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Increasing cell culture productivity of recombinant proteins via process improvements is the primary focus for research groups within biologics manufacturing. Any recommendations to improve a manufacturing process obviously must be effective, but also be robust, scalable, and with product quality comparable to the original process. In this study, we report that three different GS−/− CHO cell lines developed in media containing a standard concentration of the selection agent methionine sulfoximine (MSX), but then exposed to increased MSX concentrations during seed train expansion, achieved titer increases of 10–19%. This result was observed in processes already considerably optimized. Expanding the cells with a higher MSX concentration improved cell line production stability with increased culture age. Production cultures in 500‐L and 1000‐L bioreactors replicated laboratory results using 5‐L bioreactors, demonstrating process robustness and scalability. Furthermore, product quality attributes of the final drug substance using the higher MSX process were comparable with those from cells expanded in media with the standard selection MSX concentration. Subsequent mechanistic investigations confirmed that the cells were not altered at the genetic level in terms of integration profiles or gene copy number, nor transcriptional levels of glutamine synthetase, heavy chain, or light chain genes. This study provides an effective and applicable strategy to improve the productivity of therapeutic proteins for biologics manufacturing.

In this article, we present four sets of data from high-throughput screening (HTS) studies of different chemically defined media using an industrially relevant Chinese hamster ovary (CHO) cell line. While complex hydrolysate media was used in the early phase process development and manufacturing of a monoclonal antibody (mAb), here we seek to determine an appropriate chemically defined media for late phase process development. Over 150 combinations of chemically defined basal media, feed media, and basal and feed media supplements, such as polyphenolic flavonoid antioxidants (including rosmarinic acid (RA)), were evaluated in four HTS studies to replace the complex hydrolysate media. Specifically, these four screening studies incorporated custom design of experiment (DOE), one-factor-at-a-time (OFAT), and definitive screening design methodologies for titer improvement. Titer was improved two fold compared to the early phase process using the addition of RA to chemically defined media. This dataset exemplifies how HTS can be used as an effective approach to systematically and statistically determine media composition and supplementation to increase mAb titer. These data were presented in connection with a published paper [1].

Manufacturability of immunoglobulin G4 (IgG4) antibodies from the Chemistry, Manufacture, and Controls (CMC) perspective has received little attention during early drug discovery. Despite the success of protein engineering in improving antibody biophysical properties, a clear gap still exists between rational design of IgG4 candidates and their manufacturing suitability. Here, we illustrate that undesirable two-peak elution profiles in cation-exchange chromatography are attributed to the S228P mutation (in IgG4 core-hinge region) intentionally designed to prevent Fab-arm exchange. A new scaffolding platform for engineering IgG4 antibodies amenable to bioprocessing and bioanalysis is proposed by introducing an “IgG1-like” single-point mutation in the hinge or CH1 region of IgG4S228P. This work offers insight into the design, discovery, and development of innovative therapeutic antibodies that are well suited for robust biomanufacturing and quality control.

The use of antioxidants in Chinese hamster ovary (CHO) cell cultures to improve monoclonal antibody production has been a topic of great interest. Nevertheless, the antioxidants do not have consistent benefits of production improvement, which might be cell line specific and/or process specific. In this work, we investigated how treatment with the antioxidant rosmarinic acid (RA) improved cell growth and titer in CHO cell cultures using transcriptomics. In particular, transcriptomics analysis indicated that RA treatment modified gene expression and strongly affected the MAPK and PI3K/Akt signaling pathways, which regulate cell survival and cell death. Moreover, it was observed that these signaling pathways, which had been identified to be up-regulated on day 2 and day 6 by RA, were also up-regulated over time (from initial growth phase day 2 to slow growth or protein production phase day 6) in both conditions. In summary, this transcriptomics analysis provides insights into the role of the antioxidant RA in industrial cell culture processes. The current study also represents an example in the industry of how omics can be applied to gain an in-depth understanding of CHO cell biology and to identify critical pathways that can contribute to cell culture process improvement and cell line engineering.

In this article, we provide four data sets for an industrial Chinese Hamster Ovary (CHO) cell line producing antibodies during a 14-day bioreactor run. This cell line was selected for further evaluation because of its significant titer loss as the cells were passaged over time. Four conditions that differed in cell bank ages were run for this dataset. Specifically, cells were passaged to passage 12, 21, 25, and 37 and then used in this experiment. Once the run commenced the following datasets were gathered: 1). Glycosylation data for each reactor 2). Size Exclusion Chromatography (SEC) data for the antibodies produced which allowed for the identification of high and low molecular weight species in the samples (N-Glycan and SEC data was taken on day 14 only). 3/4). Metabolites levels measured using Nuclear Magnetic Resonance (NMR) and liquid chromatography-mass spectroscopy (LC-MS) for all reactors over the time course of days 1, 4, 6, 8, 12, and 14. We also provide a graph of the glutamine levels for cells of different ages as an example of the utility of the data. These metabolomics data provide relative amounts for 36 metabolites (NMR) and 109 metabolites (LC-MS) over the 14-day time course. These data were collected in connection with a co-submitted paper [1].

