Mechanisms involved in the swelling of erythrocytes caused by Pacific and Caribbean ciguatoxins

The mechanisms underlying the swelling of frog red blood cells (RBC), induced by Pacific (P-CTX-1) and Caribbean (C-CTX-1) ciguatoxins (CTXs), were investigated by measuring the length, width and surface of their elliptic shape. P-CTX-1 (0.5 to 5 nM) and C-CTX-1 (1 mu M) induced RBC swelling within 60 min. The CTXs-induced RBC swelling was blocked by apamin (1 mu M) and by Sr2+ (1 mu M). P-CTX-1-induced RBC swelling was prevented and inhibited by H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one(27 mu M), an inhibitor Of Soluble guanylate cyclase (sGC), and NOS blockade by NG methyl-L-arginine (L-NMA; 10 mu M). Cytochalasin D (cytD, 10 mu M) increased RBC surface and mimicked CTX effect but did not prevent the P-CTX-1-induced L-NMA-sensitive extra increase. Calculations revealed that P-CTX-1 and cytD increase RBC total surface envelop and volume. These data strongly suggest that the molecular mechanisms underlying CTXs-induced RBC swelling involve the NO pathway by an activation of the inducible NOS, leading to sGC activation which modulates intracellular cGMP and regulates L-type Ca2+ channels. The resulting increase in intracellular Ca2+ content, in turn, disrupts the actin cytoskeleton, which causes a water influx and triggers a Ca2+-activated K+ current through SK2 isoform channels. (c) 2005 Elsevier Inc. All rights reserved.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.

Identification of a novel frameshift mutation in the giant muscle filament titin in a large Australian family with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is an etiologically heterogeneous cardiac disease characterized by left ventricular dilation and systolic dysfunction. Approximately 25-30% of DCM patients show a family history of mainly autosomal dominant inheritance. We and others have previously demonstrated that mutations in the giant muscle filament titin (TTN) can cause DCM. However, the prevalence of titin mutations in familial DCM is unknown. In this paper, we report a novel heterozygous 1-bp deletion mutation (c.62890delG) in TTN that cosegregates with DCM in a large Australian pedigree (A3). The TTN deletion mutation c.62890delG causes a frameshift, thereby generating a truncated A-band titin due to a premature stop codon (p.E20963KfsX10) and the addition of ten novel amino acid residues. The clinical phenotype of DCM in kindred A3 demonstrates incomplete penetrance and variable expressivity. Finally, protein analysis of a skeletal muscle biopsy sample from an affected member did not reveal the predicted truncated titin isoform although the aberrant mRNA was present, suggesting posttranslational modification and degradation of the truncated protein. The identification of a novel disease-causing mutation in the giant titin gene in a third large family with DCM indicates that mutations in titin may account for a significant portion of the genetic etiology in familial DCM.

Polymorphisms in the vitamin D receptor and their associations with risk of schizophrenia and selected anthropometric measures

The association between vitamin D levels and skeletal growth has long been recognized. However, exposure to low levels of vitamin D during early life is also known to alter brain development, and is a candidate risk factor for schizophrenia. This study examines the association between four polymorphisms in the vitamin D receptor (VDR) and 1) risk of schizophrenia, and 2) three anthropometric variables (height, head size, and head shape). Four single-nucleotide polymorphisms (SNPs; rs10735810/FokI, rsl544410/BsmI, rs7975232/ApaI, and rs731236/TaqI) in the VDR gene were genotyped in 179 individuals with schizophrenia and 189 healthy controls. No significant associations were detected between any of the four VDR SNPs and risk of schizophrenia. Patients were slightly but significantly shorter compared to controls. Of the four SNPs, only rs10735810/FokI was associated with any of the anthropometric measures: the M4 isoform of this SNP was significantly associated with larger head size (P = 0.002). In light of the evidence demonstrating a role for vitamin D during brain development, the association between polymorphisms in VDR and brain development warrants closer scrutiny.

Subcellular localization of mammalian type II membrane proteins

Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

Protein kinase C activation and its role in kidney disease

Protein kinase C (PKC) comprises a superfamily of isoenzymes, many of which are activated by cofactors such as diacylglycerol and phosphatidylserine. In order to be capable of activation, PKC must first undergo a series of phosphorylations. In turn, activated PKC phosphorylates a wide variety of intracellular target proteins and has multiple functions in signal transduced cellular regulation. A role for PKC activation had been noted in several renal diseases, but two that have had most investigation are diabetic nephropathy and kidney cancer. In diabetic nephropathy, an elevation in diacylglycerol and/or other cofactor stimulants leads to an increase in activity of certain PKC isoforms, changes that are linked to the development of dysfunctional vasculature. The ability of isoform-specific PKC inhibitors to antagonize diabetes-induced vascular disease is a new avenue for treatment of this disorder. In the development and progressive invasiveness of kidney cancer, increased activity of several specific isoforms of PKC has been noted. It is thought that this may promote the kidney cancer's inherent resistance to apoptosis, in natural regression or after treatments, or it may promote the invasiveness of renal cancers via cellular differentiation pathways. In general, however, a more complete understanding of the functions of individual PKC isoforms in the kidney, and development or recognition of specific inhibitors or promoters of their activation, will be necessary to apply this knowledge for treatment of cellular dysregulation in renal disease.

