DNA alignment and Bayesian trees of partial sequences of COI, COIII and the haemocyanin functional unit f-g of 28 octopod species

Background: Octopods have successfully colonised the world's oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods. Results: Analysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence. Conclusions: This study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Effects of high temperature and CO2 on intracellular DMSP in the cold-water coral Lophelia pertusa

Significant warming and acidification of the oceans is projected to occur by the end of the century. CO2 vents, areas of upwelling and downwelling, and potential leaks from carbon capture and storage facilities may also cause localised environmental changes, enhancing or depressing the effect of global climate change. Cold-water coral ecosystems are threatened by future changes in carbonate chemistry, yet our knowledge of the response of these corals to high temperature and high CO2 conditions is limited. Dimethylsulphoniopropionate (DMSP), and its breakdown product dimethylsulphide (DMS), are putative antioxidants that may be accumulated by invertebrates via their food or symbionts, although recent research suggests that some invertebrates may also be able to synthesise DMSP. This study provides the first information on the impact of high temperature (12 °C) and high CO2 (817 ppm) on intracellular DMSP in the cold-water coral Lophelia pertusa from the Mingulay Reef Complex, Scotland (56°49' N, 07°23' W), where in situ environmental conditions are meditated by tidally induced downwellings. An increase in intracellular DMSP under high CO2 conditions was observed, whilst water column particulate DMS + DMSP was reduced. In both high temperature treatments, intracellular DMSP was similar to the control treatment, whilst dissolved DMSP + DMS was not significantly different between any of the treatments. These results suggest that L. pertusa accumulates DMSP from the surrounding water column; uptake may be up-regulated under high CO2 conditions, but mediated by high temperature. These results provide new insight into the biotic control of deep-sea biogeochemistry and may impact our understanding of the global sulphur cycle, and the survival of cold-water corals under projected global change.

An Automatic Quantification and Registration Strategy to Create a Gene Expression Atlas of Zebrafish Embryogenesis

In order to properly understand and model the gene regulatory networks in animals development, it is crucial to obtain detailed measurements, both in time and space, about their gene expression domains. In this paper, we propose a complete computational framework to fulfill this task and create a 3D Atlas of the early zebrafish embryogenesis annotated with both the cellular localizations and the level of expression of different genes at different developmental stages. The strategy to construct such an Atlas is described here with the expression pattern of 5 different genes at 6 hours of development post fertilization.

La ciudad editada: de Las Vegas a Nueva York (1972-1978): Venturi / Scott Brown / Koolhaas

