Repetitive spinal motor neuron discharges following single transcranial magnetic stimulation: relation to dexterity.

Transcranial magnetic stimulation allows to study the properties of the human corticospinal tract non-invasively. After a single transcranial magnetic stimulus, spinal motor neurons (MNs) sometimes fire not just once, but repetitively. The biological significance of such repetitive MN discharges (repMNDs) is unknown. To study the relation of repMNDs to other measures of cortico-muscular excitability and to physiological measures of the skill for finely tuned precision movements, we used a previously described quadruple stimulation (QuadS) technique (Z'Graggen et al. 2005) to quantify the amount of repMNDs in abductor digiti minimi muscles (ADMs) on both sides of 20 right-handed healthy subjects. Skillfulness for finger precision movements of both hands was assessed using a finger tapping task. In 16 subjects, a follow-up examination was performed after training of either precision movements (n = 8) or force (n = 8) of the left ADM. The size of the QuadS response (amplitude and area ratios) was greater in the dominant right hand than in the left hand (QuadS amplitude ratio: 47.1 +/- 18.1 versus 37.7 +/- 22.0%, Wilcoxon test: P < 0.05; QuadS area ratio: 49.7 +/- 16.2% versus 36.9 +/- 23.0%, Wilcoxon test: P < 0.05), pointing to a greater amount of repMNDs. Moreover, the QuadS amplitude and area increased significantly after finger precision training, but not after force training. This increase of repMNDs correlated significantly with the increase in performance in the finger tapping task. Our results demonstrate that repMNDs are related to handedness and therefore probably reflect supraspinal excitability differences. The increase of repMNDs after skills training but not after force training supports the hypothesis of a supraspinal origin of repMNDs.

A review of neuromuscular blockade in the critically ill patient.

The continuous administration of neuromuscular blocking agents is thought to be associated with a number of adverse effects and complications, including post-paralytic syndrome (characterized by persistent paralysis), muscle weakness, and the inability to wean from the ventilator despite discontinuation of the therapy. Consequently, clinical objectives emphasize administering only the dose necessary to optimize the effect of the drug and for the shortest possible time. This article provides an overview of the administration of neuromuscular blocking agents, from the perspective of a critical care pharmacist and critical care nurses. The complexities associated with pharmacological paralysis in critically ill patients warrants the comprehensive approach to care that multidisciplinary team members can provide.

Production of platelet-derived growth factor receptor (PDGFR-beta) in E. coli. Mapping ligand binding domain.

Portions of the extracellular domain of the platelet-derived growth factor receptor beta (PDGFR-beta) were expressed as fusion proteins with a hexa His tag in E. coli. Following purification by Ni chelate chromatography, the recombinant receptors were tested in cross-competition studies with 125I-labelled PDGF-AA and -BB. Although of lower affinity than the native receptor (IC50 values of 10(-8) M) the recombinant molecules retained ligand binding specificity and neutralized the mitogenic effect of PDGF-BB. These data indicate that the ligand binding region lies within the first four immunoglobulin-like domains on PDGFR-beta. This E. coli expression system could be further used as a rapid and economical means to produce mutated receptors and map the ligand binding domain.

Topological similarities in TGF-beta 2, PDGF-BB and NGF define a superfamily of polypeptide growth factors.

BACKGROUND: The development of functional diversity through gene duplication and subsequent divergent evolution can give rise to proteins that have little or no sequence similarity, but retain similar topologies. RESULTS: The crystal structures of nerve growth factor, transforming growth factor-beta 2 and platelet-derived growth factor-BB show that all three are based on a cystine-knot plus beta-strands topology. There is very little sequence identity between the three proteins and the relationship between the structures had not been deduced from sequence comparisons. Each growth factor is usually active as a dimer; each exists as a dimer in the crystal, but the relative orientations of the protomers are different in each case. CONCLUSION: The structural motif of disulphide bonds and hydrogen-bonded beta-strands unexpectedly found in these three growth factors acts as a stable framework for elaboration of loops of low sequence similarity that contain the specificity for receptor interaction.

Platelet-derived growth factor AA and AB dimers are present in normal human epithelial lining fluid.

