Imaging haematopoietic cells recruitment to an acute wound in vivo identifies a role for c-Met signalling.

We have used a direct in vivo imaging strategy to investigate the role of c-Met signalling and kinase activity during the immune response to wounding. Our assay utilizes the optical translucent properties of the zebrafish embryo and demonstrates the versatility of microscopy-based approach to the screening of compounds for inhibition of the wounding response. We have focussed on the c-Met pathway as little is known about the influence of c-Met signalling in immune responses, although it has been suggested that the c-Met tyrosine kinase receptor signalling pathway may be involved in cytokine secretion and directional migration in immune cells. Using both imaging of fixed zebrafish embryos in combination with digital time lapse microscopy of neutrophils recruited to a wound site, we find that pharmacological inhibition of c-Met, using a specific inhibitor, modulates the immune response in zebrafish embryos. We have found that inhibition of c-Met does not prevent the inflammatory response but does appear to limit recruitment and retention of immune cells at the wound site. This work demonstrates the versatility of using direct imaging assays for inhibitor studies and suggests that the HGF/c-Met signalling cascade plays an important role in the migration of haematopoietic cells in vivo.

Increased use of laparoscopy in acute right iliac fossa pain - is it good for patients?

AIM: The use of laparoscopy, with or without appendicectomy, is becoming more common in the management of acute right iliac fossa (RIF) pain, but little is known of the 'unintended' consequences of this change. This study aimed to evaluate the impact of increased use of laparoscopy on the number and type of patients treated surgically and on the rate of negative appendicectomy. METHOD: A prospective audit was carried out of admissions to a teaching hospital over two, 3-month periods during 2007 and 2008. The management, investigations and outcome of patients presenting with RIF pain were studied. RESULTS: Admissions were stable over the two time-periods. There was a significant increase in the number of laparoscopic operations performed, from 22.5% (14/62) in 2007 to 85.7% (72/84) in 2008 (P < 0.0001), and the percentage of patients undergoing surgery rose from 55.4% (n = 62) in 2007 to 71.2% (n = 84) in 2008 (P < 0.01). In 2008, female patients were more likely to have surgery, an increase from 37.1% to 66.2% (P < 0.001), and were more likely to have a laparoscopic procedure, an increase from 50% to 98% (P < 0.0001). The rate of histologically confirmed appendicitis did not increase significantly (50/122 vs 57/118; P = 0.25), but the number of patients with a normal appendix either left in situ because it was macroscopically normal or found to be histologically normal following excision, increased significantly, from 9.01% in 2007 to 21.2% in 2008 (P < 0.01). The diagnostic value of pelvic ultrasound decreased from 75.6% of examinations in 2007 to 54.5% in 2008 (P = 0.039). CONCLUSION: An increase in laparoscopic procedures has resulted in more operations in women, an associated higher negative appendicectomy rate and decreased usefulness of pelvic ultrasound. Increased use of laparoscopy needs to be balanced against the diagnostic benefits of 'negative' laparoscopy.

Integration of biochemical and topographic cues for the formation and spatial distribution of invadosomes in nasopharyngeal epithelial cells.

Invadosomes are invasive protrusions generated by cells which can secrete matrix metalloproteinases for focal digestion of extracellular matrix. They also aid invasive cancer cells in their transmigration through vascular endothelium. However, how the physical and chemical cues in a three-dimensional (3D) system signal the spatial localization of invadosomes remains largely unknown. Here we study the topographic guidance of invadosome formation in invasive nasopharyngeal cells under the stimulation of an inflammatory cytokine, TGF-ß1, using engineered gratings with different width and depth. We first report that TGF-ß1 can act as an external signal to upregulate the formation of invadosomes with a random distribution on a plane 2D surface. When the cells were seeded on parallel 3D gratings of 5 µm width and 1 µm depth, most of the invadosomes aligned to the edges of the gratings, indicating a topographic cue to the control of invadosome localization. While the number of invadosomes per cell were not upregulated when the cells were seeded on 3D topography, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges when the cells move at a lower speed and are guided along narrow gratings. The invadosomes forming at 3D edges also have a longer half-life than those forming on a plane surface. These data suggest that there are integrated biochemical and 3D geometric cues underlying the spatial regulation of invasive structures so as to elicit efficient invasion or metastasis of cells. STATEMENT OF SIGNIFICANCE: Nasopharyngeal cells were integrated with the biological cues and matrix topography to govern the activity and spatial distribution of invadosomes. The biochemical induction of invadosome formation by TGF-ß1 in nasopharyngeal cells was observed. When the cells were seeded on parallel 3D gratings, most of the invadosomes aligned to the edges of the gratings due to topographical induced invadosome localization. While the number of invadosomes per cell were not upregulated, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges with a higher stability when the cells are guided along narrow gratings. The integrated biochemical and 3D geometric cues could elicit efficient invasion or metastasis of cells.

Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts.

Using a sensitive and quantitative adhesion assay, we have studied the initial stages of the intercellular adhesion of the C2 mouse myoblast line. After dissociation in low levels of trypsin in EDTA, C2 cells can rapidly reaggregate by Ca2+-independent mechanisms to form large multicellular aggregates. If cells are allowed to recover from dissociation by incubation in defined media, this adhesive system is augmented by a Ca2+-dependent mechanism with maximum recovery seen after 4 h incubation. The Ca2+-independent adhesion system is inhibited by preincubation of cell monolayers with cycloheximide before dissociation. Aggregation is also reduced after exposure to monensin, implicating a role for surface-translocated glycoproteins in this mechanism of adhesion. In coaggregation experiments using C2 myoblasts and 3T3 fibroblasts in which the Ca2+-dependent adhesion system was inactivated, no adhesive specificity between the two cell types was seen. Although synthetic peptides containing the RGD sequence are known to inhibit cell-substratum adhesion in various cell types, incubation of C2 myoblasts with the integrin-binding tetrapeptide, RGDS, greatly stimulated the Ca2+-independent aggregation of these cells while control analogs had no effect. These results show that a Ca2+-independent mechanism alone is sufficient to allow for the rapid formation of multicellular aggregates in a mouse myoblast line, and that many of the requirements and perturbants of the Ca2+-independent system of intercellular myoblast adhesion are similar to those of the Ca2+-dependent adhesion mechanisms.

Fibronectin glycosylation modulates fibroblast adhesion and spreading.

The role of the carbohydrate residues of fibronectin concerning the specificities of that glycoprotein to interact with fibroblastic cell surfaces, gelatin, and heparin was examined. Tunicamycin was used to produce carbohydrate-depleted fibronectin; it was synthesized by cultured fibroblasts. Unglycosylated and glycosylated fibronectins were analyzed for their ability to bind gelatin and heparin, using affinity columns. Fibronectin-coated surfaces were used to quantitatively measure cell adhesion and spreading. The results showed that the lack of carbohydrates significantly increased the interaction of the protein with gelatin and markedly enhanced its ability to promote adhesion and spreading of fibroblasts. In contrast, the binding of fibronectin to heparin was not influenced by glycosylation. The composite data indicate that the Asn-linked oligosaccharides of fibronectin act as modulators of biological functions of the glycoprotein.

A role for Cdc42 in macrophage chemotaxis.

Three members of the Rho family, Cdc42, Rac, and Rho are known to regulate the organization of actin-based cytoskeletal structures. In Bac1.2F5 macrophages, we have shown that Rho regulates cell contraction, whereas Rac and Cdc42 regulate the formation of lamellipodia and filopodia, respectively. We have now tested the roles of Cdc42, Rac, and Rho in colony stimulating factor-1 (CSF-1)-induced macrophage migration and chemotaxis using the Dunn chemotaxis chamber. Microinjection of constitutively activated RhoA, Rac1, or Cdc42 inhibited cell migration, presumably because the cells were unable to polarize significantly in response to CSF-1. Both Rho and Rac were required for CSF-1-induced migration, since migration speed was reduced to background levels in cells injected with C3 transferase, an inhibitor of Rho, or with the dominant-negative Rac mutant, N17Rac1. In contrast, cells injected with the dominant-negative Cdc42 mutant, N17Cdc42, were able to migrate but did not polarize in the direction of the gradient, and chemotaxis towards CSF-1 was abolished. We conclude that Rho and Rac are required for the process of cell migration, whereas Cdc42 is required for cells to respond to a gradient of CSF-1 but is not essential for cell locomotion.

L-asparaginase-deficient mutants of yeast.

Yeast L-asparaginase is a multimeric enzyme for which only a single structural gene has been found. Fourteen mutants deficient in L-asparaginase have been isolated, and they have been located at one site on the genetic map of Saccharomyces cerevisiae. The L-asparaginase gene (aspl) is located about 18 centimorgans from a gene governing tryptophan synthesis (trp4) on fragment 2 of the map.

