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3031Background: Stromal signature regulated by TGF-β pathway is one of the major mechanisms of tumor immune surveillance, leading to resistance to immune checkpoint inhibitors (ICI). Moreover, TGF-β responsive signatures (TBRS) of stromal cells have been associated with poor prognosis. Vactosertib (TEW-7197) is a potent, highly selective, oral inhibitor of TGFBR1. The safety, anti-tumor effect of vactosertib and its association with TBRS levels were evaluated in patients with advanced solid tumors. Based on in-house TCGA analysis, high fibroblast TBRS (F-TBRS) levels were seen in pancreatic, lung, colorectal (CMS4 subtype), and stomach (GS subtype) cancers in association with poor prognosis (adjusted hazard ratio 1.27; P = 1.06 x 10-8). Methods: 29 patients were enrolled in a phase I modified 3+3 dose-escalating study (NCT02160106) and received vactosertib once daily at the dose range of 30~340 mg for 5 days with 2 days off. RNA sequencing of pre-treatment tumor samples in 16 patients were analyzed to eva...

Among the various phenotypes seen in Down syndrome (DS), mental retardation is the most common and most debilitating condition suffered by individuals with DS. The DYRK1A gene on human chromosome 21q22.2 encodes a subfamily of protein kinases that displays dual substrate specificities and is known to play a critical role in neurodevelopment. To study DS mental retardation, we have generated transgenic mice that contain only one copy of the complete human DYRK1A gene in a bacterial artificial chromosome. The transgenic mice showed significant impairment in hippocampal-dependent memory tasks in a Morris water maze. Interestingly, we observed shifts in both long-term potentiation and long-term depression, which suggests a role for DYRK1A in bidirectional synaptic plasticity. These mice represent the most clinically relevant DYRK1A mouse model to date and provide us a valuable tool for the in vivo study of mechanisms that underlie the learning and memory deficit in DS.

RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.

The human thyroid hormone receptor beta (hTR beta) is capable of both transcriptional silencing and hormone-dependent activation. However, the detailed mechanism of this transcriptional regulation remains to be elucidated. One possibility is that hTR beta interacts directly with factors of the basal transcriptional machinery, thereby modulating basal promoter activity in a direct manner, as has been shown for other transcription factors. Here, we show that hTR beta interacts specifically with the human basal transcription factor TFIIB. Deletion analysis revealed two contact sites in the receptor: one is located in the N terminus, while the other is part of the ligand-binding domain (LBD) and is located at the C terminus. Interestingly, each receptor contact site interacts with different sites in TFIIB. Cotransfection experiments revealed that, when fused to the DNA-binding domain of yeast transcription factor GAL4, the C-terminal interaction site of hTR beta was transcriptionally inactive; however, when it was cotransfected with the remaining part of the LBD on a separate molecule, silencing function was restored. In agreement with that, we show that thyroid hormone is able to significantly decrease the interaction of its receptor LBD with TFIIB. Our data suggest that hTR beta acts as a transcriptional silencer by interacting with TFIIB and that thyroid hormone may act in part by preventing transcriptional repression at this level.

How eukaryotic promoter-specific activator proteins (activators) stimulate transcription is a central question. We have previously shown that an acidic activator can directly interact with the general transcription factor TFIIB and increase its stable assembly into a preinitiation complex1,2. We have proposed that this increase in TFIIB assembly is at least part of the mechanism by which an acidic activator functions1,2. A prediction of this hypothesis is that a TFIIB mutant unable to interact with an acidic activator could not support activated transcription, and here we present experiments that verify this prediction. In conjunction with previous studies, our results argue that interaction between an acidic activator and TFIIB is necessary for transcriptional activation.

