A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1ß, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1ß, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.

Multimodal analysis in acute and chronic experimental autoimmune encephalomyelitis.

Different experimental autoimmune encephalomyelitis models (EAE) have been developed. However, due to the different experimental conditions applied, observations simultaneously considering different pathological targets are still scarce. Using EAE induced in Dark Agouti rats with syngenic whole spinal cord homogenate suspended in incomplete Freund's adjuvant, we here analyze neurosteroidogenic machinery, cytokine levels, microglial cells, infiltration of inflammatory cells, myelin proteins and Na(+), K(+)-ATPase pump activity in the spinal cord. Data obtained in the acute phase of the disease confirmed that neurological signs were accompanied by the presence of perivascular infiltrating T cells (CD3(+) cells) and activated monocytic/microglial cells (ED1(+) and MHC-II(+)) in the spinal cord. In particular, the number of MHC-II(+) cells was significantly increased in association with increased expression of pro- (i.e., TNF-α, IL-1ß) and anti-inflammatory (i.e., TGF-ß) cytokines as well as with decreased expression of proteolipid protein and myelin basic protein. During the chronic phase of the disease, the number of MHC-II(+) cells was still increased, although less than in the acute phase. Changes in the number of MHC-II(+) cells were associated with decreased Na(+),K(+)-ATPase enzymatic activity. A general decrease in the levels of neuroactive steroids, with the exception of an increase in tetrahydroprogesterone and 17ß-estradiol, was detected in the acute phase. These changes were maintained or reverted in the chronic phase of EAE. In conclusion, we report that modifications in the neuroimmune response in the acute and chronic phases of EAE are associated with specific changes in myelin proteins, Na(+),K(+)-ATPase pump and in the levels of neuroactive steroids.

Blood glutathione peroxidase-1 mRNA levels can be used as molecular biomarkers to determine dietary selenium requirements in rats.

Transcript (mRNA) levels are increasingly being used in medicine as molecular biomarkers for disease and disease risk, including use of whole blood as a target tissue for analysis. Development of blood molecular biomarkers for nutritional status, too, has potential application that parallels opportunities in medicine, including providing solid data for individualized nutrition. We previously reported that blood glutathione peroxidase-1 (Gpx1) mRNA was expressed at levels comparable to major tissues in rats and humans. To determine the efficacy of using blood Gpx1 mRNA to assess selenium (Se) status and requirements, we fed graded levels of Se (0-0.3 microg Se/g as selenite) to weanling male rats. Se status was determined by liver Se concentration and selenoenzyme activity, and selenoprotein mRNA abundance in liver and blood was determined by ribonuclease protection analysis. Liver Se and plasma glutathione peroxidase-3 and liver Gpx1 activities indicated that minimal Se requirements were at 0.08 microg Se/g diet. When total RNA was isolated from whole blood, Gpx1 mRNA in Se-deficient rats decreased to 10% of levels in Se-adequate (0.2 microg Se/g diet) rats. With Se supplementation, blood Gpx1 mRNA levels increased sigmoidally to a plateau with a minimum Se requirement of 0.08 microg Se/g diet, whereas glutathione peroxidase-4 mRNA levels were unaffected. Similarly, Gpx1 mRNA in RNA isolated from fractionated red blood cells decreased in Se-deficient rats to 23% of Se-adequate levels, with a minimum Se requirement of 0.09 microg Se/g diet. Additional studies showed that the preponderance of whole blood Gpx1 mRNA arises from erythroid cells, most likely reticulocytes and young erythrocytes. In summary, whole blood selenoprotein mRNA levels can be used as molecular biomarkers for assessing Se requirements, illustrating that whole blood has potential as a target tissue in development of molecular biomarkers for use in nutrition as well as in medicine.

Screening and identification of natural antisense transcripts in Helicobacter pylori by a novel approach based on RNase I protection assay.