Normal lung architecture is related to the presence of mesenchymal cells (fibroblasts and smooth muscle cells), and to the production by these cells of extracellular matrix. The turnover of mesenchymal cells is under a fine regulation due, at least in part, to the local presence of different mediators acting on their cell cycle. Since normal human alveolar macrophages obtained by bronchoalveolar lavage (BAL) spontaneously release platelet-derived growth factor (PDGF), a cytokine with chemotactic and growth activity on mesenchymal cells, we evaluated normal human epithelial lining fluid (ELF) for the presence of PDGF. Active only as a dimer, PDGF is a glycoprotein composed of two chains (A and B) and, thus, can be present in three forms: AA, AB, and BB dimers. Interestingly, normal ELF contains PDGF AA dimers, and to a lesser extent AB dimers, while no significant level of BB dimers is detected. Furthermore, ELF PDGF is biologically active and responsible for a significant part of the chemotactic activity and the "competence" growth activity for mesenchymal cells present in normal ELF. These findings suggest that ELF PDGF has a role in normal lung structure maintenance and tissue repair.

Crystal structure of human platelet-derived growth factor BB.

The crystal structure of the homodimeric BB isoform of human recombinant platelet-derived growth factor (PDGF-BB) has been determined by X-ray analysis to 3.0 A resolution. The polypeptide chain is folded into two highly twisted antiparallel pairs of beta-strands and contains an unusual knotted arrangement of three intramolecular disulfide bonds. Dimerization leads to the clustering of three surface loops at each end of the elongated dimer, which most probably form the receptor recognition sites.

Presence of elevated levels of platelet-derived growth factor (PDGF) in lung adenocarcinoma pleural effusions.

Significant tumor stroma development is a specific feature of adenocarcinoma of the lung in comparison to small-cell lung cancer (SCLC). The fibrotic component of tumor stroma is thought to result from the migration and local replication of mesenchymal cells in response to the presence of cytokines. One of them, platelet-derived growth factor (PDGF), is a chemotactic and growth factor for mesenchymal cells. Since several lung adenocarcinoma cell lines, but not SCLC cell lines, have been shown in vitro to express PDGF genes, we evaluated pleural effusions for the presence of PDGF in patients with adenocarcinoma of the lung, SCLC, or nonmalignant pleural effusions. In adenocarcinoma of the lung, PDGF levels in pleural effusions were higher than in SCLC and in nonmalignant pleural effusions and were associated with the presence of a growth-promoting activity for fibroblasts due, in part, to the presence of PDGF. This observation suggests the role of PDGF in tumor stroma formation in adenocarcinoma of the lung.

Effects of homo- and heterodimeric isoforms of PDGF on signalling events in rat renal mesangial cells.

Platelet-derived growth factor (PDGF) exists in three dimeric isoforms, AA, BB and AB. Mesangial cells exclusively bound the BB homodimer and responded only to the BB isoform in terms of DNA synthesis and phosphoinositide hydrolysis. PDGF-BB stimulated a dose-dependent formation of inositol trisphosphate (InsP3). Neither pertussis toxin nor short-term (10 min) treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the PDGF-BB-evoked production of InsP3. In contrast, the response to PDGF-BB was attenuated in cells in which protein kinase C has been down-regulated by long-term (24 h) treatment with TPA. In parallel to the generation of InsP3, there was a biphasic increase in 1,2-diacylglycerol (DAG). The second peak of DAG generation was associated with a concomitant 2-fold increase in choline formation. In addition, PDGF-BB stimulated the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity, when 1-propanol was added to mesangial cells. Stimulation of mesangial cells with PDGF-BB caused a dose-dependent formation of prostaglandin E2. Furthermore, mesangial cells secreted PDGF-AA into the culture supernatant.

Specific growth stimulation of cultured smooth muscle cells from spontaneously hypertensive rats by platelet-derived growth factor A-chain homodimer.

Cultured vascular smooth muscle cells (VSMC)1 from spontaneously hypertensive rats (SHR) possess specific cell surface receptors for both homodimeric forms of platelet-derived growth factor (PDGF-AA and PDGF-BB), in contrast to cells from normotensive Wistar Kyoto (WKY) animals, which express receptors only for the B-chain form of PDGF. Stimulation of quiescent VSMC from SHR with PDGF-AA resulted in activation of S6-kinase and induction of phosphoinositide catabolism, as well as cellular proliferation when cultures were maintained for prolonged periods with daily supplementation of the growth factor. WKY-derived VSMC showed no response to PDGF-AA, which was consistent with their lack of specific receptors for this homodimer. The responsiveness of quiescent cells from SHR and WKY to the B-chain homodimer was similar. The enhanced growth responsiveness of SHR-derived cells to fetal calf serum, as compared with cells from their normotensive counterparts, may be accounted for in part by their expression of receptors for the AA homodimer of PDGF.

