Uridine 5-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

No abstract

As part of a search for novel inhibitors of HIV-1 reverse transcriptase, the acetone extract of the giant African snail, Achatina fulica, was shown to be active. Fractionation of the extract yielded inophyllums A, B, C, and E and calophyllolide (1a, 2a, 3a, 3b, and 6), previously isolated from Calophyllum inophyllum Linn., a known source of nutrition for A. fulica. From a methanol/methylene chloride extract of C. inophyllum, the same natural products in considerably greater yield were isolated in addition to a novel enantiomer of soulattrolide (4), inophyllum P (2b), and two other novel compounds, inophyllums G-1 (7) and G-2 (8). The absolute stereochemistry of inophyllum A (1a) was determined to be 10(R), 11(S), 12(S) from a single-crystal X-ray analysis of its 4-bromobenzoate derivative, and the relative stereochemistries of the other inophyllums isolated from C. inophyllum were established by a comparison of their 1H NMR NOE values and coupling constants to those of inophyllum A (1a). Inophyllums B and P (2a and 2b) inhibited HIV reverse transcriptase with IC50 values of 38 and 130 nM, respectively, and both were active against HIV-1 in cell culture (IC50 of 1.4 and 1.6 microM). Closely related inophyllums A, C, D, and E, including calophyllic acids, were significantly less active or totally inactive, indicating certain structural requirements in the chromanol ring. Altogether, 11 compounds of the inophyllum class were isolated from C. inophyllum and are described together with the SAR of these novel anti-HIV compounds.

The sponge Batzella sp. from Jamaica contains four new alkaloids, batzelladines F−I (1-4), that induce the p56lck−CD4 dissociation. Batzelladines F−I were isolated using a bioassay-directed fractionation scheme and were identified by interpretation of spectroscopic data. Batzelladines F, G, and H + I were active in the p56lck−CD4 dissociation assay at micromolar concentrations.

Chromatographic methods were developed for the separation and characterization of acidic (sialylated) and neutral (asialo-complex and high-mannose) oligosaccharides released from glycoproteins with peptide Nglycosidase F, endo-P-N-acetylglucosaminidase F and endo-P-N-acetylglucosaminidase H using a carbohydrate analyzer (Dionex BioLC). All the carbohydrate separations were carried out on a polymeric pellicular anionexchange column HPIC-AS6/CarboPac PA-1 (Dionex) using only two eluants namely, 0.5 M NaOH and 3% acetic acid/NaOH pH 5.5, which were mixed with water to generate various gradients. Developed conditions for quantitative detection of carbohydrates with pulsed amperometry were necessary to obtain steady baselines at 0.1 -0.3 pA output with suitable sensitivity (< 5 pmol) in separations employing a variety of acidic and alkaline sodium acetate gradients.Oligosaccharides released from heat-denatured and trypsin-treated glycoproteins were purified initially from large-scale digestions (> 0.1 g) by extraction of peptide material into phenol/chloroform and finally by ionexchange chromatography of the aqueous phase. Oligosaccharides isolated from the peptide N-glycosidase digests of bovine fetuin, human transferrin and al-acid glycoprotein gave multiple peaks in each charge group in separations based on the charge content at pH 5.5. Alkaline sodium acetate gradients were developed to obtain oligosaccharide maps of the glycoproteins within 60 min, in which separated oligosaccharides eluted in the order of neutral, mono-, di-, tri-and tetra-sialylated species based on both charge, size and structure. Baseline separations were obtained with neutral oligosaccharide types but mixtures of high-mannose and complex types were poorly resolved. The high-mannose peaks were eliminated specifically from complex oligosaccharides by digesting with a-mannosidase. Treatment with P-galactosidase, P-N-acetylglucosaminidase and a-mannosidase resulted in a decrease of the oligosaccharide elution times corresponding to the number of sugar residues lost, the profile of changes was highly reproducible. In contrast, treatment with a-L-fucosidase, endo-P-N-acetylglucosaminidase F and endo-P-N-acetylglucosaminidase H resulted in an increase in their corresponding oligosaccharide retention times similar to the presence of an additional sugar residue.Conditions developed for separation of the reduced oligosaccharides and also a mixture of monosaccharide to oligosaccharide containing about 15 sugar residues within 30 min were useful in determining the effect of endoand exo-glycosidases on porcine thyroglobulin oligosaccharides. Changes in elution time of the oligosaccharides following specific glycosidase digestions combined with methylation analysis provided a rapid and sensitive tool for confirmation of the carbohydrate primary structures present in thyroglobulin.Knowledge of carbohydrate structures in addition to protein primary structure is a pre-requisite for studying structure/ function relationships in glycoproteins. Primary structu...

