Demonstration of an epidermal growth factor-dependent 58 kDa phosphoprotein secreted by rat kidney fibroblasts

Epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate increased the amount of 32Pi found as phosphoserine in a major, hitherto not described 58 kDa phosphoprotein (pp58) secreted by normal rat kidney fibroblasts. Platelet-derived growth factor, insulin, nerve growth factor and fibroblast growth factor did not affect pp58 while transforming growth factor beta decreased the accumulation of radioactivity into pp58. Cycloheximide, actinomycin D and ammonium chloride suppressed the labelling of pp58.     

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

A Mechanism of Energy Dissipation in Cyanobacteria

When grown under a variety of stress conditions, cyanobacteria express the isiA gene, which encodes the IsiA pigment-protein complex. Overexpression of the isiA gene under iron-depletion stress conditions leads to the formation of large IsiA aggregates, which display remarkably short fluorescence lifetimes and thus a strong capacity to dissipate energy. In this work we investigate the underlying molecular mechanism responsible for chlorophyll fluorescence quenching. Femtosecond transient absorption spectroscopy allowed us to follow the process of energy dissipation in real time. The light energy harvested by chlorophyll pigments migrated within the system and eventually reaches a quenching site where the energy is transferred to a carotenoid-excited state, which dissipates it by decaying to the ground state. We compare these findings with those obtained for the main light-harvesting complex in green plants (light-harvesting complex II) and artificial light-harvesting antennas, and conclude that all of these systems show the same mechanism of energy dissipation, i.e., one or more carotenoids act as energy dissipators by accepting energy via low-lying singlet-excited S₁ states and dissipating it as heat.     

The turbulence of images: On imagery,media and ethnographic discourse

As the anthropological theory of politeness has put it, people have universally two kinds of social want. They wish to be close to others, liked and accepted, but they also want some distance, freedom from imposition and respect of mutual difference. These wants, which one may also call basic human rights, are however often violated, and images play an important role in this.

Images are ultimately generated in the human mind and may find verbal as well as written and pictorial representation. Tyler [1978] has proposed a typology of mental images based on criteria of conscious control, completeness, abstraction, media and autonomy. These criteria also prove useful when probing into prototypical usages of intrusive imagery such as cursing, blessing, magic, witchcraft and the evil eye. Also the criteria help one understand the subtle process by which imagery can lead to the articulation and even discovery of peoples’ selves.

Ethnographic discourse has always been to a large extent about images, but until recently the ethnographer stood between his or her audience and the people of whom he or she was speaking or writing. Now, as sound recordings, film and video have arrived, this allows for richer varieties of cooperation where the non‐literate counterparts of the ethnographer can make more expressive and coherent contributions than before.

Also it seems that in the face of suppression people welcome the new media as a support in their ongoing battle for a recognition of their cultural and social worthiness.     

Genetic and morphometric differentiation among island populations of two Norops lizards (Reptilia: Sauria: Polychrotidae) on independently colonized islands of the Islas de Bahia (Honduras)

Aim Anole lizards (Reptilia: Sauria: Polychrotidae) display remarkable morphological and genetic differentiation between island populations. Morphological differences between islands are probably due to both adaptive (e.g. differential resource exploitation and intra‐ or interspecific competition) and non‐adaptive differentiation in allopatry. Anoles are well known for their extreme diversity and rapid adaptive speciation on islands. The main aim of this study was to use tests of morphological and genetic differentiation to investigate the population structure and colonization history of islands of the Islas de Bahia, off the coast of Honduras. Location Five populations of Norops bicaorum and Norops lemurinus were sampled, four from islands of the Islas de Bahia and one from the mainland of Honduras. Methods Body size and weight differentiation were measured in order to test for significant differences between sexes and populations. In addition, individuals were genotyped using the amplified fragment length polymorphism technique. Bayesian model‐based and assignment/exclusion methods were used to study genetic differentiation between island and mainland populations and to test colonization hypotheses. Results Assignment tests suggested migration from the mainland to the Cayos Cochinos, and from there independently to both Utila and Roatán, whereas migration between Utila and Roatán was lacking. Migration from the mainland to Utila was inferred, but was much less frequent. Morphologically, individuals from Utila appeared to be significantly different in comparison with all other localities. Significant differentiation between males of Roatán and the mainland was found in body size, whereas no significant difference was detected between the mainland and the Cayos Cochinos. Main conclusions Significant genetic and morphological differentiation was found among populations. A stepping‐stone model for colonization, in combination with an independent migration to Utila and Roatán, was suggested by assignment tests and was compatible with the observed morphological differentiation.     

Immunoaffinity-purified DNA polymerase alpha displays novel properties

The purification and characterization of a novel and more intact form of the DNA polymerase alpha-primase complex from calf thymus are described. The polymerase-primase was enriched 10,000-fold to apparent homogeneity by chromatography on phosphocellulose, heparin-Sepharose, and an immobilized anti-human DNA polymerase alpha monoclonal antibody [SJK287-38; Tanaka, S., Hu, S., Wang, T. S.-F., & Korn, D. (1982) J. Biol. Chem. 257, 8386-8390]. A quantitative elution from the antibody column was achieved by shifting the pH from neutrality to between 12.5 and 13. From 1 kg of calf thymus, the procedure yields 1-2 mg of polymerase-primase with a specific activity of 30,000-40,000 units/mg for the polymerase and 15,000-20,000 units/mg for the primase. The complex sediments at 9 S through a sucrose gradient and exhibits a Stokes radius of 6.0 nm, yielding a native molecular mass of 335,000. Denaturing gel electrophoresis of the complex gives bands of Mrs 180,000, 155,000, 148,000, 73,000, 59,000, and 48,000 with a relative abundance of the two smallest subunits. Primase activity was partially resolved from the complex by centrifugation through sucrose gradients. The primer-forming activity was found to be associated with the Mr 59,000 and 48,000 polypeptides. In contrast to conventional preparations, the immunopurified polymerase displays several features which show it is the most intact form of the enzyme known to date. The deoxynucleoside triphosphate Km values are all within the range of 0.6-0.9 microM. The Km for binding to a single RNA primer on M13 DNA is 3.5 nM; the Ki for nonspecific binding to unprimed DNA is 70 microM (nucleotide).(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis.

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.     

Hydrodynamic and circular dichroic analysis of mammary-derived growth inhibitor (MDGI)

The mammary-derived growth inhibitor exists in solution as a monomeric molecule with a molar mass of 14,500 +/- 400 g/mol. The largest diameter and the height of the polypeptide chain were estimated to be 3.75 +/- 0.25 nm and 2.01 +/- 0.13 nm respectively. This is in good agreement with the structurally related bovine peripheral myelin P2 protein (about 70% amino acid sequence homology). CD measurements have revealed MDGI to be a protein with about 50% beta structure and less than 20% alpha helix similarly as in fatty acid-binding proteins. Removal of endogenous long-chain fatty acid by lipidex or storage in the frozen state lead to a destabilization of the active MDGI conformation which is accompanied by a loss of its activity with regard to growth inhibition of Ehrlich Ascites cells.     

An inexpensive insoluble chromogenic substrate for the determination of proteolytic activity

Summary A new insoluble chromogenic substrate for the determination of proteolytic activity was developed. This substrate was prepared by incorporating black drawing ink into casein and heating this complex at 200°C for 4 h. It is especially suitable for determining the activity of alkaline bacterial proteinases.