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This study investigated the effect of corrosion pits on the mechanical degradation of steel Q345 under strong acid corrosion. The experiment on a total of 27 steel Q345 specimens corroded by 36% industrial hydrochloric acid for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h, respectively, is conducted to study failure mode and stress-strain curves. After that, a noncontact topography scanner (DSX500) scanned geometric parameters of corrosion pits in steel Q345 to establish its mechanical degradation. The surface morphology and the corresponding degradation law of corroded steel are also revealed. In addition, the steel plastic fracture criterion taking into account equivalent plastic fracture strain and average stress triaxiality T is adopted to propose the uniform pit model. The analysis results show that the failure mode gradually changes from ductile to brittle. It is noted that the depth and relative size of the corrosion pit are the main factors affecting the increase of the maximum pit coefficient. Further, FEM based on uniform corrosion pits is found by the flexible damage evolution criterion. Its calculation results are entirely consistent with experimental results, indicating that this model can mirror the surface morphology of steel suffering strong corrosion, and simulated accurately stress flow law and necking failure of strong corrosion steel. The proposed criterion is validated with verification results and can provide good references for the design of steel bridges under strong corrosion.

Mechanisms underlying spontaneous locomotor recovery after spinal cord injury (SCI) remain unclear. Using adult zebrafish with complete SCI, we show that V2a interneurons regrow their axon to bridge the lesioned spinal segments in a subclass-specific and chronological order. Early after SCI, reestablishment of a unitary high-rhythm locomotor circuit is driven merely by axon-regrown fast V2a interneurons. Later, the reestablished intraspinal de novo circuit is organized into a modular design by axon-regrown fast and slow V2a interneurons rostral to the lesion, selectively driving caudal fast V2a/motor neurons and slow V2a/motor neurons, respectively. This orderly circuitry reestablishment determines the stepwise restoration of locomotor repertoire and recapitulates developmental processes. This progress can be interrupted by ablation of calretinin, a fast module-related protein, and accelerated by physical training. These findings suggest that promotion of axon regrowth of propriospinal V2a interneurons and establishment of de novo intraspinal circuits underpin the effectiveness of physical training in patients after SCI.

Animals use a precisely timed motor sequence to escape predators. This requires the nervous system to coordinate several motor behaviors and execute them in a temporal and smooth manner. We here describe a neuronal circuit that faithfully generates a defensive motor sequence in zebrafish larvae. The temporally specific defensive motor sequence consists of an initial escape and a subsequent swim behavior and can be initiated by unilateral stimulation of a single Mauthner cell (M-cell). The smooth transition from escape behavior to swim behavior is achieved by activating a neuronal chain circuit, which permits an M-cell to drive descending neurons in bilateral nucleus of medial longitudinal fascicle (nMLF) via activation of an intermediate excitatory circuit formed by interconnected hindbrain cranial relay neurons. The sequential activation of M-cells and neurons in bilateral nMLF via activation of hindbrain cranial relay neurons ensures the smooth execution of escape and swim behaviors in a timely manner. We propose an existence of a serial model that executes a temporal motor sequence involving three different brain regions that initiates the escape behavior and triggers a subsequent swim. This model has general implications regarding the neural control of complex motor sequences.

Aiming at the problems of long lip feature coding and model training time in lip reading research, a distributed deep learning model based on spark is proposed. Firstly, we introduce the Ch-LipNet model structure, and then turn the cyclic layer LSTM network of the Ch-LipNet model into a ConvLSTM network to train and recognize the improved model. Secondly, a deep learning framework based on the keras library in the spark environment is built, and then the Ch-LipNet model and the improved model are built separately. Finally, the training and recognition of distributed deep learning models are carried out. On the lip language dataset NSTDB, the improved model in this paper is compared with the Ch-LipNet model in the local environment, and the recognition accuracy is increased by 15.6%. At the same time, the two models were tested separately in the spark environment, and the results showed that the distributed deep learning model can effectively improve the accuracy and training efficiency of lip reading.

