Signaling by higher inositol polyphosphates. Synthesis of bisdiphosphoinositol tetrakisphosphate ("InsP8") is selectively activated by hyperosmotic stress

Evidence has accumulated that inositol pyrophosphates (diphosphoinositol pentakisphosphate (PP-InsP5) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4)) are intracellular signals that regulate many cellular processes including endocytosis, vesicle trafficking, apoptosis, and DNA repair. Yet, in contrast to the situation with all other second messengers, no one studying multicellular organisms has previously described a stimulus that acutely and specifically elevates cellular levels of PP-InsP5 or [PP]2-InsP4. We now show up to 25-fold elevations in [PP]2-InsP4 levels in animal cells. Importantly, this does not involve classical agonists. Instead, we show that this [PP]2-InsP4 response is a novel consequence of the activation of ERK1/2 and p38MAPalpha/beta kinases by hyperosmotic stress. JNK did not participate in regulating [PP]2-InsP4 levels. Identification of [PP]2-InsP4 as a sensor of hyperosmotic stress opens up a new area of research for studies into the cellular activities of higher inositol phosphates.     

Purification, sequencing, and molecular identification of a mammalian PP-InsP5 kinase that is activated when cells are exposed to hyperosmotic stress

Mammalian cells utilize multiple signaling mechanisms to protect against the osmotic stress that accompanies plasma membrane ion transport, solute uptake, and turnover of protein and carbohydrates (Schliess, F., and Haussinger, D. (2002) Biol. Chem. 383, 577-583). Recently, osmotic stress was found to increase synthesis of bisdiphosphoinositol tetrakisphosphate ((PP)2-InsP4), a high energy inositol pyrophosphate (Pesesse, X., Choi, K., Zhang, T., and Shears, S. B. (2004) J. Biol. Chem. 279, 43378-43381). Here, we describe the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIP5K) that synthesizes (PP)2-InsP4. Partial amino acid sequence, obtained by mass spectrometry, matched the sequence of a 160-kDa rat protein containing a putative ATP-grasp kinase domain. BLAST searches uncovered two human isoforms (PPIP5K1 (160 kDa) and PPIP5K2 (138 kDa)). Recombinant human PPIP5K1, expressed in Escherichia coli, was found to phosphorylate diphosphoinositol pentakisphosphate (PP-InsP5) to (PP)2-InsP4 (Vmax = 8.3 nmol/mg of protein/min; Km = 0.34 microM). Overexpression in human embryonic kidney cells of either PPIP5K1 or PPIP5K2 substantially increased levels of (PP)2-InsP4, whereas overexpression of a catalytically dead PPIP5K1(D332A) mutant had no effect. PPIP5K1 and PPIP5K2 were more active against PP-InsP5 than InsP6, both in vitro and in vivo. Analysis by confocal immunofluorescence showed PPIP5K1 to be distributed throughout the cytoplasm but excluded from the nucleus. Immunopurification of overexpressed PPIP5K1 from osmotically stressed HEK cells (0.2 M sorbitol; 30 min) revealed a persistent, 3.9 +/- 0.4-fold activation when compared with control cells. PPIP5Ks are likely to be important signaling enzymes.     

Turnover of bis-diphosphoinositol tetrakisphosphate in a smooth muscle cell line is regulated by beta2-adrenergic receptors through a cAMP-mediated, A-kinase-independent mechanism.

Bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') is a 'high-energy' inositol phosphate; we report that its metabolism is receptor-regulated in DDT1 MF-2 smooth muscle cells. This conclusion arose by pursuing the mechanism by which F- decreased cellular levels of [PP]2-InsP4 up to 70%. A similar effect was induced by elevating cyclic nucleotide levels, either with IBMX or by application of either Bt2cAMP (EC50 = 14.7 microM), Bt2cGMP (EC50 = 7.9 microM) or isoproterenol (EC50 = 0.4 nM). Isoproterenol (1 microM) decreased [PP]2-InsP4 levels 25% by 5 min, and 71% by 60 min. This novel, agonist-mediated regulation of [PP]2-InsP4 turnover was very specific; isoproterenol did not decrease the cellular levels of either inositol pentakisphosphate, inositol hexakisphosphate or other diphosphorylated inositol polyphosphates. Bradykinin, which activated phospholipase C, did not affect [PP]2-InsP4 levels. Regulation of [PP]2-InsP4 turnover by both isoproterenol and cell-permeant cyclic nucleotides was unaffected by inhibitors of protein kinases A and G. The effectiveness of the kinase inhibitors was confirmed by their ability to block phosphorylation of the cAMP response element-binding protein. Our results indicate a new signaling action of cAMP, and furnish an important focus for future research into the roles of diphosphorylated inositol phosphates in signal transduction.     

