Design and properties of hepatitis C virus antisense oligonucleotides for liver specific drug targeting

Different backbone modified antisense oligonucleotides (AS-ODNs) directed against the hepatitis C virus genome were 5'-conjugated to cholesterol, cholic acid or taurocholic acid to enhance liver specific drug targeting and hepatocellular uptake. The lipophilic character of modified AS-ODNs was determined from RP-HPLC retention times and duplex stability was correlated with Tm-values from UV melting curves.     

Synthesis and properties of bile acid phosphoramidites 5'-tethered to antisense oligodeoxynucleotides against HCV.

Recently, we synthesized antisense oligonucleotides (AS-ODNs) directed against the non-coding-region (NCR) and the adjacent core region of the hepatitis C virus (HCV) RNA. Backbone modifications like phosphorothioates, methyl- and benzylphosphonates were introduced three at each end of the sequence. For improvement of liver specific drug targeting and/or hepatocellular uptake efficient AS-ODNs were covalently conjugated to biomolecules such as cholesterol or bile acids. The use of base-labile alkylphosphonates afforded mild conditions for deprotection of bile acid conjugated AS-ODNs. Here, we describe a convenient synthesis of new cholic acid and taurocholic acid phosphoramidites. Derivatization to taurocholic acid was effected directly before phosphitylation reaction, which is the last step of the phosphoramidite synthesis. These building blocks were coupled to the 5'-position of AS-ODNs in the last step of solid-phase synthesis. After mild deprotection, purification and characterization the properties of these modified AS-ODNs like their lipophilicity or their ability to form stable duplices to DNA and RNA were investigated. Enhanced lipophilicity and formation of stable duplices and heteroduplices makes bile acid conjugated AS-ODNs interesting as antiviral antisense therapeutics against HCV.     

Induction of potent cell growth inhibition by schizophyllan/K-ras antisense complex in combination with gemcitabine

Antisense oligonucleotides (AS-ODNs) specifically hybridize with target mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. In our previous reports, we demonstrated that β-glucan schizophyllan (SPG) can form a complex with AS-ODNs attached with oligo deoxyadenosine dA₄₀ (AS-ODN-dA₄₀/SPG), and that this complex can be recognized by β-glucan receptor Dectin-1 on antigen presenting cells and lung cancer cells. In many types of cancer cell, activating K-ras mutations related to malignancy are frequently observed. In this study, we first designed 78 AS-ODNs for K-ras to optimize the sequence for highly efficient gene suppression. The selected AS-ODN (K-AS07) having dA₄₀ made a complex with SPG. The resultant complex (K-AS07-dA₄₀/SPG) showed an effect of silencing the ras gene in the cells (PC9: human adenocarcinoma differentiated from lung tissue) expressing Dectin-1, leading to the suppression of cell growth. Furthermore, the cytotoxic effect was enhanced when used in combination with the anticancer drug gemcitabine. Gemcitabine, a derivative of cytidine, was shown to interact with dA₄₀ in a sequence-dependent manner. This interaction did not appear to be so strong, with the gemcitabine being released from the complex after internalization into the cells. SPG and the dA₄₀ part of K-AS07-dA₄₀ play roles in carriers for K-AS07 and gemcitabine, respectively, resulting in a strong cytotoxic effect. This combination effect is a novel feature of the AS-ODN-dA₄₀/SPG complexes. These results could facilitate the clinical application of these complexes for cancer treatment.     

Characterization of therapeutic oligonucleotides using liquid chromatography with on-line mass spectrometry detection

A method for the analysis and characterization of therapeutic and diagnostic oligonucleotides has been developed using a combination of liquid chromatography and mass spectrometry (LC-MS). The optimized ion-pairing buffers permit a highly efficient separation of native and chemically modified antisense oligonucleotides (AS-ODNs) from their metabolites or failure synthetic products. The mobile phases were MS compatible, allowing for direct and sensitive analysis of components eluting from the column. The method was applied for the quantitation and characterization of AS-ODNs, including phosphorothioates and 2'-O-methyl-modified phosphorothioates. Tandem LC-MS analysis confirmed the identity of the oligonucleotide metabolites, failure products, the presence of protection groups not removed after synthesis, and the extent of depurination or phosphorothioate backbone oxidation.     

