Zinc supplementation ameliorates sorafenib-induced cognitive impairment through ROS/JNK signaling pathway.

Sorafenib, a multiple kinase inhibitor, is widely used in cancer patients. Recently, clinical studies highlighted the relationship between cognitive deficits and sorafenib exposure. Zinc abundant in the body has been reported to exert neuroprotective activities. However, the effects of zinc supplementation on sorafenib-induced cognitive impairment are still unknown. In the current study, we verified that mice challenged with sorafenib displayed characteristic features of cognitive impairment. However, zinc treatment effectively improved these changes. Histopathological staining also showed that zinc significantly alleviated hippocampal microstructural and ultrastructural damages induced by sorafenib. Meanwhile, zinc significantly reduced sorafenib-induced ROS production and neuronal cells apoptosis in vivo and vitro. Additionally, we also investigated whether zinc protected against sorafenib-induced neuronal cells apoptosis via ROS/JNK pathway through treating SH-SY5Y cells with the NAC or the specific JNK activator anisomycin. The results indicated that NAC performed the same protective effects as zinc in sorafenib-challenged SH-SY5Y cells and activation of JNK by anisomycin partly abolished the protective effects of zinc. Collectively, the present study suggested that inhibition of oxidative stress and the JNK pathway might contribute to the protective effects of zinc against sorafenib-caused cognitive impairment in vivo and vitro.

Modulation of the p38 MAPK Pathway by Anisomycin Promotes Ferroptosis of Hepatocellular Carcinoma through Phosphorylation of H3S10.

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor worldwide. Ferroptosis is emerging as an effective target for tumor treatment as it has been shown to potentiate cell death in some malignancies. However, it remains unclear whether histone phosphorylation events, an epigenetic mechanism that regulates transcriptional expression, are involved in ferroptosis. Our study found that supplementation with anisomycin, an agonist of p38 mitogen-activated protein kinase (MAPK), induced ferroptosis in HCC cells, and the phosphorylation of histone H3 on serine 10 (p-H3S10) was participated in anisomycin-induced ferroptosis. To investigate the anticancer effects of anisomycin-activated p38 MAPK in HCC, we analyzed cell viability, colony formation, cell death, and cell migration in Hep3B and HCCLM3 cells. The results showed that anisomycin could significantly suppress HCC cell colony formation and migration and induce HCC cell death. The hallmarks of ferroptosis, such as abnormal accumulation of iron and elevated levels of lipid peroxidation and malondialdehyde, were detected to confirm the ability of anisomycin to promote ferroptosis. Furthermore, coincubation with SB203580, an inhibitor of activated p38 MAPK, partially rescued anisomycin-induced ferroptosis. And the levels of p-p38 MAPK and p-H3S10 were successively increased by anisomycin treatment. The relationship between p-H3S10 and ferroptosis was revealed by ChIP sequencing. The reverse transcription PCR and immunofluorescence results showed that NCOA4 was upregulated both in mRNA and protein levels after anisomycin treatment. And by C11-BODIPY staining, we found that anisomycin-induced lipid reactive oxygen species was reduced after NCOA4 knockdown. In conclusion, the anisomycin-activated p38 MAPK promoted ferroptosis of HCC cells through H3S10 phosphorylation.

A valepotriate-enriched fraction from Valeriana glechomifolia decreases DNA methylation and up-regulate TrkB receptors in the hippocampus of mice.

