Functional studies of the deubiquitinating enzymes USP19, USP4 and UCH-L1

El marcaje de proteínas con ubiquitina, conocido como ubiquitinación, cumple diferentes funciones que incluyen la regulación de varios procesos celulares, tales como: la degradación de proteínas por medio del proteosoma, la reparación del ADN, la señalización mediada por receptores de membrana, y la endocitosis, entre otras (1). Las moléculas de ubiquitina pueden ser removidas de sus sustratos gracias a la acción de un gran grupo de proteasas, llamadas enzimas deubiquitinizantes (DUBs) (2). Las DUBs son esenciales para la manutención de la homeostasis de la ubiquitina y para la regulación del estado de ubiquitinación de diferentes sustratos. El gran número y la diversidad de DUBs descritas refleja tanto su especificidad como su utilización para regular un amplio espectro de sustratos y vías celulares. Aunque muchas DUBs han sido estudiadas a profundidad, actualmente se desconocen los sustratos y las funciones biológicas de la mayoría de ellas. En este trabajo se investigaron las funciones de las DUBs: USP19, USP4 y UCH-L1. Utilizando varias técnicas de biología molecular y celular se encontró que: i) USP19 es regulada por las ubiquitin ligasas SIAH1 y SIAH2 ii) USP19 es importante para regular HIF-1α, un factor de transcripción clave en la respuesta celular a hipoxia, iii) USP4 interactúa con el proteosoma, iv) La quimera mCherry-UCH-L1 reproduce parcialmente los fenotipos que nuestro grupo ha descrito previamente al usar otros constructos de la misma enzima, y v) UCH-L1 promueve la internalización de la bacteria Yersinia pseudotuberculosis.

Spermiogenesis particularities of a sperm storage species: Helicolenus dactylopterus (Teleostei: Scorpaenidae)

A study of the spermiogenesis and spermatozoa of Helicolenus dactylopterus was conducted. Females of this species have the capacity to store sperm within their ovaries, and male gametes have a considerable cytoplasmic mass surrounding their heads to survive the long period of intraovarian sperm storage. Our observations show that early spermatids are round-shaped cells and have a spherical nucleus with diffuse chromatin. The nuclear volume decreases as a result of progressive chromatin condensation during spermiogenesis, causing the nucleus to take on a U-shape. Flagellar insertion is not central to the nucleus but consistently occurs at an oblique angle towards one side of it. The flagellum is inserted into the nuclear fossa, without subsequent nuclear rotation. In mature spermatozoa, the flagellum is adjacent to the nucleus. A comparison of the spermatozoa in the testicular lobules and those in the intraovarian storage structures suggests that the increase in volume of the cytoplasmic mass may occur in the posterior region of the testis, in the testicular duct. Spermatozoa enter the ovary in groups that reach the ovarian lumen and are surrounded by the ovarian epithelium for storage in sperm storage crypts

Anàlisi microscòpica de l'esperma ejaculada i de la maduració epididimària dels espermatozoides de Sus domesticus