胶质细胞谷氨酸转运体-1反义寡核苷酸对脑缺血预处理神经保护效应的抑制作用

本研究应用胶质细胞谷氨酸转运体-1(glial glutamate transporter-1,GLT-1)的反义寡核苷酸(antisense oligo-deoxynucleotides,AS-ODNs)抑制Wistar大鼠GLT-1蛋白的表达,观察其对脑缺血预处理(cerebral ischemic preconditioning.CIP)增强脑缺血耐受作用的影响,探讨GLT-1在CIP诱导的脑缺血耐受中的作用.将凝闭双侧椎动脉的Wistar大鼠随机分为7组:(1)Sham组:只暴露双侧颈总动脉,不阻断血流;(2)CIP组:夹闭双侧颈总动脉3 min;(3)脑缺血打击组:夹闭双侧颈总动脉8 min;(4)CIP 脑缺血打击组:夹闭双侧颈总动脉3 min作为CIP,再灌注2 d后,夹闭双侧颈总动脉8min;(5)双蒸水组:于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射双蒸水,每次5 μL,其它同sham组;(6)AS-ODNs组:于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射GLT-1 AS-ODNs溶液,每次5 μL,其它同sham组,再根据AS-ODNs的剂量进一步分为9 nmol和18 nmol 2个亚组;(7)AS-ODNs CIP 脑缺血打击组:于CIP前12 h、后12 h及后36 h右侧脑室注射GLT-1 AS-ODNs溶液,每次5 μL,其它同CIP 脑缺血打击组,根据AS-ODNs的剂量进一步分为9 nmol和18 nmol 2个亚组.Western blot分析法观察GLT-1蛋白的表达,硫堇染色观察海马CA1区锥体神经元迟发性死亡(delayed neuronal death,DND)情况.Western blot分析显示,侧脑室注射GLT-1 AS-ODNs可剂量依赖性地抑制大鼠海马CA1区GLT-1蛋白表达.硫堇染色显示,sham组和CIP组海马CA1区未见明显的DND;脑缺血打击组海马CA1区有明显的DND:预先给予CIP可显著对抗脑缺血打击引起的DND,表明CIP可以诱导海马CA1区神经元产生缺血性耐受,对抗脑缺血打击引起的DND;而在GLT-1 AS-ODNs CIP 脑缺血打击组,侧脑室注射GLT-1 AS-ODNs后,大鼠海马CA1区出现了明显的DND,表明GLT-1 AS-ODNs通过抑制大鼠GLT-1蛋白表达从而减弱CIP对抗脑缺血打击的神经保护作用.以上结果进一步证实了GLT-1参与CIP诱导的脑缺血耐受.     

Antisense activity detection by inhibition of fluorescence resonance energy transfer.

Use of antisense nucleic acids to modulate expression of particular genes is a promising approach to the therapy of human papillomavirus type 16 (HPV-16)-associated cervical cancer. Understandably, evaluation of the in vivo performance of synthetic antisense oligodeoxynucleotides (AS-ODNs) or ribozymes is of ultimate importance to development of effective antisense tools. Here we report the use of a bacterial reporter system based on the inhibition of fluorescence resonance energy transfer (FRET) to measure the interaction of AS-ODNs with HPV-16 target nt 410-445, using variants of the green fluorescent protein (GFP). An optimal FRET-producing pair was selected with GFP as the donor and yellow fluorescent protein (YFP) as the acceptor molecule. Hybridization of AS-ODNs with a chimaeric mRNA containing the antisense target site flanked by GFP variants resulted in the inhibition of the FRET effect. Use of different linkers suggested that the amino acid content of the linker has no significant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target-specific and mutant AS-ODNs, suggested a specific effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins confirming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS-ODNs activity in vivo.     

