Purification of a cytosolic factor from rat liver that stimulates transcription in isolated nuclei and its action on purified RNA polymerase II-DNA system.

A cytosolic factor that stimulates transcription in isolated nuclei was purified approximately 4000-fold to near homogeneity from rat liver. The molecular weight of the factor was determined as 47 000 by SDS-polyacrylamide gel electrophoresis. The factor had no detectable deoxyribonuclease and protease activity but showed ribonuclease inhibitor activity. The factor could stimulate transcription in isolated nuclei by 50% at about 3.0 ng and the maximal stimulation was about 100%. When [gamma-S]ATP and [gamma-S]GTP were included in the reaction, the factor stimulated the synthesis of RNA which was able to bind to a mercury-Sepharose column and about 80% of the bound RNA was sensitive to a low concentration of alpha-amanitin. When heparin was added before initiation to preincubation mixture containing RNA polymerases II and DNA, a small but definite incorporation of [14C]UTP was observed. The factor alone had no stimulatory effect on the heparin-resistant incorporation of [14C]UTP but, in the presence of two rat liver nuclear fractions, phosphocellulose 0.5 and 1 M KCl step fractions, could stimulate the incorporation above the level with the combination of the two nuclear fractions. Antibody raised against the factor inhibited accurate transcription from the adenovirus 2 major late promoter in a nuclear lysate from Ehrlich ascites tumor cells, and the inhibition was neutralized by the factor.

Differential response of type I and type II cyclic AMP-dependent protein kinases in submandibular gland of isoproterenol-treated rats.

The cytosolic protein kinase isozymes were studied in relation to the secretion of saliva and the proliferative process induced by isoproterenol. At the same time as salivary secretion began, cytosolic protein kinase was activated and, in the absence of cyclic AMP, increased to a maximum level at 10 min after administration of the drug. The active state of the enzyme was maintained for 1-2 h. Chromatographic analysis of the cytosol enzyme revealed that in an activated state type II kinase easily dissociates into the catalytic and regulatory subunits while type I kinase hardly dissociates. Thus we conclude that type I kinase, at least in submandibular gland, is less dissociable in the presence of cyclic AMP than type II kinase but highly active in a ternary complex (R2-4 cAMP-C2). The presence of cyclic AMP-independent protein kinase, which co-chromatographed with protein kinase II, was demonstrated by use of a heat-stable inhibitor from bovine muscle, which totally inhibited the cyclic AMP-dependent enzymes, but stimulated the cyclic AMP-independent protein kinase. The level of protein kinase I decreased to its lowest value, about half that of the control value, at 10 h after isoproterenol injection and then increased slightly at 15 h, whereas type II kinase remained virtually unchanged, suggesting that the two protein kinase isozymes are independently regulated in the proliferating cells.

Development of a fully automated network system for long-term health-care monitoring at home.

Daily monitoring of health condition at home is very important not only as an effective scheme for early diagnosis and treatment of cardiovascular and other diseases, but also for prevention and control of such diseases. From this point of view, we have developed a prototype room for fully automated monitoring of various vital signs. From the results of preliminary experiments using this room, it was confirmed that (1) ECG and respiration during bathing, (2) excretion weight and blood pressure, and (3) respiration and cardiac beat during sleep could be monitored with reasonable accuracy by the sensor system installed in bathtub, toilet and bed, respectively.

Analysis of electron spin resonance spectra of alkyl spin labels in excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes.

The electron spin resonance (ESR) spectra of alkyl spin labels were observed in the excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes. The spectrum of 5-doxylstearic acid (5-NS) in the stratum corneum and order parameter obtained from the spectrum, indicated that the spin label was present in highly ordered lipid lamella. On the other hand, the spectrum of methyl ester of 5-NS (5-NMS) and its apparent rotational correlation time calculated from the spectrum, showed only a weakly immobilized component in the stratum corneum as well as in the whole excised skin. The ester spin label seemed to be scarcely present in the rigid lipid lamella, but mainly in the relatively fluid environment. On the other hand, cationic alkyl spin labels showed quite different spectra depending on their alkyl chain lengths. Long-chain 4-(N,N-dimethyl-N,-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-15) seemed to be present in the protein region of the stratum corneum as we recently reported, whereas hydrophilic quaternary ammonium spin label 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-1) seemed to be present in the bulk water of the skin, even in delipidized skin. These findings indicated that the different interaction and different localization of the alkyl spin labels depended on their electronic charge as well as their alkyl chain lengths.

Effects of n-alkyltrimethylammonium on skin permeation of benzoic acid through excised guinea pig dorsal skin.

