Neutrophils are highly specialized innate immune effector cells that evolved for antimicrobial host defense. In response to inflammatory stimuli and pathogens, they form neutrophil extracellular traps (NETs), which capture and kill extracellular microbes. Deficient NET formation predisposes humans to severe infection, but, paradoxically, dysregulated NET formation contributes to inflammatory vascular injury and tissue damage. The molecular pathways and signaling mechanisms that control NET formation remain largely uncharacterized. Using primary human neutrophils and genetically manipulated myeloid leukocytes differentiated to surrogate neutrophils, we found that mammalian target of rapamycin ( IntroductionNeutrophils (polymorphonuclear leukocytes, PMNs) are key effector cells in infection, inflammation, and tissue injury. 1 Formation of neutrophil extracellular traps (NETs), first identified with human PMNs, is a function of neutrophils. 2 NETs are complex lattices of decondensed chromatin that trap and kill bacteria, fungi, and some parasites by exposing them to high concentrations of NETassociated microbicidal factors. 3,4 Rapidly evolving studies indicate that NETs effect extracellular microbial killing while limiting the spread of pathogens in vivo. 3,5 The intracellular signaling pathways that regulate NET formation by PMNs remain largely unknown. There is evidence that generation of reactive oxygen species (ROS) is a key event. 4,6 Nevertheless, we showed in primary human PMNs that NET formation requires signaling events and regulatory pathways in addition to ROS generation. 4 Consistent with our results, subsequent studies in human HL-60 myeloid leukocytes and genetically altered mice indicate that activity of peptidylarginine deiminase 4, an enzyme responsible for chromatin decondensation, is also required. 5,7 Recent observations further suggest that NET formation requires enzymatic activity of neutrophil elastase (NE) and myeloperoxidase to initiate degradation of core histones that lead to chromatin decondensation before plasma membrane rupture. 8 Furthermore, ROS generation and NET formation can be dissociated under some conditions. 9,10 Thus, molecular regulation of NET formation is complex and may involve multiple signaling pathways and effector events, depending on the neutrophil agonists and inflammatory context.The mammalian target of rapamycin (mTOR) is a highly conserved PI3K-like serine/threonine kinase with functional homologs found in all studied eukaryotic organisms. 11 mTOR integrates nutrient, energy, oxygen sensing, and mitogenic input signals. 12 We found that mTOR also responds to inflammatory signals and mediates a previously unrecognized pathway of posttranscriptional gene regulation in human PMNs. 13 These results identified a new mechanism by which mTOR can regulate innate, as well as, adaptive, immune responses. Immunoregulatory activities of mTOR are now increasingly recognized. 14 Recent observations indicate that hypoxia inducible factor 1␣ (HIF-1␣), the regulated subunit of ...

The mammalian gastrointestinal tract harbors a highly diverse microbial population that plays a major role in nutrition, metabolism, protection against pathogens, and development of the immune system. It is estimated that at least 1000 different bacterial species cohabit the human intestinal tract. Most recently, the Human Microbiome Project, using new genomic technologies, has started a catalog of specific microbiome composition and its correlation with health and specific diseases. Herein we provide a brief review of the intestinal microbiome, with a focus on new studies showing that there is an important link between the microbes that inhabit the intestinal tract and the developing brain. With future research, an understanding of this link may help us to treat various neurobehavioral problems such as autism, schizophrenia, and anxiety.

Neurabin I, a neuronal actin-binding protein, binds protein phosphatase 1 (PP1) and p70 ribosomal S6 protein kinase (p70S6K), both proteins implicated in cytoskeletal dynamics. We expressed wild-type and mutant neurabins fused to green fluorescent protein in Cos7, HEK293, and hippocampal neurons. Biochemical and cellular studies showed that an N-terminal F-actin-binding domain dictated neurabin I localization at actin cytoskeleton and promoted disassembly of stress fibers. Deletion of the C-terminal coiled-coil and sterile alpha motif domains abolished neurabin I dimerization and induced filopodium extension. Immune complex assays showed that neurabin I recruited an active PP1 via a PP1-docking sequence, 457 Cross talk between protein kinases and phosphatases regulates synaptic strength and information processing in mammalian brain (33). Prior studies identified protein phosphatase 1 (PP1) as a key regulator of activity-dependent changes in synaptic function underlying the two major forms of plasticity known as long-term potentiation (3) and long-term depression (LTD) extensively studied for hippocampal neurons (19). LTD-inducing stimuli promoted distribution of PP1 to dendritic spines (18), where it associated with the actin-rich cytoskeletal structure known as the postsynaptic density or PSD (30). This localization was critical for effective dephosphorylation of PP1 substrates such as Ca 2ϩ /calmodulin-dependent protein kinase II (30) and DL-␣-amino-3-hydroxy-5-methylisoxazolepropionic acid receptors (34) and down-regulation of synaptic function. This has focused attention on the cellular mechanisms that target PP1 to the neuronal actin cytoskeleton (32).Neurabin I (NrbI), identified by F-actin binding (20), shares structural homology with spinophilin (1) or neurabin II (NrbII) (25), which was isolated as a PP1-binding protein. PP1 complexes containing both neurabins have been demonstrated in extracts from rat brain (16). In addition, the PDZ (PSD-95/ Dlg/ZO-1 homology) domain in NrbI recruited p70 ribosomal S6 protein kinase (p70S6K) (4) and kalirin-7 (24), a GTPexchange factor, molecules that have been implicated in the control of neuronal morphology. The C terminus of NrbI contained coiled-coil and SAM (sterile alpha motif) domains, shown to mediate homodimerization and heterodimerization of other proteins (14, 27), which may contribute to the in vitro actin-cross-linking or bundling activity of neurabins (20). The C terminus of NrbI also bound the trans-Golgi protein TGN38 (28) and suggested that NrbI was a multifunctional protein scaffold that regulated both membrane and cytoskeletal functions.By immunohistochemistry, NrbII was localized to dendritic spines and thus called spinophilin (1). In contrast, NrbI was present in both spines and growth cones (20). Subcellular fractionation showed that both neurabins are present in highly purified preparations of PSD (25, 32) and growth cones (R. T. Terry-Lorenzo and S. Shenolikar, unpublished observations). Ectopic expression of NrbI (20) and NrbII (25) in cul...

Opitz syndrome (OS) is a human genetic disease characterized by deformities such as cleft palate that are attributable to defects in embryonic development at the midline. Gene mapping has identified OS mutations within a protein called Mid1. Wild-type Mid1 predominantly colocalizes with microtubules, in contrast to mutant versions of Mid1 that appear clustered in the cytosol. Using yeast two-hybrid screening, we found that the ␣4-subunit of protein phosphatases 2A͞4͞6 binds Mid1. Epitope-tagged ␣4 coimmunoprecipitated endogenous or coexpressed Mid1 from COS7 cells, and this required only the conserved C-terminal region of ␣4. Localization of Mid1 and ␣4 was influenced by one another in transiently transfected cells. Mid1 could recruit ␣4 onto microtubules, and high levels of ␣4 could displace Mid1 into the cytosol. Metabolic 32 P labeling of cells showed that Mid1 is a phosphoprotein, and coexpression of full-length ␣4 decreased Mid1 phosphorylation, indicative of a functional interaction. Association of green fluorescent protein-Mid1 with microtubules in living cells was perturbed by inhibitors of MAP kinase activation. The conclusion is that Mid1 association with microtubules, which seems important for normal midline development, is regulated by dynamic phosphorylation involving MAP kinase and protein phosphatase that is targeted specifically to Mid1 by ␣4. Human birth defects may result from environmental or genetic disruption of this regulatory cycle.