DNA methylation, an epigenetic modification that mediates gene silencing, has been shown to play a role in the neurobiology of major depression. Studies suggested that terpenes inhibit DNA methylation and increase gene expression. The present study investigated the involvement of DNA methylation in the antidepressant-like activity of diene valepotriates, non-glicosilated carbocyclic iridoids that comprise a family of terpenes obtained from Valeriana glechomifolia. The antidepressant-like effect of diene valepotriates acute administration (5 mg/kg, p.o.) in mice submitted to the forced swimming test was followed by a decrease in global DNA methylation in animals' hippocampus (but not in the pre-frontal cortex). Mice pretreatment with anysomicin (a protein synthesis inhibitor) and K252a (an inhibitor of Trk receptors) attenuated diene valepotriates-induced antidepressant-like effect in the forced swimming test. Diene valepotriates elicited an upregulation in the TrkB receptor and a tendency to increase BDNF levels in mice hippocampus. These results demonstrate that DNA methylation could be an in vivo molecular target of diene valepotriates. The diene valepotriates-triggered reduction in hippocampal DNA methylation is accompanied by increased protein synthesis, which is involved in its antidepressant-like activity. Furthermore, BDNF-mediated TrkB signaling may contribute for diene valepotriates antidepressant-like effect.

Inhibition of LONP1 Suppresses Pancreatic Cancer Progression Via c-Jun N-Terminal Kinase Pathway-Meditated Epithelial-Mesenchymal Transition.

OBJECTIVE: The aim of this study was to investigate the role of LONP1 in the progression of pancreatic cancer. METHODS: Lentivirus was used to silence LONP1 in PANC-1 cells. Colony formation assay, cell counting kit (CCK8) assay, cell scratch-wound assay, and transwell assay were used to assess the effects of our strategy on inhibiting cancer growth, migration, and invasion. Protein expression was detected by Western blot analysis. RESULTS: The expression of LONP1 in pancreatic carcinoma tissues was higher than that in adjacent normal pancreatic tissues. Downregulation of LONP1 suppressed the proliferation, migration, and invasion of PANC-1 cells. Knockdown of LONP1 in PANC-1 cells inhibited epithelial-mesenchymal transition and matrix metalloprotein (MMP) 2/9 by downregulation of vimentin, snail, slug, MMP2, and MMP9 and upregulation of claudin-1. The c-Jun N-terminal kinase pathway was inactivated in LONP1 knockdown PANC-1 cells. Activation of the c-Jun N-terminal kinase pathway by anisomycin treatment significantly reversed the changes in epithelial-mesenchymal transition markers and MMP2/9 induced by ablation of LONP1 in PANC-1 cells. CONCLUSIONS: LONP1 plays a vital role in the proliferation and metastasis of pancreatic cancer, which provides a potential therapeutic target for the treatment of pancreatic cancer.

High-Throughput Screening of Entamoeba Identifies Compounds Which Target Both Life Cycle Stages and Which Are Effective Against Metronidazole Resistant Parasites.

Neglected tropical diseases, especially those caused by parasites, are significantly underserved by current drug development efforts, mostly due to the high costs and low economic returns. One method for lowering the costs of drug discovery and development for these diseases is to repurpose drugs developed for other indications. Here, we present the results of a screen of five repurposed drug libraries to identify potential new lead compounds to treat amebiasis, a disease that affects tens of millions of people and causes ~100,000 deaths annually. E. histolytica, the causative agent of amebiasis, has two major life cycle stages, the trophozoite and the cyst. The current primary treatment for amebiasis, nitroimidazole compounds, do not eliminate parasites from the colonic lumen, necessitating a multi-drug treatment regimen. We aimed to address this problem by screening against both life stages, with the aim of identifying a single drug that targets both. We successfully identified eleven compounds with activity against both cysts and trophozoites, as well as multiple compounds that killed trophozoites with improved efficacy over existing drugs. Two lead compounds (anisomycin and prodigiosin) were further characterized for activity against metronidazole (MNZ) resistant parasites and mature cysts. Anisomycin and prodigiosin were both able to kill MNZ resistant parasites while prodigiosin and its analog obatoclax were active against mature cysts. This work confirms the feasibility of identifying drugs that target both Entamoeba trophozoites and cysts, and is an important step toward developing improved treatment regimens for Entamoeba infection.

A role for the interoceptive insular cortex in the consolidation of learned fear.

