Dynamic exchange between stabilized conformations predicted for hyaluronan tetrasaccharides: comparison of molecular dynamics simulations with available NMR data

Studies of the hyaluronan (HA) tetrasaccharides are important for understanding hydrogen-bonding in the HA polymer, as they are probably the smallest oligomers in which characteristics of the constituent monosaccharides and the polymer are simultaneously exhibited. Here we present extensive molecular dynamics simulations of the two tetrasaccharides of HA in dilute aqueous solution. These simulations have confirmed the existence of intramolecular hydrogen-bonds between the neighboring sugar residues of HA in solution, as proposed by Scott (1989). However, our simulations predict that these intramolecular hydrogen-bonds are not static as previously proposed, but are in constant dynamic exchange on the sub-nanosecond time-scale. This process results in discrete internal motion of the HA tetrasaccharides where they rapidly move between low energy conformations. Specific interactions between water and intramolecular hydrogen-bonds involving the hydroxymethyl group were found to result in differing conformations and dynamics for the two alternative tetrasaccharides of HA. This new observation suggests that this residue may play a key role in the entropy and stability of HA in solution, allowing it to stay soluble up to high concentration. The vicinal coupling constants3 J NHCH of the acetamido groups have been calculated from our aqueous simulations of HA. We found that high values of 3J NHCH approximately 8 Hz, as experimentally measured for HA, are consistent with mixtures of both trans and cis conformations, and thus3 J NHCH cannot be used to imply a purely trans conformation of the acetamido. The rapid exchange of intramolecular hydrogen-bonds indicates that although the structure is at any moment stabilized by these hydrogen-bonds, no one hydrogen-bond exists for an extended period of time. This could explain why NMR often fails to provide evidence for intramolecular hydrogen-bonds in HA and other aqueous carbohydrate structures.      

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.     

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.     

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.     

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.     

Oxidative metabolites of diethylstilbestrol in the fetal Syrian golden hamster

14C-Diethylstilbestrol was administered orally, intraperitoneally, and intrafetally to 15-day pregnant hamsters at a dose of 20 mg/kg body weight, and the radioactivity was determined in the fetus, placenta, and maternal liver after 6 hours. Significant amounts of radioactivity were found in these tissues in every case, indicating maternal-fetal and fetal-maternal transfer of diethylstilbestrol. Part of the radioactivity found in the tissues could not be extracted even after excessive washing. This implied the presence of reactive metabolites. In the fetal and placental extracts, eight oxidative metabolites of diethylstilbestrol were identified by mass fragmentography as hydroxy- and methoxy-derivatives of diethylstilbestrol, pseudodiethylstilbestrol, and dienestrol. The presence of oxidative metabolites in the hamster fetus and the covalent binding to tissue macromolecules are possibly associated with the fetotoxic effects of diethylstilbestrol.     

The liver is an organ site for the release of inosine metabolized by non-glycolytic pig red cells

The metabolic energy source used by the pig red cell, which is unable to metabolize blood-borne glucose, was examined. Potential physiological substrates include adenosine, inosine, ribose, deoxyribose, dihydroxyacetone and glyceraldehyde, of which inosine was previously implicated. A net ATP synthesis by red cells occurs during in situ perfusion through the adult miniature pig liver. HPLC analysis of the perfusate revealed the presence primarily of inosine and hypoxanthine. Inosine production by the liver was 0.015 mumol/g per min. Moreover, red cells maintain ATP when suspended in a balanced salt medium during a 6 h incubation at 38 degrees C, in which inosine is continuously infused to give an external concentration of no more than 3 mumol/l, mimicking its plasma level. Inosine consumption under these infusion conditions was 56 nmol/ml cell per h, which is two orders of magnitude lower than when inosine is present in millimolar concentration. The total red cell inosine consumption of 9.63 mumol/h is much less than the total liver inosine production of 212 mumol/h. These findings suggest that the liver is an organ site elaborating inosine, and that maintenance of a 3 mumol/l inosine in plasma is sufficient to meet the energy requirements of the pig red cells.     

Cardiovascular investigations of an endogenous digoxin-like factor

A circulating factor with digoxin immunoreactivity has been demonstrated. Elevated levels of this substance appear to be present after volume expansion and salt loading, and in some forms of hypertension. The potentially causative role for this factor in hypertension can be demonstrated by the normalization of blood pressure after antidigoxin antibody infusions in low-renin and sodium-dependent hypertension. The possibility that renal excretory defects may be the initiating event to elevate endogenous digoxin is suggested by studies with normotensive humans and monkeys with renal disease. In the latter case cardiovascular deficits were noted that were analogous to those detected in renal hypertensive monkeys with elevated endogenous digoxin. Considered together, these results suggest the existence of a natriuretic and hypertensive substance that plays a role in body fluid homeostasis and blood pressure regulation.     

Role of the Alternaria alternata Blue-Light Receptor LreA (White-Collar 1) in Spore Formation and Secondary Metabolism

Alternaria alternata is a filamentous fungus that causes considerable loss of crops of economically important feed and food worldwide. It produces more than 60 different secondary metabolites, among which alternariol (AOH) and altertoxin (ATX) are the most important mycotoxins. We found that mycotoxin production and spore formation are regulated by light in opposite ways. Whereas spore formation was largely decreased under light conditions, the production of AOH was stimulated 2- to 3-fold. ATX production was even strictly dependent on light. All light effects observed could be triggered by blue light, whereas red light had only a minor effect. Inhibition of spore formation by light was reversible after 1 day of incubation in the dark. We identified orthologues of genes encoding the Neurospora crassa blue-light-perceiving white-collar proteins, a cryptochrome, a phytochrome, and an opsin-related protein in the genome of A. alternata. Deletion of the white-collar 1 (WC-1) gene (lreA) resulted in derepression of spore formation in dark and in light. ATX formation was strongly induced in the dark in the lreA mutant, suggesting a repressing function of LreA, which appears to be released in the wild type after blue-light exposure. In addition, light induction of AOH formation was partially dependent on LreA, suggesting also an activating function. A. alternata ΔlreA was still able to partially respond to blue light, indicating the action of another blue-light receptor system.