Kinetics of formation of fibrin oligomers. III. Ligation kinetics concurrent with and subsequent to oligomer assembly

Human fibrinogen was treated with thrombin in the presence of fibrinoligase (Factor XIIIa) and calcium ion at pH 8.5, ionic strength 0.45, and the ensuing polymerization was interrupted at various time intervals (t) both before and after the clotting time (t_c) by solubilization with a solution of sodium dodecyl sulfate and urea. Aliquots of the solubilized protein were subjected to gel electrophoresis on polyacrylamide gels after disulfide reduction by dithiothreitol and on agarose gels without reduction. The degree of γ-γ ligation was determined from the former. The latter provided the size distribution of ligated end-to-end sequences produced by splitting the ligated staggered overlapped oligomers down the middle, for degrees of polymerization, x, from 1 to 10. Addition of fibrinoligase (in which the activating thrombin had been inhibited by p-nitrophenyl-p′-guanidinobenzoate, NPGB) to Kabi fibrinogen showed the presence of small amounts of ligatable oligomers. Addition of fibrinoligase to a polymerizing mixture in which the action of thrombin had been stopped before clotting by NPGB produced the same distribution of ligated end-to-end sequences that was obtained when fibrinoligase was originally present, at least for reaction times up to 0.7 of the clotting time. The kinetics of γ-γ ligation by fibrinoligase acting on a polymerized mixture stabilized by NPGB were followed. The reaction was first order in the concentration of ligatable γ-γ junctions and the initial velocity was proportional to the enzyme concentration. The time evolution of size distribution of ligated end-to-end sequences agreed with a theory based on random ligation of ligatable junctions.     

Molecular-Mechanical studies of the mismatched base analogs of d(CGCGAATTCGCG)2:d(CGTGAATTCGCG)2, d(CGAGAATTCGCG)2, d(CGCGAATTCACG)2, d(CGCGAATTCTCG)2, and d(CGCAGAATTCGCG)·d(CGCGAATTCGCG)

Molecular-mechanical simulations have been carried out on “mismatched base” analogs of the DNA double-helical structure d(CGCGAATTCGCG)₂, in which the base pairs CG at the 3 and 10 positions have been replaced by CA, AG, TC, and TG base pairs, as well as an insertion analog in which an extra adenine has been incorporated into one strand of the above structure between bases 3 and 4. The results of these simulations (calculated relative stabilities, structures, and nmr ring-current shifts) have been compared with calorimetric and nmr data. The calculated relative stabilities of the double-helical parent dodecamer and the various “wobble” base pairs qualitatively correlate with the experimental melting temperatures. The base-pairing structure for the GT wobble pair is in agreement with that previously determined from nmr experiments. For the GA base pair, the structure with both bases anti has a slightly more favorable energy from base pairing and stacking than a structure with non-Watson-Crick H-bonding with adenine syn, in agreement with nmr experiments. The CA wobble base is calculated to favor an adenine 6NH₂ …? cytosine N3 H-bond over cytosine 4NH₂ …? adenine N1, again, in agreement with nmr experiments. There is no definitive experimental data on the TC base pair, but the existence of (somewhat long and weak) H-bonds involving cytosine 4NH₂ …? thymine 4CO and cytosine N3 …? thymine HN3 seems reasonable. We find a structure in which the extra adenine base of the insertion analogs sits “inside” the double helix.     

Sexual Interference and Sexual Defense in the Smooth Newt,Triturus vulgaris (Amphibia,Urodela, Salamandridae)

• 1 When a male smooth newt encounters a ♀ who is already engaged in courtship, he may mimic her behaviour during the spermatophore deposition and transfer stages of the courtship. He thereby usurps the courting ♂ and may inseminate the ♀ himself. Such sexual interference depresses the short-term, and perhaps long-term, mating success of the courting ♂.
• 2 In the presence of a potential rival, the courting ♂ alters certain aspects of his sexual behaviour. He displays more intensely to the ♀ and attempts to draw her away from the rival by increasing the duration of his display. He may also “check” that it is the ♀, and not the rival, who will elicit the deposition of a spermatophore from him. These changes in the behaviour of the courting ♂ are interpreted as defense against sexual interference.
• 3 Female smooth newts may be multiply inseminated as a consequence of sexual interference; this may result in sperm competition. However, ♀♀ seem to find competitive interactions between ♂ ♂ “aversive”.
• 4 Sexual interference by ♀-mimicry and associated defensive behaviour patterns are common in the urodele amphibians. Interference can be thought of as a “side-payment” conditional mating strategy.

