El prisma sedimentario submarino ligado al Younger Dryas en la plataforma continental de Benidorm (Alicante, SE de España)

Con el objetivo de ampliar los conocimientos en relación a los cambios recientes del nivel en la plataforma continental de Alicante se ha procedido al estudio de diferentes perfiles sísmicos de alta resolución. En el presente trabajo se ha identificado un prisma sedimentario cuyo techo se ubica alrededor de los 60 metros por debajo del nivel del mar actual. Comparando los resultados con la curva de variaciones del nivel del mar de los últimos 20 milenios, se puede confirmar que éste se formó durante un periodo en que el nivel del mar permaneció relativamente constante o disminuyó levemente entre el final del Bølling Warming y tras el evento frío Younger Dryas. Un total de tres unidades sísmicas (1, 2 y 3 de base a techo) se pueden diferenciar en el prisma. La subunidad 1, depositada entre el final del Bølling Warming y antes del Younger Dryas, indicando una mayor ralentización de la tasa de ascenso del nivel del mar. La subunidad 2, formada durante el evento frío Younger Dryas, reflejando un episodio de detención del nivel del mar alrededor de los 60 metros, seguido de una caída relativa mismo. La subunidad 3, formada tras este periodo frío y que se caracteriza por un incremento de la tasa de ascenso del nivel del mar.

Lessons from an estivating frog: sparing muscle protein despite starvation and disuse

Long (6- to 9-mo) bouts of estivation in green-striped burrowing frogs lead to 28% atrophy of cruralis oxidative fibers (P < 0.05) and some impairment of in vitro gastrocnemius endurance (P < 0.05) but no significant deficit in maximal twitch force production. These data suggest the preferential atrophy of oxidative fibers at a rate slower than, but comparable to, laboratory disuse models. We tested the hypothesis that the frog limits atrophy by modulating oxidative stress. We assayed various proteins at the transcript level and verified these results for antioxidant enzymes at the biochemical level. Transcript data for NADH ubiquinone oxidoreductase subunit 1 (71% downregulated, P < 0.05) and ATP synthase (67% downregulated, P < 0.05) are consistent with mitochondrial quiescence and reduced oxidant production. Meanwhile, uncoupling protein type 2 transcription (P < 0.31), which is thought to reduce mitochondrial leakage of reactive oxygen species, was maintained. Total antioxidant defense of water-soluble (22.3 +/- 1.7 and 23.8 +/- 1.5 mu M/mu g total protein in control and estivator, respectively, P = 0.53) and membrane-bound proteins (31.5 +/- 1.9 and 42.1 +/- 7.3 mu M/mu g total protein in control and estivator, respectively, P = 0.18) was maintained, equivalent to a bolstering of defense relative to oxygen insult. This probably decelerates muscle atrophy by preventing accumulation of oxidative damage in static protein reserves. Transcripts of the mitochondrially encoded antioxidant superoxide dismutase type 2 ( 67% downregulated, P < 0.05) paralleled mitochondrial activity, whereas nuclear-encoded catalase and glutathione peroxidase were maintained at control values (P = 0.42 and P = 0.231), suggesting a dissonance between mitochondrial and nuclear antioxidant expression. Pyruvate dehydrogenase kinase 4 transcription was fourfold lower in estivators (P = 0.11), implying that, in contrast to mammalian hibernators, this enzyme does not drive the combustion of lipids that helps spare hypometabolic muscle.

Studies of molecular interactions using physical techniques

The investigations described in this thesis concern the molecular interactions between polar solute molecules and various aromatic compounds in solution. Three different physical methods were employed. Nuclear magnetic resonance (n.m.r.) spectroscopy was used to determine the nature and strength of the interactions and the geometry of the transient complexes formed. Cryoscopic studies were used to provide information on the stoichiometry of the complexes. Dielectric constant studies were conducted in an attempt to confirm and supplement the spectroscopic investigations. The systems studied were those between nitromethane, chloroform, acetonitrile (solutes) and various methyl substituted benzenes. In the n.m.r. work the dependence of the solute chemical shift upon the compositions of the solutions was determined. From this the equilibrium quotients (K) for the formation of each complex and the shift induced in the solute proton by the aromatic in the complex were evaluated. The thermodynamic parameters for the interactions were obtained from the determination of K at several temperatures. The stoichiometries of the complexes obtained from cryoscopic studies were found to agree with those deduced from spectroscopic investigations. For most systems it is suggested that only one type of complex, of 1:1 stiochiometry, predominates except that for the acetonitrile-benzene system a 1:2 complex is formed. Two sets of dielectric studies were conducted, the first to show that the nature of the interaction is dipole-induced dipole and the second to calculate K. The equilibrium quotients obtained from spectroscopic and dielectric studies are compared. Time-averaged geometries of the complexes are proposed. The orientation of solute, with respect to the aromatic for the 1:1 complexes, appears to be the one in which the solute lies symmetrically about the aromatic six-fold axis whereas for the 1:2 complex, a sandwich structure is proposed. It is suggested that the complexes are formed through a dipole-induced dipole interaction and steric factors play some part in the complex formation.

