Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato.

A cDNA encoding solanidine glucosyltransferase (SGT) was isolated from potato. The cDNA was selected from a yeast expression library using a positive selection based on the higher toxicity of steroidal alkaloid aglycons relative to their associated glycosylated forms. The cDNA contained an open reading frame encoding a 56 kDa polypeptide with regions of similarity to previously characterized UDP-glucosyltransferases. The enzyme activity and reaction products of recombinant SGT in yeast were consistent with those observed for the endogenous enzyme from potato. SGT mRNA and protein accumulated in tubers in response to wounding. The time course for SGT mRNA accumulation paralleled that of 3-hydroxy-3-methylglutaryl-coenzymeA isoform 1 (hmg1) mRNA. Steady-state SGT mRNA levels also increased transiently upon wounding of leaves.

Hair methylmercury levels in U.S. women.

The scientific community has recently focused its concerns on possible developmental delays in infants exposed to methylmercury via maternal fish consumption. In this study, the authors reported levels of methylmercury in hair specimens that corresponded to 2820 monthly seafood consumption diaries recorded by U.S. women of childbearing age. In this study, the geometric mean hair methylmercury level for diarists who reported some seafood consumption was 0.36 ppm (one geometric standard deviation [GSD] range = 0.14-0.90 ppm); the corresponding value for diarists who reported no seafood consumption was 0.24 ppm (one GSD range = 0.09-0.62 ppm). Therefore, the mean hair methylmercury level associated with seafood consumption was 0.12 ppm (one GSD range = 0.05-0.32 ppm). The results of this study provide evidence that levels of methylmercury in the U.S. population are quite low. There is a significant contribution to hair methylmercury from sources other than seafood. It is not likely that maternal hair methylmercury levels in the range found in our study would be associated with adverse health effects in children.

Cloning and expression of transaldolase from potato.

We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.

The kinetics of intravenously administered methyl mercury in man.

We describe a compartmental pharmacokinetic model for methyl mercury and its metabolite mercuric mercury in humans. A tracer dose of 203Hg-labeled methyl mercury was administered iv to seven healthy young adult male volunteers. Blood samples were obtained periodically and urine and feces were collected throughout the 70 days of the study. The blood contained predominantly methyl mercury, while the excreta contained principally inorganic mercury. The behavior of both methyl mercury and inorganic mercury in the body was modeled with the simplest compartmental model which fit the data. This five-compartment model shows that inorganic mercury accumulates in the body and at longer times is the predominant form of mercury present. The biological half-life of methyl mercury in the body is 44 days and 1.6% of the body burden is lost each day by both metabolism and excretion. This rate of loss is 60% greater than that currently accepted (1.0% per day). Thus, the risk associated with dietary methyl mercury may have been overestimated.

Physiological model for the pharmacokinetics of methyl mercury in the growing rat.

We describe a physiological pharmacokinetic model for methyl mercury and its metabolite mercuric mercury in the growing rat. Demethylation appears to occur in both host tissues and gastrointestinal flora with elimination dominated by biliary secretion of inorganic mercury and by transport of methyl mercury into the gut lumen followed by substantial bacterial metabolism. Biliary transport of both organic and inorganic mercury is modeled in terms of the known secretion of glutathione from the hepatic pool. At 98 days following an oral tracer dose of 203Hg-labeled methyl mercury chloride, 65% of the administered dose had been recovered in the feces as inorganic mercury and 15% as organic mercury. Urinary excretion is a minor elimination route, accounting for less than 4% of the dose as methyl mercury and 1% of the dose as inorganic mercury. Irreversible incorporation of the mercurials into hair is a significant route of elimination. Ten percent of the administered dose was contained in the hair shed during the 98 days and over 12% of the dose (almost 90% of the body burden) remained in the hair at the end of that time period. Apparent ingestion of hair by the rats during grooming represents a novel form of toxin recirculation. Transport of both chemical species between blood and tissues is bidirectional and symmetric with relatively slow movement into and out of the brain. Transport mechanisms for both mercurial species are discussed in the context of capillary transport physiology and the blood-brain barrier to small molecules and proteins.

Partial amino acid sequence of potato solanidine UDP-glucose glucosyltransferase purified by new anion-exchange and size exclusion media.

Solanidine UDP-glucose glucosyltransferase (SGT) is involved in the biosynthesis of steroidal glycoalkaloids in potatoes. This enzyme is present at an extremely low level, is inherently unstable, and copurifies with the major storage protein patatin during isolation. We describe an improved method for isolating SGT from greening potato peel using two new chromatographic supports, Macro-Prep 50 Q anion-exchange and Superdex 75HR size exclusion media, under medium-pressure conditions at room temperature. The enzyme preparation was further resolved by SDS-PAGE and the proteins transferred to PVDF membrane (Immobilon-P). Two protein bands corresponding to active forms of SGT (36 and 37 kDa) were excised and cleaved with cyanogen bromide in trifluoroacetic acid. The resultant peptide mixtures were then separated by Tricine-SDS-PAGE and transferred to a PVDF membrane (Pro-Blott). The two major peptide bands observed in both digests (17 and 19 kDa) were sequenced. Identical N-terminal sequences were obtained from the 19-kDa peptides from both digests.