     

A comparison of the structures of apo dogfish M4 lactate dehydrogenase and its ternary complexes

Details are recorded of the X-ray diffraction data collection, heavy atom refinement and preliminary structure refinement for two different dogfish M₄ lactate dehydrogenase structures. One of these is the 2.0 Å resolution apoenzyme structure; the other is a 3.0 Å resolution abortive ternary complex. Two other ternary substrate inhibitory complexes (LDHase²: NAD: oxalate and LDHase: NADH: oxamate), isomorphous with the abortive ternary complex (LDHase: NAD-pyruvate), have also been examined. The apo-LDHase and LDHase: NAD-pyruvate structures are systematically compared to determine significant differences in their conformation. These are related to differences in structure amongst the three studied ternary complexes. These differences all occur in regions of the protein around the active site, particularly the flexible loop covering the active center pocket and the C-terminal helix αH. The changes are suggestive of a domino effect whereby the closing of the loop on binding coenzyme and substrate triggers the critical reactive residues into assuming their catalytically active positions.     

Spontaneously arising mutL mutators in evolving Escherichia coli populations are the result of changes in repeat length

Over the course of thousands of generations of growth in a glucose-limited environment, 3 of 12 experimental populations of Escherichia coli spontaneously and independently evolved greatly increased mutation rates. In two of the populations, the mutations responsible for this increased mutation rate lie in the same region of the mismatch repair gene mutL. In this region, a 6-bp repeat is present in three copies in the gene of the wild-type ancestor of the experimental populations but is present in four copies in one of the experimental populations and two copies in the other. These in-frame mutations either add or delete the amino acid sequence LA in the MutL protein. We determined that the replacement of the wild-type sequence with either of these mutations was sufficient to increase the mutation rate of the wild-type strain to a level comparable to that of the mutator strains. Complementation of strains bearing the mutator mutations with wild-type copies of either mutL or the mismatch repair gene uvrD rescued the wild-type mutation rate. The position of the mutator mutations-in the region of MutL known as the ATP lid-suggests a possible deficiency in MutL's ATPase activity as the cause of the mutator phenotype. The similarity of the two mutator mutations (despite the independent evolutionary histories of the populations that gave rise to them) leads to a discussion of the potential adaptive role of DNA repeats.     

Genome-wide linkage analysis of longitudinal phenotypes using σ<Superscript>2</Superscript><Subscript>A</Subscript> random effects (SSARs) fitted by Gibbs sampling

The study of change in intermediate phenotypes over time is important in genetics. In this paper we explore a new approach to phenotype definition in the genetic analysis of longitudinal phenotypes. We utilized data from the longitudinal Framingham Heart Study Family Cohort to investigate the familial aggregation and evidence for linkage to change in systolic blood pressure (SBP) over time. We used Gibbs sampling to derive sigma-squared-A-random-effects (SSARs) for the longitudinal phenotype, and then used these as a new phenotype in subsequent genome-wide linkage analyses. Additive genetic effects (sigma2A.time) were estimated to account for approximately 9.2% of the variance in the rate of change of SBP with age, while additive genetic effects (sigma2A) were estimated to account for approximately 43.9% of the variance in SBP at the mean age. The linkage results suggested that one or more major loci regulating change in SBP over time may localize to chromosomes 2, 3, 4, 6, 10, 11, 17, and 19. The results also suggested that one or more major loci regulating level of SBP may localize to chromosomes 3, 8, and 14. Our results support a genetic component to both SBP and change in SBP with age, and are consistent with a complex, multifactorial susceptibility to the development of hypertension. The use of SSARs derived from quantitative traits as input to a conventional linkage analysis appears to be valuable in the linkage analysis of genetically complex traits. We have now demonstrated in this paper the use of SSARs in the context of longitudinal family data.     

