine residue DNA oligomer fully covers the DNA-binding surface of XPA and that there may be an energetic advantage to binding DNA in the 3'-->5' direction rather than in the 5'-->3' direction (relative to XPA-MBD alpha-helix-3).

Base Sequence↗
PubMed2001

Human nucleotide excision repair protein XPA: NMR spectroscopic studies of an XPA fragment containing the ERCC1-binding region and the minimal DNA-binding domain (M59-F219).

XPA is a central protein component of nucleotide excision repair (NER), a ubiquitous, multi-component cellular pathway responsible for the removal and repair of many structurally distinct DNA lesions from the eukaryotic genome. The solution structure of the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) has recently been determined and chemical shift mapping experiments with 15N-labeled XPA-MBD show that XPA binds DNA along a basic surface located in the C-terminal loop-rich subdomain. Here, XPA-DNA interactions are further characterized using an XPA fragment containing the minimal DNA-binding domain plus the ERCC1-binding region (XPA-EM: M59-F219). The 15N/1H HSQC spectrum of XPA-EM closely maps onto the 15N/1H HSQC spectrum of XPA-MBD, suggesting the DNA-binding domain is intact in the larger XPA fragment. Such a conclusion is corroborated by chemical shift mapping experiments of XPA-EM with a single strand DNA oligomer, dCCAATAACC (d9), that show the same set of 15N/1H HSQC cross peaks are effected by the addition of DNA. However, relative to DNA-free XPA-MBD, the 15N/1H HSQC cross peaks of many of the basic residues in the loop-rich subdomain of DNA-free XPA-EM are less intense, or gone altogether, suggesting the acidic ERRC1-binding region of XPA-EM may associate transiently with the basic DNA-binding surface. While the DNA-binding domain in XPA-EM is structured and functional, 15N-edited NOESY spectra of XPA-EM indicate that the acidic ERRC1-binding region is unstructured. If the structural features observed for XPA-EM persist in XPA, transient intramolecular association of the ERCC1-binding domain with the DNA-binding region may play a role in the sequential assembly of the NER components.

Binding Sites↗
PubMed2001

Positive and negative regulation of squalene synthase (ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae.

To identify regulatory cis-elements in the proximal promoter of the yeast ERG9 squalene synthase gene, promoter deletion analysis was performed. This approach identified two regulatory elements, one an upstream repressing cis-element (URS), and the other an upstream activating cis-element (UAS). Electromobility shift assays (EMSAs) demonstrated that distinct proteins bind each element. Genetic screens were performed to identify yeast mutants that altered expression of ERG9 promoter-reporter gene fusions. Three non-ergosterol biosynthetic pathway genes were identified. A mutation in TPO1(YLL028W) led to a 5.5-fold increase in ERG9 expression while mutations in YER064C and SLK19 (YOR195W) led to a 3.1- and 5.6-fold decrease, respectively. Deletion analysis of these genes demonstrated that TPO1 and SLK19 specifically regulated ERG9 expression when tested with several different promoter-reporter gene fusions. Additionally, EMSAs demonstrated that extracts derived from the TPO1 deletion strain was unable to shift the repressing cis-element while protein extracts from the SLK19 deletion strain had a reduced shift of the activating cis-element. Furthermore, these two mutants showed quantitative differences in sterols and antifungal drug susceptibilities consistent with their role in regulating ERG9 expression.

Base Sequence↗
PubMed2001

Solution structure of Pyrobaculum aerophilum DsrC, an archaeal homologue of the gamma subunit of dissimilatory sulfite reductase.

The solution structure of DsrC, an archaeal homologue of the gamma subunit of dissimilatory sulfite reductase, has been determined by NMR spectroscopy. This 12.7-kDa protein from the hyperthermophilic archaeon Pyrobaculum aerophilum adopts a novel fold consisting of an orthogonal helical bundle with a beta hairpin along one side. A portion of the structure resembles the helix-turn-helix DNA-binding motif common in transcriptional regulator proteins. The protein contains two disulfide bonds but remains folded following reduction of the disulfides. DsrC proteins from organisms other than Pyrobaculum species do not contain these disulfide bonds. A conserved cysteine next to the C-terminus, which is not involved in the disulfide bonds, is located on a seven-residue C-terminal arm that is not part of the globular protein and is likely to dynamically sample more than one conformation.

