Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

Modulation of cerebral acetylcholine metabolism by the dorsal diencephalic conduction system in the rat

We investigated the effects of interruption of the impulse flow in the habenulopeduncular pathways by local infusion of tetrodotoxin on the acetylcholine and choline content in selected dopamine rich regions in the forebrain and midbrain in rats. The tetrodotoxin infusion caused a marked increase in acetylcholine content in the medial frontal cortex, striatum and ventral tegmental area+interpeduncular nucleus, but not in the limbic area or the substantia nigra, whereas choline content was reduced only in both the striatum and ventral tegmental area+interpeduncular nucleus. There was an increase in 3,4-dihydroxyphenylacetic acid content in the striatum after the manipulation. These findings suggest that the dorsal diencephalic conduction system may be involved in the integration of the activity of cholinergic neurons in the forebrain and midbrain regions and striatal dopanine neurons may play a role in the modulation of cholinergic neurons.     

Increased expression in vivo and in vitro of foreign genes directed by A-type inclusion body hybrid promoters in recombinant vaccinia viruses.

We constructed A-type inclusion body (ATI) hybrid promoters, that is, late ATI promoters followed by tandemly repeated early regions of the promoter for the 7.5-kDa protein (the 7.5-kDa promoter). The repetition of the whole early promoter sequence of the 7.5-kDa gene, including the upstream consensus sequence and initiation region, efficiently increased the early expression of the bacterial chloramphenicol acetyltransferase gene in recombinant vaccinia virus. Recombinant vaccinia virus could express influenza virus hemagglutinin via the hybrid promoter more efficiently, induced higher levels of neutralizing antibody and cytotoxic T lymphocytes, and consequently protected mice more efficiently against challenge with influenza virus than did recombinant vaccinia virus containing the widely used 7.5-kDa promoter.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).     

Immobilization ofβ-fructofuranosidase fromAureobasidium on DEAE-cellulose

Summary -Fructofuranosidase, which produces fructo-oligosaccharides (1-kestose and nystose) from sucrose, was purified fromAureobasidium and immobilized on DEAE-cellulose at especially high efficiency (95%). The enzymatic profiles of the immobilized enzyme were almost identical to those of the native form except that the stability was slightly improved. The immobilized enzyme was stable during long-term continuous reaction for up to 360 h.     

Embryonic Development and Postnatal Changes in Free d-Aspartate and d-Serine in the Human Prefrontal Cortex

Abstract: We have analyzed free chiral amino acids (aspartate and serine) in the human frontal cortex at different ontogenic stages (from 14 weeks of gestation to 101 years of age) by HPLC with fluorometric detection after derivatization with N-tert -butyl-oxycarbonyl- l -cysteine and o -phthaldialdehyde. Exceptionally high levels of free d -aspartate and d -serine were demonstrated in the fetal cortex at gestational week 14. The ratios of d -aspartate and of d -serine to the total corresponding amino acids were also high, at 0.63 and 0.27, respectively. The concentration of d -aspartate dramatically decreased to a trace level by gestational week 41 and then remained very low during all postnatal stages. In contrast, the frontal tip contained persistently high levels of d -serine throughout embryonic and postnatal life, whereas the d -amino acid content in adolescents and aged individuals was about half of that in the fetuses. Because d -aspartate and d -serine are known to have selective actions at the NMDA-type excitatory amino acid receptor, the present data suggest that these d -amino acids might play a pivotal role in cerebral development and functions that are related to the NMDA receptor.     

Two flavonol glycosides from Vancouveria hexandra.

In addition to two known glycosides, ikarisoside F and epimedin A, two new glycosides of a flavonol with a gamma, gamma-dimethylallyl group were isolated from the underground and the aerial parts of Vancouveria hexandra. The structures were determined to be des-O-methylanhydroicaritin 3,7-diglucoside and anhydroicaritin 3-glucosyl (1----3)rhamnoside-7-glucoside by means of spectral analysis.     

Flavonol glycoside production in callus cultures of Epimedium diphyllum.

Callus cultures of Epimedium diphyllum produced a large amount of epimedoside A in addition to a small amount of diphylloside B, ikarisoside C, epimedoside E, diglycosides of des-O-methylanhydroicaritin (8-gamma, gamma-dimethylallylkaempfero). Icariin, epimedins A-C, which are glycosides of anhydroicaritin, were also produced in the callus cultures. Contents of the flavonol glycosides in callus tissue were higher than those of mother plants, but the composition of each flavonol glycoside mixture in the callus cultures was different from that of the original plants. The time-course experiments showed that an inverse relationship existed between cell growth and flavonol glycoside production. Effects of hormonal factors on cell growth and flavonol glycoside production indicated that 2,4-dichlorophenoxyacetic acid was needed for the production of flavonol glycosides.     

Purification and properties of extracellularβ-fructofuranosidases fromAureobasidium sp. ATCC 20524

Summary Two extracellular -fructofuranosidases (E-1 andE-2) fromAureobasidium sp. ATCC 20524, producing 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, were purified to homogeneity. Molecular weights of the enzymes were estimated to be about 304000 (E-1) and 315000 (E-2) Da by gel filtration. The enzymes contained 33% (w/w) (E-1) and 27% (w/w) (E-2) carbohydrate. TheK _m values for sucrose ofE-1 andE-2 andE-2 were 0.34 and 0.28 M, respectively. were 0.34 and 0.28 M, respectively. The enzymatic profiles of these enzymes were almost identical to intracellular enzymesP-1 andP-2 except for the differences in carbohydrate content andK _m values ofE-2 andP-2.     

Y-chromatin positive cells in the smear preparations of the gonad from an XX male

Summary An ambisexual male infant showed a normal female karyotype by the leukocyteculture technique in two different occasions. Cultured fibroblasts from the right gonad had also a 46,XX complement. They were Y-chromatin negative. To our surprise, Y-chromatin was observed in 96% of the nuclei in smears from the uncultured gonad. It is emphasized that the examination of the uncultured gonad by the quinacrine technique is mandatory in the study of XX males.