Multi-focal Stimulation of the Cortico-cerebellar Loop During the Acquisition of a Novel Hand Motor Skill in Chronic Stroke Survivors.

Impairment of hand motor function is a frequent consequence after a stroke and strongly determines the ability to regain a self-determined life. An influential research strategy for improving motor deficits is the combined application of behavioral training and non-invasive brain stimulation of the motor cortex (M1). However, a convincing clinical translation of the present stimulation strategies has not been achieved yet. One alternative and innovative approach is to target the functionally relevant brain network-based architecture, e.g., the dynamic interactions within the cortico-cerebellar system during learning. Here, we tested a sequential multifocal stimulation strategy targeting the cortico-cerebellar loop. Anodal transcranial direct current stimulation (tDCS) was applied simultaneously to a hand-based motor training in N = 11 chronic stroke survivors during four training sessions on two consecutive days. The tested conditions were: sequential multifocal (M1-cerebellum (CB)-M1-CB) vs. monofocal control stimulation (M1-sham-M1-sham). Additionally, skill retention was assessed 1 and 10 days after the training phase. Paired-pulse transcranial magnetic stimulation data were recorded to characterize stimulation response determining features. The application of CB-tDCS boosted motor behavior in the early training phase in comparison to the control condition. No faciliatory effects on the late training phase or skill retention were detected. Stimulation response variability was related to the magnitude of baseline motor ability and short intracortical inhibition (SICI). The present findings suggest a learning phase-specific role of the cerebellar cortex during the acquisition of a motor skill in stroke and that personalized stimulation strategies encompassing several nodes of the underlying brain network should be considered.

Dissociative ionization and Coulomb explosion of CH4 in two-color asymmetric intense laser fields.

Directional fragment ejection from a tetrahedral molecule CH4 in linearly polarized two-color (ω and 2ω) asymmetric intense laser fields (50 fs, 1.4 × 1014 W cm-2, 800 nm and 400 nm) has been studied by three-dimensional ion coincidence momentum imaging. The H+ fragment produced from dissociative ionization, CH4 → H+ + CH3 + e-, is preferentially ejected on the larger amplitude side of the laser electric fields. Comparison with theoretical predictions by weak-field asymptotic theory shows that the observed asymmetry can be understood by the orientation selective tunneling ionization from the triply degenerated highest occupied molecular orbital (1t2) of CH4. A similar directional ejection of H+ was also observed for the low kinetic energy components of the two-body Coulomb explosion, CH4 → H+ + CH3+ + 2e-. On the other hand, the fragment ejection in the opposite direction were observed for the high energy component, as well as H2+ produced from the Coulomb explosion CH4 → H2+ + CH2+ + 2e-. Possible origins of the characteristic fragmentation are discussed.

Helicity-dependent dissociative tunneling ionization of CF4 in multicycle circularly polarized intense laser fields.

Dissociative tunneling ionization of tetrafluoromethane (CF4) in circularly polarized ultrashort intense laser fields (35 fs, 0.8 × 1014 W cm-2, 1035 nm), CF4 → CF4+ + e- → CF3+ + F + e-, has been studied by three-dimensional electron-ion coincidence momentum imaging. The photoelectron angular distribution in the recoil frame revealed that the dissociative tunneling ionization occurs efficiently when the laser electric field points from F to C. The obtained results are qualitatively consistent with the theoretical predictions by the weak-field asymptotic theory (WFAT) for tunneling ionization from the highest and next-highest occupied molecular orbitals, HOMO (1t1), and HOMO-1 (4t2), respectively. On the other hand, the angular distribution shows clear dependences on the polarization helicity, indicating that the breaking of the C-F bonds is sensitive to the helicity of the multicycle circularly polarized laser fields.

Theory of high harmonic generation for probing time-resolved large-amplitude molecular vibrations with ultrashort intense lasers.

We present a theory that incorporates the vibrational degrees of freedom in a high-order harmonic generation (HHG) process with ultrashort intense laser pulses. In this model, laser-induced time-dependent transition dipoles for each fixed molecular geometry are added coherently, weighted by the laser-driven time-dependent nuclear wave packet distribution. We show that the nuclear distribution can be strongly modified by the HHG driving laser. The validity of this model is first checked against results from the numerical solution of the time-dependent Schrödinger equation for a simple model system. We show that in combination with the established quantitative rescattering theory this model is able to reproduce the time-resolved pump-probe HHG spectra of N(2)O(4) reported by Li et al. [Science 322, 1207 (2008)].

