A subfamily of P-type ATPases with aminophospholipid transporting activity.

The appearance of phosphatidylserine on the surface of animal cells triggers phagocytosis and blood coagulation. Normally, phosphatidylserine is confined to the inner leaflet of the plasma membrane by an aminophospholipid translocase, which has now been cloned and sequenced. The bovine enzyme is a member of a previously unrecognized subfamily of P-type adenosine triphosphatases (ATPases) that may have diverged from the primordial enzyme before the separation of the known families of ion-translocating ATPases. Studies in Saccharomyces cerevisiae suggest that aminophospholipid translocation is a general function of members of this family.

An assessment of the carcinogenic potential of ezetimibe using nonclinical data in a weight-of-evidence approach.

Ezetimibe blocks intestinal absorption of sterols via interaction with the Neimann-Pick C1-Like 1 (NPC1L1) transporter and is approved for use in the treatment of primary hyperlipidemia (heterozygous familial and non-familial), homozygous familial hypercholesterolemia, and homozygous sitosterolemia. A recently completed randomized clinical trial [simvastatin and ezetimibe in aortic stenosis (SEAS)] testing the effectiveness of Vytorin (a combination of simvastatin and ezetimibe) in patients with aortic stenosis reported an unexpected safety finding: an increase in overall cancer incidence and cancer-associated mortality (all types) in the treated groups relative to the placebo control. A subsequent meta-analysis utilizing a much larger database from two ongoing clinical trials indicated that the observed findings in the SEAS trial were likely due to chance and not a true drug-induced effect. Nonetheless, it has been suggested by various commentators on the SEAS trial that ezetimibe may be carcinogenic. The extensive nonclinical database for ezetimibe was used to test the hypothesis that ezetimibe may be a direct or indirect carcinogen. Using two different in silico approaches, ezetimibe showed no structural alerts for genetic toxicity or carcinogenicity. Ezetimibe was not genotoxic in two reverse mutation assays, one in vitro clastogenicity assay, and two mouse micronucleus assays. No evidence of proliferative lesions was observed in three species in studies of 1-12 months in duration. Ezetimibe was not carcinogenic in standard 2-year bioassays in mice and rats. Additionally, in these 2-year bioassays, no drug-related non-neoplastic lesions were noted. The absence of drug-induced non-neoplastic or proliferative lesions in these studies indicates that ezetimibe treatment was not associated with findings characteristic of carcinogens (i.e., DNA reactivity or cell proliferation) Administration of pharmacologic doses of ezetimibe to mice did not alter hepatic expression patterns of genes associated with apoptosis, cell proliferation, or epithelial-mesenchymal transition. No evidence of drug-induced tumors was observed in mice in which the molecular target of ezetimibe (NPC 1L1) was knocked out over the life span of the animal. In conclusion, the nonclinical data do not support the proposed hypothesis based on the single observation from the SEAS trial and, rather, support the conclusion that ezetimibe does not represent a carcinogenic hazard to humans using this drug in a therapeutic setting.

Imaging macrophages in trehalose with SIMS.

Phagocytosis is a major component of the animal immune system where apoptotic cellular material, metabolites, and waste are safely processed. Further, efficient phagocytosis by macrophages is key to maintaining healthy vascular systems and preventing atherosclerosis. Single-cell images of macrophage phagocytosis of red blood cells, RBCs, and polystyrene microspheres have been chemically mapped with TOF-SIMS. We demonstrate here cholesterol and phosphocholine localizations as relative to time and activity.

Differential expression of putative transbilayer amphipath transporters.

The aminophospholipid translocase transports phosphatidylserine and phosphatidylethanolamine from one side of a bilayer to another. Cloning of the gene encoding the enzyme identified a new subfamily of P-type ATPases, proposed to be amphipath transporters. As reported here, mammals express as many as 17 different genes from this subfamily. Phylogenetic analysis reveals the genes to be grouped into several distinct classes and subclasses. To gain information on the functions represented by these groups, Northern analysis and in situ hybridization were used to examine the pattern of expression of a panel of subfamily members in the mouse. The genes are differentially expressed in the respiratory, digestive, and urogenital systems, endocrine organs, the eye, teeth, and thymus. With one exception, all of the genes are highly expressed in the central nervous system (CNS); however, the pattern of expression within the CNS differs substantially from gene to gene. These results suggest that the genes are expressed in a tissue-specific manner, are not simply redundant, and may represent isoforms that transport a variety of different amphipaths.