Fluoropolymer is a type of dielectric material that have been used in fabricating Organic Thin Film Transistor (OTFT). Dielectric layer is one of the layers that make OTFT. It is the layer that sort the electric field that can be used. Fluoropolymer have unique characteristics that can be utilized for fabrication of OTFT. Fluoropolymer is low dielectric contact material which is needed in ultra-large-scale integration (ULSI) to breakdown the parasitic capacitance that affect the value of current flow. Drop cast technique has been used as depositing method on interdigitated electrode (IDE) on glass substrate. The length of the channel is varied to study the IV characteristic. Current- voltage (I-V) measurements have been used to measure the resistance at the different channel length. The value of resistivity can give some impact to the OFET device.

The proximal urethra and urinary bladder trigone play important roles in continence. We have previously shown that PGD2 is released from guinea pig bladder urothelium/suburothelium and can inhibit detrusor contractile responses. We presently wished to investigate PGD2 actions in guinea pig out-flow region and the distribution of DP1 /DP2 receptors. The effects of PGD2 on urothelium-intact trigone and proximal urethra contractility were studied in organ bath experiments. Expression of DP1 /DP2 receptor proteins was analysed by western blot. Immunohistochemistry was used to identify distribution of DP1 /DP2 receptors. PGD2 in a dose-dependent manner inhibited trigone contractions induced by electrical field stimulation (EFS) and inhibited spontaneous contractions of the proximal urethra. PGD2 was equally (trigone) or slightly less potent (urethra) compared with PGE2 . Expression of DP1 and DP2 receptors was found in male guinea pig bladder trigone, neck and proximal urethra. In the trigone and proximal urethra, DP1 receptors were found on the membrane of smooth muscle cells and weak immunoreactivty was observed in the urothelium. DP2 receptors were distributed more widespread, weakly and evenly in the urothelium and smooth muscles. Inhibitory effects by PGD2 on motor activity of guinea pig trigone and proximal urethra are consistent with finding DP1 and DP2 receptors located in the urothelium and smooth muscle cells of the trigone and proximal urethra, and PGD2 may therefore be a modulator of the bladder out-flow region, possibly having a function in regulation of micturition and a role in overactive bladder syndrome.

Background The human sulfatase 1 (SULF1) and sulfatase 2 (SULF2) genes modulate cell signaling and homeostasis in many tissues. Gene expression analyses have implicated SULF2 in disease pathogenesis, including Alzheimer's disease (AD), but changes in brain SULF2 expression have not been directly established. Objective To investigate the expression of SULF1 and SULF2 in brain tissues from AD cases and cognitively normal controls. Methods Autopsy tissue from AD cases (n = 20) and age-and gender-matched cognitively normal controls (n = 20) were identified from the Mayo Clinic Alzheimer's Disease Patient Registry neuropathology database. Tissue slides were stained for SULF1 and SULF2 protein expression in the hippocampus and frontal lobe and an expression score computed from the proportion of cells stained and the intensity of staining (range 0 [no expression] to 9 [marked expression]). Results SULF2 expression was reduced in AD cases. Compared to cognitively normal controls, SULF2 expression in AD cases was significantly decreased in the hippocampal Cornu Ammonis (CA) (mean score of 6.5 in cases versus 8.3 in controls; p = 0.003), in the gray matter of the parahippocampal gyrus (5.6 in cases versus 7.6 in controls; p = 0.003), and in the frontal lobe gray matter (5.4 in cases versus 7.4 in controls; p = 0.002). There was no difference in SULF1 expression in the hippocampus or frontal lobe of AD cases and controls. As expected there were no differences in SULF1 or SULF2 expression in white matter in AD cases compared to cognitively normal controls. Conclusion Decresed SULF2 in specific regions of the brain occurs in AD.

