Microarray analysis detects novel Pax3 downstream target genes.

Pax3 is a transcription factor that is required for the development of embryonic neural tube, neural crest, and somatic derivatives. Our previous study (Mayanil, C. S. K., George, D., Mania-Farnell, B., Bremer, C. L., McLone, D. G., and Bremer, E. G. (2000) J. Biol. Chem. 275, 23259-23266) reveals that overexpression of Pax3 in a human medulloblastoma cell line, DAOY, resulted in an up-regulation in alpha-2,8-polysialyltransferase (STX) gene expression and an increase in polysialic acid on neural cell adhesion molecule. This finding suggests that STX might be a previously undescribed downstream target of Pax3. Because Pax3 is important in diverse cellular functions during development, we are interested in the identification of additional downstream targets of Pax3. We utilized oligonucleotide arrays and RNA isolated from stable Pax3 transfectants to identify potential target genes. A total of 270 genes were altered in the Pax3 transfectants as compared with the vector control and parental cell line. An independent analysis by cDNA expression array and real-time quantitative polymerase chain reaction of several genes confirmed the changes observed by the oligonucleotide microarray data. Of the genes that displayed significant changes in expression, several contain paired and homeodomain binding motifs of Pax3 in their promoter regions. Using promoter-luciferase reporter transfection assays and electromobility shift assays, we showed at least one previously undescribed downstream target, STX, to be a biological downstream target of Pax3. Thus we report several previously undescribed candidate genes to be potential downstream targets of Pax3.

Proteoglycan synthesis during intramembranous bone regeneration following avulsive wounding in guinea pig long bones.

Information on proteoglycan synthesis by bone cells and tissue is largely limited to studies of developing fetal bone. The present investigation focuses on proteoglycan synthesis during the intramembranous type of bone regeneration seen within avulsive (puncture-type) defects placed in guinea pig tibiae. [35S] Sulfate-labeled proteoglycans were extracted from tissue within regenerating tibial avulsive defects seven days following surgical wounding and also from xiphisternal cartilage utilized as an internal control. Labeled proteoglycans in 4M guanidine HCl extracts of regenerating bone and cartilage were purified by DEAE-Sephacel chromatography and further analyzed by chromatography and appropriate enzyme digestions. Regenerating bone tissue contained a proteoglycan relatively small in size (Kav = 0.56 following chromatography on Sepharose CL-2B) compared to proteoglycan from xiphisternal cartilage (Kav = 0.17). Alkaline borohydride treatment degraded this bone proteoglycan (Kav = 0.4 on Sepharose CL-6B), indicating an average molecular weight of glycosaminoglycan chains approximating 50,000. Enzymatic digestions followed by Sepharose CL-6B chromatography showed that glycosaminoglycan side chains of regenerating bone proteoglycan contained dermatan sulfate, with 60% chondroitinase AC II-resistant but chondroitinase ABC-sensitive material. This bone proteoglycan did not interact with hyaluronic acid to form aggregates under conditions where such aggregates were formed by xiphisternal cartilage proteoglycan. The regenerating bone proteoglycans are therefore similar to other bone proteoglycans in hydrodynamic size and glycosaminoglycan chain size, but differ in the per cent of iduronic acid within glycosaminoglycan side chains. This guinea pig bone proteoglycan may be associated with the large mesenchymal cell population noted histologically within the bone defects at seven days of regeneration.

A histologic comparison of Hydron and zinc oxide-eugenol as endodontic filling materials in the primary teeth of dogs.

Currently available endodontic filling materials for primary teeth demonstrate several inadequacies. Therefore, this study histologically evaluates Hydron (2-hydroxyethylmethacrylate) and zinc oxide and eugenol (ZOE) as primary tooth endodontic filling materials in dogs to compare the materials' biocompatibility and to determine whether they undergo resorption in a physiologic manner. Primary molars of six mongrel dogs, 2 to 3 months old, received one-step endodontic therapy. By random selection, two thirds of the molars were filled with either Hydron or ZOE and the remainder were used as unoperated controls. Block sections taken during physiologic root resorption were used to obtain serial hematoxylin and eosin sections of periapical regions. Hydron was phagocytosed by mononuclear macrophages in a physiologic manner and at a rate comparable to that of root tissues. In contrast, ZOE demonstrated delayed resorption and transitory inflammation. Neither material showed cytotoxicity. Hydron may therefore be a suitable endodontic material for human primary teeth.

Biosynthesis of chondroitin sulphate by a Golgi-apparatus-enriched preparation from cultures of mouse mastocytoma cells.

Mouse mastocytoma cells grown in suspension culture produce chondroitin 4-sulphate. A Golgi-apparatus-enriched fraction from these cells was prepared and examined for chondroitin-synthesizing activity. When Golgi-apparatus-enriched fractions were incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine, they demonstrated a greater than 13-fold increase in chondroitin-synthesizing activity over cell homogenates. Similar incubations with the addition of a pentasaccharide from chondroitin sulphate resulted in a greater than 40-fold increase in [14C]glucuronic acid-incorporating activity over cell homogenates. Other membrane fractions had much less activity, suggesting that the Golgi apparatus is the most active location for chondroitin biosynthesis. Products of the incubations indicated the formation of [14C]chondroitin glycosaminoglycan on endogenous primers and formation of [14C]-hexasaccharide and somewhat larger [14C]oligosaccharides on exogenous pentasaccharide acceptors. There was, however, a significant amount of large [14C]-chondroitin glycosaminoglycan formed on pentasaccharide, indicating that some pentasaccharide did serve as a true primer for polysaccharide synthesis.

Galactosyl transferase of a Golgi fraction from cultured neoplastic mast cells.

Suspension cultures of neoplastic mouse mast cells were used to obtain large quantities of a homogeneous cell population as starting material for cell fractionation. A Golgi fraction was prepared by slight modification of established techniques and identified by electron microscopy. Assay of galactosyl transferase activity using ovalbumin, desialylated degalactosylated orosomucoid, and N-acetylglucosamine as galactose acceptors showed that the Golgi fraction was enriched in specific activity over the homogenate. The Golgi galactosyl transferase was examined in detail. Acceptor concentrations for optimal galactose incorporation were determined, and substrate inhibition effects were shown with higher concentrations of all three acceptors. Manganese was shown to be necessary for galactose incorporation. A higher concentration of manganese afforded some protection from substrate inhibition by acceptors, but at the same time was itself inhibitory. All three acceptors competed with one another for galactose incorporation, indicating that a single enzyme catalyzed the transfer of galactose for all acceptors.

A micro method for simultaneous determination of galactosyltransferase and 5'-nucleotidase activities in cell fractions.

Galactosyltransferase and 5'-nucleotidase were assayed in the same reaction mixture, with ovalbumin as exogenous acceptor of (14-C)galactose and with (3-H)AMP as the substrate for the 5'-nucleotidase assay. The substrates and reaction products of either assay had no significant effect on the activity of the other enzyme.