Method for determination of methadone in exhaled breath collected from subjects undergoing methadone maintenance treatment

At present drugs of abuse testing using exhaled breath as specimen is only possible for alcohol. However, we recently discovered that using modern liquid chromatography–mass spectrometry technique amphetamine and methamphetamine is detectable in exhaled breath following intake in drug addicts. We therefore undertook to develop a method for determination of methadone in exhaled breath from patients undergoing methadone maintenance treatment. Exhaled breath was collected from 13 patients after intake of the daily methadone dose. The compounds were trapped by filtering the air through a C18 modified silica surface. After elution of any trapped methadone the extract was analysed by a combined liquid chromatography–tandem mass spectrometry method. Recovery of trapped methadone from the filter surface was 96%, no significant matrix effect was observed, and the quantification using methadone-d3 as an internal standard was accurate (<10% bias) and precise (coefficient of variation 1.6–2.0%). Methadone was indisputably identified by means of the mass spectrometry technique in exhaled breath samples from all 13 patients. Identification was based on monitoring two product ions in selected reaction monitoring mode with correct relative ratio (±20%) and correct retention time. Excretion rates ranged from 0.39 to 78 ng/min. No methadone was detected in 10 control subjects. This finding confirms that breath testing is a new possibility for drugs of abuse testing. Collection of exhaled breath specimen is likely to be more convenient and safe as compared to other matrices presently in use.     

Prevalence of Obesity Among Children With Chronic Conditions

New evidence suggests that children with chronic conditions may be predisposed to overweight and obesity. This study provides prevalence estimate of obesity for children and adolescents with select chronic conditions. We analyzed reported height and weight and the corresponding BMI from 46,707 subjects aged 10–17 years collected by the National Survey of Children's Health (NSCH‐2003). Our main outcome measure was the prevalence of obesity (defined as ≥95th percentile of the sex‐specific BMI for age growth charts), adjusted for underlying demographic and socioeconomic factors. We found that the prevalence of obesity among children 10–17 years of age without a chronic condition was 12.2% (95% confidence interval (CI) 11.5–13.0); the prevalence of obesity for children with asthma was 19.7% (19.5–19.9); with a hearing/vision condition was 18.4% (18.2–18.5); with learning disability was 19.3% (19.2–19.4); with autism was 23.4% (23.2–23.6); and with attention‐deficit/hyperactivity disorder was 18.9% (18.7–19.0). Our findings suggest that children 10–17 years of age with select chronic conditions were at increased risk for obesity compared to their counterparts without a chronic condition.     

Reducing the impact of pesticides on biological control in Australian vineyards: pesticide mortality and fecundity effects on an indicator species, the predatory mite Euseius victoriensis (Acari: Phytoseiidae)

Laboratory bioassays on detached soybean, Glycine max (L.) Merr., leaves were used to test 23 fungicides, five insecticides, two acaricides, one herbicide, and two adjuvants on a key Australian predatory mite species Euseius victoriensis (Womersley) in "worst-case scenario" direct overspray assays. Zero- to 48-h-old juveniles, their initial food, and water supply were sprayed to runoff with a Potter tower; spinosad and wettable sulfur residues also were tested. Tests were standardized to deliver a pesticide dose comparable with commercial application of highest label rates at 1,000 liter/ha. Cumulative mortality was assessed 48 h, 4 d, and 7 d after spraying. Fecundity was assessed for 7 d from start of oviposition. No significant mortality or fecundity effects were detected for the following compounds at single-use application at 1,000 liter/ha: azoxystrobin, Bacillus thuringiensis (Bt) subsp. kurstaki, captan, chlorothalonil, copper hydroxide, fenarimol, glyphosate, hexaconazole, indoxacarb, metalaxyl/copper hydroxide, myclobutanil, nonyl phenol ethylene oxide, phosphorous acid, potassium bicarbonate, pyraclostrobin, quinoxyfen, spiroxamine, synthetic latex, tebufenozide, triadimenol, and trifloxystrobin. Iprodione and penconazole had some detrimental effect on fecundity. Canola oil as acaricide (2 liter/100 liter) and wettable sulfur (200 g/100 liter) had some detrimental effect on survival and fecundity and cyprodinil/fludioxonil on survivor. The following compounds were highly toxic (high 48-h mortality): benomyl, carbendazim, emamectin benzoate, mancozeb, spinosad (direct overspray and residue), wettable sulfur (> or = 400 g/100 liter), and pyrimethanil; pyrimethanil had no significant effect on fecundity of surviving females. Indoxacarb safety to E. victoriensis contrasts with its toxicity to key parasitoids and chrysopid predators. Potential impact of findings is discussed.