A standard fed-batch fermentation process using 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) induction at 37 °C in complex batch and feed media had been developed for manufacturing of a therapeutic protein (TP) expressed in inclusion bodies (IBs) by E. coli BL21 (DE3) driven by T7 promoter. Six unauthentic TP N-terminal variants were identified, of which methionylated TP (Met-TP) ratio was predominant. We hypothesized that lowering metabolic and protein production rates would reduce the Met-TP ratio while improving TP titer. The standard process was surprisingly auto-induced without added IPTG due to galactose in the complex media. Without changing either the clone or the batch medium, a new process was developed using lower feed rates and auto-induction at 29 °C after glucose depletion while increasing induction duration. In comparison to the standard process, the new process reduced the unauthentic Met-TP ratio from 23.6 to 9.6 %, increased the TP titer by 85 %, and the specific production yield from 210 to 330 mg TP per gram of dry cell weight. Furthermore, the TP recovery yield in the purified IBs was improved by ~20 %. Adding together, ~105 % more TP recovered in the purified IBs from per liter of fermentation broth for the new process than the standard process. The basic principles of lowering metabolic and production rates should be applicable to other recombinant protein production in IBs by fed-batch fermentations.

We examined the impact of coexpressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, synapse-associated protein (SAP) 97, and determined that coexpression of these proteins caused an approximately twofold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channel conductance, or channel open probability. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3, leading to prominent colocalization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was ∼13 pS, whereas coexpression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16, 29, and 42 pS. These changes occurred without altering channel open probability, current rectification properties, or pH sensitivity. Thus association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kir2.3 COOH-terminal domain and altering channel conformation.

A novel 2986-base transcript encoded by the antisense strand of the HRES-1 human endogenous retrovirus was isolated from peripheral blood lymphocytes. This transcript codes for a 218-amino acid protein, termed HRES-1/Rab4, based on homology to the Rab4 family of small GTPases. Antibody 13407 raised against recombinant HRES-1/Rab4 detected a native protein of identical molecular weight in human T cells. HRES-1 nucleotides 2151-1606, located upstream of HRES-1/Rab4 exon 1, have promoter activity when oriented in the direction of HRES-1/Rab4 transcription. The human immunodeficiency virus, type 1 (HIV-1), tat gene stimulates transcriptional activity of the HRES-1/Rab4 promoter via trans-activation of the HRES-1 long terminal repeat. Transfection of HIV-1 tat into HeLa cells or infection of H9 and Jurkat cells by HIV-1 increased HRES-1/Rab4 protein levels. Overexpression of HRES-1/Rab4 in Jurkat cells abrogated HIV infection, gag p24 production, and apoptosis, whereas dominant-negative HRES-1/Rab4(S27N) had the opposite effects. HRES-1/Rab4 inhibited surface expression of CD4 and targeted it for lysosomal degradation. HRES-1/Rab4(S27N) enhanced surface expression, recycling, and total cellular CD4 content. Infection by HIV elicited a coordinate down-regulation of CD4 and up-regulation of HRES-1/Rab4 in PBL. Moreover, overexpression of HRES-1/Rab4 reduced CD4 expression on peripheral blood CD4+ T cells. Stimulation by HIV-1 of HRES-1/Rab4 expression and its regulation of CD4 recycling reveal novel coordinate interactions between an infectious retrovirus and the human genome.

Fertility of spermatozoa depends on maintenance of the mitochondrial transmembrane potential (Δψm), which is generated by the electron-transport chain and regulated by an oxidation–reduction equilibrium of reactive oxygen intermediates, pyridine nucleotides, and glutathione (GSH). Here, we report that male mice lacking transaldolase (TAL)−/−are sterile because of defective forward motility. TAL−/−spermatozoa show loss of Δψmand mitochondrial membrane integrity because of diminished NADPH, NADH, and GSH. Mitochondria constitute major Ca2+stores; thus, diminished mitochondrial mass accounts for reduced Ca2+fluxing, defective forward motility, and infertility. Reduced forward progression of TAL-deficient spermatozoa is associated with diminished mitochondrial reactive oxygen intermediate production and Ca2+levels, intracellular acidosis, and compensatory down-regulation of carbonic anhydrase IV and overexpression of CD38 and γ-glutamyl transferase. Microarray analyses of gene expression in the testis, caput, and cauda epididymidis of TAL+/+, TAL+/−, and TAL−/−littermates confirmed a dominant impact of TAL deficiency on late stages of sperm-cell development, affecting the electron-transport chain and GSH metabolism. Stimulation ofde novoGSH synthesis by oralN-acetyl-cysteine normalized the low fertility rate of TAL+/−males without affecting the sterility of TAL−/−males. Whereas TAL−/−sperm failed to fertilize TAL+/+oocytesin vitro, sterility of TAL−/−sperm was circumvented by intracytoplasmic sperm injection, indicating that TAL deficiency influenced the structure and function of mitochondria without compromising the nucleus and DNA integrity. Collectively, these data reveal an essential role of TAL in sperm-cell mitochondrial function and, thus, male fertility.