Trafficking of intracerebroventricularly injected antisense oligonucleotides in the mouse brain

Intracerebroventricular (icv) delivery of therapeutic molecules directly into the brain parenchyma has attracted considerable attention because of the advantage of bypassing the blood-brain barrier. Exogenous icv administration of antisense oligodeoxynucleotides (AS-ODNs) has been implicated in modifying gene expression within the targeted brain area. The biodistribution, tissue penetration, and stability of exogenously administered AS-ODNs are the major determinants with regard to their potential utility as agents for modifying gene expression. This report examined the distribution and clearance of labeled AS-ODNs with the aim of exploring the feasibility of icv administration of AS-ODNs as a targeted treatment approach to Alzheimer's disease. A single icv injection of fluorescein-labeled 2'-O-(methoxy) ethyl (2'MOE) ribosyl-modified AS-ODNs directed at the beta-secretase cleavage site of beta-amyloid precursor protein (APP) mRNA into the mouse brain showed rapid uptake by 15 minutes, overall gradual spread and retention by 30 minutes to 3 hours, and complete clearance by 8 hours postinjection. Labeled AS-ODNs were observed to penetrate across the cell membrane and accumulate in both nuclear and cytoplasmic compartments of neuronal and nonneuronal cell populations. Current study provides a basic pattern of uptake, distribution, and stability of AS-ODNs in the mouse brain.     

Polyethylenimine-based antisense oligodeoxynucleotides of IL-4 suppress the production of IL-4 in a murine model of airway inflammation

BACKGROUND: Interleukin-4 (IL-4) plays a crucial role as an inflammatory mediator in allergic asthma via inducing Th2 inflammation and IgE synthesis. To develop an effective therapeutic agent which specifically inhibits production of IL-4, antisense oligodeoxynucleotides (AS-ODNs) against murine IL-4 mRNA were generated and complexed with polyethylenimine (PEI) to improve intracellular delivery. METHODS: AS-ODNs were generated against the translation initiation region of murine IL-4 mRNA, and complexed with linear PEI. In vitro efficacy of AS-ODNs/PEI complexes was tested by measuring IL-4 production in the D10.G4.1 cell line, and cytotoxicity was tested by XTT assay. Physicochemical properties of polyplexes were examined using atomic force microscopy (AFM) and DNase I protection assay. In vivo effects of IL-4 AS-ODNs/PEI complexes were tested in a murine model of airway inflammation. IL-4 concentrations in the bronchoalveolar lavage (BAL) fluid and circulating IgE levels were measured by ELISA, and histological analysis of lung tissues was performed. RESULTS: IL-4 AS-ODNs/PEI complexes were spheres with an average diameter of 98 nm and resistant to DNase I-mediated degradation. IL-4 AS-ODNs/PEI complexes showed up to 35% inhibition of IL-4 production in D10.G4.1 cells without causing any toxicity, while naked ODNs gave less than 1% reduction. Furthermore, IL-4 AS-ODNs/PEI complexes were effective in suppressing secretion of IL-4 (up to 30% reduction) in the BAL fluid in an ovalbumin-sensitized murine model of airway inflammation. Circulating IgE levels were decreased, and airway inflammation was alleviated by treatment with IL-4 AS-ODNs polyplexes. CONCLUSIONS: These data demonstrate that complexation of IL-4 AS-ODNs with PEI provides a potential therapeutic tool in controlling inflammation associated with allergic asthma, and further presents an opportunity to the development of clinical therapy based on combination of multiple AS-ODNs of cytokines and/or signaling effectors involved in Th2 inflammation and eosinophilia.     

Inhibition of the Proliferative Cycle and Apoptotic Events in WiDr Cells after Down-Regulation of the Calcium-Binding Protein Calretinin Using Antisense Oligodeoxynucleotides

The colon adenocarcinoma cell line WiDr expresses the calcium-binding protein calretinin (CR). In order to deduce possible functions of calretinin in these cells we decreased its concentration by antisense techniques. Treatment of WiDr cells with phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) led to a drop in calretinin expression, as evidenced by immunohistochemical staining of WiDr cells and Western blot analysis of cytosolic cell extracts. The morphology of these epithelial cells changed from polygonal to spherical and they formed dense cell clusters. Cells displaying morphological alterations typical for apoptotic cells were observed after incubation with AS-ODNs, as evidenced by phase-contrast and electron microscopy. The mitotic rate of AS-ODN-treated cells dropped significantly, as demonstrated by mitotic labeling and time-lapse microcinematography. Furthermore, an accumulation of cells in phase G1 and a reduction of [³H]thymidine-labeled cells was observed in antisense-treated cells. The basal level of [Ca²⁺]_iwas not influenced by the down-regulation of calretinin. WiDr cells incubated with the nonsense, reverse-sense, or with an oligodeoxynucleotide with a totally unrelated sequence did not show any significant differences when compared to control cells. We conclude that calretinin levels have an impact on the progression of the cell cycle of WiDr cells.     

