Characterization of high osmolarity-induced vacuole fragmentation in "Saccharomyces c."

La majorité des organelles d'une cellule adaptent leur nombre et leur taille pendant les processus de division cellulaire, de trafic vésiculaire ou suite à des changements environnementaux par des processus de fusion et de fragmentation membranaires. Ceci est valable notamment pour le golgi, les mitochondries, les péroxisomes et les lysosomes. La vacuole est le compartiment terminal de la voie endocytaire dans la levure Saccharomyces cerevisiae\ elle correspond aux lysosomes des cellules mammifères. Suite à un choc hyperosmotique, la vacuole se fragmente en plusieurs petites vésicules. Durant ce projet, cette fragmentation a été étudiée en utilisant la technique de microscopie confocale in vivo. J'ai observé que la division de la vacuole se produit d'une façon asymétrique. La première minute après le choc osmotique, les vacuoles rétrécissent et forment des longues invaginations tubulaires. Cette phase est dépendante de la protéine Vps1, un membre de la famille des protéines apparentées à la dynamine, ainsi que d'un gradient transmembranaire de protons. Pendant les 10-15 minutes qui suivent, des vésicules se détachent dans les régions où l'on observe les invaginations pendant la phase initiale. Cette deuxième phase qui mène à la fission des nouveaux compartiments vacuolaires dépend de la production du lipide PI(3,5)P2 par la protéine Fab1. J'ai établi la suite des événements du processus de fragmentation des vacuoles et propose la possibilité d'un rôle régulateur de la protéine kinase cycline-dépendante Pho85.¦En outre, j'ai tenté d'éclaircir plus spécifiquement le rôle de Vps1 pendant la fusion et fission des vacuoles. J'ai trouvé que tous les deux processus sont dépendants de l'activité GTPase de cette protéine. De plus l'association avec la membrane vacuolaire paraît régulée par le cycle d'hydrolyse du GTP. Vps1 peut lier la membrane sans la présence d'un autre facteur protéinique, ce qui permet de conclure à une interaction directe avec des lipides de la membrane. Cette interaction est au moins partiellement effectuée par le domaine GTPase, ce qui est une nouveauté pour un membre de cette famille de protéines. Une deuxième partie de Vps1, nommée insert B, est impliquée dans la liaison à la vacuole, soit par interaction directe avec la membrane, soit par régulation du domaine GTPase. En assumant que Vps1 détienne deux régions capables de liaison aux membranes, je conclus qu'elle pourrait fonctionner comme facteur de « tethering » lors de la fusion des vacuoles.¦-¦La cellule contient plusieurs sous-unités, appelées organelles, possédant chacune une fonction spécifique. Dépendant des processus qui s'y déroulent à l'intérieur, un environnement chimique spécifique est requis. Pour maintenir ces différentes conditions, les organelles sont séparées par des membranes. Lors de la division cellulaire ou en adaptation à des changements de milieu, les organelles doivent être capables de modifier leur morphologie. Cette adaptation a souvent lieu par fusion ou division des organelles. Le même principe est valable pour la vacuole dans la levure. La vacuole est une organelle qui sert principalement au stockage des aliments et à la dégradation des différents composants cellulaires. Alors que la fusion des vacuoles est un processus déjà bien décrit, la fragmentation des vacuoles a jusqu'ici été peu étudiée. Elle peut être induit par un choc osmotique: à cause de la concentration de sel élevé dans le milieu, le cytosol de la levure perd de l'eau. Par un flux d'eau de la vacuole au cytosol, la cellule est capable d'équilibrer celui-ci. Quand la vacuole perd du volume, elle doit réadapter le rapport entre surface membranaire et volume, ce qui se fait efficacement par une fragmentation d'une grande vacuole en plusieurs