Human HepG2 and Rat Fao Hepatic-Derived Cell Lines Show Different Responses to Ciprofibrate, a Peroxisome Proliferator: Analysis by Flow Cytometry

Peroxisome proliferators, and especially hypolipidemic drugs such as ciprofibrate, are known to be hepatocarcinogens in rodents, but their effect in humans is controversial. In an attempt to investigate the effects of ciprofibrate at a cellular level, the analysis of individual whole cells was performed by flow cytometry on samples from two hepatic-derived cell lines: the rat Fao cell line and the human HepG2 cell line. The increase of light scatter signals in rat Fao cells treated for 3 days with ciprofibrate at 250 μMwas related to modifications of intrinsic cellular parameters, such as size and cytoplasmic granularity. Conversely, no variations appeared in human HepG2-treated cells. Moreover, the study of the cell cycle distribution of asynchronously growing cells showed an increase in the percentage of proliferative cells in Fao-treated cells, but not in HepG2-treated cells. In order to give a simultaneous assessment of changes in cellular parameters and cell metabolism, these flow cytometric experiments were completed with the measurements of the palmitoyl–CoA oxidase activity, used as a marker of peroxisome proliferation. The cellular modifications in the rat Fao cell line were accompanied by a great increase in this enzymatic activity, whereas the human HepG2 cell line, which failed to exhibit changes of cytometric data, presented no, or weak, increase in this oxidase activity. The cellular modifications observed in the rat Fao cell line may be related to the well-known hepatocarcinogenicity of ciprofibrate in rodents, whereas the absence of response of HepG2 cells is in favor of the noncarcinogenicity of this drug in humans. This report validates another methodological approach for the investigation of the safety of peroxisome proliferators in humans.     

Insulin Receptor Sub-Types in a Human Lymphoid-Derived Cell Line (IM-9): Differential Regulation by Insulin,Dexamethasone and Monensin

Abstract

The cells of the human IM-9 lymphocyte-derived line contain a sub-population of insulin binding sites which differ from classical insulin binding sites in their higher binding affinity for insulin-like growth factor II (IGF-II) and insulin-like growth factor I (IGF-I). These atypical insulin binding sites are identified on IM-9 cells by [¹²⁵I]IGF-II binding.

To determine whether the atypical and classical insulin receptors of IM-9 cells were subject to different modes of in vivo regulation, we treated IM-9 cells with agents known to alter the surface expression of insulin receptors - insulin, dexamethasone and monensin. We then measured insulin and IGF-II binding to the surface of the washed cells.

Pretreatment of IM-9 cells with 1 μM insulin for 20 h at 37°C induced a 44–48% decrease in the number of high affinity insulin binding sites, but no change in the number of IGF-II binding sites. In contrast, the surface expression of both insulin and IGF-II binding sites (classical and atypical insulin receptors) increased 1.3 to 1.7-fold after treatment with dexamethasone (200 nM) and decreased 30 to 45% after monensin (1 μM). These results suggest that atypical and classical insulin receptors are differentially susceptible to down-regulation by insulin.     

Miropin,a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia,Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

All prokaryotic genes encoding putative serpins identified to date are found in environmental and commensal microorganisms, and only very few prokaryotic serpins have been investigated from a mechanistic standpoint. Herein, we characterized a novel serpin (miropin) from the human pathogen Tannerella forsythia, a bacterium implicated in initiation and progression of human periodontitis. In contrast to other serpins, miropin efficiently inhibited a broad range of proteases (neutrophil and pancreatic elastases, cathepsin G, subtilisin, and trypsin) with a stoichiometry of inhibition of around 3 and second-order association rate constants that ranged from 2.7 × 10⁴ (cathepsin G) to 7.1 × 10⁵ m⁻¹s⁻¹ (subtilisin). Inhibition was associated with the formation of complexes that were stable during SDS-PAGE. The unusually broad specificity of miropin for target proteases is achieved through different active sites within the reactive center loop upstream of the P1-P1′ site, which was predicted from an alignment of the primary structure of miropin with those of well studied human and prokaryotic serpins. Thus, miropin is unique among inhibitory serpins, and it has apparently evolved the ability to inhibit a multitude of proteases at the expense of a high stoichiometry of inhibition and a low association rate constant. These characteristics suggest that miropin arose as an adaptation to the highly proteolytic environment of subgingival plaque, which is exposed continually to an array of host proteases in the inflammatory exudate. In such an environment, miropin may function as an important virulence factor by protecting bacterium from the destructive activity of neutrophil serine proteases. Alternatively, it may act as a housekeeping protein that regulates the activity of endogenous T. forsythia serine proteases.     

