Terminal deoxynucleotidyltransferase forms a ternary complex with a novel chromatin remodeling protein with 82 kDa and core histone

BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances the Ig and TcR gene diversity in the N region at the junctions of variable (V), diversity (D) and joining (J) segments in B- and T-cells. TdT synthesizes the N region in concert with many proteins including DNA-PKcs, Ku70 and Ku86. To elucidate the molecular mechanism of the N region synthesis, we first attempted to isolate the genes with products that directly interact with TdT. RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 2 (TdIF2). The confined region of the C-terminal in TdIF2 is involved in specific interaction with the entire C-terminal in TdT. TdIF2 contains an acidic region comprised of 42 residues. TdIF2 was shown to bind specifically to a core histone by pull down assay using specific antibodies against TdIF2. When a TdT/TdIF2 complex was applied on to a DNA-cellulose column, only TdT bound to the column while TdIF2 passed through. TdIF2 reduces the TdT activity to 46% of its maximum value in vitro assay system using activated DNA as primer. CONCLUSIONS: TdIF2 binds directly to TdT and core histone. Furthermore, TdT, TdIF2 and core histone form a ternary complex. TdIF2 liberates H2A/H2B from a core histone in correlation with PCNA. The enzymatic consequence of the TdIF2/TdT complex is the reduction of TdT activity in vitro. TdIF2 would function as a chromatin remodeling protein at the N region synthesis.     

Direct evidence for granuloma-inducing activity of interleukin-1. Induction of experimental pulmonary granuloma formation in mice by interleukin-1-coupled beads.

Pulmonary granulomas were induced in BALB/c mice by the intratracheal injection of Sephadex G-50 and latex beads. Very large granulomas developed around Sephadex G-50 beads. Minimal inflammation was produced in mice given latex beads. Aqueous extracts prepared from pulmonary granuloma lesions induced in mice by Sephadex G-50 beads contained high levels of interleukin-1 (IL-1) activity but not interleukin-2 (IL-2) activity. IL-1 activity in the extracts correlated with granuloma size. In a subsequent step, large granulomas were induced by the intratracheal injection of Sepharose 4B beads coupled to fractions of the extracts containing IL-1 activity (ie, granuloma-derived IL-1) prepared from Sephadex G-50-induced granulomatous lungs. In addition, large granulomas were induced by the intratracheal injection of recombinant IL-1-coated Sepharose 4B beads. In contrast, very small granulomas were seen when IL-2-coated or plain Sepharose 4B beads were injected into mice. These results indicate that IL-1 participates in the induction and/or expression of granulomas.     

pH-dependent fusion of synaptosomal membrane studied by fluorescence quenching method

We have found that both the synaptic vesicles (SV) and synaptic plasma membrane vesicles (SPM) have an activity to fuse with phosphatidylcoline/phosphatidylserine liposomes in a pH-dependent manner. The activity increases with decreases in extravesicular pH. At a pH lower than 4.0, the activity is almost steady at its maximum value, and there was a rapid drop around pH 5.5. The pH-dependent fusion was inhibited by proteolysis with trypsin; hence, at least in part, some membrane proteins play an important role in these pH-dependent fusion processes. To find specific markers, we screened various protein modifiers and found that anion channel blockers, stilbene derivatives (DIDS and SITS) and glibenclamide, affected the fusion process. DIDS and SITS decreased the fusion activity with an IC50 of 180 and 300 microM, respectively, whereas glibenclamide, on the contrary, increased it. From the results of an autoradiogram using 3H-tagged DIDS, a 30 kDa DIDS-binding protein was identified in the synaptic plasma membrane, which is possible to be responsible for the pH-dependent fusion.     

Effects of short-term cholestyramine feeding on cholesterol metabolism in differently aged rats

The effects of short-term (7 days) feeding of a diet containing cholestyramine (5%) on the cholesterol metabolism were studied in male Sprague-Dawley rats at ages of 5 weeks (young) and 9-10 months (adult). Cholestyramine significantly enhanced the activities of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase both in young and adult rats; however, the absolute values were significantly higher in the former. The time-courses of changes in the activities of these enzymes after cessation of cholestyramine were comparable in both groups of rats. The rate of incorporation of mevalonate into sterol was also higher in young than in adult rats, while the stimulating effect of cholestyramine was markedly greater in adult rats. Hepatic acyl-CoA:cholesterol acyltransferase activity was comparable, but cholestyramine significantly decreased it only in adult rats. In adult rats hepatic cholesterol was decreased significantly by the resin while it remained uninfluenced in young rats. The serum cholesterol level tended to be higher in adult rats regardless of the dietary manipulation. The results indicate an appreciable age-dependent change in the hepatic cholesterol metabolism in response to the interruption of enterohepatic circulation of bile acids.     

Balloon cell nevus of the soft palate: an immunohistochemical and ultrastructural study

An ultrastructural analysis of oral balloon cell nevus of intramucosal type complemented with an immunohistochemical study was performed for the first time. The lesion was composed of large balloon cells with an admixture of small nevus cells and melanophages at the periphery. Balloon cells showed cytoplasmic accumulation of vacuoles of varying sizes and the presence of microgranular and vacuolated melanosomes were found. Residual cytoplasm contained no identifiable organelles. A spectrum of transitional forms between balloon cells and conventional nevus cells with microvacuoles was readily observed. Both cells exhibited intense immunoreactivity to multiple melanocytic markers. Ballooning phenomenon was not evident in melanophages containing a large amount of melanosome complex. It can be inferred, from the present and previous observations, that progressive vacuolization of melanosomes in nevomelanocytes may be responsible for the formation of peculiar ballooning appearance, suggesting an aberrant melanogenesis.