Estudi estructural, ultraestructural i histoquímic de les glàndules sexuals accessòries del mascle reproductor porcí (Sus domesticus)

El present treball analitza al microscopi òptic i al microscopi electrònic de transmissió les glàndules sexuals accessòries de Sus domesticus (raça Landrace - varietat anglesa) a partir de mascles reproductors porcins adults i sans. Un millor coneixement dels patrons estructural i ultraestructural normals de las glàndules sexuals accessòries permetrà diagnosticar amb facilitat quina ha estat l'estructura o la funció glandular afectada en mascles en els que s'observa una disminució de la qualitat del semen. Per altra banda, els estudis anatomopatològics s'han de complementar amb tècniques histoquímiques que generalment permeten confirmar o excloure un diagnòstic histopatològic previ. Les glàndules sexuals accessòries del mascle reproductor porcí estan molt desenvolupades i inclouen les glàndules vesiculars, la pròstata i les glàndules bulbouretrals. L'epiteli secretor de les glàndules vesiculars està format per cèl·lules columnars, cèl·lules basals i mastòcits. Les cèl·lules columnars es caracteritzen per presentar tres morfologies diferents que es consideren diferents estadis d'un mateix tipus cel·lular: les cèl·lules principals, les cèl·lules clares i les cèl·lules denses. Les cèl·lules principals secreten activament glicoproteïnes N- i O- glicosilades amb residus d'α-L-fucosa, α(16)fucosa, α-D-mannosa, α-D-glucosa, α- i -D-N-acetilgalactosamina, -D-galactosa-(13)-D-N-acetilgalactosamina, α-D-galactosa, galactosa-(14)-N-acetilglucosamina, D-N-acetilglucosamina i àcid neuramínic. Aquestes glicoproteïnes afavoreixen les interaccions entre l'espermatozoide i l'occit i regulen la permeabilitat de la membrana espermàtica. La pròstata està formada per dues porcions glandulars, el cos de la pròstata (BP) y la pròstata disseminada (DP), entre las quals s'observen diferencies estructurals, ultraestructurals, histoquímiques i funcionals. En ambdues porcions, l'epiteli secretor està constituït per cèl·lules columnars principals, denses i cèl·lules basals, i també per cèl·lules cúbiques en el BP i per cèl·lules mucoses en la DP. En ambdues porcions glandulars, se sintetitzen i secreten N- i O- glicoproteïnes neutres i àcides. Aquestes glicoproteïnes s'alliberen mitjançant un mecanisme regulat en el BP y mitjançant un mecanisme regulat i un constitutiu en la DP. Les glucoproteïnes luminals del BP contenen residus de fucosa, mannosa, α- i -D-N-acetilgalactosamina, galactosa-(14)-N-acetilglucosamina, D-N-acetilglucosamina i àcid neuramínic. En la DP les glicoproteïnes presenten, a més, -D-galactosa-(13)-D-N-acetilgalactosamina i α-D-galactosa. Les glicoproteïnes secretades en el BP i en la DP per via regulada, participen en el control de l'estabilitat del plasmalemma dels espermatozoides, eviten la resposta immune uterina i l'aglutinació dels espermatozoides i afavoreixen la seva motilitat progressiva. Les glicoproteïnes secretades per via constitutiva en la DP protegeixen i lubrifiquen la uretra pelviana. L'epiteli secretor de les glàndules bulbouretrals està format per cèl·lules piramidals principals i denses. Les cèl·lules principals sintetitzen i secreten principalment O-glicoproteïnes àcides carboxilades i sulfatades amb residus glicosídics d'N-acetilgalactosamina, -D-galactosa-(13)-D-N-acetilgalactosamina, α-D-galactosa, D-N-acetilglucosamina i àcid neuramínic. Aquests residus proporcionen resistència a la proteolisi a les O-glicoproteïnes secretades, les quals participen en la lubrificació y protecció de l'epiteli, i intervenen en el control de la permeabilitat del plasmalemma dels espermatozoides i en el transport d'ions al seu través.

Tumour necrosis factor alpha induces rapid reduction in AMPA receptor-mediated calcium entry in motor neurones by increasing cell surface expression of the GluR2 subunit: relevance to neurodegeneration

The AMPA receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) have both been implicated in motor neurone vulnerability in Amyotrophic Lateral Sclerosis/Motor Neurone Disease. TNF alpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNF alpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/ml, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using Fura-2 AM microfluorimetry we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggests that TNF alpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in ALS

Zea mays annexins modulate cytosolic free Ca2+ and generate a Ca2+-permeable conductance

Regulation of reactive oxygen species and cytosolic free calcium ([Ca2+](cyt)) is central to plant function. Annexins are small proteins capable of Ca2+-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+] cyt when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca2+-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca2+-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd3+ and had a Ca2+-to-K+ permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca2+ influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport pathways, regulate [Ca2+] cyt, and may function as peroxidases in vitro.