A growing body of evidence suggests that learned fear may be related to the function of the interoceptive insular cortex. Using an auditory fear conditioning paradigm in rats, we show that the inactivation of the posterior insular cortex (pIC), the target of the interoceptive thalamus, prior to training produced a marked reduction in fear expression tested 24h later. Accordingly, post-training anisomycin infused immediately, but not 6h after, also reduced fear expression tested the following day, supporting a role for the pIC in consolidation of fear memory. The long-term (ca. a week) and reversible inactivation of the pIC with the sodium channel blocker neosaxitoxin, immediately after fear memory reactivation induced a progressive decrease in the behavioral expression of conditioned fear. In turn, we observed that fear memory reactivation is accompanied by an enhanced expression of Fos and Zif268, early genes involved in neural activity and plasticity. Taken together these data indicate that the pIC is involved in the regulation of fear memories.

Curcumol suppresses RANKL-induced osteoclast formation by attenuating the JNK signaling pathway.

Osteoclasts, derived from hemopoietic progenitors of the monocyte/macrophage lineage, have a unique role in bone resorption, and are considered a potential therapeutic target in the treatment of such pathologic bone diseases as osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we demonstrate that curcumol, one of the major components of the essential oil of Rhizoma Curcumae, exhibits an inhibitory effect on receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast differentiation with both bone marrow-derived macrophages and RAW264.7 cells in a dose-dependent manner. In addition, RANKL-induced mRNA expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K, is prominently reduced in the presence of curcumol. Furthermore, the molecular mechanism of action was investigated, and curcumol inhibited osteoclastogenesis by specifically impairing RANKL-induced c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling, which was further identified in rescue studies by means of anisomycin, a JNK signaling-specific activator. Taken together, these findings suggest that curcumol suppresses RANKL-induced osteoclast differentiation through the JNK/AP-1 signaling pathway, and may be useful as a therapeutic treatment for bone resorption-associated diseases.

Baicalein reduces the invasion of glioma cells via reducing the activity of p38 signaling pathway.

Baicalein, one of the major flavonids in Scutellaria baicalensis, has historically been used in anti-inflammatory and anti-cancer therapies. However, the anti-metastatic effect and related mechanism(s) in glioma are still unclear. In this study, we thus utilized glioma cell lines U87MG and U251MG to explore the effect of baicalein. We found that administration of baicalein significantly inhibited migration and invasion of glioma cells. In addition, after treating with baicalein for 24 h, there was a decrease in the levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 expression as well as proteinase activity in glioma cells. Conversely, the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 was increased in a dose-dependent manner. Moreover, baicalein treatment significantly decreased the phosphorylated level of p38, but not ERK1/2, JNK1/2 and PI3K/Akt. Combined treatment with a p38 inhibitor (SB203580) and baicalein resulted in the synergistic reduction of MMP-2 and MMP-9 expression and then increase of TIMP-1 and TIMP-2 expression; and the invasive capabilities of U87MG cells were also inhibited. However, p38 chemical activator (anisomycin) could block these effects produced by baicalein, suggesting baicalein directly downregulate the p38 signaling pathway. In conclusion, baicalein inhibits glioma cells invasion and metastasis by reducing cell motility and migration via suppression of p38 signaling pathway, suggesting that baicalein is a potential therapeutic agent for glioma.

Memory recall and modifications by activating neurons with elevated CREB.

Memory is supported by a specific ensemble of neurons distributed in the brain that form a unique memory trace. We previously showed that neurons in the lateral amygdala expressing elevated levels of cAMP response-element binding protein are preferentially recruited into fear memory traces and are necessary for the expression of those memories. However, it is unknown whether artificially activating just these selected neurons in the absence of behavioral cues is sufficient to recall that fear memory. Using an ectopic rat vanilloid receptor TRPV1 and capsaicin system, we found that activating this specific ensemble of neurons was sufficient to recall established fear memory. Furthermore, this neuronal activation induced a reconsolidation-like reorganization process, or strengthening of the fear memory. Thus, our findings establish a direct link between the activation of specific ensemble of neurons in the lateral amygdala and the recall of fear memory and its subsequent modifications.

