Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization oftrans-4-hydroxytamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 muM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 muM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 muM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 muM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

Regulation of the mouse Nas1 promoter by vitamin D and thyroid hormone

The renal sodium-sulfate cotransporter, NaSi-1, a protein implicated to control serum sulfate levels, has been shown to be regulated in vivo by 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) and tri-iodothyronine (T-3). Recently, we cloned the mouse NaSi-1 gene (Nas1) and in the present study identified a 1,25-(OH)(2)D-3- and T-3-responsive element located within the Nas1 promoter. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3-responsive element (IR0 T3RE) at -436 to -425 which conferred 1,25(OH)(2)D-3 and T3 responsiveness, respectively. In summary, we have identified responsive elements that mediate the enhanced transcription of Nas1 by 1,25-(OH)(2)D-3 and T-3, and these mechanisms may provide important clues to the physiological control of sulfate homeostasis.

Transcriptional regulation of the sodium-sulfate cotransporter NaSi-1 gene

Inorganic sulfate is one of the most abundant anions in mammalian plasma and is essential for proper cell growth and development, as well as detoxification and activation of many biological compounds. To date, little is understood how physiological levels of sulfate are maintained in the body. Our studies, and of others, have identified the NAS(i)-1 protein to be a functional sulfate transporter in the kidney and intestine, and due to this localization, constitutes a strong candidate gene for maintaining body sulfate homeostasis. Several factors, including hormones and metabolic conditions, have been shown to alter NAS(i)-1 mRNA and protein levels in vivo. In this study, we describe the transcriptional regulation of NaSi-1, with a focus on the mouse NaSi-1 gene (Nas1) that was recently cloned in our laboratory. Vitamin D (1,25-(OH)(2)D-3) and thyroid hormone (T-3) led to an increase in Nas1 promoter activity in OK cells. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3 responsive element (IRO T3RE) at -426 to -425 which conferred 1,25-(OH)(2)D-3 and T-3 responsiveness respectively. These findings suggest for vitamin D and thyroid hormone regulation of NaSi-1, may provide important clues to the physiological control of sulfate homeostasis.

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [H-3]zeatin riboside ([H-3]ZR), a water-soluble blue dye or [H-3]H2O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within I h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [H-3]ZR and of blue dye in the same bud position was increased 3- to 10-fold relative to intact plants, whereas content of [H-3]H2O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [H-3]H2O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [H-3]ZR injected at physiological concentrations. Within 1 h, 80-95% of [H-3]ZR was converted to other active CKs (mainly zeatin riboside-5'phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O-acetylZR, O-acetylZRMP and a compound correlated with sites of high CK-concentrations) and inactive catabolites (adenosine, adenine, 5'AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.

Use of web-based formative assessment to support student learning in metabolism/nutrition unit

In 2002, an integrated basic science course was introduced into the Bachelor of Dental Sciences programme at the University of Queensland, Australia. Learning activities for the Metabolism and Nutrition unit within this integrated course included lectures, problem-based learning tutorials, computer-based self-directed learning exercises and practicals. To support student learning and assist students to develop the skills necessary to become lifelong learners, an extensive bank of formative assessment questions was set up using the commercially available package, WebCT®. Questions included short-answer, multiple-choice and extended matching questions. As significant staff time was involved in setting up the question database, the extent to which students used the formative assessment and their perceptions of its usefulness to their learning were evaluated to determine whether formative assessment should be extended to other units within the course. More than 90% of the class completed formative assessment tasks associated with learning activities scheduled in the first two weeks of the block, but this declined to less than 50% by the fourth and final week of the block. Patterns of usage of the formative assessment were also compared in students who scored in the top 10% for all assessment for the semester with those who scored in the lowest 10%. High-performing students accessed the Web-based formative assessment about twice as often as those who scored in the lowest band. However, marks for the formative assessment tests did not differ significantly between the two groups. In a questionnaire that was administered at the completion of the block, students rated the formative assessment highly, with 80% regarding it as being helpful for their learning. In conclusion, although substantial staff time was required to set up the question database, this appeared to be justified by the positive responses of the students.

