Temporal Association Between Pulmonary Inflammation and Antioxidant Induction Following Hyperoxic Exposure of the Preterm Guinea Pig

The time course and nature of the pulmonary inflammatory and antioxidant responses, both during and after hyperoxic-induced acute lung injury were studied in the preterm guinea pig. Three-day preterm (65 days gestation) guinea pigs were randomly exposed to either 21% O₂ (control) or 95% O₂ (hyperoxia) for 72 hours. All pups were then maintained in ambient conditions for up to a further 11 days, during which time lung damage was monitored. In animals exposed to hyperoxia, evidence of acute lung injury and inflammation was characterized by a marked increase in microvascular permeability and elevated numbers of neutrophils in bronchoalveolar lavage fluid. Protein concentration, elastase-like activity and elastase-inhibitory capacity in lavage fluid were at a maximum at the end of the 72 hours hyperoxic exposure. Four days later, all values had returned to control levels. In contrast, increased numbers of neutrophils, macrophages and lymphocytes were recovered in the lavage fluid during this early recovery period. Coinciding with the influx of inflammatory cells, there was a significant increase in glutathione peroxidase, manganese superoxide dismutase and catalase activities in immature lung. Lung copper/zinc superoxide dismutase activity remained unchanged during both experimental periods. The strong temporal relationship between the influx of inflammatory cells to the lung and the induction of pulmonary antioxidant enzyme defences suggests that a common mechanism underlies both responses. These findings have led us to regard inflammation in the hyperoxic-injured immature lung as a beneficial event and not, as previously suggested, as part of the injurious process.     

Receptor and Biological Response to Estriol in the Fetal Uterus of Guinea Pig

Abstract

The binding of ³H-estriol was examined in the fetal uterus of guinea pig. The physico-chemical characteristics of the binding of ³H-estriol to macromolecules are similar to the typical receptor protein for estrogens. Different estrogens (estriol, estradiol, estrone and diethylstilbestrol) compete with this binding but progesterone and testosterone have no effect. The binding affinity has a Kd of 5.5 ± 1.6 ± 10^?10M. By ultra-centrifugation in sucrose gradient, two specific components with sedimentation coefficients of 8 and 45 are found. Competition studies suggest that the same specific binding sites may be present for estriol (E₃) and for estradiol. The s.c. administration of E₃ to the pregnant guinea pig (1 mg/day per kg body weight for 3 days) provokes two biological responses in the fetal uterus: a uterotrophic effect and a significant increase in the progesterone receptor. The increase in the fetal uterine weight is 50–70% in relation to the non-treated animals and the progesterone receptor concentration is 10–14 times higher than in the control animals. These effects are similar (or slightly higher) than in animals primed with equimolecular quantities of estradiol. In contrast, single daily injections of E₃ to newborn guinea pig, results only in weak uterotrophic activity.

It is concluded that estriol is capable of causing a biological response in the uterus during intra-uterine life.     

三个品种豚鼠血液蛋白多态性的比较分析

目的比较分析白毛黑眼（WHBE）豚鼠和DHP豚鼠、花色豚鼠三个品种豚鼠在13个血液蛋白位点上的多态性。方法采用垂直板浓度和pH均不连续的聚丙烯酰胺凝胶电泳法对WHBE豚鼠、DHP豚鼠和花色豚鼠的66只个体的后白蛋白（Po）、前转铁蛋白1（Prt1）、前转铁蛋白2（Prt2）、转铁蛋白1（Tf1）、转铁蛋白2（Tf2）、后转铁蛋白（Ptf）、慢α球蛋白（Sag）、红细胞酯酶（Es）、血清酯酶1（Est1）、血清酯酶3（Est3）、血红蛋白α（Hbα）、血红蛋白β（Hbβ）和白蛋白（Alb）共13个蛋白位点进行了电泳及染色,再利用电泳图谱对各蛋白位点基因频率、平均杂合度和遗传距离进行计算,然后结合聚类分析。结果 Tf1、Tf2、Ptf、Est1和Es在三个豚鼠品种中表现为多态,其中Tf1可作为识别WHBE豚鼠的遗传标记。Po、Prt1、Prt2、Sag、Est3、Hbα、Hbβ和Alb等位点在三个豚鼠品种中的表型一致。Hardy-Weinberg平衡状态分析表明,Es为DHP豚鼠的高度不平衡位点。Ptf为花色豚鼠的高度不平衡位点。在WHBE豚鼠中,Tf1为高度不平衡位点,Est1为不平衡位点。在三个豚鼠品种中,所检测的13个蛋白位点的平均杂合度的排列顺序为：花色豚鼠（0.350 1）〉WHBE豚鼠（0.339 0）〉DHP豚鼠（0.313 5）。聚类分析结果表明,花色豚鼠和WHBE豚鼠的遗传遗传距离最近（0.064 3）,DHP豚鼠与花色豚鼠的遗传距离最远（0.179 2）。结论利用这些蛋白位点可以有效鉴别WHBE豚鼠、DHP豚鼠和花色豚鼠血液蛋白的遗传多态性。     

