Determination of the apparent functional molecular mass of the hepatocellular sodium-dependent taurocholate transporter by radiation inactivation

The apparent target size of the sodium-dependent taurocholate transporter in basolateral rat liver plasma membrane vesicles, showing overshooting taurocholate uptake in the presence of sodium was estimated by radiation inactivation. Radiation at -105 to -120 degrees C and 2.5 Mrad/min causes a dose-dependent monoexponential reduction of the overshoot of taurocholate uptake in the presence of sodium. In contrast, taurocholate transport in the absence of sodium and taurocholate permeation at 4 degrees C remained totally unaffected by the radiation dose, indicating that the passive permeability of the membrane towards taurocholate remained unaffected. Radiation inactivation by high-energy electrons provides information about the size of the functional unit of the transporter in situ. The target size determined represents the size of the radiation-sensitive mass which is compact enough for significant energy transfer to occur within all parts of the transport system. The minimal function molecular mass was determined to be 170 kDa for the sodium-dependent taurocholate transporter. To prove the validity of radiation inactivation data four internal standard enzymes were tested under identical conditions.     

Protein structure calculation with data imputation: the use of substitute restraints

The amount of experimental restraints e.g., NOEs is often too small for calculating high quality three-dimensional structures by restrained molecular dynamics. Considering this as a typical missing value problem we propose here a model based data imputation technique that should lead to an improved estimation of the correct structure. The novel automated method implemented in AUREMOL makes a more efficient use of the experimental information to obtain NMR structures with higher accuracy. It creates a large set of substitute restraints that are used either alone or together with the experimental restraints. The new approach was successfully tested on three examples: firstly, the Ras-binding domain of Byr2 from Schizosaccharomyces pombe, the mutant HPr (H15A) from Staphylococcus aureus, and a X-ray structure of human ubiquitin. In all three examples, the quality of the resulting final bundles was improved considerably by the use of additional substitute restraints, as assessed quantitatively by the calculation of RMSD values to the “true” structure and NMR R-factors directly calculated from the original NOESY spectra or the published diffraction data.     

A primary genetic linkage map for human chromosome 12

A primary genetic map for human chromosome 12 has been constructed from data on 23 restriction fragment length polymorphic systems collected in 38 normal families with large sibships. Linkage analysis of the genotypic data has ordered 16 loci into a continuous genetic map of 111 cM in males and 258 cM in females. Although most of the genetic map reflects a higher rate of recombination in females relative to males, significantly more frequent recombination was observed in males than in females in intervals between loci on the distal portion of the short arm of the chromosome. The mapping data shown here will serve as a first step toward a high-resolution genetic map for human chromosome 12.     

Photochemical crosslinking of protein and DNA in chromatin. II. Synthesis and application of psoralen-cystamine-arylazido photocrosslinking reagents

The synthesis and testing of a new type of nucleic acid-protein photocrosslinking reagent is described. The reagents are composed of a psoralen ligand for nucleic acid photoattachment, which is linked to an azidobenzoyl group, for protein photoattachment. The linker contains a disulfide bridge which can be opened by reduction with mercaptans. The reagents were tested in a chromatin system, where it was found that they induced cleavable crosslinks between the histones and the DNA upon irradiation with long-wavelength ultraviolet light (lambda greater than 300 nm).     

In vivo atrial peptide-venodilation: minimal potency relative to nitroglycerin in dogs

Venodilation is thought to contribute to the hemodynamic actions of atrial peptides. Therefore, we measured the effective vascular compliance (EVC) as a parameter of overall venous tone in 7 pentobarbital anesthetized dogs under autonomic blockade during i.v. infusions of rat atriopeptin II (AP II, up to 100 pmol/kg/min), rat alpha-atrial natriuretic factor, and nitroglycerin (GTN). AP II lowered mean arterial pressure by reducing peripheral vascular resistance with a threshold between 3 and 10 pmol/kg/min (but was ineffective in anesthetized or conscious dogs without autonomic blockade). Neither atrial peptide altered EVC, while GTN augmented EVC and caused a 4.6-fold larger reduction of central venous pressure than AP II at equihypotensive dosage. These findings, with infusion rates probably close to endogeneous release, reveal a vasodilator potency of atrial peptides, which is restricted to systemic arterioles without affecting venous tone.     

