RE: J Kiffer et al. - Letter to the Editor regarding F Schwarz et al. - Are single fractions of radiotherapy suitable for plantar fasciitis?
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Chronic Disease↗
Biomedical subjects
R J Fisher
Publications and source records attributed to R J Fisher.
Coupling of oscillatory activity between muscles is strikingly reduced in a deafferented subject compared with normal controls.
Oscillatory activity in the primate motor cortex has been shown to be phase locked to oscillations in contralateral hand and forearm muscle activity in the 15- to 30-Hz frequency range. Recent studies have shown that the degree of coupling between the cortex and the periphery is strongly influenced by the type and degree of movements of the digits. It has also been suggested that changes in corticomuscular and muscle-muscle coherence could be modulated by peripheral sensory inputs. In the current study, we investigated task-dependent changes in the coherent coupling of electromyographic (EMG) activity recorded from different intrinsic (abductor pollicis brevis and first dorsal interosseous) and two extrinsic (flexor digitorum superficialis and extensor digitorum communis) hand muscles during performance of a precision-grip task by normal subjects and by a single subject who has a total loss of touch, vibration, pressure, and kinesthetic sensation below the neck. The task required a hold-move-hold pattern of grip force to be exerted on a compliant object with the dominant right hand. We found significant task-related modulation of 15- to 30-Hz coherence between EMG activity in hand muscles in the control subjects. In contrast, the deafferented subject showed very low levels of significant coherence in the 15- to 30-Hz range and no peak at this frequency in the power spectra of her EMG activity. These results suggest that the presence of sensory afferent signals are necessary for the modulation of 15- to 30-Hz oscillations in the motor system.
Adult↗
Effects of combined cortical and acoustic stimuli on muscle activity.
Hitherto, it has proven difficult to investigate interactions between cerebral and brainstem motor systems in the human. We hypothesised that transcranial magnetic stimulation (TMS) centred over the dorsal premotor and primary motor cortices might elicit net facilitatory cortico-reticular effects that could interact at the level of the brainstem with a habituated startle to give a reticulospinal discharge and electromyographic (EMG) response with a longer latency than the direct corticospinal response. Conversely, any reticulo-cortical activity evoked by a habituated startle should influence the size of the direct response to cortical TMS. EMG was recorded from active left deltoid muscle in nine healthy volunteers. Acoustic stimulation was delivered binaurally through headphones and repeated until the startle response was habituated. When TMS was centred over the right dorsal premotor or primary motor cortices and delivered 50 ms after the habituated acoustic stimulus, the contralateral direct motor evoked potential was inhibited, compared with the response elicited by TMS alone. The contralateral silent period was shortened and associated with less of a decrease in EMG levels relative to TMS alone. Indeed, an actual increase in EMG over baseline levels occurred in the later half of the silent period in all subjects. We conclude that both cortico-reticular and reticular-cortical effects could be elicited in deltoid through the combination of acoustic stimulation and TMS at short interstimulus intervals. Effects were similar with TMS over premotor and primary motor cortex.
Acoustic Stimulation↗
Stimulation of in vivo angiogenesis by cytokine-loaded hyaluronic acid hydrogel implants.
Crosslinked hyaluronic acid (HA) hydrogels were evaluated for their ability to elicit new microvessel growth in vivo when preloaded with one of two cytokines, vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF). HA film samples were surgically implanted in the ear pinnas of mice, and the ears retrieved 7 or 14 days post implantation. Histologic analysis showed that all groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p < 0.01). Moreover, aqueous administration of either growth factor produced substantially more vessel growth than an HA implant with no cytokine. However, the most striking result obtained was a dramatic synergistic interaction between HA and VEGF. Presentation of VEGF in crosslinked HA generated vessel density of NI = 6.7 at day 14, where NI is a neovascularization index defined below, more than twice the effect of the sum of HA alone (NI = 1.8) plus VEGF alone (NI=1.3). This was twice the vessel density generated by co-addition of HA and bFGF (NI=3.4, p<0.001). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, synergistic angiogenic response produced by release of VEGF from crosslinked HA films.