Superinduction of leptin mRNA in mouse hypothalamic neurons.

In marked contrast to several other species, including rats and humans, leptin gene expression is undetectable in mouse brain. This unexpected finding may reflect unique energy regulation pathways in the mouse. We investigated possible mechanisms by which leptin (ob) gene expression is suppressed in mouse brain: (a) the possibility that ob mRNA levels might be detectable in vitro through the superinduction of gene expression following protein synthesis inhibition and (b) whether chromatin modification of the ob gene was responsible for this repression. Experiments were conducted on mouse hypothalamic neurons in vitro. Cells were treated with (a) protein synthesis inhibitors: cycloheximide (CHX; 25 µg/ml); puromycin (50 µg/ml); anisomycin (5 µM); (b) trichostatin A (histone deacetylase inhibitor; 500 nM); and (c) 5-aza-2'-deoxycytidine (DNA methylation inhibitor; 5 µM). Following the incubations, cells were harvested for the preparation of RNA and ob mRNA was detected using real-time reverse transcription PCR. Protein synthesis inhibitors induced a rapid increase in ob mRNA levels in mouse hypothalamic neurons in vitro. For example CHX stimulation of ob mRNA was detectable at 60 min after treatment and reached a maximum between 4 and 6 h. A dose-response analysis, with concentrations of CHX of 1, 2, 10, 25, and 50 µg/ml, indicated that CHX was already effective at 1.0 µg/ml, with a maximal effect by 25 µg/ml. In contrast, incubation with trichostatin A and 5-aza-2'-deoxycytidine had no effect and ob mRNA remained undetectable. These data show that leptin gene expression is superinduced in ob-negative mouse hypothalamic neurons following inhibition of protein synthesis. They confirm that the previously reported absence of leptin mRNA in mouse brain is probably because of an active repressive mechanism, although this may not involve chromatin modification.

Repeated preconditioning with hyperbaric oxygen induces neuroprotection against forebrain ischemia via suppression of p38 mitogen activated protein kinase.

We previously reported in rats that preconditioning with hyperbaric oxygen (HBO; 100% O(2) 3.5-atomsphere absolute (ATA), 1 h/day for 5 days) provided neuroprotection against transient (8 min) forebrain ischemia possibly through protein synthesis relevant to neurotrophin receptor and inflammatory-immune system. A recent report suggested that HBO-induced neuroprotection is relevant to brain derived neurotrophic factor and its downstream event involving suppression of p38 mitogen activated protein kinase (p38) activation. In the present study, we first performed a dose comparison (1, 2, and 3.5 ATA) of HBO-induced neuroprotection and then investigated pharmacological modification by 10 mg/kg anisomycin (a protein synthesis inhibitor and potent activator for p38) and 200 microg/kg SB203580 (a p38 inhibitor), which were given intraperitoneally 60 and 30 min before every 3.5 ATA-HBO treatment, respectively. Most prominent protective effect on hippocampal CA1 neurons was observed with 3.5 ATA-HBO (survived neurons: 69% [62-73%] vs. untreated: 3.9% [2-8%], 1 ATA: 8.8% [0-26%], 2 ATA-HBO: 46% [22-62%] (median [range]) (7 days after ischemia). Anisomycin abolished a neuroprotective effect (survived neuron: 1.2% [0-7%]). SB203580, when given between administration of anisomycin and HBO treatment, resumed a neuroprotective effect (survived neuron: 52% [37-62%]). The level of phosphorylated p38 at 10-min reperfusion was significantly decreased in 3.5 ATA-HBO group (32% [12-53%] of sham). Single pretreatment with 100 and 200 microg/kg of SB203580 exerted a similar neuroprotective effect (39% [25-51%] and 59% [50-72%]) to 2 and 3.5 ATA-HBO preconditioning, respectively. It is concluded that suppression of p38 phosphorylation plays a key role in HBO-induced neuroprotection and that pretreatment with a p38 inhibitor (SB203580) can provide similar neuroprotection.

Altered protein synthesis is a trigger for long-term memory formation.

