Partial characterization of a non-proteinaceous, low molecular weight antigen of Eimeria tenella

A low molecular weight (LMW) antigen of Eimeria tenella, initially identified using a murine monoclonal antibody (mAb C₃4F₁) raised against E. tenella sporozoites, was partially characterized using enzymatic degradation, solvent extraction, and immunization into various inbred lines of mice. The LMW antigen could be isolated using Folch extraction (methanol/chloroform/water) and the epitope recognized by mAb C₃4F₁ was resistant to degradation by α-amylase, pronase, and proteinase K, but was sensitive to sodium m-periodate treatment or digestion using mixed glycosidases (from Turbo cornutus). These observations suggest that the antigenic epitope recognized by mAb C₃4F₁ is carbohydrate-dependent and, based on our ability to isolate the LMW antigen by Folch extraction, the epitope probably resides on a polar glycolipid. The inability of sporozoite-immunized nude mice to elicit a serum antibody response to this molecule indicates that it acts as a T-dependent antigen. Furthermore, sporozoite-immunized male CBA/N mice (with an X-linked immunodeficiency) also failed to elicit a serum antibody response to this molecule, which is consistent with a carbohydrate antigenic epitope. We propose that this antigenic molecule be designated ET-GL1 to reflect its origin and probable structure (E. tenella glycolipid 1). Received: 30 June 1999 / Accepted: 2 December 1999     

Dithiothreitol prevents age-associated decrease in oocyte/conceptus viability in vitro

The present study was designed to ascertain whether the negative effects on reproductive potential of post-ovulatory ageing in vitro of oocytes can be prevented by antioxidant therapy. Mouse metaphase II (MII) oocytes were aged in vitro for 12 h prior to insemination in the presence of varying concentrations of L-ascorbic acid, 6-methoxy- 2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), L-cystine dihydrochloride, ethylenediaminetetraacetic acid (EDTA), beta- mercaptoethanol and DL-dithiothreitol (DTT). In-vitro ageing of oocytes was associated with lower fertilization rate, higher proportion of concepti exhibiting cellular fragmentation at 24 h post-insemination and lower percentage of concepti reaching the blastocyst stage. Ascorbic acid, Trolox and EDTA had no effect on cellular fragmentation or potential of oocytes for development. However, the probability of an oocyte reaching the blastocyst stage was decreased (P < or = or = 0.05) in oocytes incubated in the presence of L-cystine (50 and 500 microM) and beta-mercaptoethanol (5, 50 and 500 microM) when compared to control aged oocytes. Age-associated cellular fragmentation at 24 h post-insemination was partially prevented (P < or = 0.05) by incubating oocytes in the presence of beta-mercaptoethanol (500 microM). DTT (50 and 500 microM) increased (P < or = 0.05) fertilization rate and number of cells at 81 h post-insemination to levels similar to those exhibited by control oocytes. Furthermore, both age-associated fragmentation at 24 h post-insemination (P < or = 0.05) and decreased potential of oocytes for development to the blastocyst stage (P < or = 0.05) were prevented, at least in part, by culturing oocytes in the presence of DTT (50 microM). Although the mechanism by which DTT exerts its beneficial effects on aged oocytes remains to be elucidated, it may protect oocytes by preventing oxidation of free thiol groups and/or altering a redox-independent signalling pathway that mediates cellular fragmentation and death.      

Regulation of the replication initiator protein p65cdc18 by CDK phosphorylation

Cyclin-dependent kinases (CDKs) promote the initiation of DNA replication and prevent reinitiation before mitosis, presumably through phosphorylation of key substrates at origins of replication. In fission yeast, the p65^cdc18 protein is required to initiate DNA replication and interacts with the origin recognition complex (ORC) and the p34^cdc2 CDK. Here we report that p65^cdc18 becomes highly phosphorylated as cells undergo the G₁→S phase transition. This modification is dependent on p34^cdc2 protein kinase activity, as well as six consensus CDK phosphorylation sites within the p65^cdc18 polypeptide. Genetic interactions between cdc18⁺ and the S-phase cyclin cig2⁺ suggest that CDK-dependent phosphorylation antagonizes cdc18⁺ function in vivo. Using site-directed mutagenesis, we show that phosphorylation at CDK consensus sites directly targets p65^cdc18 for rapid degradation and inhibits its replication activity, as strong expression of a constitutively hypophosphorylated mutant form of p65^cdc18 results in large amounts of DNA over-replication in vivo. Furthermore, the over-replication phenotype produced by this mutant p65^cdc18 is resistant to increased mitotic cyclin/CDK activity, a known inhibitor of over-replication. Therefore, p65^cdc18 is the first example of a cellular initiation factor directly regulated in vivo by CDK-dependent phosphorylation and proteolysis. Regulation of p65^cdc18 by CDK phosphorylation is likely to contribute to the CDK-driven “replication switch” that restricts initiation at eukaryotic origins to once per cell cycle.     

