The Distributed Annotation System

Background  

Currently, most genome annotation is curated by centralized groups with limited resources. Efforts to share annotations transparently among multiple groups have not yet been satisfactory.     

Effects of angiotensin II microinjected into medial amygdala on male sexual behavior in rats

Research was undertaken to study the role of central angiotensin in the modulation of male sexual behavior, testing the effect of angiotensin II (Ang II) injections into the medial amygdaloid nucleus (MeA). The sexual behavior of adult male Wistar rats was evaluated, 15 min after bilateral intra-amygdaloid microinjection (0.3 microl) of saline and 5 doses of Ang II: 10; 25; 50; 100, and 150 fmol. The effects of the Ang II receptor blockade were also studied. We tested the effect of coinjection of Ang II (50 fmol) with the AT1 antagonist, losartan (20 pmol) and the AT2 antagonist, CGP 42112 (1 pmol). Ang II inhibited sexual behavior and this inhibition was prevented by the coinjection of AT1 antagonist, losartan, or the AT2 antagonist, CGP 42112. Results show that Ang II has a powerful effect on male sexual behavior, which may be mediated by both AT1 and AT2 receptors.     

Effects of fasting on the control of fatty-acid synthesis in hepatoma 7777 and host liver. Role of long-chain fatty acyl-CoA,, the mitochondrial citrate transporter and pyruvate dehydrogenase activity.

The effects of fasting on the rate of fatty acid synthesis, the properties of the mitochondrial citrate transporter and on pyruvate dehydrogenase activity were investigated in "poorly-differentiated" tmorris hepatoma 7777 and in host liver preparations. The properties of the citrate transporter from hepatoma mitochondria were similar to those of host liver mitochondria, with the exception that the Km for the liver mitochondrial citrate transporter was 248 plus or minus 20 mu M while that in hepatoma mitochondria was less than 75 mu M. The acid-insoluble CoA content was 180 plus or minus 20 pmol/mg protein in the hepatoma and remained essentially unchanged in the fasted state, while the acid-insoluble CoA levels in livers from fed rats was 720 plus or minus 80 pmol/mg protein and were increased to 1050 plus or minus 50 pmol/mg protein during fasting. After a 36-h fast, the rate of lipogenesis and the percentage of pyruvate dehydrogenase present in the active form were each decreased by approximately 80% in host liver preparations. In contrast, the rate of lipogenesis by hepatoma slices did not decrease during fasting, and essentially all pyruvate dehydrogenase present was in the active form of hepatomas obtained from either fed or fasted animals. Implications concerning the identification of possible regulatory sites in the control of lipogenesis were discussed in relation to the above observations.     

Comparison of Proteoglycans Extracted by Saline and Guanidinium Chloride from Cultured Chick Retinas

Abstract: To compare the loosely associated sulfated proteoglycans with those tightly bound to membranes, retinas from 14-day chick embryos were subjected to progressively disruptive techniques. The most easily removed proteoglycans were isolated from the medium in which the tissue was labeled with [³⁵S]sulfate. On the average, 25% of the glycosaminoglycans were in the labeling medium, 39% were in proteoglycans extracted from the tissue in the balanced salt solution, 32% were in a 4 m -guanidinium chloride (GuCl) fraction, and 4% remained unextracted. These glycosaminoglycans contained, respectively, 28, 28, 40, and 4% of the incorporated [³⁵S]sulfate. On the basis of electrophoretic mobility and TLC of chondroitinase digests, the ratio of ³⁵S in chondroitin sulfate to that in heparan sulfate was 4–7 times higher in the medium and balanced salt extracts than in the GuCl extracts. In both extracts there was more ³⁵S in chondroitin-6-sulfate than in chondroitin-4-sulfate. Dialysis of the extracts against 0.5 M-NaCl resulted in the precipitation of about 12% of the glycosaminoglycans in the saline extracts and about 40% in GuCl extract. These subfractions, which were relatively enriched in heparan sulfate, were largely soluble in dithiothreitol in 8 m -urea (DTT). Similarities between the proteoglycans in the medium and those extracted by balanced salt solutions suggest that the saline-extracted proteoglycans were for the most part loosely associated with cell surfaces or extracellular matrices, whereas the GuCl-extracted proteoglycans probably were bound to membranes.     

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.     

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.