FGF receptor dependent regulation of Lhx9 expression in the developing nervous system.

LIM-homeodomain (LIM-hd) proteins form a multifunctional family of transcription factors that plays critical roles in the development of progenitor and post-mitotic cells. Considerable work has focused on what regulates their expression post-mitotically in the spinal cord. However, little is known about what regulates LIM-hd genes at earlier developmental stages. To address this question, we explored the role of fibroblast growth factor (FGF) signalling in regulating the expression of a Xenopus laevis Lhx9 orthologue (XLhx9). XLhx9 is first expressed in the eye field and hindbrain, and when FGF receptor (FGFR) activation was inhibited prior to its onset, both brain and eye field expression was diminished. However, when FGFRs were inhibited after XLhx9 onset, retinal expression remained strong and brain expression was again diminished. These data suggest that while FGF signalling initiates and maintains brain XLhx9 expression, in the eye primordium the requirement of FGFs for expression is rapidly lost.

TSAd interacts with Smad2 and Smad3.

Smad-dependent signalling initiated by TGFbeta superfamily members can be modulated by a variety of interacting proteins. Using yeast two-hybrid, co-immunoprecipitation, and GST pull-down assays we identified T-cell SH2 adapter (TSAd) as a protein that interacts with Smad2 and Smad3. TSAd is an adapter protein thought to participate in many different signalling pathways. The objective of this study was to elucidate the domains important for interaction between TSAd and Smad proteins. Our results suggest a model for TSAd-Smad interaction that is facilitated by multiple TSAd domains, but primarily through the TSAd type I SH2 domain. Interestingly, we also found that both Smad2 and Smad3 interact with the Lck type I SH2 domain, but not the PI3K type III SH2 domain. This research raises the possibility that interaction between SH2-containing proteins and Smad proteins may represent another method to modulate Smad-dependent signalling.

Ca(v)3.1 splice variant expression during neuronal differentiation of Y-79 retinoblastoma cells.

A decrease in transient-type calcium channel current, Ca(v)3.1 protein and the mRNA encoding these channels has been reported during differentiation of human retinoblastoma cells. In this study, we examined splice variants of Ca(v)3.1 before and after neuronal differentiation of the Y-79 retinoblastoma cell line to investigate the potential contribution of Ca(v)3.1 to Y-79 differentiation. In Ca(v)3.1, alternative splicing induces variations in three cytoplasmic regions, e.g. the link between domains II and III (producing isoforms e+ and e-), the link between domains III and IV (producing isoforms a, b, ac and bc) and the carboxy terminal region (producing isoforms f and d). Our results demonstrate that Ca(v)3.1e was not expressed in either undifferentiated or differentiated retinoblastoma cells. Splice variants Ca(v)3.1ac; Ca(v)3.1bc and Ca(v)3.1b were all identified in undifferentiated retinoblastoma cells, while expression of these variants in differentiated cells was restricted to the Ca(v)3.1bc isoform. The carboxy terminal variant Ca(v)3.1f is expressed independently of the differentiation status of retinoblastoma cells with or without Ca(v)3.1d. Examination of the functional contribution of Ca(v)3.1 protein to Y-79 cell differentiation revealed that in Y-79 cells transfected with Ca(v)3.1 antisense oligodeoxynucleotides, knockdown of Ca(v)3.1 did not alter the time-course of differentiation or neuritogenesis. The changes in Ca(v)3.1 splice variants were not required for the initiation of differentiation but may be associated with tissue-specific expression or localized alterations in Ca(2+) signaling that are essential for establishment of the mature differentiated phenotype.

Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2'-bipyridine) ruthenium (II).

The paper shows the ability of the fluorochrome tris(2,2'-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.

Insight into the profibrinolytic activity of heparin: effects on the activation of plasminogen mediated by urokinase.

The aim of this work was to clarify the role of urokinase-type plasminogen activator (uPA) on the profibrinolytic activity of heparin, chemically modified heparins [partially: N-desulfated (N-des), N-desulfated N-acetylated (N-des N-ac), O-desulfated (O-des), O/N-desulfated N-acetylated (O/N-des N-ac)] and heparan sulfate. Binding competition assays of plasminogen and uPA to heparin-sepharose demonstrated that heparin bound to both enzymes. Moreover, in the presence of increasing amounts of heparin, plasminogen activation mediated by uPA occurred as a bell-shaped curve, suggesting the formation of a ternary complex. In contrast, all chemically-modified heparins lacked this cofactor activity, although N-des and heparan sulfate partially retained the uPA binding capacity, and O-des partially bound to both plasminogen and uPA. Plasmatic euglobulins from mice treated with heparin, as well as with modified heparins with uPA binding capacity, presented a 2-fold enhancement of 47 kDa lytic band, as assessed by zymographic analysis. Western blotting analysis anti-uPA (47 kDa) showed that the enhanced uPA activity correlated with a true increase in uPA protein levels. These results suggest that the profibrinolytic activity of heparin mediated by uPA could be caused by an increase in uPA protein levels rather than by a cofactor activity mediated by a formation of ternary complexes.