Background and Purpose We have described a urothelium-dependent release of PGD2-like activity which had inhibitory effects on the motility of guinea pig urinary bladder. Here, we have pharmacologically characterized the receptors involved and localized the sites of PGD2 formation and of its receptors. Experimental Approach In the presence of selective DP and TP receptor antagonists alone or combined, PGD2 was applied to urothelium-denuded diclofenac-treated urinary bladder strips mounted in organ baths. Antibodies against PGD2 synthase and DP1 receptors were used with Western blots and for histochemistry. Key Results PGD2 inhibited nerve stimulation -induced contractions in strips of guinea pig urinary bladder with estimated pIC50 of 7.55 ± 0.15 (n = 13), an effect blocked by the DP1 receptor antagonist BW-A868C. After blockade of DP1 receptors, PGD2 enhanced the contractions, an effect abolished by the TP receptor antagonist SQ-29548. Histochemistry revealed strong immunoreactivity for PGD synthase in the urothelium/suburothelium with strongest reaction in the suburothelium. Immunoreactive DP1 receptors were found in the smooth muscle of the bladder wall with a dominant localization to smooth muscle membranes. Conclusions and Implications In guinea pig urinary bladder, the main effect of PGD2 is an inhibitory action via DP1 receptors localized to the smooth muscle, but an excitatory effect via TP receptors can also be evoked. The urothelium with its suburothelium might signal to the smooth muscle which is rich in PGD2 receptors of the DP1 type. The results are important for our understanding of regulation of bladder motility.

The urothelium of the bladder has long been recognized as a protective barrier between detrusor and urine. In recent years, it has become more evident that the urothelium plays a role as an active source of mediators. The urothelium can release neurotransmitters and modulators such as acetylcholine, ATP, nitric oxide, prostaglandins and neuropeptides. They exert both excitatory and inhibitory effects in modulating urinary tract motility. In addition, several studies have reported the existence of an urothelium-derived unknown inhibitory factor in the urinary bladder. By the use of a new serial cascade superfusion bioassay on guinea pig ureter, recent studies confirm that the guinea pig bladder urothelium releases a substance with inhibitory bioactivity, which was resistant to treatment with nitric oxide synthase inhibitor and cyclooxygenase inhibitor and to adenosine A1/A2 receptor blockade. Lately, a marked and quickly inactivated novel release of PGD2 from the bladder urothelium was discovered, together with localization of prostaglandin D synthase therein. PGD2 was found to have an inhibitory influence on nerve-induced contractions in guinea pig urinary bladder and on spontaneous contractions in the out-flow region. An altered release of excitatory and inhibitory factors is likely to play an important part in bladder motility disturbances, of which the prostanoids are a notable group. Due to the fact that the bladder is relaxed 99% of the time, not only excitatory mechanisms in the bladder are necessary to study, but also inhibitory mechanisms need considerable attention, which will contribute to the discovery of new targets to treat bladder motility disorders.

The proximal urethra and urinary bladder trigone play important roles in continence. We have previously shown that PGD 2 is released from guinea pig bladder urothelium/suburothelium and can inhibit detrusor contractile responses. We presently wished to investigate PGD 2 actions in guinea pig out‐flow region and the distribution of DP 1/DP 2 receptors. The effects of PGD 2 on urothelium‐intact trigone and proximal urethra contractility were studied in organ bath experiments. Expression of DP 1/DP 2 receptor proteins was analysed by western blot. Immunohistochemistry was used to identify distribution of DP 1/DP 2 receptors. PGD 2 in a dose‐dependent manner inhibited trigone contractions induced by electrical field stimulation (EFS) and inhibited spontaneous contractions of the proximal urethra. PGD 2 was equally (trigone) or slightly less potent (urethra) compared with PGE 2. Expression of DP 1 and DP 2 receptors was found in male guinea pig bladder trigone, neck and proximal urethra. In the trigone and proximal urethra, DP 1 receptors were found on the membrane of smooth muscle cells and weak immunoreactivty was observed in the urothelium. DP 2 receptors were distributed more widespread, weakly and evenly in the urothelium and smooth muscles. Inhibitory effects by PGD 2 on motor activity of guinea pig trigone and proximal urethra are consistent with finding DP 1 and DP 2 receptors located in the urothelium and smooth muscle cells of the trigone and proximal urethra, and PGD 2 may therefore be a modulator of the bladder out‐flow region, possibly having a function in regulation of micturition and a role in overactive bladder syndrome.

