Extensive precursor-product relationship between gangliosides formed from exogenous glucosylceramide in rat liver

Glucosylceramide, radiolabelled on the glucose residue, was administered to rats and the radioactive gangliosides formed at different time points were chemically characterized. They were identified as GM3, GM1, GD1a and GD1b, each one carrying only radioactive glucose. The time course of each individual ganglioside showed that the simpler gangliosides were formed earlier but were consumed earlier than the more complex ones, resulting in radioactivity patterns that were different at each time point. Only 30 h after injection did it resemble that of endogenous rat liver gangliosides. These results indicate that an extensive precursor-product relationship actually exists in the course of ganglioside biosynthesis.     

Inhibition of the soluble guanylate cyclase from rat lung by sulfated polyanions

Soluble guanylate cyclase was partially purified from rat lung homogenates, and shown to be inhibited by the following sulfated polyanions, with the I₅₀ in μg/ml in parentheses: Polyvinyl sulfate (0.33), 40,000-dalton dextran sulfate (0.45), polyanetholesulfonate (0.63) 500,000-dalton dextran sulfate (1.8), λ-carrageenan (2.9), τ-carrageenan (6.1), κ-carrageenan (48.0), heparin (68.0). There was a good correlation between inhibitory potency and sulfate content (as total sulfur). Inhibition by heparin and the carrageenans (but not the others) was potentiated by Mn²⁺, but not Ca²⁺ or Mg²⁺, when [Mn²⁺] exceeded [GTP]. Mn²⁺-potentiation could be blocked by high Na⁺. Heparin-agarose shows promise as an affinity matrix for guanylate cyclase.     

Anasterocerebroside A,a new glucosylceramide from the Patagonian starfish Anasterias minuta

Eight glucosylceramides (1-8) were isolated from the water-insoluble lipid fraction of a methylene chloride/methanol/water extract of the Patagonian starfish Anasterias minuta. One of the constituents was identified as a new glucosylceramide, anasterocerebroside A (1), while the known glucosylceramide 7 was isolated and characterized for the first time as a pure compound. The structures of 1 and 7 were established by spectroscopic and chemical methods.     

A new sulfated beta-galactan from clams with anti-HIV activity

A new polysaccharide composed of galactan sulfate with a beta-(1-->3)-glycosidic linkage has been isolated from the marine clam species Meretrix petechialis. The polysaccharide was homogeneous in its composition containing D-galactose. The glycosidic linkage was examined by 2D DQF-COSY and 2D NOESY spectroscopy. The coupling constant of anomeric proton was 7.8 Hz, suggesting a beta-galacto configuration. The downfield shift of H-2 of galactose residue demonstrated the presence of 2-O-sulfonate group. TQF-COSY confirmed that the C-6 position was substituted with a sulfonate group. The anti-HIV activity of the polysaccharides has been evaluated by the inhibition of syncytia formation. The fusion index and percentage fusion inhibition of sulfated galactan were 0.34 and 56% at 200 micrograms/mL.     

Phosphodiesterase activator from rat kidney cortex.

Incubation of homogenates of rat renal cortex at 4 degrees resulted in increased cAMP phosphodiesterase activity; the increase was much more rapid in hypotonic medium than in one of physiological tonicity. cAMP phosphodiesterase activity did not increase with incubation of supernatant fractions (48,000 x g, 20 min) prepared from isotonic homogenates. Extraction of the isotonic particulate fraction with hypotonic buffer released an activator which increased cAMP phosphodiesterase activity of the supernatant fraction. The kidney phosphodiesterase activator differed from a heat-stable, calcium-dependent protein activator of phosphodiesterase in that it was destroyed by heating (90 degrees for 10 min) and was not inhibited by EGTA. The phosphodiesterases of rat renal cortex were partially resolved by chromatography on DEAE-Bio-Gel, and a cAMP phosphodiesterase that is sensitive to the kidney activator was identified. This phosphodiesterase was separable from that affected by a calcium-dependent phosphodiesterase activator from bovine brain and from cGMP-stimulated cAMP phosphodiesterase. As determined by sucrose density gradient centrifugation, after incubation with the kidney activator, the activated form of phosphodiesterase had a lower sedimentation velocity than did the unactivated form.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

