Bovine Brain Extract Research Articles

L-Glutamate Biosensor for In Vitro Investigations: Application in Brain Extracts

Investigations of L-glutamate release in living organisms can help to identify novel L-glutamate-related pathophysiological pathways, since abnormal transmission of L-glutamate can cause many neurological diseases. For the first time, a nitrogen-modified graphene oxide (GO) sample (RGO) is prepared through a simple and facile one-pot hydrothermal reduction of GO in the presence of 20 wt.% of the dye malachite green and is used for amperometric biosensing. The biosensor demonstrates adequate stability and is easy to prepare and calibrate. The biosensor detects the current generated during the electrooxidation of hydrogen peroxide released in the L-glutamate that is converted to the alpha-ketoglutarate catalyzed by L-glutamate oxidase. The biosensor consists of a semipermeable membrane, with L-glutamate oxidase (EC 1.4.3.11) immobilized in albumin and RGO and the working Pt electrode. First, the basic version of the L-glutamate biosensor is examined in PBS to investigate its sensitivity, reliability, and stability. To demonstrate the applicability of the L-glutamate biosensor in the analysis of complex real samples, quantification of L-glutamate in bovine brain extract is performed and the accuracy of the biosensor is confirmed by alternative methods. The enhanced version of the L-glutamate biosensor is applied for L-glutamate release investigations in a newly developed strain of rats (DAT-knockout, DAT-KO).

The first evidence of biological activity for free Hypusine, an enigmatic amino acid discovered in the '70s.

Hypusine amino acid [Nε-(4-amino-2-hydroxybutyl)-lysine] was first isolated in 1971 from bovine brain extracts. Hypusine originates from a post-translational modification at the eukaryotic translation initiation factor 5A (eIF5A), a protein produced by archaebacteria and eukaryotes. The eIF5A protein is the only one described containing the hypusine residue, which is essential for its activity. Hypusine as a free amino acid is a consequence of proteolytic degradation of eIF5A. Herein, we showed, for the first time, evidence of biological activity for the free hypusine. C6 rat glioma cells were treated with hypusine, and different cellular parameters were evaluated. Hypusine treatment significantly reduced C6 cell proliferation and potently suppressed their clonogenic capacity without leading to apoptosis. Hypusine also decreased the Eif5A transcript content and the global protein synthesis profile that may occur due to negative feedback in response to high hypusine concentration, controlling the content of newly synthesized eIF5A, which can affect the translation process. Besides, hypusine treatment also altered cellular metabolism by changing the pathways for energy production, reducing cellular respiration coupled with oxidative phosphorylation, and increasing the anaerobic metabolism. These observed results and the relationship between eIF5A and tumor processes led us to test the combination of hypusine with the chemotherapeutic drug temozolomide. Combining temozolomide with hypusine reduced the MTT conversion to the same levels as those observed using double temozolomide dosage alone, demonstrating a synergetic action between the compounds. Thus, since 1971, this is the first study showing evidence of biological activity for hypusine not associated with being an essential component of the eiF5A protein. Finding out the molecular targets of hypusine are the following efforts to completely characterize its biological activity.

Experimental Bovine Spongiform Encephalopathy in Squirrel Monkeys: The Same Complex Proteinopathy Appearing after Very Different Incubation Times.

Incubation periods in humans infected with transmissible spongiform encephalopathy (TSE) agents can exceed 50 years. In humans infected with bovine spongiform encephalopathy (BSE) agents, the effects of a “species barrier,” often observed when TSE infections are transmitted from one species to another, would be expected to increase incubation periods compared with transmissions of same infectious agents within the same species. As part of a long-term study investigating the susceptibility to BSE of cell cultures used to produce vaccines, we inoculated squirrel monkeys (Saimiri sp., here designated SQ) with serial dilutions of a bovine brain suspension containing the BSE agent and monitored them for as long as ten years. Previously, we showed that SQ infected with the original “classical” BSE agent (SQ-BSE) developed a neurological disease resembling that seen in humans with variant CJD (vCJD). Here, we report the final characterization of the SQ-BSE model. We observed an unexpectedly marked difference in incubation times between two animals inoculated with the same dilution and volume of the same C-BSE bovine brain extract on the same day. SQ-BSE developed, in addition to spongiform changes and astrogliosis typical of TSEs, a complex proteinopathy with severe accumulations of protease-resistant prion protein (PrPTSE), hyperphosphorylated tau (p-tau), ubiquitin, and α-synuclein, but without any amyloid plaques or β-amyloid protein (Aβ) typical of Alzheimer’s disease. These results suggest that PrPTSE enhanced the accumulation of several key proteins characteristically seen in human neurodegenerative diseases. The marked variation in incubation periods in the same experimental TSE should be taken into account when modeling the epidemiology of human TSEs.