This study investigated the effect of macelignan on the P-glycoprotein-mediated drug efflux as well as CYP3A4-mediated drug metabolism and subsequently its in vivo implication on the bioavailability of paclitaxel. The inhibition effect of macelignan on the CYP3A4-mediated metabolism was negligible over the concentration range of 0.01-100muM in rat liver microsome while approximately 33% inhibition was observed at 100muM in human liver microsome, implying that the interaction of macelignan with CYP3A4 might be insignificant at the physiologically achievable concentrations. In contrast, macelignan (20muM) increased the cellular accumulation of paclitaxel by approximately 1.7-fold in NCI/ADR-RES cells overexpressing P-gp, while it did not alter the cellular accumulation of paclitaxel in OVCAR-8 cells lacking P-gp. The effect of macelignan on the systemic exposure of paclitaxel was also examined in rats after the intravenous and oral administration of paclitaxel in the presence and the absence of macelignan. The concurrent use of macelignan significantly (p

The effect of macelignan, a phytoestrogen, on P-gp function was investigated using multidrug resistant cancer cells overexpressing P-gp (NCI/ADR-RES) and the fluorescent P-gp substrates, daunorubicin and rhodamine 123. Macelignan (40 microM) increased the cellular accumulation of daunorubicin by approximately threefold in NCI/ADR-RES cells, whereas it did not alter the cellular accumulation of daunorubicin in MCF-7/sensitive cells. Similarly, the presence of macelignan also enhanced significantly (P < 0.05) the cellular accumulation of rhodamine 123 in a concentration-dependent manner in NCI/ADR-RES cells. Furthermore, cancer cells were more susceptible to the cytotoxicity of vinblastine, a P-gp substrate, in the presence of macelignan. Those results suggest that macelignan has inhibitory effects on P-gp mediated cellular efflux. However, P-gp activity did not affect the cellular accumulation of macelignan itself. Taken all together, macelignan was identified as a novel inhibitor of P-gp activity and may be a promising lead compound for the rational design of more efficacious drugs to reverse multidrug resistance in cancer.

Previous studies have shown that macelignan has anti-inflammatory and neuroprotective effects. Subsequently, in the current study, we demonstrate that oral administrations of macelignan reduce the hippocampal microglial activation induced by chronic infusions of lipopolysaccharide (LPS) into the fourth ventricle of Fisher-344 rat brains. A Morris water maze was used to evaluate the status of the hippocampal-dependent spatial learning in control rats with an artificial cerebrospinal fluid infusion, rats with chronic LPS infusions, and rats with chronic LPS infusions and oral administrations of macelignan. The rats with chronic LPS infusions showed spatial memory impairments relative to the control rats in the performance of the memory task. Daily administration of macelignan reduced the spatial memory impairments induced by the chronic LPS infusions. The results indicate that macelignan may possess therapeutic potential for the prevention of Alzheimer's disease.

A high-performance liquid chromatographic method was developed for the determination of macelignan in rat plasma and applied to the pharmacokinetic study of macelignan in rats. Chromatographic separation was achieved on a conventional ODS column with the mobile phase of water: acetonitrile: methanol = 35:32.5:32.5 (v/v/v %). The flow rate of isocratic elution was 1 mL/min and peaks were detected at 240 nm. The limit of detection was 10 ng/mL and the limit of quantitation was 20 ng/mL. The calibration curve was linear over the concentration range of 50–5000 ng/mL. Intra-and inter-day precision for the assay over the concentration range was below 10 % and the accuracy ranged between 96.0–107% for intra-day and 98.8–114% for inter-day, respectively. The method was applied to the single dose pharmacokinetic study of macelignan in rats and the results showed that this HPLC method was adequate to support the in vivo pharmacokinetic study of macelignan.

The vesicular glutamate transporter (VGLUT) transports glutamate into pre-synaptic vesicles. Three isoforms of VGLUT have been identified in humans, but their functional differences remain largely unknown. EAT-4 is the only homologue of human VGLUT in C. elegans. Here we report that mutants of eat-4 exhibit hyperforaging behavior and that each of the isoforms of human VGLUT functionally rescues the defects in eat-4 worms.