Elicitation of biological responses by estrogen in target tissues requires the presence of ER as well as receptor-active ligand in the local microenvironment. Though much attention has been devoted to the study of the receptor in estrogen target tissues, the concept is emerging that tissue estrogen sensitivity may also be regulated by ligand availability through metabolic transformation in situ. Here, we show that targeted disruption, in the mouse, of an estrogen metabolic enzyme, estrogen sulfotransferase (EST), causes structural and functional lesions in the male reproductive system. EST catalyzes the sulfoconjugation and inactivation of estrogen and is expressed abundantly in testicular Leydig cells. Although knockout males were fertile and phenotypically normal initially, they developed age-dependent Leydig cell hypertrophy/hyperplasia and seminiferous tubule damage. Development of these lesions in the testis could be recapitulated by exogenous E2 administration in younger knockout mice, suggesting that they arose in older knockout mice from chronic estrogen stimulation. Older knockout mice were also found to have reduced testis and epididymis weights but increased seminal vesicle/coagulating gland weight because of tissue swelling. Furthermore, total and forward sperm motility of older knockout mice was reduced by 60% and 80%, respectively, and these mice produced smaller litters compared with age-matched wild-type males. These findings establish a role for EST in the male reproductive system and indicate that intracrine and paracrine estrogen activity can be modulated by a ligand transformation enzyme under a physiological setting. Thus, inhibition of estrogen metabolic enzymes by environmental chemicals, as has been demonstrated recently for the human EST, may constitute a novel mechanism of endocrine disruption in vivo.

Multidrug resistance protein 1 (MRP1) is capable of actively transporting a wide range of conjugated and unconjugated organic anions. The protein can also transport additional conjugated and unconjugated compounds in a GSH- or S-methyl GSH-stimulated manner. How MRP1 binds and transports such structurally diverse substrates is not known. We have used [3H]leukotriene C4 (LTC4), a high affinity glutathione-conjugated physiological substrate, to photolabel intact MRP1, as well as fragments of the protein expressed in insect cells. These studies revealed that: (i) LTC4 labels sites in the NH2- and COOH-proximal halves of MRP1, (ii) labeling of the NH2-half of MRP1 is localized to a region encompassing membrane-spanning domain (MSD) 2 and nucleotide binding domain (NBD) 1, (iii) labeling of this region is dependent on the presence of all or part of the cytoplasmic loop (CL3) linking MSD1 and MSD2, but not on the presence of MSD1, (iv) labeling of the NH2-proximal site is preferentially inhibited byS-methyl GSH, (v) labeling of the COOH-proximal half of the protein occurs in a region encompassing transmembrane helices 14–17 and appears not to require NBD2 or the cytoplasmic COOH-terminal region of the protein, (vi) labeling of intact MRP1 by LTC4 is strongly attenuated in the presence of ATP and vanadate, and this decrease in labeling is attributable to a marked reduction in LTC4 binding to the NH2-proximal site, and (vii) the attenuation of LTC4 binding to the NH2-proximal site is a consequence of ATP hydrolysis and trapping of Vi-ADP exclusively at NBD2. These data suggest that MRP1-mediated transport involves a conformational change, driven by ATP hydrolysis at NBD2, that alters the affinity with which LTC4 binds to one of two sites composed, at least in part, of elements in the NH2-proximal half of the protein.

CD59 is a 18- to 20-kDa, GPI-anchored membrane protein that functions as a key regulator of the terminal step of the complement activation cascade. It restricts binding of C9 to the C5b-8 complex, thereby preventing the formation of the membrane attack complex (C5b-9 of complement). A single human CD59 gene has been identified, and corresponding genetic homologues from rat, mouse, and pig have been characterized in previous studies. In this study, we report the discovery and functional characterization of a separate cd59 gene in the mouse (referred to as cd59b, the previously characterized mouse cd59 gene as cd59a). Mouse cd59b is 85% and 63% identical to cd59a at the nucleotide and amino acid level, respectively. In cDNA transfection experiments with Chinese hamster ovary cells, peptide-tagged cd59b was detected on the cell surface by flow cytometry and was shown to be susceptible to phosphatidylinositol-specific phospholipase C cleavage. Chinese hamster ovary cells expressing cd59b were significantly more resistant than control cells to human and mouse complement-mediated lysis. These results suggest that cd59b encodes a GPI-anchored protein that is functionally active as a membrane attack complex inhibitor. Northern blot analysis revealed that cd59b is expressed selectively in the mouse testis. In contrast, the major transcript of cd59a was shown to be expressed at high levels in the heart, kidney, liver, and lung, but only minimally in the testis. These results revealed the existence of two distinct cd59 genes in the mouse that are differentially regulated and that may have nonoverlapping physiological functions in vivo.