UM-HACC-2A: MYB-NFIB fusion-positive human adenoid cystic carcinoma cell line

ObjectivesLimited availability of validated human adenoid cystic carcinoma (ACC) cell lines has hindered the mechanistic understanding of the pathobiology of this malignancy and the development of effective therapies. The purpose of this work was to generate and characterize a human ACC cell line. Material and MethodsImmediately after surgery, a tumor fragment from a minor salivary gland from the tongue of a female Caucasian was minced, dissociated, and a single cell suspension was plated in fibronectin-coated flasks. A culture medium containing bovine brain extract and rhEGF was optimized for these cells. Whole exome sequencing was used to evaluate the presence of MYB-NFIB translocation. ResultsThe University of Michigan-Human Adenoid Cystic Carcinoma (UM-HACC)-2A cells showed continuous growth in monolayers for at least 180 in vitro passages while maintaining epithelial morphology. Short-tandem repeat (STR) profiling confirmed a 100% match to patient DNA. Whole exome sequencing revealed the presence of the MYB-NFIB fusion in UM-HACC-2A cells, which was confirmed by PCR analysis. Western blots revealed high expression of epithelial markers (e.g. E-cadherin, EGFR, pan-cytokeratin) and proteins associated with ACC (e.g. c-Myb, p63). Developmental therapeutic studies showed that UM-HACC-2A cells were resistant to cisplatin (IC50 = 44.7 µM) while more responsive to paclitaxel (IC50 = 0.0006 µM). In a pilot study, we observed that UM-HACC-2A cells survived orthotopic transplantation into the submandibular gland. Notably, one of the mice injected with UM-HACC-2A cells exhibited lung metastasis after 6 months. ConclusionUM-HACC-2A is a MYB-NFIB fusion-positive ACC cell line that is suitable for mechanistic and developmental therapeutics studies.

Investigation of the composition and antioxidant activity of acetone and methanol extracts of Daphne sericea L. and Daphne gnidioides L.

The compositions of methanol and acetone extracts obtained from Daphne sericea L. and Daphne gnidioides L. were investigated. The antioxidant activities of each extract were determined by various test methods. Phenolic composition profile analysis by HPLC-DAD showed that D. gnidioides extracts contain more phenolic compounds than D. sericea extracts. Among the components, syringic acid was found to be the most abundant one in D. gnidioides extracts (42.8 and 38.4mg per g dry extract of methanol and acetone, respectively). Total flavonoid, total phenolic and total carotenoid contents of methanolic D. gnidioides extracts were found to be 244.5mg rutin/g dry weight of extract, 1219.3mg GAE/g dry weight of extract and 11.9mg/g dry weight of extract, respectively. DPPH·, ABTS·+ and O2·- assays were applied to plant extracts as radical scavenging activity tests. Methanolic extracts of D. sericea and D. gnidioides showed the highest radical scavenging activities according to DPPH· and ABTS·+ tests (61.6 and 147.2 in terms of IC50, respectively). Antioxidant activity tests for measuring lipid oxidation inhibiting capacity were carried in low density lipoprotein (LDL) and bovine brain extract (BBE). Methanolic extracts of D. gnidioides and D. sericea demonstrated remarkable lipid oxidation inhibiting capacity in LDL and BBE tests.