The g5R (D250) gene of African swine fever virus encodes a Nudix hydrolase that preferentially degrades diphosphoinositol polyphosphates.

The African swine fever virus (ASFV) g5R gene encodes a protein containing a Nudix hydrolase motif which in terms of sequence appears most closely related to the mammalian diadenosine tetraphosphate (Ap4A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap4A hydrolases, having highest activity with GTP, followed by adenosine 5'-pentaphosphate (p5A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp3A. In common with eukaryotic diadenosine hexaphosphate (Ap6A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP5 and [PP]2-InsP4. A comparison of the kinetics of substrate utilization showed that the k(cat)/K(m) ratio for PP-InsP5 is 60-fold higher than that for GTP, which allows classification of g5R as a novel diphosphoinositol polyphosphate phosphohydrolase (DIPP). Unlike mammalian DIPP, g5Rp appeared to preferentially remove the 5-beta-phosphate from both PP-InsP5 and [PP]2-InsP4. ASFV infection led to a reduction in the levels of PP-InsP5, ATP and GTP by ca. 50% at late times postinfection. The measured intracellular concentrations of these compounds were comparable to the respective K(m) values of g5Rp, suggesting that one or all of these may be substrates for g5Rp during ASFV infection. Transfection of ASFV-infected Vero cells with a plasmid encoding epitope-tagged g5Rp suggested localization of this protein in the rough endoplasmic reticulum. These results suggest a possible role for g5Rp in regulating a stage of viral morphogenesis involving diphosphoinositol polyphosphate-mediated membrane trafficking.     

Internalization-sequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreatic AR 4-2 J cells

Cholecystokinin (CCK) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas, after preincubation with 20 nM dexamethasone. At steady state binding at 37 degrees C (i.e., after a 5 min incubation), less than 10% of the radioactivity of [125I]BH-CCK-9 (3-(4-hydroxy-[125I]iodophenyl)propionyl (Thr34, Nle37) CCK(31-39)) could be washed away from intact cells with an ice-cold acidic medium, suggesting high and rapid internalization-sequestration of tracer. By contrast, more than 85% of the tracer dissociated rapidly after a similar acid wash from cell membranes prelabelled at steady state. In intact AR 4-2 J cells, internalization required neither energy nor the cytoskeleton framework. Tracer internalization was reversed partly but rapidly at 37 degrees C but slowly at 4 degrees C. In addition, two degradation pathways of the tracer were demonstrated, one intracellular and one extracellular. Intracellular degradation occurred at 37 degrees C but not at 20 degrees C and resulted in progressive intracellular accumulation of [125I]BH-Arg that corresponded, after 1 h at 37 degrees C, to 35% of the radioactivity specifically bound. This phenomenon was not inhibited by serine proteinase inhibitors and modestly only by monensin and chloroquine. Besides, tracer degradation at the external cell surface was still observable at 20 degrees C and yielded a peptide (probably [125I]BH-Arg-Asp-Tyr(SO3H)-Thr-Gly). This degradation pathway was partly inhibited by bacitracin and phosphoramidon while thiorphan, an inhibitor of endopeptidase EC 3.4.24.11, was without effect.     

PDGF stimulates transient phosphorylation of 180,000 dalton protein

Cell-free extracts of platelet-derived growth factor (PDGF) treated, density-arrested, quiescent BALB/c-3T3 cells are capable of phosphorylating a 180,000 dalton protein (PP180). The phosphorylation of PP180 was observed in SDS polyacrylamide gel electrophoresis profiles of Nonidet P-40 solubilized cell preparations that had been incubated with [gamma-32P]ATP. When quiescent BALB/c-3T3 cell cultures were incubated at 37 degrees C with PDGF, phosphorylation of PP180 in cell extracts could be detected after a 3-min exposure of the intact cells to PDGF, which was maximal after 10-15 minutes and had diminished by 30-60 min. PDGF stimulation of PP180 phosphorylation also was observed in extracts of cells that had been incubated with PDGF at 4 degrees C; however, in contrast to PDGF exposure at 37 degrees C, the ability of cell extracts to phosphorylate PP180 did not decrease even after 4 hr of cell exposure to PDGF at 4 degrees C. When cells exposed to PDGF at 4 degrees C were transferred to 37 degrees C for 30 min, the ability of cell extracts to phosphorylate PP180 decreased to a nonstimulated level. After cells stimulated by PDGF showed a diminished ability to phosphorylate PP180, immediate restimulation with PDGF did not induce the ability to phosphorylate PP180. Incubation for 11 hr at 37 degrees C was required before readdition of PDGF allowed observable phosphorylation of PP180 in cell extracts, but maximum PDGF stimulation of the phosphorylation of PP180 was found after the cells were incubated for 24 hr in culture conditions. The amount of the stimulation of PP180 phosphorylation was dependent on the concentration of PDGF. The stimulation of DNA synthesis by PDGF was correlated to the phosphorylation of PP180. This phosphorylation activity was not observed in extracts of cells that had been treated with epidermal growth factor (EGF), somatomedin C, insulin, plasma, or fibroblast growth factor (FGF). This novel experimental approach allows the investigation of a PDGF-stimulated phosphorylation activity in relation to the cell cycle and growth regulation.     

