Functional recycling of C2 domains throughout evolution: A comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches

The C2 domain is one of the most frequent and widely distributed calcium-binding motifs. Its structure comprises an eight-stranded beta-sandwich with two structural types as if the result of a circular permutation. Combining sequence, structural and modelling information, we have explored, at different levels of granularity, the functional characteristics of several families of C2 domains. At the coarsest level,the similarity correlates with key structural determinants of the C2 domain fold and, at the finest level, with the domain architecture of the proteins containing them, highlighting the functional diversity between the various subfamilies. The functional diversity appears as different conserved surface patches throughout this common fold. In some cases, these patches are related to substrate-binding sites whereas in others they correspond to interfaces of presumably permanent interaction between other domains within the same polypeptide chain. For those related to substrate-binding sites, the predictions overlap with biochemical data in addition to providing some novel observations. For those acting as protein-protein interfaces' our modelling analysis suggests that slight variations between families are a result of not only complementary adaptations in the interfaces involved but also different domain architecture. In the light of the sequence and structural genomic projects, the work presented here shows that modelling approaches along with careful sub-typing of protein families will be a powerful combination for a broader coverage in proteomics. (C) 2003 Elsevier Ltd. All rights reserved.

Phosphatidylinositol 3-kinase C2 alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2 alpha (PI3K-C2 alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2 alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2 alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2 alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2 alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2 alpha is required for acquisition of fusion competence in neurosecretion.

Expression of Bf/C2 Gene(S) in the Japanese Medaka Fish

Complement factor B and C2 are two central serine proteases of the alternative and classical complement pathways, respectively, that serve as the catalytic subunits of the C3 convertase. Research has been completed using a female Japanese medaka fish, (Oryzias latipes), and other teleost and elasmobrach species in order to isolate eDNA clones and perform linkage analysis of the Bf/C2 gene(s). To further analyze the evolution of the complement system in teleosts, different tissues than the ones from previous studies of medaka fish were analyzed for the constitutive gene expression of factor B and C2. Bf/C2 sequences were amplified by reverse transcription-polymerase chain reaction with primers corresponding to the common amino acid sequences shared by mammalian Bf and C2. Agarose gel electrophoresis was used to visualize sample bands and to calculate the concentration of gene expression of the Bf/C2 gene(s) in each tissue. All five tissue types, kidney, liver, muscle, testis, and spleen from a male medaka fish demonstrated Bf/C2 gene(s) expression, confirming that the messages of Bf/C2 gene(s) are distributed throughout the medaka fish. Tissues of the spleen, liver, and kidney contained the highest concentrations of expression of Bf/C2 gene( s ), while tissues of the muscle and testis contained the lowest concentrations. This research also determined that RT-PCR allowed for more sensitive analysis of gene expression than other molecular biology techniques such as Northern blotting analysis.

Seawater carbonate chemistry and egg production, hatching and metabolic rates of a Mediterranean copepod species (Acartia clausi) in a laboratory experiment

This study includes the first information on the combined effect of low pH and raised temperature on egg production rate (EP), hatching success (HS), excretion and respiration of the Mediterranean copepod Acartia clausi. Adult individuals of A. clausi and fresh surface seawater were collected at a coastal station in Saronikos Gulf during April 2012. Four different conditions were applied: two different pH levels (present: 8.09 and future: 7.83) at two temperature values (present: 16°C and present+4 °C= 20°C). EP and HS success decreased significantly over the duration of exposure at future pH at both temperature conditions. However, the analysis of the combined effect of pH, T, chlorophyll a and the duration of the experiments on EP and HS revealed that ocean acidification had no discernible effect, whereas warming; food and the duration of exposure were more significant for the reproductive output of A. clausi. Temperature appeared to have a positive effect on respiration and excretion. Acidification had no clear effect on respiration, but a negative effect on the A. clausi excretion was observed. Acidification and warming resulted in the increase of the excretion rate and the increase was higher than that observed by warming only. Our findings showed that a direct effect of ocean acidification on copepod's vital rates was not obvious, except maybe in the case of excretion. Therefore, the combination of acidification with the ambient oligotrophic conditions and the warming could result in species being less able to allocate resources for coping with multiple stressors.

Mesozooplankton in Bornholm Basin, Baltic Sea

The seasonal vertical distribution of mesozooplankton was investigated in the Bornholm Basin in October 1988, July 1991 and April 1992 by means of horizontal tows using a small net with a mesh size of 47 micrometers. This study shows that the vertical distribution of multivoltine copepods is linked with ontogeny and may also change seasonally, even in shallow areas such as the Baltic. Trends between copepod species, stages and seasons were discovered. Secondly, the correlation was investigated between the vertical mesozooplankton distribution on the one hand and chlorophyll a concentration and the density of medusae and fish larvae on the other hand. In several cases the comparison of the shape of the profiles led to greater differentiation than the coefficient of correlation. The abundances found allowed only scant statements about non-copepod taxa.

