raf/myc-infected erythroid cells are restricted in their ability to terminally differentiate.

A comparison was made of the in vitro erythroid colony-forming abilities of v-raf-, v-myc-, and v-raf/v-myc-containing retroviruses. In methylcellulose, v-raf efficiently produced colonies of well-differentiated hemoglobin-synthesizing erythroid cells, whereas v-raf/v-myc-infected erythroid cells were inhibited from terminally differentiating but retained the ability to replicate extensively. In contrast, v-myc was unable to stimulate the formation of erythroid colonies.     

Model system using coliphage phi X174 for testing virus removal by air filters.

Short-term (15-min-duration) and long-term (5- to 6-day-duration) test procedures have been developed for determining the efficiency of the removal of bacteriophage phi X174 by air-sterilizing filters. These procedures were sensitive enough to measure a 10(8)-fold reduction in the number of bacteriophage. A filter commonly used in industrial air sterilizations (Domnick-Hunter Bio-X borosilicate glass) effected a 10(8)-fold removal of viable phage in both short-term and long-term tests. A prototype low-flux, hollow-fiber membrane gave similar results; however, a prototype high-flux, hollow-fiber membrane removed only about 99.999% of the bacteriophage in short-term tests.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Organic matter and nutrient cycling within an endemic birch stand in the Etna massif (Sicily):Betula aetnensis Rafin

At altitudes between 1300 m to 2100 m in the Etna massif (Sicily), an endemic species of theBetula genus,Betula aetnensis Rafin, grows in a well-defined microclimatical context. Aboveground biomass and nutrient content studies within one stand revealed no significant differences from the otherBetula species, normally found in colder more temperate climate regions.Throughout the studied sites, biomass production, nutrient cycling and various structural or physiological characteristics (leaf area index) varied very little.Other researches indicate that the originality ofBetula aetnensis lies more in the histological or anatomical characteristics of its water conducting system which enables the species to adapt to Mediterranean-climate summer droughts in the Etna massif.

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Riassunto Sull'Etna, tra 1300 e 2100 m d'altitudine, in una zona microclimaticamente ben definita del versante nordorientale, si rinviene laBetula aetnensis Rafin.Dallo studio della fitomassa e della mineralomassa aerea del bosco di Monte Baracca, è emerso che non vi sono differenze notevoli con le altre specie indagate del genereBetula, più caratteristiche dei climi temperati e freddi.La produzione di biomassa, cosi come la gestione degli elementi nutritivi, è molto simile ai diversi popolamenti già indagati, cosi come certe caratteistiche strutturali e fisiologiche (leaf area index).L'originalità dellaBetula aetnensis è da ricercarsi nel vantaggio che ne ricava, a livello endogeno, sfruttando le caratteristiche istologiche ed anatomiche del suo apparato conduttore, che le consentono un efficace ed eccellente adattamento alle condizioni di siccità estive particolari del clima mediterraneo del vulcano.

     

Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction.

We have investigated the relationship between hydrolysis of phosphatidylcholine (PC) and activation of the Raf-1 protein kinase in Ras-mediated transduction of mitogenic signals. As previously reported, cotransfection of a PC-specific phospholipase C (PC-PLC) expression plasmid bypassed the block to cell proliferation resulting from expression of the dominant inhibitory mutant Ras N-17. In contrast, PC-PLC failed to bypass the inhibitory effect of dominant negative Raf mutants, suggesting that PC-PLC functions downstream of Ras but upstream of Raf. Consistent with this hypothesis, treatment of quiescent cells with exogenous PC-PLC induced Raf activation, even when normal Ras function was blocked by Ras N-17 expression. Further, activation of Raf in response to mitogenic growth factors was blocked by inhibition of endogenous PC-PLC. Taken together, these results indicate that hydrolysis of PC mediates Raf activation in response to mitogenic growth factors.     

Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.