GTP-dependent secretion from neutrophils is regulated by Cdk5

We have previously shown evidence for the existence of a calcium-independent, GTP-regulated mechanism of secretion from neutrophils, but this secretory mechanism remains to be fully elucidated. Cyclin-dependent kinase 5 (Cdk5), the various substrates of which include Munc18 and synapsin 1, has been implicated in neuronal secretion. Although the Cdk5 activator, p35, and Cdk5-p35 activity are primarily associated with neurons, we report here that p35 also exists in neutrophils and that an active Cdk5-p35 complex is present in these cells. Cdk5-p35 activity in human neutrophils is mostly localized in secretory granules, which show an increase in Cdk5-p35 level and activity upon GTP stimulation. The potent Cdk5 inhibitor, roscovitine, completely blocks GTP-stimulated granule Cdk5 activity, which accompanies lactoferrin secretion from neutrophil-specific granules. Roscovitine also inhibits GTP-induced lactoferrin secretion and surface localization of the secretion markers, CD63 and CD66b, to a certain extent. Furthermore, neutrophils from wild-type mice treated with roscovitine and neutrophils from p35(-/-) mice exhibit comparable surface expression levels of both CD63 and CD66b upon GTP stimulation. Although our data suggest that other molecules control GTP-induced secretion from neutrophils, it is clear that Cdk5-p35 is required to elicit the maximum GTP-induced secretory response. Our observation that multiple proteins in neutrophil granules serve as specific substrates of Cdk5 further supports the premise that the kinase is a key component of the GTP-regulated secretory apparatus in neutrophils.     

CCR2-dependent trafficking of F4/80dim macrophages and CD11cdim/intermediate dendritic cells is crucial for T cell recruitment to lungs infected with Mycobacterium tuberculosis

We previously reported that CCR2(-/-) mice are susceptible to Mycobacterium tuberculosis infection. Susceptibility was associated with an early and sustained macrophage trafficking defect, followed by delayed recruitment of dendritic cells (DCs) and T cells to the lungs. However, the relative importance of the lack of CCR2 expression by macrophages and DCs vs T cells in susceptibility to infection was unclear. In this study, we used mixed bone marrow transplantation to create mice in which the genotype of the T cells was either CCR2(+/+) or CCR2(-/-) while maintaining the genotype of the myeloid cells as CCR2(+/+). After infection with M. tuberculosis, we found that the genotype of the macrophages and/or DCs, but not that of the T cells, was critical for both T cell and myeloid cell migration to the lungs. Further investigation revealed a critical role for CCR2 in the recruitment of F4/80(dim) macrophages and CD11c(dim/intermediate) DCs to the infected lung.     

Targeted expression of the class II phosphoinositide 3-kinase in Drosophila melanogaster reveals lipid kinase-dependent effects on patterning and interactions with receptor signaling pathways

Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on the basis of their domain structures and the lipid signals that they generate. Functions have been assigned to the class I and class III enzymes but have not been established for the class II PI3Ks. We have obtained the first evidence for a biological function for a class II PI3K by expressing this enzyme during Drosophila melanogaster development and by using deficiencies that remove the endogenous gene. Wild-type and catalytically inactive PI3K_68D transgenes have opposite effects on the number of sensory bristles and on wing venation phenotypes induced by modified epidermal growth factor (EGF) receptor signaling. These results indicate that the endogenous PI3K_68D may act antagonistically to the EGF receptor-stimulated Ras-mitogen-activated protein kinase pathway and downstream of, or parallel to, the Notch receptor. A class II polyproline motif in PI3K_68D can bind the Drk adaptor protein in vitro, primarily via the N-terminal SH3 domain of Drk. Drk may thus be important for the localization of PI3K_68D, allowing it to modify signaling pathways downstream of cell surface receptors. The phenotypes obtained are markedly distinct from those generated by expression of the Drosophila class I PI3K, which affects growth but not pattern formation.     

Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development

Basement membranes are specialized extracellular matrices consisting of tissue-specific organizations of multiple matrix molecules and serve as structural barriers as well as substrates for cellular interactions. The network of collagen IV is thought to define the scaffold integrating other components such as, laminins, nidogens or perlecan, into highly organized supramolecular architectures. To analyze the functional roles of the major collagen IV isoform alpha1(IV)(2)alpha2(IV) for basement membrane assembly and embryonic development, we generated a null allele of the Col4a1/2 locus in mice, thereby ablating both alpha-chains. Unexpectedly, embryos developed up to E9.5 at the expected Mendelian ratio and showed a variable degree of growth retardation. Basement membrane proteins were deposited and assembled at expected sites in mutant embryos, indicating that this isoform is dispensable for matrix deposition and assembly during early development. However, lethality occurred between E10.5-E11.5, because of structural deficiencies in the basement membranes and finally by failure of the integrity of Reichert's membrane. These data demonstrate for the first time that collagen IV is fundamental for the maintenance of integrity and function of basement membranes under conditions of increasing mechanical demands, but dispensable for deposition and initial assembly of components. Taken together with other basement membrane protein knockouts, these data suggest that laminin is sufficient for basement membrane-like matrices during early development, but at later stages the specific composition of components including collagen IV defines integrity, stability and functionality.     

