Cloning and sequencing of the chromosomal DNA and cDNA encoding the mitochondrial citrate synthase of Aspergillus niger WU-2223L

The complementary DNA (cDNA) and chromosomal DNA encoding the citrate synthase (EC 4.1.3.7) gene (cit1) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of A. niger genes. The 920-bp DNA fragment was amplified by polymerase chain reaction with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone was isolated and sequenced, within which an ORF of 1425 by encoding a protein of 475 as with a molecular weight of 52,153 Da was found. Its N-terminal region contains a typical mitochondrial-targeting motif. The predicted as sequence was 82, 68, and 65% homologous with the mitochondrial citrate synthases of Neurospora crassa, Saccharomyces cerevisiae, and pig, respectively, but it showed lower homology to bacterial citrate synthases. The full-length cDNA clone was used to screen a chromosomal library of A. niger WU-2223L, and a 7.5 kb-SalI fragment containing the corresponding chromosomal gene was isolated. Comparison of the chromosomal and cDNA sequences revealed that the cit1 gene is interrupted by six introns. In the chromosomal DNA, upstream of the coding region, a CT-rich region, but not the TATAAA or CAAT motifs, was found. Escherichia coli MOB150, a citrate synthase-deficient mutant showing a glutamate-requiring phenotype, was transformed with the plasmid pKAC-35S, which is the expression vector pKK223-3 containing the cDNA fragment encoding a putative mature protein of A. niger citrate synthase. The transformant harboring pKAC-35S showed citrate synthase activity and a glutamate-nonrequiring phenotype.     

Vesicular transport and the Golgi apparatus in yeast

Eukaryotic cells have developed a complex intracellular membrane system to divide the cell into various compartments where specific biochemical reactions are efficiently conducted locally. They also have developed systems to deliver appropriate materials to each specific compartment. Vesicular transport is a delivery system that also links most of the main organelles in the cell. The Golgi apparatus occupies the central position of the traffic between the endoplasmic reticulum and the endosome/vacuole/plasma membrane by maturating and sorting delivery of materials. Every important feature of vesicular transport has been identified by studying the Golgi apparatus, and the unicellular microorganism Saccharomyces cerevisiae has been an extremely excellent material for this study. Cycles of production and consumption of the transport vesicles by sorting the cargo, budding from the donor, tethering, docking and fusion to the target can now be explained to a large extent at the molecular level. The functional and structural aspects of the Golgi have also been well studied in the last decade.     

Hepatotoxicity of diisopropyl ester of malonic acid and chloromalonic acids, disinfection by-products of the fungicide isoprothiolane

The fungicide isoprothiolane (diisopropyl 1,3-dithiolane-2-ylidenemalonate) decomposes to the diisopropyl esters of malonic acid (DM), chloromalonic acid (DCM) and dichloromalonic acid (DDCM) upon aqueous chlorination. In this study, the cytotoxicity of these compounds was examined using rat hepatocytes cultured on Matrigel. DCM and DDCM caused hepatocellular death at concentrations > 0.5 mM, while DM had no effect on the cell viability even at the maximum concentration examined (4 mM). Significant lipid peroxidation, measured as 2-thiobarbituric acid reactive substances, was observed in both DCM- and DDCM-treated hepatocyte cultures, and was significantly enhanced by pretreatment with 0.1 mM bis(p-nitrophenyl)phosphate (BNPP), a carboxylesterase inhibitor. When both BNPP and SKF-525A, a cytochrome P450 inhibitor, were present in the medium, DCM-induced cytotoxicity and lipid peroxidation were significantly suppressed compared to cultures with BNPP-treatment alone. By contrast, the DDCM-induced cytotoxicity was not affected by the combined pretreatment of SKF-525A and BNPP. These results indicate that DCM is metabolically activated by cytochrome P450 in an ester form, while DDCM is activated by a mechanism other than one involving cytochrome P450. To further elucidate the cytochrome P450 isozyme involved in the metabolic activation of DCM, microsomal lipid peroxidation was studied in vitro using microsomes from rats treated with beta-naphthoflavone, musk xylene, phenobarbital, pyrazole, or dexamethasone. Among these preparations, the microsomes from dexamethasone-treated rats showed the most extensive lipid peroxidation in the presence of DCM, and the lipid peroxidation was enhanced by BNPP as observed in hepatocyte cultures. These findings suggest the possible involvement of cytochrome P450 3A in the metabolic activation of DCM.     

Numerical and experimental study on flame propagating mechanism of a fuel droplet array

The major goals of the researches in combustion science are to provide predictive and controlling capabilities to enhance combustion technology and fire safety. In other words, the practical motivations for combustion study and application research are, owed to the widespread dependence on combustion processes in modern societies. Furthermore, environmental concerns recently dominate needs of combustion research for the realization of low-emissive, efficient energy generation and utilization. The utilization of microgravity is an exceedingly useful tool for the scientific research for realization of physics and dynamics of combustion phenomena, such as spray combustion. Many experiments have been conducted in order to investigate combustion phenomena without natural convection. Now in addition, numerical simulation becomes useful method for further understanding of combustion phenomena. From the fundamental viewpoints, the interactions among fluid dynamics, scalar transport, thermodynamics and chemical kinetics that are characteristic of combustion phenomena have been investigated by above experimental and numerical methods. This paper reports about our efforts on microgravity combustion study. In order to describe the characteristic mode of droplet-to-droplet flame propagation, we conducted the numerical simulations and dropshaft experiments comparing with theoretical prediction models of flame propagation.     

