Biodegradation and fate of ethyl tert-butyl ether (ETBE) in soil and groundwater: A review.

This review summarises the current state of knowledge on the biodegradation and fate of the gasoline ether oxygenate ethyl tert-butyl ether (ETBE) in soil and groundwater. Microorganisms have been identified in soil and groundwater with the ability to degrade ETBE aerobically as a carbon and energy source, or via cometabolism using alkanes as growth substrates. Aerobic biodegradation of ETBE initially occurs via hydroxylation of the ethoxy carbon by a monooxygenase enzyme, with subsequent formation of intermediates which include acetaldehyde, tert-butyl acetate (TBAc), tert-butyl alcohol (TBA), 2-hydroxy-2-methyl-1-propanol (MHP) and 2-hydroxyisobutyric acid (2-HIBA). Slow cell growth and low biomass yields on ETBE are believed to result from the ether structure and slow degradation kinetics, with potential limitations on ETBE metabolism. Genes known to facilitate transformation of ETBE include ethB (within the ethRABCD cluster), encoding a cytochrome P450 monooxygenase, and alkB-encoding alkane hydroxylases. Other genes have been identified in microorganisms but their activity and specificity towards ETBE remains poorly characterised. Microorganisms and pathways supporting anaerobic biodegradation of ETBE have not been identified, although this potential has been demonstrated in limited field and laboratory studies. The presence of co-contaminants (other ether oxygenates, hydrocarbons and organic compounds) in soil and groundwater may limit aerobic biodegradation of ETBE by preferential metabolism and consumption of available dissolved oxygen or enhance ETBE biodegradation through cometabolism. Both ETBE-degrading microorganisms and alkane-oxidising bacteria have been characterised, with potential for use in bioaugmentation and biostimulation of ETBE degradation in groundwater.

Influence of contaminant exposure on the development of aerobic ETBE biodegradation potential in microbial communities from a gasoline-impacted aquifer.

Aerobic biodegradation of ethyl tert butyl ether (ETBE) in a gasoline-impacted aquifer was investigated in laboratory microcosms containing groundwater and aquifer material from ETBE-impacted and non-impacted locations amended with either ETBE, or ETBE plus methyl tert butyl ether (MTBE). As sole substrate, ETBE was biodegraded (maximum rate of 0.54 day-1) without a lag in ETBE-impacted microcosms but with a lag of up to 66 days in non-impacted microcosms (maximum rate of 0.38 day-1). As co-substrate, ETBE was biodegraded preferentially (maximum rate of 0.25 and 0.99 day-1 in non-impacted and impacted microcosms, respectively) before MTBE (maximum rate of 0.24 and 0.36 day-1 in non-impacted and impacted microcosms, respectively). Further addition of ETBE and MTBE reduced lags and increased biodegradation rates. ethB gene copy numbers increased significantly (>100 fold) after exposure to ETBE, while overall cell numbers remained constant, suggesting that ethB-containing microorganisms come to dominate the microbial communities. Deep sequencing of 16S rRNA genes identified members of the Comamonadaceae family that increased in relative abundance upon exposure to ETBE. This study demonstrates the potential for ETBE biodegradation within the unsaturated and saturated zone, and that ETBE biodegrading capability is rapidly developed and maintained within the aquifer microbial community over extended timescales.

Efficient and automatic methods for flexible regression on spatiotemporal data, with applications to groundwater monitoring.

Fitting statistical models to spatiotemporal data requires finding the right balance between imposing smoothness and following the data. In the context of P-splines, we propose a Bayesian framework for choosing the smoothing parameter, which allows the construction of fully automatic data-driven methods for fitting flexible models to spatiotemporal data. An implementation, which is highly computationally efficient and exploits the sparsity of the design and penalty matrices, is proposed. The findings are illustrated using a simulation study and two examples, all concerned with the modelling of contaminants in groundwater. This suggests that the proposed strategy is more stable that competing methods based on the use of criteria such as generalised cross-validation and Akaike's Information Criterion. © 2015 The Authors. Environmetrics Published by John Wiley Sons Ltd.

The pathway from glandular fever to chronic fatigue syndrome: can the cognitive behavioural model provide the map?

