Metabolism of cerebrosides and sulfatides in subcellular fractions of developing rat brain.

Previous studies on myelinating rat brain indicated that microsomes, Golgi-enriched and cytosol fractions may process galactolipids destined for myelin. To extend these findings we labeled brain galactolipids in vivo and determined the specific radioactivity of cerebrosides and sulfatides in several subcellular fractions. 17-day-old rats were treated by intracranial injection with [14C]galactose 60 min prior to and [3H]galactose 15 min prior to killing. Subcellular fractions were prepared from brain stem, and concentrations of cerebrosides and sulfatides were determined, their radioactivity measured and the 3H/14C ratio compared. Our results showed that the heavier Golgi-enriched fraction (designated Fraction 2) is unique in its low galactolipid content and high specific radioactivities of cerebrosides and sulfatides. The low ratio of the specific activity of cerebroside to that of sulfatide in Fraction 2 compared to other fractions indicates that it may be the site of most rapid conversion of newly synthesized cerebrosides to sulfatides. The specific radioactivities of cerebrosides and sulfatides in cytosol are intermediate between those in Golgi-enriched Fraction 2 and microsomes and those in myelin, consistent with the role postulated for cytoplasmic elements in the transport of cerebrosides and sulfatides to myelin.

A change in the cerebrosides and sulfatides in a demyelinating nervous system. Development of the methodology and study of multiple sclerosis and Wallerian degeneration.

This report described a new method for the microanalysis of sphingolipids and its application for the characterization of cerebrosides and sulfatides in multiple sclerosis brain and rat sciatic nerves undergoing Wallerian degeneration. Tissue was extracted with isopropanol/hexane (20:78), and the total lipids obtained were subjected to benzoylation-desulfation. A portion of this was directly analyzed by silica-column high performance liquid chromatography for the determination of nonhydroxycerebroside, hydroxycerebroside, nonhydroxysulfatide, and hydroxysulfatide. Another portion was fractionated by thin-layer chromatography, and the spots corresponding to the sphingolipid derivatives were eluted. The material from each spot was analyzed by reverse phase high performance liquid chromatography for its homolog composition. With this new procedure the concentrations and homolog compositions of cerebrosides and sulfatides were measured in plaque, periplaque, and normal-appearing white matter from brains of multiple sclerosis patients and Wallerian degenerated rat sciatic nerves distal to the nerve transection. One piece of plaque studied contained only 1.86, 2.76, 0.60, and 0.45 nmol of nonhydroxycerebroside, hydroxycerebroside, nonhydroxysulfatide and hydroxysulfatide/mg of protein, respectively. These concentrations are less than 1% of those found in normal white matter. Periplaques were found to contain concentrations of these sphingolipids between those of plaque and normal white matter. The levels of these sphingolipids in degenerative nerves were 10-20% below normal the third day after the nerve was severed and about 70% below normal after 10 days. The rate of decrease lessened from ten days to 55 days. The homolog compositions of these sphingolipids in both multiple sclerosis brain and degenerating nerves were similar to those in the control. The implications of these findings and the advantages of this new analytical method are discussed.

Flavonoid glycosides and saponins from Astragalus shikokianus.

A new flavonol glycoside, kaempferol 3-O-alpha-L-rhamnopyranosyl -(1-->6)-[alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-galactopyranosyl-7-O-a lpha-L-rhamnopyranoside, named astrasikokioside I, was isolated from aerial part of Astragalus shikokianus, together with two flavonol glycosides, kaempferol 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-7-O-alpha-L- rhamnopyranoside, robinin, and three triterpenoid glycosides, soyasaponin I, sophoraflavoside II and robinioside E.

Steroidal glycosides from Capsicum annuum.

Four new steroidal glycosides, named capsicosides A-D together with proto-degalactotigonin, were isolated from the roots and seeds of Capsicum annuum var. conoides and Capsicum annuum var. fasciculatum. On the basis of detailed chemical and spectroscopic evidence, the structures of capsicosides A-D were shown to be furostanol glycosides corresponding to gitogenin and tigogenin oligoglycosides. Capsicoside A was regarded as the same compound as the previously described capsicoside, whose structure now needs to be revised according to the data presented in this report.

