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.

Influence of mast cells on two murine mammary adenocarcinomas.

A high content of mast cells (MC) is considered characteristic of neoplasias. Some researchers postulate MC as enhancers of tumor development, others as inhibitors. The purpose of this study was to evaluate the ability of peritoneal cavity MC to modulate the in vivo and in vitro growth of two murine mammary adenocarcinomas with low (M3) and high (MM3) metastatic capacity. MC from the peritoneal cavity of normal (NMC) or tumor-bearing mice (TMC) were used. TMC, which by histochemical methods appeared degranulated, were not able to modify the tumorigenicity of both tumors. NMC, in contrast, decreased M3 tumor incidence and cell proliferation in vitro and increased the latency period of only MM3 tumors. No changes in the number of spontaneous lung metastases could be seen in experiments carried out either with NMC or TMC. We conclude that NMC, which are rich in chemical mediators, can modulate some of the first steps of tumor development. Once tumor-mediated degranulation occurs, MC become unable to regulate it.

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.

[The respiratory system in Arctocephalus australis (Pinnipedia, Otariidae). I--Macroanatomy and microanatomy of the trachea and extrapulmonary airways]. / El sistema respiratorio en Arctocephalus australis (Pinnipedia, Otariidae). I--Macroanatomia y microanatomia de traquea y vias aereas extrapulmonares.

This paper presents the results of histological and histochemical organization of trachea and primary bronchi. The presence of a subepithelial lamina and a smooth muscle layer in the superficial corion, probably related with a functional respiratory mechanism are defined. The structure of tracheal and primary bronchi glands, the way they secretes and the type of secretion are also described.