Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins

The promoter of alcohol dehydrogenase I of the yeast Saccharomyces cerevisiae was studied using Bacillus amyloliquefaciens alpha-amylase as a marker protein. On glucose, activity of the original ADH1 promoter decreases during late exponential, ethanol production growth phase. When 1100 bp (from -414 bp to -1500 bp) of the upstream sequence are deleted, activity increases into the late ethanol consumption phase but the promoter becomes active only after ethanol production growth phase (Ruohonen et al. (1991) Yeast 7, 337-346). We have now restored 300 bp (from -414 bp to -700 bp) upstream of the deletion site and obtained expression from the ADH1 promoter throughout the yeast growth cycle. The restored sequence allowed alpha-amylase expression to start during early exponential growth phase indicating that it is required for activation of the ADH1 promoter during ethanol production growth phase, possibly through glucose induction. On ethanol, all the promoters were active, but the short promoter was temporally activated first, suggesting that the restored sequence is not required for promoter activity during early oxidative growth.     

Hardware implementation of CMAC neural network with reduced storage requirement.

The cerebellar model articulation controller (CMAC) neural network has the advantages of fast convergence speed and low computation complexity. However, it suffers from a low storage space utilization rate on weight memory. In this paper, we propose a direct weight address mapping approach, which can reduce the required weight memory size with a utilization rate near 100%. Based on such an address mapping approach, we developed a pipeline architecture to efficiently perform the addressing operations. The proposed direct weight address mapping approach also speeds up the computation for the generation of weight addresses. Besides, a CMAC hardware prototype used for color calibration has been implemented to confirm the proposed approach and architecture.     

ESD protection design for CMOS RF integrated circuits using polysilicon diodes

ESD protection design for CMOS RF integrated circuits is proposed in this paper by using the stacked polysilicon diodes as the input ESD protection devices to reduce the total input capacitance and to avoid the noise coupling from the common substrate. The ESD level of the stacked polysilicon diodes on the I/O pad is restored by using the turn-on efficient power-rail ESD clamp circuit, which is constructed by substrate-triggered technique. This polysilicon diode is fully process compatible to general sub-quarter-micron CMOS processes.     

Electrostatic discharge protection design for mixed-voltage CMOS I/O buffers

A new electrostatic discharge (ESD) protection circuit, using the stacked-nMOS triggered silicon controlled rectifier (SNTSCR) as the ESD clamp device, is designed to protect the mixed-voltage I/O buffers of CMOS ICs. The new proposed ESD protection circuit, which combines the stacked-nMOS structure with the gate-coupling circuit technique into the SCR device, is fully compatible to general CMOS processes without causing the gate-oxide reliability problem. Without using the thick gate oxide, the experimental results in a 0.35 /spl mu/m CMOS process have proven that the human-body-model ESD level of the mixed-voltage I/O buffer can be successfully increased from the original /spl sim/2 kV to >8 kV by using this proposed ESD protection circuit.     

A sec1-related vesicle-transport protein that is expressed predominantly in epithelial cells

Sec1-related proteins are involved in docking and fusion of transport vesicles in eukaryotic cells. Here we report the cloning and molecular characterization of a Sec1-related protein expressed in the MDCK epithelial cell line. This protein represents a canine counterpart of the murine Munc-18-2/Munc-18b/muSec1 protein, displays 93% amino acid identity with these proteins, has a similar tissue mRNA expression pattern, and associates in vitro with syntaxins 1A, 2, and 3. In situ hybridization analysis of embryonic mouse tissues revealed prominent expression of the munc-18-2 mRNA in the epithelia of several tissues. Cell-fractionation studies demonstrated that the majority of Munc-18-2 is membrane associated. Most of the protein is washed off the membranes by sodium carbonate, pH 11.5. However, the protein is poorly solubilized by detergent treatment. The Munc-18-2 protein was localized, by immunofluorescence microscopy, to the plasma membrane of MDCK cells, and is apically distributed in the epithelial cells of mouse tissues. When overexpressed in COS-1 cells, the protein appeared to be largely cytosolic. However, upon expression with syntaxin 1A, it displayed a shift to the plasma membrane, where the two proteins colocalized. These results identified Munc-18-2 as a predominantly epithelial vesicle-transport protein with a polarized distribution and provided novel in vivo evidence for the association of Sec1-related proteins with members of the syntaxin family.     