Natural antisense transcripts (NATs) are endogenous RNA molecules that exhibit partial or complete complementarity to other RNAs. Studies have shown that NATs may participate in a broad range of gene regulatory events. The identification of NATs in human, mouse and Escherichia coli has revealed their widespread occurrence in both eukaryotic and prokaryotic life. However, little is known about NATs in Helicobacter pylori (H. pylori), a human pathogen which is associated with gastric diseases. Here we systematically screened NATs in H. pylori by a novel experimental strategy based on RNase I protection assay. We successfully constructed a cDNA library of NATs and developed a novel poly(A)-tailed RT-PCR method to monitor the expression of NATs. After sequencing, bioinformatic analysis and expression detection, two novel NATs (NAT-39 and NAT-67) were confirmed. They were, respectively, complementary to the following genes: iron-regulated outer membrane protein (frpB) and periplasmic iron-binding protein (ceuE). Taken together, the results suggest that NAT-39 and NAT-67 may participate in the regulation of iron homeostasis in H. Pylori in a sequence complementary manner with target mRNAs.

Effects of chronic central leptin infusion on proopiomelanocortin and neurotensin gene expression in the rat hypothalamus.

Leptin signaling in the hypothalamus is critical for normal food intake and body weight regulation. While hyperleptinemia in obese people suggests a state of leptin resistance, the mechanism is not clearly understood. In a rat model of central leptin infusion in which animals develop resistance to the satiety action of leptin, orexigenic peptide producing neuropeptide Y neurons in the hypothalamus develop leptin resistance. However, it is still unknown if increased hypothalamic leptin tone caused by central leptin infusion results in the development of leptin resistance in anorexigenic peptide producing proopiomelanocortin (POMC) and neurotensin (NT) neurons. To this end, male rats were infused chronically with leptin (160 ng/h) or vehicle into the lateral cerebroventricle for 16 days. On day 4 of leptin infusion when food intake was decreased, POMC and NT mRNA levels, as determined by RNAse protection assay, were significantly increased as compared to control. By contrast, on day 16 of leptin infusion, when food intake was mostly normalized, both POMC and NT mRNA levels remained unchanged compared with control. These findings suggest the development of leptin resistance in the POMC and NT neurons following chronic elevation of hypothalamic leptin tone, which may be involved in the development of resistance to the satiety action of leptin following central infusion of this peptide hormone.

Genomic structure, transcriptional control, and tissue distribution of HERG1 and KCNQ1 genes.

The long QT syndrome genes human ether-a-go-go-related gene (HERG1) and voltage-gated K+ channel, KQT-like subfamily, member 1, gene (KCNQ1), encoding K+ channels critical to the repolarization rate and repolarization reserve in cardiac cells, and thereby the likelihood of arrhythmias, are both composed of two isoforms: HERG1a and HERG1b and KCNQ1a and KCNQ1b, respectively. Expression of these genes is dynamic, depending on the differentiation status and disease states. We identified their core promoter regions and transcription start sites. Our data suggest that HERG1a and HERG1b, and KCNQ1a and KCNQ1b, represent independent transcripts instead of being alternatively spliced variants of the same gene, for they each have their own transcription start sites and their own promoter regions. We obtained data pointing to the potential role of stimulating protein 1 (Sp1) in the transactivation of these genes. We compared expression profiling of these genes across a variety of human tissues. Consistent with the general lack of cis elements for cardiac-specific transcription factors and the presence of multiple sites for ubiquitous Sp1 sites in the core promoter regions of HERG1a/HERG1b and KCNQ1a/KCNQ1b genes, the transcripts demonstrated widespread distribution across a variety of human tissues. We further revealed that the mRNA levels of all HERG1 and KCNQ1 isoforms were asymmetrically distributed within the heart, being more abundant in the right atria and ventricles relative to the left atria and ventricles. These findings open up an opportunity for studying interventricular gradients of slow and rapid delayed rectifier K+ current and of cardiac repolarization as well. Our study might help us understand the molecular mechanisms for arrhythmias since heterogeneity of ion channel activities is an important substrate for arrhythmogenesis.

Effects of coffee and its chemopreventive components kahweol and cafestol on cytochrome P450 and sulfotransferase in rat liver.