BACKGROUND: The cognitive behavioural model of chronic fatigue syndrome (CFS) suggests that the illness is caused through reciprocal interactions between physiology, cognition, emotion and behaviour. The purpose of this study was to investigate whether the psychological factors operationalized in this model could predict the onset of CFS following an acute episode of infectious mononucleosis commonly known as glandular fever (GF). METHOD: A total of 246 patients with GF were recruited into this prospective cohort study. Standardized self-report measures of perceived stress, perfectionism, somatization, mood, illness beliefs and behaviour were completed at the time of their acute illness. Follow-up questionnaires determined the incidence of new-onset chronic fatigue (CF) at 3 months and CFS at 6 months post-infection. RESULTS: Of the participants, 9.4% met the criteria for CF at 3 months and 7.8% met the criteria for CFS at 6 months. Logistic regression revealed that factors proposed to predispose people to CFS including anxiety, depression, somatization and perfectionism were associated with new-onset CFS. Negative illness beliefs including perceiving GF to be a serious, distressing condition, that will last a long time and is uncontrollable, and responding to symptoms in an all-or-nothing behavioural pattern were also significant predictors. All-or-nothing behaviour was the most significant predictor of CFS at 6 months. Perceived stress and consistently limiting activity at the time of GF were not significantly associated with CFS. CONCLUSIONS: The findings from this study provide support for the cognitive behavioural model and a good basis for developing prevention and early intervention strategies for CFS.

A randomized controlled trial of a cognitive behavioural therapy-based self-management intervention for irritable bowel syndrome in primary care.

BACKGROUND: Recent guidelines for the treatment of irritable bowel syndrome (IBS) emphasize the need for research to facilitate home-based self-management for these patients in primary care. The aim of the current study was to test the efficacy of a manualized cognitive behavioural therapy (CBT)-based self-management programme for IBS in a pilot randomized controlled trial (RCT). METHOD: Sixty-four primary-care patients meeting Rome criteria for IBS were randomized into either self-management plus treatment as usual (TAU) (n=31) or a TAU control condition (n=33). The self-management condition included a structured 7-week manualized programme that was self-administered in conjunction with a 1-hour face-to-face therapy session and two 1-hour telephone sessions. The primary outcome measures were the Subject's Global Assessment (SGA) of Relief and the Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) assessed at baseline, end of treatment (2 months), and 3 and 6 months post-treatment. RESULTS: Analysis was by intention-to-treat. Twenty-three (76.7%) of the self-management group rated themselves as experiencing symptom relief across all three time periods compared to seven (21.2%) of the TAU controls [odds ratio (OR) 12.2, 95% confidence interval (CI) 3.72-40.1]. At 8 months, 25 (83%) of the self-management group showed a clinically significant change on the IBS-SSS compared to 16 (49%) of the control group (OR 5.3, 95% CI 1.64-17.26). CONCLUSIONS: This study provides preliminary evidence that CBT-based self-management in the form of a structured manual and minimal therapist contact is an effective and acceptable form of treatment for primary-care IBS patients.

Processes controlling the distribution and natural attenuation of dissolved phenolic compounds in a deep sandstone aquifer.

Processes controlling the distribution and natural attenuation (NA) of phenol, cresols and xylenols released from a former coal-tar distillation plant in a deep Triassic sandstone aquifer are evaluated from vertical profiles along the plume centerline at 130 and 350 m from the site. Up to four groups of contaminants (phenols, mineral acids, NaOH, NaCl) form discrete and overlapping plumes in the aquifer. Their distribution reflects changing source history with releases of contaminants from different locations. Organic contaminant distribution in the aquifer is determined more by site source history than degradation. Contaminant degradation at total organic carbon (TOC) concentrations up to 6500 mg l(-1) (7500 mg l(-1) total phenolics) is occurring by aerobic respiration NO3-reduction, Mn(IV)-/Fe(III)-reduction, SO4-reduction, methanogenesis and fermentation, with the accumulation of inorganic carbon, organic metabolites (4-hydroxybenzaldehyde, 4-hydroxybenzoic acid), acetate, Mn(II), Fe(II), S(-II), CH4 and H2 in the plume. Aerobic and NO3-reducing processes are restricted to a 2-m-thick plume fringe but Mn(IV)-/Fe(II)-reduction, SO4-reduction, methanogenesis and fermentation occur concomitantly in the plume. Dissolved H2 concentrations in the plume vary from 0.7 to 110 nM and acetate concentrations reach 200 mg l(-1). The occurrence of a mixed redox system and concomitant terminal electron accepting processes (TEAPs) could be explained with a partial equilibrium model based on the potential in situ free energy (deltaGr) yield for oxidation of H2 by specific TEAPs. Respiratory processes rather than fermentation are rate limiting in determining the distribution of H2 and TEAPs and H2 dynamics in this system. Most (min. 90%) contaminant degradation has occurred by aerobic and NO3-reducing processes at the plume fringe. This potential is determined by the supply of aqueous O2 and NO3 from uncontaminated groundwater, as controlled by transverse mixing, which is limited in this aquifer by low dispersion. Consumption to date of mineral oxides and SO4 is, respectively, <0.15% and 0.4% of the available aquifer capacity, and degradation using these oxidants is <10%. Fermentation is a significant process in contaminant turnover, accounting for 21% of degradation products present in the plume, and indicating that microbial respiration rates are slow in comparison with fermentation. Under present conditions, the potential for degradation in the plume is very low due to inhibitory effects of the contaminant matrix. Degradation products correspond to <22% mass loss over the life of the plume, providing a first-order plume scale half-life >140 years. The phenolic compounds are biodegradable under the range of redox conditions in the aquifer and the aquifer is not oxidant limited, but the plume is likely to be long-lived and to expand. Degradation is likely to increase only after contaminant concentrations are reduced and aqueous oxidant inputs are increased by dispersion of the plume. The results imply that transport processes may exert a greater control on the natural attenuation of this plume than aquifer oxidant availability.

