Autosomal dominant retinitis pigmentosa:linkage to rhodopsin and evidence for genetic heterogeneity

Retinitis pigmentosa (RP) is the most prevalent human retinopathy of genetic origin. Chromosomal locations for X-linked RP and autosomal dominant RP genes have recently been established. Multipoint analyses with ADRP and seven markers on the long arm of chromosome 3 demonstrate that the gene for rhodopsin, the pigment of the rod photoreceptors, cosegregates with the disease locus with a maximum lod score of approximately 19, implicating rhodopsin as a causative gene. Recent studies have indicated the presence of a point mutation at codon 23 in exon 1 of rhodopsin which results in the substitution of histidine for the highly conserved amino acid proline, suggesting that this mutation is a cause of rhodopsin-linked ADRP. This mutation is not present in the Irish pedigree in which ADRP has been mapped close to rhodopsin. Another mutation in the rhodopsin gene or in a gene closely linked to rhodopsin may be involved. Moreover, the gene in a second ADRP pedigree, with Type II late onset ADRP, does not segregate with chromosome 3q markers, indicating that nonallelic as well as perhaps allelic genetic heterogeneity exists in the autosomal dominant form of this disease.

Epidermolysis bullosa:evidence for linkage to genetic markers on chromosome 1 in a family with the autosomal dominant simplex form

DNA from members of a three-generation pedigree of Irish origin, displaying an autosomal dominant simplex form of epidermolysis bullosa of the epidermolytic, simplex, or Koebner variety (EBS2), was analyzed for linkage with a set of markers derived from the long arm of chromosome 1. Two-point analysis revealed positive lod scores for five of these markers, AT3 (Z = 2.107, theta = 0), APOA2 (Z = 1.939, theta = 0.15), D1S66 (Z = 1.204, theta = 0), D1S13 (Z = 1.026, theta = 0.15), and D1S65 (Z = 0.329, theta = 0.15). Multilocus analysis, incorporating the markers D1S19, D1S16, D1S13, APOA2, D1S66, AT3, and D1S65, resulted in a lod score of 3 maximizing at AT3. These data strongly support previous tentative indications of linkage between EBS2 and genetic markers on the long arm of chromosome 1.

Autosomal dominant retinitis pigmentosa (ADRP):localization of an ADRP gene to the long arm of chromosome 3

Members of a large pedigree of Irish origin presenting with early onset Type I autosomal dominant retinitis pigmentosa (ADRP) have been typed for D3S47 (C17), a polymorphic marker from the long arm of chromosome 3. Significant, tight linkage of ADRP to D3S47, with a lod score of 14.7 maximizing at 0.00 recombination, has been obtained, hence localizing the ADRP gene (RP1) segregating in this pedigree to 3q.

Do leaves of plants on phosphorus-impoverished soils contain high concentrations of phenolic defence compounds?

Prominent theories of plant defence have predicted that plants growing on nutrient-poor soils produce more phenolic defence compounds than those on richer soils. Only recently has the Protein Competition Model (PCM) of phenolic allocation suggested that N and P limitation could have different effects because the nutrients are involved in different cellular metabolic processes. 2. We extend the prediction of the PCM and hypothesize that N will have a greater influence on the production of phenolic defensive compounds than P availability, because N limitation reduces protein production and thus competition for phenylalanine, a precursor of many phenolic compounds. In contrast, P acts as a recyclable cofactor in these reactions, allowing protein and hence phenolic production to continue under low P conditions. 3. We test this hypothesis by comparing the foliar concentrations of phenolic compounds in (i) phenotypes of 21 species growing on P-rich alluvial terraces and P-depleted marine terraces in southern New Zealand, and (ii) 87 species growing under similar climates on comparatively P-rich soils in New Zealand vs. P-depleted soils in Tasmania. 4. Foliar P concentrations of plants from the marine terraces were about half those of plants from alluvial soils, and much lower in Tasmania than in New Zealand. However, foliar concentrations of N and phenolic compounds were similar across sites in both comparisons, supporting the hypothesis that N availability is a more important determinant of plant investment in phenolic defensive compounds than P availability. We found no indication that reduced soil P levels influenced plant concentrations of phenolic compounds. There was wide variation in the foliar N and P concentrations among species, and those with low foliar nutrient concentrations produced more phenolics (including condensed tannins). 5. Our study is the first trait comparison extending beyond standard leaf economics to include secondary metabolites related to defence in forest plants, and emphasizes that N and P have different influences on the production of phenolic defence compounds. © 2009 British Ecological Society.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2sigma;2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2sigma;2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca<inf>o</inf>²⁺) homeostasis. To elucidate the role of AP2sigma;2 in Ca<inf>o</inf>²⁺ regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2sigma;2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2sigma;2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2sigma;2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2sigma;2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2sigma;2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Extracting statistically dominant modes of a steel truss bridge from vehicle-induced vibrations

