Differential proteomic analysis of lymphatic,venous, and arterial endothelial cells extracted from bovine mesenteric vessels

Differential protein profiling by 2‐D PAGE is generally useful in biomarker discovery, proteome analysis and routine sample preparation prior to analysis by MS. The goal of this study was to compare 2‐D PAGE‐resolved protein profile of lymphatic endothelial cells to those of venous, and arterial endothelial cells isolated from lymphatic and blood vessels of bovine mesentery (bm). Three 2‐D PAGE electrophoretograms were produced for each of the three cell types and quantitatively analyzed. Protein identification by LC‐MS/MS was performed to identify 39 proteins found to be present at statistically significantly different levels in the three cell types (p<0.05). Most of the 39 proteins have not been previously reported in EC proteomic studies of 2‐D PAGE electrophoretograms. Three proteins, HSPA1B (HSP70 family member), HSPB1 (HSP27 family member), and UBE2D3 (a member of E2 ubiquitin‐conjugating enzymes) found to be at highest levels in bm arterial endothelial cells, bm venous endothelial cells, and bm lymphatic endothelial cells, respectively, were validated by immunoblotting with appropriate antibodies. The lack of substantial overlap between our results and those of other groups' comparative studies are discussed. Functional implications of differences in levels of various proteins identified in the three cell types are also discussed.     

Further expansion of the genus Cercopagis (Crustacea,Branchiopoda, Onychopoda) in the Baltic Sea,with notes on the taxa present and their ecology

The onychopod cladoceran Cercopagis that recently invaded the Baltic Sea is reported from new zones of the northern Baltic proper. Because of successful survival and an expanding distribution range, the addition of Cercopagis to the Baltic fauna is considered to be permanent. What has previously been cited as Cercopagis pengoi encompasses the morphology of several other species, subspecies and forms. Either a number of morphologically similar species is present, or there is a number of spurious species in Cercopagis. The last hypothesis is favoured. The spatial distribution pattern of Cercopagis, as well as that of total zooplankton, was correlated with depth. Deep (>100 m) and shallow (<10 m) stations had significantly lower abundance than stations of intermediate depth (<100 m). An overview of the distribution of C. pengoi group in fresh and brackish waters suggests a high tolerance to environmental factors, but with differences among taxa. Due to this ecological flexibility, the colonization of the Baltic is not unexpected. Increasing salinity may restrict dispersal of cercopagids to the southern areas of the Baltic and to the North Sea, but inland lakes (e.g. in Sweden) present an ecological profile suitable for colonization.     

The c-Cbl-associated protein and c-Cbl are two new partners of the SH2-containing inositol polyphosphate 5-phosphatase SHIP2

SHIP2 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains motifs susceptible to mediate protein-protein interaction. Using yeast two-hybrid, GST-pulldown, and coimmunoprecipitation studies, we isolated the CAP cDNA as a specific partner of SHIP2 proline-rich domain and showed by GST-pulldown experiments that the interaction took place with the SH3C of CAP. The interaction was not modulated in COS-7 cells stimulated by EGF neither in CHO cells overexpressing the insulin receptor in the presence or absence of insulin stimulation. We also showed that SHIP2 was able to coimmunoprecipitate with endogenous c-Cbl protein in the absence of CAP and with the insulin receptor in CHO-IR cell extracts. The presence of SHIP2 in a complex around the insulin receptor could account for the very specific increase in insulin sensitivity of SHIP2 knock-out mice.     

A unique autophosphorylation site on Tie2/Tek mediates Dok-R phosphotyrosine binding domain binding and function