Isotopic modelling of the significance of bacterial sulphate reduction for phenol attenuation in a contaminated aquifer.

A Triassic sandstone aquifer polluted with a mixture of phenolic hydrocarbons has been investigated by means of high-resolution groundwater sampling. Samples taken at depth intervals of 1 m have revealed the presence of a diving pollutant plume with a sharply defined upper margin. Concentrations of pollutant phenols exceed 4 g/l in the plume core, rendering it sterile but towards the diluted upper margin evidence for bacterial sulphate reduction (BSR) has been obtained. Groundwaters have been analysed for both delta34S-SO4 and delta18O-SO4. Two reservoirs have been identified with distinct sulphate oxygen isotope ratios. Groundwater sulphate (delta18O-SO4 = 3-5/1000) outside the plume shows a simple linear mixing trend with an isotopically uniform pollutant sulphate reservoir (delta18O-SO4 = 10-12/1000) across the plume margin. The sulphur isotope ratios do not always obey a simple mixing relation, however, at one multilevel borehole, enrichment in 34SO4 at the plume margin is inversely correlated with sulphate concentration. This and the presence of 34S-depleted dissolved sulphide indicate that enrichment in 34SO4 is the result of bacterial sulphate reduction. Delta34S analysis of trace hydrogen sulphide within the plume yielded an isotope enrichment factor (epsilon) of -9.4/1000 for present-day bacterial sulphate reduction. This value agrees with a long-term estimate (-9.9/1000) obtained from a Rayleigh model of the sulphate reduction process. The model was also used to obtain an estimate of the pre-reduction sulphate concentration profile with depth. The difference between this and the present-day profiles then gave a mass balance for sulphate consumption. The organic carbon mineralisation that would account for this sulphate loss is shown to represent only 0.1/1000 of the phenol concentration in this region of the plume. Hence, the contribution of bacterial sulphate reduction to biodegradation has thus far been small. The highest total phenolic concentration (TPC) at which there is sulphur isotope evidence of bacterial sulphate reduction is 2000 mg/l. We suggest that above this concentration, the bactericidal properties of phenol render sulphate-reducing bacteria inactive. Dissolved sulphate trapped in the concentrated plume core will only be utilised by sulphate reducers when toxic phenols in the plume are diluted by dispersion during migration.

Denitrification and phenol degradation in a contaminated aquifer.

A natural groundwater system modified by pollutant phenols and agricultural nitrate has been modelled in the laboratory by a series of sacrificial microcosm experiments. Samples of aquifer sediment and groundwater from the margin of the phenol plume were used to inoculate anaerobic microcosms enriched in nitrate and pollutant phenols. Rapid degradation of phenol and p-cresol was observed over a 35-day period leading to the generation of inorganic carbon and a number of transient intermediates. O-cresol proved to be recalcitrant on the experimental time-scale. A mass balance calculation shows that, during degradation, carbon was conserved in the aqueous phase. Groundwater-sediment interactions were monitored using carbon stable isotope data. A mass balance for solution TIC indicates thatp-cresol degradation stimulated the dissolution of sedimentary carbonate phases due to the formation of carbonic acid. Compound-specific carbon isotope analysis (GC-IRMS) was used to search for 13C enrichment in residual p-cresol. A slight enrichment trend (epsilon = -2.5/1000) was tentatively identified. The potential of this fractionation effect for obtaining in situ degradation rates is discussed. Results from the microcosm experiments help to explain the observed distribution of nitrate and phenols within the polluted aquifer.