Animals↗
Radiotherapy for painful bone metastases: an overview of the overviews.
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Bone Neoplasms↗
Digital nerve anaesthesia decreases EMG-EMG coherence in a human precision grip task.
There is increasing evidence that the primary motor cortex is involved in the generation of electromyographic (EMG) oscillations at frequencies in the range of 15-30 Hz that are observed during performance of a precision grip task. Since the level of the corticomuscular coherence varies according to the nature of the object that is gripped, it seemed possible that somatosensory inputs from the hand might affect this coherence. The aim of this study was to investigate whether interrupting cutaneous inputs from the digits would affect the coherence between hand muscles during precision grip of a compliant object. Subjects performed a precision grip hold-ramp-hold task before, during and after digital nerve anaesthesia of the index finger and thumb. There were marked deficits in the performance of the task, particularly during the initial formation of the grip and first hold period. Local digital nerve anaesthesia reduced but did not abolish 14-31 Hz coherence between EMG activity recorded from different hand and forearm muscles. Coherence was measured during the second hold phase of the task. Digital nerve anaesthesia did not affect the predominant frequencies in the EMG power spectra compiled from the same phase of the task. We conclude that during a precision grip task, cutaneous input enhances oscillatory synchrony between pairs of hand muscles.
Adult↗
Two phases of intracortical inhibition revealed by transcranial magnetic threshold tracking.
Intracortical inhibition was investigated in normal human volunteers by paired-pulse transcranial magnetic stimulation, using a new, computer-assisted threshold-tracking method. Motor threshold was defined as the stimulus amplitude required to evoke a motor evoked potential of 0.2 mV (peak-to-peak) in abductor pollicis brevis, and inhibition was measured as the percentage increase in threshold, when the test stimulus was preceded by a subthreshold conditioning stimulus. This method was used to investigate the dependence of intracortical inhibition on conditioning stimulus parameters and on voluntary activity. Interstimulus interval (ISI) was first stepped from 1 to 4.5 ms, as inhibition was measured using conditioning stimuli of fixed amplitude (50-90% resting motor threshold). Maximal inhibition was produced at ISIs of 1 and 2.5 ms. The effect of conditioning stimulus intensity was then assessed at these ISIs. Inhibition occurred at significantly lower conditioning stimulus intensities with ISI=1 ms than with ISI=2.5 ms. Voluntary activity reduced inhibition at both ISIs, but had a much greater effect on inhibition at ISI=2.5 ms. Inhibition during voluntary activity was also examined for single motor units in first dorsal interosseous by generating poststimulus time histograms. Inhibition, indicated by a reduction in the later peaks of increased firing, was observed with ISI=1 ms, but not with ISI=2.5 ms. We conclude that there are two distinct phases of inhibition, occurring at ISI=1 ms and ISI=2.5 ms, differing both in thresholds and susceptibility to voluntary activity.
Adult↗
Chemical cleavage at aspartyl residues for protein identification.
An alternative method to enzymatic digestion for protein identification by mass spectrometry has been developed that is based on chemical cleavage by formic acid. This method was tested on gel-purified apomyoglobin and BSA, as well as unknown proteins that cofractionate with Tyl-virus-like particles from Saccharomyces cerevisiae. Cleavage at aspartyl residues was found to be efficient and specific, and this specificity of cleavage lent itself easily to database searches. Parallel digestions using trypsin were also performed. The formic acid cleavage method generated comparable or better results than tryptic digestion for protein identification.
Animals↗
Modulation of HIV-like particle assembly in vitro by inositol phosphates.
