Cloning and characterization of a new isoform of skeletal muscle triadin.

We have shown that several isoforms of triadin, a protein involved in calcium release process through the ryanodine receptor, are expressed in rat skeletal muscle, and we have cloned two of these isoforms. One is the rat homolog of the 95-kDa triadin identified in rabbit skeletal muscle, and the second one, shorter, is a truncated form of the previous one, but with a new unique COOH-terminal end. We propose to name the two proteins identified here Trisk 95 and Trisk 51. We have produced antibodies specific to each isoform. Using these antibodies, we have shown that the newly identified protein, Trisk 51, is actually expressed in adult rat skeletal muscle and also in rat embryo skeletal muscle. Immunofluorescent labeling of rat skeletal muscle with anti-Trisk 95, anti-Trisk 51, or anti-ryanodine receptor antibodies shows a similar localization of these proteins, in the tissue. Transfection of L6 cells with cDNA of Trisk 51 or Trisk 95 leads to the expression of proteins with the expected molecular weight, identical to those detected in rat skeletal muscle. Both proteins appear during differentiation of satellite cells in myotubes which may indicate the involvement of these two isoforms in the building of a functional calcium release machinery.

The MS-5 murine stromal cell line and hematopoietic growth factors synergize to support the megakaryocytic differentiation of embryonic stem cells.

Murine embryonic stem (ES) cells are able to differentiate into erythroid, mast, and granulomonocytic cells by using appropriate culture conditions. Because we were interested in the regulation of tissue-specific expression of the platelet glycoprotein IIb gene, we studied the culture conditions, aiming at the reproducible production of myeloid cells that included megakaryocytes (MKs) from ES cells. We showed that even a complex cocktail of HGFs (stem cell factor, interleukin 3, IL6, IL11, granulocyte colony-stimulating factor, and erythropoietin) is unable to induce significant myeloid differentiation in day 12 embryoid bodies. Cocultures of MS-5 stromal cells with ES cells were slightly more productive than HGFs. A strong synergistic effect was observed on the growth of myeloid colonies and MKs when we used a combination of MS-5 cells plus the HGF cocktail. Conditioned medium from MS-5 cells also synergized with the HGF cocktail to produce a substantial number of mixed colonies containing MKs. The addition of fibroblast growth factor-2 (FGF-2) to the HGF cocktail plus MS-5 nearly doubled the number of myeloid progenitors, including those with MKs. Thrombopoietin (TPO) alone or in any combination with MS-5 or HGFs, did not increase the number of MK-containing colonies. However, when TPO was added to the HGF cocktail + FGF-2 + MS-5, the number of MKs in liquid cultures and mixed colonies increased, and many exhibited a "hairy" appearance resembling pseudopodial proplatelet formation. Having defined the culture conditions of ES cells that allow the production of all the myeloid lineages including MKs, we conclude that the hematopoietic differentiation model of ES cells is especially useful for studying the regulation of expression of any gene important in early hematopoiesis.

The tissue-specific transcriptional regulation of the megakaryocytic glycoprotein IIb gene is controlled by interactions between a repressor and positive cis-acting elements.

Much information on regulation of the transcription of megakaryocytic genes stems from studies on the glycoprotein IIb (GPIIb) gene, an early and specific marker of this lineage. Transcriptional activity is controlled by the association of positive promoter elements corresponding to binding sites for the transcription factor GATA-1 and a member of the Ets family. In the present study, we show that these elements are not directly involved in the control of cell specificity. In contrast, we identified a sequence located between -170 and -73 that exhibited a repressor activity based on an analysis of the transcriptional activity of 5'-deleted GPIIb promoter fragments transfected in the nonhematopoietic HeLa cells. Further analysis of this repressor by substitution mutagenesis of the -139/-63 region showed that bases -120/-116 and -102/-93 were required for full repressor activity. The repressor is able to interact differentially with GPIIb promoter elements active in the megakaryocytic HEL, the erythroid K562, the monocytic U937, or the nonhematopoietic HeLa cell lines, indicating that it controls GPIIb gene tissue specificity. In addition, direct evidence for tissue-specific interaction between this repressor and the GPIIb -598/ -406 enhancer was obtained when these elements were set in the context of a heterologous SV40 promoter. Interestingly, the same repressor element controlling tissue specificity of the GPIIb gene may also control its temporal expression during megakaryocyte differentiation, based on recent evidence obtained by Fong and Santoro (J Biol Chem 269:18441, 1994). Finally, we found that the -120/-116 GPIIb sequence was part of a consensus motif shared by promoters of other megakaryocyte-specific genes, suggesting a common repressor mechanism.