In vitro characterization of lamotrigine N2-glucuronidation and the lamotrigine-valproic acid interaction

Studies were performed to investigate the UDP-glucuronosyltransferase enzyme( s) responsible for the human liver microsomal N2-glucuronidation of the anticonvulsant drug lamotrigine ( LTG) and the mechanistic basis for the LTG-valproic acid ( VPA) interaction in vivo. LTG N2-glucuronidation by microsomes from five livers exhibited atypical kinetics, best described by a model comprising the expressions for the Hill ( 1869 +/- 1286 mu M, n = 0.65 +/- 0.16) and Michaelis-Menten ( Km 2234 +/- 774 mu M) equations. The UGT1A4 inhibitor hecogenin abolished the Michaelis-Menten component, without affecting the Hill component. LTG N2-glucuronidation by recombinant UGT1A4 exhibited Michaelis-Menten kinetics, with a K-m of 1558 mu M. Although recombinant UGT2B7 exhibited only low activity toward LTG, inhibition by zidovudine and fluconazole and activation by bovine serum albumin ( BSA) ( 2%) strongly suggested that this enzyme was responsible for the Hill component of microsomal LTG N2-glucuronidation. VPA ( 10 mM) abolished the Hill component of microsomal LTG N2-glucuronidation, without affecting the Michaelis-Menten component or UGT1A4-catalyzed LTG metabolism. K-i values for inhibition of the Hill component of LTG N2-glucuronidation by VPA were 2465 +/- 370 mu M and 387 +/- 12 mu M in the absence and presence, respectively, of BSA ( 2%). Consistent with published data for the effect of fluconazole on zidovudine glucuronidation by human liver microsomal UGT2B7, the Ki value generated in the presence of BSA predicted the magnitude of the LTG-VPA interaction reported in vivo. These data indicate that UGT2B7 and UGT1A4 are responsible for the Hill and Michaelis-Menten components, respectively, of microsomal LTG N2-glucuronidation, and the LTG-VPA interaction in vivo arises from inhibition of UGT2B7.

Neurobiological alterations in the human alcoholic brain: effect of genotype

Long-term alcohol abuse by human subjects leads to selective brain damage that is restricted in extent and variable in severity. Within the cerebral cortex, neuronal loss is most marked in the superior frontal cortex and relatively mild in motor cortex. Cirrhotic alcoholics and subjects with alcohol-related Wernicke-Korsakoff syndrome show more severe and more extensive damage than do uncomplicated cases. Accumulating evidence suggests that the likelihood of developing alcohol dependency is associated with one or more genetic markers. In previous work we showed that GABAA receptor functionality, and the subunit isoform expression that underlies this, differed in region- and disease-specific ways between alcoholics and controls. By contrast, glutamate receptor (NMDA, KA, AMPA) differences were muted or absent. Here we asked if genotype differentiated the form, pharmacology, or expression of glutamate and GABA receptors in pathologically vulnerable and spared cortical regions, with a view to determining whether such subject factors might influence the severity of alcohol-induced brain damage. Cerebrocortical tissue was obtained at autopsy under informed, written consent from uncomplicated and alcoholic-cirrhotic Caucasian (predominantly Anglo-Celtic) cases, together with matched controls and cases with cirrhosis of non-alcoholic origin. All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were processed for synaptic membrane receptor binding, mRNA analysis by quantitative RT-PCR, and protein analysis by Western blot. Genotyping was performed by PCR methods, in the main using published primers. Several genetic markers differentiated between our alcoholic and control subjects, including the GABAA receptor 2 subunit (GABB2) gene ( 2 (3) 10.329, P 0.01), the dopamine D2 receptor B1 (DRD2B) allele ( 2 (3) 10.109, P 0.01) and a subset of the alcohol dehydrogenase-3 (ADH3) alleles ( 2 (2) 4.730, P 0.05). Although neither the type-2 glutamate transporter (EAAT2) nor the serotonin transporter (5HTT) genes were significantly associated with alcoholism, only EAAT2 heterozygotes showed a significant association between ADH3 genotype and alcoholism ( 2 (3) 7.475, P 0.05). Other interactions between genotypes were also observed. DRD2A, DRD2B, GABB2, EAAT2 and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters, although in combined subjects there was a significant DRD2B X Area Interaction with glutamateNMDA receptor efficacy (F(1,57) 4.67; P 0.05), measured as the extent of glutamate-enhanced MK801 binding. In contrast, there was a significant Case-group X ADH3 X Area Interaction with glutamateNMDA receptor efficacy (F(3,57) 2.97; P 0.05). When GABAA receptor subunit isoform expression was examined, significant Case-group X Genotype X Area X Isoform interactions were found for EAAT2 with subunit mRNA (F(1,37) 4.22; P0.05), for GABB2 with isoform protein (F(1,37) 5.69; P 0.05), and for DRD2B with isoform protein (F(2,34)5.69; P0.05). The results suggest that subjects’ genetic makeup may modulate the effectiveness of amino acid-mediated transmission in different cortical regions, and thereby influence neuronal vulnerability to excitotoxicity.

