Mucins MUC5AC and MUC5B Are Variably Packaged in the Same and in Separate Secretory Granules.

Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1ß and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.

Atg5 Supports Rickettsia australis Infection in Macrophages In Vitro and In Vivo.

Rickettsiae can cause life-threatening infections in humans. Macrophages are one of the initial targets for rickettsiae after inoculation by ticks. However, it remains poorly understood how rickettsiae remain free in macrophages prior to establishing their infection in microvascular endothelial cells. Here, we demonstrated that the concentration of Rickettsia australis was significantly greater in infected tissues of Atg5flox/flox mice than in the counterparts of Atg5flox/flox Lyz-Cre mice, in association with a reduced level of interleukin-1ß (IL-1ß) in serum. The greater concentration of R. australis in Atg5flox/flox bone marrow-derived macrophages (BMMs) than in Atg5flox/flox Lyz-Cre BMMs in vitro was abolished by exogenous treatment with recombinant IL-1ß. Rickettsia australis induced significantly increased levels of light chain 3 (LC3) form II (LC3-II) and LC3 puncta in Atg5-competent BMMs but not in Atg5-deficient BMMs, while no p62 turnover was observed. Further analysis found the colocalization of LC3 with a small portion of R. australis and Rickettsia-containing double-membrane-bound vacuoles in the BMMs of B6 mice. Moreover, treatment with rapamycin significantly increased the concentrations of R. australis in B6 BMMs compared to those in the untreated B6 BMM controls. Taken together, our results demonstrate that Atg5 favors R. australis infection in mouse macrophages in association with a suppressed level of IL-1ß production but not active autophagy flux. These data highlight the contribution of Atg5 in macrophages to the pathogenesis of rickettsial diseases.

Characterization of Nipah virus infection in a model of human airway epithelial cells cultured at an air-liquid interface.

Nipah virus (NiV) is an emerging paramyxovirus that can cause lethal respiratory illness in humans. No vaccine/therapeutic is currently licensed for humans. Human-to-human transmission was previously reported during outbreaks and NiV could be isolated from respiratory secretions, but the proportion of cases in Malaysia exhibiting respiratory symptoms was significantly lower than that in Bangladesh. Previously, we showed that primary human basal respiratory epithelial cells are susceptible to both NiV-Malaysia (M) and -Bangladesh (B) strains causing robust pro-inflammatory responses. However, the cells of the human respiratory epithelium that NiV targets are unknown and their role in NiV transmission and NiV-related lung pathogenesis is still poorly understood. Here, we characterized NiV infection of the human respiratory epithelium using a model of the human tracheal/bronchial (B-ALI) and small airway (S-ALI) epithelium cultured at an air-liquid interface. We show that NiV-M and NiV-B infect ciliated and secretory cells in B/S-ALI, and that infection of S-ALI, but not B-ALI, results in disruption of the epithelium integrity and host responses recruiting human immune cells. Interestingly, NiV-B replicated more efficiently in B-ALI than did NiV-M. These results suggest that the human tracheal/bronchial epithelium is favourable to NiV replication and shedding, while inducing a limited host response. Our data suggest that the small airways epithelium is prone to inflammation and lesions as well as constituting a point of virus entry into the pulmonary vasculature. The use of relevant models of the human respiratory tract, such as B/S-ALI, is critical for understanding NiV-related lung pathogenesis and identifying the underlying mechanisms allowing human-to-human transmission.

Triazole fungicides induce adipogenesis and repress osteoblastogenesis in zebrafish.