Cold shock response in mammalian cells

Compared to bacteria and plants, the cold shock response has attracted little attention in mammals except in some areas such as adaptive thermogenesis, cold tolerance, storage of cells and organs, and recently, treatment of brain damage and protein production. At the cellular level, some responses of mammalian cells are similar to microorganisms; cold stress changes the lipid composition of cellular membranes, and suppresses the rate of protein synthesis and cell proliferation. Although previous studies have mostly dealt with temperatures below 20 degrees C, mild hypothermia (32 degrees C) can change the cell's response to subsequent stresses as exemplified by APG-1, a member of the HSP110 family. Furthermore, 32 degrees C induces expression of CIRP (cold-inducible RNA-binding protein), the first cold shock protein identified in mammalian cells, without recovery at 37 degrees C. Remniscent of HSP, CIRP is also expressed at 37 degrees C and developmentary regulated, possibly working as an RNA chaperone. Mammalian cells are metabolically active at 32 degrees C, and cells may survive and respond to stresses with different strategies from those at 37 degrees C. Cellular and molecular biology of mammalian cells at 32 degrees C is a new area expected to have considerable implications for medical sciences and possibly biotechnology.     

Determination of mitochondrial calcium content in hepatocytes by a rapid cellular-fractionation technique. Alpha-adrenergic agonists do not mobilize mitochondrial Ca2+.

A rapid cellular fractionation technique [the preceding paper, Shears & Kirk (1984) Biochem. J., 219, 375-382] was employed to separate a mitochondria-rich fraction from hepatocytes within seconds. Mitochondrial Ca was estimated to be no more than 41% of total cell Ca. At least half of the mitochondrial Ca was present in an energy-dependent pool; 20% of total cell Ca was accessible to EGTA within 10s. The alpha-adrenergic agonist phenylephrine stimulated glycogen phosphorylase activity by 100% within 0.5 min and induced a loss of 20% of total cell Ca after 10 min from the EGTA-inaccessible pool. However, between 0.5 and 10 min after the addition of phenylephrine to hepatocytes there was no significant change in the Ca content of the mitochondria-rich fraction. Hepatocytes that were preloaded with Ca2+ during 90 min incubation at 0-4 degrees C expelled this cation during 20 min incubation at 37 degrees C. After this time, phenylephrine failed to alter the Ca content of a mitochondria-rich fraction. It is concluded that alpha-adrenergic agonists do not mobilize Ca2+ from hepatocyte mitochondria.     

A novel context for the 'MutT' module, a guardian of cell integrity, in a diphosphoinositol polyphosphate phosphohydrolase.

Diphosphoinositol pentakisphosphate (PP-InsP5 or ''InsP7'') and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or ''InsP8'') are the most highly phosphorylated members of the inositol-based cell signaling family. We have purified a rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP) that cleaves a beta-phosphate from the diphosphate groups in PP-InsP5 (Km = 340 nM) and [PP]2-InsP4 (Km = 34 nM). Inositol hexakisphophate (InsP6) was not a substrate, but it inhibited metabolism of both [PP]2-InsP4 and PP-InsP5 (IC50 = 0.2 and 3 microM, respectively). Microsequencing of DIPP revealed a ''MutT'' domain, which in other contexts guards cellular integrity by dephosphorylating 8-oxo-dGTP, which causes AT to CG transversion mutations. The MutT domain also metabolizes some nucleoside phosphates that may play roles in signal transduction. The rat DIPP MutT domain is conserved in a novel recombinant human uterine DIPP. The nucleotide sequence of the human DIPP cDNA was aligned to chromosome 6; the candidate gene contains at least four exons. The dependence of DIPP''s catalytic activity upon its MutT domain was confirmed by mutagenesis of a conserved glutamate residue. DIPP''s low molecular size, Mg2+ dependency and catalytic preference for phosphoanhydride bonds are also features of other MutT-type proteins. Because overlapping substrate specificity is a feature of this class of proteins, our data provide new directions for future studies of higher inositol phosphates.     

