Challenges in interpreting the evidence for genetic predictors of ototoxicity.

There has been recent controversy regarding predictors of cisplatin-induced ototoxicity in children, as highlighted in a previous issue of this journal. We have reviewed the two articles that purport to show an association between TPMT and COMT variants and ototoxicity, as well as the related patent applications dating back to 2006. We summarize statistical issues not fully addressed by the authors that appear to have confounded the results of their studies.

Antifungal and molluscicidal saponins from Serjania salzmanniana.

An investigation of Serjania salzmanniana for biologically active substances has led to the isolation of two novel saponins, salzmannianoside A (3-O-[[beta-D- glucopyranosyl-(1-->4)]-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L- arabinopyranosyl] gypsogenin) [3] and salzmannianoside B (3-O-[[beta-D-glucopyranosyl-(1-->4)]-[alpha-L- arabinopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)] -alpha-L-arabinopyranosyl] hederagenin) (4). Two known saponins, pulsatilla saponin D (3-O-[[beta-D- glucopyranosyl-(1-->4)]-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L- arabinopyranosyl] hederagenin) (1) and 3-O-[[beta-D-glucopyranosyl-(1-->4)]-[alpha-L-rhamnopyranosyl-(1-->2)]-a lpha-L- arabinopyranosyl] oleanolic acid (2) were also isolated from this plant. The structures of 3 and 4 were elucidated by FABMS and 2D NMR techniques. All these four saponins were mollusicidal, causing 70-100% mortality at 10 ppm against Biomphalaria alexandrina, a vector of Schistosoma mansoni in the Nile Valley. The saponins also showed antifungal activity against Cryptococcus neoformans and Candida albicans at minimal inhibitory concentrations of 8 and 16 micrograms/mL, respectively.

Inhibition of the calcium paradox in isolated rat hearts by high perfusate sucrose concentrations.

If a colloid osmotic (oncotic) pressure gradient develops across the myocyte membrane during the calcium paradox, adding an oncotic agent to the perfusate should be inhibitory. After 10-min perfusion with Ca(2+)-free Krebs-Henseleit (KH) buffer under constant flow at 34 degrees C, myoglobin release was measured from Langendorff hearts reperfused with Ca(2+)-containing KH buffer. When the Ca(2+)-free medium contained 200 mM sucrose, myoglobin release was reduced to 5% of that observed in the absence of sucrose, a change that was not seen when 200 mosM NaCl, choline chloride, LiCl, or glycerol was added. Replacement of 75 mM NaCl in the perfusate with 150 mM sucrose resulted in myoglobin release values that were 4% of the control. Plots of myoglobin release against sucrose concentration under these hypertonic and isotonic conditions yielded similar though separate curves. Sucrose also inhibited increases in wet weight-to-dry weight ratio and decreases in ATP and phosphocreatine contents. These results support the hypothesis that an oncotic pressure gradient arises during the calcium paradox at the moment of increased membrane permeability and plays a major role in its development.

Isolation of echinocystic acid-3-O-sulfate, a new triterpene, from Tetrapleura tetraptera, and evaluation of the mutagenic potential of molluscicidal extracts and isolates.

A known triterpene glycoside, 3-O-[beta-D-glucopyranosyl-(1"-6')-2'-acetamido-2'-deoxy-beta-D-gluco pyranosyl]olean-12-en-28-oic acid [3], and new sulfated triterpene, echinocystic acid-3-O-sodium sulfate [4], have been isolated from the stem bark of Tetrapleura tetraptera. Compound 3 was 100% lethal to Biomphalaria glabrata at 20 ppm, while 4 was not molluscicidal at the same concentration. In a forward mutation assay utilizing Salmonella typhimurium strain TM677, T. tetraptera stem bark extracts were found to be mutagenic in the absence of a metabolic activating system (S-9). An MeOH extract of the fruit exhibited weak mutagenic activity only in the presence of S-9. The stem bark isolates, which included aridanin [1], 3-O-(2'-acetamido-2'-deoxy-beta-D-glucopyranosyl)echinocystic acid [2], and compounds 3 and 4, were not mutagenic either with or without metabolic activation.

Gluconeogenesis in the liver of tumor rats.