Triazoles are a major group of azole fungicides commonly used in agriculture, and veterinary and human medicine. Maternal exposure to certain triazole antifungal medication causes congenital malformations, including skeletal malformations. We hypothesized that triazoles used as pesticides in agriculture also pose a risk of causing skeletal malformations in developing embryos. In this study, teratogenic effects of three commonly used triazoles, cyproconazole, paclobutrazol, and triadimenol, were investigated in zebrafish, Danio rerio. Exposure to the triazole fungicides caused bone and cartilage malformations in developing zebrafish larvae. Data from whole-embryo transcriptomics with cyproconazole suggested that exposure to this compound induces adipogenesis while repressing skeletal development. Confirming this finding, the expression of selected bone and cartilage marker genes were significantly downregulated with triazoles exposure as determined by quantitative PCR. The expression of selected adipogenic genes was upregulated by the triazoles. Furthermore, exposure to each of the three triazoles induced adipogenesis and lipid droplet formation in vitro in 3T3-L1 pre-adipocyte cells. In vivo in zebrafish larvae, cyproconazole exposure caused lipid accumulation. These results suggest that exposure to triazoles promotes adipogenesis at the expense of skeletal development, and thus they expand the chemical group of bona fide bone to fat switchers.

Microphysiological Systems Evaluation: Experience of TEX-VAL Tissue Chip Testing Consortium.

Much has been written and said about the promise and excitement of microphysiological systems, miniature devices that aim to recreate aspects of human physiology on a chip. The rapid explosion of the offerings and persistent publicity placed high expectations on both product manufacturers and regulatory agencies to adopt the data. Inevitably, discussions of where this technology fits in chemical testing paradigms are ongoing. Some end-users became early adopters, whereas others have taken a more cautious approach because of the high cost and uncertainties of their utility. Here, we detail the experience of a public-private collaboration established for testing of diverse microphysiological systems. Collectively, we present a number of considerations on practical aspects of using microphysiological systems in the context of their applications in decision-making. Specifically, future end-users need to be prepared for extensive on-site optimization and have access to a wide range of imaging and other equipment. We reason that cells, related reagents, and the technical skills of the research staff, not the devices themselves, are the most critical determinants of success. Extrapolation from concentration-response effects in microphysiological systems to human blood or oral exposures, difficulties with replicating the whole organ, and long-term functionality remain as critical challenges. Overall, we conclude that it is unlikely that a rodent- or human-equivalent model is achievable through a finite number of microphysiological systems in the near future; therefore, building consensus and promoting the gradual incorporation of these models into tiered approaches for safety assessment and decision-making is the sensible path to wide adoption.

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

Hypertonic resuscitation improves neuronal and behavioral outcomes after traumatic brain injury plus hemorrhage.

BACKGROUND: : Resuscitation with hypertonic saline or hypertonic saline plus l-arginine acutely improves cerebral blood flow after traumatic brain injury (TBI) followed by hemorrhagic hypotension. The authors investigated whether hypertonic saline or hypertonic l-arginine would improve long-term neuronal survival and behavioral outcomes 15 days after TBI and hemorrhagic hypotension. METHODS: : Mean arterial pressure, arterial blood gases, pH, plasma glucose, hematocrit, and hemoglobin were measured in male Sprague-Dawley rats before and after moderate (2.0 atm) fluid percussion TBI. Rats were assigned to one of six groups: (1) sham TBI, (2) hemorrhage only, (3) TBI only, (4) TBI plus hemorrhage and resuscitation with 0.9% saline, (5) TBI plus hemorrhage and resuscitation with hypertonic saline (7.5%), or (6) TBI plus hemorrhage and resuscitation with l-arginine (100 mg/kg) in hypertonic saline. On postinjury days 1-5, vestibulomotor function was assessed using beam balance and beam walking tasks. On postinjury days 11-15, spatial memory function was assessed using the Morris water maze. After behavioral testing, neuronal counting was performed bilaterally on specific hippocampal regions. RESULTS: : Groups receiving hypertonic saline (P < 0.05, day 15 vs. day 11) or hypertonic l-arginine (P < 0.05, days 13-15 vs. day 11) showed improved performance over time on the Morris water maze, as well as significantly improved neuronal survival in the contralateral hippocampus (P < 0.05, hypertonic saline or hypertonic l-arginine vs. normal saline) compared with untreated TBI or normal saline-treated TBI plus hemorrhage groups. CONCLUSIONS: : Hypertonic saline and hypertonic l-arginine were both effective at promoting long-term neuronal survival and behavioral recovery. The slightly earlier improvement in Morris water maze performance in the hypertonic l-arginine group warrants further studies to determine whether higher doses of l-arginine provide additional improvement. This study supports the therapeutic benefits of hypertonic resuscitation after TBI plus hemorrhagic hypotension.

