Tuning cell motility : roles of nestin and vimentin in cancer cell invasion and motility

The cytoskeleton is a key feature of both prokaryotic and eukaryotic cells. Itis comprised of three protein families, one of which is the intermediate filaments (IFs). Of these, the IFs are the largest and most diverse. The IFs are expressed throughout life, and are involved in the regulation of cell differentiation, homeostasis, ageing and pathogenesis. The IFs not only provide structural integrity to the cell, they are also involved in a range of cellular functions from organelle trafficking and cell migration to signalling transduction. The IFs are highly dynamic proteins, able to respond and adapt their network rapidly in response to intra- and extra- cellular cues. Consequently they interact with a whole host of cellular signalling proteins, regulating function, and activity, and cellular localisation. While the function of some of the better-known IFs such as the keratins is well studied, the understanding of the function of two IFs, nestin and vimentin, is poor. Nestin is well known as a marker of differentiation and is expressed in some cancers. In cancer, nestin is primarily described as is a promoter of cell motility, however, how it fulfils this role remains undefined. Vimentin too is expressed in cancer, and is known to promote cell motility and is used as a marker for epithelial to mesenchymal transition (EMT). It is only in the last decade that studies have addressed the role that vimentin plays in cell motility and EMT. This work provides novel insight into how the IFs, nestin and vimentin regulate cell motility and invasion. In particular we show that nestin regulates the cellular localisation and organisation of two key facilitators of cell migration, focal adhesion kinase and integrins. We identify nestin as a regulator of extracellular matrix degradation and integrin-mediated cell invasion. Two further studies address the specific regulation of vimentin by phosphorylation. A detailed characterisation study identified key phosphorylation sites on vimentin, which are critical for proper organisation of the vimentin network. Furthermore, we show that the bioactive sphingolipids are vimentin network regulators. Specifically, the sphingolipids induced RhoA kinasedependent (ROCK) phosphorylation at vimentin S71, which lead to filament reorganisation and inhibition of cell migration. Together these studies shed new light into the regulation of nestin and vimentin during cell motility.

Networks and partnerships in third party logistics service production

Third party logistics, and third party logistics providers and the services they offer have grown substantially in the last twenty years. Even though there has been extensive research on third party logistics providers, and regular industry reviews within the logistics industry, a closer research in the area of partner selection and network models in the third party logistics industry is missing. The perspective taken in this study was of expanding the network research into logistics service providers as the focal firm in the network. The purpose of the study is to analyze partnerships and networks in the third party logistics industry in order to define how networks are utilized in third party logistics markets, what have been the reasons for the partnerships, and whether there are benefits for the third party logistics provider that can be achieved through building networks and partnerships. The theoretical framework of this study was formed based on common theories in studying networks and partnerships in accordance with models of horizontal and vertical partnerships. The theories applied to the framework and context of this study included the strategic network view and the resource-based view. Applying these two network theories to the position and networks of third party logistics providers in an industrial supply chain, a theoretical model for analyzing the horizontal and vertical partnerships where the TPL provider is in focus was structured. The empirical analysis of TPL partnerships consisted of a qualitative document analysis of 33 partnership examples involving companies present in the Finnish TPL markets. For the research, existing documents providing secondary data on types of partnerships, reasons for the partnerships, and outcomes of the partnerships were searched from available online sources. Findings of the study revealed that third party logistics providers are evident in horizontal and vertical interactions varying in geographical coverage and the depth and nature of the relationship. Partnership decisions were found to be made on resource based reasons, as well as from strategic aspects. The discovered results of the partnerships in this study included cost reduction and effectiveness in the partnerships for improving existing services. In addition in partnerships created for innovative service extension, differentiation, and creation of additional value were discovered to have emerged as results of the cooperation. It can be concluded that benefits and competitive advantage can be created through building partnerships in order to expand service offering and seeking synergies.