Annexins: multifunctional components of growth and adaptation

Plant annexins are ubiquitous, soluble proteins capable of Ca2+-dependent and Ca2+-independent binding to endomembranes and the plasma membrane. Some members of this multigene family are capable of binding to F-actin, hydrolysing ATP and GTP, acting as peroxidases or cation channels. These multifunctional proteins are distributed throughout the plant and throughout the life cycle. Their expression and intracellular localization are under developmental and environmental control. The in vitro properties of annexins and their known, dynamic distribution patterns suggest that they could be central regulators or effectors of plant growth and stress signalling. Potentially, they could operate in signalling pathways involving cytosolic free calcium and reactive oxygen species.

Cell entry by enveloped viruses: redox considerations for HIV and SARS-coronavirus

For enveloped viruses, genome entry into the target cell involves two major steps: virion binding to the cell-surface receptor and fusion of the virion and cell membranes. Virus-cell membrane fusion is mediated by the virus envelope complex, and its fusogenicity is the result of an active virus-cell interaction process that induces conformation changes within the envelope. For some viruses, such as influenza, exposure to an acidic milieu within the cell during the early steps of infection triggers the necessary structural changes. However, for other pathogens which are not exposed to such environmental stress, activation of fusogenicity can result from precise thiol/disulfide rearrangements mediated by either an endogenous redox autocatalytic isomerase or a cell-associated oxidoreductase. Study of the activation of HIV envelope fusogenicity has revealed new knowledge about how redox changes within a viral envelope trigger fusion. We discuss these findings and their implication for anti-HIV therapy. In addition, to compare and contrast the situation outlined for HIV with an enveloped virus that can fuse with the cell plasma membrane independent of the redox status of its envelope protein, we review parallel data obtained on SARS coronavirus entry.

Mapping the landscape of the lymphocytic choriomeningitis virus stable signal peptide reveals novel functional domains

The stable signal peptide (SSP) of the lymphocytic choriomeningitis virus surface glycoprotein precursor has several unique characteristics. The SSP is unusually long, at 58 amino acids, and contains two hydrophobic domains, and its sequence is highly conserved among both Old and New World arenaviruses. To better understand the functions of the SSP, a panel of point and deletion mutants was created by in vitro mutagenesis to target the highly conserved elements within the SSP. We were also able to confirm critical residues required for separate SSP functions by trans-complementation. Using these approaches, it was possible to resolve functional domains of the SSP. In characterizing our SSP mutants, we discovered that the SSP is involved in several distinct functions within the viral life cycle, beyond translocation of the viral surface glycoprotein precursor into the endoplasmic reticulum lumen. The SSP is required for efficient glycoprotein expression, posttranslational maturation cleavage of GP1 and GP2 by SKI-1/S1P protease, glycoprotein transport to the cell surface plasma membrane, formation of infectious virus particles, and acid pH-dependent glycoprotein-mediated cell fusion.

Microgemma vivaresi n. sp (Microsporidia, Tetramicridae), infecting liver and skeletal muscle of sea scorpions, Taurulus bubalis (Euphrasen 1786) (Osteichthyes, Cottidae), an inshore, littoral fish

The ultrastructure of a new microsporidian species Microgemmia vivaresi n. sp. causing liver cell xenoma formation in sea scorpions, Taurulus bubalis, is described. Stages of merogony, sporogony, and sporogenesis are mixed in the central cytoplasm of developing xenomas. All stages have unpaired nuclei. Uninucleate and multinucleate meronts lie within vacuoles formed from host endoplasmic reticulum and divide by binary or multiple fission. Sporonts, no longer in vacuoles, deposit plaques of surface coat on the plasma membrane that cause the surface to pucker. Division occurs at the Puckered stage into sporoblast mother cells, on which plaques join up to complete the surface coat. A final binary fission gives rise to sporoblasts. A dense globule, thought to be involved in polar tube synthesis, is gradually dispersed during spore maturation. Spores are broadly ovoid, have a large posterior vacuole, and measure 3.6 mu m x 2.1 pint (fresh). The polar tube has a short wide anterior section that constricts abruptly, then runs posteriad to coil about eight times around the posterior vacuole with granular contents. The polaroplast has up to 40 membranes arranged in pairs mostly attached to the wide region of the polar tube and directed posteriorty around a cytoplasm of a coarsely granular appearance. The species is placed alongside the type species Microgemmia hepaticus Ralphs and Matthews 1986 within the family Tetramicridae, which is transferred from the class Dihaplophasea to the class Haplophasea, as there is no evidence for the occurrence of a diplokaryotic phase.