Selection of effective antisense oligodeoxynucleotides with a green fluorescent protein-based assay. Discovery of selective and potent inhibitors of glutathione S-transferase Mu expression.

Antisense oligodeoxynucleotides (AS-ODNs) are frequently used for the down-regulation of protein expression. Because the majority of potential antisense sequences lacks effectiveness, fast screening methods for the selection of effective AS-ODNs are needed. We describe a new cellular screening assay for the evaluation of the potency and specificity of new antisense sequences. Fusion constructs of the gene of interest and the gene encoding the enhanced green fluorescent protein (EGFP) are cotransfected with AS-ODNs to COS-7 cells. Subsequently, cells are analysed for expression of the EGFP fusion protein by flow cytometry. With the assay, we tested the effectiveness of a set of 15 phosphorothioate ODNs against rat glutathione S-transferase Mu1 (GSTM1) and/or Mu2 (GSTM2). We found several AS-ODNs that demonstrated potent, sequence-specific, and concentration-dependent inhibition of fusion protein expression. At 0.5 microm, AS-6 and AS-8 inhibited EGFP-GSTM1 expression by 95 +/- 4% and 81 +/- 6%, respectively. AS-5 and AS-10 were selective for GSTM2 (82 +/- 4% and 85 +/- 0.4% decrease, respectively). AS-2 and AS-3, targeted at homologous regions in GSTM1 and GSTM2, inhibited both isoforms (77-95% decrease). Other AS-ODNs were not effective or displayed non-target-specific inhibition of protein expression. The observed decrease in EGFP expression was accompanied by a decrease in GSTM enzyme activity. As isoform-selective, chemical inhibitors of GSTM and GSTM knock-out mice are presently unavailable, the selected AS-ODNs constitute important tools for the study of the role of GSTM in detoxification of xenobiotics and protection against chemical-induced carcinogenesis.     

Synthesis of long chain acyl-enzyme thioesters by modified fatty acid synthetases and their hydrolysis by a mammary gland thioesterase.

The fatty acid synthetase multienzyme from lactating rat mammary gland was modified either by removal of the two thioesterase I domains with trypsin or by inhibiting the thioesterase I activity with phenylmethanesulfonyl fluoride. The modified multienzymes are able to convert acetyl-CoA, malonyl-CoA, and NADPH to long chain acyl moieties (C₁₆C₂₂), which are covalently bound to the enzyme through thioester linkage, but they are unable to release the acyl groups as free fatty acids. A single enzyme-bound, long chain acyl thioester is formed by each molecule of modified multienzyme. Kinetic studies showed that the modified multienzymes rapidly elongate the acetyl primer moiety to a C₁₆ thioester and that further elongation to C₁₈, C₂₀, and C₂₂ is progressively slower. Thioesterase II, a mammary gland enzyme which is not part of the fatty acid synthetase multienzyme, can release the acyl moiety from its thioester linkage to either modified multienzyme. Kinetic data are consistent with the formation of an enzyme—substrate complex between thioesterase II and the acylated modified multienzymes. The present study demonstrates that the ability of thioesterase II to modify the product specificity of normal fatty acid synthetase is most likely attributable to the capacity of thioesterase II for hydrolysis of acyl moieties from thioester linkage to the multienzyme.     

