A model for in vitro proliferation of undifferentiated bovine mammary epithelial cells

A method for culturing bovine undifferentiated mammary epithelial cells embedded in collagen gels is described. Cell growth was quantitated by incorporation of tritiated thymidine. Maximal rate of incorporation was achieved in media supplemented with sera, however considerable growth was observed in serum-free medium supplemented with insulin, transferrin and selenium. The growth promoting effect of insulin was maximally expressed only at high nonphysiological concentrations.     

Absorption of bioactive human growth hormone after oral administration in the common carp (Cyprinus carpio) and its enhancement by deoxycholate

Summary Recombinant human growth hormone was administered orally to carp and serum levels of absorbed bioactive hormone were investigated using a highly sensitive Nb2 rat lymphoma cell bioassay and radioimmumoassay. Serum levels of bioactive hGH reached maximum values 30 min after oral intubation and then gradually decreased. Co-administration of the hormone with deoxycholate to fasted carp resulted in up to a 1000-fold increase in absorption compared to aqueous solutions of the hormone, but had no effect on the kinetics of the absorption process. Absorption of the hormone in starved fish was significantly greater than in fed fish. A linear dose-response relationship was observed for hGH in starved fish and the level of absorption in fed fish was influenced by the time interval from the last meal. The ratio of bioactive to immunoactive hGH in fasted fish indicated little loss of bioactivity and also that deoxycholate may be protective against hGH degradation. The present study demonstrates for the first time that biologically active hGH is absorbed in the common carp after oral intubation. Furthermore, the use of a biological detergent dramatically increased the extent of hGH absorption. Additional studies are required to establish the approapriate conditions (diet composition, feeding level, and frequency, etc.) in which polypeptide hormones could be introduced orally to fish.Abbreviations hGH human growth hormone - HRP horseradish peroxidase - RIA radioimmunoassay     

DNA sequence, structure, and tyrosine kinase activity of the Drosophila melanogaster Abelson proto-oncogene homolog.

We report our molecular characterization of the Drosophila melanogaster Abelson gene (abl), a gene in which recessive loss-of-function mutations result in lethality at the pupal stage of development. This essential gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type lb 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region was expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues was observed with an antiphosphotyrosine antibody. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development.     

Emergence of large-scale cell morphology and movement from local actin filament growth dynamics

Variations in cell migration and morphology are consequences of changes in underlying cytoskeletal organization and dynamics. We investigated how these large-scale cellular events emerge as direct consequences of small-scale cytoskeletal molecular activities. Because the properties of the actin cytoskeleton can be modulated by actin-remodeling proteins, we quantitatively examined how one such family of proteins, enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), affects the migration and morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently while exhibiting a characteristic smooth-edged “canoe” shape, but may also exhibit less regular morphologies and less persistent movement. When we observed that the smooth-edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this led to changes in the morphology and movement persistence of cells within a population. Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused changes in cell morphology, which is coupled to the motile behavior of keratocytes. We also characterized the range of natural cell-to-cell variation within a population by using measurable morphological and behavioral features—cell shape, leading-edge shape, filamentous actin (F-actin) distribution, cell speed, and directional persistence—that we have found to correlate with each other to describe a spectrum of coordinated phenotypes based on Ena/VASP enrichment at the leading edge. This spectrum stretched from smooth-edged, canoe-shaped keratocytes—which had VASP highly enriched at their leading edges and migrated fast with straight trajectories—to more irregular, rounder cells migrating slower with less directional persistence and low levels of VASP at their leading edges. We developed a mathematical model that accounts for these coordinated cell-shape and behavior phenotypes as large-scale consequences of kinetic contributions of VASP to actin filament growth and protection from capping at the leading edge. This work shows that the local effects of actin-remodeling proteins on cytoskeletal dynamics and organization can manifest as global modifications of the shape and behavior of migrating cells and that mathematical modeling can elucidate these large-scale cell behaviors from knowledge of detailed multiscale protein interactions.     

Composition and dynamics of biostimulated indigenous oil-degrading microbial consortia from the Irish, North and Mediterranean Seas: a mesocosm study

Diversity of indigenous microbial consortia and natural occurrence of obligate hydrocarbon-degrading bacteria (OHCB) are of central importance for efficient bioremediation techniques. To investigate the microbial population dynamics and composition of oil-degrading consortia, we have established a series of identical oil-degrading mesocosms at three different locations, Bangor (Menai Straits, Irish Sea), Helgoland (North Sea) and Messina (Messina Straits, Mediterranean Sea). Changes in microbial community composition in response to oil spiking, nutrient amendment and filtration were assessed by ARISA and DGGE fingerprinting and 16Sr RNA gene library analysis. Bacterial and protozoan cell numbers were quantified by fluorescence microscopy. Very similar microbial population sizes and dynamics, together with key oil-degrading microorganisms, for example, Alcanivorax borkumensis, were observed at all three sites; however, the composition of microbial communities was largely site specific and included variability in relative abundance of OHCB. Reduction in protozoan grazing had little effect on prokaryotic cell numbers but did lead to a decrease in the percentage of A.?borkumensis 16S rRNA genes detected in clone libraries. These results underline the complexity of marine oil-degrading microbial communities and cast further doubt on the feasibility of bioaugmentation practices for use in a broad range of geographical locations.     

Cyt2Ba of Bacillus thuringiensis israelensis: activation by putative endogenous protease

The gene cyt2Ba of Bacillus thuringiensis subsp. israelensis was cloned for expression, together with p20, in an acrystalliferous strain. The large hexagonal crystals formed were composed of Cyt2Ba, which facilitated its purification. Crystal solubilization in the presence of endogenous proteases (with spores and cell debris) enabled quick and simple procedure to obtain rather pure and active toxin species by cleavage between amino acid residues 34 and 35, most likely by a camelysin-like protease that was discovered in association with activated Cyt2Ba. The product of this cleavage displayed haemolytic activity comparable to that of exogenously activated Cyt2Ba. The sequence of this putative protease shares high homology with the cell envelope-bound metalloprotease (camelysin) of the closely related species Bacillus cereus.