ALTERATIONS IN POLYRIBOSOMES DURING ERYTHROID CELL MATURATION

This communication presents a morphological study of the changes in ribosome content and organization which occur during the maturation of erythroid cells of the phenylhydrazine-treated rabbit. Electron micrographs of thin sectioned nucleated and non-nucleated erythroid cells have been subjected to a quantitative analysis of the distribution of ribosomes as polyribosomes of various sizes and as single ribosomes. The ribosomes of nucleated erythroid cells of marrow are virtually all arranged in the polyribosome configuration consisting of clusters of 2 to 6 individual ribosomes. These cells are the most active in the erythroid series in protein biosynthesis. During maturation to the non-nucleated reticulocyte stage, found in the circulating blood, there is a decrease in protein synthesizing capacity, a fall in total ribosome content, and, more significantly, a decrease in the number and size of polyribosomes. Maturation to the ribosome-free erythrocyte, either under in vitro or in vivo conditions, entails a further decrease in protein synthesis which correlates with a progressive disaggregation of the biosynthetically active polyribosomes into smaller clusters and inactive single ribosomes. Possible models which may account for the stability of the polyribosome and for the mechanism of polyribosome dissociation are discussed.     

Characterization of three PDI-like genes in Physcomitrella patens and construction of knock-out mutants

Plant genomes typically contain several sequences homologous to protein disulfide isomerase (PDI). PDI was first identified as an abundant enzyme in the endoplasmic reticulum, where it catalyzes the formation, reduction, and isomerization of disulfide bonds during protein folding. PDI-like proteins have also been implicated in a variety of other functions, such as the regulation of cell adhesion, and may act as elicitors of the autoimmune response in mammals. A PDI-like protein (RB60) was recently shown to be imported into chloroplasts in the unicellular green alga Chlamydomonas reinhardtii and a higher plant, Pisum sativum, where it associates with thylakoid membranes. This suggests that the different PDI-like proteins in plant and animals may have diverse biological roles. To begin to elucidate the roles of PDI-like proteins, we have cloned, characterized, and generated knock-out mutants for three PDI-like genes that have high, medium, and low levels of expression, respectively, in the moss Physcomitrella patens. Phylogenetic analysis indicates that the three PDI-like proteins cluster with RB60 and four proteins from Arabidopsis thaliana. They are typified by an N-terminal domain rich in negatively charged residues. The knock-out mutants, which are the first knock-outs available for PDI-like proteins in a multicellular organism, were found to be viable, indicating that the function of each single gene is dispensable, and suggesting that they may be functionally complementary.     

PSL, an S phase-related p55 nuclear antigen, associates transiently with chromatin

An S phase-related nuclear 55K antigen, also designated PSL, has been characterized in various mammalian cells, using a human serum from a patient with autoimmune disorders (Barque et al., EMBO j 2 (1983) 743). In this report, we show by immunoelectron microscopy that the p55 protein associates in situ with the chromatin of rat hepatocytes. This association is a transient one, as indirect immunofluorescence studies show that PSL does not bind to individualized metaphase chromosomes. Furthermore, immunoprecipitation tests indicate that the majority of PSL is in the non-chromosomal cell fraction. These results suggest that this nuclear antigen is directly involved in the DNA replication process.     

Promotion of wound healing by yeast glucan evaluated on single animals

The effectiveness of yeast glucan in the acceleration of wound healing was evaluated in mice, rats and guinea pigs. In all experiments comparison between glucan treatment in one hind leg and saline treatment as control on the other leg was made on identical wounds. The degree of healing in the two legs was evaluated macroscopically and classified as follows: 1. healing more advanced in glucan treated wound marked by (+). 2. No significant difference between the two legs marked by (0). 3. Healing more advanced in the control wound, marked by (-). During the days when the differences were most obvious, 60% to 80% of the animals showed more advanced healing in the glucan treated wound, 20% to 40% showed no significant difference; and 0 to 15% showed more advanced healing in the control, saline treated wound. The average time for complete wound healing was reduced by about 18% as a result of glucan treatment. The histological analysis shows that the acceleration of wound healing was mediated by early arrival of macrophages to the wound area in the glucan treated wounds.     