Coffee drinking appears to reduce cancer risk in liver and colon. Such chemoprevention may be caused by the diterpenes kahweol and cafestol (K/C) contained in unfiltered beverage. In animals, K/C treatment inhibited the mutagenicity/tumorigenicity of several carcinogens, likely explicable by beneficial modifications of xenobiotic metabolism, particularly by stimulation of carcinogen-detoxifying phase II mechanisms. In the present study, we investigated the influence of K/C on potentially carcinogen-activating hepatic cytochrome P450 (CYP450) and sulfotransferase (SULT). Male F344 rats received 0.2% K/C (1:1) in the diet for 10 days or unfiltered and/or filtered coffee as drinking fluid. Consequently, K/C decreased the metabolism of four resorufin derivatives representing CYP1A1, CYP1A2, CYP2B1, and CYP2B2 activities by approximately 50%. For CYP1A2, inhibition was confirmed at the mRNA level, accompanied by decreased CYP3A9. In contrast to K/C, coffee increased the metabolism of the resorufin derivatives up to 7-fold which was only marginally influenced by filtering. CYP2E1 activity and mRNA remained unchanged by K/C and coffee. K/C but not coffee decreased SULT by approximately 25%. In summary, K/C inhibited CYP450s by tendency but not universally. Inhibition of CYP450 and SULT may contribute to chemoprevention with K/C but involvement in the protection of coffee drinkers is unlikely. The data confirm that the effects of complex mixtures may deviate from those of their putatively active components.

Eosinophil recruiting chemokines are down-regulated in peripheral blood mononuclear cells of allergic patients treated with deflazacort or desloratadine.

Chemokines are cytokines with chemotactic properties on leukocyte subsets whose modulation plays a key role in allergic inflammatory processes. To better understand the possible anti-inflammatory effects of histamine-1 receptor antagonists in allergic asthma, we studied the mRNA expression of a set of chemokines known to be involved in the eosinophils-basophils activation as well as recruitment and T-cell signaling events, before and after corticosteroid or antihistamine treatment in PBMCs from allergic-asthmatic patients ex vivo. Twelve patients were enrolled, all of whom were allergic to Parietaria judaica and suffering for mild persistent asthma: six were treated with desloratadine (10 mg/day), and six with deflazacort (12 mg/day). Before and after the treatment, PBMC samples were collected from each patient and analyzed for the expression of encoding mRNAs for several chemokines, I-309 (CCL1), MCP-1 (CCL2), MIP1-alpha (CCL3), MIP1-beta (CCL4), RANTES (CCL5), IL-8 (CXCL8), IP-10 (CXCL10), Lymphotactin (XCL1). Clinical and functional improvements were seen after 3 weeks of therapy; this was associated with a reduced expression in the mRNA levels for the chemokines RANTES, MIP1-alpha and MIP1-beta with either the corticosteroid or the antihistamine, compared to the pre-treatment levels. Chemokine downregulation was statistically significant in both groups of patients. These findings suggest that certain antihistamines may act as down-modulators of allergic inflammation, possibly through a negative regulation of the chemokines involved in activation and attraction of eosinophils. Our results suggest that clinical trials with long follow-ups may be useful in evaluating histamine-1 receptor antagonists as add-on therapy to steroids in the treatment of asthma.

Expression analysis of hypothalamic and pituitary components of the growth hormone axis in fasted and streptozotocin-treated neuropeptide Y (NPY)-intact (NPY+/+) and NPY-knockout (NPY-/-) mice.

