On the role of protein phosphorylation in the ATP-dependent permeabilization of transformed cells

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [γ-³²P] ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [γ-³²P] ATP resulted in substantial formation of ³²P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the α-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.     

Specific combinations of human serum albumin introns direct high level expression of albumin in transfected COS cells and in the milk of transgenic mice

A new series of expression vectors, each comprised of the -lactoglobulin (BLG) promoter driving one of a variety of human serum albumin (HSA) minigenes or the entire gene, were evaluated for their ability to direct expression of HSAin vitro in COS tissue culture cells and into the milk of transgenic mice. Vectors directed a hierarchy of expression levelsin vitro, dependent upon the specific complement of HSA introns included. HSA introns acted in a synergistic manner. In addition, minigenes comprised of specific subsets of introns were more efficacious than the entire HSA gene with all of its introns. Transgenic mice expressed as much as 10 mg ml^–1 of HSA in their milk. Vectors comprised of specific intron subsets directed levels at 1 mg ml^–1 or greater in the milk of 20% of generated transgenics. A statistical correlation between the expression level trendin vitro with the trend of expressionin vivo (% which express) at detectable levels (p=0.0015) and at the level of greater than 0.1 mg ml^–1 (p=0.0156) was demonstrated. A weak correlation existed (p=0.0526) atin vivo levels of 1 mg ml^–1 or greater. These new vectors are expected to direct the production of high levels of HSA in the milk of a large percentage of generated transgenic dairy animals.     

Chemoreception in the green algaDunaliella tertiolecta

A capillary assay was employed to quantify positive chemotactic responses in the motile, unicellular, marine algaDunaliella tertiolecta. Among a wide range of inorganic and organic compounds tested, only ammonium ion,l-tyrosine,l-tryptophan, andl-phenylalanine were found to be major atractants for the chlorophyte.l-Methionine andl-cysteine weakly attracted the alga at 10⁻³ M. The minimum concentration of the major attractants needed to elicit an observable chemotactic response was approximately 10⁻⁶ M. The maximum response occurred when the capillaries contained 10⁻⁵ Ml-tyrosine orl-tryptophan, 10⁻⁴ Ml-phenylalanine, and 10⁻³ M ammonium chloride. The other amino acids, carbohydrates, B-vitamins, urea, and nitrate were among the chemicals that failed to attractD. tertiolecta. The alga apparently possesses one chemoreceptor that binds ammonium ion only, and another chemoreceptor that binds the three aromatic amino acids.     

Do copper ions influence the reduction of ferricytochrome C by O-2?

Recently, it was suggested that the measured rate of reduction of ferricytochrome C by O-2 below pH 8, was too high in the presence of high concentrations of formate (Koppenol, W.H., Van Buuren, K.J.H., Butler J. and Braams, R. (1976) Biochim. Biophys. Acta 449, 157-168). The high values were attributed to the presence of impurities of copper, which compete for O-2. This assumption is consistent with either a decrease in the reduction yield of ferricytochrome C in the presence of copper, or with a very fast reaction of Cu(I) with ferricytochrome C. It was previously shown by us and by others that the reduction yield of ferricytochrome C by O-2 IS 100%. We measured the rate of reduction of ferricytochrome C by Cu(I), and found that this reaction is slow: k = (1.5 +/- 0.5) . 10(3) M-1 . s-1. Therefore, our results rule out the possibility that below pH 8 copper impurities affect the measured rate constant of the reduction of ferricytochrome C by O-2.     

Studies on isoenzyme pattern during differentiation (spherulation) of Physarum polycephalum

Plasmodial homogenates of the true slime mold Physarum polycephalum grown on a liquid medium contain carbohydrates which form a complex with protein under conditions of acrylamide electrophoresis and thus make isoenzyme studies from those extracts impossible. A method, using mild homogenization and centrifugation on top of a 30% sucrose solution was developed. This treatment leaves most of the soluble cytoplasmic enzymes in the upper layer above the sucrose, which then can be used for successful isoenzyme or protein studies with polyacrylamide electrophoresis.The activity changes and isoenzyme pattern of 16 different enzymic activities were studied during differentiation (spherulation) of Physarum polycephalum, induced either by starvation or by mannitol. Only one enzyme, esterase, exhibited a conspicuous change in isoenzyme pattern during development.     

A molecular target for viral killer toxin: TOK1 potassium channels.

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes.     

Positive impacts of livestock and wild ungulate routes on functioning of dryland ecosystems

Livestock grazing is often perceived as being detrimental to the quality and functioning of dryland ecosystems. For example, a study in a semiarid Kenyan savanna proposed that cattle form bare spaces throughout the landscape, which indicate ecosystem degradation. Other studies, conducted in north‐eastern Spain, where climatic conditions range between semiarid and Mediterranean subhumid, reported that sheep and goat trails have increased the emergence of rill erosion processes. Sometimes, this negative perception is extended to include wild, large ungulate herbivores as well. Here, we challenge this perception by highlighting the generally nonadverse and even ameliorative impacts of moderate animal rate on geoecosystem functioning of hilly drylands. Specifically, trampling routes (also known as treading paths, livestock terracettes, cattle trails, migration tracks, cowtours, etc.) formed across hillslopes by grazing animals—being either domesticated livestock or native large herbivores—transform the original two‐phase vegetation mosaic of shrubby patches and interpatch spaces into a three‐phase mosaic. The animal routes increase the complexity of ecosystem, by strengthening the spatial redistribution of water and soil resources at the patch scale and decreasing hydrological connectivity at the hillslope scale. As a consequence, the animal routes improve functioning of hilly drylands and increase their resilience to long‐term droughts and climatic change. Therefore, instead of viewing the animal routes as degraded spots, they should be perceived at a wider perspective that allows to properly understand their overall role in sustaining dryland geoecosystems.