Fungi distribution in poultry feed

Feed can easily be contaminated and colonized by fungi that use up the nutrients for their own metabolism and growth, producing secondary metabolites such as mycotoxins that are not eliminated throughout the feed processing. The major problems associated with mycotoxin contaminated animal feed are metabolic disturbances resulting in poor animal productivity. In addition, handling contaminated animal feed can also raise health issues regarding workers exposure to fungi and mycotoxins. The scope of this work was to characterize fungal distribution in 11 poultry feed samples. Twenty grams of feed were suspended in 180 mL of distilled water and homogenized during 20 minutes at 200 rpm. The washed supernatant was plated in malt extract agar (MEA) and dichloran glycerol agar base (DG18) media for morphological identification of the mycobiota present. Using macro- and microscopic analysis of the colonies, fungal contamination was evident in 72.7% of the analyzed poultry feed samples. Fungal load ranged from 0 to 13140 CFU/g, and the most prevalent species/genera were F. graminearum complex (71.1%), Penicillium sp. (11.6%), Cladosporium sp. (8.8%), and Fusarium poae (3.6%). In addition to these species, we also isolated Aspergillus sections Circumdati, Nigri and Aspergilli, and Mucor and Rhizopus genus albeit at a lower abundance. The data obtained showed that, besides high fungal contamination, mycotoxins contamination is probably a reality, particularly in the final product since mycotoxins resist to all the processing operations including thermal treatment. Additionally, data claimed attention for the probable co-exposure to fungi and mycotoxins of the workers in feed industries.

The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae

The modified nucleoside 1-methyladenosine (m1A) is found at position 58 in the TΨC loop of many eukaryotic tRNAs. The absence of m1A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNAiMet). Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosylmethionine (AdoMet). These facts, plus our demonstration that gcd14Δ cells lacked m1A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m1A)methyltransferase [(m1A)MTase]. Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m1A in either total tRNA or purified tRNAiMet lacking only this modification. Kinetic analysis of the purified complex revealed KM values for AdoMet or tRNAiMet of 5.0 μM and 2.5 nM, respectively. Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m1A)MTase activity in vitro. Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.

Regulation of cyclin D1 by the BRCA1-BARD1 complex

Background BRCA1 and cyclin D₁ are both essential for normal breast development and mutation or aberration of their expression is associated with breast cancer [1,2]. Cyclin D₁ is best known as a G₁ cyclin where it regulates the G₁ to S phase transition by acting as a rate-limiting subunit of CDK4/6 kinase activity. More recently, however, Stacey has demonstrated that cyclin D₁ levels in G₂/M determine whether a cell continues to proliferate or exits the cell cycle [3]. The majority of BRCA1 in the cell is bound to BARD1 through their N-terminal RING domains. Heterodimerization is essential for the stability and correct localization of the complex and confers ubiquitin ligase activity to BRCA1. The importance of the ligase activity of BRCA1 to breast cancer development is inferred from the fact that N-terminal diseaseassociated mutations are proposed to reduce ligase activity [4]. Methods Protein–protein interactions were demonstrated using yeast-two-hybrid and coimmunoprecipitation. Protein levels were altered through overexpression, siRNA and antisense technology. The effect of proteasome inhibitors and cycloheximide treatment was also examined. Results We initially identified cyclin D₁ as a binding partner of BARD1 in a yeast-two-hybrid screen and defined the minimal binding region as the N-terminus of BARD1. This interaction was confirmed in vivo by coimmunoprecipitation. The N-terminus of BARD1 also binds BRCA1 and imparts ubiquitin ligase activity to the complex. Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Traditionally polyubiquitin chains linked through lysine 48 target proteins for degradation by the 26 S proteasome. We have demonstrated that cyclin D₁ protein levels are inversely related to BRCA1 and BARD1 levels in several model systems. Furthermore, regulation of cyclin D1 levels occurs through a post-transcriptional mechanism and requires the ligase activity of BRCA1. Interestingly, this phenomenon is cell-cycle regulated, occurring in G₂/M. Conclusion We propose that cyclin D₁ is a potential substrate for BRCA1 ubiquitination and that this targets cyclin D₁ for proteasomal-mediated degradation. Future work will focus on ascertaining the functional consequence of cyclin D₁ regulation by the BRCA1–BARD1 complex; in particular, the impact of BRCA1, mediated through regulation of cyclin D₁, on the proliferation versus differentiation decision.

Recruitment of IRAK to the interleukin 1 receptor complex requires interleukin 1 receptor accessory protein

The proinflammatory cytokine interleukin 1 (IL-1) activates the transcription of many genes encoding acute phase and proinflammatory proteins, a function mediated primarily by the transcription factor NF-κB. An early IL-1 signaling event is the recruitment of the Ser/Thr kinase IRAK to the type I IL-1 receptor (IL-1RI). Here we describe the function of a previously identified IL-1 receptor subunit designated IL-1 receptor accessory protein (IL-1RAcP). IL-1 treatment of cells induces the formation of a complex containing both IL-1RI and IL-1RAcP. IRAK is recruited to this complex through its association with IL-1RAcP. Overexpression of an IL-1RAcP mutant lacking its intracellular domain, the IRAK-binding domain, prevented the recruitment of IRAK to the receptor complex and blocked IL-1-induced NF-κB activation.

