Genotypes of Cryptosporidium species infecting fur-bearing mammals differ from those of species infecting humans

Of 471 specimens examined from foxes, raccoons, muskrats, otters, and beavers living in wetlands adjacent to the Chesapeake Bay, 36 were positive for five types of Cryptosporidium, including the C. canis dog and fox genotypes, Cryptosporidium muskrat genotypes I and II, and Cryptosporidium skunk genotype. Thus, fur-bearing mammals in watersheds excreted host-adapted Cryptosporidium oocysts that are not known to be of significant public health importance.     

Interleukin-18 enhances the production of interferon-gamma (IFN-gamma) by allergen-specific and unspecific stimulated cord blood mononuclear cells

BACKGROUND: IL-18 is a pleiotropic cytokine involved in the polarisation of T-cell response. This study was performed to determine whether or not IL-18 is detectable in phytohemagglutinin (PHA) or betalactoglobulin (BLG) stimulated supernatants of cord blood mononuclear cells (CBMC) and to study the in vitro effect of IL-18 on the interferon (IFN)-gaamma and IL-13 release of CBMC of healthy neonates. METHODS: CBMC of neonates were isolated by Ficoll density centrifugation. The cytokines IFN-gamma, IL-13 and IL-18 in the cell culture supernatants were measured using the ELISA technique following stimulation with a unspecific (PHA 20 microg/ml) and an allergen-specific stimulus (BLG 25 microg/ml). In order to study the in vitro effect of IL-18, CBMC were stimulated either with medium alone or with IL-18, IL-18 + PHA and IL-18 + BLG. RESULTS: IL-18 levels in supernatants of CBMC were low and did not vary significantly between unstimulated and PHA or BLG stimulated cell cultures (median 21.4; 23.5 and 15.5 pg/ml, respectively). IFN-gamma and IL-13 levels were significantly higher in response to PHA and BLG (PHA: IFN-gamma, 6154; IL-13, 4357; BLG: IFN-gamma, 801; IL-13, 249 pg/ml) compared to unstimulated cell cultures. The addition of IL-18 to PHA or BLG stimulated CBMC significantly enhanced the IFN-gamma release (PHA: 6154; PHA + IL-18: 13474, p = 0.0001; BLG: 801; BLG + IL-18: 1077, p = 0.008). In comparison to incubation without IL-18, the release of IL-13 was invariable or even reduced, when CBMC were stimulated with PHA + IL-18 (4026, p = 0.16) or BLG + IL-18 (124, p = 0.0001) compared to stimulation of CBMC with PHA (4357 pg/ml) or BLG (249 pg/ml) alone. CONCLUSIONS: IL-18 is detectable in supernatants of CBMC. We observed a significant effect of IL-18 + PHA as well as IL-18 + BLG on IFN-gamma release in vitro. Based on our findings we conclude that IL-18 could act as a strong TH1-inducing factor on stimulated CBMC also in vivo.     

A cephalometric comparison of skulls from different time periods--the Bronze Age, the 19th century and the present

The aim of this study was to evaluate secular trends by means of orthodontic measurements on lateral cephalograms. We use roentgenograms from three populations: 22 Bronze Age skulls from a cemetery near Hainburg/Austria, 140 soldiers who served in the Hapsburg Imperial Army in the late 19th century, and 154 contemporary recruits of the Austrian Federal Army. Using conventional morphometric analysis, no statistically significant differences could be established. But applying geometric morphometrics to the 2D-coordinates of the pentagon composed of the landmarks Sella, Nasion, Articulare, Gonion and Menton, some biologically interpretable differences were detected, the size allometry between the 19th- and 20th-century populations being the only notable one. We conclude that landmarks should be digitized directly (and many more of them) and that conventional methods used in clinical orthodontics are inappropriate for addressing the scientific questions approached here.     

IFN-alpha induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3

The anti-inflammatory cytokine IL-10 can be induced by type I IFNs, but the molecular mechanisms involved have remained elusive. With in silico analysis of the human IL-10 promoter we identified a module consisting of an IFN regulatory factor 1 (IRF-1) site and a Stat3 site. We demonstrate that IFN-alpha will induce the binding of IRF-1 and Stat3 to the respective motifs. Mutational analysis revealed that inactivation of the IRF-1 motif substantially reduces trans-activation from 5- to 2-fold and that inactivation of the Stat3 motif completely ablates trans-activation by IFN-alpha. The dominant role of Stat3 in this module was confirmed with the blockade of trans-activation by a dominant negative Stat3. By contrast, Stat1 contributes a minor proportion to the DNA binding to the Stat site, and overexpression will counteract Stat3-mediated trans-activation. The data show that IFN-alpha induces the IL-10 gene via a module consisting of interdependent IRF-1 and Stat3 motifs. Of note, LPS-induced trans-activation does not target this module, since it is independent of the IRF-1 motif but completely depends on Stat3.     