WASP and WIP regulate podosomes in migrating leukocytes.

Podosomes are specialized adhesion sites found in rapidly migrating and invasive cells, most notably in cells from the myeloid lineage that participate in immune surveillance and phagocyte defence mechanisms. In this review, we describe the nature of leukocyte podosomes and the regulation of their turnover during migration by the key regulatory molecules Wiskott-Aldrich syndrome protein and WASP-interacting protein.

Quantifying cell-matrix adhesion dynamics in living cells using interference reflection microscopy.

Focal adhesions and podosomes are integrin-mediated cell-substratum contacts that can be visualized using interference reflection microscopy (IRM). Here, we have developed automated image-processing procedures to quantify adhesion turnover from IRM images of live cells. Using time sequences of images, we produce adhesion maps that reveal the spatial changes of adhesions and contain additional information on the time sequence of these changes. Such maps were used to characterize focal adhesion dynamics in mouse embryo fibroblasts lacking one or both alleles of the vinculin gene. Loss of vinculin expression resulted in increased assembly, disassembly and/or in increased translocation of focal adhesions, suggesting that vinculin is important for stabilizing focal adhesions. This method is also useful for studying the rapid dynamics of podosomes as observed in primary mouse dendritic cells.

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.

RhoE regulates actin cytoskeleton organization and cell migration.

The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation.

Pharmacological rescue of noise induced hearing loss using N-acetylcysteine and acetyl-L-carnitine.

Despite the use of hearing protection devices (HPDs) and engineering changes designed to improve workspaces, noise-induced hearing loss continues to be one of the most common and expensive disabilities in the US military. Many service members suffer acoustic trauma due to improper use of HPDs, sound levels exceeding the protective capacity of the HPDs, or by unexpected, injurious exposures. In these cases, there is no definitive treatment for the hearing loss. This study investigated the use of the pharmacological agents N-acetylcysteine and acetyl-L-carnitine after acoustic trauma to treat cochlear injury. N-Acetylcysteine is an antioxidant and acetyl-L-carnitine a compound that maintains mitochondrial bio-energy and integrity. N-Acetylcysteine and acetyl-L-carnitine, respectively, significantly reduced permanent threshold shifts and hair cell loss compared to saline-treated animals when given 1 and 4 h post-noise exposure. It may be possible to obtain a greater therapeutic effect using these agents in combination or at higher doses or for a longer period of time to address the secondary oxidative events occurring 7-10 days after acute noise exposure.

Mechanism of Phosphodiesterase 5 inhibitor relief of prostatitis symptoms.

BACKGROUND: Chronic prostatitis is a common urological complaint without clearly defined causation or definitive treatment. HYPOTHESIS: Phosphodiesterase 5 (PDE5) Inhibitor mediated relaxation of prostatic duct smooth muscle increases washout of prostatic reflux products reducing prostatic inflammation and consequent prostatitis symptoms. RATIONALE OF HYPOTHESIS: The presence of both Nitric Oxide Synthase and Phosphodiesterase 5 in human prostatic tissue and the effect of nitric oxide donors and PDE5 inhibitors in vitro indicate PDE5 inhibitors relax prostatic smooth muscle. Significant retrograde urinary flux into prostatic ducts has been described and suggested as the mechanism of chronic prostatitis. We postulate PDE5 inhibitors alter prostatic reflux hence prostatitis symptoms. CONCLUSION: PDE5 inhibitors may represent a simple, effective treatment for chronic prostatitis.

Pyopericardium secondary to achalasia-associated squamous cell carcinoma of the oesophagus.

Patients with achalasia of the oesophagus are known to be at increased risk of oesophageal squamous cell carcinoma. To our knowledge, this is the first report of an achalasia-associated oesophageal squamous cell carcinoma presenting with acute sepsis secondary to pyopericardium.

Developing and assessing the utility of a You-Tube based clinical genetics video channel for families affected by inherited tumours.

We have designed and implemented the first worldwide You Tube channel with 22 videos covering common questions asked in familial cancer susceptibility clinics. We discuss the use of the videos including demographics of registered You Tube users, and what lessons have been learnt about how the general public uses medical information online. The most popular video on inheritance patterns has been watched on average 84 times per month. The mostly highly viewed videos include inheritance patterns, breast cancer screening and hereditary non-polyposis colorectal cancer. Registered viewers were more commonly male and the average age of the registered user was 45-54 years; similar to that seen in Genetics Clinics suggesting that age may not be a major barrier to access to this type of information for patients. The videos have been viewed in more than 140 countries confirming that there is clearly an audience for this type of information. Patient feedback questionnaires indicate that these videos provide a useful aide memoir for the clinic appointment, and most people would recommend them to others. In summary, You Tube videos are easy and cost effective to make. They have the ability to disseminate genetics education to a worldwide audience and may be a useful adjunct to clinical appointments.