Targeting the frontoparietal network using bifocal transcranial alternating current stimulation during a motor sequence learning task in healthy older adults.

BACKGROUND: Healthy older adults show a decrease in motor performance and motor learning capacity as well as in working memory (WM) performance. WM has been suggested to be involved in motor learning processes, such as sequence learning. Correlational evidence has shown the involvement of the frontoparietal network (FPN), a network underlying WM processes, in motor sequence learning. However, causal evidence is currently lacking. Non-invasive brain stimulation (NIBS) studies have focused so far predominantly on motor-related areas to enhance motor sequence learning while areas associated with more cognitive aspects of motor learning have not yet been addressed. HYPOTHESIS: In this study, we aim to provide causal evidence for the involvement of WM processes and the underlying FPN in the successful performance of a motor sequence learning task by using theta transcranial alternating current stimulation (tACS) targeting the FPN during a motor sequence learning task. METHODS: In a cohort of 20 healthy older adults, we applied bifocal tACS in the theta range to the FPN during a sequence learning task. With the use of a double-blind, cross-over design, we tested the efficacy of active compared to sham stimulation. Two versions of the motor task were used: one with high and one with low WM load, to explore the efficacy of stimulation on tasks differing in WM demand. Additionally, the effects of stimulation on WM performance were addressed using an N-back task. The tACS frequency was personalized by means of EEG measuring the individual theta peak frequency during the N-back task. RESULTS: The application of personalized theta tACS to the FPN improved performance during the motor sequence learning task with high WM load (p < .001), but not with low WM load. Active stimulation significantly improved both speed (p < .001), and accuracy (p = .03) during the task with high WM load. In addition, the stimulation paradigm improved performance on the N-back task for the 2-back task (p = .013), but not for 1-back and 3-back. CONCLUSION: The performance during a motor sequence learning task can be enhanced by means of personalized bifocal theta tACS to the FPN when WM load is high, indicating that the efficacy of this stimulation paradigm is dependent on the cognitive demand during the learning task. These data provide further causal evidence for the critical involvement of WM processes and the FPN during the execution of a motor sequence learning task in healthy older. These findings open new exciting possibilities to counteract the age-related decline in motor performance, learning capacity and WM performance.

Enhanced nonlinear double excitation of He in intense extreme ultraviolet laser fields.

Nonlinear, three-photon double excitation of He in intense extreme ultraviolet free-electron laser fields (∼24.1 eV, ∼5 TW/cm2) is presented. Resonances to the doubly excited states converging to the He+ N=3 level are revealed by the shot-by-shot photoelectron spectroscopy and identified by theoretical calculations based on the time-dependent Schrödinger equation for the two-electron atom under a laser field. It is shown that the three-photon double excitation is enhanced by intermediate Rydberg states below the first ionization threshold, giving a greater contribution to the photoionization yields than the two-photon process by more than 1 order of magnitude.

Prognostic impact of six versus eight cycles of standard regimen in patients with diffuse large B-cell lymphoma: propensity score-matching analysis.

BACKGROUND: R-CHOP-21 has been the standard treatment for diffuse large B-cell lymphoma (DLBCL), but there is a paucity of evidence focusing on the number of cycles of regimens. PATIENTS AND METHODS: We conducted a retrospective study to compare the effectiveness of six cycles of standard regimens versus eight cycles for overall survival (OS) in DLBCL patients using propensity score matching, in consideration of relative dose intensity (RDI). RESULTS: A total of 685 patients with newly diagnosed DLBCL were identified in three institutions from 2007 to 2017. Patients treated using six cycles of standard regimens were matched by propensity scores with those treated using eight cycles. A 1 : 1 propensity score matching yielded 138 patient pairs. Eight cycles did not significantly improve OS in the conventional Cox proportional hazards model (hazard ratio 0.849, 95% confidence interval 0.453-1.588, P = 0.608). Restricted cubic spline Cox models for OS confirmed that the effect of the number of cycles was not modified by total average RDI, the International Prognostic Index, and age. Occurrence of adverse events did not differ between six and eight cycles. CONCLUSION: Even considering the impact of RDI, six cycles of the initial standard regimen for DLBCL is not inferior to eight cycles.