The transcription factor GATA-1 regulates the promoter activity of the platelet glycoprotein IIb gene.

Glycoprotein IIb (GPIIb) is an early and specific marker of the megakaryocytic lineage. We have previously shown that a fragment extending 643 base pairs upstream the transcription start site of the human GPIIb promoter was able to control the tissue-specific expression of the CAT gene in transfection experiments. Four potential GATA-binding sites, located at positions -463, -376, -243, and -54 are present within this fragment. Gel shift analysis revealed that nuclear extracts from the erythroleukemic cell line HEL contain a DNA-binding protein that recognizes these GATA sites. Using an antiserum raised to an hydrophilic region of the transcription factor GATA-1, the HEL GATA-binding protein was found to be GATA-1. Point mutations of the different GATA sites indicated that they did not equally contribute to GPIIb promoter activity. The -463 GATA motif located in an enhancer region is essential for full transcription activity and was found to be dominant upon the other GATA motifs. When this site is mutated, the -54 GATA site appears to be essential for the remaining CAT activity. These results indicate that the transcription factor GATA-1 plays an important role in the regulation of the transcription of the megakaryocyte specific GPIIb gene.

Characterization of a specific erythromegakaryocytic enhancer within the glycoprotein IIb promoter.

The gene coding for glycoprotein IIb (GPIIb), the alpha subunit of platelet integrin GPIIb/IIIa is an early and specific marker of the megakaryocytic lineage. Thus, studies on the regulation of this gene may provide helpful information on the mechanisms controlling cell specificity and differentiation in this lineage. The promoter region of this gene was isolated and analyzed to understand its tissue-specific transcriptional activity. A region extending from nucleotides -414 to -554 was found to be extremely important for the promoter function. Deletion of this region results in a 70% decrease of the promoter activity, as measured in CAT assays. This region has the properties of an enhancer. It is able to activate a heterologous promoter, in a distance- and orientation-independent manner, in both megakaryocytic and erythroid cells. This enhancer contains binding sites for nuclear factors and mutation of these sites, individually or together, abolish the enhancer activity. These nuclear factors are present in megakaryocytic and erythroid cell lineages, but they are absent in the other tested cells. One of the sites, named domain D, contains a TTATC motif that may interact with the transcription factor GATA1, active in erythroid and megakaryocytic cells. These results indicate that the promoter of a megakaryocytic gene contains a tissue specific enhancer, active in both the erythroid and the megakaryocytic lineages, and may implicate the erythroid factor GATA1.

Gene regulation in megakaryocyte.

Analysis of the mechanisms which control the differentiation of the megakaryocytic lineage is a major commitment to understand the production of circulating blood platelets. One way to approach this question is to examine the promoter domain of a marker gene which is expressed exclusively in the megakaryocytic lineage and at an early stage of the differentiation process. For this purpose the gene coding for the platelet specific glycoprotein IIb was isolated and its promoter was analysed. This promoter contains positive and negative DNA responsive elements that are responsible for the cell specific expression of the gene. With this promoter region it is now possible to direct the expression of heterologous genes in vivo using the transgenic approach.

GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs.

Platelet GPIIbIIIa is only synthesized in megakaryocyte or in cell lines with megakaryocytic features. The sequence for GPIIb and GPIIIa have recently been derived from cDNAs obtained from HEL cells. The sequence of these proteins produced by the megakaryocyte, has however, not been determined yet. This study describes full length cDNAs for GPIIb and GPIIIa isolated from megakaryocyte cDNA libraries. The cDNA sequences indicate the presence of nucleotide differences, between the sequence of the GPIIIa cDNAs from HEL cells, endothelial cells and megakaryocytes. One difference was also observed between HEL and megakaryocyte GPIIb at position 633 where a cysteine in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4 kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.

Cryopreservation of human bone marrow cells by a modification of the two-step cooling technique.