Relationship of Glucocorticoid Receptor Expression in Peripheral Blood Mononuclear Cells and the Cochlea of Guinea Pigs and Effects of Dexamethasone Administration

Background

Glucocorticoids (GCs) are widely used to treat sudden sensorineural hearing loss (SSNHL) and significantly improve hearing. However, GC insensitivity has been observed in some patients of SSNHL.

Objective

To study the correlation between GR expression in peripheral blood mononuclear cells (PBMCs) and in the cochlea of guinea pigs at mRNA and protein levels.

Methods

One group of guinea pigs received dexamethasone (10 mg/kg/day) intraperitoneally for 7 consecutive days (dexamethasone group), and another group of guinea pigs received normal saline (control group). Real time PCR and Western blotting were used to detect the expression of GR mRNA and GR protein in PBMCs and the cochleae.

Results

The GR mRNA and GR protein were detected in both PBMCs and the cochlear tissue of guinea pigs. GR mRNA and GR protein levels in PBMCs were positively correlated with those in the cochlea. The expression of GR mRNA and GR protein was significantly increased in the dexamethasone group compared to the control group.

Conclusions

Levels of GR mRNA and GR protein in the PBMCs were positively correlated with those in the cochlea of guinea pigs. Systemic dexamethasone treatment can significantly up-regulate GR expression in PBMCs and in the cochlea. Measurement of the GR level in PBMCs could be used as an indicator of GR level in the cochlea.     

Identification of the Adenosine Uptake Sites in Guinea Pig Brain

Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high-affinity binding of [3H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent KD 0.075 +/- 0.012 nM; Bmax values of 0.24 +/- 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high-affinity binding. Low concentrations of cyclohexoadenosine (10-300 nM) had no effect on NBMPR binding. These properties of the high-affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [3H]NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent MW 66,000-45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [3H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzylthioguanosine.     

Thyrotropin-Releasing Hormone Receptor Binding Sites: Autoradiographic Distribution in the Rat and Guinea Pig Brain

Thyrotropin-releasing hormone (TRH) binding sites were labeled in vitro in mounted brain tissue sections from rat and guinea pig brains with [3H]methyl TRH and localized autoradiographically using 3H-sensitive film. Regional densities of TRH binding sites were measured by computer-assisted microdensitometry. The distribution of sites in both species was highly heterogeneous. In both guinea pig and rat brains, the highest densities of binding sites were seen in the amygdaloid nuclei and the perirhinal cortex. In contrast, in other brain areas, a clear difference between the distribution of sites in rat and guinea pig was found. The temporal cortex, pontine nuclei, and interpeduncular nucleus, which contained high densities of binding in the guinea pig, were scarcely labeled in the rat. The accessory olfactory bulb and the septohippocampal area presented in the rat higher concentrations of binding sites than in the guinea pig. Other brain areas showing intermediate to low densities in both species were accumbens nucleus, bed nucleus of the stria terminalis, dentate gyrus, facial and hypoglossal nuclei, and gelatinosus subnucleus of the trigeminal nerve, among others. The anterior pituitary also presented low to intermediate concentrations of receptors. The distribution of TRH sites here described does not completely correlate with that of endogenous TRH, but is in good agreement with previous biochemical data. The results are discussed in correlation to the physiological effects that appear to be mediated by TRH.     

