Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

Heat transfer mechanism of a swirling impinging jet in a stagnation region

Heat transfer characteristics of a swirling impinging jet have been experimentally examined using a combined particle image velocimetry (PIV) and laser‐induced fluorescence (LIF) technique for simultaneous measurement of velocity and temperature fields. The present study shows that the radial width of the jet stretches with increasing swirl intensity, and that the stretching phenomenon contributes to the maximum local heat transfer coefficient. At the stagnation region, the flow near the heated surface is mixed intermittently by reverse flows toward upstream, and spatial distributions of temperature are correlated with instantaneous velocity vector maps. The dynamic behavior of recirculation zones, attributed to swirl number Sw and impinging distance, mainly determines the turbulent heat transfer at the stagnation region. © 2003 Wiley Periodicals, Inc. Heat Trans Asian Res, 32(8): 663–673, 2003; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/htj.10120     

Thermal stability of interfaces between metals and InP-based materials

The thermal stability of interfaces between metals (Ni, Pt, Ti, Mo) and III-V compound semiconductors has been investigated by the application of Rutherford backscattering spectrometry. Metal diffusion and interfacial lattice disorder of the semiconductors were analyzed for various metal/semiconductor samples annealed at temperatures up to 500°C. The interfaces of Ni/GaAs and Ti/GaAs were found to be more stable than those of Ni/In-based semiconductors and Ti/ In-based semiconductors, respectively. Faster diffusion of Pt atoms was ob-served in In-and As-containing materials than in P-containing materials. Mo/ semiconductor interfaces were the most stable.     

High-speed and large noise margin tolerance E/D logic gates withLDD structure DMTs fabricated using selective RIE technology

The authors describe a novel design concept for enhancement (E) and depletion (D) mode FET formation using i-AlGaAs/n-GaAs doped-channel hetero-MISFET (DMT) and a novel self-aligned gate process technology for submicrometer-gate DMT-LSIs based on E/D logic gates. 0.5-μm gate E-DMTs (D-DMTs) with a lightly doped drain (LDD) structure show an average V_t of 0.18 (-0.46) V, a V_t standard deviation of 22.6 (24.9) mV, and a maximum transconductance of 450 (300) mS/mm. The V_t shift is less than 50 mV with a decrease in gate length down to 0.5 μm. The gate forward turn-on voltage V_f is more than 0.9 V, i.e. about 1.6 times that for MESFETs. This superiority in V _f, preserved in the high-temperature range, leads to an improvement in noise margin tolerance by a factor of three. In addition, 31-stage ring oscillators operate with a power consumption of 20 (1.0) mW/gate and a propagation delay of 4.8 (14.5) ps/gate. Circuit simulation based on the experimental data predicts 140 ps/gate and 1 mW/gate for DMT direct-coupled FET logic circuits under standard loading conditions. DMTs and the technology developed here are very attractive for realizing low-power and/or high speed LSIs     

Development of a Trans-Mucosal Controlled-Release Device for Systemic Delivery of Antianginal Drugs Pharmacokinetics and Pharmacodynamics

Oral mucosa is well-known to be one of the best routes for drug absorption. But very few R & D works have been initiated to investigate the feasibility of using this site to control drug delivery. A transmucosal controlled-release device, which is capable of achieving excellent absorption and controlled release of drugs, has been developed. The device is a tabletshaped mucoadhesive system which is composed of two layers. The upper layer is a fast-release layer and the lower layer is a sustained-release layer, and designed to be applied between buccal and gingival mucosae. Both layers are formulated from synthetic polymers to control the release of drugs.

Isosorbide dinitrate(ISDN), a well-documented antianginal drug, is known to be susceptible to extensive presystemic elimination when taken orally. It was used as the candidate drug and the systemic bioavailability was studied in human and observed to be improved by as much as 5 fold when compared to a marketed oral sustained-release tablet; On the other hand, much smaller amount of metabolites was formed. The plasma profile of ISDN has also been observed to be substantially prolonged (12 hrs as compared to less than 1 hr for sublingual tablet and spray product on the market). These observations have demonstrated that this device is capable of not only bypassing hepatic “first-pass” metabolism but also having a sustainedrelease property of prolonging the release of ISDN.

Clinical studies performed in the anginal patients for up to one year have demonstrated the therapeutic benefits of this device in achieving a substantial reduction in the frequency of anginal attacks.

This type of device was also applied to the systemic delivery of another antianginal drug, Nifedipine, by employing a formulation with longer sustained drug release property. Again, the clinical results demonstrated that a prolonged duration of therapeutic plasma concentration has also been accomplished.     