Axonal degeneration and axonal caliber alterations following combined beta,beta'-iminodipropionitrile (IDPN) and acrylamide administration.

A new model of neurofilamentous axonal abnormality is described which employs combined administration of beta,beta'-iminodipropionitrile (IDPN) and acrylamide (AC). The model was developed to test the hypothesis that IDPN-induced swelling increases the vulnerability of the distal axon to a second neurotoxic chemical insult. Rats were given a single intraperitoneal (IP) injection of IDPN (1.5 g/kg) one week before receiving a single injection of AC (75 mg/kg, IP). Axonal degeneration was observed at multiple levels along the sciatic nerve at two weeks (with reference to IDPN administration), and was not progressive up to five weeks. Quantitation of degenerating fibers demonstrated that the extent of degeneration increased distally along the sciatic nerve. Single administration of either IDPN or AC did not produce degeneration. Thus, IDPN-induced neurofilamentous swellings alter the susceptibility of the axon to AC neurotoxicity. Two variations of this model were also studied. First, rats given five daily injections of AC (30 mg/kg, IP) beginning one week following IDPN administration developed accumulations of fast axonally transported materials in IDPN-induced microtubule channels. Second, rats given chronic injections of AC (30 mg/kg, IP, five days/week, for four weeks), to reduce the delivery of neurofilaments to the proximal axon, developed less prominent axonal enlargements when challenged with IDPN. Thus, axonal atrophy can mask the development of neurofilamentous axonal swellings.

Reversible and irreversible oxidant injury to PC12 cells by hydrogen peroxide.

A simple and reproducible model to identify biochemical changes associated with the transition from reversible to irreversible oxidant injury and cell death was established using rat pheochromocytoma PC12 cells. Cells were subjected to a transient oxidative stress induced by exposure to hydrogen peroxide (H2O2). Reversible loss of high-energy phosphates, induced by exposing cells to 0.2 mM H2O2, was preceded by transient increases in cytosolic calcium with no loss of plasma membrane integrity, as indexed by release of cytosolic enzymes. In contrast, permanent loss of high-energy phosphates, induced by treating cells with 0.5 mH H2O2, was associated with sustained rises in cytosolic-free calcium and increased oxidation of pyruvate and palmitate, two mitochondrial substrates. Initial production of pyruvate and lactate was inhibited by exposure to 0.5 mM H2O2 but returned to values comparable to control values at one hour after treatment with H2O2. Compromise of the plasma membrane was a late event, occurring between 1 and 2 hours after exposure to 0.5 mM H2O2. Collectively, these data indicate that irreversible loss of high-energy phosphates and cell death caused by oxidative stress is more closely associated with altered mitochondrial function than with impaired glycolysis.

Characterization of protein kinases in mitotic and meiotic cell extracts.

A number of protein kinases have been separated and identified in extracts from mitotic and interphase culture cells and from mature and immature amphibian oocytes using nondenaturing polyacrylamide gel electrophoresis followed by in situ phosphorylation assays. Certain of these protein kinase activities appear to correlate with the biological activity of extracts, assayed by their ability to induce meiotic maturation following injection into Xenopus oocytes. These results are consistent with the notion that protein phosphorylation/dephosphorylation may be integral to the mechanisms of both nuclear membrane breakdown and chromosome condensation, events common and distinctive to mitosis and meiosis.

The molecular response to reductive stress in LLC-PK1 renal epithelial cells: coordinate transcriptional regulation of gadd153 and grp78 genes by thiols.

