Meningiomas are central nervous system tumors that originate from the meningeal coverings of the brain and spinal cord. Most meningiomas are pathologically benign or atypical, but 3–5% display malignant features. Despite previous studies on benign and atypical meningiomas, the key molecular pathways involved in malignant transformation remain to be determined, as does the extent of epigenetic alteration in malignant meningiomas. In this study, we explored the landscape of DNA methylation in ten benign, five atypical and four malignant meningiomas. Compared to the benign tumors, the atypical and malignant meningiomas demonstrate increased global DNA hypomethylation. Clustering analysis readily separates malignant from atypical and benign tumors, implicating that DNA methylation patterns may serve as diagnostic biomarkers for malignancy. Genes with hypermethylated CpG islands in malignant meningiomas (such as HOXA6 and HOXA9) tend to coincide with the binding sites of polycomb repressive complexes (PRC) in early developmental stages. Most genes with hypermethylated CpG islands at promoters are suppressed in malignant and benign meningiomas, suggesting the switching of gene silencing machinery from PRC binding to DNA methylation in malignant meningiomas. One exception is the MAL2 gene that is highly expressed in benign group and silenced in malignant group, representing de novo gene silencing induced by DNA methylation. In summary, our results suggest that malignant meningiomas have distinct DNA methylation patterns compared to their benign and atypical counterparts, and that the differentially methylated genes may serve as diagnostic biomarkers or candidate causal genes for malignant transformation.
Voluntary movement mediated by skeletal muscle relies on endplate acetylcholine receptors (AChR) to detect nerve-released ACh and depolarize themuscle fiber. Recent structural and mechanistic studies of the endplate AChR have catalyzed a leap in our understanding of the molecular steps in this chemical-to-electrical transduction process. Studies of acetylcholine binding protein (AChBP) give insight into ACh recognition, the first step in activation of the AChR. An atomic structural model of the Torpedo AChR at a resolution of 0.4 nm, together with single-ion channel recording methods, allow tracing of the link between the agonist binding event and gating of the ion channel, as well as determination of how the channel moves when it opens to allow flow of cations. Structural models of the human AChR enable precise mapping of disease-causing mutations, while studies of the speed with which single AChR channels open and close cast light on pathogenic mechanisms.
acetylcholine receptor; acetylcholine binding protein; agonist recognition; binding-gating coupling mechanism; congenital myasthenic syndrome
Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD). Three inactivated EV71 whole-virus vaccines of different strains developed by different manufacturers in mainland China have recently entered clinical trials. Although several studies on these vaccines have been published, a study directly comparing the immunogenicity and protective effects among them has not been carried out, which makes evaluating their relative effectiveness difficult. Thus, properly comparing newly developed vaccines has become a priority, especially in China.
Methods and Findings
This comparative immunogenicity study was carried out on vaccine strains (both live and inactivated), final container products (FCPs) without adjuvant, and corresponding FCPs containing adjuvant (FCP-As) produced by three manufacturers. These vaccines were evaluated by neutralizing antibody (NAb) responses induced by the same or different dosages at one or multiple time points post-immunization. The protective efficacy of the three vaccines was also determined in one-day-old ICR mice born to immunized female mice. Survival rates were observed in these suckling mice after challenge with 20 LD50 of EV71/048M3C2. Three FCP-As, in a dose of 200 U, generated nearly 100% NAb positivity rates and similar geometric mean titers (GMTs), especially at 14–21 days post-inoculation. However, the dynamic NAb responses were different among three vaccine strains or three FCPs. The FCP-As at the lowest dose used in clinical trials (162 U) showed good protective effects in suckling mice against lethal challenge (90–100% survival), while the ED50 of NAb responses and protective effects varied among three FCP-As.
These studies establish a standard method for measuring the immunogenicity of EV71 vaccines in mice. The data generated from our mouse model study indicated a clear dose-response relationship, which is important for vaccine quality control and assessment, especially for predicting protective efficacy in humans when combined with future clinical trial results.
The objective of this study was to investigate changes in active and passive biomechanical properties of the calf muscle-tendon unit induced by controlled ankle stretching in stroke survivors.
Ten stroke survivors with ankle spasticity/contracture and ten healthy control subjects received intervention of 60-min ankle stretching. Joint biomechanical properties including resistance torque, stiffness and index of hysteresis were evaluated pre- and post-intervention. Achilles tendon length was measured using ultrasonography. The force output of the triceps surae muscles was characterized via the torque-angle relationship, by stimulating the calf muscles at a controlled intensity across different ankle positions.