Altered gluconeogenesis is frequently observed in cancerous hosts. To define its derangements in the liver, we studied glucose and glycogen production in the perfused livers of tumor-bearing rats using 13C NMR spectroscopy. Nine Fischer 344 rats were inoculated with mammary adenocarcinoma. After 5 weeks, the livers were removed and perfused with Krebs buffer containing 8 mM L-[3-13C]alanine, and 13C NMR spectroscopy was performed. Nine pair-fed rats were studied as controls. The peak heights of glucose and glycogen in the 13C NMR spectra of the perfused livers and final perfusates of the two groups of rats were compared. We found comparable amounts of C1-labeled glucose and glycogen in the two groups, but C2- to C5-labeled and C6-labeled glucose and glycogen, as well as total 13C-labeled glucose and glycogen, appeared in smaller quantities in the tumor rats than in the pair-fed rats. These findings suggest that appreciable amounts of unlabeled glycerol were utilized by both groups, but less so by the tumor rats than the pair-fed rats. In addition, there was decreased production of oxaloacetate through pyruvate dehydrogenase and the Krebs cycle in the livers of the tumor rats, where the overall metabolism of alanine into glucose and glycogen was also reduced.

Alkylphosphonates as unique compounds in the metabolism of the schistosomal vector Biomphalaria glabrata.

Examination of intact freshly laid egg masses of the schistosomal snail vector Biomphalaria glabrata by both 31P nuclear magnetic resonance spectroscopy and colorimetric phosphorus analysis indicated that 98% of the phosphorus in the egg masses was present in the form of alkylphosphonic acids. At least 87% of the total phosphorus originally present was comprised of 2-aminoethylphosphonic acid. During 11 days of embryonic development, alkylphosphonate-phosphorus decreased from 98% to 50% of the total phosphorus, indicating a possible role for phosphonate-phosphorus in the embryonic nutrition of these snails. Considering the relative uniqueness of the alkylphosphonates in nature, the very high concentration of these compounds in freshly laid eggs, and the reduction in their amount during embryonic development, the anabolic and catabolic pathways of alkylphosphonates may serve as sites for highly specific analogues in the control of these schistosomal vectors.

Metabolism of 2-aminoethylphosphonic acid during embryonic development of the schistosomal vector Biomphalaria glabrata.

1. Egg masses of the Planorbid snail Biomphalaria glabrata contain 2-aminoethylphosphonic acid (AEP) in three different chemical environments, as determined by 31P nuclear magnetic resonance spectroscopy, giving signals at 20.9, 21.0 and 23.4 delta. 2. The signal at 21.0 delta decreased in intensity during embryonic development, whereas the other two did not change significantly. 3. The following relationship is suggested: Extraembryonic AEP--------Intraembryonic AEP--------Phosphates. 4. pH Titration behavior of macromolecularly-bound AEP and synthetic derivatives of AEP was examined and indicates that AEP is found in the egg masses linked to other molecules in the following ways: (a) R2-NH-CH2CH2-P(O)(OH)(OR1), (b) R3-NH-CH2-P(O)(OR2)(OR1), (c) NH2-CH2CH2P(O)(OH)(OR1).

2-aminoethylphosphonic Acid metabolism during embryonic development of the planorbid snail helisoma.

In freshly laid egg masses of Helisoma sp., more than 95 percent of the phosphorus is found in alkylphosphonic acids, as determined by phosphorus-31 nuclear magnetic resonance spectroscopy. These compounds are metabolized during embryonic development, as shown by differential acid hydrolysis and experiments with phosphorus-33-labeled phosphoric acid. Further, nuclear magnetic resonance spectroscopy indicates phosphonic acid involvement in related snail families, including the schistosomal vector Biomphalaria glabrata.

ATP binding to human hemoglobin in the presence and absence of magnesium ions investigated with 31P NMR spectroscopy and ultrafiltration.

The addition of 3 mM hemoglobin to 1-10 mM ATP solutions at pH 6.75 resulted in a linear relationship between the change in chemical shift (deltadelta) of the gamma-phosphate of ATP and the percent ATP bound to hemoglobin. The data points obtained with oxyhemoglobin and deoxyhemoglobin fell on the same straight line. In the presence of 3 mM Mg2+, the delta delta decreased curvilinearly as the percent ATP bound was raised. In this case, the percent ATP bound to deoxyhemoglobin was greater than to oxyhemoglobin at the same delta delta value, indicating that the extra ATP binding occurs through groups other than phosphate.