Tie2/Tek is an endothelial cell receptor tyrosine kinase that induces signal transduction pathways involved in cell migration upon angiopoietin-1 (Ang1) stimulation. To address the importance of the various tyrosine residues of Tie2 in signal transduction, we generated a series of Tie2 mutants and examined their signaling properties. Using this approach in conjunction with a phosphorylation state-specific antibody, we identified tyrosine residue 1106 on Tie2 as an Ang1-dependent autophosphorylation site that mediates binding and phosphorylation of the downstream-of-kinase-related (Dok-R) docking protein. This tyrosine residue is contained within a unique interaction motif for the phosphotyrosine binding domain of Dok-R, and the pleckstrin homology domain of Dok-R further contributes to Tie2 binding in a phosphatidylinositol 3'-kinase-dependent manner. Introduction of a Tie2 mutant lacking tyrosine residue 1106 into endothelial cells interferes with Dok-R phosphorylation in response to Ang1. Furthermore, this mutant is unable to restore the migration potential of endothelial cells derived from mice lacking Tie2. Together, these findings demonstrate that tyrosine residue 1106 on Tie2 is critical for coupling downstream cell migration signal transduction pathways with Ang1 stimulation in endothelial cells.     

MiRNA Expression May Account for Chronic but Not for Acute Regulation of mRNA Expression in Human Thyroid Tumor Models

Background

For thyroid tumorigenesis, two main human in vitro models are available: primary cultures of human thyrocytes treated with TSH or EGF/serum as models for autonomous adenomas (AA) or papillary thyroid carcinomas (PTC) respectively, and human thyroid tumor derived cell lines. Previous works of our group have assessed properties of those models, with a special emphasis on mRNA regulations. It is often assumed that miRNA may be one of the primary events inducing these mRNA regulations.

Methods

The purpose of this study was to investigate the representativity of those models to study microRNA regulations and their relation with mRNA expression. To achieve this aim, the miRNA expressions profiles of primary cultures treated with TSH or EGF/serum and of 6 thyroid cancer cell lines were compared to the expression profiles of 35 tumor tissues obtained by microarrays.

Results

Our data on primary cultures have shown that the TSH or EGF/serum treatment did not greatly modify the microRNA expression profiles, which is contrary to what is observed for mRNA expression profiles, although they still evolved differently according to the treatment. The analysis of miRNA and mRNA expressions profiles in the cell lines has shown that they have evolved into a common, dedifferentiated phenotype, closer to ATC than to the tumors they are derived from.

Conclusions

Long-terms TSH or EGF/serum treatments do not mimic AA or PTC respectively in terms of miRNA expression as they do for mRNA, suggesting that the regulations of mRNA expression induced by these physiological agents occur independently of miRNA. The general patterns of miRNA expression in the cell lines suggest that they represent a useful model for undifferentiated thyroid cancer. Mirna probably do not mediate the rapid changes in gene expression in rapid cell biology regulation.     

Alteration of 5'-Nucleotidase and Na+, K+-ATPase in Central and Peripheral Nervous Tissue from Dysmyelinating Mutants (jimpy, quaking, Trembler, shiverer, and mld). Comparison with CNPase in the Developing Sciatic Nerve from Trembler

Abstract: 5'Nucleotidase and Na⁺,K⁺-ATPase are very probably myelin-associated enzymes, although not specific for this membrane. Thus, it is important to determine their activity in dysmyelinating mutants in either CNS (quaking, jimpy, shiverer, and mld) or PNS (Trembler). CNS: The activity of 5'nucleotidase was lower in mouse than in rat (10.5 and 28.0 nmol/min/mg protein in brain, respectively). In mouse myelin, the activity was 30 nmol/min/mg protein (and 72 in rat myelin). In mutants, the brain activity was very close to normal. In contrast, ATPase, the activity of which was higher in myelin as compared with forebrain homogenate, presented a reduced activity in various 21-day-old and adult mutants, except Trembler. It was normal in 8-day-old quaking and in cerebella from mutants. PNS: ATPase was lower than in brain and reduced in most mutants, this being expected for Trembler and quaking but not for shiverer and mld. 5'-Nucleotidase activity was higher compared with that in brain homogenate (relatively stable between 10-day postnatal and adult). It was affected in the mutants; in Trembler it was nearly normal in young animals but increased during development. Thus in Trembler, two different myelin-related enzymes and a myelin-specific enzyme (CNPase) presented different developmental patterns: ATPase was always reduced, 5'-nucleotidase was normal, and CNPase was slightly below normal in young (68% of the control value); CNPase activity declined during development but 5'-nucleotidase increased (42% and 190% of the control in 60-day-old animals). It is necessary to consider these results in parallel with alterations in the PNS because of Schwann cell abnormalities. Thus, determination of these two enzymes will provide a useful tool to study myelination and myelin assembly under both normal and pathological conditions.     

