Substrate specificity and some other enzymatic properties of dihydroceramide desaturase (ceramide synthase) in fetal rat skin

Dihydroceramide desaturase, which catalyzes the introduction of a double bond at the 4,5-position of the sphingosine base in a dihydroceramide, was assayed in vitro using radiolabeled D-erythro-C18-dihydroceramide (N-stearoyl sphinganine) and homogenates of fetal rat skin, and some enzymatic properties, including substrate specificity, were determined. The ceramide structure, as the enzymatic product, was confirmed by (i) oxidation of the product with 2,3-dicyano-5,6-dichlorobenzoquinone, which revealed the conversion to 3-ketoceramide (3,3'-didehydroceramide), indicating that a double bond was introduced at the adjacent to the C-3 hydroxyl residue of sphinganine, and (ii) mass spectrometry of a long chain base released from the enzymatic product, which revealed a spectrum identical to that of authentic sphingenine. A short chain dihydroceramide, which was radiolabeled at sphinganine through a newly established method, having a C2- or C6-fatty acid was not desaturated by the skin enzyme, whereas that having a C10-, C14-, or C18-acid was desaturated, maximal reactivity being observed for the C14-dihydroceramide. Other enzymatic properties were confirmed: NAD(H) or NADP(H) and a detergent were required for elevation of the activity; the optimum pH was approximately 6.7; and metal cations were not essential, but Zn2+, Cu2+, and Fe2+ were rather inhibitory. These properties of rat skin desaturase were partly similar to those of rat liver microsomes, as reported recently, however, their substrate specificities were different.     

Formation of a complex containing ATP, Mg2+, and spermine. Structural evidence and biological significance

The conformation of ATP in the presence of Mg2+ and/or spermine was studied by 31P and 1H NMR, to clarify how polyamines interact with ATP. Spermine predominantly interacted with the beta- and gamma-phosphates of ATP in the presence of Mg2+. A conformational change of the beta- and gamma-phosphate of ATP with spermine could not be observed in the absence of Mg2+ by 31P NMR. It was found by 1H NMR that the conformation of adenosine moiety of ATP was not influenced significantly by spermine. The binding of Mg2+ to ATP was slightly inhibited by spermine and vice versa. The results indicate that the binding sites of Mg2+ and spermine on ATP only partially overlap. The PotA protein, an ATP-dependent enzyme, was used as a model system to study the biological role of the ATP-Mg2+-spermine complex. The ATPase activity of PotA was greatly enhanced by spermine. Double reciprocal plots at several concentrations of spermine as an activator indicate that spermine interacts with ATP, but not with PotA. The activity of protein kinase A was also stimulated about 2-fold by spermine. The results suggest that a ternary complex of ATP-Mg2+-spermine may play an important role in some ATP-dependent reactions in vivo and in the physiological effects of endogenous polyamines.     

Genetic analysis of hydatidiform moles utilizing the oligonucleotide-DNA typing of the HLA-DRB gene

The genetic origin of hydatidiform moles was analysed utilizing HLA-DNA typing. Using HLA-DR type-specific oligonucleotide probes, the DRB types of seven moles were determined and compared with the parental DRB types to determine the paternal and/or maternal origin of the moles. In four cases, the molar tissues showed single DRB types of paternal origin, although in one, the molar DRB type was also possessed by the mother. These four moles were, therefore, considered to be androgenetic in origin. Chromosomal karyotyping was carried out for three of these cases and confirmed the DR-DNA typing results. Two moles demonstrated a DRB-type triplet, which strongly suggested triploidy. Although one mole showed a heterozygous DRB type, karyotyping indicated triploidy (69, XXX) and suggested that this mole was caused by dispermy-fertilization, in which both of the sperms had the same DRB type. Although the majority (about 80%) of partial hydatidiform moles have been reported to be triploid as a result of dispermy, four of the moles analysed in this study (cases 1, 2, 3 and 4), diagnosed as partial macroscopically and/or histopathologically, were found to be androgenetic in origin using karyotyping and DR-DNA typing. Therefore, HLA-DR DNA typing, combined in some cases with karyotyping, provides an accurate method for diagnosing androgenesis and triploidy in complete and partial hydatidiform moles.     

Malignant transformation by overproduction of translation initiation factor eIF4G

N4G3, a cell line that overexpresses translation initiation factor eIF4G, one of the components of eIF4F, was made by stable transfection of the human eIF4G cDNA into NIH3T3 cells. The cells expressed 80-100 times greater levels of eIF4G mRNA than did NIH3T3 cells. N4G3 cells formed transformed foci on a monolayer of cells, showed anchorage-independent growth, and formed tumors in nude mice. These results indicate that overexpression of eIF4G caused malignant transformation of NIH3T3 cells. It is also known that overexpression of eIF4E, another component of eIF4F, causes transformation of NIH3T3 cells. However, there was no difference in the amount of eIF4E protein between N4G3 and NIH3T3 cells, indicating that cell transformation does not involve a change in eIF4E levels. The results may be due to an effect of eIF4G on translational control of protein synthesis directed by mRNAs having long 5'-untranslated region.     