Characterization of Microwave Extracted Essential Oil from Taxus cuspidata Stem and Determination of Its Phenolic Content, Antioxidant and Free Radical Scavenging Activities

This study was outlined to probe chemical composition, antioxidant and free radical scavenging activities of Taxus cuspidata stem essential oil (TCEO) using different antioxidant models followed by determination of its total phenolic content. The TCEO was obtained by hydrodistillation of stem material using a microwave-assisted extraction technique. Gas chromatography-Mass spectrometry analysis of the TCEO resulted in the determination of 87 different compounds, representing 92.3% of total oil. The oil contained monoand sesquiterpene hydrocarbons, oxygenated monoand sesquiterpenes, diterpenes, phenolics, hydroxy derivatives, steroids, indole derivatives, and fatty acids as its major constituents. The TCEO showed antioxidant capacity as the inhibition of DPPH radical by 92.8%. Also the TCEO had potent inhibitory effect on nitric oxide, superoxide, and hydroxyl radicals by 80.0%, 71.7%, and 73.7%, respectively. Moreover, the TCEO displayed concentration-dependent reducing power ability and remarkable ferric ion-induced lipid peroxidation inhibitory effect in bovine brain extract. In addition, the TCEO yielded 6.5 ± 0.5 mg GAE/g sample equivalent phenolic content. These findings confirm the biological efficacy of T. cuspidata stem as a potential source of natural antioxidant.

Chemical Composition, Antioxidant, Lipid Peroxidation Inhibition and Free Radical Scavenging Activities of Microwave Extracted Essential Oil from Allium sativum

Various chronicle diseases are associated with oxidative stress caused by free radicalsand reactive oxygen species in the living cell system. In the present study, the chemical composition and antioxidant capacity of Allium sativumessential oil (ASEO) were investigated using different scavenging models including 1,1-diphenyl-2-picryl hydrazyl (DPPH), nitric oxide, superoxide, and hydroxyl radicals. Also the ASEO was evaluated for its lipid peroxidation inhibition efficacy, reducing power ability and total phenolic contents. The ASEO was obtained by the hydrodistillation of the dried rhizomes of A. sativum using a microwave-assisted extraction technique. Gas chromatography-mass spectrometry analysis of the ASEO resulted in the determination of 17 different compounds, representing 92.46 % of the total oil. The major components in the ASEO were phenolics, saturated fatty acids, sugar alcohols, esters, hydrazine, quinoline and isoquinoline derivatives along with some other essential phytochemicals. The ASEO showed the antioxidant capacity as the inhibition of DPPH, nitric oxide, superoxide, and hydroxyl radicals by 72.06 %, 79.96 %, 72.66 %, and 71.21 %, respectively. Moreover, the ASEO displayed concentration-dependent reducing power ability and remarkable ferric ion-induced lipid peroxidation inhibitory effect in bovine brain extract. In addition, the ASEO yielded 5.75±0.21 mg/ g gallic acid equivalent phenolic content. The findings of the present study confirm the efficacy of A. sativumas a potential source of natural antioxidant.

Phytochemical analysis, antioxidant and antilipid peroxidation effects of a medicinal plant, Adhatoda vasica

Many diseases are associated with oxidative stress caused by reactive oxygen species and free radicals generated in living cells. This study deals with finding naturally occurring antioxidants of plant origin. The study investigated the phytochemistry, antioxidant and free radical scavenging potential of ethanolic leaf extract of Adhatoda vasica (ELEAV) using different antioxidant models. The phytochemical analysis of ELEAV revealed the presence of alkaloids, flavonoids, terpenoids, saponins, phenols and steroids. ELEAV also showed an antioxidant capacity, with inhibition of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical by 69.23%. ELEAV had potent inhibitory effects on scavenging nitric oxide, superoxide and hydroxyl radicals, by 65.24%, 61.54% and 61.24%, respectively. Moreover, ELEAV displayed a concentration-dependent reducing power activity and potent inhibitory effects on ferric ion-induced lipid peroxidation (68.26%) in bovine brain extract. These findings confirm the biological efficacy of A. vasica as a potential source of natural antioxidants.

