Revisiting the structure of the Vps10 domain of human sortilin and its interaction with neurotensin

Sortilin is a multifunctional receptor involved in sorting and apoptosis. We have previously reported a 2.0‐Å structure of the Vps10 ectodomain in complex with one of its ligands, the tridecapeptide neurotensin. Here we set out to further characterize the structural properties of sortilin and its interaction with neurotensin. To this end, we have determined a new 2.7 Å structure using a crystal grown with a 10‐fold increased concentration of neurotensin. Here a second peptide fragment was observed within the Vps10 β‐propeller, which may in principle either represent a second molecule of neurotensin or the N‐terminal part of the molecule bound at the previously identified binding site. However, in vitro binding experiments strongly favor the latter hypothesis. Neurotensin thus appears to bind with a 1:1 stoichiometry, and whereas the N‐terminus does not bind on its own, it enhances the affinity in context of full‐length neurotensin. We conclude that the N‐terminus of neurotensin probably functions as an affinity enhancer for binding to sortilin by engaging the second binding site. Crystal packing differs partly from the previous structure, which may be due to variations in the degree and pattern of glycosylations. Consequently, a notable hydrophobic loop, not modeled previously, could now be traced. A computational analysis suggests that this and a neighboring loop may insert into the membrane and thus restrain movement of the Vps10 domain. We have, furthermore, mapped all N‐linked glycosylations of CHO‐expressed human sortilin by mass spectrometry and find that their locations are compatible with membrane insertion of the hydrophobic loops.     

Visualization of the eEF2-80S ribosome transition-state complex by cryo-electron microscopy

In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-Å cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the γ-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.     

Effect of dietary supplements on lean mass and strength gains with resistance exercise: a meta-analysis.

The purpose of this study was to quantify which dietary supplements augment lean mass and strength gains during resistance training. Peer-reviewed studies between the years 1967 and 2001 were included in the analysis if they met a predetermined set of experimental criteria, among which were at least 3-wk duration and resistance-training 2 or more times a week. Lean mass and strength were normalized for meta-analysis by conversion to percent change per week and by calculating the effect size for each variable. Of the 250 supplements examined, only 6 had more than 2 studies that met the criteria for inclusion in the meta-analysis. Creatine and beta-hydroxy-beta-methylbutyrate (HMB) were found to significantly increase net lean mass gains of 0.36 and 0.28%/wk and strength gains of 1.09 and 1.40%/wk (P < 0.05), respectively. Chromium, dehydroepiandrosterone, androstenedione, and protein did not significantly affect lean gain or strength. In conclusion, two supplements, creatine and HMB, have data supporting their use to augment lean mass and strength gains with resistance training.     

Influence of feeding the leucine metabolite β-hydroxy-β-methylbutyrate (HMB) on the non-specific cellular and humoral defence mechanisms of rainbow trout (Oncorhynchus mykiss)

Previous studies have shown that leucine metabolite β‐hydroxy‐β‐methylbutyrate (HMB) increases the immune function in animals as measured by cellular and humoral immune responses. In the present study the influence of feeding HMB on the nonspecific cellular and humoral defence mechanisms and protection against furunculosis in rainbow trout (Oncorhynchus mykiss) was examined. HMB was fed in a pelleted ration at either 0 (control), 10, 25 or 50 mg  kg^?1 bw day^?1 for 8 weeks. Blood and pronephros cells were taken at random from 10 fish in each group for the analyses. The respiratory burst activity (RBA) and potential killing activity (PKA) of phagocytes, lymphocyte proliferation stimulated by either concanavalin (ConA) or lipopolysaccharide (LPS), lysozyme activities and total immunoglobulin (Ig) levels in plasma were analysed at 0, 1, 2, 3, 4, 6 and 8 weeks. After 4 weeks of feeding HMB, a challenge test was performed by injection of Aeromonas salmonicida into the fish. HMB approximately doubled the respiratory burst activity and potential killing activity ability of polymorphonuclear (PMN) and morphonuclear (MN) cells (P < 0.01) and increased the mitogen‐stimulated lymphocyte proliferation (P < 0.01) when compared with the control group. HMB feeding also increased (P < 0.01) the lysozyme activity in plasma and total Ig levels in serum. During the 14‐day challenge test, mortality was decreased (P < 0.01) by up to 62% in HMB‐fed fish compared with the control group mortality. In conclusion, the non‐specific immune enhancement by HMB resulted in protection against furunculosis in the rainbow trout.     

