Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

Effects of serum type on growth and permeability properties of cultured endothelial cells

Serum is frequently added to defined basal media as a source of certain nutrients and macromolecular growth factors essential for cell growth. The many different sera commercially available may not be equally suitable for all cell types. The effects of four sera, fetal bovine serum (FBS), calf bovine serum (CS), equine serum (ES-1), and plasma-derived equine serum (ES-2), on growth and permeability properties of cultured porcine endothelial cells were determined. The rate of DNA synthesis, measured as [3H]thymidine incorporation, reached a peak at around 24 h, regardless of serum type, and was most marked with ES-1- or ES-2-treated cells. However, when estimated by total DNA, FBS, CS, or ES-1 treatment resulted in greater cell proliferation than ES-2. Based on protein synthetic rate and total cell protein, both FBS and CS appeared to be most growth supporting. At 72 h after cell plating, albumin passage across cultured endothelial monolayers was elevated in ES-1- and ES-2-treated cells compared with FBS- or CS-treated cells. "Leaky" cell monolayers were most marked with ES-1-treated cells. Cells grown in ES-2- and particularly in ES-1-enriched media were larger and more spindle-shaped compared with the typical cobblestone appearance of cells cultured in media enriched with either FBS or CS. These data suggest that CS, but not ES-1 or ES-2, is an excellent substitute for FBS to support desirable growth properties of macrovascular endothelial cells in culture.     

Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.     

Characterization of the long terminal repeats of micropia elements microdissected from the Y-chromosomal lampbrush loops "threads" of Drosophila hydei

Four micropia elements from Drosophila melanogaster and D. hydei have been analysed by sequencing. Two elements, from D. hydei, micropia-DhMiF8 and -DhMiF2, were recovered by cloning microdissected Y-chromosomal lampbrush loops "threads". This method allows isolation of repetitive sequences from defined chromosomal positions, but recovery of large and overlapping inserts is difficult. In case of the Y-chromosomal micropia elements it was not possible to define the endpoints of their long terminal repeat sequences precisely. Comparison of these locus-defined micropia elements to complete micropia elements isolated from D. melanogaster allowed identification of micropia-DhMiF8 and micropia-DhMiF2 long terminal repeats (LTRs). LTR sequences from the two Drosophila species are not conserved except for a few short sequences found at comparable positions that are believed to have functional significance. In contrast, the Leu-tRNA primer binding site and plus strand primer binding site are conserved between D. melanogaster and D. hydei.     

Localization of the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei by fluorescence in situ hybridization

We have used fluorescence in situ hybridization to map the positions of the different repetitive DNA sequences from the region forming the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei. This region harbours a megabase cluster of tandemly organized repeats of the Y-specific ay1 family and a megabase cluster of tandem repeats of the related Y-specific YsI family. In addition, ay1 repeats also occur in short blocks that are interspersed by other repetitive DNA sequences that we call Y-associated, since they have additional copies on other chromosomes. Using specific probes for ay1, YsI and Y-associated DNA sequences, we show that there is one large proximal cluster of YsI repeats and one, more distally located, large cluster of ay1 repeats. The Y-chromosomal copies of the Y-associated sequences are located in the most distal part of the ay1 cluster. This is consistent with the juxtaposition of ay1 and Y-associated sequences in more than 300 kb of cloned genomic DNA. Since both ay1 and Y-associated sequences have been shown to be transcribed in the Nooses, the lampbrush loop is formed in a distal region of the short arm of the Y chromosome, adjacent to the terminally located nucleolus organizer region. The clusters of homogeneous ay1 and YsI repeats are of no functional significance for the formation of the lampbrush loop.     

Subtypes of non-transformed human mammary epithelial cells cultured in vitro: histo-blood group antigen H type 2 defines basal cell-derived cells

Abstract. Normal (non-transformed) human mammary epithelial cell lines derived from reduction mammoplasties were analyzed by immunocytochemistry with more than 80 monoclonal antibodies (mAbs) and other specific reagents to tissue-specific and developmentally regulated antigens at different passage levels. A subpopulation of poorly differentiated, proliferating epithelial cells, corresponding to the 'selected' cell type of late passages, is shown to be characterized by a new marker, the histo-blood group antigen H type 2, probably carried on a membrane-bound glycolipid. These cells also express a number of other onco-developmental carbohydrate antigens [Le^y, Le^x, sialosyl-Le^a, precursor of Thomsen Friedenreich antigen (T_n), but not Thomsen-Friedenreich antigen and sialosyl-T_n]. Their cytokeratin (CK) phenotype, as assessed by reactivity with monospecific mAbs and two-dimensional gel electrophoresis, is CK 5, 6, 14 and 17, with CK 19 being consistently absent, and varying minor amounts of CK 7, 8 and 18, as well as 15 and 16. The reactivity of these cells with a panel of 11 mAbs specific for CK 18 varies considerably even after cloning, indicating heterogeneity of epitope expression or accessibility. Our data strongly suggest that the H type 2⁺ cells develop from the basal cell layer of the mammary gland.     

Interconversion of the salicylic acid signal and its glucoside in tobacco

Salicylic acid (SA) has been proposed to play a role in the induction of pathogenesis-related (PR) proteins and systemic acquired resistance (SAR) in tobacco. Since SA is rapidly converted to salicylic acid β-glucoside (SAG) in tobacco, we have attempted to assess the role of SAG in pathogenesis by application of chemically synthesized SAG to tobacco leaves. SAG was as active as SA in induction of PR-1 gene expression. This induction was preceded by a transient release of SA, which occurred in the extracellular spaces. The existence of a mechanism that releases SA from SAG suggests a possible role for SAG in SAR.