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PubMed1999

Urocortin expression in rat brain: evidence against a pervasive relationship of urocortin-containing projections with targets bearing type 2 CRF receptors.

Histochemical and axonal transport methods were used to clarify the central organization of cells and fibers that express urocortin (UCN), a recently discovered corticotropin-releasing factor (CRF)-related neuropeptide, which has been proposed as an endogenous ligand for type 2 CRF receptors (CRF-R2). Neurons that display both UCN mRNA and peptide expression were found to be centered in the Edinger-Westphal (EW), lateral superior olivary (LSO), and supraoptic nuclei; lower levels of expression are seen in certain cranial nerve and spinal motoneurons and in small populations of neurons in the forebrain. Additional sites of UCN mRNA and peptide expression detected only in colchicine-treated rats are considered to be minor ones. UCN-immunoreactive projections in brain are predominantly descending and largely consistent with central projections attributed to the EW and LSO, targeting principally accessory optic, precerebellar, and auditory structures, as well as the spinal intermediate gray. Although neither the EW nor LSO are known to project to the forebrain, UCN-ir neurons in the EW were identified that project to the lateral septal nucleus, which houses a prominent UCN-ir terminal field. Although substantial UCN-ir projections were observed to several brainstem cell groups that express CRF-R2, including the dorsal raphe and interpeduncular nuclei and the nucleus of the solitary tract (NTS), most prominent seats of CRF-R2 expression were found to contain inputs immunopositive for piscine urotensin I, but not rat UCN. The results define a central UCN system whose organization suggests a principal involvement in motor control and sensorimotor integration; its participation in stress-related mechanisms would appear to derive principally by virtue of projections to the spinal intermediolateral column, the NTS, and the paraventricular nucleus. Several observations, including the lack of a pervasive relationship of UCN-ir projections with CRF-R2-expressing targets, support the existence of still additional CRF-related peptides in mammalian brain.

Amino Acid Sequence↗

PubMed1998

Cardiac endothelin-1 plays a critical role in the functional deterioration of left ventricles during the transition from compensatory hypertrophy to congestive heart failure in salt-sensitive hypertensive rats.

BACKGROUND: To investigate whether endogenous ET-1 participates in an adaptive process of left ventricular hypertrophy (LVH) or a maladaptive process from LVH to congestive heart failure (CHF), we used a Dahl salt-sensitive (DS) rat model, in which systemic hypertension caused compensated concentric LVH at the age of 11 weeks followed by marked LV dilatation and global hypokinesis at the age of 17 weeks. METHODS AND RESULTS: By specific sandwich enzyme immunoassay, serum and myocardial ET-1 levels at the LVH stage were not elevated compared with age-matched Dahl salt-resistant (DR) rats, despite the marked increase of LV/body weight ratio (LV/BW). However, at the CHF stage, serum and LV ET-1 levels increased by 3. 8-fold and 5.4-fold, respectively. LV ET-1 contents had close relationships with the fractional shortening (r=0.763) and the systolic wall stress (r=0.858) measured by in vivo transthoracic echocardiography. Immunohistochemistry demonstrated that the remarkably increased ET-1 in LV is located mainly in cardiomyocytes. By competitive reverse transcriptase-polymerase chain reaction, LV prepro-ET-1 mRNA levels increased by 4.1-fold in CHF rats. We randomized 11-week-old LVH rats to chronic treatment with the endothelin receptor antagonist bosentan (Bos, 100 mg. kg-1. d-1, n=14), the alpha1-receptor antagonist doxazosin (Dox, 1 mg. kg-1. d-1, n=12), or vehicle (Cont, n=14). Bos treatment did not alter the LV geometry and function at 15 weeks; however, it attenuated the decrease of LV fractional shortening by 51% (P<0.01) without reducing the LV/BW at 17 weeks. Conversely, Dox, which decreased the blood pressure to the same extent as Bos, did not affect the progression of LV dysfunction. Bos (93%; P<0.0001 versus Cont) but not Dox (42%; P=0.8465 versus Cont) ameliorated the survival rate at 17 weeks (Cont; 36%). CONCLUSIONS: The accelerated myocardial synthesis of ET-1 contributes directly to LV contractile dysfunction during the transition from LVH to CHF. Unelevated levels of LV ET-1 at the established LVH stage and lack of effects on LV mass by chronic bosentan treatment suggest that myocardial growth is mediated through alternative pathways. These studies indicate that chronic ET antagonism may provide an additional strategy for heart failure therapy in humans.

