The involvement of the docking protein Gab1 in mitogenic signalling induced by EGF and HGF in rat hepatocytes

Grb2-assosiated binder (Gab) family proteins are docking molecules that can interact with receptor tyrosine kinases (RTKs) and cytokine receptors and bind several downstream signalling proteins. Studies in several cell types have shown that Gab1 may have a role in signalling mediated by the two RTKs epidermal growth factor (EGF) receptor (EGFR) and Met, the receptor for hepatocyte growth factor (HGF), but the involvement of Gab1 in EGFR and Met signalling has not been directly compared in the same cell. We have studied mechanisms of activation and role in mitogenic signalling of Gab1 in response to EGF and HGF in cultured rat hepatocytes. Gab1, but not Gab2, was expressed in the hepatocytes and was phosphorylated upon stimulation with EGF or HGF. Depletion of Gab1, using siRNA, decreased the ERK and Akt activation, cyclin D1 expression, and DNA synthesis in response to both EGF and HGF. Studies of mechanisms of recruitment to the receptors showed that HGF induced co-precipitation of Gab1 and Met while EGF induced binding of Gab1 to Grb2 but not to EGFR. Gab1 activation in response to both EGF and HGF was dependent on PI3K. While EGF activated Gab1 and Shc equally, within the same concentration range, HGF very potently and almost exclusively activated Gab1, having only a minimal effect on Shc. Collectively, our results strongly suggest that although Gab1 interacts differently with EGFR and Met, it is involved in mitogenic signalling mediated by both these growth factor receptors in hepatocytes.     

Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

Background

Previous studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44^mapk) and ERK2 (p42^mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca²⁺ and Ca²⁺-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F_2α.

Results

Pretreatment of the cells with the Ca²⁺ chelators BAPTA-AM or EGTA, as well as the Ca²⁺ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca²⁺/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF_2α.

Conclusion

The present data indicate that Ca²⁺ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.     

Scar tissue classification using nonlinear optical microscopy and discriminant analysis

This paper addresses the scar tissue maturation process that occurs stepwise, and calls for reliable classification. The structure of collagen imaged by nonlinear optical microscopy (NLOM) in post‐burn hypertrophic and mature scar, as well as in normal skin, appeared to distinguish these maturation steps. However, it was a discrimination analysis, demonstrated here, that automated and quantified the scar tissue maturation process. The achieved scar classification accuracy was as high as 96%. The combination of NLOM and discrimination analysis is believed to be instrumental in gaining insight into the scar formation, for express diagnosis of scar and surgery planning. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Mortality of vertebrates on a road crossing the Biebrza Valley (NE Poland)

Mortality of vertebrates was monitored on a local road running across Poland’s Biebrza River Valley during 2 years (August 2005–July 2006). On the basis of distance from the river and surrounding habitats, the road (of total length 2,510 m) was divided into three stretches. The road was monitored on foot by two people every month, over a few consecutive days. A total of 1,892 road kills representing at least 47 species were found. Of these, 90.7% were amphibians, 4.2% mammals, 3.1% birds and 2.0% reptiles. Most (70%) of the amphibians were anurans, with the common toad, common frog and moor frog among them together accounting for 82% of the total. Mortality among amphibians differed between months, most anurans dying in May and August, while a majority of Urodela are lost in October. The peaks in mortality were connected with the migration of adult amphibians in spring and juveniles in summer and autumn. The number of amphibians killed was greatest on the (wettest) stretch adjacent to the river and decreased with distance from it. Mortality among birds was highest in July—probably in association with the dispersal of young individuals. Among recorded mammalian road kills, there was a prevalence of small rodents (mainly voles) and insectivores (mainly shrews). Medium-sized mammals were found only accidentally. Mortality in general was conditioned by the number of anurans killed.     