Studies on phytochemical analysis, antioxidant and lipid peroxidation inhibitory effects of a medicinal plant, Coleus forskohlii

Oxidative stress caused by reactive oxygen species and free radicals generated in living cells may cause various chronic, non-communicable diseases. Current research is directed towards finding naturally occurring antioxidants of plant origin. This study investigated the phytochemistry, antioxidant and free radical scavenging potential of ethanolic root extract of Coleus forskohlii (ERECF) using different antioxidant models. The phytochemical analysis of ERECF revealed the presence of alkaloids, glycosides, terpenoids, saponins, phenols and steroids. ERECF showed antioxidant capacity with the inhibition of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical by 71.21%. ERECF also had a potent inhibitory effect on scavenging nitric oxide, superoxide and hydroxyl radicals, by 66.23%, 61.22% and 61.68%, respectively. Moreover, ERECF displayed a concentration-dependent reducing power ability and a remarkable ferric ion-induced lipid peroxidation inhibitory effect (69.24%) in bovine brain extract. These findings confirm the biological efficacy of C. forskohlii as a potential source of natural antioxidants.

Studies on Phytochemicals, Antioxidant, Free Radical Scavenging and Lipid Peroxidation Inhibitory effects of Trachyspermum ammi seeds

Abstract: Objective: Reactive oxygen species and free radicals generate oxidative stress in the living cells associated with number of chronic diseases. Current research is directed towards finding naturally occurring antioxidants of plant origin. Hence, this study was aimed to determine phytochemical analysis, antioxidant and free radical scavenging potential of ethanolic seed extract of Trachyspermum ammi (ESETA). Methods: A preliminary phytochemical screening was carried out for the analysis of important phytochemicals present in ESETA. Antioxidant potential of ESETA was evaluated by various scavenging models including DPPH, nitric oxide, superoxide, and hydroxyl radical as well as its lipid peroxidation ability in bovine brain extract. Results: The phytochemical analysis of ESETA revealed the presence of various phytoconstituents including alkaloids, glycosides, terpenoids, saponins, phenols and steroids. The ESETA showed the antioxidant capacity as the inhibition of DPPH radical by 73.41%. Also the ESETA had potent inhibitory effect on scavenging nitric oxide, superoxide, and hydroxyl radicals by 67.33%, 63.22% and 62.48% respectively. Moreover, the ESETA displayed concentration-dependent reducing power ability and remarkable ferric ion-induced lipid peroxidation inhibitory effect (69.22%) in bovine brain extract. Conclusion: These findings confirm the pharmacological efficacy of T. ammi as a potential source of natural antioxidant. Key words: Trachyspermum ammi, Phytochemicals, Reactive oxygen species, Free radicals, Antioxidant activity.

Profiling and relative quantitation of phosphoinositides by multiple precursor ion scanning based on phosphate methylation and isotopic labeling.

Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PtdIns), are key regulators of many fundamental biological processes, including cell growth, proliferation, and motility. Here, we present a novel method for rapid, sensitive, and simultaneous profiling of phosphatidylinositol trisphosphate (PtdInsP3), phosphatidylinositol bisphosphate (PtdInsP2), and phosphatidylinositol phosphate (PtdInsP) of different fatty acid compositions. This method is based on a technique called "charged diacylglycerol fragment ion-specific multiple precursor ion scanning" (DAG(+)-specific MPIS), coupled with prior phosphate methylation. Using DAG(+)-specific MPIS, we were able to identify 32 PtdIns, 28 PtdInsP, 30 PtdInsP2, and 3 PtdInsP3 molecular species from bovine brain extracts or prostatic cancer cell lines in an efficient and time-saving manner. Our analysis revealed a large range of fatty acyl compositions in phosphoinositides not obtained previously from mammalian samples. We also developed a method that involves isotopic labeling of endogenous phosphoinositides with deuterated diazomethane (CD2N2) for quantitation of phosphoinositides. CD2N2 was generated in situ through acid-catalyzed H/D exchange and methanolysis of trimethylsilyl diazomethane (TMS-diazomethane). Phosphoinositides, extracted from a PC3 prostatic cancer cell line, were labeled either with CH2N2 or CD2N2 and mixed in known proportions for DAG(+)-specific MPIS-based mass spectrometry (MS) analysis. The results indicate that isotopic labeling is capable of providing accurate quantitation of PtdInsP3, PtdInsP2, and PtdInsP with adequate linearity as well as high reproducibility with an average coefficient variation of 18.9%. More importantly, this new methods excluded the need for multiple phosphoinositide internal standards. DAG(+)-specific MPIS and isotopic labeling based MS analysis of phosphoinositides offers unique advantages over existing approaches and presents a powerful tool for research of phosphoinositide metabolism.

