Differential antiproliferative activities of IFNs alpha, beta and gamma: kinetics of establishment of their antiproliferative effects and the rapid development of resistance to IFNs alpha and beta

Interferons have been recognized to have potent in vitro antiproliferative activities in mouse and human systems. To further investigate the kinetics of development of interferons' antiproliferative activities, mouse B-16 melanoma cells were treated with MuIFN-alpha, MuIFN-beta or MuIFN-gamma for various initial periods of time during an 8 day cloning assay. With MuIFN-alpha and MuIFN-beta treatments, maximal expression of antiproliferative activity was attained with 2 to 4 days of interferon treatment. In contrast, with MuIFN-gamma treatment, expression of antiproliferative activity increased with progressively longer periods of time of MuIFN-gamma treatment. These results suggested that B-16 melanoma cells were initially sensitive to all three of the interferons but rapidly became resistant to MuIFN-alpha and MuIFN-beta after 2 to 4 days of treatment. This suggestion was confirmed by cell growth kinetics experiments. The cells which were resistant to the antiproliferative activity of the MuIFN-alpha remained sensitive to the antiviral activity of MuIFN-alpha, suggesting that MuIFN-alpha and MuIFN-beta regulate their antiviral and antiproliferative responses via different mechanisms. The cells which were resistant to the antiproliferative activities of MuIFN-alpha and MuIFN-beta remained sensitive to MuIFN-gamma, suggesting that they were not generally resistant to antiproliferative effects. The cells which were resistant to the antiproliferative activities of the interferons gradually lost their resistance with a half-life of 11 days when they were cultured in the absence of interferons. The differential antiproliferative actions of alpha, beta and gamma interferons observed with murine B-16 melanoma were confirmed in the human system with G-361 melanoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-mediated gene transfer of a mutant regulatory subunit of cAMP-dependent protein kinase

We have used DNA-mediated gene transfer of genomic DNA to introduce into wild-type Chinese hamster ovary (CHO) cells a mutant gene that confers resistance to the growth inhibitory effect of cAMP. This dominant mutation in CHO cell line 10248 is responsible for an alteration in the RI subunit (RI*) of the type I cAMP-dependent protein kinase (Singh, T. J., Hochman, J., Verna, R., Chapman, M., Abraham, I., Pastan, I.H., and Gottesman, M.M. (1985) J. Biol. Chem. 260, 13927-13933). The transformant 11564 which was studied in detail, has the same characteristics as the original mutant 10248 including continued growth in medium containing 8-Br-cAMP, an increase in the Ka for cAMP activation of the kinase, a greatly reduced amount of type II protein kinase activity, an altered incorporation of the photoaffinity label 8-N3[32P]cAMP into the RI* subunit of PKI, and an absence of cAMP-dependent phosphorylation of a Mr = 52,000 protein in intact cells. In addition, analysis of the DNA of the transformant indicates the presence of an increased amount of DNA for the RI gene. These results are consistent with the transfer of a mutant gene for the RI* subunit of the cAMP-dependent protein kinase and its phenotypic expression in the transformant and also support the hypothesis that the mutation responsible for the defect in cell line 10248 is due to an alteration in the gene for RI.     

Site-directed mutagenesis of the yeast PRP20/1SRM1 gene reveals distinct activity domains in the protein product

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.     

Site-directed mutagenesis of the yeast PRP20/1SRM1 gene reveals distinct activity domains in the protein product

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.     

Nuclear PRP20 protein is required for mRNA export.

The yeast PRP20 protein is highly homologous in structure and function to the RCC1 protein of higher eukaryotes. The RCC1 protein is involved in the regulation of the onset of mitosis, whereas the PRP20 protein was shown to be required for accurate and efficient mRNA metabolism. The first observable phenotype in mutant prp20 cells when shifted from permissive to non-permissive temperature is a loss of nuclear PRP20 protein. Concomitantly, an accumulation of poly(A)+ RNA in the nucleus is observed. The temperature-sensitive RCC1 allele in the mutant hamster cell line tsBN2 leads to a similar accumulation of mRNA in the nucleus.     

Microbial Colonization of Beech and Spruce Litter—Influence of Decomposition Site and Plant Litter Species on the Diversity of Microbial Community

The present study was conducted to investigate the effect of decomposition site and plant litter species on the colonizing microbial communities. For this, litter bag technique using beech and spruce litter was combined with RNA-based fingerprinting and cloning. Litter bags were incubated for 2 and 8 weeks in the A_h horizon of beech and beech–spruce mixed forest sites. Although sugars and starch were rapidly lost, lignin content increased by more than 40% for beech and more than doubled for spruce litter at both soil sites at the end of the experiment. Denaturing gradient gel electrophoresis analysis of 16S and 18S rRNA RT–PCR products was used for screening of differences between bacterial and fungal communities colonizing the two litter types. Development of the microbial community over time was observed to be specific for each litter type and decomposition site. RT–PCR products from both litter types incubated in beech–spruce mixed forest site were also cloned to identify the bacterial and fungal colonizers. The 16S rRNA clone libraries of beech litter were dominated by γ-proteobacterial members, whereas spruce libraries were mainly composed of α-, β-, and γ-proteobacterial members. Ascomycota members dominated the 18S rRNA clone libraries. Clones similar to Zygomycota were absent from spruce, whereas those similar to Basidiomycota and Glomeromycota were absent from beech libraries. Selective effects of litter quality were observed after 8 weeks. The study provides an insight into the bacterial and fungal communities colonizing beech and spruce litter, and the importance of litter quality and decomposition site as key factors in their development and succession.     

Integration and religiosity among the Turkish second generation in Europe: a comparative analysis across four capital cities

Drawing on recent cross-national surveys of the Turkish second generation, we test hypotheses of secularization and of religious vitality for Muslim minorities in Europe. Secularization predicts an inverse relationship between structural integration and religiosity, such that the Turkish second generation would be less religious with higher levels of educational attainment and intermarriage. The religious vitality hypothesis predicts the maintenance of religion in the second generation, highlighting the role of religious socialization within immigrant families and communities. Taking a comparative approach, these hypotheses are tested in the context of different national approaches to the institutionalization of Islam as a minority religion in four European capital cities: Amsterdam, Berlin, Brussels and Stockholm. Across contexts, religious socialization strongly predicts second-generation religiosity, in line with religious vitality. The secularization hypothesis finds support only among the second generation in Berlin, however, where Islam is least accommodated.