This study is intended to investigate the validity of the stability diagram (SD) aided multivariate autoregressive (MAR) analysis for identifying modal parameters of a real truss bridge. The MAR models are adopted to fit the time series of the dynamic accelerations recorded from a number of observation points on the bridge; then the modal parameters are extracted from the MAR model coefficient matrix. The SD is adopted to determine statistically dominant modes. In plotting the SD, a number of stability criteria are further adopted for filtering out those modes with unstable modal parameters. By the present method, the first five modal frequencies and mode shapes are identified with very high precision, while the damping ratios are identified with high precision for the 1st mode but with poorer precision for higher modes. Moreover, the ability of the SD in selecting structural modes without getting involved in any model-order optimization problem is highlighted through a comparison study.

CD4+ T Cells Targeting Dominant and Cryptic Epitopes from Bacillus anthracis Lethal Factor

Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called 'cryptic' or 'subdominant' epitopes. We analyzed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISpot assays we characterized epitopes that elicited a response following immunization with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, as a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 transgenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were influenced by the specific HLA alleles presenting the peptide, and imply that construction of future epitope string vaccines which are immunogenic across a wide range of HLA alleles could benefit from a combination of both cryptic and immunodominant anthrax epitopes.

Quantification of HER2 heterogeneity in breast cancer-implications for identification of sub-dominant clones for personalised treatment

Breast cancer is a heterogeneous disease, at both an inter- and intra-tumoural level. Appreciating heterogeneity through the application of biomarkers and molecular signatures adds complexity to tumour taxonomy but is key to personalising diagnosis, treatment and prognosis. The extent to which heterogeneity exists, and its interpretation remains a challenge to pathologists. Using HER2 as an exemplar, we have developed a simple reproducible heterogeneity index. Cell-to-cell HER2 heterogeneity was extensive in a proportion of both reported 'amplified' and 'non-amplified' cases. The highest levels of heterogeneity objectively identified occurred in borderline categories and higher ratio non-amplified cases. A case with particularly striking heterogeneity was analysed further with an array of biomarkers in order to assign a molecular diagnosis. Broad biological complexity was evident. In essence, interpretation, depending on the area of tumour sampled, could have been one of three distinct phenotypes, each of which would infer different therapeutic interventions. Therefore, we recommend that heterogeneity is assessed and taken into account when determining treatment options.

Computerized key to the genus Bursaphelenchus Fuchs, analysis of species clusters based on morphology, using information of insect vectors and associated plants, with a revision of the genus.

The genus Bursaphelenchus includes B. xylophilus (Steiner et Buhrer, 1934) Nickle, 1981, which is of world economic and quarantine importance. Distinction among several species of the pinewood nematodes species complex (PWNSC) is often difficult. Besides standard morphology, morphometrics and molecular biology, new tools are welcome to better understand this group. The computerized (or e-) key of this genus, presented in this communication, includes 74 species (complete list of valid species of the world fauna) and 35 characters, that were used by the taxonomic experts of this group, in the original descriptions. Morphology of sex organs (male spicules and female vulval region) was digitized and classified to distinguish alternative types. Several qualitative characters with overlapping character states (expressions) were transformed into the morphometric indices with the discontinuous ranges (characters of ratios of the spicule dimensions). Characters and their states (expressions) were illustrated in detail and supplied by brief user-friendly comments. E-key was created in the BIKEY identification system (Dianov & Lobanov, 1996-2004). The system has built-algorithm ranging characters depending on their diagnostic values at each step of identification. Matrix of species and the character states (structural part of the e-key database) may be easily transformed using statistical packages into the dendrograms of general phenetic similarities (UPGMA, standard distance: mean character difference). It may be useful in the detailed analysis of taxonomy and evolution of the genus and in its splitting to the species groups based on morphology. The verification of the dendrogram using the information on the species links with insect vectors and their associated plants, provided an opportunity to recognize the five clusters (xylophilus, hunti, eremus sensu stricto, tusciae and piniperdae sensu stricto), which seem to be the natural species groups. The hypothesis about the origin and the first stages of the genus evolution is proposed. A general review of the genus Bursaphelenchus is presented.