In the fasted and the streptozotocin (STZ)-induced diabetic male rat, hypothalamic growth hormone (GH)-releasing hormone (GHRH) mRNA levels, and pulsatile GH release are decreased. These changes are believed to be due to a rise in hypothalamic neuropeptide Y (NPY) that inhibits GHRH expression. To directly test if NPY is required for metabolic regulation of hypothalamic neuropeptides important in GH secretion, NPY, GHRH and somatostatin (SRIH) mRNA levels were determined in fasted (48 h) and STZ-treated wild-type (NPY(+/+)) and NPY-knockout (NPY(-/-)) mice by ribonuclease protection assay. In addition, pituitary receptor mRNA levels for GHRH (GHRH-R), ghrelin (GHS-R) and SRIH (sst2) were assessed by RT-PCR. Under fed conditions the GH axis of NPY(+/+) and NPY(-/-) did not differ. In the NPY(+/+) mouse, fasting resulted in a 23% weight loss and >250% increase in NPY mRNA accompanied by a significant reduction in both GHRH and SRIH mRNA. These changes were associated with increases in pituitary expression of GHRH-R and GHS-R and a concomitant suppression of sst2. In the NPY(-/-) mouse, fasting also resulted in a 23% weight loss and comparable changes in GHRH-R and sst2, but failed to alter GHRH, SRIH and GHS-R mRNA levels. Fasting resulted in an overall increase in circulating GH, which reached significance in the fasted NPY(-/-) mouse. Induction of diabetes in NPY(+/+) mice, using a single, high-dose, STZ injection (150 mg/kg), resulted in modest weight loss (5%), and a 158% increase NPY expression which was associated with reciprocal changes in pituitary GHS-R and sst2 expression, similar to that observed in the fasted state, but no change in hypothalamic GHRH or SRIF expression was observed. Induction of diabetes in NPY(+/+) and NPY(-/-) mice, using a multiple, low-dose, STZ paradigm (5 consecutive daily injections of 40 mg/kg), did not alter body weight, hypothalamic neuropeptide expression or pituitary receptor expression, with the exception that sst2 mRNA levels were suppressed and GH levels did rise in the NPY(-/-) mouse. These observations demonstrate that NPY is not required for basal regulation of the GH axis, but is required for fasting-induced suppression of GHRH and SRIH expression, as well as fasting-induced augmentation of pituitary GHS-R mRNA. In contrast to the rat, fasting clearly did not suppress circulating GH levels in mice, but resulted in an overall rise in mean GH levels, similar to that observed in other mammalian species. The fact that many of the fasting-induced changes in the GH axis were observed in the high-dose STZ-treated mice, but were not observed in the multiple, low-dose paradigm, suggests STZ-mediated modulation of GH axis function is dependent on the severity of the catabolic state and not hyperglycemia.

Cyclooxygenase 2 expression in the spared nerve injury model of neuropathic pain.

Cyclooxygenase-2 (COX-2) after induction peripherally, and within the CNS, plays an important role in producing inflammatory pain. However, its role in neuropathic pain models is controversial. Recently a robust and persistent model of partial nerve injury pain, the spared nerve injury (SNI) model, has been developed. The aim of the present study was to examine the regulation of COX-2 in the rat SNI model and to evaluate the effectiveness of the selective COX-2 inhibitor rofecoxib in preventing neuropathic allodynia and hyperalgesia. RNase protection assays revealed only a very small and transient increase in COX-2 mRNA in the dorsal horn of the spinal cord in the SNI model with a maximum change at 24 h. Immunohistochemical analysis showed a small increase in COX-2 protein in the deep layers of the dorsal horn 10 h following SNI surgery. Rofecoxib (100 microM) did not affect spontaneous excitatory postsynaptic currents or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propanoic acid (AMPA) and N-methyl-d-aspartate (NMDA) responses in lamina II neurons from spinal cords of animals with SNI indicating no detectable action on transmitter release or postsynaptic activity. Furthermore, rofecoxib treatment (1 and 3.2 mg/kg for 5 and 3 days respectively starting on the day of surgery) failed to modify the development of allodynia and hyperalgesia in the SNI model. However, rofecoxib significantly reduced inflammatory hypersensitivity evoked by injection of complete Freund's adjuvant into one hindpaw, indicating that the doses used were pharmacologically active. The pain hypersensitivity produced by the SNI model is not COX-2-dependent.

High-sodium intake prevents pregnancy-induced decrease of blood pressure in the rat.

