The stimulation of mitogenic signaling pathways by N-POMC peptides

The N-terminal fragment of pro-opiomelancortin (POMC) has been shown previously to act as an adrenal mitogen. However, little is known about the molecular mechanisms by which mitogenesis is stimulated, although it has been shown that N-POMC1-28 Stimulates the ERK pathway in human H295R cells. We have investigated signaling stimulated by N-POMC1-28 and N-POMC1-49 in the mouse Y1 cell line and found that both peptides stimulate ERK phosphorylation with maximal stimulation being achieved within 5 min. Similar results were observed for both MEK and c-Raf phosphorylation, although N-POMC1-49 stimulated the phosphorylation of Akt more robustly than N-POMC1-28. We also investigated the expression of tyrosine kinase receptors in adrenal cells. PCR utilizing degenerate primers was performed on cDNA from both Y1 cells and rat adrenal tissue. Sequencing of 114 clones from each cDNA population revealed the expression of a number of receptors, several of which have not been described previously in the adrenal. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Mechanisms of G protein activation via the D-2 dopamine receptor: evidence for persistent receptor/G protein interaction after agonist stimulation

Background and purpose: The aim of this report is to study mechanisms of G protein activation by agonists. Experimental approach: The association and dissociation of guanosine 5'-O-(3-[S-35] thio) triphosphate ([S-35] GTP gamma S) binding at G proteins in membranes of CHO cells stably transfected with the human dopamine D-2short receptor was studied in the presence of a range of agonists. Key results: Binding of [S-35] GTPgS was dissociable in the absence of agonist and dissociation was accelerated both in rate and extent by dopamine, an effect which was blocked by the dopamine D-2 receptor antagonist raclopride and by suramin, which inhibits receptor/G protein interaction. A range of agonists of varying efficacy increased the rate of dissociation of [S-35] GTPgS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre-bound [S-35] GTPgS, leaving a component which may not be accessible to the agonist-bound receptor. The dissociable component of the [S-35] GTPgS binding was reduced with longer association times and increased [S-35] GTPgS concentrations. Conclusions and implications: These data are consistent with [S-35] GTPgS binding being initially to receptor-linked G proteins and then to G proteins which have separated from the agonist bound receptor. Under the conditions used typically for [S-35] GTPgS binding assays, therefore, much of the agonist-receptor complex remains in proximity to G proteins after they have been activated by agonist.

Optokinetic stimulation induces illusory movement of both out-of-the-body and on-the-body hand-held visual objects

The coding of body part location may depend upon both visual and proprioceptive information, and allows targets to be localized with respect to the body. The present study investigates the interaction between visual and proprioceptive localization systems under conditions of multisensory conflict induced by optokinetic stimulation (OKS). Healthy subjects were asked to estimate the apparent motion speed of a visual target (LED) that could be located either in the extrapersonal space (visual encoding only, V), or at the same distance, but stuck on the subject's right index finger-tip (visual and proprioceptive encoding, V-P). Additionally, the multisensory condition was performed with the index finger kept in position both passively (V-P passive) and actively (V-P active). Results showed that the visual stimulus was always perceived to move, irrespective of its out- or on-the-body location. Moreover, this apparent motion speed varied consistently with the speed of the moving OKS background in all conditions. Surprisingly, no differences were found between V-P active and V-P passive conditions in the speed of apparent motion. The persistence of the visual illusion during the active posture maintenance reveals a novel condition in which vision totally dominates over proprioceptive information, suggesting that the hand-held visual stimulus was perceived as a purely visual, external object despite its contact with the hand.

Switching curve control of functional electrical stimulation assisted rowing exercise in paraplegia

An indoor rowing machine has been modified for functional electrical stimulation (FES) assisted rowing exercise in paraplegia. To perform the rowing manoeuvre successfully, however, the voluntarily controlled upper body movements must be co-ordinated with the movements of the electrically stimulated paralysed legs. To achieve such co-ordination, an automatic FES controller was developed that employs two levels of hierarchy. At the upper level, a finite state controller identifies the state or phase of the rowing cycle and activates the appropriate lower-level controller, in which electrical stimulation to the paralysed leg muscles is applied with reference to switching curves representing the desired seat velocity as a function of the seat position. In a pilot study, the hierarchical control of FES rowing was shown to be intuitive, reliable and easy to use. Compared with open-loop control of stimulation, all three variants of the closed-loop switching curve controllers used less muscle stimulation per rowing cycle (73% of the open-loop control on average). Further, the closed-loop controller that used switching curves derived from normal rowing kinematics used the lowest muscle stimulation (65% of the open-loop control) and was the most convenient to use for the client.