Brain penetration effects of microelectrodes and DBS leads in STN or GPi.

OBJECTIVE: To determine how intraoperative microelectrode recordings (MER) and intraoperative lead placement acutely influence tremor, rigidity, and bradykinesia. Secondarily, to evaluate whether the longevity of the MER and lead placement effects were influenced by target location (subthalamic nucleus (STN) or globus pallidus interna (GPi)). BACKGROUND: Currently most groups who perform deep brain stimulation (DBS) for Parkinson disease (PD) use MER, as well as macrostimulation (test stimulation), to refine DBS lead position. Following MER and/or test stimulation, however, there may be a resultant "collision/implantation" or "microlesion" effect, thought to result from disruption of cells and/or fibres within the penetrated region. These effects have not been carefully quantified. METHODS: 47 consecutive patients with PD undergoing unilateral DBS for PD (STN or GPi DBS) were evaluated. Motor function was measured at six time points with a modified motor Unified Parkinson Disease Rating Scale (UPDRS): (1) preoperatively, (2) immediately after MER, (3) immediately after lead implantation/collision, (4) 4 months following surgery-off medications, on DBS (12 h medication washout), (5) 6 months postoperatively-off medication and off DBS (12 h washout) and (6) 6 months-on medication and off DBS (12 h washout). RESULTS: Significant improvements in motor scores (p<0.05) (tremor, rigidity, bradykinesia) were observed as a result of MER and lead placement. The improvements were similar in magnitude to what was observed at 4 and 6 months post-DBS following programming and medication optimisation. When washed out (medications and DBS) for 12 h, UPDRS motor scores were still improved compared with preoperative testing. There was a larger improvement in STN compared with GPi following MER (p<0.05) and a trend for significance following lead placement (p<0.08) but long term outcome was similar. CONCLUSION: This study demonstrated significant acute intraoperative penetration effects resulting from MER and lead placement/collision in PD. Clinicians rating patients in the operating suite should be aware of these effects, and should consider pre- and post-lead placement rating scales prior to activating DBS. The collision/implantation effects were greater intraoperatively with STN compared with GPi, and with greater disease duration there was a larger effect.

Requirement of Saccharomyces cerevisiae Ras for completion of mitosis.

In the yeast Saccharomyces cerevisiae, Ras regulates adenylate cyclase, which is essential for progression through the G1 phase of the cell cycle. However, even when the adenosine 3',5'-monophosphate (cAMP) pathway was bypassed, the double disruption of RAS1 and RAS2 resulted in defects in growth at both low and high temperatures. Furthermore, the simultaneous disruption of RAS1, RAS2, and the RAS-related gene RSR1 was lethal at any temperature. The triple-disrupted cells were arrested late in the mitotic (M) phase, which was accompanied by an accumulation of cells with divided chromosomes and sustained histone H1 kinase activity. The lethality of the triple disruption was suppressed by the multicopies of CDC5, CDC15, DBF2, SPO12, and TEM1, all of which function in the completion of the M phase. Mammalian ras also suppressed the lethality, which suggests that a similar signaling pathway exists in higher eukaryotes. These results demonstrate that S. cerevisiae Ras functions in the completion of the M phase in a manner independent of the Ras-cAMP pathway.

Asef, a link between the tumor suppressor APC and G-protein signaling.

The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. Here the APC gene product is shown to bind through its armadillo repeat domain to a Rac-specific guanine nucleotide exchange factor (GEF), termed Asef. Endogenous APC colocalized with Asef in mouse colon epithelial cells and neuronal cells. Furthermore, APC enhanced the GEF activity of Asef and stimulated Asef-mediated cell flattening, membrane ruffling, and lamellipodia formation in MDCK cells. These results suggest that the APC-Asef complex may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function.

A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions.