Controlled delivery of antisense oligodeoxynucleotide from cationically modified phosphorylcholine polymer films

Antisense strategy is a promising approach for the prevention of in-stent restenosis if therapeutic agents such as antisense oligodeoxynucleotides (AS-ODNs) can be successfully delivered to the implant site. Optimizing the routes and conditions for controlled loading and release of therapeutic agents from a biocompatible polymer coating is still required. In this study, phosphorylcholine (PC) polymer films bearing different cationic charge densities were deposited onto smooth silicon substrates. The thickness of these films was determined by spectroscopic ellipsometry (SE). Human c-myc AS-ODNs were incorporated into the PC polymer films by immersion in concentrated AS-ODN solution and eluted into PBS under physiological conditions. The elution profile was monitored by UV spectrometry and gel electrophoresis. Cellular uptake of the eluted AS-ODN into vascular smooth muscle cells (VSMCs) was evaluated by fluorescence microscopy. The results showed that ODN loading capacities increased with film thickness and were also strongly dependent on the cationic charge density. AS-ODN release was characterized by a slight initial burst in the first half hour followed by a period of sustained release up to 8 days. Gel electrophoresis demonstrated DNA integrity, and different transfection efficiencies were observed when the eluted ODNs were transfected into VSMCs. These results demonstrated that cationically modified PC polymers are capable of delivery of antisense ODNs in a controlled manner and that they are well suited for specific biomedical devices such as DNA-eluting stents.     

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: Functional coupling of the sphingosine-1-phosphate receptor to PLC-xβ in Xenopus oocytes

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca²⁺-dependent oscillatory Cl⁻ currents by acting through membrane-bound receptors. External application of 50 μM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-μM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca²⁺-dependent Cl⁻ currents were observed in the absence of extracellular Ca²⁺, were blocked by intracellular injection of the Ca²⁺ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca²⁺ ATPase. Intracellular Ca²⁺ release appeared to be from inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store, because Cl⁻ currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xβ), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xβ, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein α subunits that were identified in Xenopus laevis; G_qα, G₁₁α, G₀α, and G_i1α. Among AS-ODNs against the Gαs tested, AS-G_qα and AS-G_i1α to S1P and AS-G_qα and AS-G₁₁α to LPA specifically reduced current responses, respectively, to about 20–30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca²⁺ from the IP₃-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes. J. Cell. Physiol. 176:412–423, 1998. © 1998 Wiley-Liss, Inc.     

Specific post-transcriptional inhibition of mRNA for ligand binding chain of IgE high affinity receptor

IgE high affinity receptor (FcεRI) plays an important role in triggering type I allergic reactions. In this study, we have investigated the ability of four synthetic and sequence-specific RNA interfering antisense oligodeoxynucleotides (AS-ODNs) to reduce the expression of FcεRIα gene in granulocytes of allergy sufferers in vitro. Only AS1 out of four AS-ODNs specifically inhibited the FcεRIα gene expression and the dose response assay revealed that AS1 was capable of specific inhibition of target mRNA expression over a linear concentration range without affecting the expression of house keeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Together, these results indicate that sequence-specific RNA interfering ODNs can be effectively used to silence the expression of key genes like IgE high affinity receptor that are involved in chronic inflammatory diseases.     

Determination of L-ascorbic acid levels in culture medium: Concentrations in commercial media and maintenance of levels under conditions of organ culture

Summary The method of Deutsch and Weeks was modified to provide a reliable and reasonably quick method for assaying the L-ascorbic acid content of culture medium. The modified method was used to determine the decay of L-ascorbic acid under various conditions of culture and the concentration of the vitamin in commercially prepared media. The half-life of L-ascorbic acid in a modified New circulator gassed with 95% O₂+5% CO₂ was 1.5 hr.; and when gassed with 20% O₂+5% CO₂+75% N₂, about 2 hr. In Petri dishes gassed with 20% O₂+5% CO₂+75% N₂, the half-life of L-ascorbic acid was 0.9 hr. About 4% of the L-ascorbic acid was lost per day when medium was stored at 0°C and about 9% per day when stored at 5°C. When medium with an initial content of 300 μg per ml was stored at room temperature, the half-life was found to be 15.5 hr. The L-ascorbic acid in five commercially available media, which contain the vitamin in their formulations, was assayed immediately after their delivery to the laboratory. The values of L-ascorbic acid measured in these media were in all cases far lower than prescribed. A continuous-flow organ culture system has been designed which allows the provision of a relatively constant level of L-ascorbic acid to an explant by taking advantage of the slow oxidation of L-ascorbic acid at 0°C.     