Si hubiese un denominador común entre todas las artes en lo que ha venido llamándose postmodernidad, éste tendría mucho que ver con el final del origen de la obra. Desde la literatura y la música hasta las artes plásticas y la arquitectura, la superación de la modernidad ha estado caracterizada por la sustitución del concepto de creación por el de intervención artística, o lo que es lo mismo, la interpretación de lo que ya existe. A principios del siglo XX los conceptos modernos de creación y origen implicaban tener que desaprender y olvidar todo lo anterior con el ánimo de partir desde cero; incluso en un sentido material Mies sugería la construcción literal de la materia y su movimiento de acuerdo a unas leyes. A partir de la segunda mitad de siglo los planteamientos historicistas empezaron a surgir como reacción ante la amnesia y la supuesta originalidad de los modernos. En este contexto surgen los libros Learning from Las Vegas, 1972 y Delirious New York, 1978, ambos deudores en muchos aspectos con el anterior libro de Venturi, Complexity and Contradiction in Architecture, 1966. Estos dos libros sobre ciudades, alejándose decididamente de las tendencias historicistas de la época, proponían utilizar el análisis crítico de la realidad existente como vehículo para la teoría y el proyecto de manera simultánea, convirtiéndose indirectamente en Manifiestos. Si en un primer momento Venturi, Rossi y otros planteaban acabar con los límites formales establecidos por la modernidad, así como por cualquiera de los cánones anteriores, tomando la totalidad de la obra construida como sistema de referencia, - al igual que hiciera Eliot en literatura, - los libros de Las Vegas y Nueva York sugerían directamente borrar los límites de la propia disciplina, llegando a poner en duda ¿Qué puede ser considerado arquitectura? Sin embargo, debido precisamente a la ausencia total de límites y a la inmensidad del sistema referencial planteado, “todo puede ser arquitectura”, como apuntaba Hans Hollein en 1968, los libros proponen al mismo tiempo definir el campo de actuación de cada cual de manera individual. Los escritos sobre Las Vegas y Nueva York suponen por un lado la eliminación de los limites disciplinares y por otro, la delimitación de ámbitos de trabajo concretos para sus autores: los propios de cada una de las ciudades interpretadas. La primera parte de la Tesis, Lecciones, se ocupa del necesario proceso de aprendizaje y experimentación previo a la acción crítica propiamente dicha. Los arquitectos contemporáneos necesitan acumular material, conocimiento, documentación, experiencias... antes de lanzarse a proponer mediante la crítica y la edición; y al contrario que ocurría con los modernos, cuanto más abundante sea ese bagaje previo más rica será la interpretación. Las ciudades de Roma, Londres y Berlín se entienden por tanto como experiencias capaces de proporcionar a Venturi, Scott Brown y Koolhaas respectivamente, sus “personales diccionarios”, unas interminables imaginerías con las que posteriormente se enfrentarían a los análisis de Las Vegas y Nueva York. La segunda parte, Críticas, se centra en la producción teórica en sí: los dos libros de ciudades analizados en estrecha relación con el Complexity and Contradiction. El razonamiento analógico característico de estos libros ha servido de guía metodológica para la investigación, estableciéndose relaciones, no entre los propios escritos directamente, sino a través de trabajos pertenecientes a otras disciplinas. En primer lugar se plantea un importante paralelismo entre los métodos de análisis desarrollados en estos libros y los utilizados por la crítica literaria, observando que si el new criticism y el nuevo periodismo sirvieron de guía en los escritos de Venturi y Scott Brown, la nouvelle critique y su propuesta de identificación poética fueron el claro referente de Koolhaas al abordar Nueva York. Por otro lado, la relevancia ganada por la actividad de comisariado artístico y la aparición de la figura del curator, como autoridad capaz de utilizar la obra de arte por encima de las intenciones de su propio autor, sirve, al igual que la figura del editor, como reflejo de la acción transformadora y de apropiación llevada a cabo tanto en Learning from Las Vegas, como en Delirious New York. Por último y a lo largo de toda la investigación las figuras de Bergson y Baudelaire han servido como apoyo teórico. A través de la utilización que de sus ideas hicieron Venturi y Koolhaas respectivamente, se ha tratado de mostrar la proximidad de ambos planteamientos desde un punto de vista ideológico. La Inclusión propuesta por Venturi y la ironía utilizada por Koolhaas, la contradicción y la paradoja, no son sino el reflejo de lógicas que en ambos casos reaccionan al mismo tiempo contra idealismo y materialismo, contra modernidad y antimodernidad, en un continuo intento de ser lo uno y lo otro simultáneamente. ABSTRACT If there was a common denominator among all the arts in what has been called postmodernism, it would have much to do with the end of the origin of the artwork. From literature and music to fine arts and architecture, overcoming modernity has been characterized by replacing the concept of artistic creation by the one of intervention, in other words, the interpretation of what already exists. In the early twentieth century modern concepts of creation and origin involved unlearning and forgetting everything before with the firm intention of starting from scratch. Even in a material sense Mies suggested the literal construction of matter and its motion according to laws. From the mid-century historicist approaches began to emerge in response to the amnesia and originality alleged by moderns. In this context appeared the books Learning from Las Vegas, 1972 and Delirious New York, 1978, both debtors in many respects to the previous book by Venturi, Complexity and Contradiction in Architecture, 1966. These two books on cities, which broke away decidedly with the historicist trends of the time, proposed using critical analysis of the existing reality as a vehicle for theory and projecting at the same time, indirectly becoming manifests. If at first Venturi, Rossi and others pose to erase the formal limits set by modernity, as well as any of the canons before, taking the entire work built as a reference system, - as did Eliot in literature - the books on Las Vegas and New York proposed directly erasing the boundaries of the discipline itself, coming to question what could be considered architecture? However, and precisely because of the absence of limits and the immensity of the established framework, - “everything could be architecture” as Hans Hollein pointed in 1968, - the books suggested at the same time the definition of a field of action for each one individually. The cities of Las Vegas and New York represented on the one hand the elimination of disciplinary limits and on the other, the delimitation of specific areas of work to its authors: Those on each of the cities interpreted. The first part of the thesis, Lessons, attend to the necessary process of learning and experimentation before the critical action itself. Contemporary architects need to accumulate material, knowledge, information, experiences... before proposing through criticism and editing; and unlike happened with moderns, the most abundant this prior baggage is, the richest will be the interpretation. Rome, London and Berlin are therefore understood as experiences capable of providing Venturi, Scott Brown and Koolhaas respectively, their “personal dictionaries”, interminable imageries with which they would later face the analysis of Las Vegas and New York. The second part, Critiques, focuses on the theoretical production itself: the two books on both cities analyzed closely with the Complexity and Contradiction. The analogical reasoning characteristic of these books has served as a methodological guide for the research, establishing relationships, not directly between the writings themselves, but through works belonging to other disciplines. First, an important parallel is set between the methods of analysis developed in these books and those used by literary criticism, noting that if the new criticism and new journalism guided Venturi and Scott Brown´s writings, the nouvelle critique and its poetic identification were clear references for Koolhaas when addressing New York. On the other hand, the relevance gained by curating and the understanding of the figure of the curator as an authority capable to use artworks above the intentions of their authors, like the one of the Editor, reflects the appropriation and processing actions carried out both in Learning from Las Vegas, and Delirious New York. Finally and over all the research Bergson and Baudelaire figures resonate continuously. Through the use of their ideas done by Venturi and Koolhaas respectively, the research has tried to show the proximity of both approaches from an ideological point of view. Inclusion, as posed by Venturi and irony, as used by Koolhaas, contradiction and paradox are reflections of the logic that in both cases allow them to react simultaneously against idealism and materialism, against modernism and anti-modernism.