Glycosides of 19-formylthevetiogenin and 5 alpha-thevetiogenin from Thevetia neriifolia.

C-nor-D-homo-homologues of cannogenin and uzarigenin glycosides were isolated along with known cardenolide glycosides from the frozen fresh leaves of Thevetia neriifolia. A bisdesmosidic tetraoside of 3 beta,14,21-trihydroxy-5 beta,14 beta-pregnan-20-one was also obtained from the polar fraction and the structure established.

Flavonol sinapoyl glycosides from leaves of Thevetia peruviana.

From the leaves of Thevetia peruviana four new flavonol glycosides, kaempferol 3-glucosyl(1 --> 4) [6"'-sinapoylglucosyl] (1 --> 2) galactoside and 3-[2""-sinapoylglucosyl](1 --> 4)[6"'-sinapoylglucosyl](1 --> 2)galactoside and kaempferol and quercetin 3-[6"'-sinapoylglucosyl](1 --> 2)galactoside were isolated, together with the known compounds, kaempferol and quercetin 3-glucosyl(1 --> 2)galactoside. The glycosides were characterized from FAB mass, 1H and 13C NMR and UV spectral data.

Ergostane glycosides from Petunia hybrida.

Six new ergostane glycosides, designated as petunioside A, petunioside B, 24-epipetunioside B, petunioside C, 24-epipetunioside C and petunioside D, were isolated from the methanolic extract of the fresh aerial parts of Petunia hybrida. Their structures were determined by means of spectroscopic analysis and single crystal X-ray analysis.

Cardenolides from Erysimum cheiranthoides.

Three new cardiac glycosides were isolated along with two known cardenolides from the seeds of Erysimum cheiranthoides. The new ones were characterized by spectral methods as 16 beta-hydroxystrophanthidin (strophadogenin) 3-O-beta-glucopyranosyl-(1-->4)-beta-boiviopyranoside, strophadogenin 3-O-alpha-rhamnopyranosyl-(1-->4)-beta-digitoxopyranoside and strophanthidin 3-O-alpha-rhamnopyranosyl-(1-->4)-beta-digitoxopyranoside, named cheiranthosides I, II and III, respectively.

Topostatin, a novel inhibitor of Topoisomerases I and II produced by Thermomonospora alba strain No. 1520. II. Physico-chemical properties and structure elucidation.

Topostatin is a new topoisomerase inhibitor isolated from the culture filtrate of Thermononospora alba strain No. 1520. The inhibitor inhibits topoisomerases I and II, and it has neither ability to stabilize the cleavable complex nor ability to intercalate into DNA strands. The molecular formula of topostatin was determined as C36H58N4O11S based on the FAB-MS analyses, and the structure was elucidated to be a novel 14-membered ring containing peptide and terpenoid by various NMR spectroscopies.

A streptothricin-like antibiotic mixture, A-269A (and A-269A').

A streptothricin-like antibiotic, A-269A (referred to as A-269A hereafter) was isolated from the culture broth of Streptomyces sp., strain No. A-269. In this paper, the characterization of the producer, and the production, isolation, physico-chemical properties and biological properties of A-269A are reported. The structure of the antibiotic was also examined by 1H NMR, 13C NMR and fast atom bombardment mass spectra studies. From the spectroscopic data, A-269A was assumed to be a mixture of antibiotic LL-BL 136 and an isomeric compound in which the carbamoyl group is substituted on C-12' hydroxyl instead of C-10 hydroxyl.

A new 68Ge/68Ga generator system using an organic polymer containing N-methylglucamine groups as adsorbent for 68Ge.

A macroporous styrene-divinylbenzene copolymer containing N-methylglucamine groups was selected for a new 68Ge/68Ga generator system. This resin packed into a column effectively adsorbed the parent nuclide 68Ge. The daughter 68Ga was eluted from the resin with a solution of a low-affinity gallium chelating ligand such as citric or phosphoric acid. The 68Ge leakage was less than 0.0004% of the 68Ge adsorbed on the resin. By simple mixing of transferrin and desferoxamine conjugated HSA and IgG with the eluate from the column, 68Ga-labeling was completed in high yield.

