Methanogens rapidly transition from methane production to iron reduction.

Methanogenesis, the microbial methane (CH4 ) production, is traditionally thought to anchor the mineralization of organic matter as the ultimate respiratory process in deep sediments, despite the presence of oxidized mineral phases, such as iron oxides. This process is carried out by archaea that have also been shown to be capable of reducing iron in high levels of electron donors such as hydrogen. The current pure culture study demonstrates that methanogenic archaea (Methanosarcina barkeri) rapidly switch from methanogenesis to iron-oxide reduction close to natural conditions, with nitrogen atmosphere, even when faced with substrate limitations. Intensive, biotic iron reduction was observed following the addition of poorly crystalline ferrihydrite and complex organic matter and was accompanied by inhibition of methane production. The reaction rate of this process was of the first order and was dependent only on the initial iron concentrations. Ferrous iron production did not accelerate significantly with the addition of 9,10-anthraquinone-2,6-disulfonate (AQDS) but increased by 11-28% with the addition of phenazine-1-carboxylate (PCA), suggesting the possible role of methanophenazines in the electron transport. The coupling between ferrous iron and methane production has important global implications. The rapid transition from methanogenesis to reduction of iron-oxides close to the natural conditions in sediments may help to explain the globally-distributed phenomena of increasing ferrous concentrations below the traditional iron reduction zone in the deep 'methanogenic' sediment horizon, with implications for metabolic networking in these subsurface ecosystems and in past geological settings.

Squirrel monkey immunophilin FKBP51 is a potent inhibitor of glucocorticoid receptor binding.

Squirrel monkeys have high circulating cortisol to compensate for expression of low-affinity glucocorticoid receptors (GRs). We have demonstrated that the FK506-binding immunophilin FKBP51 is elevated in squirrel monkey lymphocytes (SML) and, in preliminary studies, have shown that squirrel monkey FKBP51 is inhibitory to GR binding. In this report, we have demonstrated that elevated FKBP51 is the unequivocal cause of glucocorticoid resistance in SML in the following ways: 1) FK506 increased GR binding in cytosol from SML in a concentration-dependent manner, an effect reproduced by rapamycin but not cyclosporin A. The apparent K6 (6.1 nM) and rank-order of steroid displacement of [3H]dexamethasone binding in FK506-treated SML cytosol are characteristic of high-affinity GR binding. 2) cytosol from COS-7 cells expressing squirrel monkey FKBP51 inhibited GR binding in cytosol from human lymphocytes by 74%. Cytosol from COS-7 cells expressing human FKBP51 inhibited GR binding by 23%. 3) expression of squirrel monkey FKBP51 increased the median effective concentration (EC50) for dexamethasone in GR transactivation studies in COS-7 cells by approximately 17-fold, compared with the EC50 in control cells. The expression of human FKBP51 increased the EC50 for dexamethasone in COS-7 cells by less than 3-fold, compared with control. Squirrel monkey FKBP51 shares 94% overall amino acid homology with human FKBP51, with 92% and 99% homology with human FKBP51 in the peptidyl-prolyl isomerase and the tetratricopeptide repeat domains, respectively. Amino acid differences in the more variable N- or C-terminal regions or in regions which join the highly homologous functional domains may be responsible for its more potent inhibitory activity.

Cell-specific expression of the rat secretogranin II promoter.

Secretogranin II (SgII) is a member of the granin family of secretory proteins, which are selectively expressed in neuroendocrine cells. As a first step in understanding the molecular basis for cell type-specific expression of SgII, we isolated a 12-kb clone from a rat genomic library that contained the entire rat SgII coding region, the transcription initiation site, and approximately 3 kb of 5'-flanking region. Within 75 bp of the transcription start site (+1) we located a TATA box and a consensus cAMP responsive element. Within the 5'-flanking region, a number of potential cis-acting elements were identified, including 2 Pit-1 binding sites, 15 E box motifs, and near-perfect matches for AP-1 and AP-2 sites. To demonstrate cell type-specific expression the rat SgII gene, a plasmid containing 2.6 kb of the 5'-flanking region of the SgII gene fused to the luciferase reporter gene (p2774Luc) was transfected into rat pheochromocytoma PC-12 cells, rat pituitary GH4C1 (GH) cells, human BE(2)-M17 (M17) neuroblastoma cells, and mouse fibroblast NIH/3T3 cells. The promoter activity was 6- to 36-fold higher in neuroendocrine cells than in NIH/ 3T3 cells. Progressive deletions in the 5'-flanking region to 61 bp upstream of the start site (p223Luc) had no effect on promoter activity in PC-12 cells. On the other hand, a 5'-deletion in the SgII promoter to -1032 increased promoter activity 3.8-fold in GH cells. This level of expression was maintained when the SgII promoter was further truncated to -189, whereas truncation to -61 resulted in a 2.6-fold reduction in promoter activity. These results suggest that the sequence between -61 and +162 bp is sufficient for SgII promoter activity in PC-12 cells. However, other elements in the 5'-flanking region contribute to both positive and negative regulation of the rat SgII gene in GH cells.

