RACK1 targets the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to link integrin engagement with focal adhesion disassembly and cell motility

The extracellular signal-regulated kinase (ERK) cascade is activated in response to a multitude of extracellular signals and converts these signals into a variety of specific biological responses, including cell differentiation, cell movement, cell division, and apoptosis. The specificity of the biological response is likely to be controlled in large measure by the localization of signaling, thus enabling ERK activity to be directed towards specific targets. Here we show that the RACK1 scaffold protein functions specifically in integrin-mediated activation of the mitogen-activated protein kinase/ERK cascade and targets active ERK to focal adhesions. We found that RACK1 associated with the core kinases of the ERK pathway, Raf, MEK, and ERK, and that attenuation of RACK1 expression resulted in a decrease in ERK activity in response to adhesion but not in response to growth factors. RACK1 silencing also caused a reduction of active ERK in focal adhesions, an increase in focal adhesion length, a decreased rate of focal adhesion disassembly, and decreased motility. Our data further suggest that focal adhesion kinase is an upstream activator of the RACK1/ERK pathway. We suggest that RACK1 tethers the ERK pathway core kinases and channels signals from upstream activation by integrins to downstream targets at focal adhesions.     

MEK partner 1 (MP1): regulation of oligomerization in MAP kinase signaling

Specificity in signal transduction can be achieved through scaffolds, anchors, and adapters that assemble generic signal transduction components in specific combinations and locations. MEK Partner-1 (MP1) was identified as a potential "scaffold" protein for the mammalian extracellular signal-regulated kinase (ERK) pathway. To gain insight into the interactions of MP1 with the ERK pathway, we analyzed the ability of MP1 to bind to MEK1, ERK1, and to itself, and the regulation of these interactions. Gel filtration of cell lysates revealed two major MP1 peaks: a broad high molecular weight peak and a 28 kDa complex. An MP1 mutant that lost MEK1 binding no longer enhanced RasV12-stimulated ERK1 activity, and functioned as a dominant negative, consistent with the concept that MP1 function depends on facilitating these oligomerizations. Activation of the ERK pathway by serum or by RasV12 did not detectably affect MP1-MP1 dimerization or MP1-MEK1 interactions, but caused the dissociation of the MP1-ERK1 complex. Surprisingly, pharmacological inhibition of ERK activation did not restore the complex, suggesting that regulation of complex formation occurs independently of ERK phosphorylation. These results support the concept that MP1 functions as a regulator of MAP kinase signaling by binding to MEK1 and regulating its association with a larger signaling complex that may sequentially service multiple molecules of ERK.     

Structure-activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters

Sulfonic acid esters are considered as potentially alkylating agents that may exert genotoxic effects in bacterial and mammalian cell systems. One possible source of human exposure stems from drug synthesis when the salt-forming agents methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid are used together with alcoholic solvents such as methanol, ethanol and propanol. In this study computer-assisted structural considerations and in vitro approaches (Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, and the micronucleus test using L5178Y mouse lymphoma cells) were used to assess the genotoxic properties of 19 sulfonic esters. While all esters may be principally active as genotoxicants based on the presence of the sulfonate moiety, the statistical correlative multiple computer automated structure evaluation (MCASE) system (MC4PC version 1.0) using the Ames mutagenicity A2I module (version 1.54), rank-ordered the activity of the benzenesulfonic acid esters in the Ames test negligible due to an inactivating modulator and a deactivating fragment, whereas the methane- and toluenesulfonic acid esters were predicted to be positive in this test. In the Ames test, with the exception of the p-toluenesulfonic acid ethyl and iso-butyl esters, all compounds came out positive in Salmonella strain TA100. Methanesulfonic iso-propyl, sec-butyl and benzenesulfonic acid iso-propyl ester also showed mutagenic potential in strain TA98. In general, differences between results seen in Ames tests performed with or without metabolic activation were rather small. In L5178Y mouse lymphoma cells, benzenesulfonic acid n- and iso-butyl ester and p-toluenesulfonic acid iso-butyl ester did not increase the number of cells containing micronuclei. The other esters were positive in this micronucleus test; however, methanesulfonic acid iso-butyl ester was found to be only weakly positive at excessively cytotoxic concentrations. These compounds were generally found to be more potent with regard to micronucleus induction when tested without metabolic activation (20 h treatment). In conclusion, the iso-propyl esters of the three sulfonic acids under study were found to be the strongest mutagens, either when tested in the Ames test or the micronucleus assay, whereas p-toluenesulfonic acid iso-butyl ester was the only compound shown to be devoid of a genotoxic potential in both tests.     

Yolk formation in Locusta migratoria and Schistocerca gregaria: Related ligands and oocyte receptors

During vitellogenesis the transport of yolk precursor proteins, the vitellogenins (VTG), from the hemolymph into the oocyte is achieved by receptor-mediated endocytosis. Recently the receptor for the VTG of Locusta migratoria has been isolated. Now a new protocol has been developed for the purification of the VTG receptor of this locust from ovarian membranes. By CHAPS solubilization of the membranes followed by ion exchange and immunoaffinity chromatography, a 100-fold purification of the VTG receptor was achieved. The amino acid composition of the receptor protein has been determined. However, first attempts to sequence the receptor failed due to the N-terminal blocking of the molecule. With the same methods the VTG receptor of another locust, Schistocerca gregaria, has been isolated, purified, and characterized. This receptor has an apparent M_r of 186 kDa under nonreducing conditions. It recognizes L. migratoria VTG and vice versa. However, in cross-competition experiments in which the Schistocerca VTG competed with Locusta VTG for binding to the Locusta VTG receptor, the Schistocerca VTG was less efficient. Furthermore, the VTG receptor proteins of S. gregaria and L. migratoria are immunologically related as revealed by Western blotting with anti-Locusta VTG receptor antibodies. It appears that important structural elements required for efficient and specific endocytosis of VTG have been conserved. © 1994 Wiley-Liss, Inc.     