El present treball analitza la morfologia espermàtica de l'ejaculat de Sus domesticus, la histologia del conducte epididimari i la qualitat de l'esperma epididimari. El material d'estudi prové de mascles reproductors porcins de les races Landrace i Pietrain, sans i sexualment madurs. La metodologia emprada es basa en l'examen al microscopi òptic (camp dar, contrast de fases i contrast interferencial) i al microscopi electrònic (de rastreig i de transmissió). Per a l'anàlisi estadística de les dades s'ha utilitzat el test de la X2 de Pearson (p<0,01). L'estudi de la morfologia espermàtica de l'ejaculat permet distingir diversos tipus de gàmetes que s'han classificat en tres grups: espermatozoides madurs, espermatozoides immadurs i espermatozoides aberrants, així com algunes cèI.lules somàtiques. L'espermatozoide madur de Sus domesticus és un gàmeta típic de mamífer (format per tres parts: cap, peça de connexió i cua) en que destaquen: la forma oval i plana del cap, el desenvolupament d'una protuberància acrosòmica apical en una de les cares del cap i la presencia dels cossos laminars en la peça de connexió. L'espermatozoide immadur es caracteritza per la presencia de la gota citoplasmàtica, el major desenvolupament de la protuberància acrosòmica apical i per la flexibilitat del cap. Els espermatozoides aberrants es descriuen i classifiquen segons la morfologia externa i la morfologia interna, distingint-se una amplia gama de malformacions que afecten les diverses parts de l'espermatozoide. Les cèl·lules somàtiques presents en l'ejaculat ofereixen les característiques pròpies d'un macròfag i se les ha observat englobant espermatozoides immadurs. L'estudi de l'estructura i la ultraestructura de les tres regions anatòmiques de l'epidídim (caput, corpus i cauda) revela que: a) l'epiteli epididimari és pseudoestratificat amb esterocilis, b) cada regió epididimària presenta uns valors característics en relació al diàmetre intern del conducte, a l'alçada de l'epiteli, a la longitud dels esterocilis i al nombre de cèl·lules somàtiques luminals, i c) l'epiteli epididimari esta format per cinc tipus cel·lulars: les cèl·lules principals, les cèl·lules basals, les cèl·lules dares, les cèl·lules estretes i les cèl·lules basòfiles. Dels resultats obtinguts es pot deduir que: a) aquests cinc tipus cel·lulars es distribueixen al llarg del conducte epididimari de forma no homogènia, b) les cèl·lules basals, les cèl·lules principals, les cèI.Iules dares i les cèl·lules estretes són diversos estadis del desenvolupament d'un mateix tipus cel·lular especialitzat en la secreció i reabsorció cel·lular, i c) les cèl·lules basòfiles són les precursores de les cèl·lules somàtiques luminals. La qualitat de l'esperma procedent de les tres regions de l'epidídim ha estat analitzada a partir dels següents paràmetres espermàtics: vitalitat, resistència osmòtica dels acrosomes, estabilitat cefàlica, morfologia, malformacions i aglutinació. La vitalitat espermàtica disminueix progressivament al llarg del conducte epididimari. La resistència osmòtica dels acrosomes s'assoleix en la regió corporal de l'epidídim. L'estabilitat cefàlica dels espermatozoides és més elevada en les dues primeres regions de l'epidídim que en la regió caudal. Cada regió de l'epidídim es caracteritza per una morfologia espermàtica específica: a) el caput es caracteritza per l'elevat percentatge d'espermatozoides immadurs amb gota citoplasmàtica proximal, b) el corpus es caracteritza per l'elevat percentatge d'espermatozoides immadurs amb gota citoplasmàtica distal, i c) el cauda es caracteritza per l'elevat percentatge d'espermatozoides madurs. S'han estudiat les següents malformacions d'origen epididimari: espermatozoides de cua doblegada per l'anell de Jensen (origen en el cauda), espermatozoides de cua enrotllada i espermatozoides de cues fusionades (origen en el corpus). Els espermatozoides perden la capacitat de doblegar la cua per la peça intermèdia a mesura que avancen pel conducte epididimari. L'aglutinació espermàtica tendeix a augmentar progressivament al llarg del conducte epididimari, si bé, no s'han observat variacions significatives en els diversos tipus d'aglutinació. La maduració epididimaria dels espermatozoides de Sus domesticus és un procés lent i complex, i la qualitat de l'ejaculat depèn de que aquesta maduració hagi estat completa. La presencia en l'esperma ejaculat de formes gamètiques pròpies de l'esperma epididimari és un signe d'una incompleta maduració dels espermatozoides; i, pot considerar-se com un paràmetre indicador d'estrés del mascle reproductor, tant més quant més s'assembli a la morfologia espermàtica de la regió cefàlica de l'epidídim.

L'emmagatzematge intraovàric d'esperma en Helicolenus dactylopterus (Pisces: Scorpaeniformes)

El penegal, Helicolenus dactylopterus dactylopterus (Pisces: Scorpaeniformes), és una espècie que habita profunditats d'entre els 200 i 1000 m i presenta una clara distribució batimètrica en funció de la seva talla. Presenta fecundació interna i a l'interior de l'ovari conté estructures d'emmagatzematge que permeten emmagatzemar l'esperma durant períodes de temps considerablement llargs. Les cèl·lules sexuals masculines es mantenen viables gràcies a diverses substàncies nutritives que obtenen de la bossa citoplasmàtica i de l'epiteli criptal que delimita les estructures d'emmagatzematge. Aquest epiteli és també responsable de la seva protecció. Un cop els ous han assolit la maduresa, els espermatozoides són alliberats al lumen ovàric i es dóna la fertilització. És una espècie zigòpara: allibera òvuls fecundats que han estat retinguts al tracte reproductiu femení durant un curt període de temps i, per tant, els embrions són alliberats en estadis molt primerencs de desenvolupament. Així, el penegal presenta una estratègia reproductiva evidentment eficaç.

