Demonstration of five major glycoproteins in myelin and myelin subfractions.

Myelin was found to contain five major glycoproteins with molecular weights of 120000, 95000, 88000, 43000 and 38000. Light myelin contained only 5-7% of the amount of these glycoproteins in whole myelin, whereas heavy myelin and the membrane fraction contained amounts nearly identical with whole myelin. Since all the major and minor glycoproteins, with the exception of 120000-mol-wt. glycoprotein, were detected only after treating the myelin membrane with neuraminidase, N-acetylneuraminic acid is a terminal sugar residue in these glycoproteins.     

Exploring beliefs behind support for and opposition to wildlife management methods: a qualitative study

Wildlife management methods such as culling (lethal control) and fencing can be controversial in some circumstances. Such controversy can be problematic for decision-makers or those managing decision-making processes and can lead to management delays or inertia. Understanding the reasons why people support or oppose specific management methods is therefore an important objective for researchers. Attitudes towards methods are in part based on individual beliefs about those methods, the species of wildlife being managed and other associated phenomena. This paper adopts a qualitative approach to develop understanding of these beliefs. We conducted 17 focus-groups on wild deer management at two locations in Britain, with both ‘professional’ land manager and ‘public’ participants (n?=?103). We identified a number of individual beliefs which are grouped into five categories: naturalness, overabundance, impacts, effectiveness and animal welfare. Our findings suggest that potentially controversial management methods will receive most support where the objective is to maintain a ‘natural’ environment, at sites where impacts are evident, and when using targeted and effective methods.     

Comparison of standardized mortality ratios (SMRs) obtained from use of reference populations based on a company-wide registry cohort to SMRs calculated against local and national rates

The DuPont Company has maintained a mortality registry for all active and pensioned U.S. employees since 1957. Standardized mortality ratios (SMRs) for each plant site in the U.S. can be calculated based on the comparison with the entire U.S. DuPont population or with a regional subset of DuPont employees. We compared the SMRs derived from a large, international cohort mortality study of chloroprene workers (IISRP study) with those derived from the entire DuPont Registry and appropriate subpopulations of the registry for two U.S. neoprene plants--Louisville (Kentucky) and Pontchartrain (Louisiana). SMRs from the IISRP study for the Louisville cohort based on national rates for all causes of death, all cancers, respiratory cancer, and liver cancer are higher than those based on local mortality rates. Both the national and local comparisons (several counties surrounding each plant) for all-cancer SMRs are lower than 1.0, the local comparison being statistically significantly reduced. In contrast, the SMRs based on the total U.S. DuPont worker mortality rates for all causes of death (1.13), all cancers (1.11), and respiratory cancers (1.37) are statistically significantly increased. The SMR for liver cancer (1.27), although elevated, is not statistically significant. SMRs based on DuPont Region 1 were closer to 1.0, and the SMR for all cancers was no longer significant. Stratification of the Louisville subcohort of males using the same cumulative exposure categories used in the IISRP study yielded SMRs calculated against DuPont Region 1 that were generally higher than those calculated against U.S. and local rates. Only the third exposure category showed SMRs statistically significantly above 1.0 for all cancers and for cancer of bronchus, trachea, and lung. However, there does not appear to be an exposure-response trend. The SMRs from the IISRP study for the Pontchartrain cohort based on national rates are higher than those based on local rates for all causes of death, but all are less than 1.0. The all-cause SMRs for both local and national comparisons are significantly reduced. There were no deaths from liver cancers observed in this cohort. Comparisons of the Pontchartrain cohort against the total U.S. DuPont worker mortality rates resulted in higher SMRs for all causes of death (0.98), all cancers (1.03), and respiratory cancer (1.08), but none were statistically significant. SMRs based on DuPont Region 2 showed very little change from those based on the total registry. The use of reference rates based on regional workers in the same large company produces SMRs lower than those based on the entire company population (regional socio-cultural effects) but higher than those based on geographically closer local general populations (healthy worker effect). The healthy worker effect is seen in cancer mortality rates as well as in other chronic diseases.     

Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study)

Objective

to assess the levels and determinants of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) in a healthy Caucasian population.

Methods

population sample of 2884 men and 3201 women aged 35 to 75. IL-1β, IL-6 and TNF-α were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay.

Results

Spearman rank correlations between duplicate cytokine measurements (N = 80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48–3.90) pg/ml for IL-1β; 1.47 (0.71–3.53) pg/ml for IL-6; 2.89 (1.82–4.53) pg/ml for TNF-α and 1.3 (0.6–2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1β levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-α levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity.