Identity of a Tilapia Pheromone Released by Dominant Males that Primes Females for Reproduction

Knowledge of the chemical identity and role of urinary pheromones in fish is scarce, yet it is necessary in order to understand the integration of multiple senses in adaptive responses and the evolution of chemical communication [1]. In nature, Mozambique tilapia (Oreochromis mossambicus) males form hierarchies, and females mate preferentially with dominant territorial males, which they visit in aggregations or leks [2]. Dominant males have thicker urinary bladder muscular walls than subordinates or females and store large volumes of urine, which they release at increased frequency in the presence of subordinate males or preovulatory, but not postspawned, females [3–5]. Females exposed to dominant-male urine augment their release of the oocyte maturation-inducing steroid 17α,20β-dihydroxypregn-4-en-3-one (17,20β-P) [6]. Here we isolate and identify a male Mozambique tilapia urinary sex pheromone as two epimeric (20α- and 20β-) pregnanetriol 3-glucuronates. We show that both males and females have high olfactory sensitivity to the two steroids, which cross-adapt upon stimulation. Females exposed to both steroids show a rapid, 10-fold increase in production of 17,20β-P. Thus, the identified urinary steroids prime the female endocrine system to accelerate oocyte maturation and possibly promote spawning synchrony. Tilapia are globally important as a food source but are also invasive species, with devastating impact on local freshwater ecosystems [7, 8]. Identifying the chemical cues that mediate reproduction may lead to the development of tools for population control [9–11].

Phytochelatin signal tranduction and cadmium chelation in plants

As plantas utilizam diversas estratégias de sinalização para reconhecer e responder aos stresses ambientais. A maioria das vias de transdução de sinais partilham um sinal genérico, normalmente a modulação dos níveis intracelulares de Ca2+. Esta por sua vez pode iniciar uma cascata de fosforilação proteica que finalmente afecta as proteínas directamente envolvidas na protecção celular ou culmina em factores de transcrição que vão determinar a resposta fisiológica ao stresse. A percepção destes sinais e a compreensão de como estes podem activar as respostas adaptativas são factores-chave para a tolerância das plantas a stresses abióticos. Um dos principais stresses abóticos que restrigem o crescimento das plantas é a presença de metais pesados. A produção de fitoquelatinas e a subsequente quelação dos metais é o mecanismo mais conhecido de tolerância ao stresse metálico em plantas. Fitoquelatinas (PCs) são péptidos com grupos tiol que são sintetizados através da transpeptidação da glutationa (GSH), pela acção da enzima fitoquelatina sintase (PCS). No entanto, até ao momento, as vias de sinalização que levam à síntese de fitoquelatinas e à percepção do stresse metálico são pouco compreendidas. Dentro deste contexto, o presente trabalho foi elaborado com o intuito de elucidar a via de sinalização através da qual o cádmio é detectado pelas células vegetais e induz a síntese de PCs. Quase todos, os estudos de stresses abióticos em plantas apontam para o facto de a sua sinalização se basear nos mesmos tipos de sinais moleculares, nomeadamente a sinalização por cálcio, a fosforilação proteica e a indução de espécies reactivas de oxigénio (ROS). Trabalhos recentes sugerem que a sinalização de PCs poderá envolver todos estes parâmetros. Assim, uma primeira abordagem foi efectuada para compreender a síntese de PCs na espécie Arabidopsis thaliana, através da monitorizaçção da actividade de enzimas relacionadas, a γ-EC sintetase, GSH sintetase e a PC sintase (PCS), assim como o tempo necessário para o elongamento das PCs e a sua acumulação. Seguidamente, ao longo deste processo foi analisada a expressão de sinais específicos, associados com sinais de cálcio, fosforilação proteica e sinalização por ROS. A importância destes factores na síntese de PCs foi também avaliada através do uso de moduladores farmacológicos de cálcio e fosfatases proteicas e também pela indução de stresse oxidativo. Os resultados demonstraram novos dados sobre o papel do cálcio e da fosforilação proteica na produção de PCs e na síntese de GSH, revelando que a actvidade da PCS é regulada por fosforilação e que a sinalização de cálcio pode mediar a síntese de GSH. O envolvimento da sinalização de ROS na síntese de GSH, atráves de crosstalk com a sinalização de cálcio também foi proposta. Assim, os resultados aqui apresentados descrevem uma possível via de sinalização de cádmio nas plantas e da indução de fitoquelatinas. Este trabalho poderá ser portanto muito útil na implementação de novas metodologias de agricultura sustentável e práticas de fitorremediação em solos contaminados com metais pesados.

