Permeation and metabolism of Alternaria mycotoxins with perylene quinone structure in cultured Caco-2 cells

The absorption of four Alternaria toxins with perylene quinone structures, i.e. altertoxin (ATX) I and II, alteichin (ALTCH) and stemphyltoxin (STTX) III, has been determined in the Caco-2 cell Transwell system, which represents a widely accepted in vitro model for human intestinal absorption and metabolism. The cells were incubated with the four mycotoxins on the apical side, and the concentration of the toxins in the incubation media of both chambers and in the cell lysate were determined by liquid chromatography coupled with diode array detection and mass spectrometry (LC-DAD-MS) analysis. ATX I and ALTCH were not metabolised in Caco-2 cells, but ATX II and STTX III were partly biotransformed by reductive de-epoxidation to the metabolites ATX I and ALTCH, respectively. Based on the apparent permeability coefficients (P_app), the following ranking order for the permeation into the basolateral compartment was obtained: ATX I > ALTCH >> ATX II > STTX III. Total recovery of the four toxins decreased in the same order. It is assumed that the losses of STTX III, ATX II and ALTCH in Caco-2 cells are caused by covalent binding to cell components due to the epoxide group and/or the α,β-unsaturated carbonyl group present in these toxins. We conclude from this study that ATX I and ALTCH are well absorbed from the intestinal lumen into the portal blood in vivo. For ATX II and STTX III, intestinal absorption of the parent toxins is very low, but these toxins are partly metabolised to ATX I and ALTCH, respectively, in the intestinal epithelium and absorbed as such.     

Statistical Alignment with a Sequence Evolution Model Allowing Rate Heterogeneity along the Sequence

We present a stochastic sequence evolution model to obtain alignments and estimate mutation rates between two homologous sequences. The model allows two possible evolutionary behaviors along a DNA sequence in order to determine conserved regions and take its heterogeneity into account. In our model, the sequence is divided into slow and fast evolution regions. The boundaries between these sections are not known. It is our aim to detect them. The evolution model is based on a fragment insertion and deletion process working on fast regions only and on a substitution process working on fast and slow regions with different rates. This model induces a pair hidden Markov structure at the level of alignments, thus making efficient statistical alignment algorithms possible. We propose two complementary estimation methods, namely, a Gibbs sampler for Bayesian estimation and a stochastic version of the EM algorithm for maximum likelihood estimation. Both algorithms involve the sampling of alignments. We propose a partial alignment sampler, which is computationally less expensive than the typical whole alignment sampler. We show the convergence of the two estimation algorithms when used with this partial sampler. Our algorithms provide consistent estimates for the mutation rates and plausible alignments and sequence segmentations on both simulated and real data.     

Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a pK of 7.2 +/- 0.1. By analogy to other serine proteases this pK is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D2Okcat/Km=2.9+/-0.2 and a D2Okcat=1.7+/-0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A "burst" of product release during pre steady-state Gly-Pro-pNA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (approximately 15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly (kon) depends upon the ionization of an enzyme residue with a pK of 6.2 +/- 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a pK of 7.9 +/- 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme-inhibitor complex was reversible and dissociated with a koff of (5.5 +/- 0.4) x 10(-5) s(-1), thus yielding a Ki* (as koff/kon) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors (phi) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme-inhibitor complex.     

Physiological responses to low dose infusions of atrial peptide in conscious dogs

Mongrel dogs prepared with chronic catheters in their femoral artery and vein and urinary bladder received 60 minute infusions of atrial peptide ranging from 5 to 100 ng/kg/min. Infusion of atrial peptides caused dose dependent increases in plasma atrial peptide concentration with doses of 25 ng/kg/min or less increasing plasma concentrations to levels observed in normal animals during stimulation of endogenous atrial peptide secretion. Atrial peptide infusion at doses of 10 ng/kg/min and above caused significant decreases in mean arterial pressure which were not accompanied by statistically significant changes in heart rate. Atrial peptide infusion at doses of 25 ng/kg/min and above increased urinary sodium excretion and urine flow rate. Atrial peptide infusion was without effect on plasma vasopressin, ACTH and corticosterone concentrations. However, atrial peptide infusion resulted in dose dependent decreases in plasma aldosterone concentration and plasma renin activity, but the decreases were only significant with the high physiologic (25 ng/kg/min) and pharmacologic doses (50 & 100 ng/kg/min). These data show that atrial peptide infusions in conscious dogs have minimal effects when infused in small doses that mimic endogenous atrial peptide release. At higher doses, significant effects on the cardiovascular, renal and endocrine systems can be observed but their physiological significance is unclear.     