Development and experimental identification of a biomechanical model of the trunk for functional electrical stimulation control in paraplegia

Objectives. Theoretic modeling and experimental studies suggest that functional electrical stimulation (FES) can improve trunk balance in spinal cord injured subjects. This can have a positive impact on daily life, increasing the volume of bimanual workspace, improving sitting posture, and wheelchair propulsion. A closed loop controller for the stimulation is desirable, as it can potentially decrease muscle fatigue and offer better rejection to disturbances. This paper proposes a biomechanical model of the human trunk, and a procedure for its identification, to be used for the future development of FES controllers. The advantage over previous models resides in the simplicity of the solution proposed, which makes it possible to identify the model just before a stimulation session ( taking into account the variability of the muscle response to the FES). Materials and Methods. The structure of the model is based on previous research on FES and muscle physiology. Some details could not be inferred from previous studies, and were determined from experimental data. Experiments with a paraplegic volunteer were conducted in order to measure the moments exerted by the trunk-passive tissues and artificially stimulated muscles. Data for model identification and validation also were collected. Results. Using the proposed structure and identification procedure, the model could adequately reproduce the moments exerted during the experiments. The study reveals that the stimulated trunk extensors can exert maximal moment when the trunk is in the upright position. In contrast, previous studies show that able-bodied subjects can exert maximal trunk extension when flexed forward. Conclusions. The proposed model and identification procedure are a successful first step toward the development of a model-based controller for trunk FES. The model also gives information on the trunk in unique conditions, normally not observable in able-bodied subjects (ie, subject only to extensor muscles contraction).

The application of implant technology for cybernetic systems

Objective: To assess the usefulness, compatibility, and long-term operability of a microelectrode array into the median nerve of the left arm of a healthy volunteer, including perception of feedback stimulation and operation of an instrumented prosthetic hand. Setting: The study was carried out from March 14 through June 18, 2002, in England and the United States. Results: The blindfolded subject received feedback information, obtained from force and slip sensors on the prosthetic hand, and subsequently used the implanted device to control the hand by applying an appropriate force to g rip an unseen object. Operability was also demonstrated remotely via the Internet, with the subject in New York, NY, and the prosthetic hand in Reading, England. Finally, the subject was able to control an electric wheelchair, via decoded signals from the implant device, to select the direction of travel by opening and closing his hand. The implantation did not result in infection or any perceivable loss of hand sensation or motion control. The implant was finally extracted because of mechanical fatigue of the percutaneous connection. Further testing after extraction has not indicated any measurable long-term defects in the subject. Conclusions: This implant may allow recipients to have abilities they would otherwise not possess. The response to stimulation improved considerably during the trial, suggesting that the subject learned to process the incoming information more effectively.

The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

Neuromuscular junction morphology, fiber-type proportions, and satellite-cell proliferation rates are altered in MyoD(-/-) mice

Gene compensation by members of the myogenic regulatory factor (MRF) family has been proposed to explain the apparent normal adult phenotype of MyoD(-/-) mice. Nerve and field stimulation were used to investigate contraction properties of muscle from MyoD(-/-) mice, and molecular approaches were used to investigate satellite-cell behavior. We demonstrate that MyoD deletion results in major alterations in the organization of the neuromuscular junction, which have a dramatic influence on the physiological contractile properties of skeletal muscle. Second, we show that the lineage progression of satellite cells (especially initial proliferation) in the absence of MyoD is abnormal and linked to perturbations in the nuclear localization of beta-catenin, a key readout of canonical Wnt signaling. These results show that MyoD has unique functions in both developing and adult skeletal muscle that are not carried out by other members of the MRF family.

Coppicing shifts CO2 stimulation of poplar productivity to above-ground pools: a synthesis of leaf to stand level results from the POP/EUROFACE experiment

A poplar short rotation coppice (SRC) grown for the production of bioenergy can combine carbon (C) storage with fossil fuel substitution. Here, we summarize the responses of a poplar (Populus) plantation to 6 yr of free air CO2 enrichment (POP/EUROFACE consisting of two rotation cycles). We show that a poplar plantation growing in nonlimiting light, nutrient and water conditions will significantly increase its productivity in elevated CO2 concentrations ([CO2]). Increased biomass yield resulted from an early growth enhancement and photosynthesis did not acclimate to elevated [CO2]. Sufficient nutrient availability, increased nitrogen use efficiency (NUE) and the large sink capacity of poplars contributed to the sustained increase in C uptake over 6 yr. Additional C taken up in high [CO2] was mainly invested into woody biomass pools. Coppicing increased yield by 66% and partly shifted the extra C uptake in elevated [CO2] to above-ground pools, as fine root biomass declined and its [CO2] stimulation disappeared. Mineral soil C increased equally in ambient and elevated [CO2] during the 6 yr experiment. However, elevated [CO2] increased the stabilization of C in the mineral soil. Increased productivity of a poplar SRC in elevated [CO2] may allow shorter rotation cycles, enhancing the viability of SRC for biofuel production.

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases

OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase, endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.