Ultrasonographic assessment of human skeletal muscle size

The measurement of human muscle size is essential when assessing the effects of training, disuse and ageing. The considered gold standard for cross-sectional area measurements of muscle size is magnetic resonance imaging (MRI). However, MRI is costly and often inaccessible. The aim of the present study was to test the reproducibility and validity of a more accessible alternative method using ultrasonography (ULT). We examined the cross-sectional areas in the vastus lateralis muscle of six individuals. Axial-plane ULT scans were taken at given levels along the entire muscle length. The ULT scanning was repeated on different days (reliability) and validated against MRI-based measurements. Mean intraclass correlation coefficients were 0.998 for the reliability of ULT and 0.999 for the validity of ULT against MRI. The coefficient of variation values for cross-sectional area measurements assessed by six different experimenters were 2.1% and 0.8% for images obtained with ULT and MRI, respectively. The ULT method is a valid and reliable alternative tool for assessing cross-sectional areas of large individual human muscles. The present findings justify the application of the ULT method for the detection of changes throughout large muscles in response to training, disuse or as a consequence of sarcopenia.     

Dissecting natural killer cell activation pathways through analysis of genetic mutations in human and mouse

Summary:  Natural killer (NK) cell cytotoxicity is mediated by multiple germ line-encoded activating receptors that recognize specific ligands expressed by tumor cells and virally infected cells. These activating receptors are opposed by NK inhibitory receptors, which recognize major histocompatibility complex class I molecules on potential targets, raising the threshold for NK cell activation. Once an abnormal cell has been detected, NK cells are the sentinel source of cytolytic mediators, such as granzymes and perforins, as well as interferon-γ, which can polarize the immune response to a T-helper 1 cell type. Activation signals are transmitted by adhesion-dependent pathways, immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathways, DAP10 ITAM-independent pathways, and by signaling through immunoreceptor tyrosine-based switch motifs. These pathways activate downstream signaling partners to trigger NK cell cytotoxicity. Some of these downstream molecules are unique to the various pathways, and some of these molecules are shared. Because of the complexity of signals involved in NK cell–target cell interaction, the generation of mice with targeted mutations in signaling molecules involved in adhesion, activation, or inhibition is essential for a precise dissection of the mechanisms regulating NK cell effector functions. Here we review recent advances in the genetic analysis of the signaling pathways that mediate NK cell killing.     

Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator

In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N- terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.      

Nkx2.5‐negative myocardium of the posterior heart field and its correlation with podoplanin expression in cells from the developing cardiac pacemaking and conduction system

Recent advances in the study of cardiac development have shown the relevance of addition of myocardium to the primary myocardial heart tube. In wild‐type mouse embryos (E9.5–15.5), we have studied the myocardium at the venous pole of the heart using immunohistochemistry and 3D reconstructions of expression patterns of MLC‐2a, Nkx2.5, and podoplanin, a novel coelomic and myocardial marker. Podoplanin‐positive coelomic epithelium was continuous with adjacent podoplanin‐ and MLC‐2a‐positive myocardium that formed a conspicuous band along the left cardinal vein extending through the base of the atrial septum to the posterior myocardium of the atrioventricular canal, the atrioventricular nodal region, and the His‐Purkinje system. Later on, podoplanin expression was also found in the myocardium surrounding the pulmonary vein. On the right side, podoplanin‐positive cells were seen along the right cardinal vein, which during development persisted in the sinoatrial node and part of the venous valves. In the MLC‐2a‐ and podoplanin‐positive myocardium, Nkx2.5 expression was absent in the sinoatrial node and the wall of the cardinal veins. There was a mosaic positivity in the wall of the common pulmonary vein and the atrioventricular conduction system as opposed to the overall Nkx2.5 expression seen in the chamber myocardium. We conclude that we have found podoplanin as a marker that links a novel Nkx2.5‐negative sinus venosus myocardial area, which we refer to as the posterior heart field, with the cardiac conduction system. Anat Rec, 290:115–122, 2007. © 2006 Wiley‐Liss, Inc.