Frugivory on Margaritaria nobilis L.f. (Euphorbiaceae): poor investment and mimetism

Dehiscent fruits of Euphorbiaceae usually have two stages of seed dispersal, autochory followed by myrmecochory. Two stages of Margaritaria nobilis seed dispersal were described, the first stage autochoric followed by ornithocoric. Their dehiscent fruits are green and after they detached from the tree crown and fall on the ground, they open and expose blue metallic cocas. We studied the seed dispersal system of Margaritaria nobilis in a semi-deciduous forest in Brazil. In 80 h of focal observations, we recorded only 12 visits of frugivores, however the thrush Turdus leucomelas was the only frugivore that swallowed the fruits on the tree crown. Pitylus fuliginosus (Fringilidae) and Pionus maximiliani (Psittacidae) were mainly pulp eaters, dropping the seeds below the tree. On the forest floor, after fruits dehiscence, jays (Cyanocorax chrysops), guans (Penelope superciliaris), doves (Geotrygon montana) and collared-peccaries (Pecari tajacu) were observed eating the blue diaspores of M. nobilis. Experiments in captivity showed that scaly-headed parrots (Pionus maximiliani), toco toucans (Ramphastos toco), jays (Cyanochorax chrysops), and guans (Penelope superciliaris) consumed the fruits and did not prey on the seeds before consumption. The seeds collected from the feces did not germinate in spite of the high viability. The two stages of seed dispersal in M. nobilis resembles the dispersal strategies of some mimetic species. However M. nobilis seeds are associated with an endocarp, it showed low investment in nutrients, and consistent with this hypothesis, M. nobilis shared important characteristics with mimetic fruits, such as bright color display, long seed dormancy and protection by secondary compounds.

Microscopic variability in mechanical defence and herbivory response in microphyllous leaves of tropical herb species from Serra do Cipó, Brazil

Tropical high altitude grasslands present several species with both microphyllous and highly sclerophyllous leaves, and co-occur in specific soil patches, thus exposed to identical environments. In this article we describe herbivory among co-occurring microphyllous species in a tropical high altitude grassland ecosystem of Serra do Cipó, Minas Gerais state, and we tested the effect of variable anatomic traits on leaf herbivory patterns. Leaf anatomical traits were investigated for Baccharis imbricata Heering , Lavoisiera imbricata DC. and L. subulata Triana (focal species). Herbivory was measured from branches and leaves of individual plants and compared among co-occurring species within one multispecific shrub patch and among L. subulata individuals from this patch and an adjacent monospecific patch. For all present plant species and individuals we estimated the proportion of leaves with different levels of area lost. For the focal species, six leaves were sorted and taken for histological sectioning, in order to allow precise measures of defensive structures. Relative mean leaf area lost differed significantly among the six species found in the multispecific patch. Lavoisiera subulata individuals were more attacked in the multispecific than in the monospecific patch. Leaf margin protection traits in both B. imbricata and L. imbricata showed significant effect against herbivory. Data suggest that some anatomic traits have direct effect against herbivory but their effect are not clearly perceptible among branches within individual plants or among plants within the same species.

Are there evidences of a complex mimicry system among Asclepias curassavica (Apocynaceae), Epidendrum fulgens (Orchidaceae), and Lantana camara (Verbenaceae) in Southern Brazil?

The goal of this paper was to test the presence of mimicry in Asclepias curassavica L., Epidendrum fulgens Brong., and Lantana camara L. The study was carried out at the Parque Estadual de Itapeva, RS, southern Brazil, from 2004 to 2006. Flowering period of each of the three species was followed up; focal observations of butterflies visiting flowers, from fixed point and during random walks were carried out. We also estimated the frequency of pollinaria removal in the orchid, as well as its mode of reproduction. All these variables were important for testing the mimicry hypothesis. Despite some temporal coincidences in the flowering period of two plants in the system, there was no statistical association among the three plants as to flowering period. Twenty-nine species of butterflies, as potential pollinators, were recorded, particularly Agraulis vanillae maculosa, Dryas iulia alcionea, Urbanus simplicius, Tegosa claudina, and Heliconius erato phyllis, which were the more frequent visitors of the three plants. There was association between the number of visits to L. camara and E. fulgens, based on Pearson correlation (r = 0.4603; n = 19; P = 0.0473). Pollinaria removal of E. fulgens was low, as measured by the percentage of removal (range: 0 - 10%). The analysis of the mode of reproduction of this orchid showed its pollinator-dependence, since no fruits were formed by spontaneous self-pollination. In contrast, the percentage of fruit set that resulted from geitonogamy and xenogamy was, in average, 86%. The results here shown are not conclusive as to the occurrence of a mimicry system among the three plants.