EAAT2 splice variants in Alzheimer disease

Regional atrophy caused by neuronal loss is a characteristic of Alzheimer Disease (AD). Excitatory amino acid transporter-2 (EAAT2) is the major carrier responsible for clearing glutamate from the synaptic cleft in mammalian CNS. A localized attenuation of glutamate transport via reduced expression of functional forms of EAAT2 might contribute to regional excitotoxicity. The EAAT2 gene spans over 100 kb and encodes a 12-kb message. Several groups have identified alternative splice variants of EAAT2 in human brain tissue. These variants can affect transport by altering wild-type EAAT2 protein expression, localization, or transport efficiency. Alternative EAAT2 mRNA transcripts reportedly elicit a dominant-negative effect on glutamate uptake in cell culture. A 50% reduction in the expression in AD cortex of the truncated EAAT2 C-terminal isoform, EAAT2b, has been reported. We obtained cerebral cortex tissue, under informed written consent from the next of kin, from pathologically confirmed control, AD, and non-AD dementia cases. We aimed to determine the distribution and expression patterns of EAAT2 subtypes in susceptible and spared brain regions. We detected five alternate transcripts of EAAT2, two of which had not previously been reported. The relative contributions of novel variants, wild-type EAAT2, and previously discovered splice variants was investigated using Real-time PCR in AD, non-AD dementia, and age-matched control cortex. Our aim is to survey the relationship between these expression patterns and those of markers such as tau, GFAP, and b-amyloid, and to assess the correlation between variant-transporter expression and the level of cell loss.

Genes and gene expression in human alcoholic brain

Chronic alcoholism leads to localized brain damage, which is prominent in superior frontal cortex but mild in motor cortex. The likelihood of developing alcohol dependence is associated with genetic markers. GABA-A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the localized expression of glutamate N-methyl-D-aspartate (NMDA) and GABA-A receptors to influence the severity of alcohol-induced brain damage. Cerebral cortex tissue was obtained at autopsy from alcoholics without disease comorbid with alcoholics, alcoholics with cirrhosis, and matched controls. DRD2A, DRD2B, GABRB2, SLC1A2, and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters. In contrast, a specific alcohol dehydrogenase (ADHIC) genotype interacted significantly with NMDA efficacy and affinity in a region-specific manner SLC1A2 (glutamate transporter-2) genotype interacted significantly with local GABAA receptor b subunit mRNA expression, and ADHIC, DRD2B, SLC1A2, and APOE genotypes with b subunit isoform protein expression. In the latter instance, possession of the alcoholism- associated allele altered b isoform protein expression patterns toward a less-efficacious form of the GABA-A receptor in the pathologically vulnerable region. GABRB2 and GRIN2B (NMDA receptor 2B subunit} Genotypes were associated with significant regional difference in the pattern of b subunit protein isoform expression, but this was not influenced by alcoholism status. Genotype may modulate amino acid transmission locally so as to mediate neuronal vulnerability. This has implications for the effectiveness of pharmacological interventions aimed at ameliorating brain damage and, possibly, dependence.

Interactions between serotonergic genotype and amino acid transmitter receptor expression in human alcoholics