HIV-1 Gag protein assembles into 100- to 120-nm diameter particles in mammalian cells. Recombinant HIV-1 Gag protein assembles in a fully defined system in vitro into particles that are only 25-30 nm in diameter and that differ significantly in other respects from authentic particles. However, particles with the size and other properties of authentic virions were obtained in vitro by addition of inositol phosphates or phosphatidylinsitol phosphates to the assembly system. Thus, the interactions between HIV-1 Gag protein molecules are altered by binding of inositol derivatives; this binding is apparently essential for normal HIV-1 particle assembly. This requirement is not seen in a deleted Gag protein lacking residues 16-99 within the matrix domain.
Amino Acid Sequence↗
Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models.
The hepatocyte growth factor (HGF/SF) receptor, Met, regulates mitogenesis, motility, and morphogenesis in a cell type-dependent fashion. Activation of Met via autocrine, paracrine, or mutational mechanisms can lead to tumorigenesis and metastasis and numerous studies have linked inappropriate expression of this ligand-receptor pair to most types of human solid tumors. To prepare mAbs to human HGF/SF, mice were immunized with native and denatured preparations of the ligand. Recloned mAbs were tested in vitro for blocking activity against scattering and branching morphogenesis. Our results show that no single mAb was capable of neutralizing the in vitro activity of HGF/SF, and that the ligand possesses a minimum of three epitopes that must be blocked to prevent Met tyrosine kinase activation. In vivo, the neutralizing mAb combination inhibited s.c. growth in athymic nu/nu mice of tumors dependent on an autocrine Met-HGF/SF loop. Importantly, growth of human glioblastoma multiforme xenografts expressing Met and HGF/SF were markedly reduced in the presence of HGF/SF-neutralizing mAbs. These results suggest interrupting autocrine and/or paracrine Met-HGF/SF signaling in tumors dependent on this pathway is a possible intervention strategy.
Animals↗
Control of fusion pore dynamics during exocytosis by Munc18.
Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. All vesicle transport steps also have an essential requirement for a member of the Sec1 protein family, including the neuronal Munc18-1 (also known as nSec1) in regulated exocytosis. Here, in adrenal chromaffin cells, we expressed a Munc18 mutant with reduced affinity for syntaxin, which specifically modified the kinetics of single-granule exocytotic release events, consistent with an acceleration of fusion pore expansion. Thus, Munc18 functions in a late stage in the fusion process, where its dissociation from syntaxin determines the kinetics of postfusion events.
Action Potentials↗
Regulation of kiss-and-run exocytosis.
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Animals↗
Control of membrane fusion dynamics during regulated exocytosis.
The study of regulated exocytosis uniquely allows the direct measurement of intracellular membrane fusion events in real time. We have exploited this to examine factors that regulate not only the extent but also the dynamics of single fusion/release events. The general strategy used has been to assess exocytosis in transiently transfected PC12 or adrenal chromaffin cells. We aimed to design mutant constructs based on in vitro biochemistry, in some cases informed by knowledge of protein structure. Using this approach we have demonstrated an inhibitory role for the putative Rab3 effector Noc2 that requires interaction with Rab3. Using carbon-fibre amperometry on adrenal chromaffin cells, we have demonstrated regulation of the kinetics of single granule release events consistent with changes in fusion pore dynamics and switches between full fusion and 'kiss-and-run' fusion. These studies have demonstrated a late role for cysteine string protein in exocytosis. In addition, they have focused attention on a key role for Munc18 in the regulation of post-fusion events that affect fusion pore dynamics.
Adrenal Medulla↗
Trophic factor withdrawal: p38 mitogen-activated protein kinase activates NHE1, which induces intracellular alkalinization.
Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.
Activating Transcription Factor 2↗
Analysis of human immunodeficiency virus type 1 containing HERV-K protease.