Cellular mechanotransduction is crucial for physiological function in the lower urinary tract. The bladder is highly dependent on the ability to sense and process mechanical inputs, illustrated by the regulated filling and voiding of the bladder. However, the mechanisms by which the bladder integrates mechanical inputs, such as intravesicular pressure, and controls the smooth muscles, remain unknown. To date no tools exist that satisfactorily mimic in vitro the dynamic micromechanical events initiated e.g. by an emerging inflammatory process or a growing tumour mass in the urinary tract. More specifically, there is a need for tools to study these events on a single cell level or in a small population of cells. We have developed a micromechanical stimulation chip that can apply physiologically relevant mechanical stimuli to single cells to study mechanosensitive cells in the urinary tract. The chips comprise arrays of microactuators based on the electroactive polymer polypyrrole (PPy). PPy offers unique possibilities and is a good candidate to provide such physiological mechanical stimulation, since it is driven at low voltages, is biocompatible, and can be microfabricated. The PPy microactuators can provide mechanical stimulation at different strains and/or strain rates to single cells or clusters of cells, including controls, all integrated on one single chip, without the need to preprepare the cells. This paper reports initial results on the mechano-response of urothelial cells using the micromechanical stimulation chips. We show that urothelial cells are viable on our microdevices and do respond with intracellular Ca2+ increase when subjected to a micro-mechanical stimulation.

We have described a urothelium-dependent release of PGD2 -like activity which had inhibitory effects on the motility of guinea pig urinary bladder. Here, we have pharmacologically characterized the receptors involved and localized the sites of PGD2 formation and of its receptors.In the presence of selective DP and TP receptor antagonists alone or combined, PGD2 was applied to urothelium-denuded diclofenac-treated urinary bladder strips mounted in organ baths. Antibodies against PGD2 synthase and DP1 receptors were used with Western blots and for histochemistry.PGD2 inhibited nerve stimulation -induced contractions in strips of guinea pig urinary bladder with estimated pIC50 of 7.55 ± 0.15 (n = 13), an effect blocked by the DP1 receptor antagonist BW-A868C. After blockade of DP1 receptors, PGD2 enhanced the contractions, an effect abolished by the TP receptor antagonist SQ-29548. Histochemistry revealed strong immunoreactivity for PGD synthase in the urothelium/suburothelium with strongest reaction in the suburothelium. Immunoreactive DP1 receptors were found in the smooth muscle of the bladder wall with a dominant localization to smooth muscle membranes.In guinea pig urinary bladder, the main effect of PGD2 is an inhibitory action via DP1 receptors localized to the smooth muscle, but an excitatory effect via TP receptors can also be evoked. The urothelium with its suburothelium might signal to the smooth muscle which is rich in PGD2 receptors of the DP1 type. The results are important for our understanding of regulation of bladder motility.

Background While studying a urothelium-derived inhibitory factor in guinea pig urinary bladders we observed considerable release of prostanoids, including PGD 2 -like activity. The present study was carried out to identify the prostanoids and to study their roles in modulating guinea pig urinary bladder motility. Methods Release of PGE 2 and PGD 2 in isolated guinea pig urinary bladder preparations was analyzed by high performance liquid chromatography (HPLC) combined with bioassay on bladder strips. Isolated urothelium-intact (UI) or -denuded (UD) bladder strips were subjected to electrical field stimulation (EFS) and applications of PGE 2 and PGD 2 . Results A resting release of 95 ± 9 (n = 5) ng g tissue − 1 h − 1 PGE 2 -like activity and 210 ± 34 (n = 4) ng g tissue − 1 h − 1 PGD 2 -like activity was found, where PGD 2 -like was subject to marked spontaneous inactivation during isolation. Prostanoids release was decreased by 70–90% by the cyclo-oxygenase inhibitor diclofenac in UI preparations. Urothelium removal decreased prostanoids release by more than 90%. PGE 2 increased basal tone and spontaneous contractions, whereas PGD 2 had little or no effect on these. Exogenous PGE 2 enhanced and PGD 2 inhibited contractile responses to EFS, exogenous acetylcholine- and ATP, whereas PGD 2 caused marked dose-dependent inhibition. PGE 2 and PGD 2 effects were more pronounced in diclofenac-treated UD tissues. Conclusions PGD 2 and PGE 2 are released from guinea pig bladder urothelium and PGD 2 has inhibitory effects on bladder motility, mainly through a postjunctional action on smooth muscle responsiveness. General significance The release and inhibitory effects merit further studies in relation to normal biological function as well as overactive bladder syndrome.