Differential expression of the eukaryotic release factor 3 (eRF3/GSPT1) according to gastric cancer histological types

Background: There are now several lines of evidence to suggest that protein synthesis and translation factors are involved in the regulation of cell proliferation and cancer development. Aims: To investigate gene expression patterns of eukaryotic releasing factor 3 (eRF3) in gastric cancer. Methods: RNA was prepared from 25 gastric tumour biopsies and adjacent non-neoplastic mucosa. Real time TaqMan reverse transcription polymerase chain reaction (RT-PCR) was performed to measure the relative gene expression levels. DNA was isolated from tumour and normal tissues and gene dosage was determined by a quantitative real time PCR using SYBR Green dye. Results: Different histological types of gastric tumours were analysed and nine of the 25 tumours revealed eRF3/GSPT1 overexpression; moreover, eight of the 12 intestinal type carcinomas analysed overexpressed the gene, whereas eRF3/GSPT1 was overexpressed in only one of the 10 diffuse type carcinomas (Kruskal-Wallis Test; p , 0.05). No correlation was found between ploidy and transcript expression levels of eRF3/GSPT1. Overexpression of eRF3/GSPT1 was not associated with increased translation rates because the upregulation of eRF3/GSPT1 did not correlate with increased eRF1 levels. Conclusions: Overexpression of eRF3/GSPT1 in intestinal type gastric tumours may lead to an increase in the translation efficiency of specific oncogenic transcripts. Alternatively, eRF3/GSPT1 may be involved in tumorigenesis as a result of its non-translational roles, namely (dis)regulating the cell cycle, apoptosis, or transcription.

Risk of colorectal cancer associated with the C677T polymorphism in 5,10-methylenetetrahydrofolate reductase in Portuguese patients depends on the intake of methyl-donor nutrients

Background: Polymorphisms located in genes involved in the metabolism of folate and some methyl-related nutrients are implicated in colorectal cancer (CRC). Objective: We evaluated the association of 3 genetic polymorphisms [C677T MTHFR (methylene tetrahydrofolate reductase), A2756G MTR (methionine synthase), and C1420T SHMT (serine hydroxymethyltransferase)] with the intake of methyl-donor nutrients in CRC risk. Design: Patients withCRC(n 196) and healthy controls (n 200) matched for age and sex were evaluated for intake of methyl-donor nutrients and the 3 polymorphisms. Results: Except for folate intake, which was significantly lower in patients (P 0.02), no differences were observed in the dietary intake of other methyl-donor nutrients between groups. High intake of folate ( 406.7 g/d) was associated with a significantly lower risk of CRC (odds ratio: 0.67; 95% CI: 0.45, 0.99). The A2756G MTR polymorphism was not associated with the risk of developing CRC. In contrast, homozygosity for the C677TMTHFRvariant (TT) presented a 3.0-fold increased risk of CRC (95% CI: 1.3, 6.7). Similarly, homozygosity for the C1420T SHMT polymorphism also had a 2.6-fold increased risk (95% CI: 1.1, 5.9) of developing CRC. When interactions between variables were studied, low intake of all methyl-donor nutrients was associated with an increased risk ofCRC in homozygous participants for the C677T MTHFR polymorphism, but a statistically significant interaction was only observed for folate (odds ratio: 14.0; 95% CI: 1.8, 108.5). No significant associations were seen for MTR or SHMT polymorphisms. Conclusion: These results show an association between the C677T MTHFR variant and different folate intakes on risk of CRC.