Flocculation onset in Saccharomyces cerevisiae: effect of ethanol, heat and osmotic stress

AIMS: To examine the effect of different stress conditions on the onset of flocculation in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]; plasma membrane integrity was accessed using propidium iodide and the staining of the yeast cell wall was performed using calcofluor white M2R. Cells in exponential phase of growth were subjected to different stress conditions. The addition of 1%, 3% and 5% (v/v) ethanol, 1% and 3% (v/v) isopropanol or a brief heat shock (52 degrees C, 5 min), did not induce an early flocculation phenotype when compared with control cells. The addition of 10% (v/v) ethanol, a continuous mild heat-stress (37 degrees C) or an osmotic stress (0.5 or 1 mol l(-1) of NaCl) did not induce a flocculent phenotype. CONCLUSIONS: Flocculation seems not to be induced as a response to different chemical (ethanol and isopropanol) and physical (heat and osmotic) stress conditions. Conversely, osmotic and ethanol [10% (v/v)] stress, as well as a continuous mild heat shock (37 degrees C), have a negative impact on the phenotype expression of flocculation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to the elucidation of the control of yeast flocculation. This information might be useful to the brewing industry, as the time when the onset of flocculation occurs can determine the fermentation performance and the beer quality, as well as in other biotechnological industries where flocculation can be used as a cell separation process.     

Reptilian melanomacrophages function under conditions of hypothermia: observations on phagocytic behavior

Melanomacrophages (MMs) were removed from livers of turtles of three North American families and cultured. J774 mammalian macrophages were similarly cultured and the MMs were exposed to E. coli(fluor) at approximately 2, 7, 27, and 37 degrees C. At least one third of the MMs continued to function at the low temperatures where less than 2% of the mammalian cells incorporated bacteria deeply into the cytoplasm. In most instances, when the bacteria were not internalized deeply into the cytoplasm, they became stationary just inside, or within, the cell membrane. The MMs were significantly less efficient than the mammalian cells at 37 degrees C and significantly more efficient at 2 and 7 degrees C. In general, it appears that MMs are never as efficient as mammalian macrophages under the most ideal temperatures for the cell but they are capable of functioning at reasonable levels at temperature extremes. The observations are suggestive of a genetic mechanism functioning in the MMs that is rarely expressed in J774 cells under conditions of hypothermia. MMs in vitro and probably in vivo consume bacteria, fungi, attach to helminth eggs, and consume old erythrocytes.     

Two subpopulations of alpha 1-adrenergic receptors with high and low affinity for agonists: short-term exposure to agonists reduced the high-affinity sites

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including beta-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in alpha 1-adrenergic receptors induced by agonists. alpha 1-receptors were studied on DDT1 MF-2 smooth muscle cells (DDT1-MF-2 cells) by specific [3H]prazosin binding. In competition binding on membranes and on intact cells at 4 degrees C or at 37 degrees C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37 degrees C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [3H]prazosin binding inhibited by 20 microM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [3H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4 degrees C, but only 30% at 37 degrees C. On DDT1-MF-2 cells preequilibrated with [3H]prazosin at 4 degrees C, and then shifted to 37 degrees C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4 degrees C it is the native form of alpha 1-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37 degrees C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 microM norepinephrine. The nature of this low-affinity form was further investigated. On DDT1-MF-2 cells preincubated with the agonist and then extensively washed at 4 degrees C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [3H]prazosin at 4 degrees C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4 degrees C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37 degrees C may be due to the agonist-induced sequestration of alpha 1-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.     

Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of tetanus toxin to somatic neural hybrid cells with varying ganglioside composition