Neuronal and endothelial nitric oxide synthase gene knockout mice

Targeted disruption of the neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) genes has led to knockout mice that lack these isoforms. These animal models have been useful to study the roles of nitric oxide (NO) in physiologic processes. nNOS knockout mice have enlarged stomachs and defects in the inhibitory junction potential involved in gastrointestinal motility. eNOS knockout mice are hypertensive and lack endothelium-derived relaxing factor activity. When these animals are subjected to models of focal ischemia, the nNOS mutant mice develop smaller infarcts, consistent with a role for nNOS in neurotoxicity following cerebral ischemia. In contrast, eNOS mutant mice develop larger infarcts, and show a more pronounced hemodynamic effect of vascular occlusion. The knockout mice also show that nNOS and eNOS isoforms differentially modulate the release of neurotransmitters in various regions of the brain. eNOS knockout mice respond to vessel injury with greater neointimal proliferation, confirming that reduced NO levels seen in endothelial dysfunction change the vessel response to injury. Furthermore, eNOS mutant mice still show a protective effect of female gender, indicating that the mechanism of this protection cannot be limited to upregulation of eNOS expression. The eNOS mutant mice also prove that eNOS modulates the cardiac contractile response to ß-adrenergic agonists and baseline diastolic relaxation. Atrial natriuretic peptide, upregulated in the hearts of eNOS mutant mice, normalizes cGMP levels and restores normal diastolic relaxation.

Head-to-head comparison of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia in an animal model of coronary artery stenosis

To compare the sensitivity of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia we produced a physiologically significant stenosis in the left circumflex artery of 14 open-chest dogs (range: 50 to 89% reduction in luminal diameter). In each study, dobutamine (5 to 40 µg kg-1 min-1 in 3-min stages) and pacing (20 bpm increments, each 2 min, up to 260 bpm) were performed randomly, and then followed by dipyridamole (up to 0.84 mg/kg over 10 min). The positivity of stress echocardiography tests was quantitatively determined by a significant (P<0.05) reduction of or failure to increase absolute and percent systolic wall thickening in the stenotic artery supplied wall, as compared to the opposite wall (areas related to the left anterior descending artery). Systolic and diastolic frozen images were analyzed off-line by two blinded observers in the control and stress conditions. The results showed that 1) the sensitivity of dobutamine, dipyridamole and pacing stress tests was 57, 57 and 36%, respectively; 2) in animals with positive tests, the mean percent change of wall thickening in left ventricular ischemic segments was larger in the pacing (-19 ± 11%) and dipyridamole (-18 ± 16%) tests as compared to dobutamine (-9 ± 6%) (P = 0.05), but a similar mean reduction of wall thickening was observed when this variable was normalized to a control left ventricular segment (area related to the left anterior descending artery) (pacing: -16 ± 7%; dipyridamole: -25 ± 16%; dobutamine: -26 ± 10%; not significant), and 3) a significant correlation was observed between magnitude of coronary stenosis and left ventricular segmental dysfunction induced by ischemia in dogs submitted to positive stress tests. We conclude that the dobutamine and dipyridamole stress tests showed identical sensitivities for the detection of myocardial ischemia in this one-vessel disease animal model with a wide range of left circumflex artery stenosis. The pacing stress test was less sensitive, but the difference was not statistically significant. The magnitude of segmental left ventricular dysfunction induced by ischemia was similar in all stress tests evaluated.