Characterization of the effects of antiepileptic drugs on bone metabolism: in vitro studies with human osteoblastic and osteoclastic cells

Bone is constantly being molded and shaped by the action of osteoclasts and osteoblasts. A proper equilibrium between both cell types metabolic activities is required to ensure an adequate skeletal tissue structure, and it involves resorption of old bone and formation of new bone tissue. It is reported that treatment with antiepileptic drugs (AEDs) can elicit alterations in skeletal structure, in particular in bone mineral density. Nevertheless, the knowledge regarding the effects of AEDs on bone cells are still scarce. In this context, the aim of this study was to investigate the effects of five different AEDs on human osteoclastic, osteoblastic and co-cultured cells. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood and were characterized for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. Osteoblastic cell cultures were obtained from femur heads of patients (25-45 years old) undergoing orthopaedic surgery procedures and were then studied for cellular proliferation/viability, ALP activity, histochemical staining of ALP and apoptosis rate. Also the expression of osteoblast-related genes and the involvement of some osteoblastogenesis-related signalling pathways on cellular response were addressed. For co-cultured cells, osteoblastic cells were firstly seeded and cultured. After that, PBMC were added to the osteoblastic cells and co-cultures were evaluated using the same osteoclast and osteoblast parameters mentioned above for the corresponding isolated cell. Cell-cultures were maintained in the absence (control) or in the presence of different AEDs (carbamazepine, gabapentin, lamotrigine, topiramate and valproic acid). All the tested drugs were able to affect osteoclastic and osteoblastic cells development, although with different profiles on their osteoclastogenic and osteoblastogenic modulation properties. Globally, the tendency was to inhibit the process. Furthermore, the signaling pathways involved in the process also seemed to be differently affected by the AEDs, suggesting that the different drugs may affect osteoclastogenesis and/or osteoblastogenesis through different mechanisms. In conclusion, the present study showed that the different AEDs had the ability to directly and indirectly modulate bone cells differentiation, shedding new light towards a better understanding of how these drugs can affect bone tissue.

Multiple skin neoplasms in subjects under 40 years of age in Goiania, Brazil

OBJECTIVE To describe the trend for malignant skin neoplasms in subjects under 40 years of age in a region with high ultraviolet radiation indices.METHODS A descriptive epidemiological study on melanoma and nonmelanoma skin cancers that was conducted in Goiania, Midwest Brazil, with 1,688 people under 40 years of age, between 1988 and 2009. Cases were obtained fromRegistro de Câncer de Base Populacional de Goiânia(Goiania’s Population-Based Cancer File). Frequency, trends, and incidence of cases with single and multiple lesions were analyzed; transplants and genetic skin diseases were found in cases with multiple lesions.RESULTS Over the period, 1,995 skin cancer cases were observed to found, of which 1,524 (90.3%) cases had single lesions and 164 (9.7%) had multiple lesions. Regarding single lesions, incidence on men was observed to have risen from 2.4 to 3.1/100,000 inhabitants; it differed significantly for women, shifting from 2.3 to 5.3/100,000 (Annual percentage change – [APC] 3.0%, p = 0.006). Regarding multiple lesions, incidence on men was observed to have risen from 0.30 to 0.98/100,000 inhabitants; for women, it rose from 0.43 to 1.16/100,000 (APC 8.6%, p = 0.003). Genetic skin diseases or transplants were found to have been correlated with 10.0% of cases with multiple lesions – an average of 5.1 lesions per patient. The average was 2.5 in cases without that correlation.CONCLUSIONS Skin cancer on women under 40 years of age has been observed to be increasing for both cases with single and multiple lesions. It is not unusual to find multiple tumors in young people – in most cases, they are not associated with genetic skin diseases or transplants. It is necessary to avoid excessive exposure to ultraviolet radiation from childhood.

