Increase In Wet Weight Research Articles

NPY Y2 receptor agonist PYY(3-36) inhibits diarrhea by reducing intestinal fluid secretion and slowing colonic transit in mice

Peptide YY (PYY)(3-36), a neuropeptide Y (NPY) Y2 receptor agonist, is a powerful inhibitor of intestinal secretion. Based on this anti-secretory effect, NPY Y2 receptor agonists may be useful as novel anti-diarrheal agents, but anti-diarrheal efficacy has yet to be determined. We therefore examined the anti-diarrheal efficacy of PYY(3-36) and a selective Y2 receptor agonist, N-acetyl-[Leu28, Leu31]-NPY(24-36), in experimental mouse models of diarrhea. Intraperitoneal administration of PYY(3-36) (0.01–1 mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10 mg/kg) significantly inhibited diarrhea (increase in wet fecal weight and diarrhea score) induced by dimethyl-prostaglandin E2, 5-hydroxytryptamine, and castor oil. Anti-diarrheal activities of PYY(3-36) and N-acetyl-[Leu28, Leu31]-NPY(24-36) were comparable to the effects of loperamide (1 mg/kg), a widely used anti-diarrheal drug. To clarify the anti-diarrheal mechanisms of NPY Y2 receptor agonists, we investigated the effects of PYY(3-36) and N-acetyl-[Leu28, Leu31]-NPY(24-36) on intestinal fluid secretion and colonic transit. PYY(3-36) (1 mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10 mg/kg) significantly reduced dimethyl-prostaglandin E2-induced intestinal fluid accumulation in conscious mice, suggesting that NPY Y2 receptor agonists inhibit diarrhea, at least in part, by reducing intestinal secretion. In addition, PYY(3-36) (0.01–1 mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10 mg/kg) potently inhibited normal fecal output, suggesting that NPY Y2 receptor activation inhibits colonic motor function and NPY Y2 receptor agonists inhibit diarrhea partly by slowing colonic transit. These results indicate that NPY Y2 receptor agonists inhibit diarrhea in mice by not only reducing intestinal fluid secretion, but also slowing colonic transit, and illustrate the therapeutic potential of NPY Y2 receptor agonists as effective treatments for diarrhea.

Loss of MicroRNA Leads to Abnormal Function and Development of the Adult Mouse Uterus.

MicroRNAs (miRNA) are 19-22 nucleotide long, non-coding RNA molecules that post-transcriptionally regulate gene expression by base-pairing with the 3′untranslated region of complementary messenger RNA targets. To date, over 700 miRNAs have been identified in animal cells and have been reported to play a role in numerous vital processes, including: embryonic development, cellular differentiation and proliferation, and apoptosis. Our laboratory recently demonstrated that conditional deletion of Dicer in ovarian granulosa cells and derivatives of the Mullerian duct (i.e. oviduct, uterus, and cervix) by Cre/loxP recombination (Dicerfl/fl;Amhr2-Cre, Dicer-cKO) led to female infertility. This was shown to be the result of a severe oviductal developmental defect that prevented fertilized embryos from entering the uterus. While the uterus of Dicer-cKO females was also disrupted, as evidenced by shortened uterine horns and reduced numbers of uterine glands, its functional ability to establish and maintain pregnancy is not yet known. We first evaluated whether the uterus of pseudopregnant mice could undergo a decidual reaction. To test uterine decidualization, sesame oil was injected into one uterine horn of 3.5 day pseudopregnant mice, while the contralateral horn served as the non-injected control. We next completed an embryo transfer experiment, where 5 blastocysts collected from naturally mated CD-1 mice on day 3.5 (day 0.5 = plug) were transferred into each uterine horn of day 2.5 pseudopregnant adult female Dicer-cKO (n=13) and wild-type mice (Dicerfl/fl or Dicerfl/wt littermates lacking the Amhr2-Cre allele, n=7). Recipient females were then sacrificed on day 7 of pregnancy and the number of implantation sites per mouse recorded. Results of the decidualization experiment indicated that uteri of Dicer-cKO mice (n=11) failed to exhibit a normal decidualization response when compared to wild-type mice (n=7), which exhibited a 3.6-fold increase (p<0.05) in uterine wet weight (mg) after body weight (g) correction. The uteri were 11.8 ± 4.3 mg and 19.7 ± 3.8 mg for the non-injected and 15.0 ± 6.1 mg and 70.8 ± 28.0 mg for sesame oil injected, for the Dicer cKO and wild-type mice, respectively. In the embryo transfer experiment, implantation occurred in 4 of 13 Dicer-cKO recipient females. Interestingly, all implantation sites in the pregnant Dicer-cKO mice were located at the cervical end of the uterine horns. Implantation sites in Dicer-cKO mice appeared less vascular, as evidenced by a blanched color in comparison to the dark red implantation sites observed in wild-type mice. Embryos failed to space appropriately in the two Dicer-cKO recipients with multiple embryos. Histological analysis, however, indicated that the uteri of the pregnant Dicer-cKO mice were able to mount what appeared to be a normal decidualization response with normal embryonic development. We next examined adult female Dicer-cKO uterine tissue histology at 60, 180, 230, and 330 days of age. Uteri from Dicer-cKO mice exhibited adenomyosis, a condition of abnormal endometrial growth within the myometrial layer, which progressively worsened as these animals aged. In conclusion, uterine function in Dicer-cKO mice was compromised and miRNA mediated post-transcriptional gene regulation appears to play a significant role in normal uterine development and function. (poster)