Estrogenic regulation of host immunity against an estrogen receptor-negative human breast cancer.

PURPOSE: The risk of developing breast cancer is positively correlated with exposure to increased levels of estrogen and/or an increased duration of estrogen exposure. Many different mechanisms have been proposed to explain the association of estrogens with breast cancer risk; however, the well-documented immune modulatory properties of estrogen have received little attention. In part, this is due to a lack of suitable models for studying this relationship. EXPERIMENTAL DESIGN: We have developed an animal model using estrogen receptor (ER)-negative human breast cancer cell line, MDA-MB-468, xenografted into severe combined immunodeficient (SCID) mice. We also generated the ER-alpha knockout (ER-alphaKO) mice on the SCID background and then tested the ability of 17beta-estradiol to stimulate growth of xenografted ER-negative human breast cancer tumors in wild-type and ER-alphaKO SCID mice. We quantified vascularization of tumors, macrophage recruitment to the tumor site by immunocytochemistry, and inflammatory cytokine production. RESULTS: We show that estrogen treatment of C57BL/6/SCID mice promotes the growth of xenografted ER-negative tumors in wild-type mice and this estrogen-induced tumor growth is abrogated in ER-alphaKO mice. Tumor neovascularization of estrogen-treated mice was unchanged versus control; however, estrogen treatment of the C57BL/6/SCID host suppressed macrophage recruitment to and inflammatory cytokine production at the tumor site. CONCLUSIONS: These data are consistent with estrogen modulation of the inflammatory response as a contributing factor in estrogen-stimulated growth of an ER-negative tumor. This effect on the host innate immune response was mediated by ER-alpha.

Biotransport kinetics and intratumoral biodistribution of malonodiserinolamide-derivatized [60]fullerene in a murine model of breast adenocarcinoma.

[60]Fullerene is a highly versatile nanoparticle (NP) platform for drug delivery to sites of pathology owing to its small size and both ease and versatility of chemical functionalization, facilitating multisite drug conjugation, drug targeting, and modulation of its physicochemical properties. The prominent and well-characterized role of the enhanced permeation and retention (EPR) effect in facilitating NP delivery to tumors motivated us to explore vascular transport kinetics of a water-soluble [60]fullerene derivatives using intravital microscopy in an immune competent murine model of breast adenocarcinoma. Herein, we present a novel local and global image analysis of vascular transport kinetics at the level of individual tumor blood vessels on the micron scale and across whole images, respectively. Similar to larger nanomaterials, [60]fullerenes displayed rapid extravasation from tumor vasculature, distinct from that in normal microvasculature. Temporal heterogeneity in fullerene delivery to tumors was observed, demonstrating the issue of nonuniform delivery beyond spatial dimensions. Trends in local region analysis of fullerene biokinetics by fluorescence quantification were in agreement with global image analysis. Further analysis of intratumoral vascular clearance rates suggested a possible enhanced penetration and retention effect of the fullerene compared to a 70 kDa vascular tracer. Overall, this study demonstrates the feasibility of tracking and quantifying the delivery kinetics and intratumoral biodistribution of fullerene-based drug delivery platforms, consistent with the EPR effect on short timescales and passive transport to tumors.

A novel rat medial prefrontal cortical slice preparation to investigate synaptic transmission from amygdala to layer V prelimbic pyramidal neurons.