Conclusion

Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.     

Analysis of gene function of bacteriophage phi 29 of Bacillus subtilis: identification of cistrons essential for viral assembly.

Restrictive infection of Bacillus subtilis by suppressor-sensitive (sus) mutants of phi 29 has been used to search for cistrons that function in viral assembly. The products of cistrons 7, 9, 10, and 16 are necessary for head morphogenesis. The neck upper collar protein P10 and the tail protein P9 must be present for DNA packaging to occur. The protein P7 must be present for phage-related particles to form. A prohead-like particle has been isolated during 16-restrictive infection. The particle is composed of the proteins Hd, P10, F, and P7. P16 must function for DNA-filled particles to accumulate. A DNA-containing particle produced in the absence of the cistron 11 product may be an intermediate in the phi 29 assembly pathway. The protein P13 interacts with P9 and P11 to form a stable DNA-filled particle. The products of cistrons 2 and 3 are essential for viral DNA synthesis, and in their absence virus-related particles are not detected.     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

Asymmetry of the acceleration and deceleration capacity of heart rate is strongly dependent on ventricular premature complexes.

In this letter, we explain the role of acceleration and deceleration capacities as novel risk predictors after myocardial infarction and their relation to the occurrence of ventricular premature complexes.     

Operational Sex Ratio and Individual Mating Frequencies in Two Bushcricket Species (Orthoptera,Tettigonioidea, Poecilimon)

During mating the male bushericket transfers a large spermatophore which is consumed by the female after copulation. Such a ‘nuptial gift' is likely to have strong effects on the mating frequency of both males and females and thus on the operational sex ratio (OSR): For the male a longer time period will be required in which he is prevented from further matings. For the female consumption may be advantageous increasing interest in matings. To quantify these effects field studies were carried out on two species of the genus Poecilimon. Both species differed widely in the mean number of matings observed per day (19% of all females in P. veluchianus compaied to 42% in P. affinis; Fig. 2) and thus in the distribution of female mating intervals (P. veluchianus 2 days, P. affinis 1 day (modes); Fig. 3). Separate experiments on the minimum male mating interval and the spermatophore weight also revealed distinct differences: the mean spermatophore weight was 26% of the male body weight in P. veluchianus and 15% in P. affinis (Fig. 6). Accordingly the refractory period was longer in P. veluchianus; 3 days after a previous mating nearh all males were ready to remate, compared to 2 days in P. affinis (Fig. 5). These differences, however, compensated one another in that the amount of spermatophylax material transferred per day was very similar in both species in relation to female body weight. Using data on female mating frequency per day and male remating ability, an upper estimate for the OSR was calculated. A lower limit for the OSR was given taking into account the most frequent female mating interval (big. 7). In both species the OSR remained relatively constant for large parts of the season. The medians (upper/lower estimate for the ratio males to females ready to mate: 2.95/1.19 in P. veluchianus; 2.22 0.88 in P. affinis) were very low, comparable to that in explosive breeding frog species. Thus in the bushcricket mating system, females may be able to demand large male mating efforts so that the operational sex ratio can come close to unity.     

Population genomic analysis of a bacterial plant pathogen: novel insight into the origin of Pierce's disease of grapevine in the U.S

Invasive diseases present an increasing problem worldwide; however, genomic techniques are now available to investigate the timing and geographical origin of such introductions. We employed genomic techniques to demonstrate that the bacterial pathogen causing Pierce's disease of grapevine (PD) is not native to the US as previously assumed, but descended from a single genotype introduced from Central America. PD has posed a serious threat to the US wine industry ever since its first outbreak in Anaheim, California in the 1880s and continues to inhibit grape cultivation in a large area of the country. It is caused by infection of xylem vessels by the bacterium Xylella fastidiosa subsp. fastidiosa, a genetically distinct subspecies at least 15,000 years old. We present five independent kinds of evidence that strongly support our invasion hypothesis: 1) a genome-wide lack of genetic variability in X. fastidiosa subsp. fastidiosa found in the US, consistent with a recent common ancestor; 2) evidence for historical allopatry of the North American subspecies X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa; 3) evidence that X. fastidiosa subsp. fastidiosa evolved in a more tropical climate than X. fastidiosa subsp. multiplex; 4) much greater genetic variability in the proposed source population in Central America, variation within which the US genotypes are phylogenetically nested; and 5) the circumstantial evidence of importation of known hosts (coffee plants) from Central America directly into southern California just prior to the first known outbreak of the disease. The lack of genetic variation in X. fastidiosa subsp. fastidiosa in the US suggests that preventing additional introductions is important since new genetic variation may undermine PD control measures, or may lead to infection of other crop plants through the creation of novel genotypes via inter-subspecific recombination. In general, geographically mixing of previously isolated subspecies should be avoided.     

