Kohei Hosaka

Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase (PDE1) Is a Pharmacological Target of Differentiation-Inducing Factor-1, an Antitumor Agent Isolated from Dictyostelium

The differentiation-inducing factor-1 (DIF-1) isolated from Dictyostelium discoideum is a potent antiproliferative agent that induces growth arrest and differentiation in mammalian cells in vitro. However, the specific target molecule(s) of DIF-1 has not been identified. In this study, we have tried to identify the target molecule(s) of DIF-1 in mammalian cells, examining the effects of DIF-1 and its analogs on the activity of some candidate enzymes. DIF-1 at 10–40 μm dose-dependently suppressed cell growth and increased the intracellular cyclic AMP concentration in K562 leukemia cells. It was then found that DIF-1 at 0.5–20 μm inhibited the calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (PDE1) in vitro in a dose-dependent manner. Kinetic analysis revealed that DIF-1 acted as a competitive inhibitor of PDE1 versus the substrate cyclic AMP. Because DIF-1 did not significantly affect the activity of other PDEs or CaM-dependent enzymes and, in addition, an isomer of DIF-1 was a less potent inhibitor, we have concluded that PDE1 is a pharmacological and specific target of DIF-1.

Identification of genes affecting lipid content using transposon mutagenesis in Saccharomyces cerevisiae.

Genes involved in lipid accumulation were identified in Saccharomyces cerevisiae using transposon insertion mutagenesis. Five ORFs, such as SNF2, IRA2, PRE9, PHO90, and SPT21 were found from the analysis of the insertion sites in transposon insertion mutants with higher lipid content. Since these ORFs are not directly involved in storage lipid biosynthesis, we speculate that they are involved in carbon fluxes into storage lipids in response to nutrient conditions. Lipid analysis of disruptants of these ORFs indicated that the Δsnf2, and Δira2 disruptants had significantly higher lipid content. Cultivation in a nitrogen-limited medium increased the lipid content in all disruptants, among which the Δpre9 disruptant was the most sensitive to nitrogen limitation. We then focused on the Δsnf2 disruptant due to its higher lipid content and its function as a regulator of phospholipid synthesis. Lipid class analysis indicated that triacylglycerol and free fatty acids contributed to the increase in total lipids of the Δsnf2 disruptant. The addition of exogenous fatty acids was not so effective at increasing the lipid content in the Δsnf2 disruptant as it was in the wild type. It should be noticed that exogenous free linoleic acid was much higher in the Δsnf2 disruptant than in the wild type, as in the case of endogenous free fatty acids. In addition, the incorporation of exogenous fatty acids into cells increased in the disruptant, suggesting that fatty acid transporters were regulated by SNF2. The results suggest that metabolic fluxes into storage lipids, which are activated in the Δsnf2 disruptant, is repressed by the incorporation of exogenous fatty acids. They provide new insight into the biosynthesis of storage lipids in yeast.

Zinc inhibits calcineurin activity in vitro by competing with nickel

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM)-dependent protein serine/threonine phosphatase that contains Zn(2+) in its catalytic domain and can be stimulated by divalent ions such as Mn(2+) and Ni(2+). In this study, the role of exogenous Zn(2+) in the regulation of CN activity and its relevance to the role of Ni(2+) was investigated. Zn(2+) at a concentration range of 10nM-10 micro M inhibited Ni(2+)-stimulated CN-activity in vitro in a dose-dependent manner and approximately 50% inhibition was attained with 0.25 micro M Zn(2+). Kinetic analysis showed that Zn(2+) inhibited the activity of CN by competing with Ni(2+). Interaction of CN and CaM was not inhibited with Zn(2+) at 10 micro M. Zn(2+) never affected the activity of cAMP phosphodiesterase 1 or myosin light-chain kinase (CaM-dependent enzymes) and rather activated alkaline phosphatase. The present results indicate that Zn(2+) should be a potent inhibitor for CN activity although this ion is essential for CN.

Pharmacological evidence that stalk cell differentiation involves increases in the intracellular Ca(2+) and H(+) concentrations in Dictyostelium discoideum.