petites vésicules. Comment ce processus se déroule d'un point de vue morphologique n'a pas été décrit jusqu'à présent. En analysant la fragmentation vacuolaire par microscopie, j'ai trouvé que celle-ci se déroule en deux phases. Pendant la première minute suivant le choc osmotique, les vacuoles rétrécissent et forment des longues invaginations tubulaires. Cette phase dépend de la protéine Vps1, un membre de la famille des protéines apparentées à la dynamine, ainsi que du gradient transmembranaire de protons. Ce gradient s'établit par une pompe membranaire, la V-ATPase, qui transporte des protons dans la vacuole en utilisant l'énergie libérée par hydrolyse d'ATP. Après cette phase initiale, la formation de nouvelles vésicules vacuolaires dépend de la synthèse du lipide PI(3,5)P2.¦Dans la deuxième partie de l'étude, j'ai tenté de décrire comment Vps1 lie la membrane pour effectuer un remodelage de la vacuole. Vps1 est nécessaire pour la fusion et la fragmentation des vacuoles. J'ai découvert que tous les deux processus dépendent de sa capacité d'hydrolyser du GTP. Ainsi l'association avec la membrane est couplée au cycle d'hydrolyse du GTP. Vps1 peut lier la membrane sans la présence d'une autre protéine, et interagit donc très probablement avec les lipides de la membrane. Deux parties différentes de la protéine sont impliquées dans la liaison, dont une, inattendue, le domaine GTPase.¦-¦Numerous organelles undergo membrane fission and fusion events during cell division, vesicular traffic, or in response to changes in environmental conditions. Examples include Golgi (Acharya et al., 1998) mitochondria (Bleazard et al., 1999) peroxisomes (Kuravi et al., 2006) and lysosomes (Ward et al., 1997). In the yeast Saccharomyces cerevisiae the vacuole is the terminal component of the endocytic pathway and corresponds to lysosomes in mammalian cells. Yeast vacuoles fragment into multiple small vesicles in response to a hypertonic shock. This rapid and homogeneous reaction can serve as a model to study the requirements of the fragmentation process. Here, I investigated osmotically induced fragmentation by time-lapse microscopy. I observe that the small fragmentation products originate directly from the large central vacuole by asymmetric scission rather than by consecutive equal divisions and that fragmentation occurs in two distinct phases. During the first minute, vacuoles shrink and generate deep invaginations, leaving behind tubular structures. This phase requires the dynamin-like GTPase Vps1 and the vacuolar proton gradient. In the subsequent 10-15 minutes, vesicles pinch off from the tubular structures in a polarized fashion, directly generating fragmentation products of the final size. This phase depends on the production of phosphatidylinositol- 3,5-bisphosphate by the Fab1 complex. I suggest a possible regulation of vacuole fragmentation by the CDK Pho85. Based on my microscopy study I established a sequential involvement of the different fission factors.¦In addition to the morphological description of vacuole fragmentation I more specifically aimed to shed some light on the role of Vps1 in vacuole fragmentation and fusion. I find that both functions are dependent on the GTPase activity of the protein and that also the membrane association of the dynamin-like protein is coupled to the GTPase cycle. I found that Vps1 has the capacity for direct lipid binding on the vacuole and that this lipid binding is at least partially mediated through residues in the GTPase domain, a complete novelty for a dynamin family member. A second stretch located in the region of insert Β has also membrane-binding activity or regulates the association with the vacuole through the GTPase domain. Under the assumption of two membrane-binding regions I speculate on Vps1 as a possible tethering factor for vacuole fusion.