Electrophysiological recordings from identified synapses in CNS slice preparations in vitro provide important information regarding the connectivity of neuronal circuits and the underlying cellular mechanisms responsible for neuronal excitability and synaptic transmission. We present an anatomical, electrophysiological, and pharmacological characterization of a novel brain slice preparation (BLA-mPFC) to investigate basolateral amygdala synaptic input to rat layer V medial prefrontal cortex pyramidal neurons. A fluorescent tracer (DiI) unilaterally infused in vivo into the basolateral amygdala was used to detect amygdala efferent fibers innervating layer V of the prelimbic and infralimbic cortices within prefrontal cortex slices. In vitro, evoked synaptic responses elicited by stimulating identified basolateral amygdala pathway terminals within the acute BLA-mPFC slice preparation yielded monosynaptic excitatory postsynaptic responses in layer V pyramidal neurons from the prelimbic cortex as determined by extracellular and intracellular recordings. The BLA-mPFC preparation provides essential knowledge of amygdaloid input to the medial prefrontal cortex where information from various brain areas is integrated and returned to subcortical structures, such as the amygdala itself. In addition to investigating normal synaptic function, this preparation provides opportunities to investigate this synapse in animals which have received drugs chronically or have been manipulated genetically to model specific mental diseases known to involve prefrontal cortex and/or amygdala pathology (e.g., schizophrenia, addiction, anxiety, and depression).

Role of aldehyde dehydrogenase isozymes in the defense of rat lens and human lens epithelial cells against oxidative stress.

PURPOSE: 4-Hydroxynonenal (HNE), a metastable lipid peroxidation product, is highly toxic to various cell types if not detoxified. Because of its constant exposure to light, the ocular lens continuously generates reactive oxygen species which, under conditions of oxidative stress, may lead to excessive lipid peroxidation and consequent formation of lipid-derived aldehydes (LDAs) such as HNE. The contribution of various isozymes of aldehyde dehydrogenase (ALDH) to the oxidation of LDAs has never been systematically investigated in the lens. The present study was undertaken to ascertain the role of ALDH1A1 and -3A1 in HNE metabolism and HNE-induced toxicity in cultured human lens epithelial cells (HLECs) and in rat and mouse lenses. METHODS: The metabolism of 3H-HNE was studied in ALDH3A1-knockout mouse lens and in HLECs transfected with ALDH1A1- or -3A1-specific antisense RNA and short interfering (Si)RNA. Appropriate controls were used, including wild-type mouse lens, scrambled oligonucleotides, and a transfection reagent. Transfected HLECs were exposed to oxidative stress (Fenton reaction) or HNE (30 microM) for 3 hours. Toxicity parameters, such as cell viability, apoptosis, and protein-HNE adducts and oxidation of exogenously added 3H-HNE were measured. Rat lenses were transfected with the SiRNA specific to ALDH1A1, and oxidation of 3H-HNE and the susceptibility of the transfected lenses to oxidation-induced opacification were measured. RESULTS: Rat lenses transfected with ALDH1A1-specific SiRNA, or cultured in the presence of the ALDH inhibitor cyanamide/disulfiram and subjected to oxidative stress displayed accelerated loss of transparency and a diminished capacity to oxidize HNE. Similarly, inhibition of ALDH1A1 in HLECs by ALDH1A1-specific antisense RNA or SiRNA was associated with decreased oxidation of 3H-HNE and increased susceptibility of the cells to oxidative damage, including apoptosis. Furthermore, 3H-HNE metabolism and HNE-induced toxicity were not affected in ALDH3A1-specific SiRNA- or antisense RNA-treated rat lenses, HLECs, or ALDH3A1-null mouse lenses. CONCLUSIONS: The results suggest that, under oxidative stress, HNE produced in the lens epithelium can cause toxicity and thus contribute to oxidation-induced cataractogenesis. Furthermore, the studies indicate that ALDH1A1 is a critical isozyme for maintaining clarity in human, rat, and mouse lenses.

A New Imaging Platform for Visualizing Biological Effects of Non-Invasive Radiofrequency Electric-Field Cancer Hyperthermia.