Transformation of Medicago truncatula with the arginine decarboxylase gene to modify polyamine metabolism toward water deficit resistance

Dissertation submitted to obtain a Ph.D. (Doutoramento) degree in Biology at the Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa

Susceptibilidade genética para cancro da mama

RESUMO: O cancro da mama é a patologia oncológica mais frequente nas mulheres sendo o responsável pela maior taxa de mortalidade por cancro no sexo feminino. Contudo, as causas inerentes a esta patologia permanecem por esclarecer. Nos últimos anos tem-se verificado que o risco para patologia neoplásica depende de factores ambientais e genéticos, estando estes últimos associados à variabilidade genética inter-individual. Polimorfismos genéticos em genes envolvidos no metabolismo de hormonas sexuais, de cancerígenos ambientais e na reparação da lesão genética, são potenciais candidatos a estarem associados à susceptibilidade individual para esta patologia. Assim, neste trabalho desenvolveram-se estudos de associação caso-controlo na população Portuguesa, com vista a avaliar-se o papel atribuído aos polimorfismos na susceptibilidade para cancro da mama. Foram seleccionados polimorfismos em genes envolvidos em diferentes vias mecanicistas: destoxificação de cancerígenos, metabolismo de estrogénios, reparação por excisão de bases, reparação por excisão de nucleótidos, reparação mismatch e reparação por recombinação homóloga. Os resultados obtidos revelaram associação entre os seguintes polimorfismos e a susceptibilidade individual para cancro da mama: os dois SNPs estudados no gene XRCC1 (Arg194Trp e Arg399Gln) e o SNP no gene XRCC3 (Thr241Met) após estratificação pelo status menopausico. Mediante estratificação por status de amamentação os SNPs identificados nos genes MnSOD (Val16Ala) e XRCC2 (Arg118His); um SNP no gene MLH3 (Leu844Pro), e por fim como resultado de interacção gene-gene as interacções descritas por MSH3 Ala1045Thr/MSH6 Gly39Glu e MSH4 Ala97Thr/MLH3 Leu844Pro. Os resultados obtidos e apresentados na presente dissertação, revelam que o estudo de polimorfismos pode representar um papel determinante na etiologia do cancro da mama. No entanto, mais estudos envolvendo estes mesmos polimorfismos em populações casuisticamente superiores serão uma mais-valia nos estudos de associação para esta neoplasia. Adicionalmente, a utilização da metodologia de Pools de DNA, poderá ser uma ferramenta útil na pré-selecção dos polimorfismos mais relevantes a estudar, na medida em que permite estimar a frequência alélica de cada SNP numa determinada população.-----------------------------------ABSTRACT: Breast cancer is the most common form of cancer among women, being the responsible for the highest mortality rate from cancer among the female sex. However, the main causes related to this pathology remain unclear. The risk of neoplasic disease has been connected with genetic and environmental factors. In fact, genes and the environment share the stage for most, if not all, common non-familial cancers, and are related to individual susceptibility. Genetic polymorphisms identified in genes encoding enzymes involved in estrogen metabolism, xenobiotics and DNA repair pathways are believed to be candidates for associations with breast cancer. Therefore, it was our intention to develop case-control studies among the Portuguese population, in order to evaluate the potential role of several genetic polymorphisms in breast cancer susceptibility. We selected polymorphisms in genes involved in different pathways: carcinogenic detoxification, estrogen metabolism, base excision repair, nucleotide excision repair, mismatch repair and double strand break repair by homologous recombination. The results obtained revealed potential associations between some polymorphisms studied and individual susceptibility to breast cancer. Regarding this fact, our results suggest the potential involvement of two XRCC1 gene polymorphisms (Arg194Trp and Arg399Gln) and XRCC3 gene polymorphism (Thr241Met) after stratification to menopausal status and after stratification to breastfeeding status an association of MnSOD gene polymorphism (Val16Ala) and XRCC2 (Arg188His) with the disease. The SNP identified in MLH3 gene (Leu844Pro), and the interaction gene-gene described by MSH3 Ala1045Thr/MSH6 Gly39Glu and MSH4 Ala97Thr/MLH3 Leu844Pro were also related to breast cancer susceptibility. The results shown in the present dissertation have revealed the potential role of polymorphisms in breast cancer etiology. However, further studies will be needed with larger populations to confirm these results. Additionally, the use of DNA pools methodology, as a pre-selection tool, could allow the identification of the most relevant polymorphisms to be studied, estimating the allelic frequency of each SNPs in different populations.