A Complex Containing RNA Polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p Plays a Role in Protein Kinase C Signaling

Yeast contains at least two complex forms of RNA polymerase II (Pol II), one including the Srbps and a second biochemically distinct form defined by the presence of Paf1p and Cdc73p (X. Shi et al., Mol. Cell. Biol. 17:1160–1169, 1997). In this work we demonstrate that Ccr4p and Hpr1p are components of the Paf1p-Cdc73p-Pol II complex. We have found many synthetic genetic interactions between factors within the Paf1p-Cdc73p complex, including the lethality of paf1Δ ccr4Δ, paf1Δ hpr1Δ, ccr4Δ hpr1Δ, and ccr4Δ gal11Δ double mutants. In addition, paf1Δ and ccr4Δ are lethal in combination with srb5Δ, indicating that the factors within and between the two RNA polymerase II complexes have overlapping essential functions. We have used differential display to identify several genes whose expression is affected by mutations in components of the Paf1p-Cdc73p-Pol II complex. Additionally, as previously observed for hpr1Δ, deleting PAF1 or CDC73 leads to elevated recombination between direct repeats. The paf1Δ and ccr4Δ mutations, as well as gal11Δ, demonstrate sensitivity to cell wall-damaging agents, rescue of the temperature-sensitive phenotype by sorbitol, and reduced expression of genes involved in cell wall biosynthesis. This unusual combination of effects on recombination and cell wall integrity has also been observed for mutations in genes in the Pkc1p-Mpk1p kinase cascade. Consistent with a role for this novel form of RNA polymerase II in the Pkc1p-Mpk1p signaling pathway, we find that paf1Δ mpk1Δ and paf1Δ pkc1Δ double mutants do not demonstrate an enhanced phenotype relative to the single mutants. Our observation that the Mpk1p kinase is fully active in a paf1Δ strain indicates that the Paf1p-Cdc73p complex may function downstream of the Pkc1p-Mpk1p cascade to regulate the expression of a subset of yeast genes.     

Activation of ERK by Ca2+ store depletion in rat liver epithelial cells

In rat liver epithelial (WB) cells,Ca²⁺ pool depletion induced by twoindependent methods resulted in activation of extracellular signal-regulated protein kinase (ERK). In the first method,Ca²⁺ pool depletion bythapsigargin increased the activity of ERK, even when rise in cytosolicCa²⁺ was blocked with theCa²⁺ chelator BAPTA-AM. For thesecond method, addition of extracellular EGTA at a concentration shownto deplete intracellular Ca²⁺pools also increased ERK activity. In each instance, ERK activation, asmeasured by an immunocomplex kinase assay, was greatly reduced by thetyrosine kinase inhibitor genistein, suggesting thatCa²⁺ store depletion increased ERKactivity through a tyrosine kinase pathway. The intracellularCa²⁺-releasing agent thapsigarginincreased Fyn activity, which was unaffected by BAPTA-AM pretreatment,suggesting that Fyn activity was unaffected by increased cytosolic freeCa²⁺. Furthermore, depletion ofintracellular Ca²⁺ with EGTAcaused inactivation of protein phosphatase 2A and protein tyrosinephosphatases. ANG II-induced activations of Fyn, Raf-1, and ERK wereaugmented in cells pretreated with BAPTA-AM, but ANG II-inducedexpression of the dual-specificity phosphatase mitogen-activatedprotein kinase phosphatase-1 was blocked by BAPTA-AM pretreatment.Together these results indicate that ERK activity is regulated by thebalance of phosphorylation vs. dephosphorylation reactions in intactcells and that the amount of Ca²⁺stored in intracellular pools plays an important role in this regulation.

     

The Resistance of Retroviral Vectors Produced from Human Cells to Serum Inactivation In Vivo and In Vitro Is Primate Species Dependent

The ability to deliver genes as therapeutics requires an understanding of the vector pharmacokinetics similar to that required for conventional drugs. A first question is the half-life of the vector in the bloodstream. Retroviral vectors produced in certain human cell lines differ from vectors produced in nonhuman cell lines in being substantially resistant to inactivation in vitro by human serum complement (F. L. Cosset, Y. Takeuchi, J. L. Battini, R. A. Weiss, and M. K. Collins, J. Virol. 69:7430-7436, 1995). Thus, use of human packaging cell lines (PCL) may produce vectors with longer half-lives, resulting in more-efficacious in vivo gene therapy. However, survival of human PCL-produced vectors in vivo following systemic administration has not been explored. In this investigation, the half-lives of retroviral vectors packaged by either canine D17 or human HT1080 PCL were measured in the bloodstreams of macaques and chimpanzees. Human PCL-produced vectors exhibited significantly higher concentrations of circulating biologically active vector at the earliest time points measured (>1, 000-fold in chimpanzees), as well as substantially extended half-lives, compared to canine PCL-produced vectors. In addition, the circulation half-life of human PCL-produced vector was longer in chimpanzees than in macaques. This was consistent with in vitro findings which demonstrated that primate serum inactivation of vector produced from human PCL increased with increasing phylogenetic distance from humans. These results establish that in vivo retroviral vector half-life correlates with in vitro resistance to complement. Furthermore, these findings should influence the choice of animal models used to evaluate retroviral-vector-based therapies.     