Despite an increase of circulatory volume and of renin-angiotensin-aldosterone system (RAAS) activity, pregnancy is paradoxically accompanied by a decrease in blood pressure. We have reported that the decrease in blood pressure was maintained in pregnant rats despite overactivation of RAAS following reduction in sodium intake. The purpose of this study was to evaluate the impact of the opposite condition, e.g., decreased activation of RAAS during pregnancy in the rat. To do so, 0.9% or 1.8% NaCl in drinking water was given to nonpregnant and pregnant Sprague-Dawley rats for 7 days (last week of gestation). Increased sodium intakes (between 10- and 20-fold) produced reduction of plasma renin activity and aldosterone in both nonpregnant and pregnant rats. Systolic blood pressure was not affected in nonpregnant rats. However, in pregnant rats, 0.9% sodium supplement prevented the decreased blood pressure. Moreover, an increase of systolic blood pressure was obtained in pregnant rats receiving 1.8% NaCl. The 0.9% sodium supplement did not affect plasma and fetal parameters. However, 1.8% NaCl supplement has larger effects during gestation as shown by increased plasma sodium concentration, hematocrit level, negative water balance, proteinuria, and intrauterine growth restriction. With both sodium supplements, decreased AT1 mRNA levels in the kidney and in the placenta were observed. Our results showed that a high-sodium intake prevents the pregnancy-induced decrease of blood pressure in rats. Nonpregnant rats were able to maintain homeostasis but not the pregnant ones in response to sodium load. Furthermore, pregnant rats on a high-sodium intake (1.8% NaCl) showed some physiological responses that resemble manifestations observed in preeclampsia.

Effect of 17beta-estradiol on mRNA expression of large- conductance, voltage-dependent, and calcium-activated potassium channel alpha and beta subunits in guinea pig.

Large-conductance, voltage- and calcium-activated potassium (MaxiK) channels play a key role in cell excitability. MaxiK channels are composed of a pore-forming alpha-subunit and a regulatory beta-subunit, of which four (beta1-4) genes have been identified. Previous findings suggested that MaxiK channel activity is regulated by estradiol. However, the underlying mechanisms have remained incompletely documented. Therefore, we used reverse transcriptase polymerase chain reaction to clone four cDNA fragments that were specific to the guinea pig alpha, beta1, beta2, and beta4 genes. Using a sensitive ribonuclease protection assay, we found that the alpha and beta4 mRNAs were the most abundant mRNAs in the brain and pituitary, whereas in the aorta, the alpha-subunit was coexpressed with the beta1-subunit. Moreover, there was a significant upregulation of the alpha- but not the beta1-subunit mRNA and the alpha-subunit protein in the aorta of the estrogenvs oil-treated ovariectomized animals. In specific brain areas including preoptic area, ventral hypothalamus, hippocampus, and amygdala, and in the pituitary, neither the alpha- nor beta4-subunit mRNAs were affected by estrogen. These findings suggest that estrogen may not affect the mRNA expression of MaxiK channels in the brain and pituitary. However, estrogen causes increased expression of MaxiK alpha in the aorta, which may explain some of the cardioprotective effects of estrogen in women.

GABA-glutamate interaction in the control of BDNF expression in hypothalamic neurons.

Brain derived-neurotrophic factor (BDNF) belongs to the neurotrophin family and regulates the survival, differentiation and maintenance of function in different neuronal populations. We previously reported that glutamate increases the expression of BDNF mRNA, its four transcripts and the BDNF peptide in fetal hypothalamic neurons, essentially through NMDA receptor activation. In the present study, we investigated whether GABA interacts with glutamate in the regulation of BDNF gene expression. BDNF and Trk B (BDNF receptor) mRNAs were determined by RNAse protection assay. BDNF transcripts expression levels were evaluated by semi-quantitative RT-PCR. BDNF peptide content was analyzed by enzyme immunoassay (ELISA).We found that picrotoxin (a GABA(A) receptor antagonist) stimulated BDNF mRNA expression and that GABA decreased the glutamate-induced augmentation with no effect on the expression of mRNA encoding the BDNF receptor, Trk B. Measurements of BDNF transcripts levels showed that transcripts containing exons I and III were increased by picrotoxin, whereas those containing exons II and IV were unchanged. GABA solely diminished the glutamate-stimulated expression of transcripts containing exon III. In addition, GABA also inhibited the stimulatory effect of glutamate on BDNF peptide content. Our findings show an interaction between glutamate and GABA on BDNF expression (mRNA, transcripts and peptide) in fetal hypothalamic neurons.