Alkaline phosphatase. 31P NMR probes of the mechanism

31P NMR signals from substrates and products of alkaline phosphatase have been adapted to measure the rates and product ratios for the hydrolysis and phosphotransferase reactions from pH 6 to 10. Below pH 8, glycerol is a poorer acceptor than H2O (glycerol phosphates:Pi = 0.5). Tris is a more effective acceptor below pH 8, showing a maximum acceptor efficiency at pH 8 (Tris phosphate:Pi = 2). Phosphotransferase efficiencies are in the order expected for the pKaS of the alcohol groups, Tris less than glycerol Cl, C3 less than glycerol C2. Tris and glycerol induce chemical shifts in 113Cd(II) present at the A site but not the B or C sites of the metal triad present at each active center of Cd(II)6 alkaline phosphatase, suggesting that the alcoxides of the acceptors coordinate the A site metal and become the nucleophiles attacking the phosphoseryl residue (E-P) in the second step of the mechanism. The interaction is through the oxygen of Tris. The transferase activity of the amino alcohol shows a bell-shaped pH dependency. Aliphatic alcohol acceptors show small increases in acceptor activity between pH 6 and 8, with 5-fold increases from pH 8 to 10 (at pH 10, glycerol phosphates:Pi = 2.5). 31P NMR inversion transfer has been used to measure the koff for Pi dissociation from the noncovalent enzyme complex (E . P). For the Zn(II)4 alkaline phosphatase koff is essentially pH independent at approximately 35 s-1. For Cd(II) or Mg(II) at the B site in place of Zn(II), koff less than or equal to 1 s-1 X Cl-ion, which appears to coordinate the A site metal ion, enhances koff, suggesting that both Cl- and HPO2-4 can coordinate the A site metal ion in a 5-coordinate intermediate. pH control of the alkaline phosphatase mechanism appears to reside in the stability of E-P and not the dissociation of E . P, compatible with the hypothesis that the activity-linked pKa is that of a H2O molecule coordinated to the A site metal, which in the hydroxide form becomes the nucleophile attacking the phosphoseryl group (E-P).     

Proposal for a uniform designation of zearalenone and its metabolites

The Fusarium mycotoxin zearalenone is a frequent contaminant of food and feed. Up to now, different abbreviations and counting systems for the numerous positions of this macrocyclic ß-resorcylic acid lactone and its metabolites have been used. As the number of identified fungal and mammalian metabolites of zearalenone is still growing, the lack of a uniform designation makes the literature on these important toxins confusing and complicated. Here, we propose a logical set of abbreviations and a simple counting system, in order to facilitate future research communications on zearalenone and its congeners.     

Macro- and microgeographic genetic structure in an ant species with alternative reproductive tactics in sexuals

The genetic structure of social insect populations is influenced by their social organization and dispersal modes. The ant Hypoponera opacior shows diverse reproductive behaviours with regular cycles of outbreeding via winged sexuals and inbreeding via within-nest mating wingless sexuals that reproduce by budding. This unusual life cycle should be reflected in the genetic population structure, and we studied this on different scales using microsatellites. On a macrogeographic scale, populations were considerably structured and migration rates within the Chiricahuas were higher than those in between mountain ranges. On a local scale, our analyses revealed population viscosity through dependent colony foundation and a high genetic diversity with a multicolonial structure. The latter was also evident from recognition trials revealing consistent aggression between non-nestmates. Within-nest matings led to high inbreeding coefficients. Finally, the observed seasonal changes in relatedness can be explained by variation in queen number and differential dispersal of the two reproductive morphs.     

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.     

Disposition of 17β-trenbolone in humans

The urinary excretion and metabolic pattern of 17β-trenbolone, a synthetic anabolic steroid hormone used as a growth promotor for beef cattle in several countries, has been studied in a human subject. For the separation of the metabolites of 17β-trenbolone, a reversed-phase high-performance liquid chromatographic method was established. The method was tested with metabolites obtained from incubation of 17β-trenbolone with rat liver microsomes. Fifteen metabolites could be well separated in one run by using a concave acetonitrile—water—methanol gradient. After ingestion of the tracer-labelled hormone at a dose of 0.04 mg/kg body weight 54% of the administered radioactivity was found in the urine after 26 h and 63% after 72 h. Of the urinary material 54% was present as glucuronides, which contained mostly 17α-trenbolone, 17β-trenbolone and trendione. At least five other polar metabolites, presumably hydroxylated products, were found in smaller amounts, mostly in the unconjugated and sulphated fractions. Thus, the disposition of 17β-trenbolone in humans differs significantly from that in rats, which may have a bearing on the toxicological evaluation of the hormone.     

Building homogeneous time-resolved fluorescence resonance energy transfer assays for characterization of bivalent inhibitors of an inhibitor of apoptosis protein target

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is a central apoptosis regulator that blocks cell death by inhibiting caspase-3, caspase-7, and caspase-9 via binding interactions with the XIAP BIR2 and BIR3 domains (where BIR is baculovirus IAP repeat). Smac protein, in its dimeric form, effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Here we describe the development of highly sensitive homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assays to measure binding affinities of potent bivalent peptidomimetic inhibitors of XIAP. Our results indicate that these assays can differentiate Smac-mimetic inhibitors with a wide range of binding affinities down to the picomolar range. Furthermore, we demonstrate the utility of these fluorescent tools for characterization of inhibitor off-rates, which as a crucial determinant of target engagement and cellular potency is another important parameter to guide optimization in a structure-based drug discovery effort. Our study also explores how increased inhibitor valency can lead to enhanced potency at multimeric proteins such as IAP.