Degradative and mechanical properties of a novel resorbable plating system during a 3-year follow-up in vivo and in vitro

We tested the tissue reactions and mechanical strength of a novel biodegradable craniomaxillofacial plating system, Inion CPS™, in the course of degradation. Plates and screws composed of l-lactide, d-lactide and trimethylene carbonate were implanted to the mandible and dorsal subcutis of 12 sheep. The animals were sacrificed at 6–156 weeks. Histological evaluation was done using paraffin and methylmetacrylate techniques. Degradative and mechanical properties during the follow-up were measured both of in vivo and in vitro implants. In light microscopy, the in vivo implant material began to fragment at 52 weeks and could not be detected at 104 weeks. No significant foreign body reactions were seen in the mandibles. The dorsal subcutis disclosed mild reactions, which were, however, not of clinical significance. The implants in vitro maintained their entire mass for 26 weeks and lost 63–80% of the mass by week 104. The inherent viscosity of the implants in vitro and in vivo diminished uniformly. The screws retained their shear strength for 12–16 weeks. The plates maintained their tensile strength for at least 6 weeks. The maximum capacity of the plates in 3-point bending tests diminished gradually by 87% in 26 weeks. In conclusion, the plates and screws examined maintain adequate strength for the healing period of a bone fracture or osteotomy, producing no harmful foreign body reactions. Dr Nieminen is a consultant for Inion Ltd., while the other co-authors do not have any conflicts of interest. Inion Ltd. has financed the costs related to the study sheep, including their housing.     

Functional inactivation of the conserved Sem1p in yeast by intrabodies

Intrabody technology was applied to characterize the function and intracellular localization of a highly conserved Saccharomyces cerevisiae Sem1 protein. DSS1, the mammalian homologue of Sem1p, is functionally conserved between yeast and mammalian cells, and in mammalian cells physically interacts with the strong tumour supressor BRCA2. Yeast and the generated intrabodies are thus expected to offer a useful system for studies on Sem1p/DSS1 function. Sem1p-specific antibody isolated from a phage display library was expressed intracellularily and targeted to either the cytosol or the nucleus of yeast cells. Analysis of the applicability of different antibody fragments as intrabodies showed that the Fab intrabody was expressed most efficiently. Expression of nuclear-targeted anti-Sem1p Fab intrabodies inhibited the growth of the sigma1278b yeast strain in a manner similar to deletion of the SEM1 gene. This indicates that the Fab intrabodies interact in vivo with Sem1p and result in inactivation of Sem1p. Localization of the Fab intrabody with or without the nuclear localization signal to the nucleus in Sem1p-dependent manner suggests that Sem1p mediates the nuclear transport of the intrabody without any targeting signal. Our results suggest that Sem1p function in yeast cells is in part manifested in the nucleus.     

Overview of on-chip electrostatic discharge protection design with SCR-based devices in CMOS integrated circuits

An overview on the electrostatic discharge (ESD) protection circuits by using the silicon controlled rectifier (SCR)-based devices in CMOS ICs is presented. The history and evolution of SCR device used for on-chip ESD protection is introduced. Moreover, two practical problems (higher switching voltage and transient-induced latchup issue) limiting the use of SCR-based devices in on-chip ESD protection are reported. Some modified device structures and trigger-assist circuit techniques to reduce the switching voltage of SCR-based devices are discussed. The solutions to overcome latchup issue in the SCR-based devices are also discussed to safely apply the SCR-based devices for on-chip ESD protection in CMOS IC products.     

Design on the low-leakage diode string for using in the power-railESD clamp circuits in a 0.35-μm silicide CMOS process

A new design of the diode string with very low leakage current is proposed for use in the ESD clamp circuits across the power rails. By adding an NMOS-controlled lateral SCR (NCLSCR) device into the stacked diode string, the leakage current of this new diode string with six stacked diodes at 5 V (3.3 V) forward bias can be reduced to only 2.1 (1.07) nA at a temperature of 125°C in a 0.35 μm silicide CMOS process, whereas the previous designs have a leakage current in the order of mA. The total blocking voltage of this new design with NCLSCR can be linearly adjusted by changing the number of the stacked diodes in the diode string without causing latch-up danger across the power rails. From the experimental results, the human-body-model ESD level of the ESD clamp circuit with the proposed low-leakage diode string is greater than 8 kV in a 0.35 μm silicide CMOS process by using neither ESD implantation nor the silicide-blocking process modifications     

A state prediction scheme for discrete time nonlinear dynamic systems

A state prediction scheme is proposed for discrete time nonlinear dynamic systems with non-Gaussian disturbance and observation noises. This scheme is based upon quantization, multiple hypothesis testing, and dynamic programming. Dynamic models of the proposed scheme are as general as dynamic models of particle predictors, whereas the nonlinear models of the extended Kalman (EK) predictor are linear with respect to the disturbance and observation noises. The performance of the proposed scheme is compared with both the EK predictor and sampling importance resampling (SIR) particle predictor. Monte Carlo simulations have shown that the performances of the proposed scheme, EK predictor, and SIR particle predictor are all model-dependent, that is, one performs better than the others for a given example. Some examples, for which the proposed scheme performs better than the others do, are also given in the paper.