Opioid receptors altered binding nature in guinea-pig brain following the development of morphine dependence

Morphine is well known to produce tolerance and dependence. The mechanisms for these phenomena are not clearly understood and there are a number of conflicting reports that chronic morphine administration decreases, increases, or leaves unchanged the number of opioid binding sites. We examined the potency of MScontin (oral controlled-release preparation of morphine) to induce morphine dependence and also determined the change of mu, delta and kappa opioid receptor types in brain homogenates obtained from morphine-dependent guinea-pigs. 1. Guinea-pigs were implanted subcutaneously with MScontin (300 mg.kg-1) and naloxone was employed to precipitate jumping behavior of withdrawal symptoms at various times. The highest degree of physical dependence was observed on the 2nd day after implantation. Therefore, this period was chosen to investigate opioid receptor binding assay. 2. Two days after implantation, the binding of 3H-DAGO (mu agonist), 3H-DPDPE (delta agonist) and 3H-U69593 (kappa agonist) to brain membranes prepared from morphine dependent and control guinea-pigs was determined. Scatchard plot of the saturation binding data revealed an increase in Bmax values (maximum specific binding) and no change in the Kd values (equilibrium dissociation constants) of 3H-opioid ligand bindings obtained from morphine-dependent animals as compared to controls. These results indicate that brain mu, delta and kappa opioid receptors are up-regulated in morphine dependent guinea-pigs.     

Fine structures and fracture processes in plastic/rubber two -phase polymer systems. I. Observation of fine structures under the electron microscope

Fine structures of several plastic/rubber two -phase polymer systems were studied by means of direct observations of ultrathin sections under the electron microscope using osmium tetroxide staining and a hardening procedure developed recently by Kato. Samples used are several types of both ABS polymers and high -impact polystyrenes, and several PVC/rubber blends and the results were discussed in relation to their dynamic viscoelastic properties. It is suggested that these studies may fruitfully be extended to clarify the structure -to property relationships by use of this method.     

Changes in the Peeled Surfaces of Copper Thin Films Deposited on Polyimide Substrates After Heat Treatment

Peel strength between a copper (Cu) thin film and a polyimide (pyromellitic dianhydride-oxydianiline, or PMDA-ODA) substrate is reduced by heat treatment at 150°C in air. In this work, we investigated the peel strength, the morphology of the interface between Cu films and polyimide substrates using optical microscopy and electron microscopy, and chemical change of the interface using Auger electron spectroscopy (AES) and micro X-ray photoelectron spectroscopy (XPS). The analysis showed that CuO “lumps” were present on the peeled surface of PMDA-ODA after heat treatment at 150°C in air. The peeled surfaces of other polyimide substrates were also analyzed: biphenyl dianhydride-para phenylene diamine (BPDA-PDA) and biphenyl dianhydride-oxydianiline (BPDA-ODA). CuO lumps were present on the peeled surface of BPDA-ODA after the heat treatment, but not that of BPDA-PDA. Compared with the adhesion strength for the Cu thin film, the adhesion strength was high for the Cu/PMDA-ODA and Cu/BPDA-ODA laminates, but the adhesion strength was very low for the Cu/BPDA-PDA laminate. This low strength is the reason that CuO lumps were not detected on the peeled surface of the BPDA-PDA substrate. These CuO lumps were related to the adhesion degradation of the Cu/polyimide laminates after the heat treatment.     

Treatment of domestic sewage by a metal membrane bioreactor

A submerged flat metal MBR (membrane bioreactor) was used to treat synthetic domestic sewage in this study. The experiment was continued for 270 days and ran under two modes as AMBR (aerobic membrane bioreactor) and A/O-MBR (anoxic/aerobic membrane bioreactor) at a permeate flux of 0.4-1 m3/(m2 d). PVA (polyvinyl alcohol) gel beads were added to the aeration tank with a volume ratio of 10% at the end of the A/O-MBR mode. The mean COD and TN removal efficiencies achieved 96.69 and 32.12% under the AMBR mode, and those were 92.17 and 72.44% under the A/O-MBR mode, respectively. SND (simultaneous nitrification and denitrification) occurred at high MLSS (mixed liquor suspended solids) concentration. The metal membranes reduced effluent COD during filtration. The system ran stably for 115 days at a permeate flux of 0.8-1 m3/(m2 d) without changing membranes under the AMBR mode, but the membrane filterability decreased gradually under high MLSS or A/O-MBR mode, and the addition of PVA worsened the membrane filterability on the contrary. PSD (particle size distribution) and sludge fractions had evident influence on membrane fouling. The main fouling mechanism was cake formation under the AMBR mode, and that was pore blocking under the A/O-MBR mode.