L1 endocytosis is controlled by a phosphorylation-dephosphorylation cycle stimulated by outside-in signaling by L1

Dynamic regulation of the cell surface expression of adhesion molecules is an important mechanism for controlling neuronal growth cone motility and guidance. Clathrin-mediated vesicular internalization of L1 via the tyrosine-based endocytosis motif YRSL regulates adhesion and signaling by this Ig superfamily molecule. Here, we present evidence that tyrosine-1176 (Y1176) of the YRSL motif is phosphorylated in vivo. The nonreceptor tyrosine kinase (p60src) is implicated in L1-mediated neurite outgrowth, and we find that p60src phosphorylates Y1176 in vitro. Phosphorylation of Y1176 prevents L1 binding to AP-2, an adaptor required for clathrin-mediated internalization of L1. mAb 74-5H7 recognizes the sequence immediately NH2-terminal to the tyrosine-based motif and binds L1 only when Y1176 is dephosphorylated. 74-5H7 identifies a subset of L1 present at points of cell-cell contact and in vesicle-like structures that colocalize with an endocytosis marker. L1-L1 binding or L1 cross-linking induces a rapid increase in 74-5H7 immunoreactivity. Our data suggest a model in which homophilic binding or L1 cross-linking triggers transient dephosphorylation of the YRSL motif that makes L1 available for endocytosis. Thus, the regulation of L1 endocytosis through dephosphorylation of Y1176 is a critical regulatory point of L1-mediated adhesion and signaling.     

Specific ablation of the nidogen-binding site in the laminin gamma1 chain interferes with kidney and lung development

Basement membrane assembly is of crucial importance in the development and function of tissues and during embryogenesis. Nidogen 1 was thought to be central in the assembly processes, connecting the networks formed by collagen type IV and laminins, however, targeted inactivation of nidogen 1 resulted in no obvious phenotype. We have now selectively deleted the sequence coding for the 56 amino acid nidogen-binding site, gamma1III4, within the Lamc1 gene by gene targeting. Here, we show that mice homozygous for the deletion die immediately after birth, showing renal agenesis and impaired lung development. These developmental defects were attributed to locally restricted ruptures in the basement membrane of the elongating Wolffian duct and of alveolar sacculi. These data demonstrate that an interaction between two basement membrane proteins is required for early kidney morphogenesis in vivo.     

spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories.     

Neutrophil elastase induces mucin production by ligand-dependent epidermal growth factor receptor activation

Neutrophil products are implicated in hypersecretory airway diseases. To determine the mechanisms linking a proteolytic effect of human neutrophil elastase (HNE) and mucin overproduction, we examined the effects of HNE on MUC5AC mucin production in human airway epithelial (NCI-H292) cells. Stimulation with HNE for 5-30 min induced MUC5AC production 24 h later, which was prevented by HNE serine active site inhibitors, implicating a proteolytic effect of HNE. MUC5AC induction was preceded by epidermal growth factor receptor (EGFR) tyrosine phosphorylation and was prevented by selective EGFR tyrosine kinase inhibitors, implicating EGFR activation. HNE-induced MUC5AC production was inhibited by a neutralizing transforming growth factor-alpha (TGF-alpha, an EGFR ligand) antibody and by a neutralizing EGFR antibody but not by oxygen free radical scavengers, further implicating TGF-alpha and ligand-dependent EGFR activation in the response. HNE decreased pro-TGF-alpha in NCI-H292 cells and increased TGF-alpha in cell culture supernatant. From these results, we conclude that HNE-induced MUC5AC mucin production occurs via its proteolytic activation of an EGFR signaling cascade involving TGF-alpha.     

Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4–S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4–IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the ‘common’ kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.     

Effects of moxifloxacin in zymogen A or S. aureus stimulated human THP-1 monocytes on the inflammatory process and the spread of infection

Antimicrobial agents have been reported to exhibit immunomodulatory and anti-inflammatory activities, both in vivo and in vitro (e.g., in human lymphocytes, macrophages and monocytes). The effects of moxifloxacin on cytokine immunomodulatory mediators, free radical generation and hydrolytic enzyme activities in zymogen A-stimulated human THP-1 monocytes were evaluated. An increase in c-AMP levels, protein kinase C activity, and the release of nitric oxide and hydrogen peroxide with a decrease in pH occurred within the first hour. Further, the effects of moxifloxacin were reduced by agents which blocked the oxygen burst, lysosome-phagosome fusion, and the energy generation within the cell. After 4 h, there was a decrease in NAG and cathepsin D activities, lipid peroxidation and the release of pro-inflammatory cytokines. These data indicate that moxifloxacin may modify the acute-phase inflammatory responses through inhibition of cytokine release in monocytes. Moxifloxacin inhibited the release of TNFalpha, IL-1, IL-6, and IL-8 in a concentration-dependent manner across a range of 0.004 to 4 microg/mL. After 4 h, there was a decrease in the release of these cytokines, thus interfering with the inflammation process to reduce infection and its spread. The effects of moxifloxacin appear initially to activate monocytes to kill bacteria through the innate immune process by releasing ROS and lysosomal hydrolytic enzymes as well as phagocytosis of the organism. At a later time the bacteria are killed through a Bacterialstatic mechanism of protein synthesis inhibition and there is a reversal of the effects of moxifloxacin on cytokine release, free radical generation and hydrolytic enzymes so that lipid peroxidation and tissue destruction by the infection process is suppressed.     

The intestinal protozoan parasite Entamoeba histolytica contains 20 cysteine protease genes,of which only a small subset is expressed during in vitro cultivation

Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.     

A virus-like particle-based vaccine selectively targeting soluble TNF-alpha protects from arthritis without inducing reactivation of latent tuberculosis

Neutralization of the proinflammatory cytokine TNF-alpha by mAbs or soluble receptors represents an effective treatment for chronic inflammatory disorders such as rheumatoid arthritis, psoriasis, or Crohn's disease. In this study, we describe a novel active immunization approach against TNF-alpha, which results in the induction of high titers of therapeutically active autoantibodies. Immunization of mice with virus-like particles of the bacteriophage Qbeta covalently linked to either the entire soluble TNF-alpha protein (Qbeta-C-TNF(1-156)) or a 20-aa peptide derived from its N terminus (Qbeta-C-TNF(4-23)) yielded specific Abs, which protected from clinical signs of inflammation in a murine model of rheumatoid arthritis. Whereas mice immunized with Qbeta-C-TNF(1-156) showed increased susceptibility to Listeria monocytogenes infection and enhanced reactivation of latent Mycobacterium tuberculosis, mice immunized with Qbeta-C-TNF(4-23) were not immunocompromised with respect to infection with these pathogens. This difference was attributed to recognition of both transmembrane and soluble TNF-alpha by Abs elicited by Qbeta-C-TNF(1-156), and a selective recognition of only soluble TNF-alpha by Abs raised by Qbeta-C-TNF(4-23). Thus, by specifically targeting soluble TNF-alpha, Qbeta-C-TNF(4-23) immunization has the potential to become an effective and safe therapy against inflammatory disorders, which might overcome the risk of opportunistic infections associated with the currently available TNF-alpha antagonists.