Stage‐specific effects of bone morphogenetic proteins on the oligodendrocyte lineage

Oligodendrocyte maturation is regulated by multiple secreted factors present in the brain during critical stages of development. Whereas most of these factors promote oligodendrocyte proliferation and survival, members of the bone morphogenetic protein family (BMPs) recently have been shown to inhibit oligodendrocyte differentiation in vitro. Oligodendrocyte precursors treated with BMPs differentiate to the astrocyte lineage. Given that cells at various stages of the oligodendrocyte lineage have distinct responses to growth factors, we hypothesized that the response to BMP would be stage‐specific. Using highly purified, stage‐specific cultures, we found that BMP has distinct effects on cultured oligodendrocyte preprogenitors, precursors, and mature oligodendrocytes. Oligodendrocyte preprogenitors (PSA‐NCAM+, A2B5−) treated with BMP2 or BMP4 developed a novel astrocyte phenotype characterized by a morphological change and expression of glial fibrillary acidic protein (GFAP) but little glutamine synthetase expression and no labeling with A2B5 antibody. In contrast, treating oligodendrocyte precursors with BMPs resulted in the accumulation of cells with the traditional type 2 astrocyte phenotype (GFAP+, A2B5+). However, many of the cells with an astrocytic morphology did not express GFAP or glutamine synthetase unless thyroid hormone was present in the medium. The addition of fibroblast growth factor along with BMP to either oligodendrocyte preprogenitor or the oligodendrocyte precursor cells inhibited the switch to the astrocyte lineage, whereas platelet‐derived growth factor addition had no effect. Treatment of mature oligodendrocytes with BMP elicited no change in morphology or expression of GFAP. These data suggest that as cells progress through the oligodendrocyte lineage, they show developmentally restricted responses to the BMPs. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 1–17, 2000     

Unlocking avian museum collections to enable and advance environmental change research

The rate and magnitude of contemporary changes in natural systems is unprecedented in the Earth's history. Studies of wild birds have been critically important in helping us understand and address these environmental changes. Avian collections provide a potentially unique perspective on change through time, but their role in environmental change research is limited by the availability of collections data. Here we describe how avian collections might be unlocked to enable environmental change research, and discuss the opportunities and constraints associated with this. We use the concept of the extended specimen to describe the types of data that could be unlocked from basic data for discoverability to enhanced data that might be directly applied to environmental change questions. We illustrate the type of environmental change research these data might support. We argue that data creation and access is currently limited by funding for digitization, a rather patchy understanding of the needs of the research community and less than adequate data-sharing by institutions and researchers. We develop a blueprint for addressing these issues which includes (1) improvements in sharing the data we are already creating and (2) building a better case for digitization at scale. As one of the largest avian collections in the world, the Natural History Museum, UK, is committed to unlocking our collections, but we will need input and support from the avian research community to do so.     

Interpretation of the osmotic behavior of sickle cell hemoglobin solutions: Different interactions among monomers and polymers

It has long been known that a simple hard particle model quantitatively explains the osmotic properties of monomeric hemoglobin near its isoelectric point. However, we find that a hard particle model is not consistent with the osmotic properties of polymerized hemoglobin and that substantial soft repulsions are indicated. With allowance for different interactions among monomers and among polymers, a self-consistent quantitative fit to the experimental data is obtained. The results suggest that the decreasing “solubility” of deoxy sickle cell hemoglobin with increasing temperature from 20 to 37°C is due to weaker repulsions between polymers at higher temperatures rather than stronger polymerization. The temperature dependence of these variables indicates that the aggregation of monomers is enthalpically and entropically driven (the latter effect being stronger), while the approach of polymers toward each other is enthalpically disfavored and entropically favored (with the former dominating). In both cases, the entropic contribution suggests that water is released. © 1998 John Wiley & Sons, Inc. Biopoly 45: 299–306, 1998     

An improved histochemical method for measuring nitric oxide synthase activity

The previous quantitative histochemical method for measuring nitric oxide synthase (NOS) activity in tissue sections involved the loss of about 15 per cent of the NOS, presumably from the section into the reaction medium. Two changes are now described. The first is concerned with the preparation in the laboratory of the active reagent, lead ammonium citrate/acetate (LACA). The second change involves an improvement of the procedure for measuring NOS activity. The new method appears to retain all the measurable NOS activity inside the section. Copyright © 1999 John Wiley & Sons, Ltd.