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.     

In vitro folding,functional characterization,and disulfide pattern of the extracellular domain of human GLP-1 receptor

The N-terminal, extracellular domain of the receptor for glucagon-like peptide 1 (GLP-1 receptor) was expressed at a high level in E. coli and isolated as inclusion bodies. Renaturation with concomitant disulfide bond formation was achieved from guanidinium-solubilized material. A soluble and active fraction of the protein was isolated by ion exchange chromatography and gel filtration. Complex formation with GLP-1 was shown by cross-linking experiments, surface plasmon resonance measurements, and isothermal titration calorimetry. The existence of disulfide bridges in the N-terminal receptor fragment was proven after digestion of the protein with pepsin. Further analysis revealed a disulfide-binding pattern with links between cysteines 46 and 71, 62 and 104, and between 85 and 126.     

Activation of pyk2/related focal adhesion tyrosine kinase and focal adhesion kinase in cardiac remodeling

Cellular remodeling during progression of dilation involves focal adhesion contact reorganization. However, the signaling mechanisms and structural consequences leading to impaired cardiomyocyte adhesion are poorly defined. These events were studied in tropomodulin-overexpressing transgenic mice that develop dilated cardiomyopathy associated with chronic elevation of intracellular calcium. Analysis of tropomodulin-overexpressing transgenic hearts by immunoblot and confocal microscopy revealed activation and redistribution of signaling molecules known to regulate adhesion. Calcium-dependent pyk2/related focal adhesion tyrosine kinase (RAFTK) showed changes in expression and phosphorylation state, similar to changes observed for a related downstream target molecule of pyk2/RAFTK termed focal adhesion kinase. Paxillin, the target substrate molecule for focal adhesion kinase phosphorylation, was redistributed in tropomodulin-overexpressing transgenic hearts with enhanced paxillin phosphorylation and cleavage. Certain aspects of the in vivo signaling phenotype including increased paxillin phosphorylation could be recapitulated in vitro using neonatal rat cardiomyocytes infected with recombinant adenovirus to overexpress tropomodulin. In addition, increasing intracellular calcium levels with ionomycin induced pyk2/RAFTK phosphorylation, and adenovirally mediated expression of wild-type pyk2/RAFTK resulted in increased phospho-pyk2/RAFTK levels and concomitant paxillin phosphorylation. Collectively, these results delineate a cardiomyocyte signaling pathway associated with dilation that has potential relevance for cardiac remodeling, focal adhesion reorganization, and loss of contractility.     

Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration

RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of Tyr31/118 caused enhanced activation of RhoA and premature formation of stress fibers with substantial loss of efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells. These phenotypes were similar to those induced by RhoA(G14V) in parental cells, and could be abolished by expression of RhoA(T19N), Rac1(G12V), or p190RhoGAP in the mutant-expressing cells. Phosphorylated Tyr31/118 was found to bind to two src homology (SH)2 domains of p120RasGAP, with coprecipitation of endogenous paxillin with p120RasGAP. p190RhoGAP is known to be a major intracellular binding partner for the p120RasGAP SH2 domains. We found that Tyr31/118-phosphorylated paxillin competes with p190RhoGAP for binding to p120RasGAP, and provides evidence that p190RhoGAP freed from p120RasGAP efficiently suppresses RhoA activity during cell adhesion. We conclude that Tyr31/118-phosphorylated paxillin serves as a template for the localized suppression of RhoA activity and is necessary for efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells.     

Cranial integration in Homo: singular warps analysis of the midsagittal plane in ontogeny and evolution

This study addresses some enduring issues of ontogenetic and evolutionary integration in the form of the hominid cranium. Our sample consists of 38 crania: 20 modern adult Homo sapiens, 14 sub-adult H. sapiens, and four archaic Homo. All specimens were CT-scanned except for two infant H. sapiens, who were imaged by MR instead. For each specimen 84 landmarks and semi-landmarks were located on the midsagittal plane and converted to Procrustes shape coordinates. Integration was quantified by the method of singular warps, a new geometric-statistical approach to visualizing correlations among regions. The two classic patterns of integration, evolutionary and ontogenetic, were jointly explored by comparing analyses of overlapping subsamples that span ranges of different hypothetical factors. Evolutionary integration is expressed in the subsample of 24 adult Homo, and ontogenetic integration in the subsample of 34 H. sapiens. In this data set, vault, cranial base, and face show striking and localized patterns of covariation over ontogeny, similar but not identical to the patterns seen over evolution. The principal differences between ontogeny and phylogeny pertain to the cranial base. There is also a component of cranial length to height ratio not reducible to either process. Our methodology allows a separation of these independent processes (and their impact on cranial shape) that conventional methods have not found.