Proton magnetic resonance studies of the conformational changes of dideoxynucleoside ethyl phosphotriesters

PMR investigations on the diastereomeric phosphate methyl protons of the dinucleoside ethyl phosphotriesters Tp(C₂H₅)T, dA, and dIp(C₂H₅)dI have been used to study the conformational changes of these dimersin solution. In D₂O (273°K), the diastereomeric phosphate-methly groups of Tp(C₂H₅)T appear as a triplet. The methyl resonances of dIp(C₂H₅)dI and dAp(C₂H₅)dA appear as two sets of triplets and their chemical shift differences (δ₁ ? δ₂), decrease with increasing temperature, finally becoming zero at 292°K and 333°K, respectively. The same phenomenon is observed for dAp(C₂H₅)dA in CD₃OD; in this detacking solvent, the difference (δ₁ ? δ₂) diminishes to zero at a lower temperature (261°K). At room temperature in D₂O, the chemical shift of the phosphate methyl of Tp(C₂H₅)T appears at lower field than those of dIp(C₂H₅)dI or dAp(C₂H₅)dA. The differences between the chemical shifts of these groups (δ_I ? δ_T or δ_A ? δ_T) increase with increasing temperature, and reach maximal values at 301°K and 333°K, respectively. The results suggest that at low temperature the largest fraction of the dimer population exists in a stacked state, with the phosphate-ethyl groups outside the stack. Increasing temperature causes an oscillation of the bases and a shift in the dimer population away from the stacked state. Finally at high temperature, the planar bases rorate with respect to one another and in the case of dIp(C₂H₅)dI and dAp(C₂H₅)dA, the ethyl groups experience shielding by the anisotropic ring current of the five-membered ring of the bases. Thus, the current pmr studies and those reported earlier from our laboratory support an “oscillation-rotation model” for the unstacking process of the dimers. The relationship of this model and the “two-state model” is discussed.     

Characterization of Escherichia coli mutants tolerant to bacteriocin JF246: two new classes of tolerant mutants

Several hundred independent bacteriocin-tolerant mutants have been isolated without mutagenesis from three strains of Escherichia coli. On the basis of patterns of sensitivity to eight different colicins, over 85% of these mutants could be grouped into four classes. Two classes of mutants, class A and class B, are equivalent to tolA and tolB type mutants. We found tolA and tolB mutants were sensitive to the antibiotic bacitracin. The other two classes of bacteriocin-tolerant mutants, class F and class G, are distinguished from other types of colicin-tolerant mutants on the basis of sensitivity to colicins, dyes, detergents, antibiotics, and chelating agents. The mutation in class F and class G mutants is located between 21 to 23 min on the E. coli chromosome. We propose to designate the loci of these mutations as tolF and tolG, respectively.     

Mechanoelectrical transduction in hyaluronic acid salt solution is an entropy-driven process.

An electrical potential develops between the ends of a column of hyaluronic salt solution displaced from a resting position by gentle pressure. A previous study demonstrated that such displacement changes the optical rotary dispersion properties of the salt, either increasing the rotation in the direction already shown by the salt before displacement or changing and increasing the rotation in the opposite direction, depending on the direction of the displacement. The present investigation demonstrates that the loss of bound water component across a membrane separating the solution and water is corelated with the extent of the column displacement. In addition, a return of the column to the position before displacement is correlated with a return of the water component across the membrane-but not at the same rate as the exodus. The data seem consistent with the hypothesis that the hyaluronic acid salt, when strained, adopts a less entropic configuration, releasing bound water and thus increasing the entropy of water component. This change in the distribution of entropy is reversible; i.e., Eddington's "time's arrow" is reversible with respect to the water component of the solution.     

Viruses isolated from captive and free-ranging wild ruminants in Alberta.

Nasal secretions, leukocytes and preputial or vaginal swabs from a group of 15 captive wild ruminants, comprising six pronghorn antelope (Antilocapra americana), seven fallow deer (Dama dama) and two mule deer (Odocoileus hemionus), and from 50 free-ranging pronghorns in southern Alberta, were examined for viral agents. Captive animals were given injections of dexamethasone daily for 6 days in attempts to reactivate latent infections. Specimens were collected at 2-3 day intervals from days 0 to 18. Free-ranging pronghorns were sampled only once, at the time of capture. Fifteen viral isolates were obtained from the animals: six isolates of parainfluenza 3 (PI3) from nasal swabs from one fallow deer and one mule deer; five isolates of herpesvirus from leukocytes, vaginal, preputial and nasal swabs from three fallow deer; and four isolates of PI3 from nasal secretions of the 50 free-ranging pronghorns.     

