Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion

Transforming growth factor beta (TGF-beta) proteins play important roles in morphogenesis of many craniofacial tissues; however, detailed biological mechanisms of TGF-beta action, particularly in vivo, are still poorly understood. Here, we deleted the TGF-beta type I receptor gene Alk5 specifically in the embryonic ectodermal and neural crest cell lineages. Failure in signaling via this receptor, either in the epithelium or in the mesenchyme, caused severe craniofacial defects including cleft palate. Moreover, the facial phenotypes of neural crest-specific Alk5 mutants included devastating facial cleft and appeared significantly more severe than the defects seen in corresponding mutants lacking the TGF-beta type II receptor (TGFbetaRII), a prototypical binding partner of ALK5. Our data indicate that ALK5 plays unique, non-redundant cell-autonomous roles during facial development. Remarkable divergence between Tgfbr2 and Alk5 phenotypes, together with our biochemical in vitro data, imply that (1) ALK5 mediates signaling of a diverse set of ligands not limited to the three isoforms of TGF-beta, and (2) ALK5 acts also in conjunction with type II receptors other than TGFbetaRII.     

Effects of signal peptide exchange on HIV-1 glycoprotein expression and viral infectivity in mammalian cells

In certain cell systems, exchange of the human immunodeficiency virus (HIV) Env signal peptide (SP) sequence with that of heterologous SPs has been shown to increase gp120 transport and secretion. Here we demonstrate that exchange of the HIV-Env-SP with those from erythropoietin or tissue plasminogen activator in the proviral context does not increase wild-type membrane-bound Env expression or incorporation into released virions. In fact, virion infectivity was decreased. These infectivity decreases were largely due to effects on Env transport and/or function and only to a minor extent to cis effects as a result of the sequence exchanges themselves. Thus, in fact, it is not advantageous to employ heterologous SPs to achieve high-level expression of functional cell surface membrane- or virion-associated HIV-Env.     

Assessing the fidelity of ancient DNA sequences amplified from nuclear genes

To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from environments ranging from permafrost to desert, we demonstrate the presence of miscoding lesion damage in both the mtDNA and nuDNA, resulting in insertion of erroneous bases during amplification. Interestingly, no significant differences in the frequency of miscoding lesion damage are recorded between mtDNA and nuDNA despite great differences in cellular copy numbers. For both mtDNA and nuDNA, we find significant positive correlations between total sequence heterogeneity and the rates of type 1 transitions (adenine --> guanine and thymine --> cytosine) and type 2 transitions (cytosine --> thymine and guanine --> adenine), respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and thymine) is the main cause of miscoding lesions in both ancient mtDNA and nuDNA sequences. We argue that the problems presented by postmortem damage, as well as problems with contamination from exogenous sources of conserved nuclear genes, allelic variation, and the reliance on single nucleotide polymorphisms, call for great caution in studies relying on ancient nuDNA sequences.     

Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments

Diagenesis was studied in DNA obtained from Siberian permafrost (permanently frozen soil) ranging from 10,000 to 400,000 years in age. Despite optimal preservation conditions, we found the sedimentary DNA to be severely modified by interstrand crosslinks; single- and double-stranded breaks; and freely exposed sugar, phosphate, and hydroxyl groups. Intriguingly, interstrand crosslinks were found to accumulate approximately 100 times faster than single-stranded breaks, suggesting that crosslinking rather than depurination is the primary limiting factor for ancient DNA amplification under frozen conditions. The results question the reliability of the commonly used models relying on depurination kinetics for predicting the long-term survival of DNA under permafrost conditions and suggest that new strategies for repair of ancient DNA must be considered if the yield of amplifiable DNA from permafrost sediments is to be significantly increased. Using the obtained rate constant for interstrand crosslinks the maximal survival time of amplifiable 120-bp fragments of bacterial 16S ribosomal DNA was estimated to be approximately 400,000 years. Additionally, a clear relationship was found between DNA damage and sample age, contradicting previously raised concerns about the possible leaching of free DNA molecules between permafrost layers.     

Thy-1, via its GPI anchor, modulates Src family kinase and focal adhesion kinase phosphorylation and subcellular localization, and fibroblast migration, in response to thrombospondin-1/hep I

Normal fibroblast subpopulations have differential surface expression of the GPI-linked raft protein Thy-1, which correlates with differences in cellular adhesion and migration in vitro. Thrombospondin-1 (TSP-1) induces an intermediate state of adhesion in fibroblasts and other cells which facilitates migration. TSP-1 and the hep I peptide derived from the amino-terminal/heparin-binding domain of TSP-1 induce disassembly of cellular focal adhesions. Our lab previously reported that the induction of focal adhesion disassembly in fibroblasts by TSP-1 or by hep I requires surface expression of Thy-1, as well as lipid raft integrity and Src family kinase (SFK) signaling. We now report that TSP-1/hep I-induced fibroblast migration requires Thy-1 expression and FAK phosphorylation, and that following TSP-1/hep I stimulation, Thy-1 associates with FAK and SFK in a lipid raft-dependent manner. Furthermore, the GPI anchor of Thy-1, which localizes the protein to specific lipid raft microdomains, is necessary for hep I-induced FAK and SFK phosphorylation, focal adhesion disassembly, and migration. This is the first report of an association between Thy-1 and FAK. Thy-1 modulates SFK and FAK phosphorylation and subcellular localization, promoting focal adhesion disassembly and migration in fibroblasts, following exposure to TSP-1/hep I.     