On the temperature dependence of the rate coefficient of formation of C2+ from C + CH+

[EN] We carry out quasi-classical trajectory caculations for theC + CH+ → C2+ + H reaction on an ad hoc computed high-level ab initio potential energy surface. Thermal rate coefficients at the temperatures of relevance in cold interstellar clouds are derived and compared with the assumed, temperature-independent estimates publicly available in kinetic databases KIDA and UDfA. For a temperature of 10 K the database value overestimates by a factor of two the one obtained by us (thus improperly enhancing the destruction route of CH+ in astrochemical kinetic models) which is seen to double in the temperature range 5–300 K with a sharp increase in the first 50 K. The computed values are fitted via the popular Arrhenius–Kooij formula and best-fitting parameters α = 1:32 X 10-9 cm3s-1, β = 0:10 and γ = 2:19 K to be included in the online mentioned databases are provided. Further investigation shows that the temperature dependence of the thermal rate coefficient better conforms to the recently proposed so-called ‘deformed Arrhenius’ law by Aquilanti and Mundim.

Envelope C2-V3-C3-specific antibodies from HIV-1 infected patients from Angola correlate with neutralization activity in plasma

Poster presented at the 2015 Keystone Symposia Conference X5: HIV Vaccines. Banff, Alberta, Canada, 22-27 March 2015

Assessment of Acartia tonsa effect on Necora puber zoeal stages

A navalheira (Necora puber) é uma das espécies mais consumidas em Portugal, sendo que a maioria dos organismos consumidos é atualmente importada de países do Norte da Europa. Para o sucesso da sua produção em aquacultura, é indispensável a administração de presas adequadas à dimensão das fases larvares e que respondam às suas necessidades nutricionais. Os benefícios da administração de copépodes têm vindo a ser reconhecidos, uma vez que os calanóides possuem um largo espectro de tamanhos, movimentos naturais que promovem a sua captura e elevados níveis de ácidos gordos essenciais. Acartia tonsa é uma das espécies de copépodes mais recomendadas para administrar nos primeiros estágios larvares, mas os seus protocolos de cultivo em grande escala ainda requerem otimização. Neste enquadramento, o presente estudo teve como objetivo a seleção da microalga mais adequada para o cultivo de A. tonsa, possibilitando a avaliação do seu efeito no desenvolvimento larval de N. puber, quando comparado com a administração de Artemia franciscana. Inicialmente, os copépodes foram submetidos a duas dietas distintas: Rhodomonas lens e Tetraselmis chuii, a uma densidade de 1x104 cell.mL-1, tendo sido avaliadas as taxas de eclosão, sobrevivência e produção de ovos. A microalga que promoveu uma melhor performance foi administrada nos cultivos contínuos de A. tonsa. Em relação ao desenvolvimento larvar de N. puber, foram avaliadas as taxas de crescimento, percentagem de estágios ao longo do tempo, bem como a correlação entre o peso e o comprimento das larvas, quando alimentadas com A. tonsa e A. franciscana. Adicionalmente, foi avaliado o perfil de ácidos gordos de ambas as espécies de microalgas e de zooplâncton. R. lens permitiu um melhor desempenho enquanto alimento para cultivos de A. tonsa, promovendo uma taxa de eclosão perto de 90%, enquanto que, com T. chuii os copépodes apenas sobreviveram até ao oitavo dia. Tais resultados poderão dever-se à dimensão celular de R. lens, bem ao seu elevado conteúdo de DHA. Em relação ao desenvolvimento larvar de N. puber, Acartia tonsa promoveu uma taxa de sobrevivência de 89 ± 1,63% em zoea V e o encurtamento da fase larvar. Com ambas as dietas foi demonstrada uma forte correlação entre o peso o tamanho. Foi observada uma melhor performance larvar a partir de zoea II, quando as larvas foram alimentadas com A. tonsa, devido à elevada presença de C 20:5 n3 e C 22:6 n3 e movimentos naturais que promovem a sua captura. Assim, foi possível concluir que as A. tonsa é uma presa adequada para larvas de pequenas dimensões, promovendo elevadas sobrevivências e redução da fase larvar.