Dimensions and dynamics in integrin function

Integrins play crucial roles in cell adhesion, migration, and signaling by providing transmembrane links between the extracellular matrix and the cytoskeleton. Integrins cluster in macromolecular complexes to generate cell-matrix adhesions such as focal adhesions. In this mini-review, we compare certain integrin-based biological responses and signaling during cell interactions with standard 2D cell culture versus 3D matrices. Besides responding to the composition of the matrix, cells sense and react to physical properties that include three-dimensionality and rigidity. In routine cell culture, fibroblasts and mesenchymal cells appear to use focal adhesions as anchors. They then use intracellular actomyosin contractility and dynamic, directional integrin movements to stretch cell-surface fibronectin and to generate characteristic long fibrils of fibronectin in "fibrillar adhesions". Some cells in culture proceed to produce dense, three-dimensional matrices similar to in vivo matrix, as opposed to the flat, rigid, two-dimensional surfaces habitually used for cell culture. Cells within such more natural 3D matrices form a distinctive class of adhesion termed "3D-matrix adhesions". These 3D adhesions show distinctive morphology and molecular composition. Their formation is heavily dependent on interactions between integrin alpha5ß1 and fibronectin. Cells adhere much more rapidly to 3D matrices. They also show more rapid morphological changes, migration, and proliferation compared to most 2D matrices or 3D collagen gels. Particularly notable are low levels of tyrosine phosphorylation of focal adhesion kinase and moderate increases in activated mitogen-activated protein kinase. These findings underscore the importance of the dimensionality and dynamics of matrix substrates in cellular responses to the extracellular matrix.

Validation of a simple and inexpensive method for the quantitation of infarct in the rat brain

A gravimetric method was evaluated as a simple, sensitive, reproducible, low-cost alternative to quantify the extent of brain infarct after occlusion of the medial cerebral artery in rats. In ether-anesthetized rats, the left medial cerebral artery was occluded for 1, 1.5 or 2 h by inserting a 4-0 nylon monofilament suture into the internal carotid artery. Twenty-four hours later, the brains were processed for histochemical triphenyltetrazolium chloride (TTC) staining and quantitation of the schemic infarct. In each TTC-stained brain section, the ischemic tissue was dissected with a scalpel and fixed in 10% formalin at 0ºC until its total mass could be estimated. The mass (mg) of the ischemic tissue was weighed on an analytical balance and compared to its volume (mm³), estimated either by plethysmometry using platinum electrodes or by computer-assisted image analysis. Infarct size as measured by the weighing method (mg), and reported as a percent (%) of the affected (left) hemisphere, correlated closely with volume (mm³, also reported as %) estimated by computerized image analysis (r = 0.88; P < 0.001; N = 10) or by plethysmography (r = 0.97-0.98; P < 0.0001; N = 41). This degree of correlation was maintained between different experimenters. The method was also sensitive for detecting the effect of different ischemia durations on infarct size (P < 0.005; N = 23), and the effect of drug treatments in reducing the extent of brain damage (P < 0.005; N = 24). The data suggest that, in addition to being simple and low cost, the weighing method is a reliable alternative for quantifying brain infarct in animal models of stroke.

Diagnosis, staging and treatment of hepatocellular carcinoma

Hepatocellular carcinomas are aggressive tumors with a high dissemination power. An early diagnosis of these tumors is of great importance in order to offer the possibility of curative treatment. For an early diagnosis, abdominal ultrasound and serum alpha-fetoprotein determinations at 6-month intervals are suggested for all patients with cirrhosis of the liver, since this disease is considered to be the main risk factor for the development of the neoplasia. Helicoidal computed tomography, magnetic resonance and/or hepatic arteriography are suggested for diagnostic confirmation and tumor staging. The need to obtain a fragment of the focal lesion for cytology and/or histology for a diagnosis of hepatocellular carcinoma depends on the inability of imaging methods to diagnose the lesion. Several classifications are currently available for tumor staging in order to determine patient prognosis. All take into consideration not only the stage of the tumor but also the degree of hepatocellular dysfunction, which is known to be the main factor related to patient survival. Classifications, however, fail to correlate treatment with prognosis and cannot suggest the ideal treatment for each tumor stage. The Barcelona Classification (BCLC) attempts to correlate tumor stage with treatment but requires prospective studies for validation. For single tumors smaller than 5 cm or up to three nodules smaller than 3 cm, surgical resection, liver transplantation and percutaneous treatment may offer good anti-tumoral results, as well as improved patient survival. Embolization or chemoembolization are therapeutic alternatives for patients who do not benefit from curative therapies.