Demographic structure and pathogen dynamics on the network of livestock movements in Great Britain

Using a novel interpretation of dynamic networks, we analyse the network of livestock movements in Great Britain in order to determine the risk of a large epidemic of foot-and-mouth disease (FMD). This network is exceptionally well characterized, as there are legal requirements that the date, source, destination and number of animals be recorded and held on central databases. We identify a percolation threshold in the structure of the livestock network, indicating that, while there is little possibility of a national epidemic of FMD in winter when the catastrophic 2001 epidemic began, there remains a risk in late summer or early autumn. These predictions are corroborated by a non-parametric simulation in which the movements of livestock in 2003 and 2004 are replayed as they occurred. Despite the risk, we show that the network displays small-world properties which can be exploited to target surveillance and control and drastically reduce this risk.     

Dynamic Proteomics of Human Protein Level and Localization across the Cell Cycle

Regulation of proteins across the cell cycle is a basic process in cell biology. It has been difficult to study this globally in human cells due to lack of methods to accurately follow protein levels and localizations over time. Estimates based on global mRNA measurements suggest that only a few percent of human genes have cell-cycle dependent mRNA levels. Here, we used dynamic proteomics to study the cell-cycle dependence of proteins. We used 495 clones of a human cell line, each with a different protein tagged fluorescently at its endogenous locus. Protein level and localization was quantified in individual cells over 24h of growth using time-lapse microscopy. Instead of standard chemical or mechanical methods for cell synchronization, we employed in-silico synchronization to place protein levels and localization on a time axis between two cell divisions. This non-perturbative synchronization approach, together with the high accuracy of the measurements, allowed a sensitive assay of cell-cycle dependence. We further developed a computational approach that uses texture features to evaluate changes in protein localizations. We find that 40% of the proteins showed cell cycle dependence, of which 11% showed changes in protein level and 35% in localization. This suggests that a broader range of cell-cycle dependent proteins exists in human cells than was previously appreciated. Most of the cell-cycle dependent proteins exhibit changes in cellular localization. Such changes can be a useful tool in the regulation of the cell-cycle being fast and efficient.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: II. Characterization of the Phosphate Starvation Inducible-Excreted Acid Phosphatase

Three-day-old suspension cultured cells of Lycopersicon esculentum transferred to a Pi-depleted medium had 2.7 times the excreted acid phosphatase (Apase) activity of cells transferred to a Pi-sufficient medium. Cell growth during this time period was identical for the two treatments. Excreted Apase activity was resolved into two fractions on a Sephadex G-150 column. Most of the phosphate starvation inducible (psi) enhancement in activity was in the lower molecular weight fraction. These two fractions exhibited different substrate versus pH activity profiles. With a native polyacrylamide gel electrophoresis assay, the lower molecular weight fraction resolved into two bands of activity. Both column fractions resolved into the same single band of activity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of this enzyme was 57 kilodalton. These data indicate that L. esculentum has at least two isozymes of the psi-excreted Apase and that these isozymes may associate to form high molecular weight aggregates. Labeling studies using [³⁵S]methionine show that the psi response in tomato cells is complex and involves changes in the steady state levels of several excreted proteins.     

ADP-dependent phosphorylation regulates RNA-binding in vitro: implications in light-modulated translation.

Light-regulated translation of chloroplastic mRNAs in the green alga Chlamydomonas reinhardtii requires nuclear encoded factors that interact with the 5'-untranslated region (5'-UTR) of specific mRNAs to enhance their translation. We have previously identified and characterized a set of proteins that bind specifically to the 5'-UTR of the chloroplastic psbA mRNA. Accumulation of these proteins is similar in dark- and light-grown cells, whereas their binding activity is enhanced during growth in the light. We have identified a serine/threonine protein phosphotransferase, associated with the psbA mRNA-binding complex, that utilizes the beta-phosphate of ADP to phosphorylate and inactivate psbA mRNA-binding in vitro. The inactivation of mRNA-binding in vitro is initiated at high ADP levels, levels that are attained in vivo only in dark-grown chloroplasts. These data suggest that the translation of psbA mRNA is attenuated by phosphorylation of the mRNA-binding protein complex in response to a rise in the stromal concentration of ADP upon transfer of cells to dark.