Distinct distribution of immunoreactive dynorphin and leucine enkephalin in various populations of isolated adrenal cromaffin cells

The distribution of immunoreactive-dynorphin (ir-Dyn) in isolated subpopulations of bovine adrenal chromaffin cells was examined and compared with that of adrenaline (A), noradrenaline (NA) and ir-Leucine-Enkephalin (ir-Leu-Enk). Using a stepwise bovine serum albumin (BSA) gradient, various populations of catecholamine-storing cells were separated and designated as cell layers I, II and III. Cell layer I contained more NA than A; cell layer II contained slightly more A than NA whereas cell layer III was highly enriched in A. The original cell preparation contained 2.9 times more ir-Leu-Enk than ir-Dyn (4.7 and 1.6 pmoles per 10(6) cells, respectively). After separation of the cells on BSA gradient, ir-Dyn was mainly detected in cell layer I (4.0 pmoles/10(6) cells) whereas ir-Leu-Enk was concentrated in cell layer III (8.3 pmoles/10(6) cells). Both peptides were secreted in response to acetylcholine (5 x 10(-5) M), but the amount secreted was in accordance with the cell content in each peptide. After subcellular fractionation of the adrenal medulla, the neuropeptides were found in close association with catecholamines in the secretory granules. These results indicate that bovine adrenal chromaffin cells can be isolated according to their specific content in A, NA and opioid peptides and are consistent with the hypothesis of distinct biosynthetic pathways for Dyn and the Enk.     

Identification of microtubule growth deceleration and its regulation by conserved and novel proteins

Microtubules (MTs) are cytoskeletal polymers that participate in diverse cellular functions, including cell division, intracellular trafficking, and templating of cilia and flagella. MTs undergo dynamic instability, alternating between growth and shortening via catastrophe and rescue events. The rates and frequencies of MT dynamic parameters appear to be characteristic for a given cell type. We recently reported that all MT dynamic parameters vary throughout differentiation of a smooth muscle cell type in intact Caenorhabditis elegans. Here we describe local differences in MT dynamics and a novel MT behavior: an abrupt change in growth rate (deceleration) of single MTs occurring in the cell periphery of these cells. MT deceleration occurs where there is a decrease in local soluble tubulin concentration at the cell periphery. This local regulation of tubulin concentration and MT deceleration are dependent on two novel homologues of human cylicin. These novel ORFs, which we name cylc-1 and -2, share sequence homology with stathmins and encode small, very basic proteins containing several KKD/E repeats. The TOG domain–containing protein ZYG-9^TOGp is responsible for the faster polymerization rate within the cell body. Thus we have defined two contributors to the molecular regulation for this novel MT behavior.     

In vitro effects of arachidonic acid and of inhibitors of its metabolism on the dog thyroid gland

Incubation of dog thyroid tissue with arachidonic acid (10 to 200 μM) led to the following events:

- low conversion to prostaglandins E₂ and F_2α: 0.07% and 0.02% per hour and 100 mg tissue, respectively

- inhibition of the stimulatory effect of low concentrations of TSH on thyroid secretion: the secretory effect of supra-maximal concentrations of TSH and of dB-cAMP was unaffected

- inhibition of the cyclic AMP accumulation induced by TSH: this effect was inhibited neither by indomethacin nor by ETYA; cyclic AMP accumulation in response to cholera toxin or PGE₁ was unaffected

- no effect on cyclic GMP level

- stimulation of thyroid proteins iodination.

ETYA, but not indomethacin, depressed the iodination of thyroid proteins in resting and stimulated tissue. These data show that arachidonic acid-or a metabolite-can modulate thyroid responsiveness to TSH and suggest that lipoxygenase-products of arachidonic acid metabolism could be involved in thyroid proteins iodination.