Escherichia coli endo IV is a bifunctional DNA repair protein, i.e., possessing both apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase activities. The former activity cleaves AP sites, whereas the latter one removes a variety of 3'-blocking groups present at single-strand breaks in damaged DNA. However, the precise reaction mechanism by which endo IV cleaves DNA lesions is unknown. To probe this mechanism, we have identified eight amino acid substitutions that alter endo IV function in vivo. Seven of these mutant proteins are variably expressed in E. coli and, when purified, show a 10-60-fold reduction in both AP endonuclease and 3'-diesterase activities. The most severe defect was observed with the one remaining mutant (E145G) that showed normal protein expression. This mutant has lost the ability to bind double-stranded DNA and showed a dramatic 150-fold reduction in enzymatic activities. We conclude that the AP endonuclease and the 3'-diesterase activities of endo IV are associated with a single active site, that is perhaps remote from the DNA binding domain.
As part of a survey of fish diseass, lake whitefish (Coregons clupeaformis) were coliected in fail 1995 from the St. Lawrence River 15 km upstream of Quebec City, Quebec, Canada, to assess the prevalence of liver lesions. A total of 141 fish were captured and necropsied, and three standard s ons of liver were taken for histological exmination. Prvlences of altered hepatocyte foci, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma were 0.7%, 2.1%, 0.7%, and 2.1%, respectively. Thus, the overall prevalence of liver neoplasia was 4.9% (71141). Hepatic tumors were only observed in fish 7 years old or older. Fish age was siganificantdy and positively correlated with the index assessing the number and size of macrophage aregtes (pcO.001; r = 0.16). Hepatocyte vacuolatin anisokaryosis, lymphocytic infilon, and bile duct hyperplasia were also observed but were not related to the age, length, sex, or condition factor of the fish. These results represent the first report on a series of hepatic tumors in a wild salmonid species.
To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, beta-actin, or beta-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with beta-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the beta-actin capping protein, betacap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with betacap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and beta-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.
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