Organic thiols are toxic to eukaryotic cells. Treatment of cells with thiols activates expression of grp78, but it is not known if, like other forms of stress, there is a battery of stress response genes that are induced by thiols. In LLC-PK1 renal epithelial cells, mRNAs for both grp78 and gadd153 were induced by thiols with similar time, concentration and structure-activity dependence. Dithiothreitol (DTT) was the most potent reductant and inducer of gene expression among the thiols tested. Nuclear run-on assays demonstrated that DTT activated both grp78 and gadd153 genes transcriptionally. A hamster gadd153 promoter construct which contains enhancer elements necessary for gadd153 activation was stably integrated into the LLC-PK1 cell genome and was activated by DTT. Although auto-oxidation of thiols can generate active oxygen species, transcriptional activation of the gadd153 promoter was not due to formation of hydrogen peroxide or superoxide since neither catalase nor superoxide dismutase prevented activation of the gadd153 promoter by DTT. The concentration dependence for activation of the gadd153 promoter correlated with inhibition of dome formation and protein synthesis, two toxic effects of DTT in LLC-PK1 cells. Thus, both grp78 and gadd153 are members of a gene battery which is responsive to reductive stress. There appears to be considerable, but not complete, overlap between the upstream signaling pathways for activation of both genes.

Linking gene expression to mechanisms of toxicity.

Activation of gene expression is one of the earliest cellular responses to toxicity. However, our understanding of the biological and biochemical signals that activate these toxicant-responsive genes as well as the consequences of gene activation to survival of the organism remains sketchy. In this article, strategies that can be used to link changes in gene expression to biochemical mechanisms of toxicity are addressed using the hsp70 and grp78 genes as examples. The data indicate that activation of hsp70 is linked to changes in thiol-disulfide redox perturbations while grp78 activation may be caused by loss of calcium from the endoplasmic reticulum. Each gene is part of a discrete feedback regulated signaling pathway designed to protect cells against the toxic signals that activate gene expression.

Biological availability of nickel arsenides: toxic effects of particulate Ni5As2.

To determine if fugitive nickel arsenides from an oil shale retort could pose a threat to living organisms, we studied the effects of particulate Ni5As2 on cultured mammalian cells. Culture growth rate was greatly reduced at the lowest suspension concentration tested (10 microM), even though much of the Ni5As2 powder remained insoluble. FCM analysis indicated Ni5As2 arrested cell-cycle traverse in G1 and G2. Cell survival studies indicated cells could overcome this toxicity if exposure levels were low (less than or equal to 25 microM in suspension) and restricted to less than or equal to 24 h. At higher powder levels, survival was greatly reduced. Transmission electron microscopy (TEM) demonstrated that cells exposed to less than or equal to 100 microM powder did not phagocytize the Ni5As2 particles. At higher concentrations, TEM X-ray microanalysis demonstrated that As was preferentially extracted from the Ni5As2 particle surface and free Ni was deposited inside the cell. These observations suggest that the toxicity of Ni5As2 particles may be caused by some soluble product of Ni5As2.

Antibody caging of a nuclear-targeting signal.

We have developed a technique for reversibly masking a peptide-targeting signal. A fluoresceinated derivative of the simian virus 40 large tumor antigen nuclear-targeting signal was synthesized and cross-linked to bovine serum albumin. The conjugated protein was efficiently transported into rat liver nuclei unless the peptide-targeting signal was sterically hindered by binding of an anti-fluorescein antibody. Addition of free 5-aminofluorescein competed for antibody binding and rapidly restored nuclear accumulation of the derivatized bovine serum albumin. General use of hapten derivatization and anti-hapten antibodies for caging portions of macromolecular surfaces can be extended to a variety of proteins, including antibodies themselves.

C-banding of Peromyscus constitutive heterochromatin persists following histone hyperacetylation.

Approx. 35% of the DNA of cultured cells from the cactus mouse, Peromuscus eremicus, is contained in highly condensed constitutive heterochromatin which can be visualized in metaphase chromosomes stained by the C-band technique. Previous studies have shown this constitutive heterochromatin to contain a large proportion of underacetylated, arginine-rich histones, the majority of which can be hyperacetylated when cells are treated with butyrate. In order to determine whether this simulation of the acetylated state of euchromatin alters the cytological properties of constitutive heterochromatin as well, chromosomes from butyrate-treated cells have been examined. Because of the paucity of cells in butyrate-treated cultures, prematurely condensed chromosomes (PCCs) were produced from butyrate-treated cells by fusion with mitotic cells. In these PCCs, both the highly condensed nature and the ability to C-band were preserved in the hyperacetylated constitutive heterochromatin, suggesting that the subset of arginine-rich histones which is refractory to acetylation in the presence of butyrate may be responsible for the maintenance of the heterochromatic state. In addition, PCC analyses indicated that butyrate arrests Peromyscus cells in both the G1 and G2 phases of the cell cycle and confirmed the late-replicating pattern of constitutive heterochromatin.