The spontaneous activity of the rat isolated colon is suppressed by prostacyclin analogues such as cicaprost (IC50=4.0 nM). Activation of prostanoid IP1-receptors located on NANC inhibitory neurones is involved. However, several non-prostanoids, which show medium to high IP1 agonist potency on platelet and vascular preparations, exhibit very weak inhibitory activity on the colon. The aim of the study was to investigate this discrepancy. Firstly, we have demonstrated the very high depolarizing potency of cicaprost on the rat isolated vagus nerve (EC50=0.23 nM). Iloprost, taprostene and carbacyclin were 7.9, 66, and 81 fold less potent than cicaprost, indicating the presence of IP1 as opposed to IP2-receptors. Three non-prostanoid prostacyclin mimetics, BMY 45778, BMY 42393 and ONO-1301, although much less potent than cicaprost (195, 990 and 1660 fold respectively), behaved as full agonists on the vagus nerve. On re-investigating the rat colon, we found that BMY 45778 (0.1–3 μM), BMY 42393 (3 μM) and ONO-1301 (3 μM) behaved as specific IP1 partial agonists, but their actions required 30–60 min to reach steady-state and only slowly reversed on washing. This profile contrasted sharply with the rapid and readily reversible contractions elicited by a related non-prostanoid ONO-AP-324, which is an EP3-receptor agonist. The full versus partial agonism of the non-prostanoid prostacyclin mimetics may be explained by the markedly different IP1 agonist sensitivities of the two rat neuronal preparations. However, the slow kinetics of the non-prostanoids on the NANC system of the colon remain unexplained, and must be taken into account when characterizing neuronal IP-receptors. Keywords: Prostacyclin, prostanoid IP-receptors, prostanoid EP3-receptors, cicaprost, iloprost, BMY 45778, ONO-1301, vagus nerve, colon, NANC innervation Introduction The non-prostanoid prostacyclin mimetics activate prostanoid IP1-receptors on platelets and vascular smooth muscle to elicit inhibitory effects characteristic of prostacyclin (see Jones et al., 1993; Meanwell et al., 1994 for reviews). One of the most potent of these agents is BMY 45778 (Figure 1), which completely inhibits aggregation in human platelet-rich plasma and has a Kd of 7 nM for the IP1-receptor on human platelet membranes (Seiler et al., 1997). It also fully relaxes human pulmonary artery rings with an IC50 of about 3 nM (Jones et al., 1997). The non-prostanoids tend to be less potent inhibitors on rat and rabbit platelet suspensions (Armstrong et al., 1989; Merritt et al., 1991). However, we were surprised by the very weak inhibitory effects of BMY 45778 on the spontaneous motility of the rat isolated colon (10–15% inhibition at 2–10 μM) in comparison to the complete inhibition induced by the prostacyclin analogue cicaprost (IC50=4 nM) (Wise et al., 1995). In addition, two other non-prostanoids, BMY 42393 (Figure 1, Seiler et al., 1994) and EP 185 (Jones et al., 1993) had minimal inhibitory activity at a concentration of 10 μM. Cicaprost's action on the rat colon is abolished by tetrodotoxin (TTX), and it appears that activation of IP-receptors on non-adrenergic non-cholinergic (NANC) neurones in the colon leads to the release of transmitters that inhibit the pacemaker/smooth muscle system (Figure 3a) (Qian & Jones, 1995). Figure 1 6a-Carba analogues of prostacyclin (upper row) and non-prostanoid prostacyclin mimetics (lower row) with differing agonist specificities. IP1/IP2 receptor selectivity is derived from ligand binding measurements in rat brain (Takechi et al., 1996) and ... Figure 3 Rat colon: (a) Schematic arrangement of the NANC neurone/pacemaker/smooth muscle system and the proposed sites of action of agonists for IP1, EP3, nicotinic (N) and β3-adrenergic receptors. β3-Adrenoceptors may also be located on the smooth ... The question therefore arose as to whether non-prostanoid prostacyclin mimetics are genuinely poor agonists at IP-receptors on rat peripheral neurones, or have other actions that interfere with IP agonist activity. There are two related reasons for considering the latter possibility. Firstly, the high lipophilicity of the non-prostanoids may affect cell membrane function, and their diaryl-heteroatomic units resemble moieties found in other pharmacologically active agents (e.g. methadone analogues and diphenylmethane antihistamines; see Foye, 1990). Secondly, the control of colonic motility through the interaction of enteric neurones, pacemaker cells (interstitial cells of Cajal) and smooth muscle cells is known to be complex (Huizinga & Thuneberg, 1997). In the case of IP agonist action, at least two inhibitory neurotransmitter systems are involved (Figure 3a); nitric oxide (NO) is one transmitter while the other remains unidentified, but does not appear to be ATP, VIP or pituitary adenylate cyclase activating peptide (PACAP) (Qian & Jones, 1995). As a result, there are a number of steps at which a non-prostanoid might exert an additional action. Consequently, we have looked for an anatomically and mechanistically simpler neuronal preparation from the rat. The rat cervical vagus nerve is depolarized by 5-HT and has been used previously to characterize excitatory 5-HT-receptors (mainly 5-HT3) involved in vagal afferent nerve activity in emesis (Ireland & Tyers, 1987; Rhodes et al., 1992; Minami et al., 1997). We now report that this preparation is depolarized by cicaprost at subnanomolar concentrations, and that BMY 45778, BMY 42393, and a third non-prostanoid prostacyclin mimetic, ONO-1301 (Figure 1, Kondo & Hamanaka, 1995; Kondo et al., 1995) also show strong depolarizing activity in the nanomolar range. These findings prompted us to reinvestigate the actions of the nonprostanoids on the rat colon preparation.