There is ongoing debate concerning whether new protein synthesis is necessary for, or even contributes to, memory formation and storage. This review summarizes a contemporary model proposing a role for altered protein synthesis in memory formation and its subsequent stabilization. One defining aspect of the model is that altered protein synthesis serves as a trigger for memory consolidation. Thus, we propose that specific alterations in the pattern of neuronal protein translation serve as an initial event in long-term memory formation. These specific alterations in protein readout result in the formation of a protein complex that then serves as a nidus for subsequent perpetuating reinforcement by a positive feedback mechanism. The model proposes this scenario as a minimal but requisite component for long-term memory formation. Our description specifies three aspects of prevailing scenarios for the role of altered protein synthesis in memory that we feel will help clarify what, precisely, is typically proposed as the role for protein translation in memory formation. First, that a relatively short initial time window exists wherein specific alterations in the pattern of proteins translated (not overall protein synthesis) is involved in initializing the engram. Second, that a self-perpetuating positive feedback mechanism maintains the altered pattern of protein expression (synthesis or recruitment) locally. Third, that other than the formation and subsequent perpetuation of the unique initializing proteins, ongoing constitutive protein synthesis is all that is minimally necessary for formation and maintenance of the engram. We feel that a clear delineation of these three principles will assist in interpreting the available experimental data, and propose that the available data are consistent with a role for protein synthesis in memory.

Low-frequency stimulation induces a pathway-specific late phase of LTP in the amygdala that is mediated by PKA and dependent on protein synthesis.

Activity-dependent changes in synaptic efficacy are thought to be the key cellular mechanism for the formation and storage of both explicit and implicit memory. Different patterns of stimulation can elicit different changes in the efficiency on excitatory synaptic transmission. Here, we examined the synaptic changes in the amygdala of adult mice produced by low-frequency stimulation (1 Hz, 15 min, LFS). We first compared the synaptic changes induced by LFS in three different synaptic pathways of amygdala: cortical-lateral amygdala, thalamic-lateral amygdala, and lateral-basolateral amygdala pathways. We find that the plastic changes induced by LFS are different between synaptic pathways. Low-frequency stimulation selectively elicits a slow onset and protein synthesis-dependent late-phase LTP in the cortical-lateral amygdala pathway, but not in the thalamic-lateral or lateral-basolateral pathways. We next analyzed LTP induced by LFS in the cortical-lateral amygdala pathway and found that three PKA-coupling neurotransmitter receptors are involved: 5-HT4, Dopamine D1, and beta-adrenergic receptors. Antagonists of these receptors block the LFS L-LTP, but the effects of agonists of these receptors are clearly different. These results indicate that the threshold for the induction of LFS L-LTP is different among these pathways and that the maintenance of LFS L-LTP requires a cross-talk among multiple neurotransmitters.

The formation of auditory fear memory requires the synthesis of protein and mRNA in the auditory thalamus.

The medial geniculate nucleus of the thalamus responds to auditory information and is a critical part of the neural circuitry underlying aversive conditioning with auditory signals for shock. Prior work has shown that lesions of this brain area selectively disrupt conditioning with auditory stimuli and that neurons in the medial geniculate demonstrate plastic changes during fear conditioning. However, recent evidence is less clear as to whether or not this area plays a role in the storage of auditory fear memories. In the current set of experiments rats were given infusions of protein or messenger RNA (mRNA) synthesis inhibitors into the medial geniculate nucleus of the thalamus 30 min prior to auditory fear conditioning. The next day animals were tested to the auditory cue and conditioning context. Results showed that rats infused with either inhibitor demonstrated less freezing to the auditory cue 24 h after training, while freezing to the context was normal. Autoradiography confirmed that the doses used were effective in disrupting synthesis. Taken together with prior work, these data suggest that the formation of fear memory requires the synthesis of new protein and mRNA at multiple brain sites across the neural circuit that supports fear conditioning.