Compilation and use of genetic toxicity historical control data

The optimal use of historical control data for the interpretation of genotoxicity results was discussed at the 2009 International Workshop on Genotoxicity Testing (IWGT) in Basel, Switzerland. The historical control working group focused mainly on negative control data although positive control data were also considered to be important. Historical control data are typically used for comparison with the concurrent control data as part of the assay acceptance criteria. Historical control data are also important for providing evidence of the technical competence and familiarization of the assay at any given laboratory. Moreover, historical control data are increasingly being used to aid in the interpretation of genetic toxicity assay results. The objective of the working group was to provide generic advice for historical control data that could be applied to all assays rather than to give assay-specific recommendations. In brief, the recommendations include:     

Momordica charantia extract, a herbal remedy for type 2 diabetes, contains a specific 11β-hydroxysteroid dehydrogenase type 1 inhibitor

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the intracellular regeneration of active cortisol from inert cortisone in key metabolic tissues, thus regulating ligand access to glucocorticoid receptors. There is strong evidence that increased adipose 11β-HSD1 activity may be an important aetiological factor in the current obesity and diabetes type 2 epidemics. Hence, inhibition of 11β-HSD1 has emerged as a promising anti-diabetic strategy, a concept that is largely supported by numerous studies in rodent models as well as limited clinical data with prototype inhibitors. Momordica charantia (also known as bitter melon, bitter gourd or karela) is traditionally used for treatment of diabetes in Asia, South America, the Caribbean, and East Africa. In the present study, we show that M. charantia extract capsules contain at least one ingredient with selective 11β-HSD1 inhibitory activity. The finding constitutes an interesting additional explanation for the well-documented anti-diabetic and hypoglycaemic effects of M. charantia.     

Decreased plasma and cerebrospinal fluid ascorbate levels in patients with septic encephalopathy

Septic encephalopathies rapidly affect brain function without the involvement of a specific area causing a broad range of reversible neurologic symptoms. Capillary leakage including dysfunction of the blood-brain barrier has been proposed as a potential pathogenic mechanism in this entity. We tested the hypothesis that oxidative stress measured in plasma and cerebrospinal fluid (CSF) of patients suffering from septic encephalopathy could be linked to the neurologic symptoms of the disease. The neurologic symptoms of eleven patients with septic encephalopathy were described semiquantitatively through a score system. The ascorbate levels were significantly lower in both plasma and CSF from patients with septic encephalopathy than controls, and in CSF but not plasma this decrease correlated with the severity of neurologic symptoms. No significant changes were found for alpha-tocopherol. Our findings suggest that the short-term oxidative stress may be an important factor in the development of septic encephalopathy, possibly through dysregulation of the blood-brain barrier.     

On the Social Behaviour of Cells

Polystyrene Petri dishes are in use in hundreds of thousands of laboratories world wide. Cell culture experiments performed in them provide fundamental information in a wide range of applications, including but not limited to testing novel biomaterials and pharmaceuticals, and stem cell research. These experiments cost billions of dollars per year. In this study we report on a potential deficiency of polystyrene Petri dishes, possibly caused by an increase in interracial pH under relevant culture condi-tions and affecting cell performance. We conclude that cell performance on Petri dishes could be improved by improving the Petri dishes. As a spin-off of our study we postulate the concept that cancer cells and stem cells are social. It is impossible to validate this concept on the basis of the model established in this paper. However. the coherence of our insights may encourage further study and lead to the development of a qualitative improvement of cell culture devices, including Petri dishes and culture flasks, to the identification of potential strategies for chemotherapy and chemoprevention that could suppress progression of metastasis, and to the establishment of improved settings for tissue engineering and stem cell research. An immediate recom-mendation of our study is to use chemically and biologically inert substrates for important cell culture experiments, for example, nanocrystalline diamond.     

Formation of sperm bundles in Pterostichus nigrita (Coleoptera: Carabidae)

The formation and structure of sperm bundles (spermiozeugmata), and the structure of the vas deferens where bundles are formed, in Pterostichus nigrita is described by light and electron microscopy. The spermiozeugmata are of the sheet-like type consisting of a central rod (about 3?mm long) of electron-dense material (the spermatostyle), to which two bundles of spermatozoa (about 95 per bundle) are attached. The spermatostyle has a spoon-shaped head, and the rod material is differentiated into an electron-dense core and a more electron-lucent cortex. Spermatozoa (about 340?µm long) are attached to the anterior portion of the rod only. Spermiozeugma formation occurs in the upper vas deferens (before the seminal vesicle region) with the secretion of rod material by epithelial cells, which are characterized by well developed rough endoplasmic reticulum with distended cisternae, abundant mitochondria and Golgi bodies. Some cells contain numerous myeloid structures thought to be precursors of rod material, and coated vesicles. During spermiozeugma development, the heads of spermatozoa become embedded in the developing rod material, the anterior of which sits in one of the many diverticula of the mid-region of the vas deferens. Elongation of the rod proceeds by addition of material posteriorly.