Although controversy remains regarding direct effects of estrogen on bone, in vivo data clearly show that estrogens suppress bone turnover, resulting in decreased bone resorption and formation activity. Selective estrogen receptor modulators (SERMs), such as raloxifene, produce effects on bone which are very similar to those of estrogen. In vitro, both raloxifene and estrogen inhibit mammalian osteoclast differentiation and bone resorption activity, but only in the presence of IL-6. Data from a number of ovariectomized rat model manipulations (i.e. hypophysectomy, low calcium diet and drug combinations) demonstrate a strong parallel between the antiosteopenic effects of raloxifene and estrogen. A characteristic action of estrogens on the skeleton is inhibition of longitudinal bone growth, an effect which is not observed with other resorption inhibitors, including calcitonin and bisphosphonates. Consistent with an estrogen-like mechanism on bone, raloxifene inhibits longitudinal bone growth in growing rats. In addition to the overall similarity of the bone activity profile in animals, estrogen and raloxifene also produce similar effects on various signaling pathways relative to the antiosteopenic effect of these two agents. For example, IL-6, a cytokine involved in high turnover bone resorption following estrogen deficiency in rats, is suppressed by both raloxifene and estrogen. Raloxifene and estrogen also produce a similar activation of TGF-β3 (a cytokine associated with inhibition of osteoclast differentiation and activity) in ovariectomized rats. Like 17β-estradiol, raloxifene binds with high affinity to both estrogen receptor-α (ERα) and estrogen receptor-β (ERβ). Crystal structure analyses have shown that 17β-estradiol and raloxifene bind to ERα with small, but important, differences in three dimensional structure. These subtle differences in the conformation of the ligand:receptor complex are likely the basis for the key pharmacological differences between estrogens and the various SERMs (i.e. raloxifene vs tamoxifen). Raloxifene also produces estrogen-like effects on serum cholesterol metabolism and the vasculature. Thus, while raloxifene exhibits a complete estrogen antagonist in mammary tissue and the uterus, it produces beneficial effects on the cardiovascular system and prevents bone loss via an estrogen receptor mediated mechanism.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator (SERM) which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. In vivo structure-activity relationships and molecular modeling studies have indicated that the orientation of the basic amine-containing side chain of 1, relative to the stilbene plane, is an important discriminating factor for the maintenance of tissue selectivity. We have constructed a series of analogues of 1 in which this side chain is held in an orientation which is orthogonal to the stilbene plane, similar to the low-energy conformation predicted for raloxifene. Herein, we report on the synthesis of these compounds and on their activity in a series of in vitro and in vivo biological assays reflective of the SERM profile. In particular, we describe their ability to (1) bind the estrogen receptor, (2) antagonize estrogen-stimulated proliferation of MCF-7 cells in vitro, (3) stimulate TGF-beta3 gene expression in cell culture, (4) inhibit the uterine effects of ethynyl estradiol in immature rats, and (5) potently reduce serum cholesterol and protect against osteopenia in ovariectomized (OVX) rats without estrogen-like stimulation of uterine tissue. These data demonstrate that one of these compounds, LY357489,4, is among the most potent SERMs described to date with in vivo efficacy on bone and cholesterol metabolism in OVX rats at doses as low as 0.01 mg/kg/d.