In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques

Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified in the nonfractionated colostrum (NF) and seven fractions (F1-F7) using six different fractionation techniques. Fractionation contributed with 69 additional proteins in the fluid phase compared with NF. Different fractionation techniques each resulted in detection of unique subsets of proteins. Whey production by high-speed centrifugation contributed most to detection of low abundant proteins. Hence, prefractionation of colostrum prior to 2D-LC-MS/MS analysis expanded our knowledge on the presence and location of low abundant proteins in bovine colostrum.     

Surrogate markers for atherosclerotic disease

PURPOSE OF REVIEW: Novel treatment modalities for cardiovascular prevention are emerging rapidly. Since it is virtually impossible to evaluate all these new compounds in long-term trials using clinical end points, there is an urgent need for validated surrogate markers of atherosclerosis to save both time and costs. Over the last decade, the use of imaging markers has been widely introduced into drug-development strategies. Here we will discuss the most commonly used techniques. RECENT FINDINGS: Whereas both testing of endothelial function, assessed as flow-mediated dilation, and assessment of carotid intima-media thickness have been shown to predict future cardiovascular events, predominantly intima-media thickness has been used successfully as a surrogate marker in intervention studies. More recently, standardization of intravascular ultrasound has also enabled reproducible assessment of coronary atheroma volume. Multidetector computed tomography and electron-beam computed tomography have proven useful in providing quantitative information on plaque burden and coronary calcium content, respectively. Although cardiovascular magnetic resonance (CMR) is improving continuously, additional technical improvements will be mandatory before this technique can be implemented in multicenter clinical studies. SUMMARY: The imaging modalities reviewed here all provide specific information on either functionality or morphology of the vasculature. The value of carotid intima-media thickness for cardiovascular risk prediction has been studied most extensively. Whereas assessment of plaque burden using intravascular ultrasound appears to be the most direct way to quantify coronary changes, its predictive value for future cardiovascular events remains to be established. Awaiting further technical improvements, CMR is expected to provide the most valuable information for the evaluation of atherosclerosis in the near future.     

Salivary hormonal values from high-speed resistive exercise workouts

Our study purpose examined salivary hormonal responses to high-speed resistive exercise. Healthy subjects (n = 45) performed 2 elbow flexor workouts on a novel (inertial kinetic exercise; Oconomowoc, WI, USA) strength training device. Our methods included saliva sample collection at both preexercise and immediately postexercise; workouts entailed two 60-second sets separated by a 90-second rest period. The samples were analyzed in duplicate for their testosterone and cortisol concentrations ([T], [C]). Average and maximum elbow flexor torque were measured from each exercise bout; they were later analyzed with a 2(gender) × 2(workout) analysis of variance (ANOVA) with repeated measures for workout. The [T] and [C] each underwent a 2(gender) × 2(time) ANOVA with repeated measures for time. A within-subject design was used to limit error variance. Average and maximum torque each had gender (men > women; p < 0.05) effects. The [T] elicited a 2-way interaction (p < 0.05), as men incurred a significant 14% increase over time, but women's values were unchanged. Yet multivariate regression revealed that 3 predictor variables (body mass and average and maximum torques) did not account for a significant amount of variance associated with the rise in male [T]. Changes in [C] were not significant. In conclusion, changes in [T] concur with the results from other studies that showed significant elevations in male [T], despite the brevity of current workouts and the rather modest volume of muscle mass engaged. Practical applications imply that salivary assays may be a viable alternative to blood draws from athletes, yet coaches and others who may administer this treatment should know that our results may have produced greater pre-post hormonal changes if postexercise sample collection had occurred at a later time point.