Disintegrins: integrin selective ligands which activate integrin-coupled signaling and modulate leukocyte functions

Extracellular matrix proteins and cell adhesion receptors (integrins) play essential roles in the regulation of cell adhesion and migration. Interactions of integrins with the extracellular matrix proteins lead to phosphorylation of several intracellular proteins such as focal adhesion kinase, activating different signaling pathways responsible for the regulation of a variety of cell functions, including cytoskeleton mobilization. Once leukocytes are guided to sites of infection, inflammation, or antigen presentation, integrins can participate in the initiation, maintenance, or termination of the immune and inflammatory responses. The modulation of neutrophil activation through integrin-mediated pathways is important in the homeostatic control of the resolution of inflammatory states. In addition, during recirculation, T lymphocyte movement through distinct microenvironments is mediated by integrins, which are critical for cell cycle, differentiation and gene expression. Disintegrins are a family of low-molecular weight, cysteine-rich peptides first identified in snake venom, usually containing an RGD (Arg-Gly-Asp) motif, which confers the ability to selectively bind to integrins, inhibiting integrin-related functions in different cell systems. In this review we show that, depending on the cell type and the microenvironment, disintegrins are able to antagonize the effects of integrins or to act agonistically by activating integrin-mediated signaling. Disintegrins have proven useful as tools to improve the understanding of the molecular events regulated by integrin signaling in leukocytes and prototypes in order to design therapies able to interfere with integrin-mediated effects.

Automated grading of left ventricular segmental wall motion by an artificial neural network using color kinesis images

The present study describes an auxiliary tool in the diagnosis of left ventricular (LV) segmental wall motion (WM) abnormalities based on color-coded echocardiographic WM images. An artificial neural network (ANN) was developed and validated for grading LV segmental WM using data from color kinesis (CK) images, a technique developed to display the timing and magnitude of global and regional WM in real time. We evaluated 21 normal subjects and 20 patients with LVWM abnormalities revealed by two-dimensional echocardiography. CK images were obtained in two sets of viewing planes. A method was developed to analyze CK images, providing quantitation of fractional area change in each of the 16 LV segments. Two experienced observers analyzed LVWM from two-dimensional images and scored them as: 1) normal, 2) mild hypokinesia, 3) moderate hypokinesia, 4) severe hypokinesia, 5) akinesia, and 6) dyskinesia. Based on expert analysis of 10 normal subjects and 10 patients, we trained a multilayer perceptron ANN using a back-propagation algorithm to provide automated grading of LVWM, and this ANN was then tested in the remaining subjects. Excellent concordance between expert and ANN analysis was shown by ROC curve analysis, with measured area under the curve of 0.975. An excellent correlation was also obtained for global LV segmental WM index by expert and ANN analysis (R² = 0.99). In conclusion, ANN showed high accuracy for automated semi-quantitative grading of WM based on CK images. This technique can be an important aid, improving diagnostic accuracy and reducing inter-observer variability in scoring segmental LVWM.

Minor segmental wall motion abnormalities detected in patients with Chagas' disease have adverse prognostic implications

Recent data from our laboratory have shown that patients with the indeterminate form of Chagas' disease can have impairment of left ventricular contractility, as evaluated by the slope of the left ventricle end-systolic pressure-dimension relationship. We also showed that Chagas' disease patients with minimal baseline wall motion abnormalities detected by two-dimensional echocardiography have more intense contractility impairment when compared to patients with the indeterminate form of the disease without this abnormality. The prognostic implications of these findings have not been established. We evaluated 59 patients (37-76 years, mean = 55 years) with different clinical forms of Chagas' disease, who had normal left ventricular global systolic function at baseline (57.6 ± 6.9%) and who had at least one additional echo during clinical follow-up (0.4-17.6; mean 4.6 years). Group 1 consisted of 14 patients with minor baseline left ventricle wall motion abnormalities and group 2 consisted of 45 patients without these abnormalities. During follow-up, global left ventricle systolic function deterioration was observed in 10 group 1 patients (71.4%) and in only 10 group 2 patients (22.2%; P < 0.005). Age and duration of follow-up were not independent determinants of left ventricular function deterioration in these patients. The present data indicate that mild segmental left ventricular wall motion abnormalities are associated with worsening of systolic function in Chagas' disease patients who have normal baseline global systolic performance.