Comparison of two rapid cortisol radioimmunoassays for use in the fetal sheep.

Cortisol radioimmunoassays (RIA's) utilizing highly specific antisera combined with a simple ethanol protein precipitation procedure (ETOH-PPT) are widely utilized to measure cortisol in human plasma. This same type of RIA has been assumed specific for measurement of cortisol in the plasma of several different species of experimental animals. In order to test this assumption as applied to fetal ovine plasma, we compared an ETOH-PPT cortisol RIA with another rapid cortisol assay which utilizes a dichloromethane extraction (DM-E) step. The DM-E assay in turn was compared with a chromatographic assay previously shown to be highly specific for measurement of fetal plasma cortisol in this species. Fetal ovine plasma cortisol concentrations determined by the DM-E method were nearly identical to the concentrations obtained by the specific chromatographic RIA procedure. On the other hand, the ETOH-PPT RIA grossly overestimated cortisol concentrations when compared with the DM-E RIA. While the rapid DM-E RIA appears to be suitable for use in fetal ovine plasma, the widely used ETOH-PPT RIA yields spuriously high and unpredictable values and must be considered unreliable. These comparisons demonstrate the need for careful reassessment of steroid assays prior to their application in experimental animals even though they have been previously documented as specific in human plasma.     

Identification of avidin and streptavidin binding motifs among peptides selected from a synthetic peptide library consisting solely of D-amino acids

Peptides consisting solely of D -amino acids (D -peptides) as opposed to their L -counterparts (L -peptides) are resistant towards proteolytic degradation in the organism and may therefore be useful in future efforts to develop new stable peptide-based drugs. Using the random synthetic peptide library technique several L - and D -peptides, capable of binding to both avidin and streptavidin, were found. The L -peptides contained the previously described HPQ/M motifis, and among the D -peptides three binding motifs could be identified, of which the most frequently found one contained an N-terminal aliphatic hydrophobic amino acid (V, L or I) and an aromatic amino acid (Y or F) on the second position. At the third position in this motif several different amino acid residues were found, although N was the most frequent. Peptides representing two of the D -motifs were synthesized as well as peptides containing the HPQ/M motifs, and their binding properties were examined. Although the D -peptides were originally selected using avidin they also inhibited binding between immobilized biotin and soluble streptavidin as well as avidin. The IC₅₀ of some of the peptides were approximately 10⁵ times higher than the IC₅₀ for biotin but some had a lower IC₅₀ than iminobiotin. The D -peptides, which were originally selected from the library using avidin, could also inhibit the binding between streptavidin and biotin. Likewise, L -peptides selected from a library screened with streptavidin, could inhibit the binding of both streptavidin and avidin to immobilized biotin. Furthermore, the D -peptide, VFSVQSGS, as well as biotin could inhibit binding of streptavidin to an immobilized L -peptide (RYHPQSGS). This indicates that the biotin-like structure mimicked by these two seemingly very different peptides may react with the same binding sites in the streptavidin molecule.     

Diving seals, ischemia-reperfusion and oxygen radicals

The cardiovascular adaptations of seals that contribute to their ability to tolerate long periods of diving asphyxial hypoxia result in episodic regional ischemia during diving and abrupt reperfusion upon termination of the dive. These conditions might be expected to result in production of oxygen-derived free radicals and other forms of highly reactive oxygen species. Seal organs vary during dives with respect to the degree and persistence of ischemia. Myocardial perfusion is reduced and intermittent; kidney circulation is vigorously vasoconstricted. Heart and kidney tissues from ringed seals (Phoca hispida) and domestic pigs (Sus scrofa) were compared in reactions to experimental ischemia. Resulting production of hypoxanthine, indicative of ATP degradation, was higher in pig than in seal tissues. Activity of superoxide dismutase (SOD), an oxygen radical scavenger, was higher in seal heart. We suggest that these results indicate enhanced protective cellular mechanisms in seals against the potential hazard of highly reactive oxygen forms. SOD activity was unexpectedly higher in pig kidney.