A potent inhibitor of prothrombin gene expression as a result of standardized target site selection and design of antisense oligonucleotides

The development of antisense oligonucleotides (AS-ODN) always had the limitation that because of complex mRNA secondary structures, not every designed AS-ODN inhibited the expression of its target. There have been many investigations to overcome this problem in the last few years. This produced a great deal of theoretical and empirical findings about characteristics of effective AS-ODNs in respect to their target regions but no standardized selection procedure of AS-ODN target regions within a given mRNA or standardized design of AS-ODNs against a specific target region. We present here a standardized method based on secondary structure prediction for target site selection and AS-ODN design, followed by validation of the antisense effect caused by our predicted AS-ODNs in cell culture. The combination of theoretical design and experimental selection procedure led to an AS-ODN that efficiently and specifically reduces prothrombin mRNA and antigen.     

In vitro somatic embryogenesis from callus culture of the timber yielding treeHardwickia binata Roxb.

Somatic embryogenesis was achieved in callus cultures derived from immature cotyledonary explants ofHardwickia binata Roxb., a multipurpose leguminous tree, on semisolid modified Murashige and Skoog's (mMS) medium containing 2900 mg/l potassium nitrate (KNO₃) supplemented with 4.64 µM kinetin (Kn) and 5.37µM a-naphthaleneacetic acid (NAA). Somatic embryos proliferated rapidly after transfer to MS basal medium supplemented with 2052.6 µM L-glutamine and 0.084 µM gibberellic acid (GA₃). Maturation of somatic embryos was achieved on half-strength MS basal medium supplemented with 1.23 µM IBA and 2% (w/v) sucrose. Histological studies confirmed different developmental stages of somatic embryogenesis inHardwickia binata. Abbreviations BA N⁶-benzyladenine - Kn kinetin - NAA a-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - mMS modified Murashige and Skoog (1962) medium     

GluR2 knockdown reveals a dissociation between [Ca2+]i surge and neurotoxicity

Reduction in GluR2 subunit expression and subsequent increases in AMPA receptor mediated Ca(2+) currents were postulated to exacerbate glutamate neurotoxicity following seizures or global ischemia. To directly test the effects of shifting the GluR1/GluR2 subunit ratio on excitotoxicity, GluR2 antisense deoxyoligonucleotides (AS-ODNs) were applied to dissociated hippocampal cultures for 1-8 days. The GluR1/GluR2 protein ratio was examined immunohistochemically and by Western blotting. [Ca(2+)](i) concentrations were determined by ratiometric imaging of Fura 2-loaded cells. The cultures were exposed to glutamate, AMPA, NMDA or kainic acid (KA) 3 days after GluR2 knockdown and cell viability was determined 1 day later by MTT reduction assay or Trypan blue exclusion. Although GluR2 AS-ODNs increased the GluR1/GluR2 protein ratio in a time dependent manner, neurons and glia appeared healthy and MTT reduction values were similar to untreated and sense controls. Basal [Ca(2+)](i) levels were unchanged but [Ca(2+)](i) was selectively increased by agonist stimulation of AMPA receptors. Unexpectedly, delayed neurotoxicity was attenuated at saturating doses of glutamate while little difference in cell viability was observed at lower doses or with the other excitotoxins at any concentration. Therefore, there was a dissociation between rises in AMPA receptor-mediated Ca(2+) influx and neurotoxicity despite marked decreases in GluR2 but not GluR1 immunoreactivity. It is proposed that a modification of AMPA receptor stochiometry that raises agonist-stimulated Ca(2+) influx during an excitotoxic insult may have eventual neuroprotective effects.     

Ester synthesis in benzene by polyethylene glycol-modified lipase from Pseudomonas fragi 22.39B

Lipase (EC 3.1.1.3) from Pseudomonas fragi 22.39B was modified with polyethylene glycol. The modified lipase was soluble in organic solvents such as benzene and chlorinated hydrocarbons, and catalyzed the synthesis of esters from fatty acids and alcohols in these solvents. The longer the chain length of fatty acid, the higher the ester synthesis activity. A similar specificity was not observed with other substrates like alcohol. Values of K_m and V_max were revealed by kinetic study on the ester synthesis reaction with the modified lipase in benzene. Fatty acids with branched carbon chain at the position neighboring the carboxyl group did not serve as substrates of ester synthesis.