Recognition and sex categorization of adults' and children's faces: examining performance in the absence of sex-stereotyped cues.

The ability of children and adults to classify the sex of children's and adults' faces using only the biologically based internal facial structure was investigated. Face images of 7- to 10-year-old children and of adults in their 20s were edited digitally to eliminate hairstyle and clothing cues to sex. Seven-year-olds, nine-year-olds, and adults classified a subset of these faces by sex and were asked, subsequently, to recognize the faces from among the entire set of faces. This recognition task was designed to assess the relationship between categorization and recognition accuracy. Participants categorized the adult faces by sex at levels of accuracy varying from just above chance (7-year-olds) to nearly perfect (adults). All participant groups performed less accurately for children's faces than for adults' faces. The 7-year-olds were unable to classify the children's faces by sex at levels above chance. Finally, the faces of children and adults were equally recognizable--a finding that has theoretical implications for understanding the relationship between categorizing and identifying faces.

Transcriptional suppression of synuclein gamma (SNCG) expression in human breast cancer cells by the growth inhibitory cytokine oncostatin M.

Previously, we have shown that synuclein gamma (SNCG), a member of the brain protein synuclein family, is highly expressed in human infiltrating breast carcinomas but not expressed in normal or benign breast tissues. The SNCG mRNA was also detected in several human breast cancer cell lines with the highest expression found in H3922, a cell line derived from an infiltrating ductal carcinoma. In this study, we show that expression of SNCG mRNA in H3922 cells is significantly decreased by treating cells with the cytokine oncostatin M (OM) who has a growth-inhibitory effect on these cells. A decrease in SNCG mRNA level can be detected as early as 30 min after OM addition. By 4 h OM treatment, the level of SNCG mRNA was decreased to 70% of control, and by 24 h the mRNA was below detectable level. Western blot analysis further demonstrated the suppression of SNCG protein expression by OM. The level of SNCG protein in H3922 cells was reduced more than 90% by OM after 2 days. Since OM-induced growth inhibition occurs after 3-4 days, the down-regulation of SNCG expression appears to proceed the effect of OM on cell growth. Additional experiments to measure the transcriptional rates of SNCG indicate that the observed OM-induced down-regulation of SNCG mRNA occurs mainly at the transcriptional level. In an attempt to examine the role of SNCG gene in the proliferation of breast cancer cells, SNCG cDNA was stably transfected into MCF-7 cells that do not express endogenous SNCG gene. Examination of cell growth under anchorage-dependent and anchorage-independent conditions demonstrates that over expression of SNCG gene significantly stimulated the growth of MCF-7 cells both in monolayer culture and in soft agar. These data together suggest that SNCG may play a role in cell proliferation.

Oncostatin M suppresses EGF-mediated protein tyrosine phosphorylation in breast cancer cells.

The effect of oncostatin M (OM) on epidermal growth factor (EGF)-mediated protein tyrosine phosphorylation in an infiltrating ductal breast carcinoma cell line, H3922, was investigated by Western blot analysis. Pretreatment of H3922 cells with OM for 72 h suppressed EGF-stimulated protein tyrosine phosphorylation signals by 77%. Interestingly, pretreatment with OM for 6 or 48 h had little effect on these signals. EGF-mediated tyrosine phosphorylation of EGF receptor (EGFR) was suppressed by 55% in 72-h OM pretreated H3922 cells. No reduction in EGFR protein expression was detected in these cells. Flow cytometric analysis verified that OM does not suppress EGFR expression. The effect of OM could not be attributed to induction of protein tyrosine phosphatases. An H3922 subclone cell line, designated H3922-8, was found to exhibit no proliferative response to treatment with EGF. However, EGF-mediated protein tyrosine phosphorylation was detected in these cells. Radioligand EGF binding studies comparing H3922 to H3922-8 cells indicated that the clonal cells apparently lack high affinity EGF receptors. The mechanism by which OM suppresses EGF-mediated tyrosine phosphorylation has not been completely characterized. However, the suppressive effect occurs regardless of whether the cells are acutely responsive (H3922) or virtually unresponsive (H3922-8) to EGF stimulation of cell growth.