We have previously purified a novel GTPase-activating protein (GAP) for Ras which is immunologically distinct from the known Ras GAPs, p120GAP and neurofibromin (M. Maekawa, S. Nakamura, and S. Hattori, J. Biol. Chem. 268:22948-22952, 1993). On the basis of the partial amino acid sequence, we have obtained a cDNA which encodes the novel Ras GAP. The predicted protein consists of 847 amino acids whose calculated molecular mass, 96,369 Da, is close to the apparent molecular mass of the novel Ras GAP, 100 kDa. The amino acid sequence shows a high degree of similarity to the entire sequence of the Drosophila melanogaster Gap1 gene. When the catalytic domain of the novel GAP was compared with that of Drosophila Gap1, p120GAP, and neurofibromin, the highest degree of similarity was again observed with Gap1. Thus, we designated this gene Gap1m, a mammalian counterpart of the Drosophila Gap1 gene. Expression of Gap1m was relatively high in brain, placenta, and kidney tissues, and it was expressed at low levels in other tissues. A recombinant protein consisting of glutathione-S-transferase and the GAP-related domain of Gap1m stimulated GTPase of normal Ras but not that of Ras having valine at the 12th residue. Expression of the same region in Saccharomyces cerevisiae suppressed the ira2- phenotype. In addition to the GAP catalytic domain, Gap1m has two domains with sequence closely related to those of the phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine kinase. These results clearly show that Gap1m is a novel Ras GAP molecule of mammalian cells.

Interaction of rat ascites hepatoma cells with cultured mesothelial cell layers: a model for tumor invasion.

Interactions of rat ascites hepatoma cells with primary cultured layers of rat mesentery-derived cells were studied. The mesentery-derived cells were isolated from rat mesentery and cultured in Eagle's minimum essential medium with a 2-fold concentration of amino acids and vitamins supplemented with 10% calf serum. The primary cultured cells, consisting mainly of mesothelial cells in polygonal shape, forms a "paving stone" sheet. Upon seeding the tumor cells on the mesentery-derived cell layers, three different types of tumor cell growth were observed. Type 1 was the formation of piled-up tumor cell nests on mesothelial cell layers. Type 2 was the formation of flattened tumor cell islands underneath mesothelial cell layers. This island formation was clearly observed under a phase contrast microscope 2 days after the tumor cell seeding. Protrusion of cellular processes of the tumor cells beneath mesothelial cells was occasionally seen. Type 3 was the growth of tumor cells in suspension. These types of tumor cell growth closely resemble those in the peritoneal cavity observed after i.p. implantation of the tumor cells. When the tumor cells recovered from the blood of tumor-bearing rats were seeded, flattened tumor cell islands were formed 15 times more frequently than when the tumor cells isolated from host peritoneal cavity were seeded. Shortly after the appearance of small flattened tumor cell islands, a distinct morphological change of mesothelial cells from polygonal to spindle shape was seen preferentially at the marginal area of the cell layers (a partial retraction of cell edges). The retraction of mesothelial cells was induced not only by seeding the tumor cells but by adding the tumor ascites fluid or the medium conditioned by the tumor cell culture. The morphological change was reversed by changing the culture medium to remove the effectors. These results indicate that the system described in this study can provide a useful model to study tumor cell invasion.

Increased expression of nucleoside diphosphate kinase/nm23 and c-Ha-ras mRNA is associated with spontaneous lung metastasis in rat-transplantable osteosarcomas.

The relationship between expression of nucleoside diphosphate kinase (NDP kinase)/nm23, c-Ha-ras, and c-myc genes and metastatic potential was assessed in rat-transplantable osteosarcoma lines, derived from spontaneous and chemical carcinogen (4-hydroxyamino quinoline 1-oxide)-induced osteosarcomas in Fischer 344/NS1c rats. These osteosarcomas possess metastatic potential and highly metastatic lines spontaneous osteosarcoma-selected lung metastatic lesions and 4-hydroxyamino-quinoline 1-oxide-induced osteosarcoma-selected lung metastatic lesions were respectively established by selectively transplanting lung metastatic lesions. Northern blot analysis revealed that the levels of NDP kinase/nm23 and c-Ha-ras gene expression were increased in line with metastatic ability; thus transcript levels were remarkably greater in both spontaneous osteosarcoma-selected lung metastatic lesions and 4-hydroxyamino-quinoline 1-oxide-induced osteosarcoma-selected lung metastatic lesions highly metastatic lines than in their respective low metastatic spontaneous and chemical carcinogen (4-hydroxyamino quinoline 1-oxide)-induced osteosarcoma counterparts. c-myc mRNA expression was observed in all tumor lines, without any correlation with metastatic ability. Southern blot analysis did not show evidence of gross rearrangement or amplification of NDP kinase/nm23, c-Ha-ras, or c-myc genes suggesting regulation of their gene expression at the transcriptional and/or posttranscriptional level. These results indicate that NDP kinase/nm23 and c-Ha-ras might be cooperatively involved in a positive manner in signal transduction processes, especially involving G-protein reactions, responsible for metastasis of rat-transplantable osteosarcomas.