Compared to healthy controls, the ankle position corresponding to the peak torque of the stroke survivors was shifted towards plantar flexion (P<0.001). Stroke survivors showed significantly higher resistance torques and joint stiffness (P<0.05), and these higher resistances were reduced significantly after the stretching intervention, especially in dorsiflexion (P = 0.013). Stretching significantly improved the force output of the impaired calf muscles in stroke survivors under matched stimulations (P<0.05). Ankle range of motion was also increased by stretching (P<0.001).
At the joint level, repeated stretching loosened the ankle joint with increased passive joint range of motion and decreased joint stiffness. At the muscle-tendon level, repeated stretching improved calf muscle force output, which might be associated with decreased muscle fascicle stiffness, increased fascicle length and shortening of the Achilles tendon. The study provided evidence of improvement in muscle tendon properties through stretching intervention.
Stroke; Ultrasound; Muscle tendon; Spasticity; Triceps surae muscles
Paclitaxel and sirolimus are the two major drugs for the treatment of coronary arterial disease in current drug eluting stents. The two drugs can effectively inhibit the in-stent restenosis through their independent pathways and show synergistic effect in preventing tumor tissue growth. We hypothesize that the combination of the two drugs in a drug eluting stent (DES) can also effectively suppress the neointima growth in the stented artery. The present work was focused on the investigation of paclitaxel/sirolimus combination release profiles from a novel biodegradable polymer (poly (D, L-lactide -co-glycolide) / amorphous calcium phosphate, PLGA/ACP) coated stent both in vitro and in vivo. For the in vitro, the drug releasing profiles were characterized by measuring the drug concentration in a drug release medium (Dulbecco’s phosphate buffered saline, DPBS, pH 7.4) at predetermined time points. For the in vivo, a rat aorta stenting model was employed. The results showed that both paclitaxel and sirolimus had a two-phase release profile both in vitro and in vivo, which is similar to the drug release profile of their individual coated DESs, and there is no evident of interference between two drugs. The data suggest that paclitaxel and sirolimus can be combined pharmacokinetically in a DES for the treatment of coronary arterial diseases.
Amorphous Calcium Phosphate; Drug-Eluting Stent; Paclitaxel; Poly (D; L-lactide -co-glycolide); Sirolimus
To characterize the human humoral immune response against enterovirus 71 (EV71) infection and map human epitopes on the viral capsid proteins.
A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3) of BJ08 strain (genomic C4) were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA) was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 μg/μl). Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel.
A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3) were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15) was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71) were identified and mapped to VP1.
These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents.
EV71; Capsid protein; Epitopes; Humoral immune response
Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain of nicotinic acetylcholine receptors (AChRs). To understand interactions of competitive antagonists at the atomic structural level, we studied binding of the curare derivatives d-tubocurarine (d-TC) and metocurine to AChBP using computational methods, mutagenesis, and ligand binding measurements. To account for protein flexibility, we used a 2-ns molecular dynamics simulation of AChBP to generate multiple snapshots of the equilibrated dynamic structure to which optimal docking orientations were determined. Our results predict a predominant docking orientation for both d-TC and metocurine, but unexpectedly, the bound orientations differ fundamentally for each ligand. At one subunit interface of AChBP, the side chain of Tyr-89 closely approaches a positively charged nitrogen in d-TC but is farther away from the equivalent nitrogen in metocurine, whereas, at the opposing interface, side chains of Trp-53 and Gln-55 closely approach the metocurine scaffold but not that of d-TC. The different orientations correspond to ~170° rotation and ~30° degree tilt of the curare scaffold within the binding pocket. Mutagenesis of binding site residues in AChBP, combined with measurements of ligand binding, confirms the different docking orientations. Thus structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.