Replication Factor C3 of Schizosaccharomyces pombe, a Small Subunit of Replication Factor C Complex, Plays a Role in Both Replication and Damage Checkpoints

We report here the isolation and functional analysis of the rfc3+ gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3+ gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3+ is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.

Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae

The Snf1 protein kinase family has been conserved in eukaryotes. In the yeast Saccharomyces cerevisiae, Snf1 is essential for transcription of glucose-repressed genes in response to glucose starvation. The direct interaction between Snf1 and its activating subunit, Snf4, within the kinase complex is regulated by the glucose signal. Glucose inhibition of the Snf1-Snf4 interaction depends on protein phosphatase 1 and its targeting subunit, Reg1. Here we show that Reg1 interacts with the Snf1 catalytic domain in the two-hybrid system. This interaction increases in response to glucose limitation and requires the conserved threonine in the activation loop of the kinase, a putative phosphorylation site. The inhibitory effect of Reg1 appears to require the Snf1 regulatory domain because a reg1Δ mutation no longer relieves glucose repression of transcription when Snf1 function is provided by the isolated catalytic domain. Finally, we show that abolishing the Snf1 catalytic activity by mutation of the ATP-binding site causes elevated, constitutive interaction with Reg1, indicating that Snf1 negatively regulates its own interaction with Reg1. We propose a model in which protein phosphatase 1, targeted by Reg1, facilitates the conformational change of the kinase complex from its active state to the autoinhibited state.

Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

Organization and chromosomal localization of the gene encoding the mouse acid labile subunit of the insulin-like growth factor binding complex.

After birth, most of insulin-like growth factor I and II (IGFs) circulate as a ternary complex formed by the association of IGF binding protein 3-IGF complexes with a serum protein called acid-labile subunit (ALS). ALS retains the IGF binding protein-3-IGF complexes in the vascular compartment and extends the t1/2 of IGFs in the circulation. Synthesis of ALS occurs mainly in liver after birth and is stimulated by growth hormone. To study the basis for this regulation, we cloned and characterized the mouse ALS gene. Comparison of genomic and cDNA sequences indicated that the gene is composed of two exons separated by a 1126-bp intron. Exon 1 encodes the first 5 amino acids of the signal peptide and contributes the first nucleotide of codon 6. Exon 2 contributes the last 2 nt of codon 6 and encodes the remaining 17 amino acids of the signal peptide as well as the 580 amino acids of the mature protein. The polyadenylylation signal, ATTAAA, is located 241 bp from the termination codon. The cDNA and genomic DNA diverge 16 bp downstream from this signal. Transcription initiation was mapped to 11 sites over a 140-bp TATA-less region. The DNA fragment extending from nt -805 to -11 (ATG, +1) directed basal and growth hormone-regulated expression of a luciferase reporter plasmid in the rat liver cell line H4-II-E. Finally, the ALS gene was mapped to mouse chromosome 17 by fluorescence in situ hybridization.

Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.

The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.

The structure of the copper(II) complex with 4,5-dichlorophthalic acid: The 1:1 complex 'adduct' salt trans-tetraaqua(2-carboxy-4,5-dichlorobenzoato)(4,5-dichlorobenzene-1,2-dicarboxylic acid)copper(II) 2-carboxy-4,5-dichlorobenzoate

The unusual (1:1) complex ‘adduct’ salt of copper(II) with 4,5-dichlorophthalic acid (H2DCPA), having formula [Cu(H2O)4(C8H3Cl2O4) (C8H4Cl2O4)] . (C8H3Cl2O4) has been synthesized and characterized using single-crystal X-ray diffraction. Crystals are monoclinic, space group P21/c, with Z = 4 in a cell with dimensions a = 20.1376(7), b =12.8408(4) c = 12.1910(4) Å, β = 105.509(4)o. The complex is based on discrete tetragonally distorted octahedral [CuO6] coordination centres with the four water ligands occupying the square planar sites [Cu-O, 1.962(4)-1.987(4) Å] and the monodentate carboxyl-O donors of two DCPA ligand species in the axial sites. The first of these bonds [Cu-O, 2.341(4) Å] is with an oxygen of a HDCPA monoanion, the second with an oxygen of a H2DCPA acid species [Cu-O, 2.418(4) Å]. The un-coordinated ‘adduct’ molecule is a HDCPA counter anion which is strongly hydrogen-bonded to the coordinated H2DCPA ligand [O… O, 2.503(6) Å] while a number of peripheral intra- and intermolecular hydrogen-bonding interactions give a two-dimensional network structure.

Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex

Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G1-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G1-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G 1 into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.