Activation of adenylate cyclase by islet amyloid polypeptide with COOH-terminal amide via calcitonin gene-related peptide receptors on rat liver plasma membranes.

Both human and rat islet amyloid polypeptide with COOH-terminal amide (IAPP-NH2) dose-dependently displaced the specific binding of 125I-labeled [Tyr0] rat alpha-calcitonin gene-related peptide (CGRP) to rat liver plasma membranes, whereas human IAPP (IAPP-COOH) had no effect. Conversely, human or rat IAPP-NH2 but not human IAPP-COOH evoked dose-dependent activation of adenylate cyclase in the membranes, and these effects were significantly inhibited by the CGRP-receptor antagonist human CGRP-1(8-37). Moreover, the dose of human or rat IAPP-NH2 necessary for producing half-maximal activation of adenylate cyclase was comparable with that for producing a half-maximal inhibition of the label binding. Thus, IAPP-NH2 but not IAPP-COOH appears to induce adenylate cyclase activation via CGRP receptors on rat liver plasma membranes.

Calcitonin gene-related peptide and induction of hyperglycemia in conscious rats in vivo.

The effect of calcitonin gene-related peptide (CGRP) on glucose metabolism was investigated in conscious and unrestrained rats in vivo. Intravenous injection of rat CGRP (5.67 and 0.567 nmol/kg) caused a significant, dose-dependent increase in plasma glucose concentration and a simultaneous dose-dependent increase in plasma insulin level. In contrast, plasma glucagon level was not changed. On the other hand, intravenous infusion of CGRP (46.6 pmol.kg-1.min-1) decreased tolerance to intragastric administration of glucose (IGGTT). Plasma insulin response to IGGTT, however, was not affected by CGRP infusion. Moreover, although intravenous injection of CGRP (5.67 nmol/kg) elicited a significant increase in plasma epinephrine and norepinephrine concentrations, concomitant administration of epinephrine and norepinephrine, inducing a more prominent rise in plasma catecholamines than those induced by CGRP, affected neither plasma glucose nor insulin levels. Finally, plasma insulin levels obtained by simulating CGRP-induced changes in plasma glucose or glucose plus catecholamine levels by infusion of glucose or glucose plus catecholamines were not different from those induced by CGRP injection. These results suggest that CGRP has a hyperglycemic action that is not mediated by sympathetic outflow in conscious rats, and inhibition of insulin secretion, if any, does not play a major role in this hyperglycemic action of CGRP. We have demonstrated specific CGRP receptors linked to adenylate cyclase activation in rat liver plasma membranes; this hyperglycemic effect of CGRP in vivo may be partly due to its direct action on the liver.

A recombination repair gene of Schizosaccharomyces pombe, rhp57, is a functional homolog of the Saccharomyces cerevisiae RAD57 gene and is phylogenetically related to the human XRCC3 gene.

To identify Schizosaccharomyces pombe genes involved in recombination repair, we identified seven mutants that were hypersensitive to both methyl methanesulfonate (MMS) and gamma-rays and that contained mutations that caused synthetic lethality when combined with a rad2 mutation. One of the mutants was used to clone the corresponding gene from a genomic library by complementation of the MMS-sensitive phenotype. The gene obtained encodes a protein of 354 amino acids whose sequence is 32% identical to that of the Rad57 protein of Saccharomyces cerevisiae. An rhp57 (RAD57 homolog of S. pombe) deletion strain was more sensitive to MMS, UV, and gamma-rays than the wild-type strain and showed a reduction in the frequency of mitotic homologous recombination. The MMS sensitivity was more severe at lower temperature and was suppressed by the presence of a multicopy plasmid bearing the rhp51 gene. An rhp51 rhp57 double mutant was as sensitive to UV and gamma-rays as an rhp51 single mutant, indicating that rhp51 function is epistatic to that of rhp57. These characteristics of the rhp57 mutants are very similar to those of S. cerevisiae rad57 mutants. Phylogenetic analysis suggests that Rhp57 and Rad57 are evolutionarily closest to human Xrcc3 of the RecA/Rad51 family of proteins.

