Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

DNA polymerase α₁, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α₁ and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α₁ activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.     

Dysmyelination of Shiverer Mutant Mice In Vivo and In Vitro

Demyelination in the CNS of shiverer mutant mice was studied in vivo and in vitro. By immunohistochemical reaction with glial fibrillary acidic protein antibody, hypertrophy of the fibrous astrocytes was observed in the white matter of shiverer cerebella. The cerebella of shiverer mice in primary culture from the day of birth showed very poor myelination under optical microscopy. Axons of Purkinje cells are thought to be the main myelinated axons in the primary culture of the cerebellum. Purkinje cells from shiverer appeared normal with regard to Bodian silver impregnation, hematoxylin and eosin staining, and P400 protein characterization of Purkinje cells. Addition of the conditioned culture medium of shiverer to the control culture did not interfere with myelination. We concluded that the demyelination in the CNS of shiverer could be caused by an intrinsic defect of the oligodendrocyte rather than by hypertrophy of the astrocytes or by diffusible factors.     

Paternal transmission of the mouse Thp mutation is lethal in some genetic backgrounds

T^hp is a large deletion on chromosome 17 which includes the maternal lethal gene Tme. Documentation of inheritance patterns suggests that Tme is an imprinted gene which is required for viability; maternal deletion is lethal while paternal deletion is viable. However, paternal transmission of T^hp is rarely the expected 50%. We show here that paternally inherited T^hp is lethal in some strains, providing evidence of an incompletely penetrant, dosage sensitive lethal allele of a locus that probably maps to the hairpin tail region of chr. 17. Interpretation of the various phenotypes associated with loss of the putative Tme gene, lgf2r, may need to be revised in view of these observations. Dev Genet 20:23–28, 1997. © 1997 Wiley-Liss, Inc.     

Suppression of Heme Oxygenase-1 mRNA Expression by Interferon-γ in Human Glioblastoma Cells

Heme oxygenase is a rate-limiting enzyme in heme catabolism that cleaves heme to form biliverdin, carbon monoxide, and iron. Heme oxygenase-1 is an inducible isozyme and is expressed in many types of cells and tissues. Large amounts of these heme degradation products may be noxious to the host, especially in the brain. We therefore searched for the factors that suppress the expression of heme oxygenase-1. Northern blot analysis showed that treatment with interferon-gamma and with interleukin-1beta for 24 h decreased the expression levels of heme oxygenase-1 mRNA to approximately 20 and approximately 50% of the control levels, respectively, in a human glioblastoma cell line, T98G. Treatment with a combination of these two cytokines additively decreased the expression levels of heme oxygenase-1 mRNA. Western blot analysis showed that the expression level of heme oxygenase-1 protein was also decreased by treatment with interferon-gamma, but not with interleukin-1beta. Moreover, pretreatment with interferon-gamma partially suppressed the induction of heme oxygenase-1 mRNA expression caused by either sodium nitroprusside, cadmium, or hemin. These findings raise the possibility that the expression of heme oxygenase-1 is down-regulated by interferon-gamma in the nervous system.     

Post-translational regulation and proteolytic activity of the metalloproteinase ADAMTS8

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs (ADAMTS)8 is a secreted protease, which was recently implicated in pathogenesis of pulmonary arterial hypertension (PAH). However, the substrate repertoire of ADAMTS8 and regulation of its activity are incompletely understood. Although considered a proteoglycanase because of high sequence similarity and close phylogenetic relationship to the proteoglycan-degrading proteases ADAMTS1, 4, 5, and 15, as well as tight genetic linkage with ADAMTS15 on human chromosome 11, its aggrecanase activity was reportedly weak. Several post-translational factors are known to regulate ADAMTS proteases such as autolysis, inhibition by endogenous inhibitors, and receptor-mediated endocytosis, but their impacts on ADAMTS8 are unknown. Here, we show that ADAMTS8 undergoes autolysis at six different sites within its spacer domain. We also found that in contrast to ADAMTS4 and 5, ADAMTS8 levels were not regulated through low-density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytosis. Additionally, ADAMTS8 lacked significant activity against the proteoglycans aggrecan, versican, and biglycan. Instead, we found that ADAMTS8 cleaved osteopontin, a phosphoprotein whose expression is upregulated in PAH. Multiple ADAMTS8 cleavage sites were identified using liquid chromatography–tandem mass spectrometry. Osteopontin cleavage by ADAMTS8 was efficiently inhibited by TIMP-3, an endogenous inhibitor of ADAMTS1, 4, and 5, as well as by TIMP-2, which has no previously reported inhibitory activity against other ADAMTS proteases. These differences in post-translational regulation and substrate repertoire differentiate ADAMTS8 from other family members and may help to elucidate its role in PAH.     

ASK1 and ASK2 differentially regulate the counteracting roles of apoptosis and inflammation in tumorigenesis

Apoptosis and inflammation generally exert opposite effects on tumorigenesis: apoptosis serves as a barrier to tumour initiation, whereas inflammation promotes tumorigenesis. Although both events are induced by various common stressors, relatively little is known about the stress‐induced signalling pathways regulating these events in tumorigenesis. Here, we show that stress‐activated MAP3Ks, ASK1 and ASK2, which are involved in cellular responses to various stressors such as reactive oxygen species, differentially regulate the initiation and promotion of tumorigenesis. ASK2 in cooperation with ASK1 functioned as a tumour suppressor by exerting proapoptotic activity in epithelial cells, which was consistent with the reduction in ASK2 expression in human cancer cells and tissues. In contrast, ASK1‐dependent cytokine production in inflammatory cells promoted tumorigenesis. Our findings suggest that ASK1 and ASK2 are critically involved in tumorigenesis by differentially regulating apoptosis and inflammation.     

<Emphasis Type="Italic">Anaconda</Emphasis>, a new class of transposon belonging to the <Emphasis Type="Italic">Mu</Emphasis> superfamily,has diversified by acquiring host genes during rice evolution

A new type of transposon, named Anaconda (Anac) has been found in rice (Oryza sativa). In this paper, we demonstrate that Anaconda elements have diversified by acquisition of host cellular genes, amplification of the elements, and substitution and deletion of short segments. We identified four Anaconda elements in studies of rice alternative oxidase (AOX) genes, and subsequently isolated an additional 23 elements based on the identity of their terminal inverted repeats (TIRs). The Anaconda elements have long TIRs (114–458 bp). They also have direct repeats of 9 or 10 bp in their flanking regions that are thought to have been generated upon transposition. These structural features reveal that the Anaconda elements belong to the Mu superfamily. The most prominent feature of the Anaconda elements is the high frequency with which they have acquired host cellular genes. Of the 27 elements found here, 19 appear to have sequences presumably derived from rice genes, for example, the genes for AOX1c (four elements), cytochrome P450 (five elements), l-asparaginase (five elements), and PCF8 (two elements). Four elements, AnacA1–A4, have both the AOX1c and P450 genes. One element, AnacB14, involves a gene similar to mudrA of maize MuDR. Database analyses revealed that the loci of 26 of the 27 Anaconda elements in the subspecies japonica are the same as those in the subspecies indica. This suggests that these elements were incorporated before the divergence of these two subspecies.