Signals from a self-antigen induce positive selection in early B cell ontogeny but are tolerogenic in adults

Positive and negative signals from self-Ags shape the B cell repertoire and the development of distinct B cell subsets, but little is known about what distinguishes these signals. To address this question, we have studied the development of anti-hen egg lysozyme MD4 Ig transgene B cells while systematically varying the level, distribution, and timing of exposure to different forms of hen egg lysozyme as a self-Ag. This process has allowed us to explore the effects of Ag independent of BCR specificity. Our findings show how the selection of autoreactive B cells is a competitive process involving immunogenic and tolerogenic forms of self-Ags. Due to a developmental switch during B cell ontogeny, autoreactive anti-hen egg lysozyme MD4 Ig transgene B cells are negatively selected by self-Ags in adult bone marrow but susceptible to positive selection by some of the same self-Ags in fetal and neonatal life. However, the persistence of B1 cells and IgM autoantibodies from early ontogeny enables autoreactive B cells from the adult bone marrow to escape negative selection. Our data suggest that this rescue may be due to the clearance or masking of self-Ag by IgM autoantibody. We discuss the implications of these findings in terms of B cell selection and the maintenance of self-tolerance during early and adult life.     

Antimicrobial peptides from chilli pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells

During the last few years, a growing number of cysteine-rich antimicrobial peptides has been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides play an important role in the protection of plants against microbial infection. In this work, proteins from chilli pepper (Capsicum annuum L.) seeds were extracted in phosphate buffer, pH 5.4 and peptides purification were performed by employing ion-exchange chromatographies on DEAE, CM-Sepharose, Sephacryl S-100 and reverse phase in HPLC. Three peptide enriched fractions, namely F1, F2 and F3, were obtained after the CM-Sepharose chromatography. The F1 fraction, mainly composed of three peptides ranging from 6 to 10 kDa, was submitted to N-terminal amino acid sequencing. The closer to 10 kDa peptide showed high sequence homology to lipid transfer proteins (LTPs) previously isolated from others seeds. F1 fraction exhibited strong fungicidal activity against Candida albicans, Saccharomyces cerevisiae and Schizosaccharomyces pombe and also promoted several morphological changes to C. albicans, including the formation of pseudohyphae, as revealed by scanning electron micrography. F1 fraction also reduced the glucose stimulated acidification of the medium mediated by H⁺-ATPase of S. cerevisiae cells in a dose-dependent manner and caused the permeabilization of yeast plasma membrane to the dye SYTOX Green, as verified by confocal laser microscopy.     

The release of GLP-1 and ghrelin, but not GIP and CCK, by glucose is dependent upon the length of small intestine exposed

Previous observations suggest that glucagon-like peptide-1 (GLP-1) is released into the bloodstream only when dietary carbohydrate enters the duodenum at rates that exceed the absorptive capacity of the proximal small intestine to contact GLP-1 bearing mucosa in more distal bowel. The aims of this study were to determine the effects of modifying the length of small intestine exposed to glucose on plasma concentrations of GLP-1 and also glucose-dependent insulinotropic peptide (GIP), insulin, cholecystokinin (CCK) and ghrelin, and antropyloric pressures. Glucose was infused at 3.5 kcal/min into the duodenum of eight healthy males (age 18-59 yr) over 60 min on the first day into an isolated 60-cm segment of the proximal small intestine ("short-segment infusion"); on the second day, the same amount of glucose was infused with access to the entire small intestine ("long-segment infusion"). Plasma GLP-1 increased and ghrelin decreased (P < 0.05 for both) during the long-, but not the short-, segment infusion. By contrast, increases in plasma CCK and GIP did not differ between days. The rises in blood glucose and plasma insulin were greater during the long- than during the short-segment infusion (P < 0.05). During the long- but not the short-segment infusion, antral pressure waves (PWs) were suppressed (P < 0.05). Isolated pyloric PWs and basal pyloric pressure were stimulated on both days. In conclusion, the release of GLP-1 and ghrelin, but not CCK and GIP, is dependent upon >60 cm of the intestine being exposed to glucose.     

Quantitative Detection and Differentiation of Free-Living Amoeba Species Using SYBR Green–Based Real-Time PCR Melting Curve Analysis

Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.