脑药物转运载体——抗转铁蛋白受体单链抗体的克隆表达及鉴定

为构建特异性的脑药物转运载体 ,分段合成了抗大鼠转铁蛋白受体的单链抗体基因 (Ox2 6 scfv) .经重叠PCR拼接成完整片段 ,克隆入pUC19载体中 ,测序正确后克隆到大肠杆菌表达载体pET 15b E .tag上 .IPTG诱导 ,表达产物分子量为 2 9kD ,约占菌体总蛋白量的 4 0 % .包涵体经 6mol L盐酸胍变性后 ,过SephacrylS 30 0HR分子筛柱复性蛋白 .免疫酶染色实验表明 ,该单链抗体能与转铁蛋白受体特异性结合 ,为建立以转铁蛋白受体为介导的血脑屏障转运载体打下了基础     

Progesterone Receptor Expression Declines in the Guinea Pig Uterus during Functional Progesterone Withdrawal and in Response to Prostaglandins

Progesterone withdrawal is essential for parturition, but the mechanism of this pivotal hormonal change is unclear in women and other mammals that give birth without a pre-labor drop in maternal progesterone levels. One possibility suggested by uterine tissue analyses and cell culture models is that progesterone receptor levels change at term decreasing the progesterone responsiveness of the myometrium, which causes progesterone withdrawal at the functional level and results in estrogen dominance enhancing uterine contractility. In this investigation we have explored whether receptor mediated functional progesterone withdrawal occurs during late pregnancy and labor in vivo. We have also determined whether prostaglandins that induce labor cause functional progesterone withdrawal by altering myometrial progesterone receptor expression. Pregnant guinea pigs were used, since this animal loses progesterone responsiveness at term and gives birth in the presence of high maternal progesterone level similarly to primates. We found that progesterone receptor mRNA and protein A and B expression decreased in the guinea pig uterus during the last third of gestation and in labor. Prostaglandin administration reduced while prostaglandin synthesis inhibitor treatment increased progesterone receptor A protein abundance. Estrogen receptor-1 protein levels remained unchanged during late gestation, in labor and after prostaglandin or prostaglandin synthesis inhibitor administration. Steroid receptor levels were higher in the non-pregnant than in the pregnant uterine horns. We conclude that the decreasing expression of both progesterone receptors A and B is a physiological mechanism of functional progesterone withdrawal in the guinea pig during late pregnancy and in labor. Further, prostaglandins administered exogenously or produced endogenously stimulate labor in part by suppressing uterine progesterone receptor A expression, which may cause functional progesterone withdrawal, promote estrogen dominance and foster myometrial contractions.     

豚鼠生长激素受体及其突变体在哺乳动物细胞中的功能性表达

采用RT PCR克隆得到豚鼠生长激素受体cDNA。序列同源比较显示生长激素受体的一些功能性保守氨基酸在豚鼠生长激素受体中被其他氨基酸所取代。例如 ,哺乳动物生长激素受体中保守的第 170位组氨酸和第 333位酪氨酸分别是受体二聚化和生长激素刺激蛋白质合成及脂生成所必需的 ,但在豚鼠生长激素受体中分别被酪氨酸和丝氨酸所取代。为此 ,采用定点突变法得到了突变体gpGHRY16 8H和 gpGHRS332Y ,并构建了表达质粒 pcDNA3 gpGHR ,pcDNA3 gpGHRY16 8H和pcDNA3 gpGHRS332Y。借助COS 7和CHO细胞 ,研究了豚鼠生长激素受体及其突变体的生物活性。实验表明转染了pcDNA3 gpGHR的COS 7细胞对牛生长激素具有高亲和性 [Ka=1.3× 10 9(mol/L) -1],并且用鼠抗生长激素受体单克隆抗体mAb2 6 3可检测到一分子量约 92kD的蛋白质。在CHO细胞中 ,虽然两个氨基酸的定点突变不影响受体与配体的结合 ,但都提高了生长激素刺激的蛋白质合成而降低生长激素刺激的脂肪生成。蛋白质印迹实验揭示突变体 gpGHRY16 8和gpGHRS332Y分别降低和提高了生长激素诱导的JAK2酪氨酸磷酸化。因此 ,报道了豚鼠生长激素受体在生长激素代谢功能中的调节作用以及受体中保守氨基酸的取代导致配体结合后信号转导的变化。     