Analytical SEM of cell polyanions stained with tris(2,2'-bipyridine)ruthenium(II).

Peritoneal mast cells and lymphocytes from mice were placed on graphite supports, fixed in methanol, stained with the new fluorochrome tris(2,2'-bipyridine)ruthenium(II) and microanalysed using scanning electron microscopy (SEM). X-ray microanalysis showed the expected signal of P and S (K alpha lines) in emission spectra of lymphocytes and mast cells. The signal of Ru (L alpha 1 and L beta 1) overlapped with that of Cl, although the peak in the corresponding region was about 7 times higher than that from unstained cells. X-ray images showed the topographic localization of P, S and Ru in mast cells and lymphocytes and confirmed the accumulation of the Ru complex in heparin- and DNA-containing structures. These results indicate that, by using suitable marker elements and detection methods, analytical SEM is a useful complement in cytochemical studies.

Formation and microscopical application of a fluorescent 1,10-phenanthroline derivative of ruthenium red.

In this work we describe the formation and microscopical application of a fluorescent derivative of Ruthenium Red (RR) obtained by heating the dye in the presence of 1,10-phenanthroline (OP). The RR-OP reaction product showed absorption maxima at 416 and 444 nm and intense fluorescence emission at 578 nm under 440 nm exciting light. Neither RR nor OP solutions alone were fluorescent when excited at 440 nm. Using fluorescence microscopy, chicken blood cell smears stained 5 min with the RR-OP derivative showed the chromatin of erythrocyte nuclei with a bright orange fluorescence under violet-blue (436 nm) exciting light.

Cytochemical application of tris (2,2'-bipyridine) ruthenium (II): fluorescence reaction with sulfated polyanions of mast cell granules.

We describe the use of tris (2,2'-bipyridine) ruthenium (II) (Rubipy) as a cationic fluorochrome for cytochemical and histochemical studies. After staining with Rubipy, mast cell granules (MCGs) and lymphocyte nuclei (LN) from mouse peritoneal cavity and human breast carcinoma showed intense orange fluorescence and no fading under blue or blue-violet exciting light. Staining at low pH (< 2) or pre-treatment with Al3+ ions strongly diminished the fluorescence of LN, whereas that of MCG was less affected. Ca2+ and Ba2+ ions only diminished MCG fluorescence. Blots of DNA, pectic acid, heparin, and other sulfated polysaccharides stained with Rubipy showed high emission, which was reduced in DNA and pectic acid staining at low pH. Studies with chemically modified heparins suggested that O-sulfates were more important than N-sulfates in Rubipy-heparin interactions. These results are in agreement with an ionic binding mode between Rubipy and heparin. A very suitable method for mast cell detection was found with Mayer's hematoxylin before Rubipy staining, which could be of great value for histopathological studies. This procedure allowed visualization of the mast cells by fluorescence microscopy, and nuclei and tissue morphology were easily visualized under brightfield illumination.

Fluorescence of mast cell granules in paraffin sections and cell smears induced by an N-quaternary oxazole scintillator.

The N-quaternized derivative of dimethyl-POPOP (termed Q4) induces a bluish-green fluorescent reaction in mast cell granules from paraffin sections and cell smears, in addition to a previously described bluish-white fluorescent reaction in chromatin DNA. The chromatin reaction was abolished by staining the samples either with Mayer's Haematoxylin before Q4 treatment or by Q4 treatment at pH 1.5. The reaction in mast cell granules was absent after substrate methylation. The staining sequence Haematoxylin-Eosin-Q4 also worked well in paraffin sections, allowing the observation of the current histological image under bright-field illumination as well as double-colour emission under fluorescence microscopy. The sequence is proposed as a new diagnostic procedure for demonstrating mast cell granules.

Growth inhibition in vitro of murine mammary adenocarcinoma cells by heparin and chemically modified heparins.

Heparin, a highly sulfated polysaccharide used as an antithrombotic and anticoagulant, inhibits proliferation of several cell types. We have investigated the effect of heparin and chemically modified heparins on the growth of a cell culture of a murine mammary adenocarcinoma (M3). We found that heparin inhibited the proliferation of M3 cells growing either with or without 2% fetal calf serum (FCS) in a dose-dependent and reversible fashion. Several heparins with different anticoagulant properties showed a similar antiproliferative effect. Histological assays showed that heparin was internalized and appeared in cytoplasmic vesicules. O-desulfated, O/N-desulfated N-acetylated and N-desulfated heparins lost their antiproliferative activity, while N-desulfated N-acetylated heparin significantly inhibited cell proliferation with or without FCS. The finding of an antiproliferative action of N-desulfated N-acetylated heparin which does not show anticoagulant activity suggests a possible therapeutic role for this compound as an antineoplastic drug.