The expression of p53 tumor suppressor gene in breast cancer cells is down-regulated by cytokine oncostatin M.

Previously (J. Liu, et al., Cell Growth Differ., 8: 667-676, 1997), we showed that oncostatin M (OM), a cytokine produced by activated T cells and macrophages, inhibited the proliferation of breast cancer cells derived from solid tumors and malignant effusions. OM-treated cells showed reduced growth rates and differentiated phenotypes. Because the p53 tumor suppressor protein plays an important role in cellular proliferation, we examined p53 protein expression in three OM-responsive breast cancer cell lines, MCF-7, MDA-MB231, and H3922. Western blot analysis showed that p53 protein levels in all three of the cell lines were decreased by OM treatment. Reduction of p53 protein was detected after 1 day of OM treatment and reached maximal suppression of 10-20% of control after 3 days in H3922 and 40% of control after 4 days in MCF-7 cells. A comparison of p53 mRNA in OM-treated cells versus untreated control cells showed that exposure to OM reduced the steady-state levels of p53 mRNA transcripts to an extent similar to that of the p53 protein levels. This observation suggests that the effect of OM on p53 protein expression does not occur at the posttranslational level. Nuclear run-on assays verified that OM decreased the number of actively transcribed p53 mRNAs, which suggests a transcriptional regulatory mechanism. The effect of OM on p53 expression seems to be mediated through the extracellular signal-regulated kinase (ERK) pathway, inasmuch as the inhibition of ERK activation with a specific inhibitor (PD98059) to the ERK upstream kinase mitogen/extracellular-regulated protein kinase kinase abrogated the OM inhibitory activity on p53 expression in a dose-dependent manner. In addition to OM, we showed that the p53 protein expression in MCF-7 cells was also decreased by phorbol 12-myristate 13-acetate treatment (PMA). Because both OM and PMA induce MCF-7 cells to differentiate, our data suggest that p53 expression in breast cancer cells is down-regulated during the differentiation process.

Cloning and characterization of human oncostatin M promoter.

Oncostatin M (OSM), an IL-6 subfamily cytokine, inhibits proliferation and causes morphological changes in many tumor cell lines. GM-CSF, phorbol-12-myristate-13-acetate (PMA), and lipopolysaccharide (LPS) induce OSM expression. To investigate the mechanisms governing OSM promoter activity, we have cloned and partially sequenced an 8.5 kb fragment of human genomic DNA immediately 5' of the OSM coding region and mapped the transcription start site. Transient transfection assays with a series of 5' deletion plasmids demonstrated maximal reporter activity in U937 cells with a minimum 304 bp construct. The 5'-proximal region of the human OSM gene contains a C/EBP consensus element around -45 bp and several GC-rich regions around -60, each of which is responsible for basal promoter activity. Electrophoretic mobility shift assay coupled with supershift analysis confirmed the presence of a cis -acting binding site for activated STAT5 complexes following GM-CSF treatment. Furthermore, transient transfection studies demonstrated a loss of GM-CSF responsiveness in reporter constructs containing mutations within this STAT element. Our results establish that C/EBP and an as yet unidentified GC-rich binding transcription factor are responsible for basal OSM promoter activity, while GM-CSF-stimulated OSM expression is driven by activated STAT5 complexes binding to a cis -acting STAT element on the OSM promoter.

Developmental course of auditory processing interactions: Garner interference and Simon interference.

Previous research suggests that with increasing age children become more efficient in inhibiting conflicting responses and in resisting interference from irrelevant information. We assessed the abilities of 100 children (ages 3-16 years) and 20 adults to resist interference during the processing of 2 auditory dimensions of speech, namely the speaker's gender and spatial location. The degree of interference from irrelevant variability in either dimension did not vary with age. Apparently, young children do not have more difficulty in resisting interference when the nontarget and the target are both perceptual attributes. We also assessed the participants' abilities to inhibit conflicting task-irrelevant information from spatial location and to resist interference from spatial variability in the context of conflict. In the presence of conflicting task-irrelevant information, both interference effects declined significantly with age. Developmental change in auditory processing seems to vary as a function of (1) the nature of the target-nontarget combination and (2) the presence/absence of conflicting task-irrelevant information.