Secondary Structural Studies of Bovine Caseins: Structure and Temperature Dependence of β-Casein Phosphopeptide (1-25) as Analyzed by Circular Dichroism, FTIR Spectroscopy, and Analytical Ultracentrifugation

The defining structural feature of all of the caseins is their common phosphorylation sequence. In milk, these phosphoserine residues combine with inorganic calcium and phosphate to form colloidal complexes. In addition, nutritional benefits have been ascribed to the phosphopeptides from casein. To obtain a molecular basis for the functional, chemical, and biochemical properties of these casein peptides, the secondary structure of the phosphopeptide of bovine β-casein (1–25) was reexamined using Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. Both methods predict secondary structures for the peptide which include polyproline II elements as well as β-extended sheet and turn-like elements. These structural elements were highly stable from 5° to 70°C. Reexamination of previously published ¹H NMR data using chemical shift indices suggests structures in accord with the CD and FTIR data. Dephosphorylation showed little or no secondary structural changes, as monitored by CD and FTIR, but the modified peptide demonstrated pronounced self-association. The polymers formed were not highly temperature sensitive, but were pressure sensitive as judged by analytical ultracentrifugation at selected rotor speeds. Molecular dynamics (MD) simulations demonstrated relatively large volume changes for the dephosphorylated peptide, in accord with the pressure dependent aggregation observed in the analytical ultracentrifuge data. In contrast the native peptide in MD remained relatively rigid. The physical properties of the peptide suggest how phosphorylation can alter its biochemical and physiological properties.     

Whole Genomic Sequence and Replication Kinetics of a New Enterovirus C96 Isolated from Guangdong,China with a Different Cell Tropism

Enterovirus 96 (EV-C96) is a newly described serotype within the enterovirus C (EV-C) species, and its biological and pathological characters are largely unknown. In this study, we sequenced the whole genome of a novel EV-C96 strain that was isolated in 2011 from a patient with acute flaccid paralysis (AFP) in Guangdong province, China and characterized the properties of its infection. Sequence analysis revealed the close relationship between the EV-C96 strains isolated from the Guangdong and Shandong provinces of China, and suggested that recombination events occurred both between these EV-C96 strains and with other EV-C viruses. Moreover, the virus replication kinetics showed EV-C96 Guangdong strain replicated at a high rate in RD cells and presented a different cell tropism to other strains isolated from Shandong recently. These findings gave further insight into the evolutionary processes and extensive biodiversity of EV-C96.     

Lysis and Lysogenization of Groups A, C, and G Streptococci by a Transducing Bacteriophage Induced from a Group G Streptococcus

A temperate bacteriophage, designated GT-234, was isolated from a group G Streptococcus after ultraviolet irradiation. After several single-plaque passages in a group G indicator strain, this phage formed plaques in 3 of 14 group A strains, in 3 of 15 group C strains, and in 4 of 13 group G strains-but not in some representatives of several other serogroups. After propagation in each of the sensitive strains, the progeny from each was shown to be the same phage by (i) adsorption and plaque formation in each of the other groups, (ii) lysogenization in each of the other groups, (iii) high titers on infection of each serogroup, regardless of the group of propagating strain, and (iv) neutralization of infection in each of the other groups by antiserum against the phage propagated in group G. Phage GT-234 is serologically related to virulent group A phage A25, from which it is morphologically indistinguishable. Like A25, it is a transducing phage. Other studies showed that A25, as well as a group A temperate transducing phage (AT-298), could also infect strains of group C and G. These results indicate a need for reassessment of group specificity and phage receptors among streptococci of groups A, C, and G and raise possibilities for intergroup transduction.     

Induction of Chlorosis,ROS Generation and Cell Death by a Toxin Isolated from Pyricularia oryzae

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.     

Germination of Bacillus cereus Spores Is Induced by Germinants from Differentiated Caco-2 Cells, a Human Cell Line Mimicking the Epithelial Cells of the Small Intestine

Spores of 11 enterotoxigenic strains of Bacillus cereus isolated from foods and humans adhered with similar efficiencies to Caco-2 cells, whereas subsequent germination triggering was observed with only 8 of these strains. Notably, Hep-2 cells did not trigger germination, while spores of all strains displayed similar germination efficiencies in brain heart infusion broth.     