Elliptic curves, group law, and efficient computation

This thesis is about the derivation of the addition law on an arbitrary elliptic curve and efficiently adding points on this elliptic curve using the derived addition law. The outcomes of this research guarantee practical speedups in higher level operations which depend on point additions. In particular, the contributions immediately find applications in cryptology. Mastered by the 19th century mathematicians, the study of the theory of elliptic curves has been active for decades. Elliptic curves over finite fields made their way into public key cryptography in late 1980’s with independent proposals by Miller [Mil86] and Koblitz [Kob87]. Elliptic Curve Cryptography (ECC), following Miller’s and Koblitz’s proposals, employs the group of rational points on an elliptic curve in building discrete logarithm based public key cryptosystems. Starting from late 1990’s, the emergence of the ECC market has boosted the research in computational aspects of elliptic curves. This thesis falls into this same area of research where the main aim is to speed up the additions of rational points on an arbitrary elliptic curve (over a field of large characteristic). The outcomes of this work can be used to speed up applications which are based on elliptic curves, including cryptographic applications in ECC. The aforementioned goals of this thesis are achieved in five main steps. As the first step, this thesis brings together several algebraic tools in order to derive the unique group law of an elliptic curve. This step also includes an investigation of recent computer algebra packages relating to their capabilities. Although the group law is unique, its evaluation can be performed using abundant (in fact infinitely many) formulae. As the second step, this thesis progresses the finding of the best formulae for efficient addition of points. In the third step, the group law is stated explicitly by handling all possible summands. The fourth step presents the algorithms to be used for efficient point additions. In the fifth and final step, optimized software implementations of the proposed algorithms are presented in order to show that theoretical speedups of step four can be practically obtained. In each of the five steps, this thesis focuses on five forms of elliptic curves over finite fields of large characteristic. A list of these forms and their defining equations are given as follows: (a) Short Weierstrass form, y2 = x3 + ax + b, (b) Extended Jacobi quartic form, y2 = dx4 + 2ax2 + 1, (c) Twisted Hessian form, ax3 + y3 + 1 = dxy, (d) Twisted Edwards form, ax2 + y2 = 1 + dx2y2, (e) Twisted Jacobi intersection form, bs2 + c2 = 1, as2 + d2 = 1, These forms are the most promising candidates for efficient computations and thus considered in this work. Nevertheless, the methods employed in this thesis are capable of handling arbitrary elliptic curves. From a high level point of view, the following outcomes are achieved in this thesis. - Related literature results are brought together and further revisited. For most of the cases several missed formulae, algorithms, and efficient point representations are discovered. - Analogies are made among all studied forms. For instance, it is shown that two sets of affine addition formulae are sufficient to cover all possible affine inputs as long as the output is also an affine point in any of these forms. In the literature, many special cases, especially interactions with points at infinity were omitted from discussion. This thesis handles all of the possibilities. - Several new point doubling/addition formulae and algorithms are introduced, which are more efficient than the existing alternatives in the literature. Most notably, the speed of extended Jacobi quartic, twisted Edwards, and Jacobi intersection forms are improved. New unified addition formulae are proposed for short Weierstrass form. New coordinate systems are studied for the first time. - An optimized implementation is developed using a combination of generic x86-64 assembly instructions and the plain C language. The practical advantages of the proposed algorithms are supported by computer experiments. - All formulae, presented in the body of this thesis, are checked for correctness using computer algebra scripts together with details on register allocations.

Broadening of transgenic adenocarcinoma of the mouse prostate (TRAMP) model to represent late stage androgen depletion independent cancer

BACKGROUND The transgenic adenocarcinoma of the mouse prostate (TRAMP) model closely mimics PC-progression as it occurs in humans. However, the timing of disease incidence and progression (especially late stage) makes it logistically difficult to conduct experiments synchronously and economically. The development and characterization of androgen depletion independent (ADI) TRAMP sublines are reported. METHODS Sublines were derived from androgen-sensitive TRAMP-C1 and TRAMP-C2 cell lines by androgen deprivation in vitro and in vivo. Epithelial origin (cytokeratin) and expression of late stage biomarkers (E-cadherin and KAI-1) were evaluated using immunohistochemistry. Androgen receptor (AR) status was assessed through quantitative real time PCR, Western blotting, and immunohistochemistry. Coexpression of AR and E-cadherin was also evaluated. Clonogenicity and invasive potential were measured by soft agar and matrigel invasion assays. Proliferation/survival of sublines in response to androgen was assessed by WST-1 assay. In vivo growth of subcutaneous tumors was assessed in castrated and sham-castrated C57BL/6 mice. RESULTS The sublines were epithelial and displayed ADI in vitro and in vivo. Compared to the parental lines, these showed (1) significantly faster growth rates in vitro and in vivo independent of androgen depletion, (2) greater tumorigenic, and invasive potential in vitro. All showed substantial downregulation in expression levels of tumor suppressor, E-cadherin, and metastatis suppressor, KAI-1. Interestingly, the percentage of cells expressing AR with downregulated E-cadherin was higher in ADI cells, suggesting a possible interaction between the two pathways. CONCLUSIONS The TRAMP model now encompasses ADI sublines potentially representing different phenotypes with increased tumorigenicity and invasiveness.