Hepatitis G virus in the general population and in patients on hemodialysis

To determine the routes of transmission of hepatitis G virus (HGV) and the relationship between HGV and hepatitis C virus (HCV) infections, we tested for HGV RNA by polymerase chain reaction and antibody to HCV (anti-HCV) in 494 hemodialysis patients, 638 inhabitants of two HCV endemic areas, and in 400 blood donors in Japan. HGV RNA was detected in 6.9% of hemodialysis patients, in 1.4% of inhabitants, and in 0.8% of donors, and anti-HCV was detected in 39.3%, 12.4%, and 1.8%, respectively. Of HGV RNA-positive hemodialysis patients, and HGV RNA-positive inhabitants, 64.7% and 11.1%, respectively, had been given blood transfusions. The prevalences of HGV RNA and anti-HCV significantly increased with the duration of hemodialysis. Of all HGV RNA positives, 74.4% were coinfected with HCV and subjects with HGV RNA alone had normal liver function. In conclusion, HGV is transmitted by blood transfusion and within the hemodialysis unit itself. HGV does not seem to injure hepatocytes.     

Flame retardant mechanism of polyamide 6-clay nanocomposites

The thermal and flammability properties of polyamide 6/clay (2 and 5% by mass fraction) nanocomposites were measured to determine their flame retardant (FR) performance. The gasification process of the nanocomposite samples at an external radiant flux of 50 kW/m² in a nitrogen atmosphere was observed, and the residues collected at various sample mass losses were analyzed by thermogravimetric analysis, transmission electron microscopy, and X-ray diffraction to determine the content of the residue and to understand the FR mechanism of the nanocomposites. The analysis of the floccules of blackened residues shows that up to 80% by mass of the residues consists of clay particles and the remainder is thermally stable organic components with possible graphitic structure. Furthermore, clay particles are stacked in the carbonaceous floccule residues and the d-spacing of the clay platelets is in the range of 1.3-1.4 nm as compared to the well exfoliated original sample. The accumulation of the initially well-dispersed clay particles in the sample on the burning/gasifying sample surface are due to two possible mechanisms. One is recession of the polymer resin from the surface by pyrolysis with the de-wetted clay particles left behind. Another mechanism is the transportation of clay particles pushed by numerous rising bubbles of degradation products and the associated convection flow in the melt from the interior of the sample toward the sample surface. Numerous rising bubbles may have another effect on the transport of clay particles. Bursting of the bubbles at the sample surface pushes the accumulated clay particles outward from the bursting area and forms the island-like floccules instead of forming a continuous net-like structure of a clay filled protective layer. Therefore, both PA6/clay nanocomposite samples did not produce sufficient amounts of protective floccules to cover the entire sample surface and vigorous bubbling was observed over the sample surface which was not covered by the protective floccules.     

Fault diagnosis of a logical circuit by use of a pseudorandom signal and a neural network

This paper describes a new method of pseudorandom testing of a digital circuit by use of a correlation method and a neural network. The authors have recently proposed a new method of fault diagnosis in a logical circuit by applying a pseudorandom M-sequence to the circuit under test, calculating the cross-correlation function between the input and the output, and comparing the cross-correlation functions with the references. This method, called the M-sequence correlation (MSEC) method, is further extended by using a neural network in order not only to detect the existence of faults, but also to find the place or location of the faults. The authors investigated the effects of using parts of the fault patterns to train the neural network to be able to detect faults. It is shown that more than 95% of faults can be detected even when only 60% of the possible training data are used.     

Thermal properties and characteristics of thin layers of nickel dithiolene complexes

Thermal properties of near infra–red–absorbing nickel dithiolene complexes have been examined by thermal gravimetric and differential scanning calorimetry analysis. The nickel complexes with relatively low melting points gave a DRAW disc with better recording characteristics, when they were used as inhibitors of the photofading of thin layers of cyanine dyes. Thin layers of the nickel complexes on polymethylmethacrylate or glass were formed by spin coating and by the radio–frequency reactive ion plating technique. Their reflectance, absorption spectra and optical recording characteristics have been recorded.     

Mass transfer correlation of NH3–H2O bubble absorption

The objectives of this paper are to study the effect of key parameters on absorption performance and to develop an experimental correlation of mass transfer coefficient for ammonia–water bubble absorption. The orifice diameter, liquid concentration and vapor velocity are considered as the key parameters. This study successfully visualized the bubble behavior and measured the volumetric diameter of bubbles during the bubble absorption process. The bubble absorption is grouped into two processes, bubble growth (process I) and bubble disappearance (process II), respectively. The following conclusions were drawn from the present study. A new experimental correlation for the volumetric bubble diameter was proposed with ±15% error band, which could be applied to calculate the mass transfer coefficient. The mass transfer coefficient increased with a decrease of the liquid concentration. In process II, the mass transfer coefficient increased with an increase of the Galileo number. Finally, experimental correlations of mass transfer coefficient were developed for processes I and II with ±18% error bands.     

Flammability of polystyrene layered silicate (clay) nanocomposites: Carbonaceous char formation

Polymer layered‐silicate (clay) nanocomposites have not only the unique advantage of reduced flammability, but also improved mechanical properties. This is a key advantage over many flame retardants, which reduce flammability but also reduce the mechanical properties of the polymer. In our efforts to further understand the mechanism of flame retardancy with polymer‐clay nanocomposites, we investigated the effect of the clay, the loading level and polymer melt viscosity on the flammability of polystyrene‐clay nanocomposites. The nanoscale dispersion of the clay in the polymer was analysed by wide‐angle X‐ray diffraction (XRD) and transmission electron microscopy (TEM). Cone calorimetry and gasification studies were used to evaluate the flammability of these nanocomposites. There were major reductions in peak heat release rates (HRRs), and increased carbonaceous char formation, for these nanocomposites. It was determined that while the viscosity of the PS nanocomposite played a role in lowering the peak HRR, the clay loading level had the largest effect on peak HRR. Finally, it was found that clay catalysed carbonaceous char formation, and the reinforcement of the char by the clay was responsible for the lowered flammability of these nanocomposites. Published in 2002 by John Wiley & Sons, Ltd.