Lipid a from lipopolysaccharide recognition: Structure,dynamics and cooperativity by molecular dynamics simulations

Molecular dynamics simulations of Lipid A and its natural precursor Lipid IVA from E.coli have been carried out free in solution, bound to the myeliod differentiation protein 2 (MD2) and in the complex of MD2 with the toll like receptor 4 (TLR4). In addition, simulations of the ligand free MD2 and MD2‐TLR4 complex were performed. A structural and energetic characterization of the bound and unbound states of Lipid A/IVA was generated. As the crystal structures depict, the main driving force for MD2‐Lipid A/IVA are the hydrophobic interactions between the aliphatic tails and the MD2 cavity. The charged phosphate groups do strongly interact with positively charged residues, located at the surface of MD2. However, they are not essential for keeping the lipids in the cavity, indicating a more prominent role in binding recognition and ionic interactions with TLR4 at the MD2/TLR4 interface. Interestingly, in the absence of any ligand MD2 rapidly closes, blocking the binding cavity. The presence of TLR4, though changing the dynamics, was not able to impede the aforementioned closing event. We hypothesize that fluctuations of the H1 region are essential for this phenomenon, and it is plausible that an equilibrium between the open and closed states exists, although the lengths of our simulations are not sufficient to encompass the reversible process. The MD2/Lipid A‐TLR4 complex simulations show that the presence of the ligand energetically stabilizes the complex relative to the ligand‐free structures, indicating cooperativity in the binding process. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Activation of c-Neu Tyrosine Kinase by o,p′-DDT and β-HCH in Cell-Free and Intact Cell Preparations from MCF-7 Human Breast Cancer Cells

It has been suggested that there is a positive correlation between increased incidence of breast cancer and the presence of organochlorine residues such as DDT and HCH in breast tissues in the United States. To study possible biochemical links between these two parameters, we have examined the effect of o,p′-DDT, the most estrogenic congener of the DDT family of chemicals and β-HCH on protein phosphorylation activities in MCF-7, a line derived from human breast cancer cells. Both of these organochlorine chemicals were found to be potent activators of protein kinases. Among kinases activated, protein tyrosine kinases (PTK) appear to be most affected as judged by the antagonistic action of genistein, a class-specific PTK inhibitor. Moreover, these organochlorines were found to activate PTK even under cell-free conditions, indicating that they are likely to interact directly with the target protein tyrosine kinase. As a result of immunoprecipitation with specific antibodies, and testing on the action of these organochlorines, we could show that the major kinase activated by o,p′-DDT is c-Neu (= c-erbB2 product protein). The concentrations of these organochlorines required to activate c-Neu were extremely low (0.1–1 nM range), whereas an inactive analog p,p′-DDT showed no stimulatory property even at 100 nM. Such an action of these organochlorine compounds were not antagonized by the presence of 1 μM tamoxifen, indicating that it is not mediated through the estrogen receptor. In addition, their c-Neu activating actions were specifically antagonized by a c-Neu antibody known to interact with the extracellular domain of c-Neu only without affecting the EGF receptor. Moreover, these chemicals did not cause downregulation of the EGF receptor during the 72 hour test period. Together these data indicate that the action of these chemicals on c-Neu kinase is very specific. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 83–92, 1998     

The Southern Churchill, Petrocephalus Wesselsi, a New Species of Mormyrid from South Africa defined by Electric Organ Discharges, Genetics, and Morphology

East African and south African churchills (Petrocephalus, Mormyridae) were synonymised in 1959 to become members of a single species of subcontinental, southern African distribution, Petrocephalus catostoma (Günther, 1866). By comparison with the type material for P. catostoma from the Ruvuma River and P. stuhlmanni from the Ruvu River, both of East African origin, we confirm the South African form of churchill to represent a new species, P. wesselsi, ranging from the northern Limpopo and Incomati systems south to the Pongola River (Natal) as its southern limit. We also compared churchills from the Sabie River (25° S, South Africa, Incomati system) with churchills from the Upper Zambezi River (17° S, Namibia), using electric organ discharges (EODs) and morphology. The duration of an EOD pulse of the South African form (N = 39; 943.2±S.E. 18.82µs) is, on average, more than twice that of the Upper Zambezi form (N = 37; 436.6±15.1µs), and the amplitude of the second head-positive phase (P2 phase relative to P1 = 1) significantly weaker (0.133 ± 0.0005 vs. 0.472 ± 0.002 for Upper Zambezi males, 0.363 ± 0.03 for Upper Zambezi females). In contrast to the Upper Zambezi form, the EOD of the South African form exhibits no difference between the sexes. Fish from the two origins differ significantly in 11 out of 14 anatomical characters studied, confirming molecular genetic differentiation on the species level.     