Derivation of soil screening values for metals in Portuguese natural soil

The increasing human activity has been responsible by profound changes and a constinuos degradation of the soil compartment in all the European territory. Some European policies are appearing focusing soil’s protection and the management of contaminated sites, in order to recover land for other uses. To regulate the risk assessment and the management of contaminated soils, many European member states adopted soil guideline values, as for example soil screnning values (SSV).These values are particularly useful for the the first tier of the Ecological Risk Assessment (ERA) processes of contaminated sites,especially for a first screening of sites requiring a more site-specific evaluation. Hence, the approriate definition of regional SSVs will have relevant economic impacts in the management of contaminated sites. Portugal is one of European Member States that still lack these soil guideline values. In this context, this study gaves a remarkable contribution in the generation of ecotoxicological data for soil microbiological parameters, terrestrial plants and invertebrates for the derivation of SSVs for uranium (U), cadmium (Cd) and copper (Cu), using a Portuguese natural soil, representative of a dominant type of soil in the Portuguese territory. SSVs were derived based on two methods proposed by the the Technical Guidance Document for Risk Assessment of the European Commission; namely the assessment factor method (AF) and the species sensitivity distribution (SSD) method (with some adaptations). The outputs of both methods were compared and discussed. Further, this study laid the foundation for a deeper reflection about the cut-off (hazard concentration for a given percentage of species - HCps) to be estimated from the SSDs, and to be selected for the derivation of SSVs, with the adequate level of protection. It was proven that this selection may vary for different contaminants, however a clear justification should be given, in each case. The SSvs proposed in this study were for: U (151.4 mg U kg-1dw), Cd (5.6 mg Cd kg-1dw), and Cu (58.5 mg Cu kg-1dw) These values should now be tested for their descriminating power of soils with different levels of contamination. However, this studies clarifies the approach that should be followed for the derivation of SSVs for other metals and organic contaminants, and for other dominant types of Portuguese natural soils.

