Schizophrenia, eye movements, and biocultural heterogeneity

Smooth-pursuit eye-tracking dysfunction is a putative genetic trait marker for schizophrenia. In this study 88 Japanese schizophrenics from Kyushu and Okinawa were examined for the marker using precise high-resolution instrumentation: 76% of the schizophrenics from Kyushu and 89% of those from Okinawa had pursuit dysfunction. The presence of the culture-neutral smooth-pursuit marker for schizophrenia in Japan demonstrates that the etic concept "schizophrenia" is cross-culturally valid. Furthermore, the ubiquity of the marker in biologically and culturally diverse populations may indicate a limit on the extent of meaningful heterogeneity likely to be discovered within the condition.     

CD48 stimulation by 2B4 (CD244)-expressing targets activates human NK cells

Human NK cells can be activated by a variety of different cell surface receptors. Members of the SLAM-related receptors (SRR) are important modulators of NK cell activity. One interesting feature of the SRR is their homophilic interaction, combining receptor and ligand in the same molecule. Therefore, SRR cannot only function as activating NK cell receptors, but also as activating NK cell ligands. 2B4 (CD244) is the only SRR that does not show homophilic interaction. Instead, 2B4 is activated by binding to CD48, a GPI-anchored surface molecule that is widely expressed in the hemopoietic system. In this study, we show that 2B4 also can function as an activating NK cell ligand. 2B4-expressing target cells can efficiently stimulate NK cell cytotoxicity and IFN-gamma production. Using soluble receptor fusion proteins and SRR-transfected cells, we show that 2B4 does not bind to any other SRR expressed on NK cells, but only interacts with CD48. Lysis of 2B4-expressing target cells can be blocked by anti-CD48 Abs and triggering of CD48 in a redirected lysis assay can stimulate NK cell cytotoxicity. This demonstrates that 2B4 can stimulate NK cell cytotoxicity and cytokine production by interacting with NK cell expressed CD48 and adds CD48 to the growing number of activating NK cell receptors.     

Mammalian Fbh1 is important to restore normal mitotic progression following decatenation stress

We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1^f/f), and the other with a homozygous disruption (Fbh1^?/?). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1^?/? cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1^f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1^f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1^f/f and Fbh1^?/? cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.     

The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.     

Solution structure of the Tn3 resolvase-crossover site synaptic complex

Tn3 resolvase is a site-specific DNA recombinase, which catalyzes strand exchange in a synaptic complex containing twelve resolvase subunits and two res sites. Hyperactive mutants of resolvase can form a simpler complex (X synapse) containing a resolvase tetramer and two shorter DNA segments at which strand exchange takes place (site I). We have solved the low-resolution solution structure of the purified, catalytically competent X synapse from small-angle neutron and X-ray scattering data, using methods in which the data are fitted with models constructed by rigid body transformations of a published crystallographic structure of a resolvase dimer bound to site I. Our analysis reveals that the two site I fragments are on the outside of a resolvase tetramer core and provides some information on the quaternary structure of the tetramer. We discuss implications of our structure for the architecture of the natural synaptic complex and the mechanism of strand exchange.     

Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol

Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP^−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG.     

Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.

Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell.     

EFFECTS OF METAL AND PETROLEUM HYDROCARBON CONTAMINATION ON BENTHIC DIATOM COMMUNITIES NEAR CASEY STATION,ANTARCTICA: AN EXPERIMENTAL APPROACH1