Oncostatin M-specific receptor expression and function in regulating cell proliferation of normal and malignant mammary epithelial cells.

Oncostatin M (OSM) is a cytokine produced by activated T lymphocytes and macrophages. OSM is structurally and functionally related to leukaemia inhibitory factor (LIF), another cytokine in the interleukin 6 (IL-6) family. The biological activities of OSM are mediated through two types of receptor complexes, the LIF/OSM shared receptor (type I) and OSM-specific receptor (OSM-R, type II), which is composed of gp130 as a binding subunit and a newly identified affinity conversion subunit, OSM-R beta. Previous research conducted in the authors' laboratory has shown that OSM inhibits the growth of several breast cancer cell lines. To investigate whether OSM has a similar effect in primary normal human mammary epithelial (HME) cells, the activity of OSM in HME cells derived from four donors was examined. OSM produced a dose-dependent inhibition of DNA synethesis in these cells. In order to determine the receptor subtypes mediating OSM activity in HME and breast cancer cells, flow cytometry analysis using anti-gp130mAb and anti-OSM-R beta mAb was performed. In these studies, the authors were able to examine expressions of gp130 and OSM-R beta. In addition, quantitative RT-PCR assays were conducted to measure expressions of the mRNAs of the subunits for type I and type II OSM receptor. The results show that HME cells and most breast cancer cell lines express both the type I and the type II OSM receptors. However, type II, OSM-specific receptors are expressed at a higher levels than type I, OSM/LIF shared receptors. Accordingly, we compared the growth regulatory activities of OSM with LIF in HME cells and in breast cancer cells. In contrast to the inhibitory activity of OSM, LIF stimulated the growth of breast cancer cells, whereas it had no effect on normal mammary epithelial cell growth. Together, these data suggest that OSM plays an inhibitory role in normal and malignant mammary epithelial cell growth in vitro. OSM activity is mediated by the OSM-specific receptor (type II), not by the OSM/LIF shared receptor.

Structural and functional analysis of the protein products derived from mutant fur alleles in an endoprotease-deficient Chinese hamster ovary cell strain.

The fur gene encodes the endoprotease, furin. We recently demonstrated mutations in both fur alleles in the mutant Chinese hamster ovary (CHO)-K1 strain, RPE.40, and hypothesized that these mutations were responsible for the endoprotease-deficient phenotype of these cells. We now present the structural and functional properties of three protein products derived from the mutant fur alleles. None of these protein products were able to process the precursor to von Willebrand factor, which is processed by wild-type furin. Pro-protein processing activity initially attributed to one of the mutant proteins was due to wild-type furin produced inadvertently from one of the expression constructs used in these experiments. None of the mutant proteins exhibited evidence of autocatalysis, consistent with the lack of activity versus the test substrate, and glycosylation patterns suggested at least two of them remained in the endoplasmic reticulum. These results confirm that RPE.40 cells are furin null mutants, as earlier evidence had suggested.

Six-month-olds' categorization of natural infant-directed utterances.

In this study, the authors demonstrated that 6-month-old infants are able to categorize natural, 650 Hz low-pass filtered infant-directed utterances. In Experiment 1, 24 male and 24 female infants heard 7 different tokens from 1 class of utterance (comforting or approving). Then, some infants heard a novel test stimulus from the familiar class of tokens; others heard a test stimulus from the unfamiliar class. Infants categorized these tokens as evidenced by response recovery to tokens from the unfamiliar class but not to novel tokens from the familiar class. Experiment 2 confirmed that the infants were able to discriminate between closely matched tokens from within each category, supporting the conclusion that the results of Experiment 1 indicated categorization. The authors discuss both a mechanism that might explain the development of this ability and the mutual adaptation seen in parent-infant communication.

Liver LDL receptor mRNA expression is decreased in human ApoB/CETP double transgenic mice and is regulated by diet as well as the cytokine oncostatin M.