Drug Effect Unveils Inter-head Cooperativity and Strain-dependent ADP Release in Fast Skeletal Actomyosin

Amrinone is a bipyridine compound with characteristic effects on the force-velocity relationship of fast skeletal muscle, including a reduction in the maximum shortening velocity and increased maximum isometric force. Here we performed experiments to elucidate the molecular mechanisms for these effects, with the additional aim to gain insight into the molecular mechanisms underlying the force-velocity relationship. In vitro motility assays established that amrinone reduces the sliding velocity of heavy meromyosin-propelled actin filaments by 30% at different ionic strengths of the assay solution. Stopped-flow studies of myofibrils, heavy meromyosin and myosin subfragment 1, showed that the effects on sliding speed were not because of a reduced rate of ATP-induced actomyosin dissociation because the rate of this process was increased by amrinone. Moreover, optical tweezers studies could not detect any amrinone-induced changes in the working stroke length. In contrast, the ADP affinity of acto-heavy meromyosin was increased about 2-fold by 1 mm amrinone. Similar effects were not observed for acto-subfragment 1. Together with the other findings, this suggests that the amrinone-induced reduction in sliding velocity is attributed to inhibition of a strain-dependent ADP release step. Modeling results show that such an effect may account for the amrinone-induced changes of the force-velocity relationship. The data emphasize the importance of the rate of a strain-dependent ADP release step in influencing the maximum sliding velocity in fast skeletal muscle. The data also lead us to discuss the possible importance of cooperative interactions between the two myosin heads in muscle contraction.Muscle contraction, as well as several other aspects of cell motility, results from cyclic interactions between myosin II motors and actin filaments. These force-generating interactions are driven by the hydrolysis of ATP at the myosin active site as outlined in Scheme 1 (1–3). In the absence of actin, the P_i and ADP release steps (k₄ and k₅) are rate-limiting for the entire cycle at high (>12 °C) and low temperatures, respectively (4–6). In the presence of actin, the rate of P_i release increases significantly, and the overall cycle is accelerated more than 2 orders of magnitude. The sliding velocity of myosin-propelled motors is generally believed to be rate-limited by actomyosin dissociation (rate constant k′₅, k′₆, or k′₂ in Scheme 1) (7). Alternatively, some studies (8, 9) have suggested that the sliding velocity is determined by the fraction of myosin heads in the weak-binding states, AM⁴ ATP and AM ADP P_i. However, it is worth emphasizing that K_T is very low under physiological conditions (1, 3) with low population of these states. For the same reason, the rate of dissociation of the AM complex is governed by K′₁ and k′₂.Open in a separate windowSCHEME 1.Simplified kinetics scheme for MgATP turnover by myosin (lower row) and actomyosin (upper row). Inorganic phosphate is denoted by P_i; MgATP is denoted by ATP, and MgADP is denoted by ADP; myosin is denoted by M. The states AM*ADP and AM ADP correspond to myosin heads with their nucleotide binding pocket in a partially closed and open conformation, respectively (7, 52). Rate constants are indicated by lowercase letters (rightward transitions, k₂ − k₅ and k′₂ − k′₅, or leftward transitions, k₋₂ − k₋₅ and k′₋₂ − k′₋₅) and equilibrium constants by uppercase letters (K₁, K′₁, K_T, K₃, K′₃, K₆, k′₆, and K_DP). The equilibrium constants are association constants except for simple bimolecular reactions where they are defined as k_i/k_−i.For the study of contractile mechanisms in both muscle and other types of cells, drugs may be useful as pharmacological tools affecting different transitions or states in the force-generating cycle. Whereas the use of drugs as tools may be less specific than site-directed mutagenesis, it also has advantages. The motor protein function may be studied in vivo, with maintained ordering of the protein components, e.g. as in the muscle sarcomere, allowing more insight into the relationship between specific molecular events and contractile properties of muscle. A drug that has been used quite extensively in this context is butanedione monoxime. The usefulness of this drug is based on firm characterization of its effect on actomyosin function on the molecular level (3, 10–13). More recently other drugs, like N-benzyl-p-toluene sulfonamide (14, 15) and blebbistatin (16), have been found to affect myosin function, and their effects at the molecular level have also been elucidated in some detail (14, 15, 17, 18). Both these drugs appear to affect the actomyosin interaction in a similar way as butanedione monoxime