Pathway-based association methods have been proposed to be an effective approach in identifying disease genes, when single-marker association tests do not have sufficient power. The analysis of quantitative traits may be benefited from these approaches, by sampling from two extreme tails of the distribution. Here we tested a pathway association approach on a small genome-wide association study (GWAS) on 653 subjects with extremely high high-density lipoprotein cholesterol (HDL-C) levels and 784 subjects with low HDL-C levels. We identified 102 genes in the sterol transport and metabolism pathways that collectively associate with HDL-C levels, and replicated these association signals in an independent GWAS. Interestingly, the pathways include 18 genes implicated in previous GWAS on lipid traits, suggesting that genuine HDL-C genes are highly enriched in these pathways. Additionally, multiple biologically relevant loci in the pathways were not detected by previous GWAS, including genes implicated in previous candidate gene association studies (such as LEPR, APOA2, HDLBP, SOAT2), genes that cause Mendelian forms of lipid disorders (such as DHCR24), and genes expressing dyslipidemia phenotypes in knockout mice (such as SOAT1, PON1). Our study suggests that sampling from two extreme tails of a quantitative trait and examining genetic pathways may yield biological insights from smaller samples than are generally required using single-marker analysis in large-scale GWAS. Our results also implicate that functionally related genes work together to regulate complex quantitative traits, and that future large-scale studies may benefit from pathway-association approaches to identify novel pathways regulating HDL-C levels.
GWAS; lipid; HDL-C; pathway analysis; cholesterol; sterol transport; sterol metabolism; genetic association
Nitric oxide (NO), produced by NO synthase (NOS), modulates the function of all retinal neurons and ocular blood vessels and participates in the pathogenesis of ocular diseases. To further understand the regulation of ocular NO release, we systematically studied the morphology, topography and light responses of NOS-containing amacrine cells (NOACs) in dark-adapted mouse retina. Immunohistological staining for neuronal NOS (bNOS), combined with retrograde labeling of ganglion cells (GCs) with Neurobiotin (NB, a gap junction permeable dye) and Lucifer yellow (LY, a less permeable dye), was used to identify NOACs. The light responses of ACs were recorded under whole-cell voltage clamp conditions and cell morphology was examined with a confocal microscope. We found that in dark-adapted conditions bNOS-immunoreactivity (IR) was present primarily in the inner nuclear layer and the ganglion cell layer. bNOS-IR somas were negative for LY, thus they were identified as ACs; nearly 6 % of the cells were labeled by NB but not by LY, indicating that they were dye-coupled with GCs. Three morphological subtypes of NOACs (NI, NII and displaced) were identified. The cell density, inter-cellular distance and the distribution of NOACs were studied in whole retinas. Light evoked depolarizing highly sensitive ON-OFF responses in NI cells and less sensitive OFF responses in NII cells. Frequent (1 to 2 Hz) or abrupt change of light-intensity evoked larger peak responses. The possibility for light to modify NO release from NOACs is discussed.
retrograde labeling; patch clamp recording; dye coupling
Radiolabeled antagonists of specific peptide receptors identify a higher number of receptor binding sites than agonists and may thus be preferable for in vivo tumor targeting. In this study, two novel radioiodinated 1,4-benzodiazepines, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (7) and (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (9), were developed. They were characterized in vitro as high affinity selective antagonists at cholecystokinin type 1 and 2 (CCK1 and CCK2) receptors using receptor binding, calcium mobilization, and internalization studies. Their binding to human tumor tissues was assessed with in vitro receptor autoradiography and compared with an established peptidic CCK agonist radioligand. The 125I-labeled CCK1 receptor-selective compound 9 often revealed a substantially higher amount of CCK1 receptor binding sites in tumors than the agonist 125I-CCK. Conversely, the radioiodinated CCK2 receptor-selective compound 7 showed generally weaker tumor binding than 125I-CCK. In conclusion, compound 9 is an excellent radioiodinated non-peptidic antagonist ligand for direct and selective labeling of CCK1 receptors in vitro. Moreover, it represents a suitable candidate to test antagonist binding to CCK1 receptor-expressing tumors in vivo.