Dynamic Studies on Estrogen Response in Fetal Guinea Pig Uterus: Effect of Estradiol Administration on Estradiol Receptor,Progesterone Receptor and Uterine Growth

Abstract

The uterus of the guinea pig fetus has been shown to respond to estradiol treatment by an increase in uterine wet weight and a stimulation of the progesterone receptor protein. A study of the kinetics of these two parameters of estrogen response in the fetal uterus was undertaken in order to correlate these responses with changes in the estrogen receptor. Administration of estradiol to pregnant guinea pigs (1 mg/kg/body weight) leads to a rapid stimulation of the progesterone receptor by 6h after treatment which reaches maximal values by 15.5h, which are increased 7-fold in estradiol-primed guinea pigs above values in untreated animals. The estradiol receptor undergoes rapid translocation from the cytosol into the nucleus by 1h after hormone treatment and is retained in the nucleus for at least 6h. At the same time, there is a 50% decrease in the total occupied and available estradiol receptor concentration at 6h after treatment. Estradiol treatment also provokes an increase in wet weight of the fetal uterus which is significantly greater after 3 consecutive days of treatment (171% ± 24 (S.D.) above wet weights of untreated uteri which were considered as 100%) than after only 1 day (121% ± 25 (S.D.)). These estrogen responses were found to be of long duration since uterine wet weights and progesterone receptor concentrations remained well above control values even 5 days after a single treatment with estradiol. In conclusion, the fetal uterus responds to estradiol treatment by a slow increase in wet weight and a rapid stimulation of the progesterone receptor protein with a concomitant loss in estradiol receptor concentration.     

胎盘膜胰岛素受体结合活性与巯基和二硫键的关系

部分纯化的人胎盘膜经DTT还原,NEM,DTNB修饰蛋白巯基后,改变了胰岛素受体的结合活性。Scatchard分析表明,当DTT浓度较低时,亲和常数基本不变;高亲和位点数略微升高,较高浓度的DTT处理时,结合位点数和亲和常数均有所下降。DTT还原膜蛋白二硫键后再用NEM,DTNB修饰巯基,胰岛素结合活性进一步下降。NEM或DTNB单独处理结合活性下降较少。胰岛素与受体结合后,用DTT洗后剩余的结合胰岛素比缓冲液洗低,表明有一部分胰岛素以二硫键与受体共价结合。在0—5mmol/L浓度范围内,随着DTT处理浓度的升高,这种以二硫键共价结合胰岛素增加。     

Impact of Cadmium Exposure during Pregnancy on Hepatic Glucocorticoid Receptor Methylation and Expression in Rat Fetus

Adverse fetal environment due to maternal undernutrition or exposure to environmental chemicals alters glucocorticoid (GC) metabolism increasing the risk of metabolic disorders in adulthood. In this study, we investigated the effects of maternal exposure to cadmium (Cd, 50 ppm) during pregnancy in the methylation of fetal hepatic glucocorticoid receptor promoter (GR) and the correlation with its expression and that of the DNA methyltransferases (DNMT1a and 3a). We also studied the expression of liver phosphoenolpyruvate carboxykinase (PEPCK) and acyl-CoA oxidase (AOX), two enzymes involved in the metabolism of carbohydrates and lipids respectively. The methylation of the rat GR gene exon 1(10) (GR1(10)) in nucleotides -2536 to -2361 was analyzed by pyrosequencing. Quantitative real time PCR was used to assess hepatic GR, PEPCK and AOX mRNA, and their protein levels using Western blotting analysis. Differential methylation was noted across groups at all CpG sites in the GR exon 1(10) in a sex-dependent manner. In males, CpG were more methylated than the controls (185±21%, p<0.001) but only CpG sites 1,6,7 and 9 showed a significantly different extent of methylation. In addition, a lower expression of GR (mRNA and protein) was found. On the contrary, in females, CpG were less methylated than the controls (62±11%, p<0.05) and overexpressed, affecting PEPCK and AOX expression, which did not change in males. The GR methylation profile correlates with DNMT3a expression which may explain epigenetic sex-dependent changes on GR1(10) promoter induced by Cd treatment. In conclusion, Cd exposure during pregnancy affects fetal liver DNMT3a resulting in sex-dependent changes in methylation and expression of GR1(10). Although these effects do not seem to be directly involved in the low birth weight and height, they may have relevant implications for long-term health.     

