Assessment of aflatoxin and cyclopiazonic acid production by Aspergillus flavus isolates from Hungary

Thirty-two isolates of Aspergillus flavus were obtained from various sources in Hungary. All isolates were morphologically identified as A. flavus and three atypical variants were confirmed as A. flavus by comparing their DNA with an ex type culture of A. flavus. None of these isolates produced aflatoxins when tested on coconut agar or grown on rice medium and culture extracts examined by thin layer chromatography. Also, none of the isolates converted sterigmatocystin, O-methyl sterigmatocystin, norsolorinic acid, or sodium acetate to aflatoxin. However, 59% of the isolates produced cyclopiazonic acid based on thin layer chromatographic analysis of culture extracts. The isolates that lack the ability to produce both aflatoxin and cyclopiazonic acid are potential candidates for use in bicontrol studies.     

In vitro and in vivo activation of murine gamma/delta T cells induces the expression of IgA, IgM, and IgG Fc receptors.

The present studies examined resting and activated murine gamma/delta T lymphocytes, in vitro and in vivo, for surface expression of FcR. Polyclonal gamma/delta TCR+ lymphocytes selectively grown from the spleen and intestine of normal mice did not express FcR when the cells were in a resting state, but when cells were activated with anti-CD3 antibody virtually all of the splenic gamma/delta lymphocytes and a large subpopulation of the intestinal gamma/delta lymphocytes expressed IgA and IgM FcR. This was confirmed by using transgenic mice. Resting gamma/delta TCR+ lymphocytes from the spleen, thymus, lymph node, and blood of gamma/delta TCR transgenic mice did not express FcR for any of the five major classes of Ig H chains. Activation of the gamma/delta TCR+ cells via the CD3/TCR complex induced high levels of IgM and IgA FcR and low levels of IgG FcR. Finally, in hepatic granulomas of schistosome-infected mice, activated gamma/delta TCR+ cells are present and express high levels of IgA and IgM FcR and low levels of IgG FcR. These investigations establish that transition of gamma/delta TCR+ lymphocytes from a resting to an activated state (triggered via the T3Ti TCR complex) is accompanied by the induction of surface membrane receptors specific for Ig H chain isotypes. The activation-linked expression of FcR on gamma/delta TCR+ lymphocytes provides potential mechanisms for coupling the functional activities of gamma/delta T lymphocytes with immune mechanisms that involve Ig molecules, such as antibody-dependent cellular cytotoxicity.     

The effect of ethanol upon early development in mice and rats. VIII. The effect of chronic consumption of some beverages upon preimplantation development in rats

The effects of chronic consumption of some beverages (plum-brandy 24% and cognac 20%) upon preimplantation development in rats were studied. The control of possible effects was performed on day 5 by usual flushing, examination and photographying of oviductal and uterine embryos. In order to evaluate the effect of the beverage applied, the following criteria were used: mean litter size, migration of the embryos from the oviduct to the uterus, the developmental stage attained by the pre-implantation embryos and the appearance of pathological embryos. The main results were the following: both beverages applied influenced the preimplantation development; with respect to the developmental rate and to the induction of pathological changes, the effect of both beverages was similar (retardation and an increased, number of pathological morulae and blastocysts); a different action could be detected as to the mean litter size and to the migration of preimplantation embryos: plum-brandy reduced more substantially the mean litter size, whereas cognac had a more marked retarding effect upon the migration of embryos from the oviduct to the uterus: all the changes detected show a more or less marked "litter-effect". The present data were compared with the corresponding effects of chronic ethanol administration observed previously in our laboratory. No obvious potentiating effect of beverage congeners could be established. The findings are discussed in connection with other experimental models of alcohol embryo and fetopathy.     

Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.     