Adaptation, Physiological↗

PubMed1994

Differential regulation of the expression of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 mRNAs in adult rat brain after intrahippocampal injection of quinolinic acid.

Intrahippocampal injection of the endogenous excitotoxin quinolinic acid (QUIN) induces seizures together with local, delayed neurodegeneration in specific cell layers. In situ hybridization histochemistry was used to study the spatio-temporal pattern of expression of neurotrophins (NTFs) after this treatment. As in other excitatory paradigms, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNA levels increased dramatically and transiently in dentate gyrus after the administration of 120 nmol of QUIN to the left hippocampus. BDNF, but not NGF, mRNA also increased in the hippocampal pyramidal cell layer, mainly in the CA1 field. Neurotrophin-3 (NT3) mRNA levels decreased in dentate gyrus, practically disappeared around 12 h after the insult and returned to basal levels four days later. A very different pattern of expression of NTFs was found locally: (a) upregulation of NGF and BDNF mRNAs expression was prevented in a spherical region of 1-2 mm diameter around the injection site, (b) a delayed increase in NT3 mRNA levels, beginning at 12 h and lasting for at least 4 days after the administration of QUIN, was found in the same region, in cell layers showing neurodegeneration. Pretreatment with the non-competitive NMDA antagonist MK-801 (2 mg/kg, 30 min before the insult), partially blocked the increase in both BDNF and NGF mRNAs, as well as the decrease in NT3, in the contralateral hippocampus. However, this treatment did not prevent the QUIN-induced local downregulation of NGF and BDNF. Treatment with the AMPA/kainate antagonist NBQX (30 mg/kg, 15 and 5 min before, and 10 min after the insult) did not influence the effect of QUIN upon NGF or BDNF mRNA levels, although it partially prevented the hippocampal contralateral decrease in NT3 mRNA. In conclusion, the present study strongly supports previous work concerning different regulation of BDNF/NGF respect to NT3 in seizure inducing paradigms. Moreover, the different and to some extent opposite regulation of NTFs in the hippocampal region contiguous to the injection site, respect to the remaining hippocampus, suggests a differential regulation of NTFs in QUIN-induced neurodegenerative and seizural processes. Finally, our pharmacological data, (i) show that the upregulation of NGF and BDNF mRNAs, indirectly induced by QUIN, is not mediated by AMPA receptors, and (ii) suggest other effects for QUIN, apart from the stimulation of NMDA receptors.

Animals↗

PubMed2000

Insulin-like growth factor-I is expressed by avian flexor tendon cells.

Cells in normal tendon are in a resting G0 state, performing maintenance functions. However, traumatic injury introduces growth factors such as platelet-derived growth factor and insulin-like growth factor from blood as well as activates endogenous growth factors. These factors stimulate migration and proliferation of tendon cells at the wound area. Tendon cells require growth-promoting factors to transit the cell cycle. To evaluate the contribution of endogenous growth factors in tendon, extracts of the epitenon and internal compartment of avian flexor tendon as well as medium of cultured cells from the epitenon (tendon surface cells) and internal tendon (tendon internal fibroblasts) were collected to assess their ability to stimulate DNA synthesis. Acid-ethanol extracts of tissues and medium were chromatographed on a P-30 molecular sieve column and assayed for mitogenic activity by quantitating [3H]thymidine incorporation into tendon cell DNA. The extract from the internal tendon compartment was more stimulatory for DNA synthesis than that from the epitenon, particularly when tested on tendon internal fibroblasts. However, conditioned medium fractions from surface epitenon cells stimulated DNA synthesis to a high degree on both tendon surface cells and tendon internal fibroblasts. Conditioned medium from tendon internal fibroblasts was also stimulatory. An anti-insulin-like growth factor-I antibody ablated most of the mitogenic activity present in both tissues and conditioned medium. The levels of acid-extractable insulin-like growth factor-I in tendon were determined by competitive radioimmunoassay as 1.48+/-0.05 ng/g tissue for the epitenon and 3.83+/-0.03 ng/g tissue for the internal compartment. Results of Western immunoblots of conditioned medium revealed insulin-like growth factor-I at the 7.5 kDa position. Cultured tendon surface cells and tendon internal fibroblasts as well as cells in intact flexor tendon expressed insulin-like growth factor-I mRNA detected by reverse transcriptase-polymerase chain reaction. In situ hybridization histochemistry positively identified insulin-like growth factor-I mRNA in tendons from 52-day-old chickens. Platelet-derived growth factor was not detected at the protein or message levels. Furthermore, tendon surface cells and tendon internal fibroblasts both expressed receptors for insulin-like growth factor-I detected by flow cytometry. These data suggest that tendon cells express insulin-like growth factor-I mRNA and synthesize insulin-like growth factor-I in both the epitenon and the internal compartment of tendon, which is present in an inactive form, most likely bound to insulin-like growth factor-binding proteins.