Adrenal Phenylethanolamine N-Methyltransferase Induction in Relation to Glucocorticoid Receptor Dynamics: Evidence that Acute Exposure to High Cortisol Levels Is Sufficient to Induce the Enzyme

Glucocorticoids (GCs) are thought to regulate, in a permissive fashion, the basal activity of adrenal medullary phenylethanolamine N-methyltransferase (PNMT). However, it is unclear whether a large short-term increase in GC release, such as occurs during an acute stress response, may also play a role in PNMT regulation. The present study investigated how the GC influence over PNMT activity varies in relation to dynamic changes in the hormone-receptor signal. Using [3H]dexamethasone (DEX) and [3H]RU 28362 as radioligands, we have confirmed the presence of GC receptors in bovine adrenal medullary cells. A concentration-dependent decline in soluble GC receptor sites and an increase in nuclear uptake of [3H]DEX were found in response to GC levels as low as 5 x 10(-8) M. The loss of soluble sites plateaued between 5 x 10(-8) and 10(-6) M cortisol, with further losses occurring at 10(-5) and at 10(-4) M. The functional consequence of GC receptor binding was confirmed by measuring PNMT activity following 3-day exposure to cortisol. The pattern of PNMT induction was similar to that seen with GC receptor occupancy; at cortisol concentrations between 10(-8) and 10(-5) M, PNMT induction was at a plateau, with a further increase in activity at 10(-4) M. The increase in PNMT activity following 3-day exposure to low (10(-7) M) and high (5 x 10(-5), 10(-5) M) cortisol was blocked by the GC receptor antagonist RU 38486, suggesting a GC receptor-mediated event. Finally, a short (2 h) pulse of GC, which mimics the time course of physiological elevation of GC following acute stress, elevated adrenal medullary PNMT activity measured 3 days later. Therefore, our results provide novel evidence that short-term exposure of adrenal medullary cells to high cortisol levels can elevate PNMT activity.     

Prenatal Alcohol Exposure Modifies Glucocorticoid Receptor Subcellular Distribution in the Medial Prefrontal Cortex and Impairs Frontal Cortex-Dependent Learning

Prenatal alcohol exposure (PAE) has been shown to impair learning, memory and executive functioning in children. Perseveration, or the failure to respond adaptively to changing contingencies, is a hallmark on neurobehavioral assessment tasks for human fetal alcohol spectrum disorder (FASD). Adaptive responding is predominantly a product of the medial prefrontal cortex (mPFC) and is regulated by corticosteroids. In our mouse model of PAE we recently reported deficits in hippocampal formation-dependent learning and memory and a dysregulation of hippocampal formation glucocorticoid receptor (GR) subcellular distribution. Here, we examined the effect of PAE on frontal cortical-dependent behavior, as well as mPFC GR subcellular distribution and the levels of regulators of intracellular GR transport. PAE mice displayed significantly reduced response flexibility in a Y-maze reversal learning task. While the levels of total nuclear GR were reduced in PAE mPFC, levels of GR phosphorylated at serines 203, 211 and 226 were not significantly changed. Cytosolic, but not nuclear, MR levels were elevated in the PAE mPFC. The levels of critical GR trafficking proteins, FKBP51, Hsp90, cyclophilin 40, dynamitin and dynein intermediate chain, were altered in PAE mice, in favor of the exclusion of GR from the nucleus, indicating dysregulation of GR trafficking. Our findings suggest that there may be a link between a deficit in GR nuclear localization and frontal cortical learning deficits in prenatal alcohol-exposed mice.     