Combined effects of chemical and natural stressors to soil organisms and plants

Nas últimas décadas, a Terra tem experimentado um aquecimento global e mudanças nos padrões de precipitação. Muitos estudos sobre a avaliação de risco de agrotóxicos em organismos não-alvo foram realizados com base em protocolos padronizados, com condições abióticas controladas. Mas, em campo, os organismos são expostos a flutuações de vários fatores ambientais, bem como a poluentes, que podem alterar os limites de tolerância dos organismos aos stressores naturais, bem como alterar a toxicidade ou biodisponibilidade do químico em causa. Considerando isso, o principal objetivo deste trabalho foi o de avaliar de que modo e em que medida os fatores ambientais (temperatura, humidade do solo e radiação UV) podem interagir uns com os outros ou afetar a toxicidade do carbaril para invertebrados do solo e plantas. Para isso, foram utilizadas quatro espécies padrão: Folsomia candida, Eisenia andrei, Triticum aestivum e Brassica rapa, e simulados diferentes cenários climáticos, com vários parâmetros letais e subletais analisados. A exposição combinada foi analisada utilizando, quando possível, a ferramenta MIXTOX, com base no modelo de referência de acção independente (IA) e possíveis desvios, assim como rácios sinergísticos/antagonísticos (a partir de valores de EC50/LC50), quando a dose-resposta de um dos stressores não foi obtida. Todos os fatores de stress aplicados isoladamente causaram efeitos significativos sobre as espécies testadas e sua exposição combinada com carbaril, apresentaram respostas diferenciadas: para as minhocas, a seca e temperaturas elevadas aumentaram os efeitos deletérios do carbaril (sinergismo), enquanto o alagamento e temperaturas baixas diminuíram sua toxicidade (antagonismo). Para os colêmbolos, o modelo IA mostrou ser uma boa ferramenta para prever a toxicidade do carbaril tanto para temperaturas altas como para as baixas. Para as duas espécies de plantas foram encontradas diferenças significativas entre elas: em termos gerais, as interações entre carbaril e os stressores naturais foram observadas, com sinergismo aparecendo como o padrão principal relacionado com a radiação UV, solos secos e temperaturas elevadas, enquanto o padrão principal relacionado com temperaturas baixas e stress de alagamento foi o antagonismo. Quando os efeitos de dois stressores naturais (radiação UV e humidade do solo) em plantas foram avaliados, uma interação significativa foi encontrada: a seca aliviou o efeito deletério da radiação UV em T. aestivum e o alagamento aumentou os seus efeitos, mas para B. rapa a adição de ambos os stresses de água causou um aumento (sinergismo) dos efeitos deletérios da radiação UV para todos os parâmetros avaliados. Portanto é necessário que as diferenças sazonais e latitudinais, bem como as mudanças climáticas globais, sejam integradas na avaliação de risco de contaminantes do solo.

Physiology and metabolic profile of Moringa olifera and Eucalyptus globulus plants subjected to climate changes

As alterações climáticas emergentes têm um grande impacto no crescimento e desenvolvimento de espécies florestais, nomeadamente em espécies de valor industrial e medicinal, como é o caso do eucalipto (Eucalyptus globulus) e da moringa (Moringa oleifera). Assim, é urgente conhecer as respostas fisiológicas e entender as variações que ocorrem nos perfis metabólicos de espécies vegetais. Neste trabalho, plantas jovens de Eucalyptus globulus foram expostas a radiação UVB (12kJ/m2) e foram avaliadas as respostas fisiológicas e o perfil metabólico, um e onze dias após a aplicação da radiação. A dose de UVB usada não afetou as reações fotoquímicas nem as trocas gasosas, contudo ao nível do metabolismo do carbono (AST e amido) e do conteúdo de pigmentos verificaram-se pequenas alterações (AST e pigmentos). Através da análise do perfil metabólico de E. globulus foram encontrados compostos voláteis e semi-voláteis pertencentes às famílias dos terpenos, sesquiterpenos e aldeídos. Em geral, os sesquiterpenos e os álcoois monoterpénicos apresentaram uma tendência para manter e, em alguns casos, diminuir com o stress, enquanto que o grupos dos aldeídos aumentou e os monoterpenos apresentaram um comportamento mais heterogéneo. O E. globulus mostrou ser uma espécie tolerante à aplicação da dose de UVB usada neste trabalho. Por outro lado, plantas jovens de M. oleifera foram expostas a défice hídrico (DH). Um grupo de plantas foi recolhido um dia após o final da exposição e o outro grupo após onze dias do final da exposição. Foi avaliado o perfil metabólico desta espécie através de GC/MS. Os dados cromatográficos indicaram que em condições de stress (DH(1) e DH(11)), as quantidades de compostos associados a vias primárias e secundárias de defesa (como os alcanos, álcoois, ácidos carboxílicos, esteróis, aminoácidos e açucares) sofreram algumas alterações. As plantas analisadas 11 dias após a remoção do stress mostraram maiores variações do perfil de metabolitos. No entanto, tanto um como onze dias após a remoção do stress, as plantas apresentaram a formação de novos rebentos. Apesar do perfil de metabolitos ter sofrido algumas alterações, por não se registarem casos de morte, conclui-se que as plantas de moringa mostraram ser tolerantes aos tratamentos aplicados.

In vitro propagation of insectivorous plants for phytochemical purposes

Dissertação mest., Engenharia Biológica, Universidade do Algarve, 2009