Phytochemicals, Antioxidant and Anti-Lipid Peroxidation Activities of Ethanolic Extract of a Medicinal Plant, A ndrographis paniculata

Various human diseases are associated with oxidative stress caused by reactive oxygen species and free radicals generated in the living cells. Current research is directed toward finding naturally occurring antioxidant components of plant origin. This study was outlined to probe phytochemical analysis, antioxidant and anti-lipid peroxidation potential of ethanolic whole plant extract of Andrographis paniculata (EWPEAP) using different antioxidant and free radical scavenging models. The phytochemical analysis of EWPEAP revealed the presence of alkaloids, glycosides, terpenoids, saponins, phenols and steroids. The EWPEAP showed the antioxidant capacity as the inhibition of 1,1-diphenyl-2-picrylhydrazyl radical by 72.4%. The EWPEAP also had potent inhibitory effect on scavenging nitric oxide, superoxide and hydroxyl radicals by 66.5%, 62.2% and 62.5%, respectively. Moreover, the EWPEAP displayed concentration-dependent reducing power ability and significant (P < 0.05) ferric ion-induced lipid peroxidation inhibitory effect in bovine brain extract. These findings confirm the biological efficacy of A. paniculata as a potential source of natural antioxidant. Practical Applications Andrographis paniculata plant is widely used in Southeast Asia as febrifuge, tonic, stomachic and anthelmintic. It is also used as a principal ingredient of several pharmaceutical preparations in various forms as well as household medicine. Although it is being used in traditional practices, A. paniculata has been found to possess several biological properties including hepatoprotective, antithrombotic, anti-inflammatory, immunostimulant, antimalarial, antihyperglycemic and cardioprotective effect. Because oxidative stress has been implicated as one of the inducing factors for the generation of various metabolic disorders, the present study has evaluated the antioxidant potential of whole plant of A. paniculata, which may prove to be beneficial against free radical generated disorders.

Interaction of protein phosphatase inhibitors with membrane lipids assessed by surface plasmon resonance based binding technique

The interaction of okadaic acid (OA), tautomycin (TM), microcystin-LR (MC-LR), cantharidin (CA), epigallocatechin-gallate (EGCG) and cyclosporin A (CsA), inhibitors of protein phosphatases, with liposome covered surfaces prepared from the lipid extracts of bovine brain, heart and liver was investigated by surface plasmon resonance (SPR) based binding technique. The SPR sensorgrams indicated reversible association or partial intercalation of the inhibitors with liposomes at 20°C or 37°C, respectively. Distinct lipid composition specificities were reflected in different saturation values of inhibitor binding in a decreasing order of liver>heart>>brain lipids. Assaying the effect of OA, TM, MC-LR, CA and EGCG on the activity of protein phosphatases in neuroblastoma B50, cardiomyoblast H9C2 and hepatocarcinoma HepG2 cells implied that the cell type specific association of phosphatase inhibitors with membrane lipids may influence their inhibitory potencies exerted on intact cells.

Identification and differentiation of PDX1 β-cell progenitors within the human pancreatic epithelium