Organic phosphate binding to hemoglobin in intact human erythrocytes determined by 31P nuclear magnetic resonance spectroscopy.

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy was used to estimate the percent of 2,3-diphosphoglycerate and ATP bound to hemoglobin in intact human erythrocytes at 37 degrees C. Binding was assessed by comparing the chemical shifts (delta) of 2,3-diphosphoglycerate and of ATP observed in intact cells with the delta values of these organic phosphates determined in model solutions closely simulating intracellular conditions, in which percent binding was directly evaluated by membrane ultrafiltration. The results showed that the percent of bound 2,3-diphosphoglycerate in intact cells varied with pH, the state of oxygenation, and 2,3-diphosphoglycerate concentration. The values ranged from 33% in cells incubated with glucose in air at an intracellular pH of 7.2 to 100% in cells incubated with inosine in N2 at a pH of 6.75. At the same 2,3-diphosphoglycerate concentration, a greater percentage of the compound appeared to be bound in erythrocytes than in the closely simulated model system. ATP was not significantly bound to hemoglobin under any condition examined, but appeared to be strongly complexed to Mg2+ inside the erythrocyte. The binding percentages for both 2,3-diphosphoglycerate and ATP in intact cells estimated by 31P NMR spectroscopy were lower than those calculated by others from individual association constants determined for the binding of different ligands to hemoglobin.

2-Aminoethylphosphonic acid metabolism in the rat.

Time course studies of the incorporation of radioactive 2-aminoethylphosphonic acid (AEP) into the tissues of rats demonstrated that maximum incorporation into the liver lipids occurred within 12 to 30 hr after injection, compared to 2 to 3 hr for the incorporation of phosphorylethanolamine. Little incorporation of AEP was observed in the other tissues investigated (heart, lung, spleen, adipose, kidney). The AEP was incorporated to the greatest extent into 1,2-diacylglyceryl-aminoethylphosphonate (diacylglyceryl-AEP), the phosphonate analogue of phosphatidylethanolamine, with some incorporation into the lyso derivative. Diacylglycerol-AEP apparently was not further metabolized by the rat; no methylation of diacylglyceryl-AEP to phosphonolecithin was observed. Subcellular fractionation was performed on the livers of rats who received (3)H-AEP 12,30,36, and 48 hr prior to sacrifice. The greatest amount of radioactivity was recovered in the soluble fractions. Lipid extraction was performed on the subcellular fractions, and most of the radioactivity present in the lipids was found in the microsomal fraction, with the next highest recovery in the mitochondrial and nuclear fractions

Interactions between hemoglobin and organic phosphates investigated with 31P nuclear magnetic resonance spectroscopy and ultrafiltration.

1. The chemical shifts (delta) of the phosphates of 2,3-diphosphoglycerate and adenosine triphosphate (ATP) were determined by phosphorus nuclear magnetic resonance (31P NMR) spectroscopy and were found to be displaced downfield following the addition of hemoglobin (3 mM) to a solution of either diphosphoglycerate (5 mM) or ATP (1 mM). 2. The binding of these compounds to hemoglobin was also determined by membrane ultrafiltration. A direct relationship was observed between the change in chemical shift ((delta delta) of the 2-P and 3-P of diphosphoglycerate and the percent diphosphoglycerate bound, when the latter was varied by altering pH, oxygenation state, or total diphosphoglycerate concentration. 3. In comparable studies with ATP binding, a linear relationship between the delta delta values of the gamma-, beta-, and alpha-P of ATP and the percent of ATP bound was not observed when the data from all of the experiments were plotted. NMR signals were not detectible in deoxyhemoglobin solutions containing 1 mM ATP but were seen in solutions containing 3.8 mM ATP. 4. The results indicate that 31P NMR spectroscopy is a promising tool for investigating organic phosphate interactions with hemoglobin.

Properties of a membrane-bound cardiolipin synthetase from Lactobacillus plantarum.