Heparin, carboxyl-reduced sulfated heparin, and Trestatin A sulfate. Antiproliferative and anticoagulant activities.

Human smooth muscle cells were used to investigate the antiproliferative activities of sulfated carbohydrates. The antiproliferative potencies of coarse heparin fractions prepared by ultrafiltration increased with the mean molecular-weight, whereas the anticoagulant activities of a high-molecular-weight fraction had submaximal values. Furthermore, the dependence of antiproliferative activity on sulfate content is discussed. Carboxyl-reduction of heparin abolished both antiproliferative and anticoagulant activities. Sulfation of this compound yielded CRS-heparin with restored antiproliferative potency but devoid of antithrombin III-mediated anticoagulant activity. Sulfation of the pseudo-nonasaccharide, Trestatin A, yielded a compound having the highest antiproliferative activity, so far observed for a low-molecular-weight compound, and having only weak anticoagulant properties.

Expression of biologically active human platelet-derived growth factor (PDGF) B-receptor in Xenopus laevis oocytes.

Platelet-derived growth factor (PDGF) consists of three different isoforms, PDGF-AA, PDGF-AB and PDGF-BB, which bind to at least two types of receptors: the B-receptor, to which only PDGF-BB binds, and the A/B receptor, to which all three isoforms bind. Microinjection of synthetic mRNA in Xenopus laevis oocytes was used to obtain cell-surface expression of the human PDGF B-receptor. The production of receptor molecules of correct size (190 kd) was demonstrated by specific immunoprecipitation; the binding properties of the membrane- associated PDGF B-receptor were investigated with highly purified recombinant [125I] labeled human PDGF-BB and -AA. Unlike Swiss mouse 3T3 cells, which possess both B- and A/B-receptors and, therefore, bind both isoforms with high affinity, the mRNA-injected oocytes bound only the BB isoform. Mock-injected oocytes showed no specific binding.

Both homodimeric isoforms of PDGF (AA and BB) have mitogenic and chemotactic activity and stimulate phosphoinositol turnover.

Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB, which were previously shown to bind to two receptors with different isoform-specificity, the A/B-type (binds all three isoforms) and the B-type (binds only PDGF-BB). Results from competition binding experiments with Swiss 3T3 cells suggest the existence of a third receptor type, which recognizes PDGF-AB and PDGF-BB. Furthermore, Swiss 3T3 cells and human dermal fibroblasts express different relative and absolute levels of these receptor types. In particular, Swiss 3T3 cells express 90,000 PDGF-AA binding sites (A/B-receptors) per cell, whereas human fibroblasts express only 20,000 A/B-receptors per cell. All three PDGF isoforms were tested in either cell type for their effect on DNA synthesis. PDGF-BB and PDGF-AA were also tested in Swiss 3T3 cells for their effect on inositol phospholipid metabolism and chemotaxis. Each isoform promoted all three processes dose-dependently, but there were differences in the maximum cellular responses elicited. These responses reflect the capacity of the cells to bind the individual isoforms. These results demonstrate that the previous distinctions in responsiveness to the different PDGF isoforms are primarily a consequence of the differences in the levels of surface expression of the various isoform-specific receptor types, rather than of the differences in the intrinsic activity of these isoforms. Furthermore, these results suggest that all types of PDGF receptors are capable of responding to their respective ligands by mediating phosphoinositide breakdown, chemotactic responses, and DNA synthesis. Whether they exhibit other functional differences remains to be seen.

Inhibitors of angiotensin-converting enzyme prevent myointimal proliferation after vascular injury.

The role of a local angiotensin system in the vascular response to arterial injury was investigated by administering the angiotensin-converting enzyme (CE) inhibitor cilazapril to normotensive rats in which the left carotid artery was subjected to endothelial denudation and injury by balloon catheterization. In control animals, by 14 days after balloon injury, the processes of smooth muscle cell (SMC) proliferation, migration of SMCs from the media to the intima, and synthesis of extracellular matrix produced marked thickening of the intima, with reduction of the cross-sectional area of the lumen. However, in animals that received continuous treatment with the CE inhibitor, neointima formation was decreased (by about 80 percent), and lumen integrity was preserved. Thus, the angiotensin-converting enzyme may participate in modulating the proliferative response of the vascular wall after arterial injury, and inhibition of this enzyme may have therapeutic applications to prevent the proliferative lesions that occur after coronary angioplasty and vascular surgery.