Calpain inhibition by peptide epoxides.

The protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex was purified greater than 1000-fold from extracts of rat liver mitochondria; the specific activity was greater than 1000 units/mg of protein (1 unit gives half-maximum re-activation of 10 munits of phosphorylated complex). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave two bands (Mr 47700 and 35300) indistinguishable from the alpha- and beta-subunits of the branched-chain dehydrogenase component of the complex. On gel filtration (Sephacryl S-300), apparent Mr was 190000. This and other evidence suggests that activator protein is free branched-chain dehydrogenase; this conclusion is provisional until identical amino acid composition of the subunits has been demonstrated. Activator protein (i.e. free branched-chain dehydrogenase) was inhibited (up to 30%) by NaF, whereas branched-chain complex was not inhibited. There was no convincing evidence for interconvertible active and inactive forms of activator protein in rat liver mitochondria. Activator protein was detected in mitochondria from liver (ox, rabbit and rat) and kidney (ox and rat), but not in rat heart or skeletal-muscle mitochondria. In rat liver mitochondrial extracts, branched-chain complex sedimented with the mitochondrial membranes, whereas activator protein remained in the supernatant. Activator protein re-activated phosphorylated (inactive) particulate complex from rat liver mitochondria, but it did not activate dephosphorylated complex. Liver and kidney, but not muscle, mitochondria apparently contain surplus free branched-chain dehydrogenase, which is bound by the complex with lower affinity than is the branched-chain dehydrogenase intrinsic to the complex. It is suggested that this functions as a buffering mechanism to maintain branched-chain complex activity in liver and kidney mitochondria.     

Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (flumazepil) in pigeons

The selective benzodiazepine receptor antagonist, Ro 15-1788, produced behavioral effects in pigeons at doses at least 100 times lower than those previously reported to possess intrinsic pharmacological activity in mammals. In contrast to its effects in mammalian species, in pigeons, Ro 15-1788 does not exhibit partial agonist activity. Key-peck responses of pigeons were studied under a multiple fixed-interval 3-min, fixed-interval 3-min schedule in which the first response after 3-min produced food in the presence of red or white keylights. In addition, every 30th response during the red keylight produced a brief electric shock (punishment). Under control conditions, punished responding was suppressed to 30% of unpunished response levels. Ro 15-1788 (0.01 mg/kg, i.m.) increased unpunished response rates by 33% without affecting rates of punished responding. Doses of 0.1 to 1.0 mg/kg Ro 15-1788 produced dose-related decreases in both punished and unpunished responding. As is characteristic of other benzodiazepines, midazolam (0.1 and 0.3 mg/kg, i.m.) markedly increased punished responding but had little effect on rates of unpunished responding. Ro 15-1788 antagonized the increases in punished responding and also reversed the rate-decreasing effects of higher doses of midazolam. However, the effectiveness of Ro 15-1788 as a benzodiazepine antagonist was limited by its intrinsic activity: rate-decreasing doses of Ro 15-1788 were unable to completely reverse behavioral effects of midazolam. Midazolam was an effective antagonist of the behavioral effects of Ro 15-1788 (up to 0.1 mg/kg) but midazolam did not influence the rate-decreasing effects of 1.0 mg/kg Ro 15-1788 across a 100-fold dose range. In the pigeon, the behavioral effects of relatively low doses of Ro 15-1788 (0.01-0.1 mg/kg) appear to be related to benzodiazepine receptor mechanisms, whereas other systems appear to be involved in the effects of higher doses.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

Purification and characterization of a high-Mr proteinase inhibitor of pro-phenol oxidase activation from crayfish plasma.

Crayfish plasma was found to contain a proteinase inhibitor, which was purified to apparent homogeneity by acid precipitation, affinity chromatography on concanavalin A-Sepharose and hydrophobic-interaction chromatography. The inhibitor is a monomeric protein with an Mr of about 155,000 and a pI in the range 4.6-4.8. It is heat-stable and tolerant to low pH. It inhibits the serine proteinases trypsin and chymotrypsin, but not thrombin or subtilisin. Furthermore, it is efficient in decreasing the activity of a proteinase from crayfish haemolymph that is involved in the activation cascade of pro-phenol oxidase and can also block pro-phenol oxidase activation by this serine proteinase. This cascade is believed to play a central role in the recognition mechanism of non-self material in crustaceans and insects. The data presented give some evidence that the new proteinase inhibitor is involved in the regulation of this system.