Effects of salinity, light and temperature on growth rates of two species of Gracilaria (Rhodophyta)

Effects of temperature, salinity and light intensity on growth rates of Gracilaria lichenoides and G. tenuistipitata var. liui Zhang et Xia were tested. Eight to ten levels of each factor were first tested separately. The best growth rate was obtained under the conditions of 32°C, 30 and 240 μmol/(m2·s) for G. lichenoides, and 24°C, 20 and 200 μmol/(m2·s) for G. tenuistipitata, respectively. Then a uniform design was used to evaluate the optimal combinations of the three factors. The best conditions for the highest daily specific growth rates (% increase in wet weight) are determined to be 31.30°C, 32.10, and 287.23 μmol/(m2·s) for G. lichenoides (16.26%/d), and 25.38°C, 21.10, and 229.07 μmol/(m2·s) for G. tenuistipitata (14.83%/d), respectively.

First spermiation and spermatozoa concentration in hemicastrated rats.

Male rats ten days of age were divided in three groups: a) hemicastrated, b) sham operated and c) control. They were daily sacrificed between 30 and 50 days of age. Wet weight and dry weight were determined in testis, seminal vesicles and epididymis. The concentration of spermatozoa was measured in testis and caput epididymis. The results show a significant increase in both wet and dry weight of testis of hemicastrated animals (40.5% at 30 days of age). The total population of psermatozoa per testis is higher in hemicastrated after 48 days of age. The first arrival of spermatozoa to the caput epididymis (first spermiation) which occurs in control at 45 days of age is delayed 48 hours in the hemicastrated group, however at 50 days of age the concentration of spermatozoa in the caput is higher than in control. We related our results to the rise in plasma FSH which occurs immediately after hemicastration and involved a larger and higher exposure of the testis to FSH.

Hepatic gene expression following consumption of soy protein isolate in female Sprague–Dawley rats differs from that produced by 17β-estradiol treatment

Although soy foods have been recognized as an excellent source of protein, there have been recent concerns regarding potential adverse effects of isoflavone phytochemicals found in soy products, which are known to bind and activate estrogen receptors. Here, we used global hepatic gene expression profiles in ovariectomized female Sprague-Dawley rats treated with 17beta-estradiol (E(2)) or fed with soy protein isolate (SPI) as a means of estimating potential estrogenicity of SPI. Female Sprague-Dawley rats were fed AIN-93G diets containing casein (CAS) or SPI starting at postnatal day (PND) 30. Rats were ovariectomized on PND 50 and infused with E(2) or vehicle in osmotic pumps for 14 d. Microarray analysis was performed on liver using Affymetrix GeneChip Rat 230 2.0. Serum E(2) levels were within normal ranges for the rat and SPI feeding did not increase uterine wet weight in the absence or presence of E(2). SPI feeding altered (P<0.05, >or=+/-1.5-fold) the expression of 82 genes, while E(2) treatment altered 892 genes. Moreover, only 4% of E(2)-affected genes were also modulated by SPI, including some whose expression was reversed by SPI feeding. The interaction between E(2) and SPI uniquely modulated the expression profile of 225 genes including the reduction of those involved in fatty acid biosynthesis or glucocorticoid signaling and an induction of those involved in cholesterol metabolism. The different hepatic gene signatures produced by SPI feeding compared with E(2) and the lack of increase in uterine wet weight in rats fed with SPI suggest that SPI is not estrogenic in these tissues.