Prostaglandin E2 upregulates EGF-stimulated signaling in mitogenic pathways involving Akt and ERK in hepatocytes

Prostaglandins (PGs) such as PGE2 enhance proliferation in many cells, apparently through several distinct mechanisms, including transactivation of the epidermal growth factor (EGF) receptor (EGFR) as well as EGFR-independent pathways. In this study we found that in primary cultures of rat hepatocytes PGE2 did not induce phosphorylation of the EGFR, and the EGFR tyrosine kinase blockers gefitinib and AG1478 did not affect PGE2-stimulated phosphorylation of ERK1/2. In contrast, PGE2 elicited EGFR phosphorylation and EGFR tyrosine kinase inhibitor-sensitive ERK phosphorylation in MH1C1 hepatoma cells. These findings suggest that PGE2 elicits EGFR transactivation in MH1C1 cells but not in hepatocytes. Treatment of the hepatocytes with PGE2 at 3 h after plating amplified the stimulatory effect on DNA synthesis of EGF administered at 24 h and advanced and augmented the cyclin D1 expression in response to EGF in hepatocytes. The pretreatment of the hepatocytes with PGE2 resulted in an increase in the magnitude of EGF-stimulated Akt phosphorylation and ERK1/2 phosphorylation and kinase activity, including an extended duration of the responses, particularly of ERK, to EGF in PGE2-treated cells. Pertussis toxin abolished the ability of PGE2 to enhance the Akt and ERK responses to EGF. The results suggest that in hepatocytes, unlike MH1C1 hepatoma cells, PGE2 does not transactivate the EGFR, but instead acts in synergism with EGF by modulating mitogenic mechanisms downstream of the EGFR. These effects seem to be at least in part G(i) protein-mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways.     

Certain aspects of resistance of plum trees to bacterial canker: Part III. The action of cell-free filtrates of Pseudomonas mors-prunorum Wormald and related phyto-pathogenic bacteria on plum trees

A technique for introducing 2-3 c.c. liquid into the bark of plum trees is described. Parallel injections with dyestuff throughout the spring failed to afford a reliable index of the invasive powers of the various test fluids introduced in this manner.
A susceptible variety of plum (Giant Prune) showed greater injury by cell-free filtrates of Ps. morsprunorum than did a resistant variety (Warwickshire Drooper), which showed negligible injury. The greatest injuries were caused by filtrates of cultures 5 weeks old, and over, especially when concentrated.
Of other bacteria tested Ps. prunicola, Ps. syringae strains, Syringa 3, Ap. No. 1, Ap. No. 2, and Bact. pruni , yielded damaging filtrates, whereas Ps. syringae , strains pear 6 and 7, Ps. tabaci, Ps. fluorescens, Ps. marginalis, Ps. phaseolicola, Ps. tumefaciens, Ps. pisi and Ps. cerasi failed to do so.
Some evidence is advanced for the view that the deleterious activity of Ps. mors-prunorum may be due in part to an endotoxin of protein nature obtainable from the dried bacterial cells by acetic acid extraction.     

Certain aspects of resistance of plum trees to bacterial canker Part I. Some biochemical characteristics of Pseudomonas morsprunorum (Wormald) and related phytopathogenic bacteria

Several strains of Pseudomonas mors-prunorum (Wormald) and Ps. prunicola (Wormald) isolated from pathological lesions of plum and cherry were studied together with the causal organism of bacterial canker of stone-fruits in California (Ps. syringae from apricot) and other phytopathogenic bacteria obtained from pear and syringa. Comparison was also made with pseudomonas forms pathogenic to pea, bean, lettuce, and tobacco, and with the common saprophytes Ps. fluorescens and Ps. pyocyaneus. With the exception of two yellow organisms (B. pruni and the Pear 8 strain—the latter, however, very occasionally showing fluorescence), all belong to the green-fluorescent group of Pseudomonas (Dowson's Group II). On the basis of their dissimilation of C and N compounds a very close relationship has been established between these fruit-tree and syringa pathogens of the green-fluorescent group. Ps. mors-prunorum is not highly specialized in its nutrient requirements but can satisfy its fundamental C and N requirements from a very large variety of simple substances. The only consistent biochemical differentiation shown by Ps. mors-prunorum (including some of the syringa strains) in comparison with Ps. prunicola (including Ps. syringae from apricot and most of the pear strains) is its more rapid production of add from sucrose. Both the mors-prunorum and prunicola varieties produce a levan from sucrose, which causes a raised gummy growth on solid sucrose-containing media. This applies also to Ps. pisi, Ps. tabaci, and Ps. phaseolicola , but is not the case with the weakly pathogenic forms— Ps. marginalis, cerasi (= trifoliorum , from bean), and the saprophytes— Ps. fluorescens and Ps. pyocyaneus.
On the basis of biochemical characteristics, considered apart from host pathogenicity, there is no justification for erecting to specific rank these various levan-forming. green-fluorescent, phytopathogenic pseudomonads.