CD97 is a newly identified, activation-associated human leucocyte antigen with seven putative transmembrane domains. It has an extended extracellular segment containing several adhesion molecule structure motifs, and has been shown to interact with the human complement regulator, decay-accelerating factor (DAF, CD55). To understand further the interaction between CD97 and DAF, as well as the structure and function of CD97 in general, we have cloned the mouse CD97 cDNA and studied the encoded protein for its membrane association property and ability to interact specifically with the murine decay-accelerating factor. The full-length mouse CD97 cDNA that we have cloned and characterized encodes a protein that is 60% identical to the three epidermal growth factor (EGF) domain-containing form of human CD97 but does not contain the Arg-Gly-Asp (RGD) motif which is present in human CD97. Two other alternatively spliced forms of mouse CD97 were also identified. These forms differ by the number of EGF-like sequence repeats present in the N-terminal region. Northern blot analysis revealed that CD97 is expressed widely in mouse tissues and in resting as well as activated cultured mouse splenocytes. Transient transfection of human embryonic kidney (HEK) 293 cells with the mouse CD97 cDNA in a green-fluorescence protein vector (pEGFP-N1) showed plasma membrane targeting of the expressed protein. Western blot analysis confirmed its membrane association and identified the existence of a processed C-terminal fragment, supporting the notion that CD97 on the cell membrane is composed of post-translationally generated subunits. Adhesion studies demonstrated that normal, but not DAF knockout mouse erythrocytes and splenocytes adhered to mouse CD97-transfected HEK cells. The interaction of CD97 and DAF was found to be species-restrictive in that human erythrocytes were unable to bind to mouse CD97-transfected HEK cells. These results indicate that the general structure, membrane association property and DAF-binding ability of CD97 are conserved and that the adhesive interaction between CD97 and DAF is independent of the RGD motif. The finding that CD97 is distributed widely among various mouse tissues suggests that CD97 may have other roles beyond lymphocyte activation.

Estrogen sulfotransferase (EST) catalyzes the specific sulfonation and inactivation of estrogens. A common site for EST expression in mammalian species is the testicular Leydig cells. In previous in vivo studies, we have shown that testicular expression of EST is under the regulation of LH. Thus, EST expression in mouse Leydig cells was abolished by hypophysectomy, but could be restored by hCG injection. In this study, we have evaluated the downstream mechanisms by which LH exerts its regulatory effect on EST. Primary mouse Leydig cells were isolated and purified by collagenase digestion and Percoll density gradient centrifugation. They were cultured in serum-free medium at 32 C and treated with various agents for 24 or 48 h, and levels of EST messenger RNA and enzyme activity were determined. Consistent with the in vivo data suggesting an essential role of LH in regulating EST expression, treatment of primary mouse Leydig cells in vitro with 100 microM 8-bromo-dibutyryl cAMP [(Bu)2cAMP] increased EST expression 3- to 5-fold. The effect of (Bu)2cAMP was attenuated by the steroidogenesis inhibitor aminoglutethimide and was mimicked by the potent androgen 5alpha-dihydrotestosterone (5-DHT). The activity of 5-DHT in stimulating EST expression was blocked by the androgen receptor antagonist, hydroxyflutamide. These data suggested the involvement of androgen in (Bu)2cAMP-induced EST expression. Further evidence came from the study with interleukin-1beta, another agent known to suppress Leydig cell steroidogenesis by down-regulating P450c17 gene expression. Treatment of Leydig cells with 0.2 ng/ml interleukin-1beta inhibited (Bu)2cAMP-induced EST expression, which was overcome by the addition of 5-DHT. Finally, in the testis-feminized mouse (Tfm) in which the androgen receptor is nonfunctional due to a frameshift mutation, testicular EST expression is completely absent, whereas messenger RNAs of steroidogenic enzymes such as P450c17 and 3beta-hydroxysteroid dehydrogenase are relatively abundant. We conclude that, by acting as an autocrine or paracrine factor, androgen plays an essential role in the regulation of estrogen sulfotransferase expression in Leydig cell by LH and cAMP.

Contraction of guinea-pig isolated aorta induced by the prostaglandin E analogue sulprostone (1-400 nM) has a lower maximum response (40%) than that of phenylephrine or U-46619 (TP-receptor agonist). A prostanoid EP3-receptor subtype is involved based on agonist potency ranking: equi-effective molar ratios (EMR) are sulprostone (EC50 approximately equal to 23 nM) 1.0, SC-46275 0.11, misoprostol 2.2, gemeprost 3.3, PGE2 5.4, 17-phenyl PGE2 6.0, GR-63799 8.9. GR-63799, which contains a bulky ester group, is relatively more potent on neuronal EP3 preparations than on the aorta. ONO-AP-324, a relative of the non-prostanoid prostacyclin mimetic series, behaves as an EP3 partial agonist on the aorta, inhibiting sulprostone responses but acting synergistically (in a similar manner to sulprostone) with phenylephrine; it may be a useful pharmacological tool for studying EP3-receptors. Sulprostone contractions are markedly suppressed in zero-Ca2+ bathing fluid containing either 2 mM EDTA or 50 microM EGTA, and by Cd2+ (500 microM), but are usually unaffected by nifedipine (0.3 microM) and verapamil (4.44 microM). Influx of Ca2+, but not through L-type Ca2+-channels, appears to be the major contractile mechanism. The guinea-pig aorta is a valuable addition to the vascular EP3 preparations available and may increase our knowledge of the mechanisms whereby Gi-coupled receptors mediate vasoconstriction (c.f. 5-HT1B/D- and alpha2-receptors). The possibility of certain EP3 agonists distinguishing EP3-receptor isoforms is discussed.