Weaning of juvenile seahorses Hippocampus erectus Perry, 1810 from live to frozen food

In this study, the weaning of juvenile seahorses Hippocampus erectus from live Artemia to frozen Mysis spp. at different ages and sizes was evaluated as a means of improving commercial culture efficacy. The incremental increases in wet weight and standard length of the 42, 49, 56, 63 and 70 day-post-partum juvenile seahorses over a 14-day period were significantly different ( F 4, 45 = 47.359, F 4, 45 = 32.108, P < 0.05), with 70 day-post-partum juveniles having highest specific growth rate (SGR) and weight gain (WG), at 5.7 ± 1.2 and 122.3 ± 18.7%, respectively. Wet weight decreased in juveniles of 42 and 49 day-post-partum at 4–6 days after being weaned. The incremental growth rate of the same age juveniles of different body sizes also differed significantly ( F 3, 36 = 27.185 in wet weight, F 3, 36 = 19.424 in standard length, P < 0.05); the larger the juveniles, the higher their SGR and WG. The difference in body size and wet weight between the large size and small size juveniles increased after weaning to Mysis spp., with daily growth rate in wet weight 0.07 ± 0.003 g/day in large juveniles and 0.02 ± 0.004 g/day in small juveniles from 63 to 112 days post-partum. Body size and wet weight of the juveniles appear to be an important factor in deciding an appropriate time for weaning in commercial seahorse culture. The present study suggests that for juvenile H. erectus, weaning to frozen food should occur at approximately 0.54–0.70 g in wet weight or 6.59–7.46 cm in standard length.

Rolipram, a specific type‐4 phosphodiesterase inhibitor, inhibits cyclophosphamide‐induced haemorrhagic cystitis in rats

To investigate the protective roles of type 4 phosphodiesterase (PDE4) inhibitor in cyclophosphamide (CYP)-induced haemorrhagic cystitis, as the PDE4 inhibitor has anti-inflammatory effects but its characterization is still unknown in urinary tract diseases. In female Sprague-Dawley rats, CYP was administered intraperitoneally and bladders were harvested 24 h after CYP injection. In another group, rolipram as a PDE4 inhibitor was administered before CYP treatment. The effects and mechanisms of CYP with/without rolipram pretreatment were evaluated by microscopic features, bladder wet weight, myeloperoxidase (MPO) activity, nitric oxide (NO)-metabolite production and expression levels of inflammation-related genes. CYP injection resulted in severe cystitis. Pretreatment with rolipram significantly reduced the increase in bladder wet weight and MPO activity, and ameliorated histological inflammatory changes caused by CYP. The levels of inflammation-related transcripts including inducible NO synthase (iNOS), interleukin-1beta and tumour necrosis factor-alpha, induced by CYP, were down-regulated significantly by pretreatment with rolipram. Also, rolipram reduced the NO-metabolite production and iNOS protein expression in the immunohistochemical examination. These results indicate that rolipram can attenuate the development of CYP-induced cystitis in rats by suppressing cytokine production and iNOS induction. Thus, treatment with PDE4 inhibitor has potential clinical implications of the prevention of bladder inflammatory diseases.

Prolonged oestrogen treatment does not correlate with a sustained increase in anterior pituitary mitotic index in ovariectomized Wistar rats.