The development of cardiac hypertrophy was studied in adult female Wistar rats following daily subcutaneous injections of isoproterenol (ISO) (0.3 mg/kg body weight). A time course was established for the change in tissue mass, RNA and DNA content, as well as hydroxyproline content. Heart weight increased 44% after 8 days of treatment with a half time of 3.4 days. Ventricular RNA content was elevated 26% after 24 h of a single injection and reached a maximal level following 8 days of therapy. The half time for RNA accumulation was 2.0 days. The total content of hydroxyproline remained stable during the first 2 days of treatment but increased 46% after 4 days of therapy. Ventricular DNA content was unchanged during the early stage (1-4 days) of hypertrophic growth but increased to a new steady-state level 19% above the controls after 8 days of treatment. Intraventricular pressures and coronary flow measures were similar for control and experimental animals following 4 days of developed hypertrophy. However, dP/dt in the ISO-treated hearts was slightly but significantly (P less than 0.05) elevated. These data indicate that the adaptive response to ISO shows an early hypertrophic phase (1-4 days) characterized by a substantial increase in RNA content and cardiac mass in the absence of changes in DNA. However, prolonged stimulation (8-12 days) appears to represent a complex integration of both cellular hypertrophy and hyperplasia within the heart.

Low ascorbate concentrations in diabetes may be secondary to inadequate dietary vitamin C intake or may relate to the varied metabolic roles of the vitamin. To determine whether inadequate dietary intake is a factor we calculated daily vitamin C intakes using both a vitamin C questionnaire and a 4-day food diary in a group of 30 patients with Type 2 diabetes (mean age 68.8 +/- 6.9 yr, 17M/13F) and in 30 community controls (mean age 68.0 +/- 5.5 yr, 12M/18F)). Measures of plasma glucose, serum fructosamine, and plasma ascorbic and dehydroascorbic acid were obtained from 20 subjects in each group. There was no significant difference in daily vitamin C intake between the two groups using both methods: food diary, 61.4 +/- 28.3 (patients) vs 69.5 +/- 33.4 (controls) mg; questionnaire, 54.0 +/- 28.9 (patients) vs 65.0 +/- 30.9 (controls) mg. Vitamin C intake derived from both methods was significantly correlated (p < 0.001). Plasma ascorbate (30.4 +/- 19.1 mumol l-1) and dehydroascorbate (27.6 +/- 6.4 mumol l-1) levels were significantly lower in patients vs in controls (68.8 +/- 36.0 and 31.8 +/- 4.8 mumol l-1, respectively), p < 0.0001 and p < 0.01. Plasma ascorbate levels were significantly correlated with vitamin C intake derived from the food diary (p < 0.01) and questionnaire (p < 0.01) methods in the diabetic group only. Low ascorbate levels in diabetes appears to be a consequence of the disease itself and not due to inadequate dietary intake of vitamin C. A short vitamin C questionnaire is a convenient and reliable estimate of vitamin C intake.(ABSTRACT TRUNCATED AT 250 WORDS)

A MeOH/CH(2)Cl(2) extract of the bud covers of Artocarpus altilis collected in Micronesia showed activity in a cathepsin K inhibition assay. In addition to the three known flavonoids isolated from the bud covers of this species, two new compounds have been identified whose structures were determined on the basis of spectral data. These compounds include a dimeric dihydrochalcone, cycloaltilisin 6 (2), and a new prenylated flavone, cycloaltilisin 7 (3). Novel compounds 2 and 3 have IC(50) values of 98 and 840 nM, respectively, in cathepsin inhibition.