Pharmacotoxicology of clinically-relevant concentrations of obeticholic acid in an organotypic human hepatocyte system.

Nonalcoholic steatohepatitis (NASH) is an emerging health crisis with no approved therapies. Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, shows promise in NASH trials. However, the precise mechanisms mediating OCA effects and impact on cholesterol metabolism are not fully understood. We explored the pharmaco-toxicological effects of OCA on patho-physiological pathways in hepatocytes using a previously described perfused organotypic liver system that allows culture in near-physiological insulin/glucose milieus, and exhibits drug responses at clinically-relevant concentrations. Primary hepatocytes experienced 48-hour exposure to OCA at concentrations approximating therapeutic (0.5µM) and supratherapeutic (10µM) levels. Global transcriptomics by RNAseq was complimented by cellular viability (MTT), CYP activity assays, and secreted FGF19 levels in the media. Dose-dependent, transcriptional effects suggested suppression of bile acid synthesis (↓CYP7A1, ↓CYP27A1) and increased bile efflux (↑ABCB4, ↑ABCB11, ↑OSTA, ↑OSTB). Pleiotropic effects included suppression of TGFß and IL-6 signaling pathways, and signatures suggestive of HDL suppression (↑SCARB1, ↓ApoAI, ↓LCAT) and LDL elevation (↑ApoB, ↓CYP7A1). OCA exhibited direct FXR-mediated effects with increased FGF19 secretion. Transcriptomics revealed regulation of metabolic, anti-inflammatory, and anti-fibrotic pathways beneficial in NASH, and predicted cholesterol profiles consistent with clinical findings. Follow-up studies under lipotoxic/inflammatory conditions would corroborate these effects in a disease-relevant environment.

A2A adenosine receptor (AR) activation inhibits pro-inflammatory cytokine production by human CD4+ helper T cells and regulates Helicobacter-induced gastritis and bacterial persistence.

Helicobacter pylori causes a lifelong infection and provides a model of bacterial adaptation and persistent colonization. Adenosine is an anti-inflammatory mediator that limits tissue damage during inflammation. We studied the role of adenosine in the T-cell-mediated regulation of gastritis and bacterial persistence. After 4 h of activation, human T helper (Th) cells increased A(2A) adenosine receptor (A(2A)AR) mRNA level (sevenfold). A(2A)AR was the predominant subtype expressed in resting and stimulated gastric or peripheral Th cells. Stimulation with ATL313, an A(2A)AR agonist, increased cyclic AMP (cAMP) accumulation and reduced interleukin-2 (IL-2) production by 20-50%. ATL313 also attenuated tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production, which was inhibited by an A(2A)AR antagonist. Infection of IL-10-deficient mice with H. pylori is cleared spontaneously due to the marked inflammation. Administration of ATL313 during infection reduced gastritis and pro-inflammatory cytokine responses while bacterial load increased. In contrast, infection of A(2A)AR-deficient mice enhanced gastritis. Thus, A(2A)AR limits the pro-inflammatory effects of Th cells and favor chronic Helicobacter infection.

Hip pain in athletes.

Hip pain in athletes involves a wide differential diagnosis. Adolescents and young adults are at particular risk for various apophyseal and epiphyseal injuries due to lack of ossification of these cartilaginous growth plates. Older athletes are more likely to present with tendinitis in these areas because their growth plates have closed. Several bursae in the hip area are prone to inflammation. The trochanteric bursa is the most commonly injured, and the lesion is easily identified by palpation of the area. Iliotibial band syndrome presents with similar lateral hip pain and may be identified by provocative testing (Ober's test). A methodical physical examination that specifically tests the various muscle groups that move the hip joint can help determine a more specific diagnosis for the often vague complaint of hip pain. A number of hip conditions are more prevalent in athletes of certain ages. Transient synovitis is a common diagnosis in the very young, Legg-Calvé-Perthes disease causes bony disruption of the femoral head in prepubescents, and slipped capital femoral epiphysis is seen most commonly in obese adolescent males. Femoral neck stress fractures are seen in adult athletes, especially those involved in endurance sports, and can progress to necrosis of the femoral head if not found early. Older athletes may be limited by degenerative joint disease but nonetheless should be encouraged to stay active.

Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein.

Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae. Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0. 4-0.7 micromol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated. By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone. Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp. Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K(MgATP)(M), V(max), and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies.

The structure and function of A1 and A2B adenosine receptors.

Of the four G protein coupled adenosine receptor (AR) subtypes, the A1 is best suited for studies of reconstitution with G proteins. Recombinant A1 receptors extended with hexahistidine and FLAG have been purified to near homogeneity. In reconstitution assays using pure recombinant G protein subunits, the composition of the gamma subunit influences coupling to purified A1ARs. The least well characterized AR is the A2B. New data indicate that A(2B)ARs can trigger the degranulation of canine and human mast cell lines. Recombinant human A(2B)ARs are blocked by the anti-asthma drugs theophylline and enprofylline at concentrations that are used therapeutically to treat asthma. Although A(2B)ARs have long been known to stimulate adenylyl cyclase, they also can activate phospholipase C and mobilize Ca2+ by signaling through Gq/11. There is great potential for new therapies based on compounds that selectively target individual AR subtypes.

Reconstitution of bovine A1 adenosine receptors and G proteins in phospholipid vesicles: betagamma-subunit composition influences guanine nucleotide exchange and agonist binding.

We have studied the interactions of purified A1 adenosine receptors and G proteins reconstituted into phospholipid vesicles to investigate how the betagamma composition of G protein heterotrimers influences coupling. Recombinant hexahistidine-tagged bovine A1 adenosine receptors were expressed in Sf9 cells and purified to homogeneity by sequential chromatography over heparin-sepharose, xanthine amino congener-agarose, and nickel-nitrilotriacetic acid columns. These receptors were reconstituted with pure recombinant G proteins of defined subunit composition. Receptor-G protein complexes containing alphai2 and beta1gamma2 or beta1gamma3 and stimulated with the agonist, (R)-phenylisopropyladenosine, exchange guanine nucleotide 2-3 times more rapidly than do complexes containing beta1gamma1. This difference is not overcome by increasing the concentration of betagamma subunits. Receptor-G protein complexes containing beta1gamma1 also bind less of the agonist, [125I]-iodoaminobenzyladenosine (125I-ABA), than do complexes containing beta1gamma3. Kinetic experiments show that 125I-ABA dissociates 2-fold more rapidly from receptor-G protein complexes containing beta1gamma1 than from complexes containing the other betagamma subunits. The affinity of the interaction between immobilized Galphai2 subunits and beta1gamma1 or beta1gamma2 measured with an optical biosensor in the absence of receptor is similar. Taken together, these data implicate the gamma-subunit in influencing the interaction between the A1 adenosine receptor and G proteins.

Reconstitution of recombinant bovine A1 adenosine receptors in Sf9 cell membranes with recombinant G proteins of defined composition.

We investigated the coupling of A1 adenosine receptors to recombinant G proteins. Recombinant baculoviruses were used to express bovine A1 adenosine receptors in Sf9 insect cells that lack endogenous adenosine receptors. Binding parameters for recombinant receptors expressed in Sf9 cell membranes using the antagonist radioligand [125I]BW-A844U ([125I]8-cyclopentyl-3-iodoaminophenethyl-1-propylxanthine) are Bmax = 2-5 pmol/mg of protein and K(D) = 0.53 +/- 0.12 nM. In competition assays, the potency order of agonists is (R)-phenylisopropyladenosine > (S)-phenylisopropyladenosine > 5'-N-ethylcarboxamidoadenosine, properties characteristic of native bovine A1 adenosine receptors. The agonist radioligand 125I-N6-4-aminobenzyladenosine binds to two affinity states of the recombinant A1 adenosine receptors with K(D) values of 0.09 and 10.4 nM. The high affinity binding site represents <10% of total sites and is increased 7-fold on reconstitution with both alpha and betagamma G protein subunits but not with either subunit alone; thus, exogenous alpha and betagamma subunits do not functionally interact with endogenous Sf9 betagamma and alpha subunits, respectively. Four different alpha subunits (alpha i1, alpha i2, alpha i3, and alpha o) and six different beta gamma subunits (beta1gamma1, beta1gamma2, beta1gamma3, beta2gamma2, beta2gamma3, and bovine brain betagamma)) increased GTP-sensitive, high affinity agonist binding. The results indicate that bovine A1 adenosine receptors couple equally well to G protein alpha i and alpha o subunits in combination with betagamma subunits containing the beta1 or beta2 subunits and gamma2 or gamma3 subunits. G protein heterotrimers that contain the beta1gamma1 dimer couple with similar potency but reduced efficacy to A1 adenosine receptors.

