Measuring enzyme motility in organic media using novel H-D exchange methodology

A novel deuterium ((2)H) NMR technique as developed for measuring the total number of deuterons exchanged by lyophilised protein samples following hydrogen-deuterium (H-D) exchange. Using this methodology differences in the H-D exchange behaviour of the proteolytic enzyme subtilisin Carlsberg hydrated either in air or an organic solvent were probed as a function of hydration. At low thermodynamic water activity (a(w)), the degree of H-D exchange increased rapidly with hydration (from anhydrous to a(w) 0.22). At a(w) 0.22, subtilisin powders hydrated in air were found to have reached an H-D exchange level comparable to that found upon aqueous dissolution and in agreement with previous studies using lysozyme. Lyophilised subtilisin hydrated in either dichloromethane (DCM) or diisopropyl ether (DIPE) showed a pattern of exchange (vs. a(w)) comparable to that found for powders hydrated in air. However, subtilisin hydrated in n-hexane showed a significant reduction in H-D exchange at all a(w) studied. Control experiments demonstrated that the reduction in H-D exchange observed for subtilisin in n-hexane was not a kinetic effect. This lower level of exchange in n-hexane implies that hydrated subtilisin Carlsberg has a lower conformational motility and more rigid protein matrix.     

Affinities and Historical Zoogeography of the New Zealand Short-Tailed Bat, Mystacina tuberculata Gray 1843, Inferred from DNA-Hybridization Comparisons

We carried out DNA-hybridization comparisons among representatives of the major groups of Chiroptera to determine the phylogenetic position of the New Zealand short-tailed bat, Mystacina tuberculata. All analyses confirmed the noctilionoid affinity of this species suggested by an earlier serological study, with support from taxon jackknifing and at bootstrap levels of 98% or higher. However, a specific association with Noctilio was not found in more than 13% of the bootstrapped trees. The most precise of the thermal-stability indices employed (T _m, the median melting temperature of hybridized sequences) demonstrated a sister-group relationship of Mystacina to all noctilionoids, with Noctilio the first branch within Noctilionoidea but separated from the Mystacina lineage by a very short internode. Our determination of the timing of the divergence of Mystacina from noctilionoids is 54 myrbp. This estimate is based on independent indications that extant bat lineages began to diversify in the latest Cretaceous and is much earlier than the tentative estimate of 35 myrbp inferred from serology. Even if the diversification of all living bats occurred as early as 83 myrbp, as some authors have suggested, separation of Mystacinidae—on that basis, at 66 myrbp—could not have taken place soon enough for this taxon to be isolated on New Zealand before New Zealand separated from the rest of Gondwanaland. However, any of these dates would allow for the distribution of the noctilionoid–mystacinid common ancestor in South America, Australia, and Antarctica before the final sundering of Australia from Antarctica and for the divergence of Mystacinidae as a possible result of that event. This hypothesis is supported by the presence of fossil mystacinids in early and mid-Miocene deposits at Bullock Creek and Riversleigh, Queensland, showing that Mystacinidae had been resident in Australia from at least 25–20 myrbp. The most obvious scenario explaining the presence of Mystacinidae in New Zealand is therefore fortuitous dispersal from Australia across the Tasman Sea.     

Insufficient Milk Supply and Breast Cancer Risk: A Systematic Review

Background

An association between insufficient milk supply, the inability of a mother''s breast milk to provide sufficiently for her infant, and breast cancer has been suggested by observations in animal models. To determine if an association has been reported in epidemiological studies of human breast cancer, a systematic review of the literature has been conducted. We also sought to identify the methodological limitations of existing studies to guide the design of any future prospective studies in this field.

Methodology/Principal Findings

PubMed, EMBASE, Web of Science, BIOSIS, and CAB abstracts were searched. We selected any study that (1) assessed breast cancer in association with breastfeeding history and (2) examined the relationship between insufficient milk supply with breast cancer. Seven relevant studies were identified that met both criteria. There was statistically significant heterogeneity among the results which likely reflects clinically significant differences in definitions of insufficient milk supply and reference groups that were used. Among premenopausal women who had experienced insufficient milk supply, odds ratios (ORs) for breast cancer risk ranged from 0.9 to 16.3. Among postmenopausal women, ORs ranged from 0.6 to 6.7. Based on the range of odds ratios obtained in the studies reported in this review, it remains unclear if there is a true association between insufficient milk supply and breast cancer.