Reduced incorporation of the influenza B virus BM2 protein in virus particles decreases infectivity

BM2 is the fourth integral membrane protein encoded by the influenza B virus genome. It is synthesized late in infection and transported to the plasma membrane from where it is subsequently incorporated into progeny virus particles. It has recently been reported that BM2 has ion channel activity and may be the functional homologue of the influenza A virus M2 protein acting as an ion channel involved in viral entry. Using a reverse genetic approach it was not possible to recover virus which lacked BM2. A recombinant influenza B virus was generated in which the BM2 AUG initiation codon was mutated to GUG. This decreased the efficiency of translation of BM2 protein such that progeny virions contained only 1/8 the amount of BM2 seen in wild-type virus. The reduction in BM2 incorporation resulted in a reduction in infectivity although there was no concomitant decrease in the numbers of virions released from the infected cells. These data imply that the incorporation of sufficient BM2 protein into influenza B virions is required for infectivity of the virus particles. (C) 2004 Elsevier Inc. All rights reserved.

Time course of gag protein assembly in HIV-1-infected cells: a study by immunoelectron microscopy

Recent biochemical studies have identified high molecular complexes of the HIV Gag precursor in the cytosol of infected cells. Using immunoelectron microscopy we studied the time course of the synthesis and assembly of a HIV Gag precursor protein (pr55gag) in Sf9 cells infected with recombinant baculovirus expressing the HIV gag gene. We also immunolabeled for pr55gag human T4 cells acutely or chronically infected with HIV-1. In Sf9 cells, the time course study showed that the first Gag protein appeared in the cytoplasm at 28-30 h p.i. and that budding started 6-8 h later. Colloidal gold particles, used to visualize the Gag protein, were first scattered randomly throughout the cytoplasm, but soon clusters representing 100 to 1000 copies of pr55gag were also observed. By contrast, in cells with budding or released virus-like particles the cytoplasm was virtually free of gold particles while the released virus-like particles were heavily labeled. Statistical analysis showed that between 80 and 90% of the gold particles in the cytoplasm were seen as singles, as doublets, or in small groups of up to five particles probably representing small oligomers. Clusters of gold particles were also observed in acutely infected lymphocytes as well as in multinuclear cells of chronically infected cultures of T4 cells. In a few cases small aggregates of gold particles were found in the nuclei of T4 lymphocytes. These observations suggest that the Gag polyprotein forms small oligomers in the cytoplasm of expressing cells but that assembly into multimeric complexes takes place predominantly at the plasma membrane. Large accumulations of Gag protein in the cytoplasm may represent misfolded molecules destined for degradation.

Dual role of the beta(2)-adrenergic receptor C terminus for the binding of beta-arrestin and receptor internalization

Homologous desensitization of beta(2)-adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind beta-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by beta-arrestins, we have investigated beta-arrestin interaction and internalization of a set of mutants of the human beta(2)-adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-beta-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of beta-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and beta-arrestin2 translocation. A beta(2)-adrenergic receptor truncated distal to residue 381 interacted normally with beta-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and beta-arrestin, but its last eight amino acids facilitate receptor internalization in concert with beta-arrestin2.

Diallyl disulphide, a beneficial component of garlic oil, causes a redistribution of cell-cycle growth phases, induces apoptosis and enhances butyrate-induced apoptosis in colorectal adenocarcinoma cells (HT-29)

Colon cancer is a leading and expanding cause of death worldwide. A major contributory factor to this disease is diet composition; some components are beneficial (e.g. dietary fibre) whilst others are detrimental (e.g. alcohol). Garlic oil is a prominent dietary constituent that prevents the development of colorectal cancer. This effect is believed to be mainly due to diallyl disulphide (DADS), which selectively induces redox stress in cancerous (rather than normal) cells which leads to apoptotic cell death. However, the detailed mechanism by which DADS causes apoptosis remains unclear. We show that DADS-treatment of colonic adenocarcinoma cells (HT-29) initiates a cascade of molecular events characteristic of apoptosis. These include a decrease in cellular proliferation, translocation of phosphatidylserine to the plasma-membrane outer-layer, activation of caspase-3, genomic-DNA fragmentation and G2/M phase cell-cycle arrest. Short-chain fatty acids (SCFAs), particularly butyrate (abundantly produced in the gut by bacterial fermentation of dietary polysaccharides), enhance colonic cell integrity but, in contrast, inhibit colonic-cancer cell growth. Combining DADS with butyrate augmented the effect of butyrate on HT-29 cells. These results suggest that the anti-cancerous properties of DADS afford greater benefit when supplied with other favourable dietary factors (SCFA/polysaccharides) that likewise reduce colonic tumour development.

Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones

Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by beta-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by beta-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK(1)R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nM, 10 min) induced interaction of NK(1)R and beta-arrestin at the plasma membrane, and the SP-NK(1)R-beta-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK(1)R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H(+)ATPase inhibitor bafilomycin A(1), which prevent endosomal SP degradation, suppressed NK(1)R recycling by >50%. Preincubation of neurones with SP (10 nM, 5 min) desensitized Ca(2+) transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK(1)R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK(1)R-beta-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK(1)R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating beta-arrestin-mediated endosomal signalling.

Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system

BACKGROUND: Bile acids (BAs) regulate cells by activating nuclear and membrane-bound receptors. G protein coupled bile acid receptor 1 (GpBAR1) is a membrane-bound G-protein-coupled receptor that can mediate the rapid, transcription-independent actions of BAs. Although BAs have well-known actions on motility and secretion, nothing is known about the localization and function of GpBAR1 in the gastrointestinal tract. METHODS: We generated an antibody to the C-terminus of human GpBAR1, and characterized the antibody by immunofluorescence and Western blotting of HEK293-GpBAR1-GFP cells. We localized GpBAR1 immunoreactivity (IR) and mRNA in the mouse intestine, and determined the mechanism by which BAs activate GpBAR1 to regulate intestinal motility. KEY RESULTS: The GpBAR1 antibody specifically detected GpBAR1-GFP at the plasma membrane of HEK293 cells, and interacted with proteins corresponding in mass to the GpBAR1-GFP fusion protein. GpBAR1-IR and mRNA were detected in enteric ganglia of the mouse stomach and small and large intestine, and in the muscularis externa and mucosa of the small intestine. Within the myenteric plexus of the intestine, GpBAR1-IR was localized to approximately 50% of all neurons and to >80% of inhibitory motor neurons and descending interneurons expressing nitric oxide synthase. Deoxycholic acid, a GpBAR1 agonist, caused a rapid and sustained inhibition of spontaneous phasic activity of isolated segments of ileum and colon by a neurogenic, cholinergic and nitrergic mechanism, and delayed gastrointestinal transit. CONCLUSIONS & INFERENCES: G protein coupled bile acid receptor 1 is unexpectedly expressed in enteric neurons. Bile acids activate GpBAR1 on inhibitory motor neurons to release nitric oxide and suppress motility, revealing a novel mechanism for the actions of BAs on intestinal motility.

Trafficking and signaling of G protein-coupled receptors in the nervous system: implications for disease and therapy

G protein-coupled receptors (GPCRs) are expressed throughout the nervous system where they regulate multiple physiological processes, participate in neurological diseases, and are major targets for therapy. Given that many GPCRs respond to neurotransmitters and hormones that are present in the extracellular fluid and which do not readily cross the plasma membrane, receptor trafficking to and from the plasma membrane is a critically important determinant of cellular responsiveness. Moreover, trafficking of GPCRs throughout the endosomal system can initiate signaling events that are mechanistically and functionally distinct from those operating at the plasma membrane. This review discusses recent advances in the relationship between signaling and trafficking of GPCRs in the nervous system. It summarizes how receptor modifications influence trafficking, discusses mechanisms that regulate GPCR trafficking to and from the plasma membrane, reviews the relationship between trafficking and signaling, and considers the implications of GPCR trafficking to drug development.

Endosomes: a legitimate platform for the signaling train

Although long regarded as a conduit for the degradation or recycling of cell surface receptors, the endosomal system is also an essential site of signal transduction. Activated receptors accumulate in endosomes, and certain signaling components are exclusively localized to endosomes. Receptors can continue to transmit signals from endosomes that are different from those that arise from the plasma membrane, resulting in distinct physiological responses. Endosomal signaling is widespread in metazoans and plants, where it transmits signals for diverse receptor families that regulate essential processes including growth, differentiation and survival. Receptor signaling at endosomal membranes is tightly regulated by mechanisms that control agonist availability, receptor coupling to signaling machinery, and the subcellular localization of signaling components. Drugs that target mechanisms that initiate and terminate receptor signaling at the plasma membrane are widespread and effective treatments for disease. Selective disruption of receptor signaling in endosomes, which can be accomplished by targeting endosomal-specific signaling pathways or by selective delivery of drugs to the endosomal network, may provide novel therapies for disease.