Apoptosis of oligodendrocytes via Fas and TNF-R1 is a key event in the induction of experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, immunization with myelin Ags leads to demyelination and paralysis. To investigate which molecules are crucial for the pathogenesis of EAE, we specifically assessed the roles of the death receptors Fas and TNF-R1. Mice lacking Fas expression in oligodendrocytes (ODCs) were generated and crossed to TNF-R1-deficient mice. To achieve specific deletion of a loxP-flanked fas allele in ODCs, we generated a new insertion transgene, expressing the Cre recombinase specifically in ODCs. Fas inactivation alone as well as the complete absence of TNF-R1 protected mice partially from EAE induced by the immunization with myelin ODC glycoprotein. The double-deficient mice, however, showed almost no clinical signs of EAE after immunization. Histological analysis revealed that demyelination was suppressed in CNS tissue and that lymphocyte infiltration was notably reduced. We conclude that the death receptors Fas and TNF-R1 are major initiators of ODC apoptosis in EAE. Although only moderate reduction of lymphocyte infiltration into CNS tissue was observed, the absence of these receptors appears to confer protection from demyelination and development of clinical disease.     

Anesthesia and post-mortem interval profoundly influence the regulatory serine phosphorylation of glycogen synthase kinase-3 in mouse brain

Glycogen synthase kinase-3 (GSK3) is a crucial enzyme contributing to the regulation of neuronal structure, plasticity and survival, is implicated as a contributory factor in prevalent diseases such as Alzheimer's disease and mood disorders and is regulated by a wide range of signaling systems and pharmacological agents. Therefore, factors regulating GSK3 in vivo are currently of much interest. GSK3 is inhibited by phosphorylation of serine-9 or serine-21 in GSK3beta and GSK3alpha, respectively. This study found that accurate measurements of phospho-Ser-GSK3 in brain are confounded by a rapid post-mortem dephosphorylation, with approximately 90% dephosphorylation of both GSK3 isoforms occurring within 2 min post-mortem. Furthermore, three anesthetics, pentobarbital, halothane and chloral hydrate, each caused large in vivo increases in the serine phosphorylation of both GSK3beta and GSK3alpha in several regions of mouse brain. Thus, studies of the phosphorylation state of GSK3 in brain, and perhaps in other tissues, need to take into account post-mortem changes and the effects of anesthetics and there is a direct correlation between anesthesia and high levels of serine-phosphorylated GSK3.     

The long-term effect of mesh bioprosthesis in inguinal hernia repair on testicular nitric oxide metabolism and apoptosis in rat testis

Polypropylene mesh is the most widely used material in inguinal hernia repair. Although polypropylene mesh is known as an inert material, it is experimentally proven that mesh generates a chronic inflammatory tissue reaction. The aim of the present study was to investigate the long-term effects of polypropylene mesh material used in inguinal hernia operations on testicular function, testicular nitric oxide (NO) metabolism and germ cell-specific apoptosis in rats. The study comprised 40 male rats that were randomly allocated into two groups. In group 1, the left spermatic cord was elevated and a 0.5 x 1 cm polypropylene mesh was placed behind the left inguinal spermatic cord and group 2 consisted of the sham-operated controls. Blood samples were taken at 6 months preoperatively and postoperatively after to assess luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels for hormonal evaluation. Testicular NO was evaluated by the Griess method, apoptosis by a TUNEL method and inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) expressions by immunohistochemical staining. Mild (+) eNOS expression was observed in all specimens. Mild (+) iNOS expression was only detected in ipsilateral testis of the mesh-implanted study group. Apoptotic cells were not detected in any samples. We are of the opinion that long-term polypropylene mesh implantation has no effect on testicular hormonal function and only a limited effect on nitric oxide levels and this effect is not sufficient to cause apoptosis in testis that could lead to infertility. It seems that mesh implantation is a reliable method in inguinal hernia repair; however, further work is required by more sensitive methods to fully elucidate the potential testicular damage.     

Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities

Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA-mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1-mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.     

Enthalpy measurement using calorimetry shows a significant difference in potential energy between the active and latent conformations of PAI-1

A central feature of the serpin inhibition mechanism is insertion of the reactive center loop into the central beta-sheet (beta-sheet A). This insertion also occurs when the reactive center loop is cleaved without protease inhibition. Using this effect, we have measured the enthalpy (DeltaH) of loop cleavage and insertion for plasminogen activator inhibitor 1 (PAI-1) as -38 kcal/mol. Because loop insertion can be blocked by incorporating a peptide into the central beta-sheet, it was possible to assign -7 kcal/mol to loop cleavage and -31 kcal/mol to loop insertion. These values are lower than values reported for the serpins alpha 1 -proteinase inhibitor and antithrombin of -53 to -63 kcal/mol, respectively, for loop insertion with negligible enthalpy for loop cleavage. A free energy difference of -9 kcal/mol has been reported between the active and spontaneously loop inserted "latent forms" of PAI-1, which is significantly smaller in magnitude than the -31 kcal/mol of enthalpy we measured for loop insertion. Because the enthalpy should relate closely to those regions of PAI-1 that have moved to lower potential energy, a difference distance matrix is presented that identifies regions of PAI-1 that move during loop insertion.     