Herein, we present a novel imaging platform to study the biological effects of non-invasive radiofrequency (RF) electric field cancer hyperthermia. This system allows for real-time in vivo intravital microscopy (IVM) imaging of radiofrequency-induced biological alterations such as changes in vessel structure and drug perfusion. Our results indicate that the IVM system is able to handle exposure to high-power electric-fields without inducing significant hardware damage or imaging artifacts. Furthermore, short durations of low-power (< 200 W) radiofrequency exposure increased transport and perfusion of fluorescent tracers into the tumors at temperatures below 41°C. Vessel deformations and blood coagulation were seen for tumor temperatures around 44°C. These results highlight the use of our integrated IVM-RF imaging platform as a powerful new tool to visualize the dynamics and interplay between radiofrequency energy and biological tissues, organs, and tumors.

Selective activation of protein kinase C delta in human neutrophils following ischemia reperfusion of skeletal muscle.

Circulatory neutrophils are known to be critical mediators of inflammation and oxidative stress during ischemia reperfusion (I/R) injury. Recent studies have shown an important role for protein kinase C (PKC) in neutrophil survival and function. Activation of specific isotypes of PKC are known to be involved in membrane alteration and motility, oxidative phosphorylation, and apoptosis modulation of neutrophils. However, the role of PKC in neutrophil responses to I/R in the clinical setting has not been studied. In this study, we examined the neutrophil activation of PKC induced by tourniquet-controlled I/R of skeletal muscle in humans. We found that I/R rapidly activates and translocates PKC delta, but not any of the classical forms of PKC (alpha or beta) from cytosol to the particulate fraction of neutrophils. Particulate translocation of PKC delta is sustained up to 4 h after reperfusion and is associated with kinase activity. Postreperfusion activation of PKC delta in neutrophils signals proapoptosis, but does not cause immediate cell death (as revealed by neutrophil morphology study and DNA-laddering assay). This study indicates that calcium-independent novel PKC delta (nPKC delta) might be predominantly involved in regulating membrane functions and survival of neutrophils associated with post-I/R-induced inflammatory oxidative stress.

The cancer drug tamoxifen: a potential therapeutic treatment for spinal cord injury.

Tamoxifen (TMX) is a selective estrogen receptor modulator that can mimic the neuroprotective effects of estrogen but lacks its systemic adverse effects. We found that TMX (1 mg/day) significantly improved the motor recovery of partially paralyzed hind limbs of male adult rats with thoracic spinal cord injury (SCI), thus indicating a translational potential for this cancer medication given its clinical safety and applicability and the lack of currently available treatments for SCI. To shed light on the mechanisms underlying the beneficial effects of TMX for SCI, we used proteomic analyses, Western blots and histological assays, which showed that TMX treatment spared mature oligodendrocytes/increased myelin levels and altered reactive astrocytes, including the upregulation of the water channels aquaporin 4 (AQP4), a novel finding. AQP4 increases in TMX-treated SCI rats were associated with smaller fluid-filled cavities with borders consisting of densely packed AQP4-expressing astrocytes that closely resemble the organization of normal glia limitans externa (in contrast to large cavities in control SCI rats that lacked glia limitans-like borders and contained reactive glial cells). Based on our findings, we propose that TMX is a promising candidate for the therapeutic treatment of SCI and a possible intervention for other neuropathological conditions associated with demyelination and AQP4 dysfunction.

Dynamic cross talk model of the epithelial innate immune response to double-stranded RNA stimulation: coordinated dynamics emerging from cell-level noise.

We present an integrated dynamical cross-talk model of the epithelial innate immune response (IIR) incorporating RIG-I and TLR3 as the two major pattern recognition receptors (PRR) converging on the RelA and IRF3 transcriptional effectors. bioPN simulations reproduce biologically relevant gene-and protein abundance measurements in response to time course, gene silencing and dose-response perturbations both at the population and single cell level. Our computational predictions suggest that RelA and IRF3 are under auto- and cross-regulation. We predict, and confirm experimentally, that RIG-I mRNA expression is controlled by IRF7. We also predict the existence of a TLR3-dependent, IRF3-independent transcription factor (or factors) that control(s) expression of MAVS, IRF3 and members of the IKK family. Our model confirms the observed dsRNA dose-dependence of oscillatory patterns in single cells, with periods of 1-3 hr. Model fitting to time series, matched by knockdown data suggests that the NF-κB module operates in a different regime (with different coefficient values) than in the TNFα-stimulation experiments. In future studies, this model will serve as a foundation for identification of virus-encoded IIR antagonists and examination of stochastic effects of viral replication. Our model generates simulated time series, which reproduce the noisy oscillatory patterns of activity (with 1-3 hour period) observed in individual cells. Our work supports the hypothesis that the IIR is a phenomenon that emerged by evolution despite highly variable responses at an individual cell level.

