Identification of 3-hydroxy-3-methylglutaric acid (HMG) as a hypoglycemic principle of Spanish moss (Tillandsia usneoides).

Bioactivity-directed fractionation, using brine shrimp lethality and murine hypoglycemia, of an ethanol extract prepared from Tillandsia usneoides, led to the isolation of four apparently bioactive compounds from the water-soluble fraction. The compounds were identified as citric acid, succinic acid, 3-hydroxy-3-methylglutaric acid (HMG), and 3,6,3',5'-tetramethoxy-5,7,4'-trihydroxyflavone-7-O-beta-D-g lucoside. The brine shrimp lethality of the acids was simply due to acidity; however, HMG elicited significant hypoglycemic responses in fasting normal mice. Ethyl and methyl esters of citric acid were prepared and tested in the murine hypoglycemic assay. Five of the predominant sugars were identified by tlc. Free thymidine was also isolated. Further evaluation of HMG and other potential inhibitors of HMG CoA lyase, in the treatment of symptoms of diabetes mellitus, is suggested.

Effect of tolbutamide and glyburide on pyruvate kinase flux in isolated rat hepatocytes.

The effects of two representative sulfonylureas, tolbutamide and glyburide, on pyruvate kinase (PK) flux were examined in fasted rat hepatocytes. PK flux was estimated by trapping 14C from NaH14CO3 in a 2 mM lactate pool, accounting for any incomplete trapping by parallel incubations with L-[1-14C]alanine. Glyburide (20 microM) and tolbutamide (1 mM) decreased glucose formation by 34.9% and 54.8%, respectively, from 2 mM lactate. This decrease in glucose formation was associated with a proportional decrease in pyruvate carboxylase (PCOX) flux (32.7% and 50.5%, respectively). Under these conditions, no net change in PK flux was observed. When hepatocytes were preincubated with lactate and/or sulfonylurea addition for 30 min prior to radiolabeling with NaH14CO3, the metabolic state of the cells changed markedly. Glyburide produced a 34.6% decrease in glucose formation and a 31.3% decrease in PCOX flux, but no change in PK flux. In contrast, tolbutamide decreased glucose formation by 12.5% and increased PK flux by 53.2%, but no change in PCOX flux was observed. Such an increase in PK flux may be linked to tolbutamide-mediated increases in fructose-1,6-bisphosphate (F16P) via fructose-2,6-bisphosphate (F26P). These findings demonstrate that tolbutamide and glyburide decrease hepatic glucose production through various alterations in carbohydrate metabolism, depending upon the metabolic state of the cell. In addition, F26P may play a larger role in the hypoglycemic mechanism of action of tolbutamide than glyburide, since pyruvate carboxylase accounted for most of the decrease in glucose formation observed with glyburide and because preincubation with tolbutamide resulted in an activation of PK.

Identification of enterotoxigenic Escherichia coli isolates with enzyme-labeled synthetic oligonucleotide probes.

Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E. coli from 100 clinical isolates. There was very good agreement between the three methods.

Enzyme-linked synthetic oligonucleotide probes: non-radioactive detection of enterotoxigenic Escherichia coli in faecal specimens.

Synthetic oligonucleotides, complementary to unique sequences in the heat stable enterotoxin gene of Escherichia coli specific for humans, were prepared with a 30-atom spacer arm and a 3' terminal sulfhydryl group which was coupled to bromoacetyl-derivatized alkaline phosphatase. The resulting direct enzyme-linked oligonucleotide probes, containing one enzyme molecule per oligonucleotide, successfully diagnosed enterotoxigenic Escherichia coli in clinical specimens by using a modified colony hybridization method and a colorimetric assay. The procedure is rapid, simple and reliable with a sensitivity equivalent to that using 5'-terminally labelled [32p]-oligonucleotide probes. The results indicate that the enzyme-labelled oligonucleotide probes should be applicable to the routine diagnosis of enterotoxigenic Escherichia coli and possess the potential for the detection of other microbial pathogens.

Growth rate of the bacterium Sphaerotilus natans in lead-containing medium and the effect of calcium ions.

Growth rate of bacteria Sphaerotilus natans in lead-containing medium (1 X 10(-4) M Pb2+) and bioaccumulation of lead in the bacterial cells were investigated together with the effect of calcium ions on these processes. A rapid increase in biomass was observed in the presence of calcium and lead whereas calcium alone had no visible effect on the bacterial growth. The increase in biomass in the presence of lead and calcium was accompanied by a pronounced drop in lead content in the bacterial cells. This suggests that calcium ions protect the bacterial cells against lead poisoning.

A phytochemical screening procedure for sweet ent-kaurene glycosides in the genus Stevia.

