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Dna2 is a multifunctional enzyme in yeast that possesses endonuclease activity well suited to remove RNA-DNA primers of Okazaki fragments, raising the question of whether endonuclease activity is essential for in vivo Dna2 function. Systematic site-directed mutations of amino acid residues in Saccharomyces cerevisiae DNA2 conserved in the central region of many eukaryotic DNA2 homologs allowed us to identify mutant dna2 alleles that were divided into three groups based on the viability of the mutant cells: (i) viable; (ii) inviable only when expression was repressed; (iii) inviable. Biochemical analyses of recombinant mutant Dna2 proteins isolated from the latter two groups revealed that they possessed normal ATPase/helicase activity, but were impaired in their endonuclease activity. Cells expressing mutant Dna2 enzymes partially impaired in endonuclease activity were viable, but were unable to grow when expression of their mutant Dna2 enzymes was further reduced. Their growth was restored when the mutant Dna2 proteins decreased in nuclease activity were induced to overexpress. In contrast, mutant Dna2 proteins lacking endonuclease activity did not allow cells to grow under any conditions tested. These in vivo and in vitro results demonstrate that the endonuclease activity of Dna2 is essential for Okazaki fragment processing.

The cDNA encoding the 471-amino acid rat 7-dehydrocholesterol reductase (DHCR), an enzyme that has been implicated in both cholesterol biosynthesis and developmental abnormalities (e.g. Smith-Lemli-Opitz syndrome) in mammals, has been cloned and sequenced, and the primary structure of the enzyme has been deduced. The DHCR gene was mapped to chromosome 8q2.1 by fluorescence in situ hybridization. Rat DHCR, calculated molecular mass of 54.15-kDa polypeptide, shares a close amino acid identity with mouse and human DHCRs (96 and 87%, respectively) as compared with its other related proteins (e.g. fungal sterol Delta14-reductase) and exhibits high hydrophobicity (>68%) with 9 transmembrane domains. Five putative sterol-sensing domains were predicted to be localized in transmembrane domains 4-8, which are highly homologous to those found in 3-hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein cleavage-activating protein, and patched protein. The polypeptide encoded by DHCR cDNA was expressed in yeast as a 55.45-kDa myc-tagged fusion protein, which was recognized with anti-myc monoclonal antibody 9E10 and shown to possess full DHCR activity with respect to dependence on NADPH and sensitivity to DHCR inhibitors. Northern blot analysis indicates that the highest expression of DHCR mRNA was detected in liver, followed by kidney and brain. In rat brains, the highest level of mRNA encoding DHCR was detected in the midbrain, followed by the spinal cord and medulla. Feeding rats 5% cholestyramine plus 0.1% lovastatin in chow resulted in both approximately a 3-fold induction of DHCR mRNA and a 5-fold increase of the enzymic activity in the liver. When rats were fed 0.1% (w/w) AY-9944 (in chow) for 14-days, a complete inhibition of DHCR activity and a significant reduction in serum total cholesterol level were observed. However, the level of hepatic DHCR mRNA fell only slightly, suggesting that AY-9944 may act more rapidly at the protein level than at the level of transcription of the DHCR gene under these conditions.

Purpose: The purpose of this study was to evaluate the usefulness of dynamic spiral CT, including multidimensional reformation, in the detection and localization of islet cell tumors of the pancreas.Material and Methods: Seven patients with histopathologically proven functioning islet cell tumors of the pancreas were studied with 2-phase contrast-enhanced spiral CT. Scanning of the arterial phase and late phase was started 30 s and 180 s, respectively, after injection of 100 ml of contrast medium at a rate of 3 mls.Results: Axial images in the arterial phase depicted the lesions in 5 patients, but in the late phase in only one patient. Multiplanar reformatted images of the arterial phase depicted the lesions in all 7 patients. Maximal intensity projection images demonstrated all lesions with information of their relationship to the vascular structure.Conclusion: Dynamic spiral CT with scanning during the arterial phase and retrospective multidimensional reformation is useful for preoperative detection and localization of small islet cell tumors of the pancreas.