Oestrogen is a powerful mitogen that is believed to exert a continuous, dose-dependent trophic stimulus at the anterior pituitary. This persistent mitotic effect contrasts with corticosterone and testosterone, changes in the levels of which induce only transient, self-limiting fluctuations in pituitary mitotic activity. To further define the putative long-term trophic effects of oestrogen, we have accurately analysed the effects of 7 and 28 days oestrogen treatment on anterior pituitary mitotic activity in ovariectomized 10-week-old Wistar rats using both bromodeoxyuridine (BrdU) and timed colchicine-induced mitotic arrest. An oestrogen dose-dependent increase in mitotic index was seen 7 days after the start of treatment as expected, representing an acceleration in gross mitotic activity from 1·7%/day in ovariectomized animals in the absence of any oestrogen replacement to 3·7%/day in the presence of a pharmacological dose of oestrogen (50 mcg/rat per day: ∼230 mcg/kg per day). Despite continued exposure to high-dose oestrogen and persistence of the increase in pituitary wet weight, the increase in mitotic index was unexpectedly not sustained. After 28 days of high-dose oestrogen treatment, anterior pituitary mitotic index and BrdU-labelling index were not significantly different from baseline. Although a powerful pituitary mitogen in the short term, responsible, presumably, for increased trophic variability in oestrus cycling females, these data indicate that in keeping with other trophic stimuli to the pituitary and in contrast to a much established dogma, the mitotic response to longer-term high-dose oestrogen exposure is transient and is not the driver of persistent pituitary growth, at least in female Wistar rats.

Effects of Various Selective Estrogen Receptor Modulators with or without Conjugated Estrogens on Mouse Mammary Gland

Selective estrogen receptor modulators (SERMs) are small molecules that, depending on the end point measured, may either function as estrogen receptor (ER) agonists or antagonize estrogens' agonist activity. A key feature of SERMs is the inhibition of ER agonist action on the uterus and mammary gland, but the degree of antagonism varies among compounds and end points. Bazedoxifene is a SERM that is being clinically evaluated both as a monotherapy for the prevention and treatment of osteoporosis and in combination with conjugated estrogens (CEs) for the treatment of menopausal symptoms and prevention of osteoporosis. The studies reported here compare the relative ER agonist and antagonist effects of three pharmacologically distinct SERMs (bazedoxifene, raloxifene, and lasofoxifene) on the ovariectomized mouse when administered alone or as a tissue-selective estrogen complex, a term used to describe the partnering of a SERM and one or more estrogens. At the minimum dose required to maximally reduce CE-stimulated uterine wet weight increase for each SERM, the degree of inhibition varied among the SERMs, with a rank order of bazedoxifene approximately raloxifene > lasofoxifene, in which only bazedoxifene was statistically similar to vehicle. In the mammary gland, in which amphiregulin mRNA and morphological effects were measured, bazedoxifene generally exhibited less agonist activity and was a more effective antagonist of CE than raloxifene or lasofoxifene. In summary, in an animal model evaluating estrogen-modulated uterine effects and mammary gland development, bazedoxifene exhibited less ER agonist activity than raloxifene or lasofoxifene, and, as a tissue-selective estrogen complex, bazedoxifene/CE demonstrated less mammary gland stimulation than raloxifene/CE and lasofoxifene/CE.

Effects of bisphenol A on fecundity, egg hatchability, and juvenile growth of Marisa cornuarietis

Recent work on the snail Marisa cornuarietis has claimed to show endocrine disruption in response to bisphenol A (BPA). The present experimental design was optimized to detect effects of BPA on fecundity, egg hatchability, and juvenile growth, with an emphasis on reproduction, since previous studies suggested this to be a sensitive endpoint. No differences in eggs/female/ month between unexposed snails and snails exposed to nominal concentrations of 0.1, 1, 25, and 640 microg BPA/L during six months of exposure were found. No effect of BPA on the percentage of eggs hatching successfully was found, as was no difference in time to hatching between the control and any BPA treatment. We observed a significant decrease in female growth and a marginal effect on female wet weight in the 640-microg/L treatment compared to the control and a significant increase in male growth rate and a marginal increase in male wet weight in the 1-microg/L treatment compared to the control. However, a much greater proportion of the variability in juvenile growth was explained by variation between pairs and between siblings from the same pair than by BPA treatment. We conclude that effects of BPA in the nominal exposure range 0.1 to 640 microg/L (measured range 0.062-696 microg/L) are unlikely to be of significance for field populations of this species. An additional adult fecundity trial at 22 degrees C (in contrast to all other experiments that were conducted at 25 degrees C) found no evidence to suggest that snails are more sensitive to BPA at the lower temperature, as has been previously claimed. The present results indicate that the sensitivity of M. cornuarietis to BPA is similar to that of other aquatic invertebrates for which data are available.