Highly diverse TCRα chain repertoire of pre-immune CD8(+) T cells reveals new insights in gene recombination.

Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8(+) T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity.

Long-term transcutaneous monitoring of oxygen tension and carbon dioxide at 42 degrees C in critically ill neonates: improved performance of the tcpo2 monitor with topical metabolic inhibition.

Whereas during the last few years handling of the transcutaneous PO2 (tcPO2) and PCO2 (tcPCO2) sensor has been simplified, the high electrode temperature and the short application time remain major drawbacks. In order to determine whether the application of a topical metabolic inhibitor allows reliable measurement at a sensor temperature of 42 degrees C for a period of up to 12 h, we performed a prospective, open, nonrandomized study in a sequential sample of 20 critically ill neonates. A total of 120 comparisons (six repeated measurements per patient) between arterial and transcutaneous values were obtained. Transcutaneous values were measured with a control sensor at 44 degrees C (conventional contact medium, average application time 3 h) and a test sensor at 42 degrees C (Eugenol solution, average application time 8 h). Comparison of tcPO2 and PaO2 at 42 degrees C (Eugenol solution) showed a mean difference of +0.16 kPa (range +1.60 to -2.00 kPa), limits of agreement +1.88 and -1.56 kPa. Comparison of tcPO2 and PaO2 at 44 degrees C (control sensor) revealed a mean difference of +0.02 kPa (range +2.60 to -1.90 kPa), limits of agreement +2.12 and -2.08 kPa. Comparison of tcPCO2 and PaCO2 at 42 degrees C (Eugenol solution) showed a mean difference of +0.91 (range +2.30 to +0.10 kPa), limits of agreement +2.24 and -0.42 kPa. Comparison of tcPCO2 and PaCO2 at 44 degrees C (control sensor) revealed a mean difference of +0.63 kPa (range 1.50 to -0.30 kPa), limits of agreement +1.73 and -0.47 kPa. CONCLUSION: Our results show that the use of an Eugenol solution allows reliable measurement of tcPO2 at a heating temperature of 42 degrees C; the application time can be prolongued up to a maximum of 12 h without aggravating the skin lesions. The performance of the tcPCO2 monitor was slightly worse at 42 degrees C than at 44 degrees C suggesting that for the Eugenol solution the metabolic offset should be corrected.

Comparison of copper-67- and iodine-125-labeled anti-CEA monoclonal antibody biodistribution in patients with colorectal tumors.

Copper-67 has comparable beta-particle emissions to that of 131I, but it displays more favorable gamma emission characteristics for application in radioimmunotherapy (RIT). This study investigates the potential of 67Cu-labeled monoclonal antibody (MAb) 35 for RIT of colorectal carcinoma. METHODS: Biokinetics of simultaneously injected 67Cu- and 125I-labeled MAb35 were studied in six patients scheduled for surgery of primary colorectal cancer. RESULTS: Whole-body clearance (T 1/2) of 67Cu, estimated from sequential anterior and posterior whole-body scans and corrected for decay of 67Cu, was 41 hr. Serum clearance of 67Cu was faster (27.41 hr) than that of 125I (38.33 hr). Mean tumor uptake of the 67Cu-labeled compound (0.0133% ID/g) exceeded that of 125I (0.0095% ID/g), and tumor-to-blood ratios were higher for 67Cu than for 125I, with averages of 6.07 and 2.41, respectively. The average 67Cu/125I ratio was 1.9 for tumor uptake, 0.7 for blood and 2.6 for tumor-to-blood ratios. Nonspecific liver uptake of 67Cu as calculated from whole-body scans was high in four patients, up to 25% of residual whole-body activity at 48 hr, but did not increase with time. We also observed some nonspecific bowel activity, as well as moderate to high uptake in benign polyps. CONCLUSION: Copper-67-labeled MAb35 is more favorable than its radioiodine-labeled counterpart for RIT of colorectal carcinoma due to higher tumor-to-blood ratios, but the problem of nonspecific liver and bowel uptake must first be overcome. The absolute accumulation of activity in tumor remains low, however, so the probability of cure with this compound alone is questionable. The use of 67Cu as one component of a multimodality adjuvant treatment seems to remain the most appropriate application for RIT.

Cathepsin X cleavage of the beta2 integrin regulates talin-binding and LFA-1 affinity in T cells.