125I-labelled tetanus toxin interaction with several somatic hybrid cell lines was investigated. Binding of toxin is most effective in NCB-20, followed by NBr-10A, NG108-C15, and SB21-B1 cells. Specific binding of toxin to NCB-20 and SB21-B1 cells is 7- and 60-fold lower, respectively, in comparison to enriched rat cerebral neuron cultures. The NCB-20, NBr-10A, and NG108-C15 clones display a complex ganglioside pattern, including the presence of [N-acetyl-neuraminyl]-galactosyl-N-acetylgalactosaminyl[ N-acetylneuraminyl]-galactosylglucosyl-ceramide (GD1a) and two unidentified [14C]galactose-labelled lipid-soluble compounds, while the SB21-B1 is most abundant in [N-acetyl-neuraminyl]-galactosylglucosyl-ceramide (GM3) and N-acetyl-galactosaminyl-[N-acetyl-neuraminyl]-galactosylglucosyl-c eramide (GM2) gangliosides. None of the cells tested contain measurable levels of [14C]galactose-labelled or resorcinol-positive bands of galactosyl-N-acetyl-galactosaminyl-[ N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl-ceramide (GD1b) and [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[ N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl-ceramide (GT1b) gangliosides. After 2 h at 37 degrees C a near plateau of toxin association with NCB-20 cells is seen. Binding in low-ionic-strength medium is 1.35-fold higher at 37 degrees C than at 4 degrees C, but is reduced by 21 and 51% at 4 degrees C and 37 degrees C, respectively, in physiologic medium. Treatment of NCB-20 cells with neuraminidase causes a partial loss (29%) of toxin-binding sites. Binding to the hybrid cells is significantly different from that of cerebral cultures with respect to temperature, salt effect, and sensitivity to neuraminidase, suggesting perhaps a different class of receptors for the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester inhibits 13-cis-retinoic acid-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in cultured murine keratinocytes: a possible negative feedback via protein kinase C-activation.

The incubation of double-labelled [( 14C]-glycerol and [3H]-myoinositol) keratinocytes with 13-cis retinoic acid induced the transient and simultaneous release of [3H]-inositol trisphosphate ([3H]-InsP3) and [14C]-diacylglycerol ([14C]-DAG) indicating that a possible mode of action of this retinoid on murine keratinocytes may be at least in part the early transient release of the two putative messengers (InsP3 and DAG) from phosphatidylinositol-4,5 bisphosphate (PtdIns4, 5P2). In contrast, the preincubation of the keratinocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to incubation with 13-cis-RA suppressed the 13-cis-RA-induced release of [3H]-InsP3 and [14C]-DAG. The specificity of the TPA effect was established by the lack of effect of the biologically inactive 4 alpha-phorbol 12, 13-didecanoate. Furthermore, the incubation of the TPA-primed keratinocytes with 13-cis-RA caused a delayed and sustained accumulation of [14C]-DAG. An exploration of the source of this late release of [14C]-DAG revealed that this [14C]-DAG was released from non-inositol containing phospholipids, particularly, phosphatidylcholine. This latter DAG released in the TPA-primed cells correlated with the translocation of the cytoplasmic protein kinase C (PKC) activity to the membrane associated PKC activity. Taken together, these results suggest that alteration of PKC activity, presumably induced by DAG released from non-inositol phospholipids, may play a major role in the TPA-induced negative feedback inhibition of 13-cis RA-induced hydrolysis of keratinocyte PtdIns4, 5P2.     

Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulations in Cultured Adrenal Chromaffin Cells in Response to Bradykinin and Histamine

In previous studies it has been shown that both bradykinin and histamine increase the formation of 3H-labeled inositol phosphates in adrenal chromaffin cells prelabelled with [3H]inositol and that both these agonists stimulate release of catecholamines by a mechanism dependent on extracellular calcium. Here, we have used mass assays of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] to investigate changes in levels of these two candidates as second messengers in response to stimulation with bradykinin and histamine. Bradykinin increased the mass of Ins(1,3,4,5)P4 despite the failure in earlier studies with [3H]inositol-labelled cells to observe a bradykinin-mediated increase in content of [3H]InsP4. Bradykinin elicited a very rapid increase in level of Ins(1,4,5)P3, which was maximal at 5-10 s and then rapidly decreased to a small but sustained elevation at 2 min. The bradykinin-elicited Ins(1,3,4,5)P4 response increased to a maximum at 30-60 s and at 2 min was still elevated severalfold above basal levels. Histamine, which produced a larger overall total inositol phosphate response in [3H]inositol-loaded cells, produced significantly smaller Ins(1,4,5)P3 and Ins(1,3,4,5)P4 responses compared with bradykinin. The bradykinin stimulation of Ins(1,4,5)P3 accumulation was partially dependent on a high (1.8 mM) extracellular Ca2+ concentration, whereas the Ins(1,3,4,5)P4 response was almost completely lost when the extracellular Ca2+ concentration was reduced to 100 nM. Changes in the inositol polyphosphate second messengers are compared with the time course of bradykinin-stimulated increases in free intracellular Ca2+ concentrations and noradrenaline release.     

Increased flexibility enhances misincorporation: temperature effects on nucleotide incorporation opposite a bulky carcinogen-DNA adduct by a Y-family DNA polymerase

The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55 degrees C and results compared with an earlier study at 37 degrees C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55 degrees C compared with 37 degrees C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55 degrees C compared with 37 degrees C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the kcat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.     

Low-temperature pausing of cultivated mammalian cells

There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells.