Studies on the interaction of ribonuclease inhibitor with pancreatic ribonuclease involving differential labeling of cysteinyl residues.

Ribonuclease inhibitor (RI) is a protein that forms a very tight complex with ribonucleases (RNases) of the pancreatic type. RI contains 30 thiol groups, some of which are important for the enzyme-inhibitor interaction. To examine which thiols are affected by the binding of RNase, differential labeling experiments were performed. Reaction of porcine RI with the cysteine-specific labeling reagent 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid resulted in labeling of an average of 7.4 of the 30 cysteinyl residues. Binding of bovine pancreatic RNase A caused a 3.2-fold reduction in the extent of modification. Peptide mapping showed that in free RI, Cys-57, -371, and -404 were labeled to the greatest extent (yield, 0.4-0.6 mol/mol). RNase A did not protect Cys-57 against modification, whereas the labeling of Cys-371 and -404 was reduced by more than 90%. A second group of residues was labeled to a lesser extent in free RI (yield, 0.04-0.2 mol/mol). Within this group 11 residues were protected by RNase A by more than 90%, 2 were not affected at all, and 7 were protected between 10 and 90%. Seven cysteinyl residues in RI that were protected in the RI.RNase A complex were no longer protected in the RI.S-protein complex. These residues were mainly present in the N-terminal region of RI. However, when the S-peptide was included to yield the RI.RNase S complex, the same pattern of labeling was obtained as with the RI.RNase A complex. Addition of the S-peptide alone had no effect on the labeling. The implications of these observations with respect to RNase binding areas of RI are discussed in relation to the results obtained from the analysis of active RI molecules that contain deletions.     

Vector competence of the Australian paralysis tick, Ixodes holocyclus, for the Lyme disease spirochete Borrelia burgdorferi

Clinical and serologic evidence of Lyme disease in Australia, including the typical rash, erythema migrans, has been reported. The vector tick transmitting Borrelia burgdorferi in Australia, however, has not been determined. The Australian paralysis tick, Ixodes holocyclus, is a logical candidate vector of the Lyme disease spirochete in Australia; therefore, we tested the ability of I. holocyclus to acquire and maintain a North American isolate of B. burgdorferi. Larval I. holocyclus ingested spirochetes, but none of 84 derived nymphs were infected. These experiments should be repeated with Australian strains of spirochetes.     

Molecular basis for methoxyamine initiated mutagenesis. 1H nuclear magnetic resonance studies of base-modified oligodeoxynucleotides.

In order to reach a more detailed understanding of the mechanism of the mutagenic action of methoxyamine and of N4-methoxycytidine and its 2'-deoxyribo-analogue, the solution structures of the self-complementary octanucleotide, d(CGAATTCG) and its analogues, d(CGAATCCG), d(CGAATMCG) and d(CGAATPCG) (designated 8mer-AT, 8mer-AC, 8mer-AM, and 8mer-AP, respectively), were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2]oxazin-7-one, in which the N-O bond is held in the anti configuration with respect to N3 of the cytosine ring. Correlated spectroscopy and nuclear Overhauser spectroscopy allowed assignment of the base, anomeric and H2'/H2" protons in 8mers-AT, -AM and -AP, and showed that all three had features consistent with a regular B-DNA duplex structure. Duplex-to-coil transition temperatures were determined to be 52(+/- 2) degrees C (8mer-AT), 51(+/- 2) degrees C (8mer-AP), 32(+/- 2) degrees C (8mer-AM); on the chemical shift timescale, the melting transition was fast for 8mer-AT and 8mer-AP, but slow for 8mer-AM. Imino proton spectra were indicative of Watson-Crick base-pairing in 8mers-AT, -AP and -AM. The 8mer-AP duplex had a structure and melting characteristics virtually identical with those of the 8mer-AT duplex. The preferred syn configuration of the methoxyl group in M had a destabilising effect on the 8mer-AM duplex. At low temperatures, the A.M base-pair was in fast equilibrium between Watson-Crick and wobble configurations, with the methoxyl function anti-oriented, but the melting transition was accompanied by isomerization of the methoxyl group to the syn conformation. This syn-anti isomerization was the rate-determining step in the duplex-to-coil transition. The 8mer-AC oligomer did not form a stable duplex.     

Intrinsic fluorescence changes and rapid kinetics of the reaction of thrombin with hirudin.

Stopped-flow fluorescence spectroscopy has been used to study the reaction of human alpha-thrombin with recombinant hirudin variant 1 (rhir) at 37 degrees C and an ionic strength of 0.125 M. A 35% enhancement in intrinsic fluorescence accompanied formation of the thrombin-rhir complex. Over one third of this enhancement corresponded to a structural change that could be induced by binding of either the NH2-terminal fragment (residues 1-51) or the COOH-terminal fragment (residues 52-65) of rhir. Three kinetic steps were detected for reaction of thrombin with rhir. At high rhir concentrations (greater than or equal to 3 microM), two intramolecular steps with observed rate constants of 296 +/- 5 s-1 and 50 +/- 1 s-1 were observed. By using the COOH-terminal fragment of rhir as a competitive inhibitor, it was possible to obtain an estimate of 2.9 x 10(8) M-1 s-1 for the effective association rate constant at low rhir concentrations. At higher ionic strengths, this rate constant was lower, which is consistent with the formation of the initial complex involving an ionic interaction. The mechanism for the reaction of both the COOH- and NH2-terminal fragments of rhir appeared to involve two steps. When thrombin was reacted with the COOH-terminal fragment at high concentrations (greater than or equal to 6 microM), the bimolecular step occurred within the dead time of the spectrometer and only one intramolecular step, with a rate constant of 308 +/- 5 s-1 was observed. At concentrations of NH2-terminal fragment below 50 microM, its binding to thrombin appeared to be a bimolecular reaction with an association rate constant of 8.3 x 10(5) M-1 s-1. In the presence of saturating concentrations of the COOH-terminal fragment, a 1.7-fold increase in this rate constant was observed. At concentrations of NH2-terminal fragment greater than 50 microM, biphasic reaction traces were observed which suggests a two-step mechanism. By comparing the reaction amplitudes and dissociation constants observed with rhir and its COOH-terminal fragment, it was possible to obtain approximate estimates for the values of the rate constants of different steps in the formation of the rhir-thrombin complex.     

Impact of protein-protein contacts on the conformation of thrombin-bound hirudin studied by comparison with the nuclear magnetic resonance solution structure of hirudin(1-51).

The impact of protein-protein interactions on the conformation of the N-terminal hirudin domain consisting of residues 1 to 51 in the X-ray crystal structure of a hirudin-thrombin complex was investigated through comparisons with the nuclear magnetic resonance solution structure of hirudin(1-51). The close overall similarity observed between these two structures contrasts with the behavior of the C-terminal 17-residue polypeptide segment of hirudin, which is flexibly disordered in solution but exhibits a defined conformation in the complex with thrombin. Localized structural differences in the N-terminal domain include that residues 1 to 3 of hirudin in the crystalline complex form a hydrogen-bonding network with thrombin that is reminiscent of a parallel beta-sheet. Moreover, the backbone conformation of residues 17 to 20 in the complex does not contain the characteristic hydrogen bond observed for the type II' reverse turn in the solution structure, and the side-chains of Ser19 and Val21 have significantly different orientations in the two structures. Most of these structural changes can be related directly to thrombin-hirudin contacts, which may also be an important factor in the mechanism of hirudin action. In this context, it is of special interest that other residues that also make numerous contacts with thrombin, e.g. Thr4, Asp5 and Asn20, have identical conformations in free hirudin and in the complex.     

Isolation and phenotypic analysis of conditional-lethal, linker-insertion mutations in the gene encoding the largest subunit of RNA polymerase II in Saccharomyces cerevisme

Summary Linker-insertion mutagenesis was used to isolate mutations in the Saccharomyces cerevisiae gene encoding the largest subunit of RNA polymerase II (RP021, also called RPBI). The mutant rpo21 alleles carried on a plamid were introduced into a haploid yeast strain that conditionally expresses RP021 from the inducible promoter pGAL10. Growth of this strain on medium containing glucose is sustained only if the plasmid-borne rpo21 allele encodes a functional protein. Of nineteen linker-insertion alleles tested, five (rpo21-4 to –8) were found that impose a temperature-sensitive (ts) lethal phenotype on yeast cells. Four of these five is alleles encode mutant proteins in which the site of insertion lies near one of the regions of the largest subunit that have been conserved during evolution. Two of the is mutants (rpo21-4 and rpo21-7) display pleiotropic phenotypes, including an auxotrophy for inositol and a decreased proliferation rate at the permissive temperature. The functional relationship between RP021 and RP026, the gene encoding the 17.9 kDa subunit shared by RNA polymerases 1, 11, and III was investigated by determining the ability of increased dosage of RP026 to suppress the is phenotype imposed by rpo21-4 to –8. Suppression of the is defect was specific for the rpo21-4 allele and was accompanied by co-suppression of the inositol auxotrophy. These results suggest that mutations in the largest subunit of RNA polymerase II can have profound effects on the expression of specific subsets of genes, such as those involved in the metabolism of inositol. In the rpo21-4 mutant, these pleiotropic phenotypes can be attributed to a defective interaction between the largest subunit and the RP026 subunit of RNA polymerase II.     

Competitive exclusion,or species aggregation?