Differentiation‐inducing factors (DIFs) are required for stalk cell formation in Dictyostelium discoideum. In the present study, in order to support our hypothesis that DIFs may function via increases in [Ca2+]c and [H+]c, we investigated the combined effects of 5,5‐dimethyl‐2,4‐oxazolidinedione (DMO, a [H+]c‐increasing agent), thapsigargin (Tg) and BHQ ([Ca2+]c‐increasing agents) on in vitro stalk cell formation in several strains. DMO, in combination with Tg or BHQ, induced stalk cell formation in a DIF‐deficient mutant HM44. Although the rates of stalk cell induction by the drugs were low in the presence of cerulenin (an inhibitor of endogenous DIF production) in HM44 and V12M2 (a wild‐type strain), the drugs succeeded in inducing sufficient stalk cell formation when a small amount of DIF‐1 was supplied. Furthermore, co‐addition of DMO, BHQ and a small amount of DIF‐1 also induced sufficient stalk cell formation in AX‐4 (an axenic strain) and HM1030 (dmtA−) but not in CT15 (dimA−). The drugs suppressed spore formation and promoted stalk cell formation in both HM18 (a sporogenous mutant) and 8‐bromo‐cAMP‐stimulated V12M2. The present results suggest that DIFs function, at least in part, via increases in [Ca2+]c and [H+]c in D. discoideum.

Dictyostelium differentiation‐inducing factor‐1 induces glucose transporter 1 translocation and promotes glucose uptake in mammalian cells

The differentiation‐inducing factor‐1 (DIF‐1) is a signal molecule that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum, while DIF‐1 and its analogs have been shown to possess antiproliferative activity inu2003vitro in mammalian tumor cells. In the present study, we investigated the effects of DIF‐1 and its analogs on normal (nontransformed) mammalian cells. Without affecting the cell morphology and cell number, DIF‐1 at micromolar levels dose‐dependently promoted the glucose uptake in confluent 3T3‐L1 fibroblasts, which was not inhibited with wortmannin or LY294002 (inhibitors for phosphatidylinositol 3‐kinase). DIF‐1 affected neither the expression level of glucose transporteru20031 nor the activities of four key enzymes involved in glucose metabolism, such as hexokinase, fluctose 6‐phosphate kinase, pyruvate kinase, and glucose 6‐phosphate dehydrogenase. Most importantly, stimulation with DIF‐1 was found to induce the translocation of glucose transporteru20031 from intracellular vesicles to the plasma membranes in the cells. In differentiated 3T3‐L1 adipocytes, DIF‐1 induced the translocation of glucose trasporteru20031 (but not of glucose transporteru20034) and promoted glucose uptake, which was not inhibited with wortmannin. These results indicate that DIF‐1 induces glucose transporteru20031 translocation and thereby promotes glucose uptake, at least in part, via a inhibitors for phosphatidylinositol 3‐kinase/Akt‐independent pathway in mammalian cells. Furthermore, analogs of DIF‐1 that possess stronger antitumor activity than DIF‐1 were less effective in promoting glucose consumption, suggesting that the mechanism of the action of DIF‐1 for stimulating glucose uptake should be different from that for suppressing tumor cell growth.

Regulation of IL-2 production in Jurkat cells by Dictyostelium-derived factors

AIMSnDifferentiation-inducing factors (DIFs) are chlorinated alkylphenones found in the cellular slime mold Dictyostelium discoideum. DIF derivatives exhibit antiproliferative activities and promote glucose consumption in mammalian cells in vitro. In this study, we assessed the ability of DIFs to regulate the immune system and investigated their mechanisms of action.nnnMAIN METHODSnWe examined the effects of 30 DIF derivatives on concanavalin A-induced IL-2 production (CIIP) in Jurkat T-cells. We also examined the effects of some DIF derivatives on the activity of AP-1 (activator protein-1), NFAT (nuclear factor of activated T-cells), and NFkappaB (nuclear factor kappa B), which are transcription factors required for CIIP.nnnKEY FINDINGSnOf the derivatives tested, some compounds suppressed CIIP as well as the known immunosuppressants cyclosporine A and FK506. A reporter gene assay revealed that 4 DIF derivatives tested suppressed CIIP, at least in part, by inhibiting the activity of AP-1, NFAT, and/or NFkappaB. Unlike cyclosporine A and FK506, the DIF derivatives had little effect on concanavalin A-induced interferon-gamma production in Jurkat cells.nnnSIGNIFICANCEnThe present results suggest that DIF derivatives could be developed as novel immunosuppressive drugs.