Logic operation at 5 Gb/s of an output interface for single-flux-quantum systems

High-speed logic operation of an output interface circuit for a single-flux-quantum (SFQ) system was demonstrated at a data rate of 5 Gb/s. Using NEC's 2.5-kA/cm/sup 2/ Nb junction process, we designed, fabricated, and tested the interface circuit consisting of a 2-b SFQ demultiplexer and two Josephson latching drivers. We verified the proper operation of the demultiplexer. The interface can convert 5-Gb/s SFQ-pulse data into two-channel 2.5-Gb/s return-to-zero data with an amplitude of approximately 6 mV.     

Experimental evidence of crystal fragmentation from highly undercooled Ni<Subscript>99</Subscript>B<Subscript>1</Subscript> melts processed on an electrostatic levitator

Employing an electrostatic levitator (ESL) equipped with a CO₂ laser heating setup, we solidified Ni₉₉B₁ bulk crystals through containerless processing at high undercoolings and observed grain-refined microstructures. The electron backscatter diffraction pattern (EBSP) and analysis of the twin directions were accomplished, from which the primary growth traces with a cellular-like structure were revealed on a macro-millimeter scale. In comparison with the strong mechanical electromagnetic stirring in a sample processed on an electromagnetic levitator, the ESL provides a quite quiescent state for the melt, which enables identification of the primary growth traces after solidification. The present observation supplied experimental evidence that the refined microstructure in the Ni₉₉B₁ alloys at the high undercooling regime was due to fragmentation of the primary growth crystal, rather than dynamic nucleation.     

Effect of G-jitter on the thermocapillary convection experiment in ISS

Effects of g-jitter on the measurement of dynamic surface deformations (DSD) caused by the oscillatory thermocapillary convection in a liquid bridge have been studied experimentally. A vibration table driven by a piezoelectric actuator is constructed to generate g-jitters of realistic amplitude and frequency spectra. A microimaging displacement meter is used to measure DSD in the presence of g-jitter. It is shown that the natural DSD in the oscillatory state of thermocapillary convection is well separated in the frequency domain from that caused by harmonic and non-harmonic g-jitters. It is concluded that the DSD measurement in the future ISS experiment is feasible even in the presence of g-jitter there.     

Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli

The structure, stability, and unfolding-refolding kinetics of Escherichia coli-expressed recombinant goat alpha-lactalbumin were studied by circular dichroism spectroscopy, X-ray crystallography, and stopped-flow measurements, and the results were compared with those of the authentic protein prepared from goat milk. The electric properties of the two proteins were also studied by gel electrophoresis and ion-exchange chromatography. Although the overall structures of the authentic and recombinant proteins are the same, the extra methionine residue at the N terminus of the recombinant protein remarkably affects the native-state stability and the electric properties. The native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively charged than the authentic one. The recombinant protein unfolded 5.7 times faster than the authentic one, although there were no significant differences in the refolding rates of the two proteins. The destabilization of the recombinant protein can be fully interpreted in terms of the increased unfolding rate of the protein, indicating that the N-terminal region remains unorganized in the transition state of refolding, and hence is not involved in the folding initiation site of the protein. A comparison of the X-ray structures of recombinant alpha-lactalbumin determined here with that of the authentic protein shows that the structural differences between the proteins are confined to the N-terminal region. Theoretical considerations for the differences in the conformational and solvation free energies between the proteins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue in the unfolded state, and also due to less exposure of hydrophobic surface on unfolding. The results suggest that when the N-terminal region of a protein has a rigid structure, expression of the protein by E. coli, which adds the extra methionine residue, destabilizes the native state through a conformational entropy effect. It also shows that differences in the electrostatic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pKa value of the N-terminal amino group between the proteins, resulting in a greater negative net charge of the recombinant protein at neutral pH.     

Study on the introduction of hazard analysis and critical control point (HACCP) concept of the water quality management in water supply systems.

In the latest revision in 2004, the 3rd edition, the Water Safety Plans (WSP) was newly introduced into the World Health Organization (WHO) Guidelines for Drinking Water Quality. The Hazard analysis and critical control point (HACCP) is a basic concept that underlies the WSPs, and is also known as the product quality management method in the field of food and the medical manufacturing industries. In the amendments of the Drinking Water Quality Standards in Japan, water suppliers are required to reasonably achieve both safe water and efficient water quality management. Therefore, the HACCP concept is focused as an adequate management method covering a whole process of water supply systems, in a systematic way. The purpose of this study is to investigate a practical procedure in introducing the HACCP into water quality management in Japan. In comparison to conventional applications of the HACCP, unmanageable variations of raw water quality, continuous treatment and supply, and numerous standards of water quality items need to be considered. The HACCP system is expected to achieve a quick response to improvements in water quality, accountability towards consumers and a decrease in accidents.