Characterization of alkylgalactolipids from calf brain by high performance liquid chromatography.

Minor nonpolar galactolipids were isolated from the total lipids of calf brain stem by column chromatography and were separated by preparative thin-layer chromatography into four groups. The material recovered from the bottom band of the thin-layer chromatography consisted of monogalactosyl diglyceride and its 1-0-alkyl isomer, alkylgalactolipid, present in a molar ratio of 11:9. After perbenzoylation, they were separated by preparative thin-layer chromatography and characterized. The fatty acid compositions of these lipids were similar to each other and to those of the ester-linked fatty acids of cerebroside esters. The major alkyl group of alkylgalactolipid was palmityl, and the other, minor componenents were oleyl, myristyl, and stearyl ethers. Perbenzoylated derivatives of these lipids were further separated by reverse-phase high performance liquid chromatography. The chromatograms from these two lipids were similar; however, most of the peaks were still mixtures of homologs containing different fatty acids or an alkyl group.

Levels and syntheses of cerebrosides and sulfatides in subcellular fractions of jimpy mutants.

During the period of brain development, the levels of nonhydroxy- and hydroxycerebrosides in the cytosol from brains of jimpy mutants were determined by high-performance liquid chromatography and compared to those in the rest of the particulates from the same brains as well as those in the littermate controls. The concentrations of cerebrosides in jimpy brain preparations were much lower than in controls at all ages. In another experiment, [U-14C]glucose was injected intraperitoneally into jimpy mutants and their littermate controls. The amounts of radioactivity incorporated into cerebrosides and sulfatides in brain cytosol, the microsome-rich fraction, and the rest of the heavier particles were determined. Although the total radioactivity incorporated into these lipids was much lower in jimpy, the specific activities were 2-3 times the control value in all subcellular fractions.

A method for the automated screening for hepatitis B surface antigen.

A reverse passive hemagglutination (RPHA-G) technique for the automated screening of hepatitis B surface antigen (HBsAg) on the Groupamatic 360 (Kontron International, Zürich, Switzerland) has been developed. It is more sensitive than manual RPHA (RPHA-M) and nearly as sensitive as radioimmunoassay (RIA); it detects 6 ng/ml of HBsAg, compared to 3 ng/ml by RIA and 25 ng/ml by RPHA-M. Although it is slightly inferior to the other two methods in respect of specificity, the incidence of false-positive reactions was only 0.34%. The test can also be performed manually without the Groupamatic.

Inhibitory activities of (-)-epigallocatechin-3-O-gallate against topoisomerases I and II.

The substitution of gallic acid at the 3 position of (-)-epigallocatechin-3-O-gallate (EGCG) increased the inhibition against topoisomerase I from calf thymus gland and topoisomerase II from human placenta, and the substitution of a hydroxyl group at the 3' position increased the inhibition against the topoisomerase I. These results suggested that the 3 and 3' positions of the EGCG molecule play important roles in the process of inhibition of topoisomerases I and II. EGCG showed strong inhibition against topoisomerases I from wheat germ, calf thymus gland and Vero cells, and showed weak or no inhibition against topoisomerases I from carcinoma cells such as A549, HeLa and COLO 201 cells. EGCG differentially inhibited the topoisomerases I from different sources.

Isoaurostatin, a novel topoisomerase inhibitor produced by Thermomonospora alba.

A novel inhibitor of topoisomerase I designated as isoaurostatin (1) was isolated from the culture filtrate of Thermomonospora alba strain No. 1520. The structure of 1 was determined to be 6,4'-dihydroxyisoaurone on the basis of spectroscopic (NMR and MS) methods. Compound 1 inhibited the relaxation activity of calf thymus topoisomerase I in a noncompetitive manner and did not inhibit the relaxation and decatenation of human placenta topoisomerase II. Compound 1 is an inhibitor belonging to cleavable complex-nonforming type without DNA intercalation.