Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.     

Identification of IL-7-dependent bone marrow-derived Thy-1-B220- lymphoid cell clones that rearrange and express both Ig and T cell receptor genes.

Bone marrow stromal cell lines and lymphoid cell lines were co-established from the Whitlock-Witte type of long term liquid cultures of MRL/1 and C57BL/10 (B10) (Thy-1.1) bone marrow cells. The present study investigates the immunologic nature of parental and cloned lymphoid cell lines. Both strains of parental lines and their clones did not grow alone but proliferated on the monolayers of co-established parental stromal cell lines from a syngeneic or alternative strain. When various lymphokines or cytokines were tested for their capacity to support the growth of these lymphoid cell clones, only IL-7 could substitute for the growth-promoting function of stromal cells. These IL-7-dependent clones expressed neither Thy-1 nor B220 Ag. However, all of them from two strains were found to rearrange synchronously H chain of Ig as well as gamma chain of TCR genes. Some of the clones transcribed a mature size of IgH mRNA. Co-expression of mRNA for lambda 5 but not for IgL chain (kappa, lambda) genes resulted in the generation of cell surface mu chain in these clones. Other clones expressed a smaller size of IgH mRNA without exhibiting surface mu chain. Irrespective of the differences in IgH rearrangements and its mRNA expression, a mature size TCR gamma mRNA was detected in all of the clones. Thus, these results demonstrate the existence of untransformed (IL-7-dependent) immature lymphoid cells rearranging both Ig and TCR genes. Their unique features concerning cell surface markers (B220- mu+), specific growth factor requirement, and various modes of Ig/TCR gene rearrangements are discussed in the context of early lymphoid development.     

Cytotoxicity and genotoxicity of xenoclauxin and desacetyl duclauxin fromPenicillium Duclauxii (delacroix)

The duclauxin derivatives xenoclauxin and desacetylduclauxin were examined for their effects on the growth of L-1210 murine leukemia cells, on the induction of DNA repair in the rat and mouse hepatocyte primary culture (HPC/DNA repair test), and on oxidative phosphorylation in mitochondria from rat livers in comparison to duclauxin. Both derivatives inhibited the growth of L-1210 culture cells as strongly as duclauxin. Duclauxin derivatives were negative in the HPC/DNA repair test. Xenoclauxin exhibited a potent uncoupling effect accompanying a marked depression of state 3 respiration of mitochondria in a similar fashion to that of duclauxin. Desacetylduclauxin significantly inhibited the state 3 respiration without causing uncoupling of oxidative phosphorylation in mitochondria. These results strongly suggest that xenoclauxin and desacetylduclauxin fromPenicillium duclauxii are not genotoxic but are cytotoxic mainly due to their potent inhibition of ATP synthesis in mitochondria.Abbreviations DNP 2,4-dinitrophenol - ETP electron transport particles - HPC hepatocyte primary culture cells - RC respiratory control - TdR thymine deoxyribonucleotide - UDS unscheduled DNA synthesis     

Inflammatory cytokine‐mediated induction of serine racemase in atopic dermatitis

Serine racemase (SR) is an enzyme that catalyses the synthesis of d ‐serine, an endogenous coagonist for N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptor in the central nervous system. Our previous study demonstrated that SR was expressed in the epidermis of wild‐type (WT) mice but not in SR knockout (KO) mice. In addition, SR immune‐reactivity was only found in the granular and cornified layers of the epidermis in WT mice. These findings suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of the skin barrier. However, its role in skin barrier dysfunction such as atopic dermatitis (AD) remains elusive. AD is a chronic inflammatory disease of skin, and the clinical presentation of AD has been reported to be occasionally associated with psychological factors. Therefore, this study examined the content of d ‐serine in stratum corneum in AD patients and healthy controls using a tape‐stripping method. Skin samples were collected from the cheek and upper arm skin of AD patient's lesion and healthy individuals. The d ‐serine content was significantly increased in the involved skin of AD in comparison with healthy individuals. An immunohistochemical analysis also revealed an increased SR expression in the epidermis of AD patients. Furthermore, the SR expression in cultured human keratinocytes was significantly increased by the stimulation with tumour necrosis factor ‐α or macrophage migration inhibitory factor. Taken together, these findings suggest that d ‐serine expressed particularly strongly in AD lesional skin and that the SR expression in the keratinocytes is linked to inflammatory cytokines.     

A luciferase complementation assay system using transferable mouse artificial chromosomes to monitor protein–protein interactions mediated by G protein-coupled receptors

G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that interact with the β-arrestin family, particularly β-arrestin 1 (ARRB1). GPCRs interact with 33% of small molecule drugs. Ligand screening is promising for drug discovery concerning GPCR-related diseases. Luciferase complementation assay (LCA) enables detection of protein–protein complementation via bioluminescence following complementation of N- and C-terminal luciferase fragments (NEluc and CEluc) fused to target proteins, but it is necessary to co-express the two genes. Here, we developed LCAs with mouse artificial chromosomes (MACs) that have unique characteristics such as stable maintenance and a substantial insert-carrying capacity. First, an NEluc-ARRB1 was inserted into MAC4 by Cre-loxP recombination in CHO cells, named ARRB1-MAC4. Second, a parathyroid hormone receptor 2 (PTHR2)-CEluc or prostaglandin EP4 receptor (hEP4)-CEluc were inserted into ARRB1-MAC4, named ARRB1-PTHR2-MAC4 and ARRB1-hEP4-MAC4, respectively, via the sequential integration of multiple vectors (SIM) system. Each MAC was transferred into HEK293 cells by microcell-mediated chromosome transfer (MMCT). LCAs using the established HEK293 cell lines resulted in 35,000 photon counts upon somatostatin stimulation for ARRB1-MAC4 with transient transfection of the somatostatin receptor 2 (SSTR2) expression vector, 1800 photon counts upon parathyroid hormone stimulation for ARRB1-PTHR2-MAC4, and 35,000 photon counts upon prostaglandin E2 stimulation for ARRB1-hEP4-MAC4. These MACs were maintained independently from host chromosomes in CHO and HEK293 cells. HEK293 cells containing ARRB1-PTHR2-MAC4 showed a stable reaction for long-term. Thus, the combination of gene loading by the SIM system into a MAC and an LCA targeting GPCRs provides a novel and useful platform to discover drugs for GPCR-related diseases.     