Interaction of the estrogen receptor/ligand complex with a DNA estrogen response element is known to regulate gene transcription. In turn, specific conformations of the receptor-ligand complex have been postulated to influence unique subsets of estrogen-responsive genes resulting in differential modulation and, ultimately, tissue-selective outcomes. The estrogen receptor ligands raloxifene and tamoxifen have demonstrated such tissue-specific estrogen agonist/antagonist effects. Both agents antagonize the effects of estrogen on mammary tissue while mimicking the actions of estrogen on bone. However, tamoxifen induces significant stimulation of uterine tissue whereas raloxifene does not. We postulate that structural differences between raloxifene and tamoxifen may influence the conformations of their respective receptor/ligand complexes, thereby affecting which estrogen-responsive genes are modulated in various tissues. These structural differences are 4-fold: ( A ) the presence of phenolic hydroxyls, ( B ) different substituents on the basic amine, ( C ) incorporation of the stilbene moiety into a cyclic benzothiophene framework, and ( D ) the imposition of a carbonyl “hinge” between the basic amine-containing side chain and the olefin. A series of raloxifene analogs that separately exemplify each of these differences have been prepared and evaluated in a series of in vitro and in vivo assays. This strategy has resulted in the development of a pharmacophore model that attributes the differences in effects on the uterus between raloxifene and tamoxifen to a low-energy conformational preference imparting an orthogonal orientation of the basic side chain with respect to the stilbene plane. This three-dimensional array is dictated by a single carbon atom in the hinge region of raloxifene. These data indicate that differences in tissue selective actions among benzothiophene and triarylethylene estrogen receptor modulators can be ascribed to discrete ligand conformations.

Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 β-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor– mediated mechanism.

Raloxifene (LY139481 HCl) is a selective estrogen receptor modulator (SERM) which blocks the effects of estrogen on some tissues, such as the breast and uterus, while mimicking estrogen in other tissues, such as bone. To study the origins of this unique pharmacology, we have prepared the major metabolites of raloxifene as chemical probes for examining the estrogen receptor function in vitro and in vivo . In human breast cancer cell (MCF-7) related assays, these glucuronide conjugates show little affinity for the estrogen receptor and are more than two orders of magnitude less potent at inhibiting cell proliferation than raloxifene. In non-traditional estrogen target tissue, such as bone, these metabolites are less effective than the parent at inhibiting cytokine-stimulated bone resorbing activity in rat osteoclasts or producing transforming growth factor beta-3 (TGF- β 3 ). In animal models, tissue distribution studies with radiolabelled metabolite indicate that conversion to raloxifene occurs readily in a variety of tissues including the liver, lung, spleen, kidney, bone and uterus. Differential conversion of metabolite in target organs, such as bone and the uterus, is not observed indicating that the origin of raloxifene's pharmacology does not result from tissue-selective deconjugation of metabolite to parent.

In our report “Identification of an estrogen response element activated by metabolites of 17β-estradiol and raloxifene” ([30 Aug., p. 1222][1]) ([1][2]), we examined regulation by raloxifene of the human transforming growth factor-β3 (TGF-β3) promoter and proposed a new pathway of gene

In view of its highly tissue-selective pharmacological properties (i.e., relatively pure antagonist in reproductive tissue with minimal agonist effects to nearly full agonist properties in bone and on cholesterol metabolism), terms used to define compounds with slightly related pharmacology (i.e., antiestrogen, partial estrogen agonist) do not adequately describe raloxifene's activity. Thus, raloxifene is distinct from agents such as tamoxifen (which does stimulate the uterus), or frank estrogen (which do not sufficiently antagonize estrogen's agonistic effects in reproductive tissue). In this regard, raloxifene and its pyrrolidine analogue, LY117018, (81) are the first representatives of a novel class of pharmacological agents, which we have termed “selective estrogen receptor modulator” (SERM). While we now have considerable evidence to distinguish estrogen recepto-mediated effects on bone from those on reproductive tissue, the precise mechanism for this tissue-specific mechanism remains an active area of investigation. Clearly, many important issues remain to be explored.

To identify genes involved in retinoic acid signaling during early embryogenesis, specifically during implantation and early postimplantation, cDNA libraries constructed from mouse embryos at e4.5 and e5.5, respectively, have been screened. Based upon DNA sequence homology, one clone has been isolated by using mouse retinoic acid receptor α (RARα) as the probe. This clone, designated as 80.3, is expressed in the embryonic portion at early egg cylinder stage, and is highly expressed in the entire embryo at e6.5. Its expression decreases in embryos older than e9.5 and can not be detected in any adult tissues. In vitro transcription/translation of this cDNA has produced a protein product with a molecular weight of approximately 50 kDa. The central to C-terminal portion of this gene is highly homologous to a human orphan receptor, TR-2. This homologous region contains a potential zinc-finger DNA binding motif followed by a putative ligand-binding domain. However, this gene is very different from TR2 in the N-terminal region and appears to be a newly identified gene with a specific pattern of expression during early embryogenesis.