Delayed Schwann cell and oligodendrocyte remyelination after ethidium bromide injection in the brainstem of Wistar rats submitted to streptozotocin diabetogenic treatment

Schwann cell disturbance followed by segmental demyelination in the peripheral nervous system occurs in diabetic patients. Since Schwann cell and oligodendrocyte remyelination in the central nervous system is a well-known event in the ethidium bromide (EB) demyelinating model, the aim of this investigation was to determine the behavior of both cell types after local EB injection into the brainstem of streptozotocin diabetic rats. Adult male Wistar rats received a single intravenous injection of streptozotocin (50 mg/kg) and were submitted 10 days later to a single injection of 10 µL 0.1% (w/v) EB or 0.9% saline solution into the cisterna pontis. Ten microliters of 0.1% EB was also injected into non-diabetic rats. The animals were anesthetized and perfused through the heart 7 to 31 days after EB or saline injection and brainstem sections were collected and processed for light and transmission electron microscopy. The final balance of myelin repair in diabetic and non-diabetic rats at 31 days was compared using a semi-quantitative method. Diabetic rats presented delayed macrophage activity and lesser remyelination compared to non-diabetic rats. Although oligodendrocytes were the major remyelinating cells in the brainstem, Schwann cells invaded EB-induced lesions, first appearing at 11 days in non-diabetic rats and by 15 days in diabetic rats. Results indicate that short-term streptozotocin-induced diabetes hindered both oligodendrocyte and Schwann cell remyelination (mean remyelination scores of 2.57 ± 0.77 for oligodendrocytes and 0.67 ± 0.5 for Schwann cells) compared to non-diabetic rats (3.27 ± 0.85 and 1.38 ± 0.81, respectively).

Bioeffects of albumin-encapsulated microbubbles and real-time myocardial contrast echocardiography in an experimental canine model

Myocardial contrast echocardiography has been used for assessing myocardial perfusion. Some concerns regarding its safety still remain, mainly regarding the induction of microvascular alterations. We sought to determine the bioeffects of microbubbles and real-time myocardial contrast echocardiography (RTMCE) in a closed-chest canine model. Eighteen mongrel dogs were randomly assigned to two groups. Nine were submitted to continuous intravenous infusion of perfluorocarbon-exposed sonicated dextrose albumin (PESDA) plus continuous imaging using power pulse inversion RTMCE for 180 min, associated with manually deflagrated high-mechanical index impulses. The control group consisted of 3 dogs submitted to continuous imaging using RTMCE without PESDA, 3 dogs received PESDA alone, and 3 dogs were sham-operated. Hemodynamics and cardiac rhythm were monitored continuously. Histological analysis was performed on cardiac and pulmonary tissues. No hemodynamic changes or cardiac arrhythmias were observed in any group. Normal left ventricular ejection fraction and myocardial perfusion were maintained throughout the protocol. Frequency of mild and focal microhemorrhage areas in myocardial and pulmonary tissue was similar in PESDA plus RTMCE and control groups. The percentages of positive microscopical fields in the myocardium were 0.4 and 0.7% (P = NS) in the PESDA plus RTMCE and control groups, respectively, and in the lungs they were 2.1 and 1.1%, respectively (P = NS). In this canine model, myocardial perfusion imaging obtained with PESDA and RTMCE was safe, with no alteration in cardiac rhythm or left ventricular function. Mild and focal myocardial and pulmonary microhemorrhages were observed in both groups, and may be attributed to surgical tissue manipulation.