by inhibiting a step before (or very early in) the myosin power stroke, leading to the inhibition of actomyosin cross-bridge formation and force production.In contrast to the reduced isometric force, caused by the above mentioned drugs, the bipyridine compound amrinone (Fig. 1A) has been found to increase the isometric force production of fast intact skeletal muscles of the frog (19, 20) and mouse (21) and also of fast (but much less slow) skinned muscle fibers of the rat (22). In all the fast myosin preparations, the effect of about 1 mm amrinone on isometric force was associated with characteristic changes of the force-velocity relationship (Fig. 1B), including a reduced maximum velocity of shortening (19–22) and a reduced curvature of the force-velocity relationship (19–22). The latter effect was accompanied (20, 21) by a less pronounced deviation of the force-velocity relationship from the hyperbolic shape (23) at high loads. There have been different interpretations of the drug effects. It has been proposed (20–22) that amrinone might competitively inhibit the MgATP binding by myosin. However, more recently, results from in vitro motility assay experiments (24) challenged this idea. These results showed that amrinone reduces the sliding velocity (V_max) at saturating MgATP concentrations but not at MgATP concentrations close to, or below, the K_m value for the hyperbolic relationship between MgATP concentration and sliding velocity. Such a combination of effects is consistent with a reduced MgADP release rate (24) but not with competitive inhibition of substrate binding. However, effects of amrinone on the MgADP release rate have not been directly demonstrated. Additionally, in view of the uncertainty about what step actually determines the sliding velocity at saturating [MgATP] (see above and Refs. 7–9), it is of interest to consider other possible drug effects that could account for the data of Klinth et al. (24). These include the following: 1) an increased drag force, e.g. because of enhancement of weak actomyosin interactions; 2) a reduced step length; and 3) effects of the drug on the rate of MgATP-induced dissociation of actomyosin.Open in a separate windowFIGURE 1.A, structure of amrinone. B, experimental force-velocity data obtained in the presence (filled symbols) and absence (open symbols) of 1.1 mm amrinone. The data, from intact single frog muscle fibers, were obtained at 2 °C and fitted by Hill''s (42) hyperbola (lines) for data truncated at 80% of the maximum isometric force. Filled line, equation fitted to control data, a/P₀* = 0.185; P₀*/P₀ = 1.196. Dashed line, amrinone, a/P₀* = 0.347; P₀*/P₀ = 1.009. Force-velocity data were obtained in collaboration with Professor K. A. P. Edman. Same data as in Fig. 8 of Ref. 20. Note a decrease in maximum sliding velocity and curvature of the force-velocity relationship at low force, in response to amrinone. Also note that amrinone caused increased isometric force and a reduced deviation of the force-velocity relationship from the Hill''s hyperbola at high force. All changes of the force-velocity relationship were statistically significant (20), and similar changes were later also observed in intact mouse muscle and skinned rat muscle fibers. Data in Fig. 1 are published by agreement with Professor K. A. P. Edman.To differentiate between these hypotheses for the amrinone effects, and to gain more general insight into fundamental aspects of muscle function (e.g. mechanisms underlying the force-velocity relationship), we here study the molecular effects of amrinone on fast skeletal muscle myosin preparations in the presence and absence of actin.In vitro motility assay studies at different ionic strengths suggest that drag forces, caused by increased fraction of myosin heads in weak binding states, are not important for the effect of amrinone on sliding velocity. Likewise, optical tweezers studies showed no effect of the drug on the myosin step length. Finally, ideas that amrinone should reduce sliding velocity by reduced rate of MgATP-induced dissociation could be discarded because the drug actually increased the rate of this process. Instead, we found an amrinone-induced increase in the MgADP affinity of heavy meromyosin (HMM) in the presence of actin. Interestingly, similar effects of amrinone were not observed using myosin S1. As discussed below, this result and other results point to an amrinone-induced reduction in the rate of a strain-dependent MgADP release step. Simulations, using a model modified from that of Edman et al. (25), support this proposed mechanism of action. The results are discussed in relation to fundamental mechanisms underlying the force-velocity relationship of fast skeletal muscle, including which step determines shortening velocity and the possible importance of inter-head cooperativity.