Transgenic mice with ectopic expression of bovine CRABP under the control of the human metallotheionein IIA promoter have shown a variety of pathological consequences. Expression of the transgene has been detected in most of the tissues examined, including heart, lung, liver, spleen, kidney, intestine, testis, and ovary, except pancreas. Two independent lines have been able to produce normal non-transgenic F1 animals of both sexes but only female transgenic progenies. All of these F1 female transgenic animals derived from both lines are sterile, and the ovaries from these animals appear to be significantly smaller as compared to their non-transgenic littermates. Histopathological examinations have shown no maturing follicles in these transgenic ovaries in which abnormal cells have been observed. Another independent line has generated transgenic F1 animals which have been growing retardly. These animals all have small spleen and liver and have become very sick at the age of 4 to 5 weeks. Histopathological examinations on these transgenic progenies have shown hepatocytes to be reduced in the cytoplasmic portion in which glycogen is highly depleted. The spleen is poorly developed as no well organized germinal centers can be observed in the spleen sections of these transgenic animals.

Our aim was to investigate whether guinea pig urothelium-derived bioactivities compatible with the existence of urothelium-derived inhibitory factor could be demonstrated by in vitro serial bioassay and whether purinergic P1 receptor agonists, nitric oxide, nitrite or prostaglandins might explain observed activities. In a cascade superfusion system, urothelium-denuded guinea pig ureters were used as bioassay tissues, recording their spontaneous rhythmic contractions in presence of scopolamine. Urothelium-intact or -denuded guinea pig urinary bladders were used as donor tissues, stimulated by intermittent application of carbachol before or during the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the adenosine/P1 nucleoside receptor antagonist 8-(p-sulfophenyl)theophylline (8-PST) or the cyclo-oxygenase inhibitor diclofenac infused to bath donor and bioassay tissues. The spontaneous contractions of bioassay ureters were unaltered by application of carbachol 1-5 µM in the presence of scopolamine 5-30 µM. When carbachol was applied over the urothelium-denuded bladder, the assay ureter contraction rate was unaltered. Introducing carbachol over the everted urothelium-intact bladder significantly inhibited the contraction frequency of the assay ureter, suggesting the transfer of an inhibitory activity from the bladder to the assay ureter. The transmissible inhibitory activity was not markedly antagonized by L-NAME, 8-PST or diclofenac, while L-NAME nearly abolished nitrite release from the urothelium-intact bladder preparations. We suggest that urothelium-derived inhibitory factor is a transmissible entity over a significant distance as demonstrated in this novel cascade superfusion assay and seems less likely to be nitric oxide, nitrite, an adenosine receptor agonist or subject to inhibition by administration of a cyclo-oxygenase inhibitor.

In this report, we demonstrate that, in contrast to most previously characterized nuclear receptors, hERR1 and hERR2 (human estrogen receptor-related protein 1 and -2) are constitutive activators of the classic estrogen response element (ERE) as well as the palindromic thyroid hormone response element (TREpal) but not the glucocorticoid response element (GRE). This intrinsically activated state of hERR1 and hERR2 resides in the ligandbinding domains of the two genes and is transferable to a heterologous receptor. In addition, we show that members of the p160 family of nuclear receptor coactivators, ACTR (activator of thyroid and retinoic acid receptors), GRIP1 (glucocorticoid receptor interacting protein 1), and SRC-1 (steroid receptor coactivator 1), potentiate the transcriptional activity by hERR1 and hERR2 in mammalian cells, and that both orphan receptors bind the coactivators in a ligand-independent manner. Together, these results suggest that hERR1 and hERR2 activate gene transcription through a mechanism different from most of the previously characterized steroid hormone receptors. (Molecular Endocrinology 13: 2151‐2162, 1999)

Phosphorylation of eukaryotic translation initiation factor-2alpha (eIF-2alpha) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2alpha kinases including mammalian double-stranded RNA-dependent eIF-2alpha kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2alpha kinases including phosphorylation of eIF-2alpha in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2alpha kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.