Serotonin can modulate the activity of neural reward pathways that are strongly implicated in mediating the effects of chronic alcohol misuse, and its treatment, in human subjects. In previous work and as discussed elsewhere at this meeting, we and others have found consistent differences in the parameters of GABA and glutamate receptors, and the expression of their component subunit transcripts and proteins, in areas of the alcoholic brain that are altered by alcoholism. We did not fi nd clear changes in GABA and glutamate transport function in such samples, but a series of microarray analyses showed consistent upregulation of the presynaptic GABA/betaine transporter SLC6A12. Microarray studies showed no signifi cant differences in the expression of transcripts associated with 5HT transmission; however, only a small number of such elements were present on the arrays. Here we partitioned GABAA and NMDA pharmacology, and subunit mRNA and protein expression, measured in samples of frontal and motor cortex obtained at autopsy from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and controls, according to 5HTTLPR (SLC6A4) and 5HT1B (HTR1B) polymorphisms. We found no effect of these genotypes on the expression of GABAA receptor gene products, but there was a signifi cant mRNA Transcript X Area X Group X 5HTTLPR Interaction with NMDA subunit isoform expression measured by Real Time PCR with GAPDH normalization. Further analysis showed the effect to be selective for alcoholics with cirrhosis, to be most marked in the pathologically vulnerable frontal cortex, and to vary with subunit transcript (F2,76 = 6.545, P = 0.002). NR1 expression was most affected, followed by NR2A, with NR2B expression least altered. Pilot data suggest 5HT1B genotype may also modulate NMDA subunit expression. Interactions between amino acid and serotonin transmission may infl uence susceptibility to alcohol dependence or pathogenesis

The effects of ethanol on PPARS in MCF-7 human breast cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors involved in various metabolic diseases. In the liver, PPARα is involved in alcohol metabolism and may lead to the development of alcoholic fatty liver and other alcohol mediated liver injuries. PPARβ modulation by ethanol induces abnormal myelin production by oligodendrocytes. PPARα and PPARβ are PPAR isoforms expressed in the human breast cell lines. Epidemiological studies show a positive correlation between alcohol intake and breast cancer risk, however, the molecular mechanisms involved are unclear. We hypothesized that ethanol would affect the expression and transactivation of human PPAR isoforms in estrogen receptor (ER) positive and ER negative breast cancer cells. Using real time RT-PCR we looked at the transcription of PPAR isoforms in the presence of increasing concentrations of ethanol and saw isoform and time dependent specific effects. Gene reporter assays enabled us to ascertain the effects of ethanol on ligand-mediated activation of human PPARα and PPARβ at concentrations equivalent to both moderate and chronic alcohol consumption. Ethanol differentially blocked the ligand-mediated activation of both PPARα and PPARβ. Since PPARα and PPARβ are involved in the differentiation and proliferation of breast cancer cells, PPARs may be a possible mechanism involved in the effect of ethanol in breast cancer.

Unraveling the molecular underpinnings of Philadelphia-negative myeloproliferative neoplasms: implications for future therapeutic strategies

The present thesis encompasses the two researches projects I conducted during my PhD program in Molecular Biology and Pathology. The common thread is represented by the analysis of the signaling pathways implicated in the pathophysiology of the two most aggressive Philadelphia-negative myeloproliferative neoplasms, namely, atypical chronic myeloid leukemia (aCML) and primary myelofibrosis (PMF). In the last decade, since the description of the JAK2V617F mutation in 2005, the field of the molecular characterization of Philadelphia-negative myeloproliferative neoplasms has experienced an astonishing implementation that led to the discovery of 16 new mutations involving signal transduction, epigenetic modifiers, cell cycle regulators. Nevertheless, their pathogenetic relevance and whether they could represent good “druggable” candidates have to be proved yet. In the first section I provide the first report of the signaling cascade down-stream the rare cytogenetic lesion t(8;9)(p22;p24)/PCM1-JAK2 associated with aCML, finding that it selectively activates the ERK1/2 signaling without affecting JAK/STAT phosphorylation. In the second part, I investigated the implication of the ε isoform of novel Protein kinase Cs (PKCs) in the pathophysiology of the aberrant megakaryocytopoiesis in PMF, concluding that the over-expression of PKCε detains a crucial relevance in the aberrant behavior of PMF megakaryocytes and its inhibition is capable to restore their normal differentiation and abrogate the anti-apoptotic signaling. Both results are discussed in the view of their therapeutic implications. In case PCM1/JAK2-related hematologic neoplasms, ERK-inhibitors rather than JAK-inhibitors (i.e. ruxolitinib) should be considered as a “tailored” drugs. In case of PMF, PKCε-inhibitors (i.e. εV1-2 peptide) configure as an appealing strategy to re-direct the megakaryocytic neoplastic clone.

Towards a polymeric binding mimic for cytochrome CYP2D6

A series of fluorescent molecularly imprinted polymers has been prepared with a view to generating material capable of mimicking the binding characteristics of the metabolically important cytochrome isoform CYP2D6. Such polymers would have the possibility to form the sensing element in a high-throughput assay for the prediction of CYP2D6 affinity. The imprinted polymers possessed binding-dependent fluorescence. They re-bound their templates and various cross-reactivities were encountered for test compound/drug recognition. One polymer in particular exhibited a rational discrimination amongst the related synthetic templates and was reasonably successful in recognising CYP2D6 substrates from a drug panel. © 2005 Elsevier B.V. All rights reserved.

Self-association of calcium-binding protein S100A4 and metastasis

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.