Cytoplasmic to nuclear translocation of RNA polymerase I is required for lipopolysaccharide-induced nucleolar RNA synthesis and subsequent DNA synthesis in murine B lymphocytes.

The synthesis of ribosomal RNA (rRNA) in murine B lymphocytes is markedly elevated in response to mitogens such as lipopolysaccharide (LPS). First, to investigate the mechanism involved, antibodies directed against RNA polymerase I, the enzyme responsible for transcription of ribosomal genes, were introduced into the cytoplasm of lymphocytes via red cell-mediated microinjection and the ability of cells to synthesize RNA was examined. Simultaneous immunofluorescence/autoradiography revealed that 7% or less of the cells injected with specific antibodies prior to stimulation were actively synthesizing rRNA 15 or 40 h following LPS addition. In contrast 19% and 27% of cells injected with control IgG were active at these times. Non-ribosomal RNA synthesis was unaffected by the presence of anti-RNA polymerase I antibodies. Since antibodies injected into the cytoplasm were limited to that compartment, these data suggest that rRNA synthesis induced by LPS requires translocation of cytoplasmic RNA polymerase I into the nucleus. Second, to test whether synthesis of rRNA is required for entry into S phase, the effect of anti-RNA polymerase I antibodies on DNA synthesis in response to LPS was evaluated. Only 7% of cells containing anti-RNA polymerase I antibodies had initiated DNA synthesis 40 h after LPS addition whereas 25% of cells containing control IgG were actively synthesizing DNA at that time. These results suggest that nuclear accumulation of RNA polymerase I and increased rRNA synthesis are required for LPS-induced DNA synthesis in B lymphocytes.

Reduction of trans-4,5-dihydroxy-1,2-dithiane by cellular oxidoreductases activates gadd153/chop and grp78 transcription and induces cellular tolerance in kidney epithelial cells.

trans-4,5-Dihydroxy-1,2-dithiane, the intramolecular disulfide form of dithiothreitol (DTTox) transcriptionally activates the stress-responsive genes gadd153(chop) and grp78. Herein, we used a renal epithelial cell line, LLC-PK1, to investigate the mechanism(s) whereby DTTox activates a molecular stress response. DTTox activated both grp78 and gadd153 transcriptionally, but gadd153 mRNA stability also increased suggesting that both transcriptional and posttranscriptional mechanisms are involved. DTTox did not activate hsp70 transcription indicating that a heat shock response was not induced. Structure-activity studies showed that DTTox analogues lacking the intramolecular disulfide were inactive. Furthermore, the ring-open intermolecular disulfide form of DTTox, 2-hydroxyethyl disulfide, was only a weak inducer of grp78 and gadd153 but was a strong inducer of hsp70 mRNA and a potent oxidant that lowered the NADPH/NADP+ ratio and depleted reduced glutathione (GSH). DTTox had little effect on the overall GSH and NADPH levels; thus cells were not undergoing oxidative stress; however, the NADPH/NADP+ ratio decreased slightly indicating that reducing equivalents were consumed. LLC-PK1 cells reduced DTTox to DTT, and the kinetics as well as the concentration dependence for reduction correlated with induction of both grp78 and gadd153 mRNA. Prior treatment with DTTox rendered cells tolerant to the potent nephrotoxicant S-(1,1,2, 2-tetrafluoroethyl)-L-cysteine. Bacitracin, an inhibitor of plasma membrane oxidoreductases, blocked DTTox reduction and gene activation as well as DTTox-induced tolerance. Thus, activation of stress genes and induction of cellular tolerance by DTTox is mediated by a novel mechanism involving cellular oxidoreductases.

Cellular compartmentalization of protein kinase activity during the cell cycle.

The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.

Multiple members of a third subfamily of P-type ATPases identified by genomic sequences and ESTs.