Human vascular smooth muscle cells have at least two distinct PDGF receptors and can secrete PDGF-AA.

Platelet-derived growth factor (PDGF), a potent mitogen and chemoattractant for smooth muscle cells and fibroblasts in culture, is believed to play an important role in the formation of proliferative lesions of arterio-sclerosis. PDGF appears as three different dimeric isoforms: AA, AB, and BB. These were recently found to bind to two different receptors, the A/B receptor (which binds all three isoforms) and the B receptor (which binds only PDGF-BB). To find out whether these receptors exhibit functional differences, we have monitored the binding and mitogenic activities of PDGF-AA and -BB in human umbilical vein smooth muscle cells (HSMCs), human dermal fibroblasts (HFs), and Swiss mouse 3T3 cells. With each cell type, there was a good correlation between the maximal levels of DNA synthesis achieved by these isoforms and the numbers of the appropriate receptor present on the cell surface: HMSCs, which have at least 32,000 B receptors but only 8,000 A/B receptors, responded well to PDGF-BB but responded poorly to PDGF-AA; whereas Swiss 3T3 cells, which have about equal numbers of B and A/B receptors (70,000 and 90,000, respectively), responded equally well to both isoforms. PDGF-AB was a more efficacious mitogen of HSMCs and HFs than was PDGF-AA and inhibited [125I]-PDGF-BB binding to HSMCs more effectively than PDGF-AA. This indicates that there may exist a third PDGF receptor type to which PDGF-BB and -AB but not PDGF-AA can bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant interferon-gamma inhibits the mitogenic effect of platelet-derived growth factor at a level distal to the growth factor receptor.

Highly purified preparations of recombinant human interferons (rIFNs)-alpha A, -beta, and -gamma all inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis in normal human dermal fibroblasts, as monitored by incorporation of [3H]-thymidine into trichloroacetic acid (TCA)-insoluble material. rIFN-gamma was the most potent, since it blocked the PDGF response by 50% at about 10 U/ml or 0.3 ng/ml, whereas with rIFN-alpha A and rIFN-beta 4000 U/ml and 600 U/ml, respectively (10 ng/ml in both cases), were required to achieve the same effect. There was a close parallelism between the ability of these rIFNs to inhibit PDGF mitogenic activity and their capacity to inhibit cell proliferation in serum-containing medium. None of the rIFNs inhibited specific binding of 125I-PDGF to fibroblasts, and none interfered with receptor internalization. The mechanism of action of rIFN-gamma was analyzed further. rIFN-gamma did not inhibit uptake of [3H]-thymidine into these cells. However, it shifted if the time point of initiation of DNA synthesis from about 14 h after stimulation with PDGF to about 18 to 21 h and decreased significantly the rate of the DNA synthesis. rIFN-gamma could be added up to 6 h following stimulation with PDGF with no loss of its inhibitory effect. rIFN-gamma also blocked the mitogenic activity of epidermal growth factor and basic fibroblast growth factor. Taken together these results implicate that rIFN-gamma exerts its antimitogenic effect by inhibiting a process that occurs late in the PDGF signaling pathway and onto which the activity pathways of other mitogens converge. In view of the important role PDGF may play in wound-healing and in the pathogenesis of the proliferative lesions of arteriosclerosis, these data point to a possible role IFN-gamma may play as a regulator of these processes in vivo.

Platelets, platelet-derived growth factor and arteriosclerosis.

Platelets participate in the pathogenesis of arteriosclerosis and in the progression of atherosclerosis by adhering to the damaged arteries and subsequently forming mural thrombi which are either swept away and embolize or are endothelialized and thus become part of the vessel wall. Rheologic considerations predict and blood perfusion experiments using flow chambers with exposed vessel wall components demonstrate that platelet participation increases with the wall shear rate and is thus particularly important in stenosed arteries (acute thrombosis) and the microvasculature (hemostasis). In addition to their involvement in thrombosis, activated platelets release growth factors, most notably a platelet-derived growth factor (PDGF) which may be the principal mediator of smooth muscle cell migration from the media into the intima and of smooth muscle cell proliferation in the intima as well as of vasoconstriction. The recent discovery that PDGF can be produced by additional cells involved in the pathogenesis of arteriosclerosis (endothelial cells, monocytes/macrophages, smooth muscle cells themselves) and that they may play a role in tumorigenesis has tremendously increased the interest in this growth factor and in potential antagonists.

Characterization of a platelet-derived growth factor receptor on Swiss 3T3 cells by affinity crosslinking.