Effects of cetyltrimethylammonium and two other n-alkyltrimethylammoniums on the permeation of benzoic acid through excised guinea pig dorsal skin were examined. Cetyltrimethylammonium markedly increased both the flux and permeability coefficient in the concentration range of 0.5 and 5 mm. However, 50mM cetyltrimethylammonium decreased them. The presence of 2 mm cetyltrimethylammonium, which induced a maximal enhancement effect, also increased the fluxes of 4-methyl, 4-ethyl and 4-n-propyl substituents of benzoic acid, but the enhancement effects were less. Analysis of the free energy of transfer of the methylene group of the substituents from the aqueous phase to skin suggested that cetyltrimethylammonium made the skin relatively more hydrophilic. Electron spin resonance spectra analysis by a long-chain quaternary alkylammonium analog, 4-(N,N-dimethyl-N-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-ox yl iodide (CAT-15), showed that the spin label was present in a nearly solid state in both excised skin and its stratum corneum. This finding suggested the high affinity of the long-chain alkylammoniums to proteins in the stratum corneum and the involvement of the interaction between the cationic surfactants and the proteins in their improvement of the hydrophilic property of the skin, as well as their marked enhancement effects on the skin permeation of relatively hydrophilic drugs.

[Annual daily intakes of Hg, PCB and arsenic from fish and shellfish and comparative survey of their residue levels in fish by body weight].

We have been surveying toxic substances in food and foodstuffs and carrying out a total diet study on the intakes of various substances since 1979 in cooperation with local public institutes in Japan. In this paper, we report the daily intakes of mercury, PCB and arsenic from foods, and the relation between the concentrations of these substance in fish and the fish body weight. The intakes of mercury and arsenic were 6.9-11.0 micrograms/ man/day and 120-230 micrograms/man/day, respectively. The intakes of these substances remained on a stable level from 1979 to 1994. On the other hand, the intake of PCB decreased from 3.1 micrograms/man/day in 1979 to 0.9 microgram/man/day in 1994. Most of the intakes of mercury, PCB and arsenic were derived from the diet group "fish and shellfish". The level of mercury in fish increased with increasing fish body weight. For PCB and arsenic, there was no correlation between these concentrations in fish and the fish body weight, except that mackerel and croaker show a higher concentration of PCB when they are small. Arsenic shows almost a constant level in each fish regardless of their body weight.

Chemical and mutagenic properties of alpha-phosphonooxynitrosamines.

The chemical and mutagenic properties of the products of solvolysis of alpha-acetoxynitrosamines in phosphate buffer were investigated. alpha-Acetoxynitrosamines decomposed in two ways: O-acyl fission yielded alpha-hydroxynitrosamines, which decomposed into aldehydes and alcohols, while O-alkyl fission gave a resonance hybrid of alpha-N-nitrosocarbonium and -iminium ions, which was trapped with phosphate and afforded alpha-phosphonooxynitrosamine. Formation of alpha-phosphonooxynitrosamines was dependent on the structure of alpha-acetoxynitrosamines; those with a secondary alpha-phosphonooxy group, including cyclic nitrosamines, were easily formed, while among those with a primary phosphonooxymethyl group, only those with an alkyl group containing a branched alpha-carbon as isopropyl, sec-butyl and tert-butyl were isolated. They were good substrates of alkaline phosphatase and showed a nuclear magnetic resonance spectrum due to the presence of a phosphorus atom. They were decomposed by acid catalysis, and the rate was dependent on the structure. They were directly mutagenic in bacterial tester strains, except for a compound with a tert-butyl group. The activity was similar or stronger in Salmonella typhimurium TA1535 and much weaker in Escherichia coli WP2 and WP2 hcr- than those of alpha-acetoxynitrosamines. Stability in neutral aqueous solution and the strong mutagenicity of alpha-phosphonooxynitrosamines suggested their possible involvement in metabolic activation as a precursor of alpha-hydroxynitrosamines, and also in the organotropic carcinogenicity of N-nitrosodialkylamines as a transport form.

Activation of N-nitrosodialkylamines by near-ultraviolet irradiation: formation of directly-acting mutagens and DNA-damaging products.

On near-ultraviolet (UVA) irradiation in a phosphate buffer, N-nitrosomorpholine (NMOR) and N-nitrosopyrrolidine (NPYR) were converted into directly-acting mutagens. The activated NPYR was fractionated, and the active product was isolated. The compound was shown to be identical to alpha-phosphonooxy NPYR on the basis of several properties: retention times in high-performance liquid chromatograms, mutagenic specificity and potency, ultraviolet spectrum, and inactivation by phosphatase treatment. Photoactivation was inhibited by superoxide dismutase, and therefore superoxide is implicated as playing a key role in mutagen formation. N-Nitrosoproline (NPRO) and eighteen other N-nitrosodialkylamines were irradiated with UVA in the presence of phi X174 RFI DNA. The DNA underwent single-strand breaks to give RFII DNA, indicating that N-nitrosodialkylamines in general have this property. DNA chain cleavage was inhibited both by superoxide dismutase and hydroxyl-radical scavengers. These results provide new information on the genotoxic mechanism of action of N-nitrosodialkylamines.