Mycorrhiza-plant colonization patterns on a subalpine glacier forefront as a model system of primary succession

Lyman glacier in the North Cascades Mountains of Washington has a subalpine forefront characterized by a well-developed terminal moraine, inconspicuous successional moraines, fluting, and outwash. These deposits were depleted of symbiotic fungi when first exposed but colonized by them over time after exposure. Four major groups of plant species in this system are (1) mycorrhiza-independent or facultative mycotrophic, (2) dependent on arbuscular mycorrhizae (AM) (3) dependent on ericoid mycorrhiza (ERM) or ectomycorrhizae (EM), and (4) colonized by dark-septate (DS) endophytes. We hypothesized that availability of mycorrhizal propagules was related to the success of mycorrhiza-dependent plants in colonizing new substrates in naturally evolved ecosystems. To test this hypothesis roots samples of 66 plant species were examined for mycorrhizal colonization. The plants were sampled from communities at increasing distances from the glacier terminus to compare the newest communities with successively older ones. Long established, secondary successional dry meadow communities adjacent to the glacier forefront, and nearby high alpine communities were sampled for comparison. DS were common on most plant species on the forefront. Nonmycorrhizal plants predominated in the earlier successional sites, whereas the proportion of mycorrhizal plants generally increased with age of community. AM were present, mostly at low levels, and nearly absent in two sites of the forefront. ERM were present in all species of Ericaceae sampled, and EM in all species of Pinaceae and Salicaceae. Roots of plants in the long established meadow and heath communities adjacent to the forefront and the high alpine community all had one or another of the colonization types, with DS and AM predominating.     

Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters

Using total internal reflection fluorescence microscopy (TIR-FM), fluorescence recovery after photobleaching (FRAP), and other light microscopy techniques, we analyzed the dynamics, the activation, and the assembly of caveolae labeled with fluorescently tagged caveolin-1 (Cav1). We found that when activated by simian virus 40 (SV40), a non-enveloped DNA virus that uses caveolae for cell entry, the fraction of mobile caveolae was dramatically enhanced both in the plasma membrane (PM) and in the caveosome, an intracellular organelle that functions as an intermediate station in caveolar endocytosis. Activation also resulted in increased microtubule (MT)-dependent, long-range movement of caveolar vesicles. We generated heterokaryons that contained GFP- and RFP-tagged caveolae by fusing cells expressing Cav1-GFP and -RFP, respectively, and showed that even when activated, individual caveolar domains underwent little exchange of Cav1. Only when the cells were subjected to transient cholesterol depletion, did the caveolae domain exchange Cav1. Thus, in contrast to clathrin-, or other types of coated transport vesicles, caveolae constitute stable, cholesterol-dependent membrane domains that can serve as fixed containers through vesicle traffic. Finally, we identified the Golgi complex as the site where newly assembled caveolar domains appeared first.     

Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo

 The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking. Isolated wheat (Triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured in a hormone-free, maltose-containing medium in the presence of ovaries serving as a conditioning factor. Embryogenesis was followed in microspores isolated from immature anthers of freshly cut tillers or from heat- and starvation-treated, excised anthers. Three types of microspore were identified on the basis of their cytological features at the start of culture. Type-1 microspores had a big central vacuole and a nucleus close to the microspore wall, usually opposite to the germ pore. This type was identical to the late microspore stage in anthers developing in vivo. Microspores with a fragmented vacuole and a peripheral cytoplasmic pocket containing the nucleus were defined as type 2. In type-3 microspores the nucleus was positioned in a cytoplasmic pocket in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores first developed into type 2 and then into type-3 microspores. After a few more days, type-3 microspores absorbed their vacuoles and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures. Apparently the three types of microspore represent stages in a continuous process and not, as previously assumed, distinct classes of responding and non-responding microspores. The first cell division of the embryogenic microspores was always symmetric. Cell tracking also revealed that the original microspore wall opened opposite to a region in the multicellular microspore which consisted of cells containing starch grains while the remaining cells were starch grain-free. The starch-containing cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detected at the root pole of the embryo. Received: 27 December 1999 / Accepted: 11 May 2000     

Hot spots for modulating toxicity identified by genomic phenotyping and localization mapping

DNA repair and checkpoint pathways protect against carcinogen-induced toxicity. Here, we describe additional, equally protective pathways discovered by interrogating 4,733 yeast proteins for their ability to diminish toxicity induced by four known carcinogens. A computational mapping strategy for global phenotypic data was developed to build a systems toxicology model detailing recovery from carcinogen exposure and identifying protein complexes that modulate toxicity. Global phenotypic data were merged with global subcellular localization and protein interactome data to generate an integrated picture of cellular recovery after carcinogen exposure. Statistically validated results from this systems-wide integration demonstrate that, in addition to the nucleus, subnetworks of toxicity-modulating proteins were overrepresented in the vacuolar membrane, endosome, endoplasmic reticulum, and mitochondrion. In addition, we show that many proteins associated with RNA polymerase II, macromolecular trafficking, and vacuole function can now be counted among the many proteins that modulate carcinogen-induced toxicity.