CHIP buffers heterogeneous Bcl-2 expression levels to prevent augmentation of anticancer drug-resistant cell population.

Many types of cancer display heterogeneity in various features, including gene expression and malignant potential. This heterogeneity is associated with drug resistance and cancer progression. Recent studies have shown that the expression of a major protein quality control ubiquitin ligase, carboxyl terminus of Hsc70-interacting protein (CHIP), is negatively correlated with breast cancer clinicopathological stages and poor overall survival. Here we show that CHIP acts as a capacitor of heterogeneous Bcl-2 expression levels and prevents an increase in the anticancer drug-resistant population in breast cancer cells. CHIP knockdown in breast cancer cells increased variation in Bcl-2 expression levels, an antiapoptotic protein, among the cells. Our results also showed that CHIP knockdown increased the proportion of anticancer drug-resistant cells. These findings suggest that CHIP buffers variation in gene expression levels, affecting resistance to anticancer drugs. In single-cell clones derived from breast cancer cell lines, CHIP knockdown did not alter the variation in Bcl-2 expression levels and the proportion of anticancer drug-resistant cells. In contrast, when clonal cells were treated with a mutagen, the variation in Bcl-2 expression levels and proportion of anticancer drug-resistant cells were altered by CHIP knockdown. These results suggest that CHIP masks genetic variations to suppress heterogeneous Bcl-2 expression levels and prevents augmentation of the anticancer drug-resistant population of breast cancer cells. Because genetic variation is a major driver of heterogeneity, our results suggest that the degree of heterogeneity in expression levels is decided by a balance between genetic variation and the buffering capacity of CHIP.

Observation of laser-induced electronic structure in oriented polyatomic molecules.

All attosecond time-resolved measurements have so far relied on the use of intense near-infrared laser pulses. In particular, attosecond streaking, laser-induced electron diffraction and high-harmonic generation all make use of non-perturbative light-matter interactions. Remarkably, the effect of the strong laser field on the studied sample has often been neglected in previous studies. Here we use high-harmonic spectroscopy to measure laser-induced modifications of the electronic structure of molecules. We study high-harmonic spectra of spatially oriented CH3F and CH3Br as generic examples of polar polyatomic molecules. We accurately measure intensity ratios of even and odd-harmonic orders, and of the emission from aligned and unaligned molecules. We show that these robust observables reveal a substantial modification of the molecular electronic structure by the external laser field. Our insights offer new challenges and opportunities for a range of emerging strong-field attosecond spectroscopies.

Calcitonin gene-related peptide stimulates adenylate cyclase activation via a guanine nucleotide-dependent process in rat liver plasma membranes.

To evaluate the functional relationship between the liver calcitonin gene-related peptide (CGRP) receptor and guanine nucleotide-binding proteins, we investigated the effects of nucleotides not only on adenylate cyclase activation by CGRP, but also on 125I-[Tyr0]rat CGRP binding to rat liver plasma membranes. In the presence of GTP, rat CGRP stimulated adenylate cyclase activity in a dose-dependent manner in rat liver plasma membranes, and this effect was reduced in the absence of GTP. Salmon calcitonin also enhanced adenylate cyclase activation in the presence of GTP, but only in higher concentrations. On the other hand, guanine nucleotides not only decreased 125I-[Tyr0]rat CGRP binding to rat liver plasma membranes, but also accelerated the dissociation of label binding, and the removal of Mg2+ from incubation medium attenuated this inhibitory action of GTP on 125I-[Tyr0]rat CGRP binding to membranes. Scatchard analysis of the data revealed that the reduction of 125I-[Tyr0]rat CGRP binding by GTP was due to the decrease in binding affinity without a significant change in binding capacity. These findings lead us to conclude that binding of CGRP to its receptors activates adenylate cyclase in rat liver plasma membranes via a guanine nucleotide-dependent process, suggesting the involvement of guanine nucleotide-binding stimulatory protein in the action of CGRP.