Exploring the binding site structure of the PPAR gamma ligand-binding domain by computational solvent mapping

Solvent mapping moves molecular probes, small organic molecules containing various functional groups, around the protein surface, finds favorable positions, clusters the conformations, and ranks the clusters based on the average free energy. Using at least six different solvents as probes, the probes cluster in major pockets of the functional site, providing detailed and reliable information on the amino acid residues that are important for ligand binding. Solvent mapping was applied to 12 structures of the peroxisome proliferator activated receptor gamma (PPARgamma) ligand-binding domain (LBD), including 2 structures without a ligand, 2 structures with a partial agonist, and 8 structures with a PPAR agonist bound. The analysis revealed 10 binding "hot spots", 4 in the ligand-binding pocket, 2 in the coactivator-binding region, 1 in the dimerization domain, 2 around the ligand entrance site, and 1 minor site without a known function. Mapping is a major source of information on the role and cooperativity of these sites. It shows that large portions of the ligand-binding site are already formed in the PPARgamma apostructure, but an important pocket near the AF-2 transactivation domain becomes accessible only in structures that are cocrystallized with strong agonists. Conformational changes were seen in several other sites, including one involved in the stabilization of the LBD and two others at the region of the coactivator binding. The number of probe clusters retained by these sites depends on the properties of the bound agonist, providing information on the origin of correlations between ligand and coactivator binding.     

Causative factors behind poloxamer 188 (Pluronic F68, Flocor)-induced complement activation in human sera. A protective role against poloxamer-mediated complement activation by elevated serum lipoprotein levels

Poloxamer 188 is a complex polydisperse mixture of non-ionic macromolecules. Adverse non-IgE-mediated hypersensitivity reactions occur in some individuals following intravenous injection of poloxamer 188-based pharmaceuticals, presumably via complement activation. Here we have delineated potential causal chemical and biological interactive factors behind poloxamer 188-induced complement activation in human serum specimens. We identified the molecular constituents inherent in poloxamer 188 preparations and studied their effect on generation of the two complement split products, SC5b-9 and Bb. Poloxamer 188 activated complement at sub-micellar concentrations and the results indicated the potential involvement of all three known complement activation pathways. The poloxamer-induced rise of SC5b-9 in human sera was abolished in the presence of a recombinant truncated soluble form of complement receptor type 1, thus confirming the role of C3/C5 convertases in the activation process. Poloxamer 188-mediated complement activation is an intrinsic property of these macromolecules and was independent of the degree of sample polydispersity, as opposed to other non-polymeric constituents. Poloxamer 188 preparations also contained unsaturated chains of diblock copolymers capable of generating SC5b-9 in human sera; this effect was terminated following the removal of double bonds by catalytic hydrogenation. By quasi-elastic light scattering, we established interaction between poloxamer and lipoproteins; interestingly, poloxamer-induced rise in SC5b-9 was significantly suppressed when serum HDL and LDL cholesterol levels were increased above normal to mimic two relevant clinical situations. This observation was consistent with previously reported data from patients with abnormal or elevated lipid profiles where no or poor complement activation by poloxamer 188 occurred. Our findings could provide the basis of novel approaches to the prevention of poloxamer-mediated complement activation.     

Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae

Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.     

The Water Dimer: Post-Hartree-Fock and Density Functional Calculations on the Potential Energy Surface

Conventional ab initio and density functional methods with extended basis sets were employed in the study of a path on the water-dimer potential energy surface. The results show that density functional methods do depend strongly on the type of exchange-correlation potential employed, as well as on the quality of the basis sets – similarly to conventional ab initio methods – and on the density of the grid. Gradient-corrected methods behave, as expected, better than uncorrected ones, the Becke–Lee–Yang–Parr (BLYP) potential being the one that gives the best results. However, too large chemical- and hydrogen-bond lengths and absolute energies, as well as too small relative total and correlation energies demonstrate that even BLYP calculations with a relative large basis set are not good as MP2 calculations of the same size. Adiabatically connected functionals (ACM), represented in this work by B3PW91, provide an improvement on the whole surface. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular cloning and characterization of horse DQB cDNA

The nucleotide sequence data reported in this paper have been submitted to the GenBank sequence database and have been assigned the accession number L33910