Inhibition of Cell Migration by PITENINs: The Role of ARF6

We have previously reported the development of small molecule phosphatidylinositol-3,4,5-trisphosphate (PIP3) antagonists (PITs) that block pleckstrin homology (PH) domain interaction, including activation of Akt, and show anti-tumor potential. Here we show that the same molecules inhibit growth factor-induced actin remodeling, lamellipodia formation and, ultimately, cell migration and invasion, consistent with an important role of PIP3 in these processes. In vivo, a PIT-1 analog displays significant inhibition on tumor angiogenesis and metastasis. ADP ribosylation factor 6 (ARF6) was recently identified as an important mediator of cytoskeleton and cell motility, which is regulated by PIP3-dependent membrane translocation of the guanine nucleotide exchange factors (GEFs) such as ADP-ribosylation factor nucleotide binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1). We demonstrate that PITs inhibit PIP3/ARNO or GRP1 PH domain binding and membrane localization, resulting in the inhibition of ARF6 activation. Importantly, we show that expression of the constitutively active mutant of Arf6 attenuates inhibition of lamellipodia formation and cell migration by PITs, confirming that inhibition of Arf6 contributes to inhibition of these processes by PITs. Overall, our studies demonstrate the feasibility of developing specific small molecule targeting PIP3 binding by PH domains as potential anti-cancer agents that can simultaneously interfere with cancer development at multiple points

Necrostatin-1 Reduces Histopathology and Improves Functional Outcome after Controlled Cortical Impact in Mice

Necroptosis is a newly identified type of programmed necrosis initiated by the activation of tumor necrosis factor alpha (TNF?)/Fas. Necrostatin-1 is a specific inhibitor of necroptosis that reduces ischemic tissue damage in experimental stroke models. We previously reported decreased tissue damage and improved functional outcome after controlled cortical impact (CCI) in mice deficient in TNF? and Fas. Hence, we hypothesized that necrostatin-1 would reduce histopathology and improve functional outcome after CCI in mice. Compared with vehicle-/inactive analog-treated controls, mice administered necrostatin-1 before CCI had decreased propidium iodide-positive cells in the injured cortex and dentate gyrus (6 h), decreased brain tissue damage (days 14, 35), improved motor (days 1 to 7), and Morris water maze performance (days 8 to 14) after CCI. Improved spatial memory was observed even when drug was administered 15 mins after CCI. Necrostatin-1 treatment did not reduce caspase-3-positive cells in the dentate gyrus or cortex, consistent with a known caspase-independent mechanism of necrostatin-1. However, necrostatin-1 reduced brain neutrophil influx and microglial activation at 48 h, suggesting a novel anti-inflammatory effect in traumatic brain injury (TBI). The data suggest that necroptosis plays a significant role in the pathogenesis of cell death and functional outcome after TBI and that necrostatin-1 may have therapeutic potential for patients with TBI

Role of 5-HT1A-mediated upregulation of brain indoleamine 2,3 dioxygenase 1 in the reduced antidepressant and antihyperalgesic effects of fluoxetine during maintenance treatment

The reduced antidepressant and antihyperalgesic effects of selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine during maintenance treatment has been reported, but little is known about the molecular mechanism of this phenomenon. In three comorbid pain and depression animal models (genetic predisposition, chronic social stress, arthritis), we showed that the fluoxetine’s antidepressant and antihyperalgesic effects were reduced during the maintenance treatment. Fluoxetine exposure induced upregulation of the 5-hydroxytryptamine 1A (5-HT1A) auto-receptor and indoleamine 2,3 dioxygenase 1 (IDO1, a rate-limiting enzyme of tryptophan metabolism) in the brainstem dorsal raphe nucleus (DRN), which shifted the tryptophan metabolism away from the 5-HT biosynthesis. Mechanistically, IDO1 upregulation was downstream to fluoxetine-induced 5-HT1A receptor expression because 1) antagonism of the 5-HT1A receptor with WAY100635 or 5-HT1A receptor knockout blocked the IDO1 upregulation, and 2) inhibition of IDO1 activity did not block the 5-HT1A receptor upregulation following fluoxetine exposure. Importantly, inhibition of either the 5-HT1A receptor or IDO1 activity sustained the fluoxetine’s antidepressant and antihyperalgesic effects, indicating that 5-HT1A-mediated IDO1 upregulation in the brainstem DRN contributed to the reduced antidepressant and antihyperalgesic effects of fluoxetine. These results suggest a new strategy to improving the therapeutic efficacy of SSRI during maintenance treatment

Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

system. mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma

A multifaceted study of White Spot Syndrome Virus (WSSV) a shrimp pathogen

xiii, 148 leavesThe white spot syndrome virus (WSSV), which has been associated with major viral epizootics, was first identified in China and Taiwan in 1992 and soon spread in shrimp farms all over the world. The mortality rates for shrimp population experiencing WSSV infections can reach IOO% within three to ten days from the onset of the symptoms. In this study, a WSSV strain was isolated from P. chinesis from Qindao, China. The morphology of this WSSV isolate was examined by electron microscope (EM) and compared with other WSSV isolates. A cDNA library was constructed from white spot syndrome virus (WSSV) infected penaeid shrimp. Among cDNA clones with WSSV sequence insert, twelve clones were sequenced and analyzed. By comparing with the DNA sequence in the GenBank, cDNA clones containing sequence identical to that of WSSV envelope protein VP28 and nucleoprotein VP15 were identified. Poly(A) sites on the mRNAs of VP28 and VP15 were identified. In the course of the study, a PCR strategy was developed for amplification of double-stranded cDNA and enrichment of abundant cDNA. A screening strategy was established to identify clones with viral cDNA insert from a cDNA library. The cDNA library approach does not require the isolation of virion, which usually is difficult, time-consuming and expensive. The cDNA library method is readily applicable to other viruses and the protocol used in this study can be used with little modification. Genes encoding major viral structural proteins VP28, VP26, VP24, VPl9 and VPl5 from five WSSV isolates were sequenced and compared with that of other sequences of WSSV strains available in the GenBank. The genes encoding these major viral structural proteins are identical among WSSV strains isolated from different shrimp species and/or geographical areas. Gene probes developed based on the DNA sequences of viral structural proteins can be used for WSSV diagnostic/identification. A truncated version of the white spot syndrome virus (WSSV) 27.5kDa envelope protein was expressed as a histidine tag fusion protein in Escherichia coli. The bacterial expression system allowed the production of up to 10 mg of purified recombinant protein per liter of bacterial culture. Antiserum from a rabbit immunized with the recombinant protein was found to recognize the 27.5kDa viral envelope protein of WSSV isolated from different geographic regions. The antiserum did not recognize any of the other known WSSV structural proteins. A sensitive immuno-dot assay for WSSV was developed using the specific rabbit polyclonal antiserum. A wet-format dipstick model was developed for simple and rapid WSSV detection. Different types of membranes were tested as solid support for dipstick assay. The conditions for antibody and microparticle coupling reaction were optimized. The present study would be of value for the development of a dry-format dipstick with detector reagent (antibody and microparticle complex) dried on a conjugate pad, which would be more desirable for field use

Detection of shrimp viral pathogens with a simple dot-blot enzyme immunoassay protocol

Dot-blot nitrocellulose enzyme immunoassays (DB-NC-EIA) were developed for the detection of two shrimp viral pathogens, white-spot virus (WSV), and yellow-head virus (YHV). The assays utilized HRP-conjugated virus-specific polyclonal antibodies to detect virus antigen present in gill homogenates of infected shrimp spotted onto nitrocellulose membrane. The DB-NC-EIA was highly specific and sensitive enough to detect the presence of viral proteins before the appearance of overt symptoms. The assays are one of the simplest and most rapid detection methods available for WSV and YHV

Genetic Analysis of the Role of Tumor Necrosis Factor Receptors in Functional Outcome after Traumatic Brain Injury in Mice

We previously reported that tumor necrosis factor-α (TNF-α) and Fas receptor induce acute cellular injury, tissue damage, and motor and cognitive deficits after controlled cortical impact (CCI) in mice (Bermpohl et al. 2007); however, the TNF receptors (TNFR) involved are unknown. Using a CCI model and novel mutant mice deficient in TNFR1/Fas, TNFR2/Fas, or TNFR1/TNFR2/Fas, we tested the hypothesis that the combination of TNFR2/Fas is protective, whereas TNFR1/Fas is detrimental after CCI. Uninjured knockout (KO) mice showed no differences in baseline physiological variables or motor or cognitive function. Following CCI, mice deficient in TNFR2/Fas had worse post-injury motor and Morris water maze (MWM) performance than wild-type (WT) mice (p < 0.05 group effect for wire grip score and MWM performance by repeated measures ANOVA). No differences in motor or cognitive outcome were observed in TNFR1/Fas KO, or in TNFR2 or TNFR1 single KO mice, versus WT mice. Additionally, no differences in propidium iodide (PI)-positive cells (at 6 h) or lesion size (at 14 days) were observed between WT and TNFR1/Fas or TNFR2/Fas KO mice. Somewhat surprisingly, mice deficient in TNFR1/TNFR2/Fas also had PI-positive cells, lesion size, and motor and MWM deficits similar to those of WT mice. These data suggest a protective role for TNFR2/Fas in the pathogenesis of TBI. Further studies are needed to determine whether direct or indirect effects of TNFR1 deletion in TNFR2/Fas KO mice mediate improved functional outcome in TNFR1/TNFR2/Fas KO mice after CCI

Gut Microbiota is critical for the induction of chemotherapy-induced pain

Chemotherapy-induced pain is a dose-limiting condition that affects 30% of patients undergoing chemotherapy. We found that the gut microbiota promotes the development of chemotherapy-induced mechanical hyperalgesia. Oxaliplatin-induced mechnical hyperalgesia was reduced in germ-free mice and in those mice pretreated with antibiotics. Restoration of the microbiota of germ-free mice abrogated this protection. These effects appear to be mediated, in part, by TLR4 expressed on hematopoietic cells, including macrophages