Animals↗

PubMed2000

Normal bronchial epithelial cell expression of glutathione transferase P1, glutathione transferase M3, and glutathione peroxidase is low in subjects with bronchogenic carcinoma.

Normal bronchial epithelial cells (NBECs) are at risk for damage from inhaled and endogenous oxidative species and from epoxide metabolites of inhaled polycyclic aromatic hydrocarbons. Epidemiological and in vitro data suggest that interindividual variation in this risk may result from variation in NBEC expression of enzymes that inactivate reactive species by conjugating them to glutathione. Quantitative competitive reverse transcription-PCR was used to measure mRNA levels of glutathione transferases (GSTs) and glutathione peroxidases (GSHPxs) in primary NBECs from subjects with or without bronchogenic carcinoma. Mean expression levels (mRNA/10(3) beta-actin mRNA) in NBECs from 23 subjects without bronchogenic carcinoma compared to those from 11 subjects with bronchogenic carcinoma respectively (in parentheses) were: mGST (26.0, 6.11), GSTM3 (0.29, 0.09), combined GSTM1,2,4,5 (0.98, 0.60), GSTT1 (0.84, 0.76), GSTP1 (287, 110), GSHPx (140, 62.1), and GSHPxA (0.43, 0.34). Levels of GSTP1, GSTM3, and GSHPx were significantly (P < 0.05) lower in NBECs from subjects with bronchogenic carcinoma. Further, the gene expression index formed by multiplying the values for mGST x GSTM3 x GSHPx x GSHPxA x GSTP1 had a sensitivity (90%) and specificity (76%) for detecting NBECs from bronchogenic carcinoma subjects that was better than any individual gene. In cultured NBECs derived from eight individuals without bronchogenic carcinoma and incubated under identical conditions such that environmental effects were minimized, the mean level of expression and degree of interindividual variation for each gene evaluated was less than that observed in primary NBECs. Data from these studies support the hypotheses that (a) interindividual variation in risk for bronchogenic carcinoma results in part from interindividual variation in NBEC expression of antioxidant genes; (b) gene expression indices will better identify individuals at risk for bronchogenic carcinoma than individual gene expression values; and (c) both hereditary and environmental exposures contribute to the level of and interindividual variation in gene expression observed in primary NBECs. Many epidemiological studies have been designed to evaluate risk associated with polymorphisms or gene expression levels of putative susceptibility genes based on measurements in surrogate tissues, such as peripheral blood lymphocytes. Based on data presented here, it will be important to include the assessment of NBECs in future studies. Measurement of antioxidant gene expression in NBECs may identify the 5-10% of individuals at risk for bronchogenic carcinoma. Bronchoscopic sampling of NBECs from smokers and ex-smokers then will allow susceptible individuals to be entered into surveillance and/or chemoprevention studies.

Adolescent↗

PubMed2006

Column experiments to assess the effects of electron donors on the efficiency of in situ precipitation of Zn, Cd, Co and Ni in contaminated groundwater applying the biological sulfate removal technology.