Microbial Biomass, Activity, and Community Structure of Water and Particulates Retrieved by Backflow from a Waterflood Injection Well

Oil field injection water was allowed to back flow from two wells at the Packard drill site in Los Angeles, Calif., and was sampled at various times to obtain information about the biomass, potential activity, and community structure of the microbiota in the reservoir formation and in the injection water. Biomass was greatest in water samples that came from the zone near the injection site and dropped off sharply in subsequent samples, which were assumed to come from zones farther away from the well. Samples obtained from near the well also had visible exopolysaccharide blankets, as seen in scanning electron microscopic preparations. In one of the wells that was sampled, rates of glucose or acetate incorporation into microbial lipids correlated with biomass; but in the other well, activities correlated with the sampling time (volume of water that back flowed). Transmission electron micrographs showed a diverse, gram-negative bacterial population in a variety of physiological states. The analysis of the phospholipid ester-linked fatty acid profiles of the samples revealed consistently large proportions of 18:1ω7c fatty acids, indicating the presence of many anaerobes, facultative organisms, or both. Proportions of cyclopropyl fatty acids and ratios of trans/cis monoenoic compounds increased with the volume of water that back flowed (analogous with the distance into the formation), while the ratio of unsaturated/saturated compounds decreased, possibly indicating higher levels of stress or starvation in the microbial communities farthest from the injection well. Greater than 90% of the total biomass was trapped on glass fiber filters, indicating that the microbiota were largely attached to particles or were clumped. These sampling techniques and analytical methods may prove useful in monitoring for problems with microbes (e.g., plugging) in waterflood operations and in the preparation of water injection wells for enhanced oil recovery by the use of microbes.     

Phosphorylation of the Three Rb Protein Family Members Is a Common Step of the cAMP-, the Growth Factor, and the Phorbol Ester-Mitogenic Cascades but Is Not Necessary for the Hypertrophy Induced by Insulin

Thyrotropin (TSH) through the cAMP cascade and in the presence of insulin induces the proliferation of dog thyroid cells. In this work, it is shown that TSH via cAMP causes the phosphorylation of the three members of the pRb family, pRb, p107, and p130, with the same kinetics as those observed when these cells are stimulated by mitogens acting through a tyrosine kinase receptor or through activation of kinase C. It is the first described point of convergence of cAMP-dependent and -independent mitogenic pathways in dog thyrocytes and suggests that the phosphorylation of the three proteins may be involved in the initiation of DNA synthesis in these cells. We also show that insulin, which induces hypertrophy and is permissive for the TSH mitogenic action, does not provoke the phosphorylation of any pRb family member, suggesting that none of these phosphorylations is required for this effect.     

Endogenous cis,trans-Abscisic Acid and Pea Seed Development: Evidence for a Role in Seed Growth from Changes Induced by Temperature

Measurements were made using GC/MS SIM¹ of the effects of temperatureon cis,trans-ABA levels in developing ovules and embryos oftwo pea genotypes contrasted in seed size. These effects werethen related to differences in the growth of the pods, seeds,embryos, and testae. In both genotypes high temperatures hastenedthe onset and rate of logarithmic and then linear growth, greatlyshortening the duration of pod and seed development but withoutgreatly altering seed size. Cis,trans-ABA was most concentratedxin the ovules immediately after fertilization. It also accumulatedin the embryo, more rapidly in the larger-seeded line, duringseed maturation. The stage when accumulation in the embryo beganwas the same irrespective of temperature. Accumulation ceasedwhen the pods started to desiccate. The effects of differentconstant temperatures on the maximum levels of embryo cis,trans-ABAwere relatively small and confounded in one genotype by variationin ovule abortion and in the other by differences in the stagewhen cis,trans-ABA accumulation ceased. However, when plantswere transferred from 13 °C to 29 °C at two differentstages during seed maturation, further seed growth was greatlyinhibited coincident with a substantial increase in embryo cis-trans-ABA.The results suggested a role for cis,trans-ABA in the controlof cotyledon enlargement during the linear phase of seed growth.     

Characterization of the Tumorlike (T) Antigen Induced by Type 12 Adenovirus : I. Purification of the Antigen from Infected KB Cells and a Hamster Tumor Cell Line

The T antigen induced by type 12 adenovirus was purified from KB cells infected in the presence of 10(-6)m 5-fluoro-2-deoxyuridine to inhibit synthesis of viral capsid antigens. The antigen was purified approximately 200-fold, and the purified product contained only negligible amounts of host-cell contaminants, as judged by the residual radioactivity from (14)C-labeled uninfected cells which had been added to infected cells at the initiation of the purification. Immunoelectrophoresis indicated that the purified T-antigen preparation contained a single antigenic species. The T antigen from a hamster cell line (HT-1) derived from a type 12 adenovirus-induced tumor was purified by the same procedure. The T antigens from the two different sources were shown to be immunologically similar by use of a rabbit antiserum prepared against the purified T antigen from infected KB cells and sera from hamsters bearing tumors induced by type 12 adenovirus.