Traumatic brain injury in vivo and in vitro contributes to cerebral vascular dysfunction through impaired gap junction communication between vascular smooth muscle cells.

Gap junctions (GJs) contribute to cerebral vasodilation, vasoconstriction, and, perhaps, to vascular compensatory mechanisms, such as autoregulation. To explore the effects of traumatic brain injury (TBI) on vascular GJ communication, we assessed GJ coupling in A7r5 vascular smooth muscle (VSM) cells subjected to rapid stretch injury (RSI) in vitro and VSM in middle cerebral arteries (MCAs) harvested from rats subjected to fluid percussion TBI in vivo. Intercellular communication was evaluated by measuring fluorescence recovery after photobleaching (FRAP). In VSM cells in vitro, FRAP increased significantly (p<0.05 vs. sham RSI) after mild RSI, but decreased significantly (p<0.05 vs. sham RSI) after moderate or severe RSI. FRAP decreased significantly (p<0.05 vs. sham RSI) 30 min and 2 h, but increased significantly (p<0.05 vs. sham RSI) 24 h after RSI. In MCAs harvested from rats 30 min after moderate TBI in vivo, FRAP was reduced significantly (p<0.05), compared to MCAs from rats after sham TBI. In VSM cells in vitro, pretreatment with the peroxynitrite (ONOO(-)) scavenger, 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron[III], prevented RSI-induced reductions in FRAP. In isolated MCAs from rats treated with the ONOO(-) scavenger, penicillamine, GJ coupling was not impaired by fluid percussion TBI. In addition, penicillamine treatment improved vasodilatory responses to reduced intravascular pressure in MCAs harvested from rats subjected to moderate fluid percussion TBI. These results indicate that TBI reduced GJ coupling in VSM cells in vitro and in vivo through mechanisms related to generation of the potent oxidant, ONOO(-).

Spatial distribution of protein kinase A activity during cell migration is mediated by A-kinase anchoring protein AKAP Lbc.

Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility.

RelA Ser276 phosphorylation is required for activation of a subset of NF-kappaB-dependent genes by recruiting cyclin-dependent kinase 9/cyclin T1 complexes.

NF-kappaB plays a central role in cytokine-inducible inflammatory gene expression. Previously we empirically determined the identity of 92 members of the genetic network under direct NF-kappaB/RelA control that show marked heterogeneity in magnitude of transcriptional induction and kinetics of peak activation. To investigate this network further, we have applied a recently developed two-step chromatin immunoprecipitation assay that accurately reflects association and disassociation of RelA binding to its chromatin targets. Although inducible RelA binding occurs with similar kinetics on all NF-kappaB-dependent genes, serine 276 (Ser(276))-phosphorylated RelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF), including Gro-beta, interleukin-8 (IL-8), and IkappaBalpha. Previous work has shown that TNF-inducible RelA Ser(276) phosphorylation is controlled by a reactive oxygen species (ROS)-protein kinase A signaling pathway. To further understand the role of phospho-Ser(276) RelA in target gene expression, we inhibited its formation by ROS scavengers and antioxidants, treatments that disrupt phospho-Ser(276) formation but not the translocation and DNA binding of nonphosphorylated RelA. Here we find that phospho-Ser(276) RelA is required only for activation of IL-8 and Gro-beta, with IkappaBalpha being unaffected. These data were confirmed in experiments using RelA(-/-) murine embryonic fibroblasts reconstituted with a RelA Ser(276)Ala mutation. In addition, we observe that phospho-Ser(276) RelA binds the positive transcription elongation factor b (P-TEFb), a complex containing the cyclin-dependent kinase 9 (CDK-9) and cyclin T1 subunits. Inhibition of P-TEFb activity by short interfering RNA (siRNA)-mediated knockdown shows that the phospho-Ser(276) RelA-P-TEFb complex is required for IL-8 and Gro-beta gene activation but not for IkappaBalpha gene activation. These studies indicate that TNF induces target gene expression by heterogeneous mechanisms. One is mediated by phospho-Ser(276) RelA formation and chromatin targeting of P-TEFb controlling polymerase II (Pol II) recruitment and carboxy-terminal domain phosphorylation on the IL-8 and Gro-beta genes. The second involves a phospho-Ser(276) RelA-independent activation of genes preloaded with Pol II, exemplified by the IkappaBalpha gene. Together, these data suggest that the binding kinetics, selection of genomic targets, and mechanisms of promoter induction by RelA are controlled by a phosphorylation code influencing its interactions with coactivators and transcriptional elongation factors.

