P2X7 receptor in the hippocampus is involved in gp120-induced cognitive dysfunction.

To investigate the role of the P2X7 receptor in learning and memory dysfunction induced by HIV-1 envelope glycoprotein gp120 (gp120), we established HIV-1-associated dementia (HAD) animal models by intracerebroventricular (ICV) infusion of gp120 in rats. We observed gp120-induced cognitive dysfunction in the radial arm water maze test. Results showed that rats in the gp120 groups had longer escape latencies and more errors compared to those in the control group. For example, the average trial time in the 50-ng/day-gp120 group on day eight (16.57 ± 1.71 s, N = 90) was significantly longer than that of control rats (9.93 ± 0.68 s, N = 90). The relative expression of P2X7 mRNA in the control, 50-, 70-, and 100-ng/day-gp120 groups were 0.43 ± 0.06, 0.48 ± 0.07, 0.83 ± 0.05, and 0.84 ± 0.10, respectively; relative P2X7 protein expression in those groups was 0.63 ± 0.07, 1.08 ± 0.06, 0.90 ± 0.07, and 1.03 ± 0.11, respectively. According to immunohistochemistry analysis, the staining intensity values for P2X7 protein expression in the control, 50-, 70-, and 100-ng/d-gp120 groups were 0.88 ± 0.07, 1.41 ± 0.12, 1.28 ± 0.13, and 1.31 ± 0.10, respectively. The above results showed that the expression of P2X7 increased significantly in the hippocampus of gp120 rats compared to that of the control group. These results suggest that ICV infusion of gp120 can successfully mimic HAD in rats, and P2X7 may be involved in gp120-induced cognitive dysfunction. This could provide a theoretical foundation and potential drug target for research and treatment of ADC.

Clinicopathologic analysis of 2736 salivary gland cases over a 11-year period in Southwest China.

OBJECTIVE: To investigate the epidemiological and clinicopathological characteristics of salivary gland tumors in southwest China in order to provide data for clinical diagnosis and other similar research. METHODS: Between March 2007 and December 2017, 2736 patients with salivary gland tumors were recruited, the clinical and pathological data were retrospectively analyzed. RESULTS: A total of 2736 patients had a ratio of males to females of about 1.02:1. The ratio of benign to malignant tumors was 3.46:1. Pleomorphic adenoma and adenoid cystic carcinoma had 50.8% and 7.2%, respectively. About 65.4% tumors occurred in the parotid gland. There was no significant difference between the tumor in the left or right parotid and the use of cell phones. There were significant differences between gender and both the characteristics and locations of salivary gland tumors (p < .05). There were also significant differences between the pathological characteristics and location of the salivary gland (p < .05). CONCLUSIONS: The salivary gland benign and malignant tumors were more common in pleomorphic adenoma and adenoid cystic carcinoma, most occurred in the parotid gland. The minor gland tumors are lower than other parts of China. The incidence of parotid gland tumors is not related to the use of cell phones.

[Correlation between high risk type human papillomavirus E6/E7 mRNA and cervical cancer].

OBJECTIVE: To investigate the correlation between the positive rate of high risk human papillomavirus(HPV)mRNA E6/E7 and cervical cancer, and provide evidence for the prevention and treatment of cervical cancer. METHODS: A total of 100 cervical cancer cases and 100 healthy controls were selected in our hospital from January 2015 to December 2015. The fluorescence quantitative PCR and pathological examination on HPV E6/E7 mRNA were carried out. The correlation between HPV E6/E7 mRNA and cervical squamous epithelial lesions were analyzed. RESULTS: In case group, the positive rate of HPV E6/E7 mRNA was 76.0%(76/100). In control group, the positive rate was 13.0%(13/100). The positive rate in case group was significantly higher than that in control group, and the difference was statistically significant(χ(2)=24.522, P<0.001). The positive predictive value and negative predictive value of the two groups were compared, and the difference was not significant(P>0.05). The positive rate of HPV E6/E7 mRNA was significantly higher than high-grade squamous intraepithelial lesion(SIL)rate(26.1%), low-grade SIL rate(17.6%)and atypical squamous cell hyperplasia rate(6.7%), the difference was statistically significant(χ(2)=7.615, P= 0.001; χ(2) =9.114, P=0.001; χ(2)=18.241, P<0.001). CONCLUSIONS: The detection rate of HPV E6/E7 mRNA in cervical cancer patients was high. And with the increased severity of cervical squamous epithelial lesions, the positive rate of HPV E6/E7 mRNA increased.

Regulation of estrogen sulfotransferase expression in Leydig cells by cyclic adenosine 3',5'-monophosphate and androgen.

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.

Targeted disruption of the mouse estrogen sulfotransferase gene reveals a role of estrogen metabolism in intracrine and paracrine estrogen regulation.

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.

Inhibition of rat colon contractility by prostacyclin (IP-) receptor agonists: involvement of NANC neurotransmission.

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.

Non-prostanoid prostacyclin mimetics as neuronal stimulants in the rat: comparison of vagus nerve and NANC innervation of the colon.

The spontaneous activity of the rat isolated colon is suppressed by prostacyclin analogues such as cicaprost (IC(50)=4.0 nM). Activation of prostanoid IP(1)-receptors located on NANC inhibitory neurones is involved. However, several non-prostanoids, which show medium to high IP(1) 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 (EC(50)=0.23 nM). Iloprost, taprostene and carbacyclin were 7.9, 66, and 81 fold less potent than cicaprost, indicating the presence of IP(1) as opposed to IP(2)-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 microM), BMY 42393 (3 microM) and ONO-1301 (3 microM) behaved as specific IP(1) 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 EP(3)-receptor agonist. The full versus partial agonism of the non-prostanoid prostacyclin mimetics may be explained by the markedly different IP(1) 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.