Summary There is a long-standing dispute over whether the analysis of species co-occurrence data, typically on islands in an archipelago, can disclose the forces at work in structuring a community. Here we present and utilise three scores S, C and T. S gives the mean number of islands shared by a species pair in the presence/absence data under study. The scores C and T are based on the way that a pair of species occurs on a pair of islands. When each species occurs on a different island, this adds to the checkerboard score C; if they occupy the same island, this increases the togetherness score T.In judging whether observed values of S, C and T are compatible with a null hypothesis assuming no species interaction, we follow Connor and Simberloff (1979) in generating a control group of (constrained) simulated incidence patterns.Presence/absence matrices can have paradoxical features, in combining a high mutual exclusion by species (checkerboardedness) with a degree of species aggregation that is also high. We show that this is in fact inevitable — that, given the usual contraints, C and T can differ only by a constant. This means that extreme checkerboardedness can be produced by forces making for species aggregation, just as well as by those making for avoidance.If we restrict our attention to a subset of species, the constraints are less rigid and the S, C and T scores are somewhat freer to vary. We consider the confamilial subsets in the Vanuatu archipelago as likely candidates for revealing any competition forces at work. Calculating the actual S, C and T scores for these subsets, we compare them with the corresponding scores in a sample of simulated colonization patterns.The actual species-distributions differ significantly from what we would expect if the colonization choices of different species were uncorrelated (save for some biological constraints). The confamilial species of the real world share more islands, and occur in a pattern less checkerboarded, and more aggregated, than their simulation counterparts. This suggests that competition pressures, if they exist, are overcome by countervailing factors.The method used is applicable in other ways, and to a wider class of problems, in analysing the forces behind community structure.     

Combined subtraction hybridization and polymerase chain reaction amplification procedure for isolation of strain-specific Rhizobium DNA sequences.

A novel subtraction hybridization procedure, incorporating a combination of four separation strategies, was developed to isolate unique DNA sequences from a strain of Rhizobium leguminosarum bv. trifolii. Sau3A-digested DNA from this strain, i.e., the probe strain, was ligated to a linker and hybridized in solution with an excess of pooled subtracter DNA from seven other strains of the same biovar which had been restricted, ligated to a different, biotinylated, subtracter-specific linker, and amplified by polymerase chain reaction to incorporate dUTP. Subtracter DNA and subtracter-probe hybrids were removed by phenol-chloroform extraction of a streptavidin-biotin-DNA complex. NENSORB chromatography of the sequences remaining in the aqueous layer captured biotinylated subtracter DNA which may have escaped removal by phenol-chloroform treatment. Any traces of contaminating subtracter DNA were removed by digestion with uracil DNA glycosylase. Finally, remaining sequences were amplified by polymerase chain reaction with a probe strain-specific primer, labelled with 32P, and tested for specificity in dot blot hybridizations against total genomic target DNA from each strain in the subtracter pool. Two rounds of subtraction-amplification were sufficient to remove cross-hybridizing sequences and to give a probe which hybridized only with homologous target DNA. The method is applicable to the isolation of DNA and RNA sequences from both procaryotic and eucaryotic cells.     

Warm-up rates during arousal from torpor in heterothermic mammals: physiological correlates and a comparison with heterothermic insects

Summary This study examines the relationship between warm-up rate, body mass, metabolic rate, thermal conductance and normothermic body temperature in heterothermic mammals during arousal from torpor. Predictions based on the assumption that the energetic cost of arousal has been minimised are tested using data for 35 species. The observation that across-species warm-up rate correlates negatively with body mass is confirmed using a comparative technique which removes confounding effects due to the non-independence of species data due to shared common ancestry. Mean warm-up rate during arousal correlates negatively with basal metabolic rate and positively with the temperature difference through which the animal warms, having controlled for other factors. These results suggest that selection has operated to minimise the overall energetic, cost of warm-up. In contrast, peak warm-up rate during arousal correlates positively with peak metabolic rate during arousal, and negatively with thermal conductance, when body mass has been taken into account. These results suggest that peak warm-up rate is more sensitive to the fundamental processes of heat generation and loss. Although heterothermic marsupials have lower normothermic body temperatures and basal metabolic rates, marsupials and heterothermic eutherian mammals do not differ systematically in warm-up rate. Pre-flight warm-up rates in one group of endothermic insects, the bees, are significantly higher than predictions based on rates of arousal of a mammal of the same body mass.Abbreviations BMR basal metabolic rate - ICM independent comparisons method - MWR mean warm-up rate - PMR peak metabolic rate - PWR peak·warm-up rate - Tb_activity body temperature during activity - Tb_torpor body temperature during torpor - T _arousal increase in body temperature during arousal     

Mechanistic studies on thrombin catalysis

The kinetic mechanism of the cleavage of four p-nitroanilide (pNA) substrates by human alpha-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates at the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of kcat/Km were bell-shaped for all substrates. The pKa values determined from the pH dependence of kcat/Km for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of kcat/Km for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA and for the binding of the competitive inhibitor N alpha-dansyl-L-arginine-4-methylpiperidine amide. The groups responsible for the observed pKa values were proposed to be His57 and the alpha-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pKa values of 6.7 and 8.6 observed in the pH profile of kcat/Km for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)