TFIID, the TATA-binding protein, was found to stimulate transcription from the adenovirus IVa2 promoter, a promoter considered to lack the TATA motif. Remarkably, a TATA-like sequence element located downstream of the transcription start site binds TFIID and is required for TFIID-dependent transcription from the IVa2 promoter. Transcription from the IVa2 and the adjacent adenovirus major late promoter (Ad-MLP) is divergent, and the cap sites are separated by 212 nucleotides. Nevertheless, the TATA motifs of the IVa2 promoter and Ad-MLP were found to be oriented in the same direction. An initiator motif around the transcription start site is located in the IVa2 promoter, and in contrast to the TATA motifs, the IVa2-initiator is in the opposite orientation with respect to the initiator of the Ad-MLP. A model is presented in which the polar nature of the initiator governs the direction of transcription. We propose that RNA polymerase II and accessory factors recognize the initiator in an orientation-dependent fashion. The recognition of the IVa2 initiator by RNA polymerase is enhanced by the binding of TFIID to the downstream TATA motif.

Although alterations in the function of the neurotransmitter system have been implicated in the pathology of Alzheimer's disease (AD), the mechanisms that underlie this pathological change are not well understood. Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is a key protease in the generation of β-amyloid, an important trigger protein in the pathogenesis of AD. The expression and activity of BACE1 are increased in the brains of sporadic AD patients, and a role for BACE1 in neurotransmission has been suggested recently. This study examines whether BACE1 plays a role in regulated exocytosis in PC12 cells. Treatment of PC12 cells with a β-secretase inhibitor reduced stimulus-dependent secretion of neurotransmitters, suggesting a potential role of BACE1 in regulated exocytosis. Using transfected human growth hormone as a reporter for a regulated secretory pathway in PC12 cells, we found that the transient overexpression of BACE1 increased basal secretion in the absence of a stimulus and reduced stimulus-dependent secretion in intact PC12 cells. In digitonin-permeabilized PC12 cells, an overexpression of BACE1 enhanced the Ca2+-independent and ATP-independent component of the secretory pathway. Furthermore, expression of the glycosylation-deficient mutant of BACE1, BACE1N354Q, led to an elevation of basal secretions over that by BACE1 wild-type, suggesting a role of BACE1 glycosylation in basal secretion. These results demonstrate an unknown role for BACE1 in secretion, and suggest that elevated levels of BACE1 in AD brains may contribute to the altered neurotransmitter pathology of AD through stimulation of spontaneous basal secretion under resting conditions.

The antioxidant and anti-inflammatory properties of the marine red alga Neorhodomela aculeate (N. aculeata) Masuda were investigated with neuronal and microglial cells. Extracts of N. aculeata had potent neuroprotective effects on glutamate-induced neurotoxicity and inhibited reactive oxygen species (ROS) generation in the murine hippocampal HT22 cell line. Also, extracts of N. aculeata inhibited H2O2-induced lipid peroxidation in rat brain homogenates. The properties of the extract as an anti-inflammatory agent were investigated in microglial activation by interferon-gamma (IFN-gamma): it reduced the inducible nitric oxide synthase that consequently resulted in the reduction of nitric oxide. These results suggest that the marine red alga N. aculeata could be considered as a potential source for reducing reactive oxygen species and inflammation related to neurological diseases.

It has been reported that inflammatory processes are associated with the pathophysiology of Alzheimer's disease (AD), and the treatment of AD using anti-inflammatory agents slows the progress of AD. Marine algae have been utilized in food products as well as in medicine products for a variety of purposes. In this study, we investigated the neuroprotective effects of methanol extracts of Ulva conglobata (U. conglobata), a marine algae, on glutamate-induced neurotoxicity in the murine hippocampal HT22 cell line and the anti-inflammatory effects on interferon gamma (IFN-gamma)-induced microglial activation in BV2 cells. U. conglobata methanol extracts significantly attenuated the neurotoxicity induced by glutamate in HT22 cells and inhibited nitric oxide production induced by IFN-gamma in BV2 cells. U. conglobata methanol extract treatments were also examined and it was found that they almost completely suppressed the expression of the proinflammatory enzyme cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). These results suggest that U. conglobata possesses therapeutic potential for combating neurodegenerative diseases associated with neuroinflammation.