The Saccharomyces cerevisiae genome contains five P-type ATPases divergent from both of the well-known subfamilies of these membrane ion transporters. This newly recognized third subfamily can be further divided into four classes of genes with nearly equal relatedness to each other. Genes of this new subfamily are also present and expressed in multicellular organisms such as Caenorhabditis elegans and mammals; some, but not all, can be assigned to the classes identified in yeast. Different classes of genes and different genes within a class are expressed differentially in tissues of the mouse. The recently cloned gene for the mammalian aminophospholipid translocase belongs to this new subfamily, suggesting that other subfamily members may transport other lipids or lipid-like molecules from one leaflet of the membrane bilayer to the other.

Identification of mitosis-specific p65 dimer as a component of human M phase-promoting factor.

Antisera raised against two mitosis-specific protein kinases from human cells recognized a single 65-kDa polypeptide (p65) that is present in similar amounts in interphase and mitotic cell extracts. Immunoblot analysis of reduced and unreduced extracts revealed that p65 exists as a 65-kDa monomer during interphase but forms a 130-kDa disulfide-linked homodimer during mitosis. Several different antibodies recognizing the p34cdc2 protein kinase and cyclin B components of M phase-promoting factor (MPF) coprecipitated p65 from mitotic but not from interphase extracts. In addition, an anti-p65 immunoaffinity column substantially depleted mitotic extracts of histon H1 kinase activity assayed under conditions diagnostic for MPF. These results suggest that active human MPF may be a complex of p34cdc2, cyclin B, and dimeric p65. A sulfhydryl cycle, proposed in the earlier literature on the biochemistry of mitosis, might underlie the dimerization of p65 and formation of active MPF.

Injected mitotic extracts induce condensation of interphase chromatin.

Although extracts from mitotic cells have been shown to induce chromosome condensation when injected into amphibian oocytes, they have not as yet been shown to induce this response in somatic interphase cells. In the experiments reported here, when mitotic extracts were injected into syncytial frog embryos, whose somatic nuclei were arrested in interphase, chromosome condensation was observed. The inability of interphase extracts, injected at similar concentrations, to induce this event demonstrates the cell cycle-specific accumulation of the factors responsible.

A widely distributed putative mammalian transcriptional regulator containing multiple paired amphipathic helices, with similarity to yeast SIN3.

The mammalian Sin3 gene (mSin3) encodes four paired amphipathic helix (PAH) motifs, three of which and an extended region beyond PAH3 share between 59 and 70% sequence similarity with the yeast transcriptional regulator, SIN3. However, mSin3/SIN3 fusion proteins were not able to substitute for the yeast molecule in complementation assays. Transcripts encoding this putative transcriptional regulator, which maps to human chromosome 15q24, were detected in multiple mouse tissues, with highest levels seen in testis, lung, and thymus. Its wide tissue distribution suggests that mSin3, like yeast SIN3, may regulate the transcription of multiple genes.

Cloning, expression, and chromosomal mapping of a human ATPase II gene, member of the third subfamily of P-type ATPases and orthologous to the presumed bovine and murine aminophospholipid translocase.

Recently, a P-type ATPase was cloned from bovine chromaffin granules (b-ATPase II) and a mouse teratocarcinoma cell line (m-ATPase II) and was shown to be homologous to the Saccharomyces cerevisiae DRS2 gene, the inactivation of which resulted in defective transport of phosphatidylserine. Here, we report the cloning from a human skeletal muscle cDNA library of a human ATPase II (h-ATPase II), orthologous to the presumed bovine and mouse aminophospholipid translocase (95.3 and 95.9% amino acid identity, respectively). Compared with the bovine and mouse counterparts, the cloned h-ATPase II polypeptide exhibits a similar membrane topology, but contains 15 additional amino acids (1163 vs 1148) located in the second intracytoplasmic loop, near the DKTGTLT-phosphorylation site. However, RT-PCR analysis performed with RNA from different human tissues and cell lines revealed that the coding sequence for these 15 residues is sometimes present and sometimes absent, most likely as a result of a tissue-specific alternative splicing event. The h-ATPase II gene, which was mapped to chromosome 4p14-p12, is expressed as a 9.5-kb RNA species in a large variety of tissues, but was not detected in liver, testis, and placenta, nor in the erythroleukemic cell line K562.