&lt;i&gt;In vivo&lt;/i&gt; and &lt;i&gt;in vitro&lt;/i&gt; effects of &lt;i&gt;Bidens pilosa&lt;/i&gt; l. (asteraceae) leaf aqueous and ethanol extracts on primed-oestrogenized rat uterine muscle.

Bidens pilosa L. is an Asteraceae growing in tropical zones, and traditionally utilized worldwide in herbal medicine. The present work is based on its traditional use during child birth as a labour facilitator. In vivo tests of acute toxicity showed a weak toxic effect for both extracts but the toxicity of the ethanol extract (LD(50)=6.15 g/kg) was upper than that of the aqueous extract (LD(50)=12.30 g/kg). The three-days uterotrophic assay on immature mice showed body weight gain followed by a concentration-dependent decrease up to 4 mg/g and a concentration dependent uterine wet weight increase. The ethanol extract exhibited the higher body weight gain representing 22.8+/-0.7%, (P < or = 0.001), at the concentration of 500 microg/g/day, while the aqueous extract was significantly more efficient on the uterine wet weight gain of 0.24+/-0.001% (P < or = 0.05), at the concentration of 1000 microg/g/day. In vitro isometric contraction measurement of oestrogen-primed rat uterine strips showed a significant high aqueous extract-induced contractile effect from 0.03-1.97 mg/ml: on the amplitude of contraction (EC(50) = 0.44+/-0.10 mg/ml, P < or = 0.05), and on the rate (1.21+/-0.25 mg/ml, P > 0.05). Inspite of the higher effect of the aqueous extract on the tonus (57.23+/-23%), the ethanol extract showed a high effect (EC(50)=0.34+/-0.09 mg/ml, P < or = 0.05). The weak toxicity, the estrogenic-like and the oxytocic-like activities observed could explain the empirical use of Bidens pilosa leaf aqueous extract as an uterotonic preparation to enhance labour, probably due to the presence of biologically active compound(s) which act directly on the uterine muscle.

On the use of dry seaweeds and condensed phytoplankton as diets for the clam Venerupis aurea, Egypt

ne of the most commercially important species of the marine clam Venerupis aurea was fed on dry preparations of seaweeds (Enteromorpha intestinalis and Ulva lactuca) and condensed preparation of phytoplankton. Daily growth rate (DGR) of clams as live wet weight increase was calculated to determine the effect of different diets. Comparing DGR of feeding bivalves with that of control, some variations were recorded. Higher growth over control was obtained for juvenile Venerupis aurea (3-6 mm) fed on phytoplankton and Enteromorpha intestinalis. For the clam size 6.1-9 mm, the bivalves fed on Enteromorpha intestinalis and Ulva lactuca showed higher growth. The largest clam size (9.1-12 mm) which was fed on the previous diets, failed to attain the same growth as control. So, the differentiation in growth was related to size of clam and also to type of the diet.

Effectiveness of liposomal- N-acetylcysteine against LPS-induced lung injuries in rodents

Acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS) are frequent complications in critically ill patients and are responsible for significant morbidity and mortality. So far, experimental evidence supports the role of oxidants and oxidative injury in the pathogenesis of ALI/ARDS. In this study, the antioxidant effects of conventional N-acetylcysteine (NAC) and liposomally entrapped N-acetylcysteine (L-NAC) were evaluated in experimental animals challenged with lipopolysaccharide (LPS). Rats were pretreated with empty liposomes, NAC, or L-NAC (25 mg/kg body weight, iv); 4 h later were challenged with LPS ( E. coli, LPS 0111:B4) and sacrificed 20 h later. Challenge of saline (SAL)-pretreated animals with LPS resulted in lung injury as evidenced by increases in wet lung weight (edema), increases in lipid peroxidation (marker of oxidative stress), decreases of lung angiotensin-converting enzyme (ACE) (injury marker for pulmonary endothelial cells) and increases in the pro-inflammatory eicosanoids, thromboxane B 2 and leukotriene B 4. The LPS challenge also increased pulmonary myeloperoxidase activity and chloramine concentrations indicative of neutrophil infiltration and activation of the inflammatory response. Pretreatment of animals with L-NAC resulted in significant increases in the levels of non-protein thiols and NAC levels in lung homogenates ( p <0.05) and bronchoalveolar lavage fluids ( p < 0.001), respectively. L-NAC was significantly ( p < 0.05) more effective than NAC or empty liposomes in attenuating the LPS-induced lung injuries as indicated by the aforementioned injury markers. Our results suggested that the delivery of NAC as a liposomal formulation improved its prophylactic effectiveness against LPS-induced lung injuries.