Modeling chemotaxis reveals the role of reversed phosphotransfer and a bi-functional kinase-phosphatase

Understanding how multiple signals are integrated in living cells to produce a balanced response is a major challenge in biology. Two-component signal transduction pathways, such as bacterial chemotaxis, comprise histidine protein kinases (HPKs) and response regulators (RRs). These are used to sense and respond to changes in the environment. Rhodobacter sphaeroides has a complex chemosensory network with two signaling clusters, each containing a HPK, CheA. Here we demonstrate, using a mathematical model, how the outputs of the two signaling clusters may be integrated. We use our mathematical model supported by experimental data to predict that: (1) the main RR controlling flagellar rotation, CheY6, aided by its specific phosphatase, the bifunctional kinase CheA3, acts as a phosphate sink for the other RRs; and (2) a phosphorelay pathway involving CheB2 connects the cytoplasmic cluster kinase CheA3 with the polar localised kinase CheA2, and allows CheA3-P to phosphorylate non-cognate chemotaxis RRs. These two mechanisms enable the bifunctional kinase/phosphatase activity of CheA3 to integrate and tune the sensory output of each signaling cluster to produce a balanced response. The signal integration mechanisms identified here may be widely used by other bacteria, since like R. sphaeroides, over 50% of chemotactic bacteria have multiple cheA homologues and need to integrate signals from different sources.

Pollen ultrastructure in anther cultures of Datura innoxia. II. The generative-cell wall.

In young pollen grains of Datura innoxia, a wall of the usual hemispherical type separates the 2 gametophytic cells initially and, in the electron microscope, appears as an electron-translucent matrix which is contiguous with the intine. Before detachment of the generative cell from the intine, the matrix decreases in thickness and in places is dispersed altogether leaving the plasmalemmae on either side of it in close apposition. A particularly prominent zone, triangular in profile, is left where the wall joins with the intine. After detachment of the cell, remnants of the matrix can be seen distributed irregularly around the cell and it is supposed that these are partly derived from material in the triangular zone as the cell is drawn away from the intine. The wall residues persist throughout the maturation phase of the pollen and are considered to be either callose resulting from incomplete digestion of the initial wall, or some other polysaccharide material which is unevenly laid down along the wall and concentrated at the junction with the intine. In pollen induced into embryogenesis by anther culture, wall material is also distributed irregularly around the detached cell in a series of discrete zones, but these are more extensive than in vivo, closer together and in many instances highly dilated. The wall profiles thus have a beaded appearance, the 'beads' being connected together by short links of the 2 apposed plasmalemmae. The contents of the swollen zones have a similar electron density to that of the matrix in vivo but also show traces of a fibrillar component. It is postulated that this unusual swelling is a prelude to dispersal of the wall by disruption of the plasmalemmal links and to the establishment of cytoplasmic continuity between the 2 cells. The significance of such binucleate pollen grains in the formation of non-haploid embryos is discussed.

Syk-Dependent Cytokine Induction by Dectin-1 Reveals a Novel Pattern Recognition Pathway for C Type Lectins

Pattern-recognition receptors (PRRs) detect molecular signatures of microbes and initiate immune responses to infection. Prototypical PRRs such as Toll-like receptors (TLRs) signal via a conserved pathway to induce innate response genes. In contrast, the signaling pathways engaged by other classes of putative PRRs remain ill defined. Here, we demonstrate that the β-glucan receptor Dectin-1, a yeast binding C type lectin known to synergize with TLR2 to induce TNFα and IL-12, can also promote synthesis of IL-2 and IL-10 through phosphorylation of the membrane proximal tyrosine in the cytoplasmic domain and recruitment of Syk kinase. syk−/− dendritic cells (DCs) do not make IL-10 or IL-2 upon yeast stimulation but produce IL-12, indicating that the Dectin-1/Syk and Dectin-1/TLR2 pathways can operate independently. These results identify a novel signaling pathway involved in pattern recognition by C type lectins and suggest a potential role for Syk kinase in regulation of innate immunity.

Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice

Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear: cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice. (C) 2009 Elsevier B.V. All rights reserved.

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G(2)/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G(2) regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G(2)/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the Go phase or asynchronously replicating cells. Our data suggested that IBV induces a G(2)/M phase arrest in infected cells to promote favorable conditions for viral replication.

Platelet PECAM-1 inhibits thrombus formation in vivo

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.

The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease

To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat ( RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT ( HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 ( AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract - but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.