Conclusions/Significance

Although some studies have shown a strong positive association, there is no consistent evidence for an effect of insufficient milk supply on breast cancer risk. Exposure definitions are in need of improvement in order to focus on primary insufficient milk supply. Reference groups consisting of women who have successfully breastfed may also introduce positive bias (inflation of the odds ratio) into study results because of the protective effect of prolonged breastfeeding in the control group.     

Multiple inserts of gene of interest and selectable marker gene are co-integrated and stably transmitted as a single genetic locus in transgenic soybean plants

Particle bombardment has been used for soybean transformation for more than 20 yr, but the integration and segregation of transgene inserts in the soybean genome have not been clearly documented. Over the past 5 yr, we processed several hundred transgenic events. In each experiment, the expression cassettes of the gene of interest (GOI) and hygromycin selectable marker gene (SMG) were co-bombarded into soybean at a 1:1 molecular ratio. More than 75% of hygromycin-resistant events also carried the GOI. Molecular analysis of transgenic plants revealed that most events carried multiple inserts of the GOI and the SMG. The GOI and the SMG were linked in selfed T1 and T2 progeny. Segregation analysis of progeny indicated that, in over 98% of the transgenic events, the multiple inserts of the GOI were integrated into the same genetic locus resulting in a 3:1 segregation ratio. Furthermore, the multiple inserts of the GOI are transmitted into succeeding generations, and no recombinants were found. These data indicate that in soybean plants, co-bombarded genes are preferentially integrated and stably segregated as a single genetic locus.     

Correlated AFM and NanoSIMS imaging to probe cholesterol-induced changes in phase behavior and non-ideal mixing in ternary lipid membranes

Cholesterol is believed to be an important component in compositionally distinct lipid domains in the cellular plasma membrane, which are referred to as lipid rafts. Insight into how cholesterol influences the interactions that contribute to plasma membrane organization can be acquired from model lipid membranes. Here we characterize the lipid mixing and phase behavior exhibited by (15)N-dilaurolyphosphatidycholine ((15)N-DLPC)/deuterated distearoylphosphatiylcholine (D(70)-DSPC) membranes with various amounts of cholesterol (0, 3, 7, 15 or 19mol%) at room temperature. The microstructures and compositions of individual membrane domains were determined by imaging the same membrane locations with both atomic force microscopy (AFM) and high-resolution secondary ion mass spectrometry (SIMS) performed with a Cameca NanoSIMS 50. As the cholesterol composition increased from 0 to 19mol%, the circular ordered domains became more elongated, and the amount of (15)N-DLPC in the gel-phase domains remained constant at 6-7mol%. Individual and micron-sized clusters of nanoscopic domains enriched in D(70)-DSPC were abundant in the 19mol% cholesterol membrane. AFM imaging showed that these lipid domains had irregular borders, indicating that they were gel-phase domains, and not non-ideally mixed lipid clusters or nanoscopic liquid-ordered domains.     

Stent implantation in acute myocardial infarction using a heparin-coated stent: a pilot study as a preamble to a randomized trial comparing balloon angioplasty and stenting

Preliminary experience with primary stenting in myocardial infarction has suggested a greater benefit in clinical outcome than has been obtained with direct balloon angioplasty. However, subacute thrombosis (SAT) remains a limitation for this new mode of therapy. In the BENESTENT II Pilot and main trials, the incidence of SAT with the heparin-coated Palmaz-Schatz stent was only 0.15%. Therefore, as a preamble to a large randomized trial, the feasibility and safety of the use of the Heparin-Coated Palmaz-Schatz trade mark Stent in Acute Myocardial Infarction (AMI) was tested in 101 patients enrolled between April and September 1996 in 18 clinical centres. In 101 stent-eligible AMI patients, as dictated by protocol, a heparin-coated stent was implanted. The primary objectives were to determine the in-hospital incidence of major adverse cardiac events (MACE: death, MI, target lesion revascularization) and bleeding complications, while the secondary objectives were the procedural success rate and the MACE, the restenosis and reocclusion rates at 6.5 months. Stent implantation (n 3 129 stents) was successful in 97 patients of the 101 who were included in this trial. During their hospital stay, two patients died and no patient experienced re-infarction, ischaemia prompting re-PTCA or CABG. Four patients suffered a bleeding complication, three major and one minor, of whom three required surgical repair. At 210 days follow-up, 81% of the patients were event free. At 6.5 months restenosis was documented in 18% of the 88 patients who underwent follow-up angiography, including three total occlusions. The results, both with respect to QCA and the occurrence of MACE, compare favourably with studies using elective stenting in both stable and unstable angina patients. As a result of this pilot study, a large randomized trial comparing direct balloon angioplasty with direct stenting in 900 patients with AMI was initiated in December 1996.