Internal force is a set of contact forces that does not disturb object equilibrium. The elements of the internal force vector cancel each other and, hence, do not contribute to the resultant (manipulation) force acting on the object. The mathematical independence of the internal and manipulation forces allows for their independent (decoupled) control realized in robotic manipulators. To examine whether in humans internal force is coupled with the manipulation force and what grasping strategy the performers utilize, the subjects (n=6) were instructed to make cyclic arm movements with a customized handle. Six combinations of handle orientation and movement direction were tested. These involved: parallel manipulations (1) VV task (vertical orientation and vertical movement) and (2) HH task (horizontal orientation and horizontal movement); orthogonal manipulations (3) VH task (vertical orientation and horizontal movement) and (4) HV task (horizontal orientation and vertical movement); and diagonal manipulations (5) DV task (diagonal orientation and vertical movement) and (6) DH task (diagonal orientation and horizontal movement). Handle weight (from 3.8 to 13.8 N), and movement frequency (from 1 to 3 Hz) were systematically changed. The analysis was performed at the thumb-virtual finger level (VF, an imaginary finger that produces a wrench equal to the sum of wrenches produced by all the fingers). At this level, the forces of interest could be reduced to the internal force and internal moment. During the parallel manipulations, the internal (grip) force was coupled with the manipulation force (producing object acceleration) and the thumb-VF forces increased or decreased in phase: the thumb and VF worked in synchrony to grasp the object more strongly or more weakly. During the orthogonal manipulations, the thumb-VF forces changed out of phase: the plots of the internal force vs. object acceleration resembled an inverted letter V. The HV task was the only task where the relative phase (coupling) between the normal forces of the thumb and VF depended on oscillation frequency. During the diagonal manipulations, the coupling was different in the DV and DH tasks. A novel observation of substantial internal moments is described: the moments produced by the normal finger forces were counterbalanced by the moments produced by the tangential forces such that the resultant moments were close to zero. Implications of the findings for the notion of grasping synergies are discussed.
Internal force; Manipulation force; Prehension; Prehension synergy; Grasping; Fingers
According to basic physics, the local effects induced by gravity and acceleration are identical and cannot be separated by any physical experiment. In contrast—as this study shows—people adjust the grip forces associated with gravitational and inertial forces differently. In the experiment, subjects oscillated a vertically-oriented handle loaded with five different weights (from 3.8 N to 13.8 N) at three different frequencies in the vertical plane: 1 Hz, 1.5 Hz and 2.0 Hz. Three contributions to the grip force—static, dynamic, and stato-dynamic fractions—were quantified. The static fraction reflects grip force related to holding a load statically. The stato-dynamic fraction reflects a steady change in the grip force when the same load is moved cyclically. The dynamic fraction is due to acceleration-related adjustments of the grip force during oscillation cycles. The slope of the relation between the grip force and the load force was steeper for the static fraction than for the dynamic fraction. The stato-dynamic fraction increased with the frequency and load. The slope of the dynamic grip force–load force relation decreased with frequency, and as a rule, increased with the load. Hence, when adjusting grip force to task requirements, the central controller takes into account not only the expected magnitude of the load force but also such factors as whether the force is gravitational or inertial and the contributions of the object mass and acceleration to the inertial force. As an auxiliary finding, a complex finger coordination pattern aimed at preserving the rotational equilibrium of the object during shaking movements was reported.
Prehension; Grasping; Stato-dynamic relation; Grip force; Load force
In a majority of studies on grasp, only normal forces were measured and only when a zero torque was exerted on a hand-held object. This study concerns finger force vectors during the torque production tasks. Subjects (n = 8) stabilized a handle with an attachment that allowed for change of external torque from −1.5 to 1.5Nm. Forces and moments exerted by the digit tips on the object were recorded. At the large (>−0.375Nm) supination torques the index/middle and ring/little pairs of fingers generated oppositely directed tangential forces. The index and middle finger produced forces in a downward direction and therefore did not support the load. At a zero torque and pronation torques, the middle, ring and little fingers produced forces along nearly the same direction. The vector of the index finger force was always directed differently from the vectors of other finger forces, the angles ranged from 19°30′ to 47°40′. The points of force application were systematically displaced with the torque, with the exception of the little finger. Tangential finger forces contributed substantially to the total torque exerted on the hand-held object.
Finger forces; Prehension; Grasping
The experimental evidence supports the validity of the principle of superposition for multi-finger prehension in humans. Forces and moments of individual digits are defined by two independent commands: “Grasp the object stronger/weaker to prevent slipping” and “Maintain the rotational equilibrium of the object”. The effects of the two commands are summed up.