Amino Acid Sequence↗
PubMed2001

Is cytochrome P450 2C9 genotype associated with NSAID gastric ulceration?

AIMS: The aim of this study was to explore whether genetic variation of cytochrome P450 2C9 (CYP2C9) contributes to NSAID-associated gastric ulceration. The hypothesis tested was that CYP2C9 poor metabolizer genotype would predict higher risk of gastric ulceration in patients on NSAIDs that are metabolized by CYP2C9, due to higher plasma NSAID concentrations. METHODS: Peripheral blood DNA samples from 23 people with a history of gastric ulceration attributed to NSAIDs metabolized by CYP2C9, and from 32 people on NSAIDs without gastropathy, were analysed to determine CYP2C9 genotype. RESULTS: The following genotypes were found: *1/*1 (wild type) in 70% of cases and 58% of controls, *1/*2 in 17% of cases and 29% of controls, *1/*3 in 13% of cases and 13% of controls. The difference between case and control nonwild-type genotype frequency was 11.5% (95% CI -14,37%), with the direction of the difference being against the hypothesis. No individuals with homozygote poor metaboliser genotype were identified. The differences in genotype frequencies between the two groups were not significant and the frequencies were similar to those in a large published population study. Ninety-five percent binomial confidence interval analysis confirms that there is no apparent clinically significant relationship between CYP2C9 genotype and risk of gastric ulceration although a small difference in risk in poor metabolizers cannot be excluded. CONCLUSIONS: These results do not support the hypothesis that gastric ulceration resulting from NSAID usage is linked to the poor metabolizing genotypes of CYP2C9.

Adult↗
PubMed2001

Genetic variation of the beta(2)-adrenoceptor: its functional and clinical importance in bronchial asthma.

Asthma is a polygenic disease for which no clear genotype-phenotype relationships have emerged. In contrast, although not associated with the diagnosis of asthma per se, variant forms of the beta(2)-adrenoceptor (beta2-AR) gene (ADRB2) display functional effects that may be clinically relevant. Single nucleotide polymorphisms (SNPs) of ADBR2 are common and result in amino acid substitutions at positions 16, 27, and 164 of the receptor as well as position 19 of its 5' upstream peptide. These SNPs influence receptor function in vitro, although evidence regarding exact relationships is conflicting. This has raised the possibility that phenotypes such as bronchial hyper-responsiveness (BHR) and responses to (beta2)-agonist drugs may be genetically determined. To date, no unequivocal relationships between SNPs and phenotype have been identified. In some studies the Gly(16) allele has been associated with increased BHR and asthma severity. In others, the Arg(16) allele has been shown to determine acute bronchodilator response and adverse events during long term beta(2)-agonist therapy. The latter may provide the basis for clinical application of this new knowledge. More recently, a small number of frequently occurring, functionally relevant ADRB2 haplotype pairs have been confirmed. These combinations of alleles may be more important in determining genotype/phenotype relationships than individual SNPs, and may explain why earlier investigations have yielded contrasting results. Future studies will be required to clarify the pharmacodynamic effects of ADRB2haplotypes both in vitro and in vivo.

Amino Acid Sequence↗
PubMed2000

Spectroscopic studies of zinc(II)- and cobalt(II)-associated Escherichia coli formamidopyrimidine-DNA glycosylase: extended X-ray absorption fine structure evidence for a metal-binding domain.