WOX1 is essential for tumor necrosis factor-, UV light-, staurosporine-, and p53-mediated cell death, and its tyrosine 33-phosphorylated form binds and stabilizes serine 46-phosphorylated p53.

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, undergoes Tyr33 phosphorylation at its first N-terminal WW domain and subsequent nuclear translocation in response to sex steroid hormones and stress stimuli. The activated WOX1 binds tumor suppressor p53, and both proteins may induce apoptosis synergistically. Functional suppression of WOX1 by antisense mRNA or a dominant negative abolishes p53-mediated apoptosis. Here, we determined that UV light, anisomycin, etoposide, and hypoxic stress rapidly induced phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (phospho-WOX1) and their binding interactions in several tested cancer cells. Mapping by yeast two-hybrid analysis and co-immunoprecipitation showed that phospho-WOX1 physically interacted with Ser46-phosphorylated p53. Knockdown of WOX1 protein expression by small interfering RNA resulted in L929 fibroblast resistance to apoptosis by tumor necrosis factor, staurosporine, UV light, and ectopic p53, indicating an essential role of WOX1 in stress stimuli-induced apoptosis. Notably, UV light could not induce p53 protein expression in these WOX1 knockdown cells, although p53 mRNA levels were not reduced. Suppression of WOX1 by dominant negative WOX1 (to block Tyr33 phosphorylation) also abolished UV light-induced p53 protein expression. Time course analysis showed that the stability of ectopic wild type p53, tagged with DsRed, was decreased in WOX1 knockdown cells. Inhibition of MDM2 by nutlin-3 increased the binding of p53 and WOX1 and stability of p53. Together, our data show that WOX1 plays a critical role in conferring cellular sensitivity to apoptotic stress and that Tyr33 phosphorylation in WOX1 is essential for binding and stabilizing Ser46-phosphorylated p53.

Caspase-8 sumoylation is associated with nuclear localization.

The cysteine protease caspase-8 plays a pivotal role in the initiation of different apoptotic pathways and controls the maturation and differentiation of various cell types including neurons, fibroblasts and lymphocytes. Specific substrates of caspase-8 are present in both the cytoplasm and the nucleus, which may determine the ultimate biological effect of caspase-8. However, the mechanisms regulating the cellular localization of caspase-8 are still unknown. We show here that, in contrast to other caspases such as caspase-9 and -3, caspase-8 can be sumoylated at lysine 156. This sumoylation (i) is associated with the nuclear localization of caspase-8 and (ii) did not impair caspase-8 activation.

Protein synthesis in the amygdala, but not the auditory thalamus, is required for consolidation of Pavlovian fear conditioning in rats.

The amygdala is an essential neural substrate for Pavlovian fear conditioning. Nevertheless, long-term synaptic plasticity in amygdaloid afferents, such as the auditory thalamus, may contribute to the formation of fear memories. We therefore compared the influence of protein synthesis inhibition in the amygdala and the auditory thalamus on the consolidation of Pavlovian fear conditioning in Long-Evans rats. Rats received three tone-footshock trials in a novel conditioning chamber. Immediately after fear conditioning, rats were infused intra-cranially with the protein synthesis inhibitor, anisomycin. Conditional fear to the tone and conditioning context was assessed by measuring freezing behaviour in separate retention tests conducted at least 24 h following conditioning. Post-training infusion of anisomycin into the amygdala impaired conditional freezing to both the auditory and contextual stimuli associated with footshock. In contrast, intra-thalamic infusions of anisomycin or a broad-spectrum protein kinase inhibitor [1-(5'-isoquinolinesulphonyl)-2-methylpiperazine, H7] did not affect conditional freezing during the retention tests. Pre-training intra-thalamic infusion of the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV), which blocks synaptic transmission in the auditory thalamus, produced a selective deficit in the acquisition of auditory fear conditioning. Autoradiographic assays of cerebral [14C]-leucine incorporation revealed similar levels of protein synthesis inhibition in the amygdala and thalamus following intra-cranial anisomycin infusions. These results reveal that the establishment of long-term fear memories requires protein synthesis in the amygdala, but not the thalamus, after auditory fear conditioning. Forms of synaptic plasticity that depend on protein synthesis, such as long-term potentiation, are likely candidates for the encoding and long-term storage of fear memories in the amygdala.