Superposition principle; Human prehension; Robotics
We studied adjustments in digit forces and moments during holding a vertically oriented handle under slow, externally imposed changes in the width of the grasp. Subjects (n = 8) grasped a customized motorized handle with five digits and held it statically in the air. The handle width either increased (expanded) or decreased (contracted) at a rate of 1.0, 1.5, or 2.0 mm/s, while the subjects were asked to ignore the handle width changes, and their attention was distracted. External torques of 0.0, 0.25, and 0.5 Nm were applied to the handle in two directions. Forces and moments at the digit tips were measured with six-component sensors. The analysis was performed at the virtual finger (VF) and individual finger (IF) levels (VF is an imagined finger that produces the same wrench, i.e., the force and moment, as several fingers combined). In all the tasks, the normal VF and thumb forces increased with the handle expansion and decreased with the handle contraction. Similar behavior was seen for the thumb tangential force. In contrast, the VF tangential force decreased with the handle expansion and increased with the handle contraction. The changes in the tangential forces assisted the perturbations in the tasks requiring exertion of the supination moments and annulled the perturbation in the pronation effort tasks. In the former tasks, the equilibrium was maintained by the changes of the moments of normal forces, whereas in the latter tasks, the equilibrium was maintained by the changes of the moments of the tangential forces. Analysis at the IF level has shown that the resultant force and moment exerted on the object could arise from dissimilar adjustments of individual fingers to the same handle width change. The complex adjustments of digit forces to handle width change may be viewed as coming from two sources. First, there are local spring-like adjustments of individual digit forces and moments caused by both mechanical properties of the digits and the action of spinal reflexes. These stiffness-like reactions mainly assist in perturbing the rotational equilibrium of the object rather than in maintaining it. Second, there are tilt-preventing adjustments defined by the common task constraints that unite the digits into a task-specific synergy. The “virtual springs theory” developed in robotics literature is insufficient for describing the phenomena observed in human grasping.
We have examined the interaction among individual finger forces in tasks that required the production of the total force by a subset of fingers in a particular direction in the flexion–extension plane. Nine subjects produced fingertip forces in a prescribed direction with a maximum voluntary contraction (MVC) effort and held the peak force for two seconds. Six finger combinations were tested, four single-finger tasks—Index (I), Middle (M), Ring (R) and Little (L)—one two-digit task (IM), and one four-digit task (IMRL). The subjects were asked to generate the finger forces in two directions, 0° (perpendicular to the surface of the transducer) and 15° toward the palm. In all task conditions, there were two experimental sessions, with and without visual feedback on the task force vector. The main findings were:
The target direction significantly affected the constant error (CE) but not the variable error (VE) while removal of the feedback resulted in an increase in VE.The direction of the forces produced by fingers that were not explicitly required to produce force (enslaved fingers) depended on the target direction.In multi-finger tasks, the individual fingers produced force in directions that could differ significantly from the target direction, while the resultant force pointed in the target direction. There was a negative co-variation among the deviations of the directions of the individual finger forces from the target direction. If a finger force vector deviated from the target, another finger force vector was likely to deviate in the opposite direction. We conclude that a multi-finger synergy is involved in the control of the finger force direction.
Synergy; Finger; Force; Error compensation; Variability; Human
We address issues of simultaneous control of the grasping force and the total moment of forces applied to a handheld object during its manipulation. Six young healthy male subjects grasped an instrumented handle and performed its cyclic motion in the vertical direction. The handle allowed for setting different clockwise (negative) or counterclockwise torques. Three movement frequencies: 1, 1.5 and 2 Hz, and five different torques: −1/3, −1/6, 0, 1/6 and 1/3 Nm, were used. The rotational equilibrium was maintained by two means: (1) Concerted changes of the moments produced by the normal and tangential forces, specifically anti-phase changes of the moments during the tasks with zero external torque and in-phase changes during the non-zero-torque tasks, and (2) Redistribution of the normal forces among individual fingers such that the agonist fingers—the fingers that resist external torque—increased the force in phase with the acceleration, while the forces of the antagonist fingers—those that assist the external torque—especially, the fingers with the large moment arms, the index and little fingers, stayed unchanged. The observed effects agree with the principle of superposition—according to which some complex actions, for example, prehension, can be decomposed into elemental actions controlled independently—and the mechanical advantage hypothesis according to which in moment production the fingers are activated in proportion to their moment arms with respect to the axis of rotation. We would like to emphasize the linearity of the observed relations, which was not prescribed by the task mechanics and seems to be produced by specific neural control mechanisms.
Prehension; Grip force; Load force; Finger; Synergies; Rotation equilibrium; Cyclic arm movement; Principle of superposition; Safety margin
The cholecystokinin (CCK1) receptor is a G protein-coupled receptor important for nutrient homeostasis. The molecular basis of CCK-receptor binding has been debated, with one prominent model suggesting occupation of the same region of the intramembranous helical bundle as benzodiazepines. Here, we used a specific assay of allosteric ligand interaction to probe the mode of binding of devazepide, a prototypic benzodiazepine ligand. Devazepide elicited marked slowing of dissociation of pre-bound CCK, only possible through binding to a topographically distinct allosteric site. This effect was disrupted by chemical modification of a cysteine in the benzodiazepine-binding pocket. Application of an allosteric model to the equilibrium interaction between a series of benzodiazepine ligands and CCK yielded quantitative estimates of each modulator’s affinity for the allosteric site, as well as the degree of negative cooperativity for the interaction between occupied orthosteric and allosteric sites. The allosteric nature of benzodiazepine binding to the CCK1 receptor provides new opportunities for small molecule drug development.