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA-1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA-1. We show that sequential cleavage of C-terminal amino acids from the β(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated β(2) tail, the cleavage of three C-terminal amino acids by CatX resulted in a 1.6-fold increase of talin binding. Removal of one more amino acid resulted in a 2.5-fold increase over the intact tail. CatX cleavage increased talin-binding affinity to the MD but not the MP talin-binding site on the β(2) tail. This was shown by molecular modeling of the β(2) tail/talin F3 complex to be a result of conformational changes affecting primarily the distal-binding site. Analysis of LFA-1 by conformation-specific mAb showed that CatX modulates LFA-1 affinity, promoting formation of high-affinity from intermediate-affinity LFA-1 but not the initial activation of LFA-1 from a bent to extended form. CatX post-translational modifications may thus represent a mechanism of LFA-1 fine-tuning that enables the trafficking of T cells.

Genetic structure of Gymnures (genus Hylomys; Erinaceidae) on continental islands of Southeast Asia: Historical effects of fragmentation

Eustatic sea level changes during Pleistocene climatic fluctuations produced several cycles of connection-isolation among continental islands of the Sunda shelf. To explore the potential effects of these fluctuations, we reconstructed a model of the vicariant events that separated these islands, based on bathymetric information. Among many possible scenarios, two opposite phylogenetic patterns of evolution were predicted for terrestrial organisms living in this region: one is based on the classical allopatric speciation mode of evolution, while the other is the outcome of a sequential dispersal colonization of the archipelago. We tested the applicability of these predictions with an analysis of sequence variation of the cytochrome b gene from several taxa of Hylomys. They were sampled throughout SE-Asia and the Sunda islands. High levels of haplotype differentiation characterize the different island taxa. Such levels of differentiation support the existence of several allopatric species, as was suggested by previous allozyme and morphological data. Also in accordance with previous results, the occurrence of two sympatric species from Sumatra is suggested by their strongly divergent haplotypes. One species, Hylomys suillus maxi, is found both on Sumatra and in Peninsular Malaysia, while the other, H. parvus, is endemic to Sumatra. Its closest relative is H. suillus dorsalis from Borneo. Phylogenetic reconstructions also demonstrate the existence of a Sundaic clade composed of all island taxa, as opposed to those from the continent. Although there is no statistical support for either proposed biogeographic model of evolution, we argue that the sequential dispersal scenario is more appropriate to describe the genetic variation found among the Hylomys taxa. However, despite strong differentiation among island haplotypes, the cladistic relationships between some island taxa could not be resolved. We argue that this is evidence of a rapid radiation, suggesting that the separation of the islands may have been perceived as a simultaneous event rather than as a succession of vicariant events. Furthermore, the estimates of divergence times between the haplotypes of these taxa suggest that this radiation may actually have predated the climatic fluctuations of the Pleistocene. Further refinement of the initial palaeogeographic models of evolution are therefore needed to account for these results.

Neutrophil transepithelial migration: role of toll-like receptors in mucosal inflammation

The symptomatic phases of many inflammatory diseases are characterized by migration of large numbers of neutrophils (PMN) across a polarized epithelium and accumulation within a lumen. For example, acute PMN influx is common in diseases of the gastrointestinal system (ulcerative colitis, Crohn's disease, bacterial enterocolitis, gastritis), hepatobiliary system (cholangitis, acute cholecystitis), respiratory tract (bronchial pneumonia, bronchitis, cystic fibrosis, bronchiectasis), and urinary tract (pyelonephritis, cystitis). Despite these observations, the molecular basis of leukocyte interactions with epithelial cells is incompletely understood. In vitro models of PMN transepithelial migration typically use N-formylated bacterial peptides such as fMLP in isolation to drive human PMNs across epithelial monolayers. However, other microbial products such as lipopolysaccharide (LPS) are major constituents of the intestinal lumen and have potent effects on the immune system. In the absence of LPS, we have shown that transepithelial migration requires sequential adhesive interactions between the PMN beta2 integrin CD11b/CD18 and JAM protein family members. Other epithelial ligands appear to be abundantly represented as fucosylated proteoglycans. Further studies indicate that the rate of PMN migration across mucosal surfaces can be regulated by the ubiquitously expressed transmembrane protein CD47 and microbial-derived factors, although many of the details remain unclear. Current data suggests that Toll-like receptors (TLR), which recognize specific pathogen-associated molecular patterns (PAMPs), are differentially expressed on both leukocytes and mucosal epithelial cells while serving to modulate leukocyte-epithelial interactions. Exposure of epithelial TLRs to microbial ligands has been shown to result in transcriptional upregulation of inflammatory mediators whereas ligation of leukocyte TLRs modulate specific antimicrobial responses. A better understanding of these events will hopefully provide new insights into the mechanisms of epithelial responses to microorganisms and ideas for therapies aimed at inhibiting the deleterious consequences of mucosal inflammation.