Progestins and danazol effect on cell-to-cell adhesion, and E-cadherin and α- and β-catenin mRNA expressions

The first step of invasion and metastasis is the detachment of cancer cells in the primary tumor, which is mainly controlled by the function in the adherens junction, consisting of E-cadherin associated proteins (E-cadherin, α- and β-catenins, vinculin, α-actinin, and actin). The cell-to-cell aggregation activity and the expressions of E-cadherin, and α- and β-catenin mRNAs in Ishikawa cells of well-differentiated endometrial cancer were significantly suppressed by estrogen. These suppressions were reversed by progesterone, medroxyprogesterone acetate (MPA) and danazol. Proteins in the adherens junction appeared to be expressed intact and to be functional in Ishikawa cells. Persistent estrogen predominant milieu might contribute to the detachment of well-differentiated endometrial cancer cells, leading to spreading of those cells, while progestins and danazol protect estrogen-induced spreading of those cells.     

Induction of endogenous tumor necrosis factor by OK-432 in ovarian cancer patients with ascites

To four ovarian cancer patients with malignant ascites, 10 KE of OK-432 was intraperitoneally administered four times at 2 day intervals for priming, and 40 KE of OK-432 was given on the 13th day after the first injection for triggering. The changes in blood monocyte and peritoneal macrophage levels and the production of tumor necrosis factor (TNF) by blood mononuclear cells (BMCs) and ascitic lymphoid cells (ALCs) were examined. In the two patients in whom TNF was induced in the ascites, TNF production by BMCs and ALCs was noted during priming. After triggering, increases in both the number of peritoneal macrophages and TNF production by ALCs were noted. In the other two patients, in whom TNF was not detected in the ascites, the ratio of peritoneal macrophages to ALCs did not change throughout the study period, and TNF production by the ALCs was not augmented. These findings suggest that OK-432 can exert a primary effect on both peritoneal macrophages and blood monocytes, and that OK-432 triggering can promote an increase in primed peritoneal macrophages and the release of TNF from these cells.     

Production of recombinant human pancreatic secretory trypsin inhibitor by Escherichia coli

A synthetic gene for human pancreatic secretory trypsin inhibitor (PSTI) was fused to the coding sequence for the amino-terminal 135 amino acid residues of human interferon-gamma (IFN-gamma) by interposing a methionine codon sequence, and the resulting hybrid gene was efficiently expressed in Escherichia coli cells. Recombinant human PSTI (rHu-PSTI) was separated from the IFN-gamma/PSTI fused protein by cleavage at the methionine residue with cyanogen bromide. Finally, rHu-PSTI was purified by affinity chromatography on a bovine trypsin-CH-Sepharose 4B column. The amino acid composition, partial amino-terminal sequence, disulfide formation, human trypsin inhibitory activity, and immunoreactivity against rabbit anti-human PSTI serum of rHu-PSTI corresponded to those of the natural form.     

Resonance Raman study on cytochrome c peroxidase and its intermediate. Presence of the Fe(IV) = O bond in compound ES and heme-linked ionization

Resonance Raman spectra of ferrous and ferric cytochrome c peroxidase and Compound ES and their pH dependences were investigated in resonance with Soret band. The Fe(IV) = O stretching Raman line of Compound ES was assigned to a broad band around 767 cm-1, which was shifted to 727 cm-1 upon 18O substitution. The 18O-isotopic frequency shift was recognized for Compound ES derived in H218O, but not in H216O. This clearly indicated occurrence of an oxygen exchange between the Fe(IV) = O heme and bulk water. The Fe(IV) = O stretching Raman band was definitely more intense and of higher frequency in D2O than in H2O as in Compound II of horseradish peroxidase, but in contrast with this its frequency was unaltered between pH 4 and 11. The Fe(II)-histidine stretching Raman line was assigned on the basis of the frequency shift observed for 54Fe isotopic substitution. From the intensity analysis of this band, the pKa of the heme-linked ionization of ferrocytochrome c peroxidase was determined to be 7.3. The Raman spectrum of ferricytochrome c peroxidase strongly suggested that the heme is placed under an equilibrium between the 5- and 6-coordinate high-spin structures. At neutral pH it is biased to the 5-coordinate structure, but at the acidic side of the transition of pKa = 5.5 the 6-coordinate heme becomes dominant. F- was bound to the heme iron at pH 6, but Cl- was bound only at acidic pH. Acidification by HNO3, H2SO4, CH3COOH, HBr, or HI resulted in somewhat different populations of the 5- and 6-coordinate forms when they were compared at pH 4.3. Accordingly, it is inferred that a water molecule which is suggested to occupy the sixth coordination position of the heme iron is not coordinated to the heme iron at pH 6 but that protonation of the pKa = 5.5 residue induces an appreciable structural change, allowing the coordination of the water molecule to the heme iron.