Sequence analysis of the sulfated rhamno-oligosaccharides derived from a sulfated rhamnan

Three sulfated rhamno-oligosaccharides, designated O1, O2 and O3, were obtained by mild acid hydrolysis of the sulfated rhamnan and purified by gel-permeation chromatography. On the basis of one- and two-dimensional nuclear magnetic resonance (1D, 2D NMR) spectroscopic analyses, the oligosaccharide O1 was characterized to be α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap. The fragmentation pattern of the homogeneous disaccharide in the product ion spectra was recognized by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation (ES-CID MS/MS). With the principles established, the sequences of the oligosaccharides O2 and O3 were deduced to be α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap, and α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap (2SO(4)), respectively. The investigation demonstrated that the sulfated rhamnan-derived oligosaccharides were novel sulfated oligosaccharides different from those of other polysaccharides-degraded from algae, and it could be possible to determine the sequence of the sulfated rhamno-oligosaccharides directly from the glycosidic cleavage fragmentation in the product ion spectra.     

Occurrence of sulfated 5alpha-cholanoates in rat bile

Bile acids in bile from male and female rats with cannulated bile ducts have been analyzed by repetitive scanning gas-liquid chromatography-mass spectrometry after initial fractionation of conjugate classes on diethylaminohydroxypropyl Sephadex LH-20. Sex differences were observed in the amounts and types of bile acids in the sulfate fraction. The proportion of total bile acids excreted as sulfates was higher in female (0.9-1.3%) than in male (0.1-0.2%) rats. Most of the sulfated bile acids had a 5alpha configuration, allochenodeoxycholic acid being the major compound in bile from female rats. This bile acid was also present in the nonsulfate fraction but could not be found in bile from male rats. The results indicate that gas-liquid chromatography-mass spectrometry has to be used to provide sufficient specificity in the bile acid analyses. Thus, compounds from the sulfate fraction having the retention times of cholic and chenodeoxycholic acid derivatives were found to be due to derivatives of the 3beta,5alpha-isomers of these bile acids.     

Formation of glucosylceramide and sterol glucoside by a UDP-glucose-dependent glucosylceramide synthase from cotton expressed in Pichia pastoris

In plants, glucosylceramide (GlcCer) biosynthesis is poorly understood. Previous investigations suggested that sterol glucoside (SG) acts as the actual glucose donor for the plant GlcCer synthase (GCS). We addressed this question by generating a Pichia pastoris double mutant devoid of GlcCer and SG. This mutant was used for heterologous expression of the plant GCS. The activity of the GCS resulted in the accumulation of GlcCer and, surprisingly, a small proportion of SG. The synthesis of GlcCer in the transformed double mutant shows that the GCS is SG-independent, while the detection of SG suggests that in addition to the sterol glucosyltransferase, also the GCS may contribute in planta to SG biosynthesis.     

Selection of lactic yeast producing glucosylceramide from cheese whey

From 2150 isolates from raw milk and milk products, yeast strains were surveyed to produce glucosylceramide from cheese whey. Most of the 54 strains that had accumulated a detectable amount of glucosylceramide were identified as Kluyveromyces lactis var. lactis. The cells of K. lactis var. lactis strain M-11 derived from domestic raw milk accumulated glucosylceramide 2.5-fold higher than K. lactis var. lactis NBRC 1267, the reference strain selected from the culture collections. Strain M-16 of K. lactis var. lactis derived from the same origin was found to synthesize a considerable amount of steryl glucoside in addition to glucosylceramide. Sequence analysis of ribosomal DNA intergenic spacer two regions revealed that strains M-11 and M-16 were diverged from a type strain of K. lactis var. lactis in the same species.