Simultaneous biliary drainage and portal vein embolization before extended hepatectomy for hilar cholangiocarcinoma: preliminary experience.

BACKGROUND: Patients with resectable hilar cholangiocarcinoma often present obstructive jaundice and a small future remnant liver (FRL) ratio. A sequential approach comprising preoperative biliary drainage followed by portal vein embolization (PVE) is usually performed but leads to long preoperative management (6-12 weeks) before patients can undergo resection. To simplify and shorten this phase of liver preparation, we developed a new preoperative approach that involves percutaneous biliary drainage and PVE during the same procedure. We report the outcomes of this combined procedure. METHODS: During 1 year, four patients underwent simultaneous biliary drainage and PVE followed 1 month later by surgical resection of hilar cholangiocarcinoma. Liver volumes were assessed by CT before, and 1, and 3 months after the combined procedure. Serum liver enzymes were assessed before and 1 month after the combined procedure. RESULTS: The combined procedure was feasible in all cases, with no related complications. After the combined procedure, transaminases remained stable or decreased, whereas gamma-glutamyl-transpeptidase, alkaline phosphatase, and bilirubin decreased. During the first month, the left lobe volume increased by +27.9 % (range 19-40.9 %). The FRL ratio increased from 24.9 to 33.2 %. All patients underwent R0 liver resection with a favorable postoperative outcome. The remnant liver volume increased by +132 % (range 78-245 %) between 1 and 3 months. CONCLUSIONS: Simultaneous percutaneous biliary drainage and PVE is feasible. This all-in-one preoperative approach greatly decreases waiting time until surgical resection. These encouraging results warrant further investigation to confirm the safety and to evaluate the reduction in the dropout rate for liver resection in this tumor with poor prognosis.

Plasmodium berghei ANKA infection induces thymocyte apoptosis and thymocyte depletion in CBA mice

Immune responses to malaria infections are characterized by strong T and B cell activation, which, in addition of potentially causing immunopathology, are of poor efficacy against the infection. It is possible that the thymus is involved in the origin of immunopathological reactions and a target during malaria infections. This work was developed in an attempt to further clarify these points. We studied the sequential changes in the thymus of CBA mice infected with Plasmodium berghei ANKA, a model in which 60-90% of the infected animals develop cerebral malaria. During the acute phase of infection, different degrees of thymocyte apoptosis were recorded: (1) starry-sky pattern of diffuse apoptosis with maintenance of cortical-medullary structure; (2) intense apoptosis with cortical atrophy, with absence of large cells; (3) severe cortical thymocyte depletion, resulting in cortical-medullary inversion. In the latter, only residual clusters of small thymocytes were observed within the framework of epithelial cells. The intensity of thymus alterations could not be associated with the degree of parasitemia, the expression of clinical signs of cerebral malaria or intensity of brain lesions. The implications of these events for malaria immunity and pathology are discussed.

Use of an immunodominant p17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

A Neospora caninum 17 kDa protein fraction (p17) has been described as an immunodominant antigen (IDA) under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17) was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT, as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86).