Changes in Purinoceptor Distribution and Intracellular Calcium Levels following Noise Exposure in the Outer Hair Cells of the Guinea Pig

Among the cells of the inner ear, the outer hair cells (OHCs) are the most important targets of noise-induced effects, being the most sensitive cell types. The aim of this study was to examine the effects of noise (50 Hz-20 kHz, 80 dB sound pressure level, 14 days) on intracellular calcium levels and on the expression pattern of purinoceptors in the membrane of the OHCs of the guinea pig and to measure the stiffness changes of the lateral membrane of these cells. In noise-exposed animals, the resting intracellular calcium concentration increased compared to nontreated animals and was slightly higher in the cells of the basal (219 ± 29 nM) than in the apical (181 ± 24 nM) turns of the cochlea. After application of 180 μM adenosine triphosphate, the intracellular calcium level rose by 60 ± 22 nM in cells from the apical and by 44 ± 10 nM in cells from the basal turns, significantly less than in nontreated animals. Expression of the P_2X1, P_2X2, P_2X4, P_2X7, P_2Y1 and P_2Y4 receptor subtypes was suppressed, while expression of the P_2Y2 subtype did not decrease in either of the two preparations. In parallel with the increase in intracellular calcium concentration, the stiffness of the lateral wall of the OHCs was increased. Noise-induced changes in intracellular calcium homeostasis and subsequently in the calcium-dependent regulatory mechanisms may modify OHC lateral wall stiffness and may lead to reduction of the efficacy of the cochlear amplifier.     

Mechanism of Resistance to Steroids: Glucocorticoid Receptor Defect in Lymphoma Cells

GLUCOCORTICOIDS cause cytolysis of normal and neoplastic lymphoid cells^1,2—a reaction which is one basis for the use of these compounds in the therapy of neoplastic, autoimmune and other diseases of the lymphoid system. Unfortunately, resistance to steroids often develops during treatment, rendering the hormones therapeutically ineffective.     

Characterization of Guinea Pig Antibody Responses to Salivary Proteins of Triatoma infestans for the Development of a Triatomine Exposure Marker

Background

Salivary proteins of Triatoma infestans elicit humoral immune responses in their vertebrate hosts. These immune responses indicate exposure to triatomines and thus can be a useful epidemiological tool to estimate triatomine infestation. In the present study, we analyzed antibody responses of guinea pigs to salivary antigens of different developmental stages of four T. infestans strains originating from domestic and/or peridomestic habitats in Argentina, Bolivia, Chile and Peru. We aimed to identify developmental stage- and strain-specific salivary antigens as potential markers of T. infestans exposure.

Methodology and Principal Findings

In SDS-PAGE analysis of salivary proteins of T. infestans the banding pattern differed between developmental stages and strains of triatomines. Phenograms constructed from the salivary profiles separated nymphal instars, especially the 5^th instar, from adults. To analyze the influence of stage- and strain-specific differences in T. infestans saliva on the antibody response of guinea pigs, twenty-one guinea pigs were exposed to 5^th instar nymphs and/or adults of different T. infestans strains. Western blot analyses using sera of exposed guinea pigs revealed stage- and strain-specific variations in the humoral response of animals. In total, 27 and 17 different salivary proteins reacted with guinea pig sera using IgG and IgM antibodies, respectively. Despite all variations of recognized salivary antigens, an antigen of 35 kDa reacted with sera of almost all challenged guinea pigs.

Conclusion

Salivary antigens are increasingly considered as an epidemiological tool to measure exposure to hematophagous arthropods, but developmental stage- and strain-specific variations in the saliva composition and the respective differences of immunogenicity are often neglected. Thus, the development of a triatomine exposure marker for surveillance studies after triatomine control campaigns requires detailed investigations. Our study resulted in the identification of a potential antigen as useful marker of T. infestans exposure.