Characterization of a prostanoid EP3-receptor in guinea-pig aorta: partial agonist action of the non-prostanoid ONO-AP-324.

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.

Potent contractile actions of prostanoid EP3-receptor agonists on human isolated pulmonary artery.

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.

A study of prostacyclin mimetics distinguishes neuronal from neutrophil IP receptors.

The prostacyclin mimetics BMY 45778 (3-[4-(4,5-diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy]acetic acid), BMY 42393 (2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid) and EP 185 (rac 5-endo-(6'-carboxyhex-2'Z-enyl)-6-exo-(p-methoxyphenyl- phenyl-methylazino)-bicyclo[2.2.2]oct-2-ene) inhibited rat neutrophil aggregation stimulated by N-formyl-methionyl-leucyl-phenylalanine (IC50 = 20, 462, and 1195 nM respectively). In contrast only BMY 45778 (1-10 microM) produced any significant inhibition (10-20%) of the spontaneous activity of rat colon. BMY 45778 (10 microM) also attenuated the inhibitory effect of the prostacyclin analogue cicaprost on rat colon, whereas BMY 42393 and EP 185 did not. BMY 45778 appears to be a low affinity partial agonist at prostacyclin receptors on rat colon and its low potency in rat colon compared with rat neutrophils suggests the presence of a different prostacyclin receptor located on enteric neurones.

[Effects of tetrandrine on rabbit platelet aggregation, thromboxane A2 generation and calmodulin activity].

The effects of tetrandrine (Tet) on platelet aggregation and thromboxane A2 (TXA2) generation were studied in rabbit platelet-rich plasma (PRP) prepared by centrifugation. The effects of Tet on calmodulin activity in platelet extracts were also investigated by measuring calmodulin-sensitive phosphodiesterase activity. ADP, collagen or arachidonic acid (AA)-induced platelet aggregation was inhibited by Tet in a dose-dependent manner. TXA2 generation in PRP treated by Tet was markedly decreased in collagen-induced group, but was not altered in AA-induced group, suggesting that the release of AA from platelet phospholipids stimulated by collagen was blocked by Tet. Further experiments showed that the effects of Tet were related to its inhibition of calmodulin-dependent phosphodiesterase activity. There was evidence that the effects originated from its anti-calmodulin properties instead of its direct action on phosphodiesterase.

Inhibitory effects of tetrandrine on Bay k 8644-stimulated contraction of isolated rabbit aortic strips.

In the presence of KCl 19 mmol.L-1, calcium agonist Bay k 8644 0.47 mumol.L-1 elicited a strong contraction of isolated rabbit aortic strips, and this contraction was concentration-dependently inhibited by tetrandrine; but this antagonism was noncompetitive. Calcium ionophore calcimycin evoked contraction was markedly depressed by tetrandrine. The results suggested that tetrandrine might not only inhibit transmembrane influx of calcium via potential-dependent channels but also interfere with other processes related to calcium.

Glutathione stimulates sulfated estrogen transport by multidrug resistance protein 1.

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.

Structural characterization of mouse CD97 and study of its specific interaction with the murine decay-accelerating factor (DAF, CD55).

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.

Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59.

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.

Characterization of binding of leukotriene C4 by human multidrug resistance protein 1: evidence of differential interactions with NH2- and COOH-proximal halves of the protein.

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 [(3)H]leukotriene C(4) (LTC(4)), 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) LTC(4) labels sites in the NH(2)- and COOH-proximal halves of MRP1, (ii) labeling of the NH(2)-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 NH(2)-proximal site is preferentially inhibited by S-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 LTC(4) is strongly attenuated in the presence of ATP and vanadate, and this decrease in labeling is attributable to a marked reduction in LTC(4) binding to the NH(2)-proximal site, and (vii) the attenuation of LTC(4) binding to the NH(2)-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 LTC(4) binds to one of two sites composed, at least in part, of elements in the NH(2)-proximal half of the protein.

Relaxant actions of nonprostanoid prostacyclin mimetics on human pulmonary artery.

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.

Genomic structure, functional comparison, and tissue distribution of mouse Cd59a and Cd59b.

CD59 is a crucial complement regulatory protein that inhibits the terminal step of the complement activation cascade by interfering with the binding of C9 to C5b-8, thus preventing the formation of the membrane attack complex (MAC). We recently reported that the mouse genome contains two Cd59 genes, while the human and rat genomes each contain only one Cd59 gene (Qian et al. 2000). Here, we describe the genomic structure, comparative activity, and tissue distribution of these two mouse genes, designated Cd59a and Cd59b. The mouse Cd59 genes encompass a total of 45.6 kb with each gene having four exons. Cd59a spans 19 kb, and Cd59b spans 15 kb, with approximately 11.6 kb of genomic DNA separating the two genes. The overall sequence similarity between Cd59a and Cd59b is approximately 60%. The sequence similarity between exon 2, exon 3, and exon 4 and the respective flanking regions between the two genes is over 85%, but exon 1 and its flanking regions are totally different. Comparative studies of the activity of both genes as inhibitors of MAC formation revealed that Cd59b has a specific activity that is six times higher than that of Cd59a. Using polyclonal antibodies specific to either Cd59a or Cd59b, we showed that Cd59a and Cd59b are both widely expressed in the kidneys, brain, lungs, spleen, and testis, as well as in the blood vessels of most mouse tissues. Interestingly, testicular Cd59a appeared to be expressed exclusively in spermatids, whereas Cd59b was expressed in more mature sperm cells. These results suggest that even though Cd59a and Cd59b are expressed in multiple tissues, they may play some different roles, particularly in reproduction.