Determination of release limits: a general methodology.

Release limits of drug dosage forms are defined as the bounds on the potency at which an individual lot can be released for marketing which will ensure that it remains within registered limits throughout its shelf life. A statistically based method is described for calculating release limits for any type of dosage form and any parameter for which the rate of change with time is predictably uniform and linear. When the mean release assay result for a specific product lot is at or within the calculated release limit bounds, assurance is provided at the specified confidence level that the average assay results obtained at any subsequent time within the shelf life will remain within registered limits.

Percutaneous retinoid absorption and embryotoxicity.

A single application of 17 micrograms/kg or 8.7 mg/kg all-trans-[10,11-3H2]-retinoic acid dissolved in acetone to shaved dorsal hamster skin resulted in rapid absorption and dose-dependent rates of elimination. An equation describing a two-compartment open model with a very brief lag time and first-order uptake and elimination was used to describe the central plasma compartment kinetics. Unchanged all-trans-retinoic acid represented less than or equal to 4% of the total circulating radio-activity. Peak circulating concentrations of parent all-trans-retinoic acid were less than those observed after an equivalent oral dose, but prolonged absorption from the skin appears to contribute to high total bioavailability of topical retinoid. Topical administration to intact skin of up to three consecutive doses of 10.5 mg/kg/d all-trans-retinoic acid or a single 5 mg/kg dose of etretinate (Ro 10-9359) during a critical stage of embryogenesis in hamsters caused erythema and/or dose-dependent epidermal hyperplasia at the site of application, but failed to induce a significant teratogenic response. Topical application of 0.01-1.0 mg/kg arotinoid Ro 13-6298 resulted in dose-dependent mucocutaneous toxicity and an increase in the numbers of dead embryos and malformed offspring. The marked skin toxicity and attenuated concentrations in maternal blood, compared to the oral route, limit the amounts of retinoid that can reach the hamster embryo. It is thus more important to compare the retinoid systemic values (absorbed dose) than it is to compare the oral or topical (applied) dose, when interpreting the results of conventional teratogenicity bioassays. The data suggest that in the human it is skin toxicity that limits the amounts of retinoid that can be applied and subsequently reach the embryo. In the rodent, overt skin toxicity under continued dosing could increase the amounts of retinoid penetrating the skin and reaching the embryo.

Hypertrophy and hyperplasia of the rat pancreas produced by short-term dietary administration of soya-derived protein and soybean trypsin inhibitor.

Feeding soy protein concentrate to weanling rats over a one-week period produced a dose-related increase in pancreatic weight due to an increase in acinar cell size. Hyperplastic changes occur simultaneously, as evidenced by an increase in mitotic activity after two days on the test diet. Similar changes were also obtained by feeding soybean Kunitz trypsin inhibitor over the same time period. The results suggest that this approach may be useful as a model to investigate the effect of plant-derived material on the pancreas, in particular proliferative lesions.

Aluminium coffee percolators as a source of dietary aluminium.

The aluminium content of coffee brewed and stored in aluminium percolators was measured using atomic absorption spectrometry. Brewing in a new aluminium pot added 0.88 (immediately after brewing ) to 1.18 mg aluminium (after a further 12-hr storage in the pot and reheating ) to each cup of coffee. Percolators which had been used repeatedly were less susceptible to mobilization of aluminium by coffee, and brewing in these increased the aluminium content of each cup of coffee by 0.40 mg immediately after brewing and by 0.58 mg after storage for 12 hr in the pot and reheating . The aluminium content of the ground coffee beans used in this work was relatively high (51.8 ppm). To demonstrate that the bulk of the aluminium measured in the percolated coffee samples were dissolved aluminium and was not part of the aluminium associated with the ground coffee, the dialysable aluminium was measured in some samples of coffee percolated in a new aluminium pot. These data indicate that 61% of the aluminium in the percolated coffee was dialysable immediately after brewing . Samples that were stored in a new aluminium percolator and reheated to 96 degrees C contained 75% dialysable aluminium. Although the levels of aluminium in percolated coffee have been measured, the bioavailability of the aluminium ingested in this way has yet to be determined.

Physical characterization of erythromycin: anhydrate, monohydrate, and dihydrate crystalline solids.

Hot-stage microscopy, thermoanalytical methods, and X-ray powder diffraction were used to demonstrate that crystalline erythromycin dihydrate converts to the crystalline anhydrate via a noncrystalline intermediate. X-ray powder diffraction, IR spectral, thermogravimetric, and differential thermal analyses were used to characterize the monohydrate material. The flow interrupt technique, a procedure recently developed to deal with low surface area samples, was employed successfully in obtaining isotherms and specific surface areas for the monohydrate and anhydrate. The relative dissolution rates of the various hydrates were determined in an aqueous solution (0.01 M phosphate buffer, pH 7.5) at 37 degrees. The results showed a significant difference in the dissolution rate of the dihydrate compared to the monohydrate and anhydrate.