Spd2 assists Spd1 in modulation of RNR architecture but does not regulate deoxynucleotide pools

In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The S. pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1-R2 holocomplex. Here, we report the characterization of Spd2, a protein with homology to Spd1. We show that Spd2 is a CRL4Cdt2 controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3ATR checkpoint dependency of CRL4Cdt2 mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4Cdt2 mutant cells to the same extent as spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, while it together with Spd2 modulates RNR architecture and sensitizes cells to DNA damage

BK channels in human glioma cells

Gliomzellen menschlicher Herkunft sind mit einer Reihe verschiedener spannungsabhängiger Ionenkanäle ausgestattet. Besonders BK Kanäle wurden in menschlichen Gliomzellinien regelmäßig beobachtet. Dies legt nahe, daß BK Kanäle auch in vivo ein fester Bestandteil solcher Zellen sind. Wir konnten das Vorkommen von BK Kanälen für 1321N1 Zellen bestätigen. Die Kanäle, die wir in unseren Experimenten charakterisierten, wiesen typische Merkmale von BK Kanälen auf : Sie öffneten erst bei Spannungen von mehr als +60 mV, waren ausgesprochen abhängig von [Ca2+]i, und liessen sich mit 100 nM IBTX sowie 1 mM TEA spezifisch blocken. Weil jedoch diese Kanäle eine so ausgeprägte Spannungsabhängigkeit zeigten, lag die Annahme nahe, daß diese Kanäle bei physiologischen Membranpotentialen geschlossen sind und deshalb auch keine physiologische Funktion haben können. Excised patch Experimente belegten jedoch, daß diese Annahme falsch ist. Anhand dieser Experimente, die eine gleichzeitige Variation von [Ca2+]i und des Membranpotentials erlauben, konnten wir zeigen, daß unter physiologischen Bedingungen diese Kanäle offen sein können. Dies ist die Bedingung für eine Funktion von BK Kanälen in menschlichen Gliomzellinien, welche bisher nicht untersucht worden war. Wir konnten beobachten, daß durch eine Erhöhung von [K+]e im Medium von 5 auf 20 mM die Zellzahl nach 6 Tagen um 20 % zunahm. Diese Zunahme der Zellzahl konnte durch spezifische BK Kanal Blocker verhindert werden. Dieser Befund deutet auf eine Rolle der BK Kanäle bei der durch bestimmte Wachstumsfaktoren ausgelösten Proliferation hin. Der genaue Mechanismus, der der Beteiligung von BK Kanälen bei der Proliferation zugrundeliegt ist noch nicht aufgeklärt. Die Untersuchungen zur Migration von 1321N1 Zellen ergaben, daß hierbei BK Kanäle keine Rolle zu spielen scheinen

Modes of nutrient uptake in Candida albicans : peptide transport and fluid phase endocytosis

Candida albicans is an important opportunistic pathogen in patients under treatments or with conditions, such as AIDS, that suppress the immune system. A basic understanding of the biology of this pathogen is essential in order to design an efficacious pathogen specific drug. The objectives of this research were to : (1) study the regulation of the peptide transport system using oxalysine-containing peptides as model compounds, (2) attempt to clone a C. albicans gene involved in peptide transport and (3) determine if fluid phase endocytosis is operative in the dimorphic fungus. An overview of the current literature related to the research presented here is given in Part I of this dissertation. Part II of this dissertation summarizes the results of the growth inhibitory effect on C. albicans of oxalysine, a lysine analog, and oxalysine-containing di-, tri-, tetra-, and pentapeptides. Several amino acids overcame ammonium ion repression and increased the toxicity of oxalysine-containing di- and tripeptides for C. albicans with little or no increase in toxicity of oxalysine or oxalysine-containing tetra- and pentapeptides. The results indicate that the dipeptide and tripeptide transport system(s) of C. albicans are regulated by micromolar amounts of amino acids in a similar fashion to regulation of peptide transport in S. cerevisiae and that multiple peptide transport systems differentially regulated by various nitrogen sources exist in C. albicans. Oxalysine is not toxic to mammalian cells and hence it may be possible to use an oxalysine-containing peptide to design an anticandidal drug. Part II of this dissertation describes the characterization of fluid phase endocytosis in C. albicans. Lucifer yellow (LY), an impermeable fluorescent dye used as a marker for fluid phase endocytosis, was internalized by C. albicans. LY was localized in vacuoles by a non-saturable, time-, temperature-, and energy-dependent process consistent with the characteristics of fluid phase endocytosis. Both the yeast and mold phases of this dimorphic fungus internalized LY, and growth in complex medium appeared to be required to enable cells to internalize LY. These studies may explain how some large molecules, such as defensins, toxins, and cationic proteins, enter C. albicans. Part IV of this dissertation describes attempts to clone a C. albicans peptide transport gene by functional complementation of a S. cerevisiae peptide transport mutant. The S. cerevisiae ptr2 mutant was transformed with a genomic C. albicans library in a multi copy vector and transformants with a PTR2+ phenotype were obtained. However, it was not possible to recover a C. albicans PTR2 homolog on a plasmid possibly due to integration of the C. albicans gene into the S. cerevisiae chromosome. Experimental approaches have been designed for the possible recovery of the putative C. albicans PTR2 homolog. Cloning of a C. albicans peptide transport gene should provide important information on the structure and regulation of the permease and facilitate the design of an anticandidal agent to be delivered to C. albicans via the peptide transport system