Xanthorrhizol, a natural sesquiterpenoid isolated from the rhizome of Curcuma xanthorrhiza Roxb (Zingiberaceae), has antibacterial activities and protective effects against cisplatin-induced hepatotoxicity. In this study, we investigated the activities of xanthorrhizol as an antioxidant or antiinflammatory agent using neuronal and microglial cells. Xanthorrhizol had potent neuroprotective effects on glutamate-induced neurotoxicity and reactive oxygen species (ROS) generation in the murine hippocampal HT22 cell line. Also, xanthorrhizol inhibited H(2)O(2)-induced lipid peroxidation in rat brain homogenates. The properties of xanthorrhizol as an antiinflammatory agent were investigated in microglial activation by lipopolysaccharide. It reduced the expression of cyclooxygenase-2 and the inducible nitric oxide synthase, which consequently resulted in the reduction of nitric oxide. The production of proinflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha in activated microglial cells, was reduced by xanthorrhizol. These results suggest that xanthorrhizol could be an effective candidate for the treatment of Alzheimer's disease- and other neurological disease-related ROS and inflammation.

Epidemiological studies suggest that the treatments of anti-inflammatory agents and anti-oxidants slow the progress of neurological diseases. Lignans are anti-oxidants and phytoestrogens found in a variety of plants. In this study, we investigated the neuroprotective effect of macelignan on glutamate-induced neurotoxicity and reactive oxygen species (ROS) in murine hippocampal HT22 cell line. Macelignan significantly attenuated the ROS production and neurotoxicity induced by glutamate in HT22 cell. Also, the properties of macelignan as an anti-inflammatory agent were investigated in microglials activation by lipopolysaccharide (LPS). It potently suppressed the expression of cyclooxygenase-2 and inducible nitric oxide synthase, that consequently resulted in the reduction of nitric oxide in LPS-treated microglial cells. It also significantly suppressed the production of pro-inflammatory cytokine tumor necrosis factor-alpha and interleukin-6. These results suggest that macelignan possesses therapeutic potentials against neurodegenerative diseases with oxidative stress and neuroinflammation.

The Caenorhabditis elegans heterochronic gene lin-14 generates a temporal gradient of the LIN-14 proteins to control stage-specific patterns of cell lineage during development. Down-regulation of LIN-14 is mediated by the lin-14 3' untranslated region (UTR), which bears seven sites that are complementary to the regulatory lin-4 RNA. Here we report molecular and genetic evidence that RNA duplexes between the lin-4 and lin-14 RNAs form in vivo and are necessary for LIN-14 temporal gradient generation. lin-4 RNA binds in vitro to a lin-14 mRNA bearing the seven lin-4 complementary sites but not to a lin-14 mRNA bearing point mutations in these sites. In vivo, the lin-4 complementary regions are necessary for lin-14 3' UTR-mediated temporal gradient formation. Based on lin-14 3' UTR sequence comparisons between C. elegans and C. briggsae, four of the seven lin-4/lin-14 RNA duplexes are predicted to bulge a lin-4 C residue, and three sites are predicted to form nonbulged RNA duplexes. Reporter genes bearing multimerized bulged C lin-4 binding sites show almost wild-type temporal gradient formation, whereas those bearing multimerized nonbulged lin-4 binding sites do not form a temporal gradient. Paradoxically, lin-4 RNA binds in vitro to nonbulged lin-14 RNA more avidly than to the bulged lin-14 RNA. This suggests that a specific secondary structure of lin-4/lin-14 RNA duplex that may be recognized by an accessory protein, rather than an RNA duplex per se, is required in vivo for the generation of the LIN-14 temporal gradient.