The first attempt to freeze human bone marrow cells with a two-step cooling method is reported. A simple and reliable way of obtaining stable first-step subzero freezing baths is described. One-milliliter samples each containing 20 X 10(6) bone marrow cells and 10% Me2SO were frozen in polypropylene cryotubes. Using these experimental conditions, the optimal freezing temperature was found to be in the range of -36 to 37.5 degrees C for BM progenitor cell (GM-CFC, CFUE, and BFUE) survival. Such temperatures were easily obtained in stable sludges of anisole or K2CO3 eutectic solution in water. The optimal holding time was 20 min before plunging tubes into liquid nitrogen. Similar or improved progenitor cell recoveries were observed compared with the conventional cooling technique. Adaptation of this two-step technique for the freezing of large volumes of BM cells for autografting is under investigation.

Reversible inhibition of the in vitro coagulation of human plasma by lectins.

Wheat germ agglutinin (WGA) and concanavalin A (Con-A) (also red kidney bean agglutinin, PHA) have been found to be inhibitors of plasma clotting in vitro. At 40 micrograms/ml and 250 micrograms/ml (4.4 MicroM and 10 microM in carbohydrate binding sites, final concentrations) respectively, WGA and Con-A are able to double the activated partial thromboplastin time of normal human control plasma. Their inhibitory effect is due to their capacity to interact with the carbohydrate portion of blood clotting factors. It is totally abolished in the presence of specific saccharides for WGA or Con-A and is attenuated in the presence of 4% (v/v, final concentration) of human erythrocytes. The action of WGA is mediated by its ability to interact with N-acetylneuraminic acid. When purified phospholipid vesicles plus kaolin are used as an activator instead of cephalidin, this effect persists to the same extent. These two lectins also prolong the plasma clotting time using Russell's viper venom plus purified phospholipid vesicles as an activator. Quick's time was also prolonged by WGA and Con-A but to a lesser extent in this case. WGA can interact directly with some purified blood clotting factors (IX, X and II) in a classical lectin-glycoprotein precipitin reaction. When assessed at individual factors level in whole plasma using clogging assays, direct inhibitions by WGA are only apparent.

[Prophylaxis of infectious complications in maxillo-facial surgery. Value of cefazolin. (A review of 400 cases)]. / Prophylaxie des complications infectieuses dans les interventions de chirurgie maxillo-faciale. Intérêt de l'utilisation de la céfazoline. (A propos de quatre cents cas).

The value of short-term prophylaxis with antibiotics in maxillo-facial surgery and plastic facial surgery is studied. 200 patients were included in the study and compared to 200 controls who were given the usual systematic antibiotic therapy, with a different antibiotic, for more than 6 days. 400 case-reports were thus retrospectively analyzed. The results show that when the surgical procedure lasts for less than three hours, short-term prophylaxis with antibiotics is more effective than the usual systematic antibiotic therapy given for more than 6 days.

Purification and cryopreservation of granulomonocytic colony forming cells (GM-CFC) from the blood of patients with chronic granulocytic leukemia (CGL) for autologous transplantation.

A simple density-cut separation technique is described for obtaining GM-CFC concentrates from the peripheral blood of patients with chronic granulocytic leukemia (CGL) for autografting at the acute blastic phase of the disease. Large numbers of granulomonocytic colony forming cells (GM-CFC) were recovered from a single procedure (68.4 X 10(6) GM-CFC). The cryopreservation of these concentrates in liquid nitrogen allowed the recovery of 46.6 +/- 33.8% viable GM-CFC. An improved yield of GM-CFC was obtained by avoiding washing the cells (65.2 +/- 41.1%). The separation technique resulted in a concentrated suspension of progenitors in a small volume of medium (mean = 15 ml) allowing a great reduction in the amount of the cryoprotective agent injected into the patient at autografting. Preliminary data obtained in 4 patients transfused in blastic crisis after chemotherapy, with or without TBI, indicated the capacity of stored concentrates to repopulate these patients.

[Irradiation of basal cell epitheliomas of the face: a handicap for the surgeon in some cases of recurrence (author's transl)]. / Le handicap pour les chirurgiens de certaines irradiations des épithéliomas baso-cellulaires de la face en cas de récidive.

A case is reported which illustrates the progressive relapsing nature and local invasive character of certain irradiated basal cell epitheliomas. One such case, that involved the tragus initially, and had been irradiated on five occasions, successively invaded the lobe of the ear, the auditory canal, the helix, the mastoid, and the zygomatotemporal region in spite of six operations and the use of cryotherapy.