Resveratrol, a polyphenolic phytoalexin present in red wine, enhances expression and activity of endothelial nitric oxide synthase.

BACKGROUND: Estrogens can upregulate endothelial nitric oxide synthase (eNOS) in human endothelial cells by increasing eNOS promoter activity and enhancing the binding activity of the transcription factor Sp1. Resveratrol, a polyphenolic phytoalexin found in grapes and wine, has been reported to act as an agonist at the estrogen receptor. Therefore, we tested the effect of this putative phytoestrogen on eNOS expression in human endothelial cells. METHODS AND RESULTS: Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells with resveratrol for 24 to 72 hours upregulated eNOS mRNA expression in a time- and concentration-dependent manner (up to 2.8-fold). eNOS protein expression and eNOS-derived NO production were also increased after long-term incubation with resveratrol. Resveratrol increased the activity of the eNOS promoter (3.5-kb fragment) in a concentration-dependent fashion, with the essential trans-stimulated sequence being located in the proximal 263 bp of the promoter sequence. In addition, eNOS mRNA was stabilized by resveratrol. The effect of resveratrol on eNOS expression was not modified by the estrogen receptor antagonists ICI 182780 and RU 58668. In electrophoretic mobility shift assays, nuclear extracts from resveratrol-incubated EA.hy 926 cells showed no enhanced binding activity of the eNOS promoter-relevant transcription factors Sp1, GATA, PEA3, YY1, or Elf-1. In addition to its long-term effects on eNOS expression, resveratrol also enhanced the production of bioactive NO in the short-term (after a 2-minute incubation). CONCLUSIONS: In concert with other effects, the stimulation of eNOS expression and activity may contribute to the cardiovascular protective effects attributed to resveratrol.

Eighteen-month-old Fischer 344 rats fed a spinach-enriched diet show improved delay classical eyeblink conditioning and reduced expression of tumor necrosis factor alpha (TNFalpha ) and TNFbeta in the cerebellum.

Diets high in antioxidant properties are known to reverse some deficits in neuronal and cognitive function that occur in aging animals. Antioxidants are also known to reduce levels of proinflammatory factors in the CNS. We report here that 6 weeks of a spinach-enriched diet ameliorates deficits in cerebellar-dependent delay classical eyeblink learning and reduces the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha) and TNFbeta in the cerebelli of eyeblink-trained animals. Eighteen-month-old Fischer 344 rats were given spinach-enriched lab chow or regular lab chow for 6 weeks. The rats were then given 6 d of 30 trials per day training using a 3 kHz tone conditioned stimulus and airpuff unconditioned stimulus. Rats were killed 3 weeks after eyeblink training. Cytokine expression was measured using RNase protection assay analysis in the eyeblink-trained animals and in a group of young control animals given regular lab chow diet. Old animals on the spinach-enriched lab chow diet learned delay eyeblink conditioning significantly faster than old animals on the regular diet. Cerebelli from older animals on the spinach-enriched diet had significantly less TNFalpha and TNFbeta than cerebelli from older animals on the control diet.

Immobilization stress rapidly modulates BDNF mRNA expression in the hypothalamus of adult male rats.

We demonstrated that short times (15 min) of immobilization stress application induced a very rapid increase in brain-derived neurotrophic factor (BDNF) mRNA expression in rat hypothalamus followed by a BDNF protein increase. The early change in total BDNF mRNA level seems to reflect increased expression of the BDNF transcript containing exon III, which was also rapidly (15 min) modified. The paraventricular and supraoptic nuclei, two hypothalamic nuclei closely related to the stress response and known to express BDNF mRNA, were analyzed by in situ hybridization following immobilization stress. In the parvocellular region of the paraventricular nucleus, BDNF mRNA levels increased very quickly as early as 15 min. In contrast, in the two other regions examined, the lateral and ventral magnocellular regions of the paraventricular nucleus, as well as in the supraoptic nucleus, signals above control were increased later, at 60 min. After stress application, plasma adrenocorticotropic hormone and corticosterone levels were strongly and significantly increased at 15 min. These studies demonstrated that immobilization stress challenge very rapidly enhanced BDNF mRNA levels as well as the protein, suggesting that BDNF may play a role in plasticity processes related to the stress response.