Altogether, 110 species of the genus Stevia, comprising both herbarium and fresh leaf samples, were screened for the presence of sweet ent-kaurene glycosides, using a combination of tlc and hplc, followed by gc/ms. Stevioside and rebaudiosides A and C were detected in a Stevia rebaudiana herbarium specimen collected in Paraguay in 1919, and stevioside was observed as a constituent of a Stevia phlebophylla herbarium specimen collected in Mexico in 1889. Steviol glycosides were not detected in any of the other 108 Stevia species studied. The phytochemical results obtained in this study are correlated with those of preliminary organoleptic tests on the sweetness of these Stevia samples, and the chemotaxonomic implications of the present findings are discussed.

Effects of Eleutherococcus senticosus extracts on hexobarbital metabolism in vivo and in vitro.

Eleutherococcus senticosus (Siberian Ginseng) is widely exported from China as a health food. Pharmacologically it has been classified as an adaptogen and enzyme induction has been proposed as its mechanism of action. To evaluate this hypothesis E. senticosus was administered to mice on an acute (40-320 mg/kg i.p., X 1 day) or chronic (80-320 mg/kg i.p., X 4-5 days) basis. Sleep latency and duration, in response to hexobarbital sodium (100 mg/kg i.p.), were determined either 1 h (acute and chronic) or 24 h (chronic) following the last E. senticosus injection. E. senticosus produced a sedative effect which decreased the sleep latency (47%) and increased sleep duration (45-228%) following acute administration. A similar effect was seen following chronic administration (125-202% increase in sleep duration). E. senticosus was also shown to produce an inhibition (66%) of hexobarbital metabolism, in vitro, as compared to controls. These data support enzyme inhibition rather than enzyme induction as a mechanism for the actions of Siberian Ginseng.

Cytotoxic effects of Eleutherococcus senticosus aqueous extracts in combination with N6-(delta 2-isopentenyl)-adenosine and 1-beta-D-arabinofuranosylcytosine against L1210 leukemia cells.

The use of the aqueous extracts of Eleutherococcus senticosus in combination with either cytarabine or N6-(delta 2-isopentenyl)-adenosine gave additive antiproliferative effects against L1210 murine leukemia. The ED50 for E. senticosus root extracts against L1210 cells was approximately 75 micrograms/mL. E. senticosus appears to be potentially useful for reducing the concentration of conventional antimetabolites used for their antiproliferative effects on tumor cells.

A class of ompA mutants of Escherichia coli K12 affected in the interaction of ompA protein and the core region of lipopolysaccharide.

A group of ompA mutants of Escherichia coli K12 are described which were sensitive to bacteriophage K3 in a background wild-type for lipopolysaccharide (LPS). With mutant LPS in vivo (lacking some core sugar residues), however, the ompA mutations gave resistance to K3. Outer membrane levels of OmpA protein were normal or near-normal when the mutations resided in either wild-type or mutant LPS backgrounds. Strains in which the mutations occurred in a wild-type LPS background adsorbed K3 phage at the same initial rate and to the same extent as a wild-type strain, but the efficiency of plaquing of the adsorbed K3 was reduced to 25-50% of wild-type levels. Under conditions where a wild-type strain irreversibly adsorbed over 90% of available phage K3 within 3 min, double mutants (ompA mutant, LPS mutant) left 90% of the phage viable after 1 h. The 10% of inactivated phage did not form plaques.

The effect of acetylsalicylic acid/salicylic acid mixtures on acetylcholinesterase activity in vitro.

An attempt was made to inhibit acetylcholinesterase activity in homogenates of rat brain and in 2% solutions of hemolyzed human or rat erythrocytes, with concentrations of acetylsalicylic acid/salicylic acid (ASA/SA) equivalent to those evident in human plasma 0--2 h after ingestion of 0.65 g of ASA. None of the 5 different ASA/SA mixtures used produced inhibition in any of the enzyme preparations while the reference compound, eserine, inhibited all. These results suggest that ingestion of ASA, at least in recommended doses, should not significantly influence acetylcholinesterase activity in vivo.

Influence of 1-methyl-3-keto-4-phenylquinuclidinium bromide on rat tissue monoamine levels in vivo.

The effects of 1-methyl-3-keto-4-phenylquinuclidinium bromide (MA540) on tissue monoamine levels in male rats have been compared to those of guanethidine. Neither compound influenced norepinephrine (NE) or 5-hydroxytryptamine (5-HT) levels in brain or epinephrine (E) levels in adrenal medulla. MA540 is about twice as potent as guanethidine as a heart NE depleting agent at both 8 and 24 h after drug administration. The durations of action of the compounds are similar. Unlike guanethidine, MA540 does not deplete NE in small intestine, which suggests that the drug may not elicit guanethidine-like intestinal side effects.