1. The possibility that prostacyclin (IP-) receptor agonists inhibit spontaneous contractions of the rat isolated colon by activating enteric neurones has been investigated. Cicaprost was used as the test agonist because of its high stability, selectivity and potency (IC50 = 3.8 nM). 2. The Na+ channel blockers saxitoxin (STX, 1 nM) and tetrodotoxin (TTX, 1 microM), whilst having little effect on resting spontaneous activity, virtually abolished the inhibitory actions of cicaprost (10 nM) and nicotine (3 microM); inhibitory responses to isoprenaline (20 nM) were not affected. Phentolamine (1 microM), propranolol (1 microM) and atropine (1 microM) had no effect on cicaprost inhibition. These data are compatible with release of inhibitory NANC transmitter(s) by cicaprost. 3. A transmitter role for nitric oxide was investigated. The nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM) inhibited the actions of both cicaprost (10 nM) and nicotine (3 microM) by 50-60%, but did not affect responses to isoprenaline (20 nM) or sodium nitroprusside (1-5 microM). The enantiomeric D-NAME (100 microM), which has negligible NOS inhibitory activity, had no effect on the action of cicaprost. 4. The involvement of purinergic transmitters was also investigated. Desensitization to the inhibitory action of ATP did not affect cicaprost responses. The P2x/P2y-receptor antagonist, suramin, at 300 microM blocked ATP responses, but not those due to adenosine; it did not affect cicaprost inhibition. The selective adenosine A1-receptor antagonist, DPCPX, used at a sufficiently high concentration (5 microM) to block adenosine A2-receptors, did not affect cicaprost inhibition. Apamin (25 nM), a blocker of calcium activated K+ channels on smooth muscle, abolished or markedly reduced the inhibitory actions of ATP and adenosine, and partially inhibited cicaprost and nicotine responses. The combination of L-NAME(100 microM) and apamin (25 nM) abolished cicaprost and nicotine responses.5. Investigation of vasoactive intestinal peptide (VIP) as a potential transmitter showed that its inhibitory action on the colon (IC50 = 50 nM) was partially inhibited by TTX (1 microM). alpha-Chymotrypsin abolished the effect of VIP but had no effect on cicaprost inhibition. Attempts to inhibit VIP responses using peptide antagonists and by agonist desensitization were unsuccessful.6. KCI (40 mM) contracted the colon and abolished spontaneous activity. Under these conditions,isoprenaline, sodium nitroprusside and ATP induced relaxation, whereas cicaprost (10-3 10 nM) had no effect. Cicaprost inhibited both the tone and the spontaneous activity induced by the EP1/EP3-receptor agonist, sulprostone (8.6 nM) but not when either TTX (1 microM) or KC1 (40 mM) was also present. On KCl-treated preparations, the prostacyclin analogue, iloprost (10-500 nM), induced contraction,presumably due to activation of EP-receptors.7. It is concluded that IP-receptor agonists inhibit the contractility of rat colon by stimulating the release of at least two transmitters from NANC enteric neurones. Nitric oxide appears to be one of the transmitters. The second transmitter mechanism is apamin-sensitive; the experimental results do not support ATP, adenosine or VIP as transmitter candidates. However, further studies using more potent and selective receptor antagonists are required.

Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1-/- and Taldo1+/- mice spontaneously developed HCC, and Taldo1-/- mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1-/- livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced beta-catenin phosphorylation and enhanced c-Jun expression in Taldo1-/- livers reflected adaptation to oxidative stress. Taldo1-/- hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1-/- mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency.

Although primarily regarded as a sex steroid, estrogen plays an important role in many other physiological processes including adipose development and disposition. Estrogen sulfotransferase (EST) regulates estrogen activity by catalyzing the sulfoconjugation and inactivation of estrogens. In the present study, we report the gender-specific expression of EST in adipose tissues of the mouse and describe contrasting mechanisms of EST regulation in the fat and liver. EST is expressed in the white adipose tissues of the male but not female mouse. Within the various fat depots of male mice, it is most abundantly expressed in the epididymal fat pad, with variable levels in other white fats and no expression in the brown fat. Fractionation of epididymal fat cells showed EST to be predominantly associated with stromal vascular cells (preadipocyte). EST expression in male mouse adipose tissues is dependent on testosterone as castration ablated, and administration of exogenous testosterone restored, EST expression. Furthermore, testosterone treatment induced abnormal EST expression in the parametrial fat of female mice. EST induction by testosterone in female mice is tissue specific because testosterone treatment had no effect on liver EST expression. Conversely, the liver X receptor agonist TO-901317 induced EST expression in female mouse liver but not in their adipose tissues. Finally, we demonstrate that male EST knockout mice developed increased epididymal fat accumulation with enlarged adipocyte size. We conclude that EST is expressed in adipose tissues in a sexually dimorphic manner, is regulated by testosterone, and plays a physiological role in regulating adipose tissue accumulation in male mice.