Detection of high-level tetracycline resistance in clinical isolates of Helicobacter pylori using PCR-RFLP

Tetracycline is one of four antibiotics commonly used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing as the incidence of tetracycline resistance is increasing. In five Brazilian tetracycline-resistant (Tet(R)) H. pylori isolates, high-level tetracycline resistance is mediated by the triple-base-pair substitution AGA(926-928)-->TTC in both 16S rRNA genes, as was previously observed in two independent high-level Tet(R) H. pylori strains. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) assay was developed for the detection of the AGA(926-928)-->TTC substitution, and confirmed the presence of the aforementioned triple-base-pair substitution in all five Brazilian Tet(R) isolates. This PCR-RFLP-based approach distinguishes the high-level Tet(R) isolates from low-level Tet(R) and Tet(S) H. pylori strains and thus allows the direct detection of Tet(R) H. pylori isolates.     

Wine polyphenols and ethanol do not significantly scavenge superoxide nor affect endothelial nitric oxide production

Epidemiological studies have shown that moderate intake of red wine reduces the risk of coronary heart disease. It has been proposed that the antiatherogenic effect be due to the scavenging of reactive oxygen species by polyphenols and ethanol or an effect on endothelial nitric oxide (NO) production. We have determined the reaction rates of superoxide with four different polyphenols and ethanol. The superoxide reaction rates were determined at 37 degrees C and pH 7.4 using competitive spin trapping and electron paramagnetic resonance (EPR) spectroscopy. Ethanol did not scavenge superoxide. For the polyphenols catechin, epicatechin, gallic acid, and quercetin, we find rate constants of respectively 2.3*10(4), 2.2*10(4), 2.3*10(3) and 1.9*10(4)(mole per second)(-1). Polyphenols can only exert a significant scavenging effect, if the plasma concentration reach sufficiently high levels. At concentrations found in vivo (low nanomolar range), the scavenging of superoxide by polyphenols and ethanol is negligible in comparison with endogenous protection against superoxide. Incubation of cultured endothelial cells with 5 micromol/L of catechin, epicatechin, gallic acid, quercetin, or ethanol 0.05% (v/v) did not influence the maximal production of NO by these cells as measured by fluorescent nitric oxide cheletropic traps (FNOCT). The observed antiatherogenic effects must be caused by a mechanism other than direct scavenging of superoxide or influence on maximal endothelial NO production.     

Human altruism: economic, neural, and evolutionary perspectives

Human cooperation represents a spectacular outlier in the animal world. Unlike other creatures, humans frequently cooperate with genetically unrelated strangers, often in large groups, with people they will never meet again, and when reputation gains are small or absent. Experimental evidence and evolutionary models suggest that strong reciprocity, the behavioral propensity for altruistic punishment and altruistic rewarding, is of key importance for human cooperation. Here, we review both evidence documenting altruistic punishment and altruistic cooperation and recent brain imaging studies that combine the powerful tools of behavioral game theory with neuroimaging techniques. These studies show that mutual cooperation and the punishment of defectors activate reward related neural circuits, suggesting that evolution has endowed humans with proximate mechanisms that render altruistic behavior psychologically rewarding.     

Symmetry elements in DNA structure important for recognition/methylation by DNA [amino]-methyltransferases

The phage T4Dam and EcoDam DNA-[adenine-N6] methyltransferases (MTases) methylate GATC palindromic sequences, while the BamHI DNA-[cytosine-N4] MTase methylates the GGATCC palindrome (which contains GATC) at the internal cytosine residue. We compared the ability of these enzymes to interact productively with defective duplexes in which individual elements were deleted on one chain. A sharp decrease in k_cat was observed for all three enzymes if a particular element of structural symmetry was disrupted. For the BamHI MTase, integrity of the ATCC was critical, while an intact GAT sequence was necessary for the activity of T4Dam, and an intact GA was necessary for EcoDam. Theoretical alignment of the region of best contacts between the protein and DNA showed that in the case of a palindromic interaction site, a zone covering the 5′-symmetric residues is located in the major groove versus a zone of contact covering the 3′-symmetric residues in the minor groove. Our data fit a simple rule of thumb that the most important contacts are aligned around the methylation target base: if the target base is in the 5′ half of the palindrome, the interaction between the enzyme and the DNA occurs mainly in the major groove; if it is in the 3′ half, the interaction occurs mainly in the minor groove.     

IkappaB kinase is a critical regulator of chemokine expression and lung inflammation in respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the major etiologic agent of severe epidemic lower respiratory tract infections in infancy. Airway mucosal inflammation plays a critical role in the pathogenesis of RSV disease in both natural and experimental infections. RSV is among the most potent biological stimuli that induce the expression of inflammatory genes, including those encoding chemokines, but the mechanism(s) that controls virus-mediated airway inflammation in vivo has not been fully elucidated. Herein we show that the inoculation of BALB/c mice with RSV results in rapid activation of the multisubunit IkappaB kinase (IKK) in lung tissue. IKK transduces upstream activating signals into the rate-limiting phosphorylation (and proteolytic degradation) of IkappaBalpha, the inhibitory subunit that under normal conditions binds to the nuclear factor (NF)-kappaB complex and keeps it in an inactive cytoplasmic form. Mice treated intranasally with interleukin-10 or with a specific cell-permeable peptide that blocks the association of the catalytic subunit IKKbeta with the regulatory protein NEMO showed a striking reduction of lung NF-kappaB DNA binding activity, chemokine gene expression, and airway inflammation in response to RSV infection. These findings suggest that IKKbeta may be a potential target for the treatment of acute or chronic inflammatory diseases of the lung.