Analysis of synaptic-like microvesicle exocytosis of B-cells using a live imaging technique.

Pancreatic β-cells play central roles in blood glucose homeostasis. Beside insulin, these cells release neurotransmitters and other signaling molecules stored in synaptic-like microvesicles (SLMVs). We monitored SLMV exocytosis by transfecting a synaptophysin-pHluorin construct and by visualizing the cells by Total Internal Reflection Fluorescence (TIRF) microscopy. SLMV fusion was elicited by 20 mM glucose and by depolarizing K(+) concentrations with kinetics comparable to insulin secretion. SLMV exocytosis was prevented by Tetanus and Botulinum-C neurotoxins indicating that the fusion machinery of these organelles includes VAMP-2/-3 and Syntaxin-1, respectively. Sequential visualization of SLMVs by TIRF and epifluorescence microscopy showed that after fusion the vesicle components are rapidly internalized and the organelles re-acidified. Analysis of single fusion episodes revealed the existence of two categories of events. While under basal conditions transient fusion events prevailed, long-lasting episodes were more frequent upon secretagogue exposure. Our observations unveiled similarities between the mechanism of exocytosis of insulin granules and SLMVs. Thus, diabetic conditions characterized by defective insulin secretion are most probably associated also with inappropriate release of molecules stored in SLMVs. The assessment of the contribution of SLMV exocytosis to the manifestation of the disease will be facilitated by the use of the imaging approach described in this study.

Identification of a novel 45-kDa cell surface molecule involved in activation of the human Jurkat T cell line.

This report describes a surface molecule, Tp45, which appears to be involved in interleukin 2 production and Ca2+ mobilization by Jurkat cells. The Tp45 molecule was identified by a monoclonal antibody, MX13, on the surface of either T3/TCR+ or T3/TCR- human T cell lines. Biochemical data showed that mAb MX13 precipitated a single polypeptide chain of 45 kDa both under reduced and nonreduced conditions from lysates of 125I-surface-labeled cells. Sequential immunodepletion experiments using lysates of 125I-labeled T3/TCR+ cells showed that Tp45 was distinct from the alpha chain of the TCR complex. However, incubation of such cells with either anti-T3 or anti-TCR monoclonal antibody induced complete modulation of both the T3/TCR complex and Tp45. Conversely, complete modulation of both Tp45 and the T3/TCR complex was observed after incubation with anti-Tp45 antibody. Functional studies showed that anti-Tp45 antibody induced high levels of interleukin 2 production in Jurkat cells. In addition, incubation of these cells with the antibody resulted in Ca2+ mobilization from internal stores. Anti-Tp45 antibody reacted with 3-19% peripheral blood (E-rosette-positive) T cells in individual donors. The magnitude of the proliferative response elicited by anti-Tp45 antibody for peripheral blood T cells was lower than that induced by an anti-T3 antibody. This observation is compatible with the idea that only a subpopulation of T cells is reactive with anti-Tp45. Multicolor flow cytometry analysis showed that the Tp45+ cells belong preferentially to the T8 subset.

Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex.

The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.

Acquired oscillopsia: potential complication after multifocal IOL implantation

A healthy 60-year-old woman had uneventful bilateral sequential cataract surgery with diffractive multifocal intraocular lens (IOL) implantation. Immediately after surgery in the first eye, the patient complained of right monocular oscillopsia during motion. Surgery in the second eye was followed by the same symptoms. Ocular motility was normal. Any movement of head or eye was accompanied by oscillopsia, disappearing immediately upon cessation of movement. Slitlamp examination revealed pseudophacodonesis, without obvious zonular laxity. We postulate that the rapid oscillation of an unsteady multifocal IOL during head or eye movement caused the optical steps to pass in front of the visual axis. Cataract surgeons must be aware of this potential, but rare, complication before deciding to implant a multifocal IOL.

Oocyst wall formation and composition in coccidian parasites

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90% protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.