Formation of nsP3-specific protein complexes during Sindbis virus replication.

Alphaviruses are arthropod-borne viruses (arboviruses) that include a number of important human and animal pathogens. Their replication proceeds in the cytoplasm of infected cells and does not directly depend on nuclei. Alphaviruses encode only four nonstructural proteins that are required for the replication of viral genome and transcription of the subgenomic RNA. However, the replicative enzyme complexes (RCs) appear to include cellular proteins and assemble on cellular organelles. We have developed a set of recombinant Sindbis (SIN) viruses with green fluorescent protein (GFP) insertions in one of the nonstructural proteins, nsP3, to further understand the RCs' genesis and structure. We studied the assembly of nsP3/GFP-containing protein complexes at different stages of infection and isolated a combination of cellular proteins that are associated with SIN nsP3. We demonstrated the following. (i) SIN nsP3 can tolerate the insertion of GFP into different fragments of the coding sequence; the designed recombinant viruses are viable, and their replication leads to the assembly of nsP3/GFP chimeric proteins into gradually developing, higher-order structures differently organized at early and late times postinfection. (ii) At late times postinfection, nsP3 is assembled into complexes of similar sizes, which appear to be bound to cytoskeleton filaments and can aggregate into larger structures. (iii) Protein complexes that are associated with nsP3/GFP contain a high concentration of cytoskeleton proteins, chaperones, elongation factor 1A, heterogeneous nuclear ribonucleoproteins, 14-3-3 proteins, and some of the ribosomal proteins. These proteins are proposed to be essential for SIN RC formation and/or functioning.

T cell receptor induced intracellular redistribution of type I protein kinase A.

The productive activation of CD4(+) T lymphocytes, leading to proliferation and cytokine secretion, requires precise temporal regulation of intracellular cyclic AMP concentrations. The major effector molecule activated by cyclic AMP in mammalian cells is the cyclic AMP-dependent protein kinase A (PKA). The type I PKA isozyme mediates the inhibitory effects of cyclic AMP on T-cell activation. Using laser scanning confocal microscopy, we demonstrated that the regulation of PKA type I activity involves spatial redistribution of PKA type I molecules following T-cell receptor (TCR) stimulation. In resting T cells, PKA type I was located in membrane proximal regions and distributed equally across the cell. Shortly after antigen engagement, T cells and antigen-presenting cells formed an area of intense contact, known as the immunological synapse. TCR concentrated at the synapse, whereas PKA type I molecules redistributed to the opposite cell pole within 10 min after T-cell stimulation. Type I PKA redistribution was solely dependent on TCR signalling, because we observed the same temporal and spatial distribution after antibody-mediated cross-linking of the TCR-associated CD3 complex. Segregation of TCR and PKA type I molecules was maintained for at least 20 min. Thirty minutes after stimulation, PKA type I partially colocalized with the TCR. After 60 min, PKA type I distribution again approached the resting state. Considering that initial TCR signals lead to increases in intracellular cyclic AMP, PKA type I molecules may be targeted towards localized cyclic AMP accumulations or transported away from these areas, depending on the requirements of the cellular response.