Cardiolipin (CL) synthetase of Lactobacillus plantarum 17-5 catalyzed the stoichiometric conversion of 2 mol of phosphatidylglycerol to 1 mol of CL. The enzyme activity was linear with time for 30 min at 37 C and with protein concentration between 20 and 200 mug of protein per ml. The enzyme was membrane associated, had a pH optimum of 5.1 in phosphate buffer, and was not stimulated by Mg2+, and the activity was not affected by the addition of ethylenediaminetetraacetic acid, cytidine diphosphate diglyceride, or cytidine triphosphate. The reaction was inhibited about 95% by Triton X-100 (0.5% final concentration) and by CL, the end product of the reaction. The activity of this enzyme was studied as a function of growth. The CL synthetase specific activity was highest during the early and midexponential growth phases, as was the cellular content of CL. The results demonstrate a correlation between enzyme-specific activity and lipid content of the cells.

Effect of inhibition of protein synthesis on lipid metabolism in Lactobacillus plantarum.

In Lactobacillus plantarum 17-5, lipid synthesis appears to be correlated with protein synthesis. Inhibition of protein synthesis by chloramphenicol (50 mug/ml) caused the nearly simultaneous inhibition of incorporation of radioactive oleic acid into polar lipids before the cessation of growth. In addition, de novo fatty acid synthesis, as determined by the incorporation of radioactive acetate into cellular lipids, was also inhibited. Removal of the antibiotic resulted in the resumption of growth, protein synthesis, and polar lipid synthesis. Inhibition of protein synthesis by leucine deprivation also produced a marked reduction in the incorporation of radioactive oleic acid into the total polar lipids at about the same time that growth stopped (30 to 60 min after the removal of leucine). However, the different classes of lipids behaved differently. For example, the incorporation of oleic acid into cardiolipin was inhibited immediately upon removal of leucine from the cultures, whereas incorporation into phosphatidyl-glycerol was maintained at near normal rates for 60 min after the removal of leucine and then ceased. In contrast, the accumulation of radioactive oleic acid in a neutral lipid identified as diglyceride occurred to a much greater extent in leucine-deprived cultures than in control (+ leucine) cultures. Upon addition of leucine to leucine-deprived cultures, the rates of synthesis of phosphatidyl-glycerol and cardiolipin returned to normal; the amount of radioactivity in the diglyceride fraction decreased to normal levels concomitantly with increased phospholipid synthesis.

31-P nuclear magnetic resonance studies on serum low and high density lipoproteins: effect of paramagnetic ion.

A paramagnetic quenching reagent, Mn-2+/EDTA (1:2.2), was developed for the purpose of investigating the phospholipid phosphate groupings of human serum low and high density lipoproteins through the quenching effect of the reagent on the 31-P nuclear magnetic resonance signals from these complexes. Systems investigated included native low and high density serum liproteins (LDL, HDL2, and HDL3), egg phosphatidylcholine vesicles together with appropriate phosphodiester model systems, diethyl phosphate in aqueous buffer, and phosphatidylcholine and sphingomyelin both in anhydrous methanol. The results of these studies indicated that ca. 50 percent of the phospholipid-phosphorus signal of LDL is quenched upon titration as compared to an 80-85 percent figure observed for HDL2 and HDL3. In all cases the spectral effects were totally reversible upon removalof the paramagnetic ion by dialysis. The results of the titration studies indicated a similar but not an identical behavior between HDL2 and HDL3. The results are consistent with model structures of HDL and LDL particles derived from low angle X-ray diffraction.

Phosphate metabolism in intact human erythrocytes: determination by phosphorus-31 nuclear magnetic resonance spectroscopy.

Whole human blood was examined by (31)P nuclear magnetic resonance spectroscopy. Individual phosphates (alpha,beta,gamma) of ATP were identifiable, and two microenvironments appeared to be present for this molecule. When sequential recordings of freshly collected blood were made, 2,3-diphosphoglycerate was observed to decrease in association with a concomitant increase in inorganic orthophosphate. When aged cells containing little 2,3-diphosphoglycerate were incubated in the presence of inosine and pyruvate, 2,3-diphosphoglycerate formation could be demonstrated. These results show that cellular metabolism can be recorded directly in intact cells by (31)P nuclear magnetic resonance.