We have investigated liver LDL receptor mRNA expression in nontransgenic, human cholesteryl ester transfer protein (CETP) transgenic, and human apolipoprotein (Apo) B/CETP double transgenic mice fed a normal chow diet and a high fat, high cholesterol diet (HFHC). Three weeks of HFHC feeding increased total serum cholesterol 1.5-fold in the nontransgenic, 3.1-fold in the CETP transgenic, and 3.4-fold in the ApoB/CETP double transgenic mice. To examine the liver LDL receptor mRNA expression among the different groups of mice fed the normal diet or fed the HFHC diet, we developed a quantitative reverse-transcribed polymerase chain reaction assay in which the LDL receptor mRNA level was normalized with the beta-actin mRNA. The results show that on the normal chow diet, the LDL receptor mRNA expression levels were lower in the ApoB/CETP mice than in the nontransgenic mice and the human CETP transgenic mice. Liver LDL receptor gene expression was lower in all groups of mice fed the HFHC diet, with the lowest level of expression in the ApoB/CETP mice. Similar results were obtained by Northern blot analysis. In addition, we have previously shown that the cytokine oncostatin M (OM) increases LDL receptor gene expression in HepG2 cells. In this study, we used the ApoB/CETP mice as the model system to examine the in vivo activity of OM on liver LDL receptor gene expression. Our data show that OM increased the level of liver LDL receptor mRNA up to 80% to 90% when the animals were fed the HFHC diet. The results from these studies demonstrate that the expression of the liver LDL receptor in the ApoB/CETP mice is suppressed compared with nontransgenic mice and that the expression of the hepatic LDL receptor gene in these mice is subjected to the normal cholesterol feedback regulation. In addition, LDL receptor gene expression in these mice is also inducible by a positive regulator.

Novel mechanism of transcriptional activation of hepatic LDL receptor by oncostatin M.

In this paper we describe a sterol-independent regulation of low density, lipoprotein receptor (LDLR) transcription by the cytokine oncostatin M (OM) in HepG2 cells. We show that OM-induced expression is independent of cholesterol regulation and occurs at the transcriptional level. To elucidate regulatory mechanism(s), we constructed a luciferase reporter system comprising either the native LDLR promoter including repeats 1, 2, and 3, or a synthetic promoter vector containing repeats 2+3 only, allowing us to directly examine OM effects on individual elements. Specific mutants in repeats 1, 2, and 3 were made to facilitate the mapping of the OM effect on the promoter. Wildtype and mutant constructs were assayed for cholesterol and OM regulation. The results show that mutation within the core SRE-1 element of repeat 2 totally abolished cholesterol regulation but had no effect on OM inducibility. More interesting, a mutation within repeat 1 reduced basal transcription activity to 10% of the native promoter, but OM induction was unaltered. However, the identical mutation engineered in repeat 3 significantly decreased OM induction of LDLR promoter activity. These results suggest a novel regulatory role for the repeat 3 element in LDLR transcription.

Oncostatin M-specific receptor mediates inhibition of breast cancer cell growth and down-regulation of the c-myc proto-oncogene.

Human oncostatin M (OM) is a M(r) 28,000 glycoprotein that has been shown to regulate cell proliferation and differentiation. The biological activities of OM can be mediated by two different heterodimeric receptor complexes, the leukemia inhibitory factor (LIF)/OM shared receptor and the OM-specific receptor. In this study, we have examined the growth-regulatory effect of OM on 10 breast cancer cell lines derived from human tumors. The cellular proliferation of seven of these breast cancer cell lines was inhibited by OM. The three cell lines that did not respond to OM treatment lacked the expression of OM receptors. The growth-inhibitory activity of OM is examined further in the H3922 breast cancer cell line, which expresses the high-affinity OM receptor at a relatively higher level. We found that the cellular proliferation of H3922 cells was induced strongly by extrogenous epidermal growth factor (EGF), EGF-like factor, and basic fibroblast growth factor. The proliferative activities of these growth factors can be abolished totally by cotreatment of H3922 cells with OM. Treatment of H3922 cells with OM for 24 h did not block EGF binding or the induction of EGF receptor tyrosine phosphorylation. This finding suggests that OM interferes with the mitogenic signal at steps distal to the EGF receptor. Examination of proto-oncogene expression demonstrated that OM down-regulates the c-myc gene in H3922 cells. The biological effects reported herein are not shared by the OM-related cytokines interleukin 6 or LIF, as demonstrated by the inability of these proteins to inhibit cell growth or modulate c-myc gene expression in breast cancer cells. Additionally, the high-affinity binding of labeled OM cannot be displaced by LIF. Together, these data suggest that OM is a growth inhibitor for breast cancer cells. The inhibitory activity is mediated predominantly through the OM-specific receptor, and activation of this receptor abrogates growth factor stimulation and down-regulates the c-myc proto-oncogene.