Formamidopyrimidine-DNA glycosylase (Fpg) is a 30.2 kDa protein that plays an important role in the base excision repair of oxidatively damaged DNA in Escherichia coli. Sequence analysis and genetic evidence suggest that zinc is associated with a C4-type motif, C(244)-X(2)-C(247)-X(16)-C(264)-X(2)-C(267), located at the C-terminus of the protein. The zinc-associated motif has been shown to be essential for damaged DNA recognition. Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc-associated protein (ZnFpg) in the lyophilized state and in 10% frozen aqueous glycerol solution show directly that the metal is coordinated to the sulfur atom of four cysteine residues. The average Zn-S bond length is 2.33 +/- 0.01 and 2.34 +/- 0.01 A, respectively, in the lyophilized state and in 10% frozen aqueous glycerol solution. Fpg was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily cobalt-associated (CoFpg). The profiles of the circular dichroism spectra for CoFpg and ZnFpg are identical, suggesting that the substitution of Co(2+) for Zn(2+) does not alter the structure of Fpg. A similar conclusion is reached upon the analysis of two-dimensional (15)N/(1)H HSQC spectra of uniformly (15)N-labeled samples of ZnFpg and CoFpg; the spectra are similar and display features characteristic of a structured protein. Biochemical assays with a 54 nt DNA oligomer containing 7, 8-dihydro-8-oxoguanine at a specific location show that CoFpg and ZnFpg are equally active at cleaving the DNA at the site of the oxidized guanine. EXAFS spectra of CoFpg indicate that the cobalt is coordinated to the sulfur atom of four cysteine residues with an average Co-S bond length of 2.28 +/- 0.01 and 2.29 +/- 0.01 A, respectively, in the lyophilized state and in 10% frozen aqueous glycerol solution. The structural similarity between CoFpg and ZnFpg suggests that it is biologically relevant to use the paramagnetic properties of Co(2+) as a structural probe.

Binding Sites↗
PubMed2000

Structure-based functional classification of hypothetical protein MTH538 from Methanobacterium thermoautotrophicum.

The structure of MTH538, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum, has been determined by NMR spectroscopy. MTH538 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized sequences from this organism. MTH538 is a so-called singleton, a sequence not closely related to any other (known) sequences. The structure of MTH538 closely resembles the known structures of receiver domains from two component response regulator systems, such as CheY, and is similar to the structures of flavodoxins and GTP-binding proteins. Tests on MTH538 for characteristic activities of CheY and flavodoxin were negative. MTH538 did not become phosphorylated in the presence of acetyl phosphate and Mg(2+), although it appeared to bind Mg(2+). MTH538 also did not bind flavin mononucleotide (FMN) or coenzyme F(420). Nevertheless, sequence and structure parallels between MTH538/CheY and two families of ATPase/phosphatase proteins suggest that MTH538 may have a role in a phosphorylation-independent two-component response regulator system.

Adenosine Triphosphatases↗
PubMed2000

Rapid and comprehensive determination of cytochrome P450 CYP2D6 poor metabolizer genotypes by multiplex polymerase chain reaction.

The liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) metabolizes numerous drugs, including many antidepressants, neuroleptics, antiarrhythmics, and antihypertensive agents. Variability in the gene that encodes this enzyme is an important factor underlying variable drug treatment responses. Some 5-10% of Caucasians lack functional CYP2D6, and the genetic basis of most of these "poor metabolizer" alleles is now well defined. As the CYP2D6 status of a patient can have profound effects on response to drug treatment, it is important to devise methods that permit rapid and economical determination of CYP2D6 genotype. We have developed a robust polymerase chain reaction method that simultaneously identifies the variants CYP2D6 *3, *4, *6, *8, *11, *12, *14, *15, *19, and *20. This constitutes most of the poor metabolizer alleles described in Caucasian and Asian populations. Separate PCR reactions or Southern blots are required for *7, the *5 deletion, and the hybrid alleles *13 and *16. The multiplex assay was validated on 100 individuals previously genotyped by specific polymerase chain reaction-restriction fragment length polymorphism analysis, and proved 100% accurate in this sample. The assay performed consistently with Taq DNA polymerases from various suppliers, within a broad range of temperatures and MgCl(2) concentrations, and using genomic DNA prepared by a range of methods including extraction from dried blood spots on card. This multiplexed, amplification refractory mutation system (ARMS) method is reliable, rapid, relatively cheap, amenable to automation, and offers the advantages of minimal sample handling with no requirement for restriction enzymes as in earlier CYP2D6 assays.

Alleles↗