The human endogenous retrovirus, type K (HERV-K) represents the most biologically active form of known retroelements present in the human genome. Several HERV-K genomes have transcriptionally active open reading frames and encode their own protease (PR). The HERV-K PR has been shown to authentically cleave human immunodeficiency virus type 1 (HIV-1) matrix-capsid peptide in the presence of HIV-1 PR inhibitors. This raised the possibility that HERV-K PR could complement HIV-1 PR function in HIV-1-infected individuals. To investigate this possibility, we fused the HIV-1 vpr gene to the HERV-K PR gene (vpr-PR). The vpr-PR expression plasmid and a PR-defective HIV-1 clone were cotransfected into 293T cells. Progeny virions were assayed for processing of the HIV-1 polyproteins by Western blot and for changes in infectivity. HERV-K PR fused to Vpr was incorporated into HIV-1 virions at a high concentration and cleaved the Gag and Pol precursor proteins. However, neither Gag nor Pol polyproteins were correctly processed. Moreover, the HERV-K PR did not restore virus infectivity. While these results do not exclude the possibility that the HERV-K PR could complement an HIV-1 PR whose function is impaired due to drugs or drug-resistant mutations, they clearly demonstrate that the HERV-K PR cannot substitute for the function of the wild-type HIV-1 PR.
Cell Line↗
A complete system for identifying inhibitors of creatine kinase B.
We have developed a complete system for discovery of lead compounds as inhibitors of creatine kinase B. In this article, we describe production and purification of the recombinant protein, conditions and features of an optimized high-throughput screening assay, and results of our implementation of the system using a diverse compound library.
Base Sequence↗
Genomic alterations (LOH, MI) on chromosome 17q21-23 and prognosis of sporadic colorectal cancer.
Genomic alterations at the long arm of chromosome 17, and in particular at the nm23 locus, are still controversial in colorectal cancer (CRC). Our aim was to investigate the possible relationship of loss of heterozygosity (LOH) and microsatellite instability (MI), at 4 microsatellite loci spanning the 17q21-23 region, to the risk of liver metastasis and nm23 protein expression. Genomic DNA extracted from 58 primary and 54 liver secondary formalin-fixed and paraffin-embedded CRCs was obtained from 82 patients. A fluorescent PCR coupled with an automated DNA sequencer was applied. Increasing fraction of loci showing LOH was positively associated with risk of liver metastases (logrank test for trend, p = 0.005); this remained independent after adjusting to T-stage (Cox regression, p = 0.022), N-stage (p = 0.007), or Dukes' stage (p = 0.012). Conversely, increasing frequency of MI was associated with a reduced risk of liver metastases in Dukes' B tumours (logrank test for trend, p = 0.032). When comparing 30 primary and matched liver secondary lesions, we found concordant genomic alteration in 72% (NME1) to 43% (D17S579). Finally, we observed a trend in association between the proportion of loci with LOH and nm23 positivity (chi2 test for trend, p = 0.024). Our findings suggest that genomic alterations in the 17q21-23 region may affect prognosis of CRC as well as regulation of the nm23 protein expression via an unknown underlying mechanism.
Adult↗
Actin-binding cellular proteins inside human immunodeficiency virus type 1.
Host proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions. To identify cellular proteins inside HIV-1, virion preparations were treated by a protease-digestion technique that removes external host proteins, allowing for the study of the proteins inside the virus. Treated HIV-1 preparations were analyzed by immunoblot, high-pressure liquid chromatography, and protein sequence analyses. These analyses identified several cellular proteins inside HIV-1: elongation factor 1alpha, glyceraldehyde-3-phosphate dehydrogenase, HS-1, phosphatidylethanolamine-binding protein, Pin1, Lck, Nm23-H1, and the C-terminal tail of CD43. Several of these proteins were found as fragments of their full-sized proteins that appear to be generated by our protease treatment of the virions, the HIV-1 protease, or a cellular protease. Recent advances in cell biology and biochemistry have identified some of these proteins as actin-binding proteins. These results support the hypothesis that actin filaments are incorporated into the virion and may provide additional clues for the understanding of the interaction between viral and cellular proteins during assembly and budding.
Actins↗