Transaldolase (TAL) is a key enzyme of the pentose phosphate pathway (PPP). TAL deficiency is a newly recognized cause of liver cirrhosis. We have developed an ion-pair LC separation combined with negative ion electrospray MS/MS detection method to assess PPP metabolites in urine samples from TAL-deficient mice. Sedoheptulose 7-phosphate (S7P), C5-polyols D-arabitol and D-ribitol, and 6-phosphogluconate (6PG) levels were markedly increased in urine of TAL-deficient mice with respect to those of wild-type and heterozygote littermates. The detection limits of S7P, D-arabitol, and 6PG were 0.15 +/- 0.015 pmol, 3.5 +/- 0.41 pmol, and 0.61 +/- 0.055 pmol, respectively. The limit of quantitation was 0.4 +/- 0.024 nmol/ml for S7P, 1.6 +/- 0.11 nmol/ml for 6PG and 10 +/- 0.7 nmol/ml for D-arabitol. Additional metabolites, hexose 6-phosphates (m/z 259), D-ribose 5-phosphate and D-xylulose 5-phosphate (m/z 229), D-fructose 1,6-diphosphate (m/z 339), C6-polyols (m/z 181) and GSSG (m/z 611), that have been positively identified in mouse urine, showed similar levels in control and TAL-deficient mice.

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent transporter of structurally diverse organic anion conjugates. The protein also actively transports a number of non-conjugated chemotherapeutic drugs and certain anionic conjugates by a presently poorly understood GSH-dependent mechanism. LY475776is a newly developed (125)I-labeled azido tricyclic isoxazole that binds toMRP1 with high affinity and specificity in a GSH-dependent manner. The compound has also been shown to photolabel a site in the COOH-proximal region of MRP1's third membrane spanning domain (MSD). It is presently not known where GSH interacts with the protein. Here, we demonstrate that the photactivateable GSH derivative azidophenacyl-GSH can substitute functionally for GSH in supporting the photolabeling of MRP1 by LY475776 and the transport of another GSH-dependent substrate, estrone 3-sulfate. In contrast to LY475776, azidophenacyl-[(35)S] photolabels both halves of the protein. Photolabeling of the COOH-proximal site can be markedly stimulated by low concentrations of estrone 3-sulfate, suggestive of cooperativity between the binding of these two compounds. We show that photolabeling of the COOH-proximal site by LY475776 and the labeling of both NH(2)- and COOH- proximal sites by azidophenacyl-GSH requires the cytoplasmic linker (CL3) region connecting the first and second MSDs of the protein, but not the first MSD itself. Although required for binding, CL3 is not photolabeled by azidophenacyl-GSH. Finally, we identify non-conserved amino acids in the third MSD that contribute to the high affinity with which LY475776 binds to MRP1.

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette (ABC) transporter that transports a range of hydrophobic xenobiotics, as well as relatively hydrophilic organic anion conjugates. The protein is present at high levels in testicular Leydig and Sertoli cells. Studies with knockout mice suggest that MRP1 may protect germ cells from exposure to some cytotoxic xenobiotics, but potential endobiotic substrates in this organ have not been identified. Previously, we have shown certain D-ring, but not A-ring, estrogen glucuronides can act as competitive inhibitors of MRP1 mediated transport, suggesting that they are potential substrates for the protein. In the case of 17 beta-estradiol-17 beta-d-glucuronide, this has been confirmed by direct transport studies. The Leydig cell is the major site of estrogen conjugation in the testis. However, the principal products of conjugation are A-ring estrogen sulfates, which are then effluxed from the cell by an unknown transporter. To determine whether MRP1/mrp1 could fulfill this function, we used membrane vesicles from MRP1-transfected HeLa cells to assess this possibility. We found that estradiol and estrone 3-sulfate alone were poor competitors of MRP1-mediated transport of the cysteinyl leukotriene, leukotriene C(4). However, in the presence of reduced glutathione (GSH), their inhibitory potency was markedly increased. Direct transport studies using [(3)H]estrone 3-sulfate confirmed that the conjugated estrogen could be efficiently transported (K(m) = 0.73 microm, V(max) = 440 pmol mg(-)1 protein min(-)1), but only in the presence of either GSH or the nonreducing alkyl derivative, S-methyl GSH. In contrast to previous studies using vincristine as a substrate, we detected no reciprocal increase in MRP1-mediated GSH transport. These results provide the first example of GSH-stimulated, MRP1-mediated transport of a potential endogenous substrate and expand the range of MRP1 substrates whose transport is stimulated by GSH to include certain hydrophilic conjugated endobiotics, in addition to previously identified hydrophobic xenobiotics.