Sexual harassment among nursing professionals: evidence and prescriptions for administrators.

The results of this study indicate that the experience of sexual harassment among nursing professionals may be more widespread than previously reported. With only one percent of respondents reporting ¿quid pro quo¿ harassment, the vast majority of harassment fell into the ¿hostile environment¿ category where the victim's perceptions are all important. Health care administrators should be cautioned that harassment and discrimination are evolving and expanding areas of the law, necessitating current knowledge of the standards for actionable conduct. To lower the risk of liability from claims of both victims and the accused, harassment and discrimination policies should be aggressive, proactive, fair, and thorough.

Expression of functional G protein beta gamma dimers of defined subunit composition using a baculovirus expression system.

The 36-kDa beta 1, 35-kDa beta 2, and 6.5-kDa gamma 2 subunits of the heterotrimeric guanine nucleotide-binding proteins have been overexpressed in Sf9 cells using a baculovirus expression system. The gamma 2 subunit expressed in Sf9 cells incorporated label derived from [3H]mevalonate and is therefore likely to be isoprenylated, as is its mammalian counterpart. Extracts of Sf9 cells doubly infected with viruses encoding a beta subunit and viruses encoding a gamma subunit are active in promoting the pertussis toxin-catalyzed ADP-ribosylation of a G protein alpha subunit. However, extracts from Sf9 cells singly infected with viruses encoding either a beta or gamma subunit are not active in this assay. Results demonstrate utility of the insect/baculovirus system for expressing G protein beta gamma subunits of defined composition.

Expression and purification of functional G protein alpha subunits using a baculovirus expression system.

The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins Gi1, Gi2, Gi3, G0, and Gs have been overexpressed in Sf9 cells using a baculovirus expression system. The Gi1 alpha, Gi2 alpha, Gi3 alpha, and G0 alpha have been purified to homogeneity from infected Spodoptera frugiperda (SF9) cells and characterized. Yields of up to 1.8 mg of purified recombinant G alpha have been obtained from 300-ml cultures of infected cells. The recombinant alpha subunits are myristoylated and are ADP-ribosylated by pertussis toxin only in the presence of beta gamma subunits. They bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) with low nM dissociation constants and stoichiometries of 0.8 mol/mol or greater. The rGi1 alpha, rGi2 alpha, and rGi3 alpha are capable of interacting with angiotensin II receptors based on their ability to restore high affinity angiotensin II binding in rat liver membranes shifted to a low affinity state with GTP gamma S.

RTA, a candidate G protein-coupled receptor: cloning, sequencing, and tissue distribution.

Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. Sequence analysis of the genomic clone further showed that the RTA gene has an intron interrupting the region encoding the amino terminus of the protein. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. RTA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. We cannot detect angiotensin binding to the RTA protein after introducing the cognate cDNA or mRNA into COS cells or Xenopus oocytes, respectively, nor can we detect an electrophysiologic response in the oocyte after application of angiotensin peptides. We conclude that RTA is not an angiotensin receptor; to date, we have been unable to identify its ligand.

Increased levels of arginine vasotocin and neurophysin during nesting in sea turtles.

Arginine vasotocin (AVT) and neurophysin (NP) levels were measured by radioimmunoassay in two species of sea turtle, the olive ridley, Lepidochelys olivacea, and the loggerhead, Caretta caretta, during the brief period of nesting and oviposition. In both species, AVT was low in animals which were not reproductively active. AVT was also low at the time animals emerged from the surf to nest, but increased significantly during oviposition and then declined as the animals returned to the water. NP increased in concert with AVT, also reaching highest levels during oviposition. In both species, however, NP levels remained elevated over prenesting levels at the time of return to the water. These findings are consistent with the hypothesis that an AVT-neurophysin complex is released from the neurohypophysis during nesting, and that AVT is a physiological regulator of oviducal contractions in sea turtles.