Mathematical model for low density lipoprotein (LDL) endocytosis by hepatocytes

Individuals with elevated levels of plasma low density lipoprotein (LDL) cholesterol (LDL-C) are considered to be at risk of developing coronary heart disease. LDL particles are removed from the blood by a process known as receptor-mediated endocytosis, which occurs mainly in the liver. A series of classical experiments delineated the major steps in the endocytotic process; apolipoprotein B-100 present on LDL particles binds to a specific receptor (LDL receptor, LDL-R) in specialized areas of the cell surface called clathrin-coated pits. The pit comprising the LDL-LDL-R complex is internalized forming a cytoplasmic endosome. Fusion of the endosome with a lysosome leads to degradation of the LDL into its constituent parts (that is, cholesterol, fatty acids, and amino acids), which are released for reuse by the cell, or are excreted. In this paper, we formulate a mathematical model of LDL endocytosis, consisting of a system of ordinary differential equations. We validate our model against existing in vitro experimental data, and we use it to explore differences in system behavior when a single bolus of extracellular LDL is supplied to cells, compared to when a continuous supply of LDL particles is available. Whereas the former situation is common to in vitro experimental systems, the latter better reflects the in vivo situation. We use asymptotic analysis and numerical simulations to study the longtime behavior of model solutions. The implications of model-derived insights for experimental design are discussed.

Characterisation of cell death in bagged baby salad leaves

Baby leaf salads are gaining in popularity over traditional whole head lettuce salads in response to consumer demand for greater variety and convenience in their diet. Baby lettuce leaves are mixed, washed and packaged as whole leaves, with a shelf-life of approximately 10 days post-processing. End of shelf-life, as determined by the consumer, is typified by bruising, water-logging and blackening of the leaves, but the biological events causing this phenotype have not been studied to date. We investigated the physiological and ultrastructural characteristics during postharvest shelf-life of two lettuce varieties with very different leaf morphologies. Membrane disruption was an important determinant of cell death in both varieties. although the timing and characteristics of breakdown was different in each with Lollo rossa showing signs of aging such as thylakoid disruption and plastoglobuli accumulation earlier than Cos. Membranes in Lollo rossa showed a later, but more distinct increase in permeability than in Cos. as indicated by electrolyte leakage and the presence of cytoplasmic fragments in the vacuole, but Cos membranes show distinct fractures towards the end of shelf-life. The tissue lost less than 25% fresh weight during shelf-life and there was little protein loss compared to developmentally aging leaves in an ambient environment. Biophysical measurements showed that breakstrength was significantly reduced in Lollo rossa, whereas irreversible leaf plasticity was significantly reduced in Cos leaves. The reversible elastic properties of both varieties changed throughout shelf-life. We compared the characteristics of shelf-life in both varieties of bagged lettuce leaves with other leafy salad crops and discuss the potential targets for future work to improve postharvest quality of baby leaf lettuce. (C) 2007 Elsevier B.V. All rights reserved.

Morphological and physiological changes induced by high hydrostatic pressure in exponential- and stationary-phase cells of Escherichia coli: relationship with cell death

The relationship between a loss of viability and several morphological and physiological changes was examined with Escherichia coli strain J1 subjected to high-pressure treatment. The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min. Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells. In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium. Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments. The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells.

Techniques for quantifying effects of dietary antioxidants on transcription factor translocation and nitric oxide production in cultured cells

Dietary antioxidants can affect cellular processes relevant to chronic inflammatory diseases such as atherosclerosis. We have used non- standard techniques to quantify effects of the antioxidant soy isoflavones genistein and daidzein on translocation of Nuclear Factor-KB (NF-KB) and nitric oxide (NO) production, which are important in these diseases. Translocation was quantified using confocal immunofluoresecence microscopy and ratiometric image analysis. NO was quantified by an electrochemical method after reduction of its oxidation products in cell culture supernatants. Activation of the RAW 264.7 murine monocyte/macrophage cell line increased the ratio of nuclear to cytoplasmic immunostaining for NF-kB. The increase was exacerbated by pre-treatment with genistein or daidzein. To show that decreases could also be detected, pre-treatment with the pine bark extract Pycnogenol (R) r was examined, and found to reduce translocation. NO production was also increased by activation, but was reduced by pre-treatment with genistein or daidzein. In the EA. hy926 human endothelial cell line, constitutive production was detectable and was increased by thrombin. The confocal and electrochemical methods gave data that agreed with results obtained using the established electromobility shift and Griess assays, but were more sensitive, more convenient, gave more detailed information and avoided the use of radioisotopes.