Crosslinking experiments with various bifunctional reagents were used to investigate the nature and fate of the platelet growth factor (PDGF) receptor on Swiss mouse 3T3 cells. With ethylene glycol bis succinimidyl succinate (EGS) two bands with Mr 205,000 and Mr 190,000 were labeled at equal intensity, while with disuccinimidyl suberate (DSS) and the photoactivatable p-azidophenylglyoxal (pAPG) almost exclusively the latter band was labeled, when analyzed by SDS polyacrylamide gel electrophoresis under reducing conditions. Evidence is presented that the Mr 190,000 band represents a Mr 175,000 receptor protein crosslinked to a single chain of the PDGF-dimer and the Mr 205,000 species the same Mr 175,000 protein crosslinked to both chains of PDGF. Pretreatment of cells with tunicamycin generated a third labeled band with Mr 150,000, while pretreatment with neuraminidase resulted in a shift of the Mr 205,000 and 190,000 bands by 5,000. This shows that the PDGF receptor is a sialoglycoprotein, consisting of a Mr approximately 135,000 proteinaceous core and a Mr approximately 40,000 carbohydrate moiety containing sialic acid. The virtually unchanged labeling intensity seen with tunicamycin and neuraminidase pretreated cells further suggests that the carbohydrate portion of the receptor is not required for PDGF binding. Finally, the crosslinking technique was used to show that at 37 degrees C performed 125I-PDGF receptor complexes disappear from the cell surface with a t1/2 approximately 8 min.

Reproducibility of noninvasive vascular laboratory measurements of the peripheral circulation.

This study reports the reproducibility of noninvasive vascular laboratory measurements for the diagnosis of peripheral arterial occlusive disease and compares their variability to other clinical measurements, including pulse rate, hemoglobin, white blood cell count, blood urea nitrogen, and creatinine. To study the reproducibility of these measurements, we considered three components that affect the repeatability; the variation associated with the measurement process, the variation associated with transient changing characteristics of the patients with time, and the variation among the patients. It is useful to consider the variation of each component relative to the variation among the patients, measured in units of standard deviation. The variation associated with the measurement process (expressed as the relative precision) was determined by repeating the measurements in pairs at random times throughout the study period. The relative precision was 0.3% for the measurements of ankle-arm systolic blood pressure ratio and treadmill walking time, 2.7% for pulsatility index, and 1.2% to 4.2% for the other clinical, hematologic, and biochemical measurements. The temporal variations were determined by repeating the measurements over a 6-week period in 15 patients. The normalized long-term fluctuation was 1.7% for ankle-arm systolic pressure ratio, 3.2% for treadmill walking time, 7.5% for pulsatility index, and 1.3% to 5.5% for the other measurements. For each test, the 95% confidence interval for an individual patient's measurements and the values above which the difference between two successive measurements can be considered significantly different (95%) have been calculated. It is concluded that the reproducibility of standard vascular laboratory measurements compares favorably to the other clinical, hematologic, and biochemical measurements evaluated in this study.

Unilateral iliac disease: the role of iliofemoral bypass.

This article reviews our experience with iliofemoral bypass during the past 10 years to better define its role in the treatment of unilateral iliac artery occlusive disease. For 50 patients, the cumulative patency rate was 96% +/- 3% at 1 year and 92% +/- 5% at 2 and 3 years. The operation was most likely to be successful if the indication was claudication (p less than 0.05). There was no significant difference when patients with or without profundaplasty were compared. It is concluded that iliofemoral bypass is a durable operation and indicated when transluminal dilatation is not possible and the common iliac artery is suitable as a source of inflow. When indicated, the procedure can be carried out safely for both high- and low-risk patients.

The current role for femorofemoral bypass.

We reviewed our experience with femorofemoral bypass during the past 10 years to define its role relative to other methods in the treatment of aortoiliac occlusive disease. The cumulative patency rate for 82 patients was 80% +/- 5% at 1 year and 67% +/- 7% at 2 and 3 years. The operation was most likely to be successful if the indication was claudication (p less than 0.05) and if the operation was performed as the primary procedure (p less than 0.01). There was no significant difference when patients with or without profundaplasty were compared. It is concluded that femorofemoral bypass is indicated to treat symptomatic unilateral iliac disease when transluminal dilatation is not possible. Femorofemoral bypass is also the procedure of choice for aortofemoral graft occlusion when the thrombosed limb cannot be reopened. Femorofemoral bypass is recommended for both high- and low-risk patients when indicated.