Characterization of anti-anti-idiotypic antibodies that bind antigen and an anti-idiotype

Two mouse monoclonal anti-anti-idiotopic antibodies (anti-anti-Id, Ab3), AF14 and AF52, were prepared by immunizing BALB/c mice with rabbit polyclonal anti-idiotypic antibodies (anti-Id, Ab2) raised against antibody D1.3 (Ab1) specific for the antigen hen egg lysozyme. AF14 and AF52 react with an “internal image” monoclonal mouse anti-Id antibody E5.2 (Ab2), previously raised against D1.3, with affinity constants (1.0 × 109 M−1 and 2.4 × 107 M−1, respectively) usually observed in secondary responses against protein antigens. They also react with the antigen but with lower affinity (1.8 × 106 M−1 and 3.8 × 106 M−1). This pattern of affinities for the anti-Id and for the antigen also was displayed by the sera of the immunized mice. The amino acid sequences of AF14 and AF52 are very close to that of D1.3. In particular, the amino acid side chains that contribute to contacts with both antigen and anti-Id are largely conserved in AF14 and AF52 compared with D1.3. Therapeutic immunizations against different pathogenic antigens using anti-Id antibodies have been proposed. Our experiments show that a response to an anti-Id immunogen elicits anti-anti-Id antibodies that are optimized for binding the anti-Id antibodies rather than the antigen.

The activation domain of the enhancer binding protein p45NF-E2 interacts with TAFII130 and mediates long-range activation of the α- and β-globin gene loci in an erythroid cell line

We have used the interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the human β-globin locus control region and the globin gene promoters as a paradigm to examine the mechanisms governing promoter/enhancer interactions in this locus. We have demonstrated that enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2, p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation domain in the NH2-terminal 80 amino acids of the protein. In contrast to the wild-type protein, expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails to reactivate expression of the endogenous β- or α-globin loci in this cell line. Protein-protein interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the transcription initiation complex, TATA binding protein associated factor TAFII130. These findings suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to the individual promoters.

Loss of the maternal H19 gene induces changes in Igf2 methylation in both cis and trans

Recent investigations have shown that the maintenance of genomic imprinting of the murine insulin-like growth factor 2 (Igf2) gene involves at least two factors: the DNA (cytosine-5-)-methyltransferase activity, which is required to preserve the paternal specific expression of Igf2, and the H19 gene (lying 90 kb downstream of Igf2 gene), which upon inactivation leads to relaxation of the Igf2 imprint. It is not yet clear how these two factors are related to each other in the process of maintenance of Igf2 imprinting and, in particular, whether the latter is acting through cis elements or whether the H19 RNA itself is involved. By using Southern blots and the bisulfite genomic-sequencing technique, we have investigated the allelic methylation patterns (epigenotypes) of the Igf2 gene in two strains of mouse with distinct deletions of the H19 gene. The results show that maternal transmission of H19 gene deletions leads the maternal allele of Igf2 to adopt the epigenotype of the paternal allele and indicate that this phenomenon is influenced directly or indirectly by the H19 gene expression. More importantly, the bisulfite genomic-sequencing allowed us to show that the methylation pattern of the paternal allele of the Igf2 gene is affected in trans by deletions of the active maternal allele of the H19 gene. Selection during development for the appropriate expression of Igf2, dosage-dependent factors that bind to the Igf2 gene, or methylation transfer between the parental alleles could be involved in this trans effect.

Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene

Thyrotropin-releasing hormone (TRH) is a brain hypothalamic hormone that regulates thyrotropin (TSH) secretion from the anterior pituitary and is ubiquitously distributed throughout the brain and other tissues including pancreas. To facilitate studies into the role of endogenous TRH, we have used homologous recombination to generate mice that lack TRH. These TRH−/− mice are viable, fertile, and exhibit normal development. However, they showed obvious hypothyroidism with characteristic elevation of serum TSH level and diminished TSH biological activity. Their anterior pituitaries exhibited an apparent decrease in TSH immunopositive cells that was not due to hypothyroidism. Furthermore, this decrease could be reversed by TRH, but not thyroid hormone replacement, suggesting a direct involvement of TRH in the regulation of thyrotrophs. The TRH−/− mice also exhibited hyperglycemia, which was accompanied by impaired insulin secretion in response to glucose. These findings indicate that TRH−/− mice provide a model of exploiting tertiary hypothyroidism, and that TRH gene abnormalities cause disturbance of insulin secretion resulting in marked hyperglycemia.

Isolation of human and mouse genes based on homology to REC2, a recombinational repair gene from the fungus Ustilago maydis

A human and a mouse gene have been isolated based on homology to a recombinational repair gene from the corn smut Ustilago maydis. The new human (h) gene, termed hREC2, bears striking resemblance to several others, including hRAD51 and hLIM15. hREC2 is located on human chromosome 14 at q23–24. The overall amino acid sequence reveals characteristic elements of a RECA-like gene yet harbors an src-like phosphorylation site curiously absent from hRAD51 and hLIM15. Unlike these two relatives, hREC2 is expressed in a wide range of tissues including lung, liver, placenta, pancreas, leukocytes, colon, small intestine, brain, and heart, as well as thymus, prostate, spleen, and uterus. Of greatest interest is that hREC2 is undetectable by reverse transcription-coupled PCR in tissue culture unless the cells are treated by ionizing radiation.

Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1β are differentially regulated in human blood mononuclear cells and mouse spleen cells

Interleukin (IL)-18, formerly called interferon γ (IFN-γ)-inducing factor, is biologically and structurally related to IL-1β. A comparison of gene expression, synthesis, and processing of IL-18 with that of IL-1β was made in human peripheral blood mononuclear cells (PBMCs) and in human whole blood. Similar to IL-1β, the precursor for IL-18 requires processing by caspase 1. In PBMCs, mature but not precursor IL-18 induces IFN-γ; in whole human blood stimulated with endotoxin, inhibition of caspase 1 reduces IFN-γ production by an IL-1β-independent mechanism. Unlike the precursor for IL-1β, precursor for IL-18 was expressed constitutively in PBMCs and in fresh whole blood from healthy human donors. Western blotting of endotoxin-stimulated PBMCs revealed processed IL-1β in the supernatants via an caspase 1-dependent pathway. However, in the same supernatants, only unprocessed precursor IL-18 was found. Unexpectedly, precursor IL-18 was found in freshly obtained PBMCs and constitutive IL-18 gene expression was present in whole blood of healthy donors, whereas constitutive IL-1β gene expression is absent. Similar to human PBMCs, mouse spleen cells also constitutively contained the preformed precursor for IL-18 and expressed steady-state IL-18 mRNA, but there was no IL-1β protein and no spontaneous gene expression for IL-1β in these same preparations. We conclude that although IL-18 and IL-1β are likely members of the same family, constitutive gene expression, synthesis, and processing are different for the two cytokines.

Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation

Histone deacetylases (HDACs) catalyze the removal of acetyl groups on the amino-terminal lysine residues of core nucleosomal histones. This activity is associated generally with transcriptional repression. We have reported previously that inhibition of HDAC activity by hydroxamic acid-based hybrid polar compounds, such as suberoylanilide hydroxamic acid (SAHA), induces differentiation and/or apoptosis of transformed cells in vitro and inhibits tumor growth in vivo. SAHA is a potentially new therapeutic approach to cancer treatment and is in Phase I clinical trials. In several tumor cell lines examined, HDAC inhibitors alter the expression of less than 1% of expressed genes, including the cell cycle kinase inhibitor p21WAF1. In T24 bladder carcinoma cells, SAHA induces up to a 9-fold increase in p21WAF1 mRNA and protein, which is, at least in part, because of an increase in the rate of transcription of the gene. SAHA causes an accumulation of acetylated histones H3 and H4 in total cellular chromatin by 2 h, which is maintained through 24 h of culture. An increase in the accumulation of acetylated H3 and H4 was detected throughout the p21WAF1 promoter and the structural gene after culture with SAHA. The level of histone acetylation did not change in chromatin associated with the actin and p27 genes, and their mRNA expression was not altered during culture of T24 cells with SAHA. Thus, the present findings indicate that the induction of p21WAF1 by SAHA is regulated, at least in part, by the degree of acetylation of the gene-associated histones and that this induced increase in acetylation is gene selective.

B7–CTLA4 interaction enhances both production of antitumor cytotoxic T lymphocytes and resistance to tumor challenge

Expression of B7-family costimulatory molecules CD80 (B7–1) and CD86 (B7–2) on tumor cells enhances host immunity. However, the role of the two B7 receptors, CD28 and CTLA4 (CD152), on T cells in antitumor immune response has not been clearly elucidated. Based on the effects of anti-CD28 and anti-CTLA4 mAbs on T cell response, it was proposed that CD28-B7 interaction promotes antitumor immunity, whereas B7-CTLA4 interaction down-regulates it. A critical test for the hypothesis is whether selective engagement of CTLA4 receptors by their natural ligands CD80 and CD86 enhances or reduces antitumor immunity. Here we used tumors expressing wild-type and mutant CD80, as well as mice with targeted mutation of CD28, to address this issue. We report that in syngeneic wild-type mice, B7W (W88>A), a CD80 mutant that has lost binding to CD28 but retained binding to CTLA4, can enhance the induction of antitumor cytotoxic T lymphocytes (CTL); B7Y (Y201>A), which binds neither CD28 nor CTLA4, fails to do so. Consistent with these observations, B7W-transfected J558 plasmocytoma and EL4 thymoma grow significantly more slowly than those transfected with either vector alone or with B7Y. Optimal tumor rejection requires wild-type CD80. Moreover, expression of a high level of CD80 on thymoma EL4 cells conveys immunity in mice with a targeted mutation of CD28 gene. Taken together, our results demonstrate that B7-CTLA4 interaction enhances production of antitumor CTL and resistance to tumor challenge and that optimal enhancement of antitumor immunity by CD80 requires its engagement of both CD28 and CTLA4.

Retinoid-related orphan receptor γ (RORγ) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis

To identify the physiological functions of the retinoid-related orphan receptor γ (RORγ), a member of the nuclear receptor superfamily, mice deficient in RORγ function were generated by targeted disruption. RORγ−/− mice lack peripheral and mesenteric lymph nodes and Peyer's patches, indicating that RORγ expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3+ and CD4+ lymphocytes is reduced 6- and 10-fold, respectively, whereas the number of circulating B cells is normal. The thymus of RORγ−/− mice contains 74.4% ± 8.9% fewer thymocytes than that of wild-type mice. Flow cytometric analysis showed a decrease in the CD4+CD8+ subpopulation. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining demonstrated a 4-fold increase in apoptotic cells in the cortex of the thymus of RORγ−/− mice. The latter was supported by the observed increase in annexin V-positive cells. RORγ−/− thymocytes placed in culture exhibit a dramatic increase in the rate of “spontaneous” apoptosis. This increase is largely associated with CD4+CD8+ thymocytes and may, at least in part, be related to the greatly reduced level of expression of the anti-apoptotic gene Bcl-XL. Flow cytometric analysis demonstrated a 6-fold rise in the percentage of cells in the S phase of the cell cycle among thymocytes from RORγ−/− mice. Our observations indicate that RORγ is essential for lymphoid organogenesis and plays an important regulatory role in thymopoiesis. Our findings support a model in which RORγ negatively controls apoptosis in thymocytes.