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the specific sulfonation of estrogens at the 3-hydroxyl position using 3'-phosphoadenosine-5'-phosphosulfate as an activated sulfate donor. Sulfated estrogens no longer bind to the estrogen receptor and are, therefore, hormonally inactive. Although liver has been considered a primary site for steroid sulfotransferase activities, we previously have cloned the mouse EST complementary DNA and found the enzyme to be expressed abundantly in the testis of normal mice. In this study we show by reverse transcription-PCR that EST is also expressed in the testes of rat and man, suggesting that testicular expression of EST may be a common phenomenon among different species. Using a purified polyclonal antibody raised against the bacterially expressed mouse EST protein, we demonstrate by immunohistochemistry that EST is localized selectively to the androgen-producing Leydig cells within the mouse testis. Additionally, we show that Leydig cell expression of EST is under the control of the pituitary hormone LH and is regulated differentially during development. In contrast to the high level of expression in mature intact animals, EST is not present in Leydig cells of hypophysectomized mice or in Leydig cells of fetal and prepubertal (day 5 or 17) mouse testes. Administration of hCG to hypophysectomized mice restored the testicular expression of EST. Together, these results suggest that testicular expression of EST may play an important role in male reproduction, conceivably by modulating the activity of locally synthesized estrogen in the testis of a sexually mature animal.

The specific prostacyclin (IP) receptor agonist cicaprost relaxed human pulmonary artery preparations precontracted with phenylephrine [50% inhibitory concentration (IC50) approximately 0.6 nM], U-46619 (IC50 approximately 0.9 nM), and K+ (approximately 40% maximal relaxation); endothelium removal had little effect on relaxant activity. Ranking of relaxant potencies for prostacyclin and five of its analogs was 17 alpha, 20-dimethyl-delta 6,6a-6a-carba PGI1 (TEI-9063) > or = cicaprost > iloprost > prostacyclin > taprostene > benzodioxane prostacyclin > 15-deoxy-16 alpha-hydroxy-16 beta,20-dimethyl-delta 6,6a-6a-carba PGI1 (TEI-3356). The potency of the isocarbacyclin TEI-3356 may have been under-estimated because of its contractile (EP3 receptor agonist) activity. The potency ranking of four nonprostanoid prostacyclin mimetics was 3-[4-(4,5-diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy] acetic acid (BMY 45778; IC50 approximately 2.5 nM) > > 2-[3-[2-(4, 5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid (BMY 42393) > octimibate > CU 23 (a novel diphenylindole). From IP receptor binding affinities obtained on human platelet membranes, it is suggested that the slightly shallower log concentration-response curves for BMY 45778, BMY 42393, and CU 23 may reflect the near-maximal receptor occupancy required for complete relaxation. A fifth nonprostanoid, CU 602, had much shallower log concentration-response curves than cicaprost against phenylephrine tone but not against U-46619 tone; this may indicate IP receptor partial agonism coupled with TP receptor antagonism. The relaxant actions of the nonprostanoid mimetics were more persistent than those of the prostacyclin analogs on washout of the organ bath; by the inhalation route, this type of compound may be retained within pulmonary tissue and thus afford greater pulmonary/systemic selectivity than currently used pulmonary vasodilators.

1. In 13 of 15 experiments, prostaglandin E2 (PGE2) and sulprostone (a prostanoid EP1/EP3-receptor agonist) contracted isolated rings of human pulmonary artery at low concentrations (> or = 5 and > or = 0.5 nM respectively). Tissue was obtained from patients undergoing surgery mainly for carcinoma of the lung. Characterization of the receptors involved was complicated by loss of sensitivity to the contractile PGE action over the experimental period. In contrast, contractile responses to KCl, phenylephrine and the specific thromboxane (TP-) receptor agonist, U-46619, did not decrease with time. 2. The relative contractile potencies for seven PGE analogues, measured during the first few hours after setting up the preparations, were as follows: sulprostone > misoprostol = gemeprost > or = PGE2 > or = GR 63799X > 17-phenyl-omega-trinor PGE2 > or = 11-deoxy PGE1. This ranking indicates that an EP3-receptor is involved. 3. The contractile action of sulprostone was not blocked by the TP-receptor antagonists, EP 169 and GR 32191, and the EP1-receptor antagonist, AH 6809. 4. In two experiments, PGE2 (50 nM) reduced basal tone and sulprostone was a weak contractile agent. Phenylephrine-induced tone was also inhibited by PGE2 (EC50 = 5-20 nM), 11-deoxy PGE1 and butaprost (a selective EP2-receptor agonist); the latter prostanoids were about 2 and 4 times less potent than PGE2 respectively. Interactions with phenylephrine were different in experiments where PGE2 alone was contractile: PGE2 induced contraction superimposed on the phenylephrine response and 11-deoxy PGE1 induced either further contraction or had no effect. Butaprost produced relaxation at high concentrations;this may not be an EP2 action since preparations were highly sensitive to relaxant actions of prostacyclin (IP-) receptor agonists (cicaprost and TEI-9063).5 The study has shown that in the majority of experiments on the human isolated pulmonary artery,the contractile EP3 system outweighed the relaxant EP2 system. However, in two experiments the reverse was true. It is not clear to what extent these differences are due to disease processes affecting the tissues.The findings are discussed in relation to the adverse cardiovascular responses occasionally encountered during treatment of postpartum haemorrhage with sulprostone, and more generally to the clinical use of EP-receptor agonists in man.