The effects of metals and petroleum hydrocarbons on benthic marine diatom communities were assessed using a manipulative field experiment at Casey Station, Antarctica. Uncontaminated, metal contaminated, and petroleum hydrocarbon contaminated sediments were deployed for 11 weeks during the 1999 austral summer. The treatments were deployed at three different locations: Brown Bay, which has elevated levels of anthropogenic contaminants, and two uncontaminated reference locations, O'Brien Bay and Sparkes Bay, the latter of which has naturally occurring high concentrations of some heavy metals. At each location, significant differences between the composition of diatom communities recruiting to control and petroleum hydrocarbon contaminated treatments were observed. Navicula directa (Smith) Ralfs occurred at lower abundances in the petroleum hydrocarbon contaminated treatments than in the control treatments. In contrast, Navicula cancellata Donkin occurred at higher relative abundances in both contaminated treatments relative to the control treatment. Interactions between treatment and location were also observed for several species, including Navicula glaciei Van Heurck. Significant differences in the overall community composition of diatom communities between control and metal contaminated treatments and metal contaminated and petroleum hydrocarbon contaminated treatments were only observed within Brown Bay. The location of deployment also had a significant influence on the composition of diatom communities. Brown Bay had higher abundances of Achnanthes brevipes Agardh but lower abundances of Navicula aff. cincta (Ehrenberg) Van Heurck than either reference locations. This experiment demonstrated that benthic diatom communities are sensitive to sediment contamination and would be suitable for future monitoring work within this and other areas of Antarctica.     

The toad fly Lucilia bufonivora: its evolutionary status and molecular identification

The blow fly genus Lucilia is composed largely of saprophages and facultative myasis agents, including the economically important species Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) and Lucilia sericata (Meigen). Only one species is generally recognized as an obligate agent of myiasis, Lucilia bufonivora Moniez, and this is an obligate parasite of toads. Lucilia silvarum (Meigen), a sister species, behaves mainly as a carrion breeder; however, it has also been reported as a facultative parasite of amphibians. Morphologically, these species are almost identical, and historically this has led to misidentification, taxonomic ambiguity and a paucity of studies of L. bufonivora. In this study, dipterous larvae were analysed from toad myiasis cases from the U.K., The Netherlands and Switzerland, together with adult specimens of fly species implicated in amphibian parasitism: L. bufonivora, L. silvarum and Lucilia elongata Shannon (from North America). Partial sequences of two genes, cox1 and ef1α, were amplified. Seven additional blow fly species were analysed as outgroups. Bayesian inference trees of cox1, ef1α and a combined‐gene dataset were constructed. All larvae isolated from toads were identified as L. bufonivora and no specimens of L. silvarum were implicated in amphibian myiasis. This study confirms L. silvarum and L. bufonivora as distinct sister species and provides unambiguous molecular identification of L. bufonivora.     

Structural properties of recombinant nonrepetitive and repetitive parts of major ampullate spidroin 1 from Euprosthenops australis: implications for fiber formation

Spider dragline silk proteins, spidroins, have a tripartite composition; a nonrepetitive N-terminal domain, a central repetitive region built up from many iterated poly-Ala and Gly rich blocks, and a C-terminal nonrepetitive domain. It is generally believed that the repetitive region forms intermolecular contacts in the silk fibers, while precise functions of the terminal domains have not been established. Herein, thermal, pH, and salt effects on the structure and aggregation and/or polymerization of recombinant N- and C-terminal domains, a repetitive segment containing four poly-Ala and Gly rich coblocks, and combinations thereof were studied. The N- and C-terminal domains have mainly alpha-helical structure, and interestingly, both form homodimers. Dimerization of the end domains allows spidroin multimerization independent of the repetitive part. Reduction of an intersubunit disulfide in the C-terminal domain lowers the thermal stability but does not affect dimerization. The repetitive region shows helical secondary structure but appears to lack stable folded structure. A protein composed of this repetitive region linked to the C-terminal domain has a mainly alpha-helical folded structure but shows an abrupt transition to beta-sheet structures upon heating. At room temperature, this protein self-assembles into macroscopic fibers within minutes. The secondary structures of none of the domains are altered by pH or salt. However, high concentrations of phosphate affect the tertiary structure and accelerate the aggregation propensity of the repetitive region. Implications of these results for dragline spidroin behavior in solution and silk fiber formation are discussed.