Incoming nucleotide binds to Klenow ternary complex leading to stable physical sequestration of preceding dNTP on DNA.

Klenow-DNA complex is known to undergo a rate-limiting, protein conformational transition from an 'open' to 'closed' state, upon binding of the 'correct' dNTP at the active site. In the 'closed' state, Mg(2+) mediates a rapid chemical step involving nucleophilic displacement of pyrophosphate by the 3' hydroxyl of the primer terminus. The enzyme returns to the 'open' state upon the release of PPi and translocation permits the next round of reaction. To determine whether Klenow can translocate to the next site on the addition of the next dNTP, without the preceding chemical step, we studied the ternary complex (Klenow-DNA-dNTP) in the absence of Mg(2+). While the ternary complex is proficient in chemical addition of dNTPs in Mg(2+), as revealed by primer extensions, the same in Mg(2+)-deficient conditions lead to non-covalent (physical) sequestration of first two 'correct' dNTPs in the ternary complex. Moreover, the second dNTP traps the first one in the DNA-helix of the ternary complex. Such a dNTP-DNA complex is found to be stable even after the dissociation of KLENOW: This reveals the novel state of the dNTP-DNA complex where the complementary base is stacked in a DNA-helix non-covalently, without the phosphodiester linkage. Further, shuttling of the DNA between the polymerase and the exonuclease site mediates the release of such a DNA complex. Interestingly, Klenow in such a Mg(2+)-deficient ternary complex exhibits a 'closed' conformation.

Leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum.

We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S(1) nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from -50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the -1 position and a BRE element with more frequent A bases at position -29 to -32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.

Constitutive expression of the steroid sulfatase gene supports the growth of MCF-7 human breast cancer cells in vitro and in vivo.

Many human breast tumors are driven by high intratumor concentrations of 17beta-estradiol that appear to be locally synthesized. The role of aromatase is well established, but the possible contribution of the steroid sulfatase (STS), which liberates estrogens from their biologically inactive sulfates, has been inadequately assessed and remains unclear. To evaluate the role of STS further, we transduced estrogen-dependent MCF-7 human breast cancer cells with a retroviral vector directing the constitutive expression of the human STS gene. Gene integration was confirmed by Southern hybridization, production of the appropriately sized messenger RNA by Northern hybridization, and expression of functional protein by metabolism of [(3)H]estrone sulfate to [(3)H]estrone. Maximum velocity estimates of estrone formation are 64.2 pmol estrone/mg protein.h in STS-transduced cells (STS Clone 20), levels comparable to those seen in some human breast tumors. Lower levels of endogenous activity are seen in MCF-7 cells (13.0 pmol estrone/mg protein.h) and in cells transduced with vector lacking the STS gene (Vector 3 cells; 12.0 pmol estrone/mg protein.h). 17beta-Estradiol sulfate induces expression of the progesterone receptor messenger RNA only in STS Clone 20 cells, whereas estrone sulfate produces the greatest stimulation of anchorage-independent growth in these cells. STS Clone 20 cells retain responsiveness to antiestrogens, which block the ability of estrogen sulfate to increase the proportion of cells in both the S and G(2)/M phases of the cell cycle. Consistent with these in vitro observations, only STS Clone 20 cells exhibit a significant increase in the proportion of proliferating tumors in nude ovariectomized mice supplemented with 17beta-estradiol sulfate. The primary activity in vivo appears to be from intratumor STS, rather than hepatic STS. Surprisingly, 17beta-estradiol sulfate appears more effective than 17beta-estradiol when both are administered at comparable concentrations. This effect, which is seen only in STS Clone 20 cells, may reflect differences in the cellular pharmacology of exogenous estrogens compared with those released by the activity of intracellular STS. These studies directly demonstrate that intratumor STS activity can support estrogen-dependent tumorigenicity in an experimental model and may contribute to the promotion of human breast tumors.