Asparaginase II of Saccharomyces cerevisiae: inactivation during the transition to stationary phase.

Asparaginase II (L-asparagine amidohydrolase, EC 3.5.1.1) activity of cells from stationary phase cultures of Saccharomyces cerevisiae is very low. When these cells are inoculated into minimal medium, asparaginase II specific activity rises rapidly and reaches a maximum after 9-10 h. During the next 2.5-3 h, a rapid decrease in asparaginase II specific activity occurs. The enzyme does not appear to be secreted into the medium or to be reabsorbed into the cell. Addition of protease inhibitors at the time of maximum activity partially or totally prevents the loss of asparaginase II. L-1-Tosylamide-2-phenylethyl chloromethyl ketone decreases the rate of loss. The sulfhydryl reagents p-hydroxymercuribenzoate and iodoacetamide inhibit the loss of asparaginase II. However, addition of EDTA causes a further increase in activity. This increase is due to de novo protein synthesis. The effect of EDTA can be reversed by the addition of Zn2+. The most likely explanation for the rapid loss of asparaginase II is proteolytic degradation by a Zn2+-dependent, thiol protease or peptidase.

Cellular signaling in macrophage migration and chemotaxis.

Whereas most cells in adult tissues are fixed in place by cell junctions, leukocytes are motile and able to migrate actively through the walls of blood vessels into surrounding tissues. The actin cytoskeleton of these cells plays a central role in locomotion, phagocytosis, and the regulation of cell shape that are crucial elements of neutrophil and monocyte/macrophage function. This review will concentrate on how macrophages in particular control the actin cytoskeleton to generate cell movement and the shape changes required for chemotaxis. It has recently become evident that a complex of seven proteins known as the Arp2/3 complex regulates the assembly of new actin filament networks at the leading front of moving cells. Proteins of the Wiskott-Aldrich Syndrome Protein (WASP) family bind directly to the Arp2/3 complex and stimulate its ability to promote the nucleation of new actin filaments. Upstream of the WASP family proteins, receptor tyrosine kinases, G-protein-coupled receptors, phosphoinositide-3-OH kinase (PI 3-kinase), and the Rho family of GTPases receive and transduce the signals that lead to actin nucleation through WASP-Arp2/3 action. Although many gaps remain in our understanding, we are now in a position to consider completing signaling pathways that are initiated from outside the cell to the actin rearrangements that drive cell motility and chemotaxis.

Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability.

Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.

Broad resistance to tobamoviruses is mediated by a modified tobacco mosaic virus replicase transgene.

Tobacco plants made transgenic to express the wild type tobacco mosaic virus (TMV) 183-kDa replicase gene were not resistant to TMV. However, transgenic plants containing essentially the same sequences, but with an additional insertion that would terminate translation in the middle of the 183-kDa gene, were highly resistant to systemic infection by TMV and other tobamoviruses. The 1.4-kbp insertion in the replicase open reading frame (ORF) of the resistant plants was shown by DNA sequencing to be an IS10-like transposable element, which apparently inserted itself into the TMV sequence at nucleotide 2875 sometime during the propagation of this replicase ORF plasmid (pREP21). Because of four stop codons, in frame with the TMV replicase ORF on the immediate 5' border of the IS insertion, REP21 effectively represents domain 1 (putative methylase domain) and a portion of domain 2 (putative helicase domain) of the TMV replicase ORF. REP21 Xanthi tobacco plants had a level of resistance to TMV similar to other reported transgenic replicase plants. No TMV was detected in upper leaves of these plants at 1-mo postinoculation. In addition, REP21 plants were resistant to an unusually broad range of tobamoviruses including tomato mosaic virus, tobacco mild green mosaic virus, TMV-U5, green tomato atypical mosaic virus, and ribgrass mosaic virus. These plants were not resistant to cucumber mosaic cucumovirus. The lack of systemic infection by TMV was due to reduced multiplication in inoculated leaves rather than complete prevention of replication.(ABSTRACT TRUNCATED AT 250 WORDS)