Allosteric modulators; cholecystokinin receptor; cooperativity; G protein-coupled receptors
Two structurally-related guanine nucleotide-binding protein-coupled receptors for two related peptides, cholecystokinin (CCK) and gastrin, have evolved to exhibit substantial diversity in specificity of ligand recognition, in their molecular basis of binding these ligands, and in their mechanisms of biochemical and cellular regulation. Consistent with this, the CCK1 and CCK2 receptors also play unique and distinct roles in physiology and pathophysiology. The paradigms for ligand recognition and receptor regulation and function are reviewed in this article, and should be broadly applicable to many members of this remarkable receptor superfamily. This degree of specialization is instructive and provides an encouraging basis for the diversity of potential drugs targeting these receptors and their actions that can be developed.
cholecystokinin; gastrin; photoaffinity labeling; GPCR; molecular modeling
Activation of guanine nucleotide-binding protein (G protein)-coupled receptors is believed to involve conformational change that exposes a domain for G protein coupling at the cytosolic surface of the helical confluence, although the mechanisms for achieving this are not well understood. This conformational change can be achieved by docking a diverse variety of agonist ligands, known to occur by interacting with different regions of these receptors. In the current study, we focus on the importance of a specific basic residue (Lys187) within the second extracellular loop of the receptor for the peptide hormone, cholecystokinin. Alanine-replacement and charge-reversal mutagenesis of this residue showed that it had no effect on the binding of natural peptide and non-peptidyl ligands of this receptor, but markedly interfered with agonist-stimulated signaling. It was demonstrated that this negative effect on biological activity could be eliminated with the truncation of the first thirty residues of the amino-terminal tail of this receptor. Complementary charge-reversal mutagenesis of each of the five conserved acidic residues within this region of the receptor in the presence of the charge-reversed Lys187 revealed that only the Asp5 mutant fully reversed the negative functional impact of the Lys187 charge reversal. Thus, we have demonstrated that a basic residue within the second extracellular loop of the cholecystokinin receptor interacts with a specific acidic residue within the amino terminus of this receptor. This residue-residue interaction is nicely accommodated within a new molecular model of the agonist-occupied cholecystokinin receptor.
G protein-coupled receptor; cholecystokinin receptor; cholecystokinin; receptor activation; charge-reversal mutagenesis
The tight coupling between load (L) and grip (G) forces during voluntary manipulation of a hand-held object is well established. The current study is to examine grip-load force coupling when motion of the hand with an object was either self-generated (voluntary) or externally generated. Subjects performed similar cyclic movements of different loads at various frequencies with three types of manipulation: (a) voluntary oscillation, (b) oscillating the right arm via the pulley system by the left leg (self-driven oscillation), (c) oscillating the arm via the pulley system by another person (other-driven oscillation). During the self-generated movements: (a) the grip forces were larger and (b) grip-load force modulation was more pronounced than in the externally generated movements. The G-L adjustments are not completely determined by the mechanics of object motion; non-mechanical factors related to movement performance, for instance perceptual factors, may affect the G-L coupling. Potentially the results of study can be used to provide hand therapists with another way for administering the Rapid Exchange Gripping Test.
grip force; load force; manipulation; voluntary; passive; feed-forward control
Stress tolerance of the heart requires high-fidelity metabolic sensing by ATP-sensitive potassium (KATP) channels that adjust membrane potential-dependent functions to match cellular energetic demand. Scanning of genomic DNA from individuals with heart failure and rhythm disturbances due to idiopathic dilated cardiomyopathy identified two mutations in ABCC9, which encodes the regulatory SUR2A subunit of the cardiac KATP channel. These missense and frameshift mutations mapped to evolutionarily conserved domains adjacent to the catalytic ATPase pocket within SUR2A. Mutant SUR2A proteins showed aberrant redistribution of conformations in the intrinsic ATP hydrolytic cycle, translating into abnormal KATP channel phenotypes with compromised metabolic signal decoding. Defective catalysis-mediated pore regulation is thus a mechanism for channel dysfunction and susceptibility to dilated cardiomyopathy.