During C. elegans development, the temporal pattern of many cell lineages is specified by graded activity of the heterochronic gene Lin-14. Here we demonstrate that a temporal gradient in Lin-14 protein is generated posttranscriptionally by multiple elements in the lin-14 3'UTR that are regulated by the heterochronic gene Lin-4. The lin-14 3'UTR is both necessary and sufficient to confer lin-4-mediated posttranscriptional temporal regulation. The function of the lin-14 3'UTR is conserved between C. elegans and C. briggsae. Among the conserved sequences are seven elements that are each complementary to the lin-4 RNAs. A reporter gene bearing three of these elements shows partial temporal gradient activity. These data suggest a molecular mechanism for Lin-14p temporal gradient formation: the lin-4 RNAs base pair to sites in the lin-14 3'UTR to form multiple RNA duplexes that down-regulate lin-14 translation.

Transcription factor IIB (TFIIB) plays a pivotal role in the formation of transcription-competent initiation complexes. TFIIB was found to interact with the TATA-binding protein, the small subunit of TFIIF, and RNA polymerase II. These interactions require distinct domains in TFIIB. Using the gel mobility-shift assay, it was found that the amino terminus of TFIIB was necessary for the formation of complexes containing RNA polymerase II and TFIIF, whereas the carboxy-terminal domain, which is composed of two imperfect direct repeats and includes a putative amphipathic alpha-helix, was sufficient for the formation of complexes containing the TATA-binding protein and TFIIB (DB complex). Protein-protein interaction analyses demonstrate that the amphipathic alpha-helix in TFIIB is important for the interaction with the TATA-binding protein. Specific residues mapping to the carboxyl terminus of the second direct repeat were found to be crucial for the interaction of TFIIB and RNA polymerase II. The interaction with the small subunit of TFIIF was mapped to the amino terminus of TFIIB, which includes a zinc finger.

We have discovered a protein termed Dr1 that interacts with the TATA-binding protein, TBP. The association of Dr1 with TBP results in repression of both basal and activated levels of transcription. The interaction of Dr1 with TBP precludes the formation of a transcription-competent complex by inhibiting the association of TFIIA and/or TFIIB with TBP. Dr1 activity is associated with a 19 kd protein. A cDNA clone encoding Dr1 was isolated. Dr1 is phosphorylated in vivo and phosphorylation of Dr1 affected its interaction with TBP. Our results suggest a regulatory role for Dr1 in repression of transcription mediated via phosphorylation.

A CENTRAL issue in eukaryotic transcriptional regulation is the mechanism by which promoter-specific transcription factors (activators) stimulate transcription. Two lines of evidence indicate that the general transcription factor TFIIB is a pivotal component in the mechanism by which an acidic activator functions. First, during assembly of the preinitiation complex TFIIB binding is a rate-limiting step enhanced by an acidic activator1. Second, the TFIIB activity in a HeLa cell nuclear extract is specifically retained on a column containing an acidic activating region1. But because our previous study monitored only TFIIB activity, it remains possible that the interaction between TFIIB and the acidic activating region is mediated through additional proteins, for example, those designated as adaptors2, coactivators3 or mediators4,5. A complementary clone encoding TFIIB has recently been isolated and shown to encode a polypeptide of relative molecular mass 35,000 (ref. 6). Here we report that TFIIB expressed in and purified from Escherichia coli (recombinant TFIIB) binds directly to the potent acidic activating region of the herpes simplex virus-1 VP16 protein.

Transcription factor IIB (TFIIB) has a central role in transcription of class II genes. The purification of the human TFIIB protein and isolation of a complementary DNA encoding TFIIB activity is reported here. The sequence of TFIIB, which seems to be encoded by a single gene, contains a repeated motif, in addition to a motif with similarity to the prokaryotic sigma-factors. The recombinant protein expressed in bacteria substituted for all the functions attributed to the human TFIIB protein.

Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.