To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm that β-cell progenitors reside within the pancreatic epithelium. Due to mesenchymal stem cell (MSC) expansion and overgrowth, progenitor cells within the pancreatic epithelium cannot be characterized in vitro, though β-cell dedifferentiation and expansion of MSC intermediates via epithelial-mesenchymal transition (EMT) may generate β-cell progenitors. Pancreatic epithelial cells from endocrine and non-endocrine tissue were expanded and differentiated in a novel pancreatic epithelial expansion medium supplemented with growth factors known to support epithelial cell growth (dexamethasone, epidermal growth factor, 3,5,3'-triiodo-l-thyronine, bovine brain extract). Cells were also infected with a single and dual lentiviral reporter prior to cell differentiation. Enhanced green fluorescent protein was controlled by the rat Insulin 1 promoter and the monomeric red fluorescent protein was controlled by the mouse PDX1 promoter. In combination with lentiviral tracing, cells expanded and differentiated in the pancreatic medium were characterized by flow cytometry (BD fluorescence activated cell sorting), immunostaining and real-time polymerase chain reaction (PCR) (7900HT Fast Realtime PCR System). In the presence of 10% serum MSCs rapidly expand in vitro while the epithelial cell population declines. The percentage of vimentin(+) cells increased from 22% ± 5.83% to 80.43% ± 3.24% (14 d) and 99.00% ± 0.0% (21 d), and the percentage of epithelial cells decreased from 74.71% ± 8.34% to 26.57% ± 9.75% (14 d) and 4.00% ± 1.53% (21 d), P < 0.01 for all time points. Our novel pancreatic epithelial expansion medium preserved the epithelial cell phenotype and minimized epithelial cell dedifferentiation and EMT. Cells expanded in our epithelial medium contained significantly less mesenchymal cells (vimentin(+)) compared to controls (44.87% ± 4.93% vs 95.67% ± 1.36%; P < 0.01). During cell differentiation lentiviral reporting demonstrated that, PDX1(+) and insulin(+) cells were localized within adherent epithelial cell aggregates compared to controls. Compared to starting islets differentiated cells had at least two fold higher gene expression of PDX1, insulin, PAX4 and RFX (P < 0.05). PDX1(+) cells were confined to adherent epithelial cell aggregates and not vimentin(+) cells (mesenchymal), suggesting that EMT is not a mechanism for generating pancreatic progenitor cells.

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane "partner" proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and Matrigel(TM). We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α(1)β(1) and α(2)β(1) integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.

Abstract 3057: CDK5 modulates the cell cycle, apoptosis and paclitaxel sensitivity in human ovarian cancer cells with wild-type p53 function

Abstract Cyclin-dependent kinase 5 (CDK5) is a ubiquitously expressed serine/threonine kinase that was initially identified in bovine brain extracts and plays an important role in normal neuron development and neurodegenerative diseases. This molecule has recently been shown to possess many cellular functions in non-neuronal tissues as well. Using a kinome siRNA screen, we identified CDK5 as a kinase that modulates paclitaxel sensitivity in human ovarian cancer cells. The aim of this report is to identify mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancer cells. Despite nucleotide sequence homology with human CDK1, a role of CDK5 in regulating the cell cycle remains unclear. Here we demonstrate dual roles of CDK5 in regulating the cell cycle and apoptosis. We have found that knockdown of CDK5 with multiple siRNAs markedly inhibits cell proliferation and increases paclitaxel sensitivity in several p53 wild type ovarian cancer cell lines using assays for cell viability and clonogenic growth. CDK5 knockdown with or without paclitaxel treatment induces G1 arrest of the cell cycle and apoptotic cell death linked to a significant induction of p53, p21Cip1 and p27Kip1 proteins. Induction of G1 arrest following CDK5 knockdown depends on induction of p21Cip1 and p27Kip1, since silencing p21Cip1 and/or p27Kip1 dramatically reduces CDK5 knockdown-induced G1 arrest. A CDK5 knockdown-induced increase of p53 expression contributes both to CDK5 knockdown-induced G1 arrest and to apoptosis, as silencing p53 markedly diminishes CDK5 knockdown-induced G1 arrest and apoptosis. As expected, a CDK5 knockdown-induced increase in p21Cip1 expression depends on p53-mediated transcriptional activation demonstrated by QRT-PCR and reporter luciferase assays. CDK5 knockdown activates caspase-3 and a pan-caspase inhibitor completely blocks CDK5 knockdown-induced apoptosis. Significant Inhibition of Bcl-2 expression is found in CDK5 knockdown-induced apoptosis and additional inhibition of Bcl-2 expression is found with combined CDK5 and paclitaxel treatment. Taken together, these data suggest that CDK5 can modulate paclitaxel sensitivity through regulating both the cell cycle and apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells with wild-type p53 function, providing a novel approach for enhancing the efficacies of paclitaxel-based regimens for low grade ovarian cancer in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3057. doi:1538-7445.AM2012-3057