The yeast URA3 gene was used as a reporter to investigate the activities of estrogenic and antiestrogenic compounds in yeast Saccharomyces cerevisiae. The control sequences of the wild type (wt) URA3 promoter were replaced with zero, two, or six copies of estrogen-response elements (ERE). Insertion of two and six copies of ERE rendered the expression of the URA3 gene to be dependent on the presence of the human estrogen receptor (ER) and the hormone 17β-estradiol (E2). Two versions of the ER genes were constructed: a full-length wild-type ER (ERa-f) and a truncated ER with domains C, D, and E (ERcde). Both forms of the ER were able to activate the ERE-URA3 reporter in a hormone-dependent manner. The growth of yeast transformants were hormone-dependent when the reporter constructs were inserted into chromosomes using yeast integrating vectors (YIp) but not with the 2μ-based episomal (high-copy number, YEp) or centromeric (low-copy number, YCp) vectors. The integrated transformants were employed to investigate the effects of estrogenic and antiestrogenic compounds. The estrogenic compounds, E2, diethylstilbestrol (DES), and estrone (EST), activated expression of the reporter genes at 1 nM concentration, which is the same concentration exhibiting activity in mammalian cells. None of the antiestrogens, at concentrations up to 1 μM, including tamoxifen (TAM), raloxifene (RAL), and ICI 164,384 (ICI) antagonized 1 nM of E2 against either form of the ER. In fact, TAM, RAL, and ICI displayed slight agonistic activity at high concentrations of 300 nM or greater to the ERcde. This system can be used to investigate or clone the missing factor(s) that is responsible for the antagonistic activity of the ER in yeast, and is also suitable for screening for the effectors of the ER.

Estrogen or raloxifene (LY156758) prevent estrogen deficiency-induced bone loss in animals and humans. We demonstrated in the rat that a 22% reduction in bone mineral density generated by ovariectomy was associated with a 2-fold reduction of transforming growth factor-beta 3 (TGF beta 3) messenger RNA expression in the femur. Administration of 17 beta-estradiol or raloxifene to ovariectomized rats restored both bone mineral density and TGF beta 3 messenger RNA expression in the femur to levels measured in intact animals. In transient transfection assays, the promoter sequence from -38 to + 110 of the human TGF beta 3 gene, which contains no palindromic estrogen response element, was sufficient to mediate 17 beta-estradiol or raloxifene induced-reporter gene expression in presence of the estrogen receptor. Raloxifene activated TGF beta 3 promoter as a full agonist at nanomolar concentrations. In the same cellular system, raloxifene inhibited the estrogen response element-containing vitellogenin promoter expression as a pure estrogen antagonist. In two well characterized osteoclast differentiation models, TGF beta 3 significantly inhibited the differentiation and bone-resorptive activities of murine and avian osteoclasts. These findings suggest that regulation of TGF beta 3 gene expression by raloxifene or estrogen in bone may be an important target to mediate bone maintenance.

The mutagenic activity of mustard gas first implicated DNA as an important site of biological alkylation in 1946.l Subsequently, other alkylating agents also have been studied in an attempt to relate the chemical nature of DNA alkylation to the observed biological changes.2~3 While cross-linking of DNA strands has been implicated as an important reaction for bifunctional agents such as mustard gas [bis(2-chloroethyl) sulfide] ,4 different mechanisms have been proposed for compounds capable of only single site attack. 2-Chloroethyl ethyl sulfide is a monofunctional alkylating agent capable of both mutagenic and lethal effects in

An unusual morphine analog was extracted and separated from an opium-like sample. The high resolution mass spectrum of the unknown substance confirmed the empirical formula as C17H18O2NCl. Mass spectra and retention factor values in thin-layer chromatography of both unknown and authentic standards were compared, which lead to the identification of the unknown as β-chloromorphide.