To gain further insights into the biological functions of Dna2, previously known as a cellular replicative helicase in Saccharomyces cerevisiae, we examined biochemical properties of the recombinant Dna2 protein purified to homogeneity. Besides the single-stranded (ss) DNA-dependent ATPase activity as reported previously, we were able to demonstrate that ssDNA-specific endonuclease activity is intrinsically associated with Dna2. Moreover, Dna2 was capable of degrading duplex DNA in an ATP-dependent fashion. ATP and dATP, the only nucleotides hydrolyzed by Dna2, served to stimulate Dna2 to utilize duplex DNA, indicating their hydrolysis is required. Dna2 was able to unwind short duplex only under the condition where the endonuclease activity was minimized. This finding implies that Dna2 unwinds only partially the 3'-end of duplex DNA and generates a stretch of ssDNA of limited length, which is subsequently cleaved by the ssDNA-specific endonuclease activity. A point mutation at the conserved ATP-binding site of Dna2 inactivated concurrently ssDNA-dependent ATPase, ATP-dependent nuclease, and helicase activities, indicating that they all reside in Dna2 itself. By virtue of its nucleolytic activities, the Dna2 protein may function in the maintenance of chromosomal integrity, such as repair or other related process, rather than in propagation of cellular replication forks.

Background:To compare half-Fourier acquisition single-shot turbo spin-echo (HASTE) magnetic resonance cholangiopancreatography (MRCP) with two-dimensional turbo spin-echo (2D TSE) MRCP for imaging pancreatobiliary diseases. Methods:Twenty-seven patients with biliary or pancreatic disease underwent MRCP on a 1.0-T scanner with a body phased-array coil. A T2-weighted HASTE sequence (18 s) and a T2-weighted 2D TSE sequence (45 s) were used during a breath-hold by the patient. The source images and maximum intensity projection images of both sequences were reviewed independently by two radiologists. Results:Motion artifacts were more severely pronounced with 2D TSE sequences than with HASTE sequences (p< 0.001). All obstructions and their sites were accurately identified with both sequences. Filling defects (calculi) in bile ducts were identified in all 22 segments (100%) with HASTE-MRCP, whereas calculi in 19 of 22 segments (86%) were identified with 2D TSE-MRCP (p= 0.25). Three missed sites on 2D TSE-MRCP were intrahepatic bile ducts. Conclusions:HASTE-MRCP is superior to 2D TSE-MRCP in terms of detecting motion artifacts and visualization of the pancreatic ducts. HASTE-MRCP is comparable to 2D TSE-MRCP for visualization of the biliary ducts and their obstruction and is superior to 2D TSE-MRCP for identification of calculi in intrahepatic bile ducts.

A new evaporative cooling system for small four-stroke engines has been developed. Performance tests of a four-stoke single cylinder engine equipped with this evaporative cooling system have been carried out in the laboratory. Air removal rates from the closed coolant loop during the starting stage of the engine have been monitored for various engine operating conditions. In addition, data of the brake horsepower, the specific fuel consumption, heat loss to the coolant, cylinder-liner wall temperature and the wall heat flux have been obtained and were compared to those of the identical engine equipped with a conventional liquid cooling system. At a fixed air fuel ratio and under the MBT condition, the brake horsepower of the engine for the evaporative cooling system is enhanced compared to that for the liquid cooling system. The heat loss through the cylinder liner is decreased when the evaporative cooling system is adopted. The test result indicates several benefits of the evaporative cooling system such as faster warm up, better fuel economy and greater engine durability.

The optimization of MOSFET performance with Hf-based high-k and TiN metal gate is discussed. MOSFET performance and mobility of metal and poly gate on both high-k and SiO2 are compared. Optimization of the thickness and composition of Hf-silicate is discussed in terms of charge trapping and boron diffusion. The effect of metal gate thickness is also investigated. Appropriate dry and wet etch processes are found to effectively eliminate the metal foot and high-k undercut. Impressive Ion- Ioff characteristics have been achieved by applying an optimized gate-first CMOS flow to the high-k/metal stack. Ion = 1100µA/µm and 540µA/µm was attained for N and PMOSFETs with Vdd = 1.2V and Ioff = 100nA/µm.