Early Growth Response 1 (Egr-1) Regulates Phosphorylation of Microtubule-associated Protein Tau in Mammalian Brain

In the normal brain, tau protein is phosphorylated at a number of proline- and non-proline directed sites, which reduce tau microtubule binding and thus regulate microtubule dynamics. In Alzheimer disease (AD), tau is abnormally hyperphosphorylated, leading to neurofibrillary tangle formation and microtubule disruption, suggesting a loss of regulatory mechanisms controlling tau phosphorylation. Early growth response 1 (Egr-1) is a transcription factor that is significantly up-regulated in AD brain. The pathological significance of this up-regulation is not known. In this study, we found that lentivirus-mediated overexpression of Egr-1 in rat brain hippocampus and primary neurons in culture activates proline-directed kinase Cdk5, inactivates PP1, promotes tau phosphorylation at both proline-directed Ser(396/404) and non-proline-directed Ser(262) sites, and destabilizes microtubules. Furthermore, in Egr-1(-/-) mouse brain, Cdk5 activity was decreased, PP1 activity was increased, and tau phosphorylation was reduced at both proline-directed and non-proline-directed sites. By using nerve growth factor-exposed PC12 cells, we determined that Egr-1 activates Cdk5 to promote phosphorylation of tau and inactivates PP1 via phosphorylation. When Cdk5 was inhibited, tau phosphorylation at both proline- and non-proline directed sites and PP1 phosphorylation were blocked, indicating that Egr-1 acts through Cdk5. By using an in vitro kinase assay and HEK-293 cells transfected with tau, PP1, and Cdk5, we found that Cdk5 phosphorylates Ser(396/404) directly. In addition, by phosphorylating and inactivating PP1, Cdk5 promotes tau phosphorylation at Ser(262) indirectly. Our results indicate that Egr-1 is an in vivo regulator of tau phosphorylation and suggest that in AD brain increased levels of Egr-1 aberrantly activate an Egr-1/Cdk5/PP1 pathway, leading to accumulation of hyperphosphorylated tau, thus destabilizing the microtubule cytoskeleton.

Functional Characterization of the In Vitro Folded Human Y1 Receptor in Lipid Environment

We describe the recombinant production of the human Y(1) receptor from inclusion bodies of E. coli cultures. The in vitro refolding was carried out in the presence of lipids from bovine brain extracts. Y(1) receptors in brain lipids compete for cellular receptors in competitive binding experiments.

Gentisic Acid, a Compound Associated with Plant Defense and a Metabolite of Aspirin, Heads a New Class of in Vivo Fibroblast Growth Factor Inhibitors

Fibroblast growth factors are key proteins in many intercellular signaling networks. They normally remain attached to the extracellular matrix, which confers on them a considerable stability. The unrestrained accumulation of fibroblast growth factors in the extracellular milieu, either due to uncontrolled synthesis or enzymatic release, contributes to the pathology of many diseases. Consequently, the neutralization of improperly mobilized fibroblast growth factors is of clear therapeutic interest. In pursuing described rules to identify potential inhibitors of these proteins, gentisic acid, a plant pest-controlling compound, an aspirin and vegetarian diet common catabolite, and a component of many traditional liquors and herbal remedies, was singled out as a powerful inhibitor of fibroblast growth factors. Gentisic acid was used as a lead to identify additional compounds with better inhibitory characteristics generating a new chemical class of fibroblast growth factor inhibitors that includes the agent responsible for alkaptonuria. Through low and high resolution approaches, using representative members of the fibroblast growth factor family and their cell receptors, it was shown that this class of inhibitors may employ two different mechanisms to interfere with the assembly of the signaling complexes that trigger fibroblast growth factor-driven mitogenesis. In addition, we obtained evidence from in vivo disease models that this group of inhibitors may be of interest to treat cancer and angiogenesis-dependent diseases.