Cloning and expression of human cDNA encoding phosphatidylinositol transfer protein β

cDNA encoding the beta isoform of human phosphatidylinositol transfer protein was cloned from a human brain cDNA library. The deduced sequence of the protein comprised 271 amino acids with a calculated molecular mass of 31,539 Da, and showed 98.1% identity to that of the beta isoform of rat phosphatidylinositol transfer protein. The cDNA hybridized to a 3.4-kb mRNA, which was widely expressed in various human tissues including brain.

Biological activities of novel derivatives of DIF-1 isolated from Dictyostelium

The differentiation-inducing factor-1 (DIF-1) is a lipophilic signal molecule (chlorinated alkylphenone) that induces stalk cell differentiation in the cellular slime mold Dictyostelium discoideum. In addition, DIF-1 and its derivatives have been shown to possess anti-leukemic activity and glucose consumption-promoting activity in vitro in mammalian cells. In this study, to assess the chemical structure-effect relationship of DIF-1, we synthesized eight derivatives of DIF-1 and investigated their stalk cell-inducing activity in Dictyostelium cells and pharmacological activities in mammalian cells. Of the derivatives, two amide derivatives of DIF-1, whose hydrophobic indexes are close to that of DIF-1, induced stalk cell differentiation as strongly as DIF-1 in Dictyostelium cells. It was also found that some derivatives suppressed cell growth in human K562 leukemia cells and promoted glucose consumption in mouse 3T3-L1 cells. These results give us valuable information as to the chemical structure-effect relationship of DIF-1.

Electrochemical assay of concanavalin A–ovalbumin binding on magnetic beads

To monitor protein-glycoprotein interactions on magnetic beads, the present study developed an electrochemical assay of the binding between concanavalin A (ConA) and ovalbumin (OVA). The system was a powerful model that could be used to evaluate cell junctions. ConA with an electroactive daunomycin was immobilized on 6 different sizes of magnetic beads (diameter: 1.0-8.9 μm) through a cross-linking agent. Six sizes of OVA-beads (diameter: 1.0-8.9 μm) were also prepared using a similar method. The binding was evaluated using an oxidation peak of ConA with daunomycin because ConA recognized OVA with α-mannose residues. When binding took place on the beads surface, the peak current was decreased due to the electroactive moieties being covered with OVA. When ConA/daunomycin-OVA binding was evaluated, the change of the peak current obtained by the beads (diameter: 8.9 μm) modified with ConA and daunomycin was the greatest in the presence of OVA-modified beads (diameter: 2.5 μm). In contrast, particle agglomeration was observed for the smallest beads (diameter: 1.0 μm) with ConA/daunomycin and OVA. The results suggested that ConA-OVA binding depended on the size of beads. Thus, this method could be applied to measure protein-glycoprotein interactions on the cell surface.

Electrochemically monitoring the binding of concanavalin A and ovalbumin.

To evaluate protein-protein interactions, a new voltammetric method was developed using a protein labeled with an electroactive compound. Concanavalin A (ConA), which is a lectin, recognizes α-mannose residues. Because the ConA was to be bound to ovalbumin (OVA), which has a high-mannose sugar chain, ConA labeled with daunomycin was prepared as the probe to monitor the binding. The binding to OVA was caused by the label modification of the ConA. As a result, the electrode response of the labeled ConA decreased as the OVA concentration increased. The electrode response of the labeled ConA was linearly over the range of 1.5×10(-10) and 1.5×10(-9)M OVA. The relative standard deviation of 1.5×10(-8)M labeled ConA and 1.5×10(-10)M OVA was 6.9% (n=5). The labeled ConA-OVA binding could then be conveniently monitored based on the change in response. In contrast, interactions between the labeled ConA and a protein with no specific sugar chain also were investigated. Incubation scarcely influenced the peak current of the labeled ConA. When several concentrations of OVA were added to a serum, good recovery determined it. Consequently, this method could be applied to the measurement of protein-protein interactions.