BACKGROUND, AIMS AND SCOPE: In a previous study, we explored the use of acetate, lactate, molasses, Hydrogen Release Compound (HRC, which is based on a biodegradable poly-lactate ester), methanol and ethanol as carbon source and electron donor to promote bacterial sulfate reduction in batch experiments, this with regards to applying an in situ metal precipitation (ISMP) process as a remediation tool to treat heavy metal contaminated groundwater at the site of a nonferrous metal work company. Based on the results of these batch tests, column experiments were conducted with lactate, molasses and HRCI as the next step in our preliminary study for a go-no go decision for dimensioning an on site application of the ISMP process that applies the activity of the endogenous population of sulfate-reducing bacteria (SRB). Special attention was given to the sustainability of the metal precipitation process under circumstances of changing chemical oxygen demand (COD) to [SO4(2-)] ratios or disrupted substrate supply. METHODS: To optimize the ISMP process, an insight is needed in the composition and activity of the indigenous SRB community, as well as information on the way its composition and activity are affected by process conditions such as the added type of C-source/ electron donor, or the presence of other prokaryotes (e.g. fermenting bacteria, methane producing Archaea, acetogens). Therefore, the biological sulfate reduction process in the column experiments was evaluated by combining classical analytical methods [measuring heavy metal concentration, SO4(2-)-concentration, pH, dissolved organic carbon (DOC)] with molecular methods [denaturing gradient gel electrophoresis (DGGE) fingerprinting and phylogenetic sequence analysis] based on either the 16S rRNA-gene or the dsr (dissimilatory sulfite reductase) gene, the latter being a specific biomarker for SRB. RESULTS AND DISCUSSION: All carbon sources tested promoted SRB activity, which resulted within 8 weeks in a drastic reduction of the sulfate and heavy metal contents in the column effluents. However, unexpected temporal decreases in the efficiency of the ISMP process, accompanied by the release of precipitated metals, were observed for most conditions tested. The most dramatic observation of the failing ISMP process was observed within 12 weeks for the molasses amended column. Subsequent lowering the COD/ SO4(2-) ratio from 1.9 to 0.4 did not alter the outcome of sulfate reduction and metal precipitation efficiency in this set-up. Remarkably, after 6 months of inactivity, bacterial sulfate reduction was recovered in the molasses set up when the original COD/ SO4(2-) ratio of 1.9 was applied again. Intentional disruption of the lactate and HRC supplies resulted in an immediate stagnation of the ISMP processes and in a rapid release of precipitated metals into the column effluents. However, the ISMP process could be restored after substrate amendment. 16S rDNA-based DGGE analysis revealed that the SRB population, in accordance with the results of the previously performed batch experiments, consisted exclusively of members of the genus Desulfosporosinus. The community of Archaea was characterized by sequencing amplicons of archaeal and methanogen-specific PCR reactions. This approach only revealed the presence of non-thermophilic Crenarchaeota, a novel group of organisms which is only distantly related to methane producing Euryarchaeota. DGGE on the dsrB genes was successfully used to link the results of the ISMP process to the community composition of the sulfate reducing bacteria. CONCLUSIONS: In the case of an intentional disruption of substrate supply, the ISMP process failed most likely because the growth and activity of the indigenous SRB community stopped due to a lack of a carbon and electron donor. On the other hand, the cause of the sudden temporal shortcomings of the ISMP process in the presence of different substrates was not immediately clear. It was first thought to be the result of competition between methanogenic prokaryotes (MP) and sulfate reducers, since the formation of small amounts of CH4 (0.01-0.03 ppm ml(-1) was detected. However, the results of molecular analyzes indicate that methanogens do not constitute a major fraction of the microbial communities that were enriched in the column experiments. Therefore, we postulate that the SRB population becomes inhibited by the formed metal sulfides. RECOMMENDATION AND PERSPECTIVE: Our results indicate that the ISMP process is highly dependent on SRB-stimulation by substrate amendments and suggest that this remedial approach might not be viable for long-term application unless substrate amendments are continued and environmental conditions are strictly controlled. This will include the removal of affected aquifer material from the metal precipitation zone at the end of the remediation process, or removal of metal precipitates when the microbial activity decreases. Additional tests are necessary to investigate what will happen when clear groundwater passes through the reactive zone while no more C-sources are amended and all indigenous carbon is consumed. Also, the effects of dramatic increases in sulfate- or HM-concentrations on the SRB-community and the concomitant ISMP process need to be studied in more detail.

Bacteria↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