The similarities and diversities of signal pathways leading to consolidation of conditioning and consolidation of extinction of fear memory.

It is generally believed that consolidation of long-term memory requires activation of protein kinases, transcription of genes, and new protein synthesis. However, little is known about the signal cascades involved in the extinction of memory, which occurs when the conditioned stimulus is no longer followed by the unconditioned stimulus. Here, we show for the first time that an intra-amygdala injection of transcription inhibitor actinomycin D at the dose that blocked acquisition failed to affect extinction of a learned response. Conversely, protein synthesis inhibitor anisomycin blocked both acquisition and extinction. Extinction training-induced expression of calcineurin was blocked by anisomycin but not by actinomycin D. NMDA receptor antagonist, phosphatidylinositol 3-kinase (PI-3 kinase), and MAP kinase inhibitors that blocked the acquisition also blocked the extinction of conditioned fear. Likewise, PI-3 kinase inhibitor blocked fear training-induced cAMP response element-binding protein (CREB) phosphorylation as well as extinction training-induced decrease in CREB phosphorylation, the latter of which was associated with calcineurin expression and could be reversed by a specific calcineurin inhibitor. Thus, molecular processes that underlie long-term behavioral changes after acquisition and extinction share some common mechanisms and also display different characteristics.

JNK1 physically interacts with WW domain-containing oxidoreductase (WOX1) and inhibits WOX1-mediated apoptosis.

Transient activation of c-Jun N-terminal kinase (JNK) promotes cell survival, whereas persistent JNK activation induces apoptosis. Bovine testicular hyaluronidase PH-20 activates JNK1 and protects L929 fibroblasts from staurosporine-mediated cell death. PH-20 also induces the expression of a p53-interacting WW domain-containing oxidoreductase (WOX1, also known as WWOX or FOR) in these cells. WOX1 enhances the cytotoxic function of tumor necrosis factor and mediates apoptosis synergistically with p53. Thus, the activated JNK1 is likely to counteract WOX1 in mediating apoptosis. Here it is demonstrated that ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts, monocytic U937 cells, and other cell types. Also, JNK1 blocked WOX1 prevention of cell cycle progression. By stimulating cells with anisomycin or UV light, JNK1 became activated, and WOX1 was phosphorylated at Tyr(33). The activated JNK1 physically interacted with the phosphorylated WOX1, as determined by co-immunoprecipitation. Alteration of Tyr(33) to Arg(33) in WOX1 abrogated its binding interaction with JNK1 and its activity in mediating cell death, indicating that Tyr(33) phosphorylation is needed to activate WOX1. A dominant negative WOX1 was developed and shown to block p53-mediated apoptosis and anisomycin-mediated WOX1 phosphorylation but could not inhibit JNK1 activation. This mutant protein bound p53 but could not interact with JNK1, as determined in yeast two-hybrid analysis. Taken together, phosphorylation of JNK1 and WOX1 is necessary for their physical interaction and functional antagonism.

A yeast assay for high throughput screening of natural anti-viral agents.

Over the last decade the yeast Saccharomyces cerevisiae has become a popular organism for studying heterologous gene expression and in vivo protein-protein interactions. Many variations of these basic systems have originated over the years. Besides these vast and varied applications of the yeast expression system, S. cerevisiae has also been used extensively in fundamental research as a model simple eukaryote. We have used the S. cerevisiae system to design a high throughput screen for anti-viral agents from natural sources. The design of the assay rests on the ability of the L-A helper virus and the M(1) satellite virus to detect small variations in -1 ribosomal frameshifting. A minor change in frameshifting efficiencies can be detected and clearly shown phenotypically in terms of zones of clearing on an agar plate. Using such a process, we have initiated a high throughput screening process for natural anti-viral agents.