The Relationship between Prenatal PCB Exposure and Intelligence (IQ) in 9-Year-Old Children

BackgroundSeveral epidemiologic studies have demonstrated relationships between prenatal exposure to polychlorinated biphenyls (PCBs) and modest cognitive impairments in infancy and early childhood. However, few studies have followed cohorts of exposed children long enough to examine the possible impact of prenatal PCB exposure on psychometric intelligence in later childhood. Of the few studies that have done so, one in the Great Lakes region of the United States reported impaired IQ in children prenatally exposed to PCBs, whereas another found no association.ObjectivesThis study was designed to determine whether environmental exposure to PCBs predicts lower IQ in school-age children in the Great Lakes region of the northeastern United States.MethodsWe measured prenatal exposure to PCBs and IQ at 9 years of age in 156 subjects from Oswego, New York. We also measured > 50 potential predictors of intelligence in children, including repeated measures of the home environment [Home Observation for Measurement of the Environment (HOME)], socioeconomic status (SES), parental IQ, alcohol/cigarette use, neonatal risk factors, and nutrition.ResultsFor each 1-ng/g (wet weight) increase in PCBs in placental tissue, Full Scale IQ dropped by three points (p = 0.02), and Verbal IQ dropped by four points (p = 0.003). The median PCB level was 1.50 ng/g, with a lower quartile of 1.00 ng/g and an upper quartile of 2.06 ng/g. Moreover, this association was significant after controlling for many potential confounders, including prenatal exposure to methylmercury, dichlorodiphenyldichloroethylene, hexachlorobenzene, and lead.ConclusionsThese results, in combination with similar results obtained from a similar study in the Great Lakes conducted 10 years earlier, indicate that prenatal PCB exposure in the Great Lakes region is associated with lower IQ in children.

Declination of Copper Toxicity in Pigeon Pea and Soil System by Growth-Promoting Proteus vulgaris KNP3 Strain

The copper-resistant (1318 microM CuSO(4).5H(2)O) strain KNP3 of Proteus vulgaris was isolated from soil near the Panki power plant, Kanpur, India, and was used to inoculate pigeon pea (Cajanus cajan var. UPS-120) seeds grown in soil for 60 days in the presence of 600 microM CuSO(4).5H(2)O. A study of siderophore production (126.34 +/- 0.52 microg ml(-1)) and its subsequent effects on plant growth promotion under in situ conditions was conducted. The parameters that were monitored included the plants' wet weight, dry weight, shoot length, chlorophyll content, and concentration of copper in plant roots and shoots. The results showed that the strain caused a significant (p < 0.05) increase in wet weight, dry weight, root length, shoot growth, and chlorophyll content (57.8%, 60%, 19.7%, 47.8%, and 36.3%, respectively) in the presence of copper. Furthermore, the strain reduced accumulation of Cu in the roots and shoots to 36.8% and 60.5%, respectively. Apart from this, copper concentration in the soil was measured on 0, 7, 15, 30, and 45 days consecutively and the results indicated that the bioinoculant KNP3 causes a significant decrease in Cu concentration in soil (55.6%), which was unlikely in the control (10.5%) treatment. The data suggested that the bacterial strain has the ability to protect plants against the inhibitory effects of copper besides reducing the copper load of the soil.

Activity of three selective estrogen receptor modulators on hormone-dependent responses in the mouse uterus and mammary gland

Selective estrogen receptor modulators (SERMs) have the unique potential to provide estrogenic effects in the skeletal and cardiovascular system, while minimizing/eliminating side effects on reproductive organs. However, despite the unifying characteristic of mixed estrogen receptor (ER) agonist/antagonist activity, compounds within this class are not interchangeable. In order to define and compare the effects of SERMs on different hormone-responsive tissues, we evaluated effects of bazedoxifene acetate (BZA), lasofoxifene (LAS) and raloxifene (RAL) in the mammary gland and uterus of the ovariectomized mouse. Endpoints measured included those regulated by estradiol alone (uterine wet weight, uterine G protein-coupled receptor 105 (GPR105) mRNA expression and mammary gland indoleamine-pyrrole 2,3 dioxygenase (INDO) mRNA expression) as well as others that required the combination of estradiol and progesterone (uterine serine protease inhibitor Kazal type 3 (Spink3) mRNA expression, mammary gland morphology and mammary gland defensin beta1 (Defbeta1) mRNA expression). The three SERMs tested had variable agonist and antagonist activity on these endpoints. In the uterus, the SERMs were mixed agonists/antagonists on estradiol-induced wet weight increase, whereas all three SERMs were estrogen receptor antagonists on GPR105 mRNA expression. However, in the presence of progesterone, BZA and RAL were agonists on Spink3 expression, while LAS was primarily an antagonist. In the mammary gland, BZA and RAL were predominantly agonists on the endpoint of mammary morphology and all three SERMs were clear agonists on Defbeta1 mRNA expression, an E+P-dependent marker. Finally, LAS and RAL had mixed agonist/antagonist activity on INDO mRNA expression, while BZA had only antagonist activity. These results demonstrate that compounds with small structural differences can elicit distinct biological responses, and that in general, SERMs tended to behave more as antagonists on endpoints requiring estrogen alone and agonists on endpoints requiring the combination of estrogen and progesterone.

Acute Inhalation Exposure to Vaporized Methamphetamine Causes Lung Injury in Mice

Methamphetamine (MA) is currently the most widespread illegally used stimulant in the United States. Use of MA by smoking is the fastest growing mode of administration, which increases concerns about potential pulmonary and other medical complications. A murine exposure system was developed to study the pulmonary affects of inhaled MA. Mice were exposed to 25–100 mg vaporized MA and assessments were made 3 h following initiation of exposure to model acute lung injury. Inhalation of MA vapor resulted in dose-dependent increases in MA plasma levels that were in the range of those experienced by MA users. At the highest MA dose, histological changes were observed in the lung and small but significant increases in lung wet weight to body weight ratios (5.656 ± 0.176 mg/g for the controls vs. 6.706± 0.135 mg/g for the 100 mg MA-exposed mice) were found. In addition, there was 53% increase in total protein in bronchoalveolar lavage (BAL) fluid, greater than 20% increase in albumin levels in the BAL fluid, greater than 2.5-fold increase in lactate dehydrogenase levels in the BAL fluid, and reduced total BAL cell numbers (approximately 77% of controls). Levels of the early response cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 were dose-dependently increased in BAL fluid of MA-exposed mice. Exposure to 100 mg MA significantly increased free radical generation in the BAL cells to 107–146% of controls and to approximately 135% of the controls in lung tissue in situ. Together, these data show that acute inhalation exposure to relevant doses of volatilized MA is associated with elevated free radical formation and significant lung injury.

244. Effects of relaxin treatment on hyaluronic synthase expression in the cervix of pregnant relaxin-deficient (Rln-/-) mice

The cervical extracellular matrix (ECM) changes extensively in a process called cervical ripening in late pregnancy. This occurs through a complex series of processes leading to collagen fibre degradation and dispersal. We tested the hypothesis that relaxin increases cervical water content by stimulating hyaluronic synthase (HAS) enzymes that regulate hyaluronic acid (HA) synthesis. HA is thought to recruit water molecules into interstitial spaces between collagen fibrils, causing them to disperse. In the first study, cervices were collected from Rln+/+ and Rln−/− mice on days 14.5, 16.5, 18.5 and 19 gestation, and day 1 postpartum (PP) to compare has expression between the genotypes by qPCR analysis. In the second study, Rln−/− mice were implanted with Alzet osmotic minipumps on day 12.5 gestation to infuse either recombinant H2 human relaxin (200 μg/mL; BAS Medical Inc.) or 0.9% saline at a continuous flow rate of 0.5 μl/h. Cervices were collected after 4 or 6 days of infusion. has1 and has2 gene expression increased significantly at term and decreased immediately postpartum in the Rln+/+ mice. Although has1 and has2 were expressed in Rln−/− mice, there was no increase in expression on day 18.5 gestation and cervix wet weight did not increase compared with Rln+/+ mice. Relaxin infusion for 4 or 6 days in pregnant Rln−/− mice significantly increased cervical has2 but decreased has1 expression compared with saline-controls. Additionally, relaxin treatment caused a 6-fold increase in cervix wet weight, a 5% increase in water content and a significant decrease in relaxin receptor (Rxfp1) expression compared with saline-controls. These data suggest that relaxin promotes cervical hydration through an has2-mediated increase in HA, and may facilitate cervical ripening by causing collagen fibril dispersal in the ECM.

Identification and Characterization of a Membrane Receptor for Proteolysis-Inducing Factor on Skeletal Muscle

Proteolysis-inducing factor (PIF) is a sulfated glycoprotein produced by cachexia-inducing tumors, which induces atrophy of skeletal muscle. PIF has been shown to bind specifically with high affinity (K(d), in nanomolar) to sarcolemma membranes from skeletal muscle of both the mouse and the pig, as well as murine myoblasts and a human muscle cell line. Ligand binding was abolished after enzymatic deglycosylation, suggesting that binding was mediated through the oligosaccharide chains in PIF. Chondroitin sulfate, but not heparan or dermatan sulfate, showed competitive inhibition (K(d), 1.1 x 10(-7) mol/L) of binding of PIF to the receptor, suggesting an interaction with the sulfated oligosaccharide chains. Ligand blotting of [(35)S]PIF to triton solublized membranes from C(2)C(12) cells provided evidence for a binding protein of apparent M(r) of approximately 40,000. Amino acid sequence analysis showed the PIF receptor to be a DING protein. Antisera reactive to a 19mer from the N-terminal amino acid residues of the binding protein attenuated protein degradation and activation of the ubiquitin-proteasome pathway induced by PIF in murine myotubes. In addition, the antisera was highly effective in attenuating the decrease in body weight in mice bearing the MAC16 tumor, with a significant increase in muscle wet weight due to an increase in the rate of protein synthesis, together with a reduction in protein degradation through attenuation of the increased proteasome expression and activity. These results confirm that the PIF binding protein has a functional role in muscle protein atrophy in cachexia and that it represents a potential new therapeutic target.

Protective effects of heme oxygenase-1 against cyclophosphamide-induced haemorrhagic cystitis in rats

To investigate the expression profiles and protective roles of the inducible isoform of heme oxygenase-1 (HO-1) in cyclophosphamide (CYP)-induced cystitis, as the HO system is involved in heme degradation and plays an important role in cellular homeostasis but its characterization is still unknown in urinary tract diseases. In female Sprague-Dawley rats CYP was administed intraperitoneally and the bladders excised at various time points. In separate experiments, hemin, an inducer of HO, was administered before CYP treatment, and bladders were harvested 24 h after CYP injection. The expressions of HO-1 and HO-2 were investigated by reverse-transcription polymerase chain reaction and immunohistochemistry. The effects of CYP with/without hemin pretreatment were evaluated by microscopic features, bladder wet weight, myeloperoxidase activity, nitric oxide (NO)-metabolite production, and expression levels of inflammation-related genes. CYP injection resulted in severe cystitis with time-dependent increases both in bladder wet weight and HO-1 mRNA expression. Pretreatment with hemin enhanced the CYP-induced expression of HO-1 mRNA and protein further, but significantly ameliorated inflammatory changes and reduced the increases in bladder oedema and myeloperoxidase activity. NO-metabolite production and inducible NO synthase (iNOS) expression, induced by CYP, were down-regulated significantly by hemin pretreatment. By contrast, HO-2 was constitutively present in the urothelium and its expression was not influenced by CYP or by CYP plus hemin. HO-1 expression is up-regulated in bladders with CYP-induced haemorrhagic cystitis, and this inducible enzyme plays cytoprotective roles in association with down-regulation of NO production and iNOS expression. Our results suggest that HO-1 induction might have therapeutic potential against inflammatory insults such as CYP-induced cystitis.