Prolactin granulogenesis is associated with increased secretogranin expression and aggregation in the Golgi apparatus of GH4C1 cells.

The GH4C1 pituitary tumor cell line (GH cells) serves as a model system to study the role of the granins in the packaging of PRL into secretory granules. The number of secretory granules containing PRL and two members of the granin family, chromogranin-B (CgB) and secretogranin-II (SgII), can be hormonally manipulated. In the present study we have investigated whether 1) granulogenesis in GH cells is preceded by condensation of the granins and PRL in the Golgi; 2) granulogenesis is preceded by an increase in granin expression in GH cells; and 3) PRL and the granins aggregate in vitro under high calcium, low pH conditions. GH cells were treated for up to 3 days with 17 beta-estradiol (1 nM), insulin (300 nM), and epidermal growth factor (10 nM) and were fixed in 4% paraformaldehyde for immunocytochemistry or harvested for RNA isolation and Northern blot analysis. After 1 day of hormone treatment, there was a significant increase in staining for PRL and the granins in the Golgi apparatus, which was identified using an antibody to MG-160. After 3 days of hormone treatment, PRL and granin staining was also found in a perinuclear region that was not stained with anti-MG-160 antibody, most likely representing secretory granules. An increase in PRL and granin expression contributed to increased Golgi staining, as the steady state levels of CgB, SgII, and PRL mRNA increased 186 +/- 14%, 203 +/- 7%, and 337 +/- 5% above control levels, respectively, within 6 h after hormone treatment. An in vitro aggregation system was used to determine whether PRL and the granins coprecipitate under high calcium, low pH conditions, which are thought to be characteristic of the trans-Golgi and secretory granules. Aggregation of the granins CgB and SgII was negligible during overnight dialysis against a buffer containing 150 mM NaCl and 10 mM 2[N-morpholino]ethanesulfonic acid-NaOH (pH 5.5) in the absence of calcium. There was significant aggregation of PRL under these conditions. When dialysis was performed in the presence of 10 mM CaCl2, PRL, CgB, and SgII coaggregated. This study indicates that increased expression and aggregation of the granins is associated with PRL granulogenesis in hormone-treated GH cells. However, the role of the granins may not be obligatory, as some cells can store PRL in the absence of detectable levels of CgB and SgII, and PRL has the capacity to self-aggregate.

Regulation of secretogranin II mRNA in rat neuronal cultures.

The regulation of SgII mRNA expression was investigated in primary cultures of neurons prepared from the hypothalamus and brainstem of 1-day-old rats. The administration of forskolin (FSK) resulted in a time- and dose-dependent increase in SgII mRNA expression, a 9-fold effect within 6 h being achieved with 10 microM FSK, which maximally increased cellular cAMP levels. SgII mRNA levels remained elevated for 24 h. Activation of protein kinase C with 100 nM phorbol 12-myristate 13-acetate (PMA) also increased SgII mRNA expression, although induction with PMA was slower and more moderate (3.8-fold above control after 24 h). Neither 10 microM 1,9-dideoxyforskolin nor 100 nM 4 alpha-phorbol 12,13-didecanoate, inactive analogues of FSK and PMA respectively, had an effect on SgII mRNA. Depolarization of neuronal cultures with 50 mM KCl had a small and variable effect on SgII mRNA levels (1.8-fold above control) in neuronal cultures and did not influence induction with FSK. To investigate whether neuron-like regulation of SgII mRNA expression could be reproduced in PC12 cells, PC12 cells were treated with 100 nM nerve growth factor (NGF) for 7 days prior to challenge with FSK or PMA. Whereas NGF alone modestly increased SgII mRNA expression in PC12 cells (1.8-fold above control), it did not uncover a stimulatory effect of FSK or PMA. These studies indicate that SgII mRNA expression is enhanced by an increase in cellular cAMP and activation of protein kinase C in primary cultures of neurons and emphasize that SgII mRNA is regulated in a cell-specific manner.

Isolation and characterization of the human secretogranin II gene promoter.

The goal of this study was to isolate and functionally characterize the human secretogranin II (SgII) gene promoter. SgII is a member of the granin family of proteins which are selectively expressed in neurosecretory cells. The human SgII promoter contains a consensus TATA box and cyclic AMP response element (CRE) 35 and 74 bp upstream of the transcription start site, respectively, elements also found in the mouse and rat SgII gene promoters. Transfection studies showed that 869 bp of the human SgII promoter were sufficient to confer cell type-specific expression of an SgII promoter-luciferase reporter gene in neurosecretory PC-12, GH and BE(2)-M17 cells. The activity of the human SgII promoter was also compared in three N-type, human neuroblastoma cell lines [BE(2)-M17, SMS-KAN and SH-SY5Y], which differ markedly in the level of SgII expression. SgII promoter activities in the neuroblastoma cell lines correlated not only with the levels of SgII but also the levels of the cyclic AMP response element-binding protein CREB which were highest in BE(2)-M17 cells and lowest in SH-SY5Y cells. To establish that the activity of the human SgII promoter in these neuroblastoma cell lines is dependent on the level of CREB, rat CREB was overexpressed in SH-SY5Y cells. SgII promoter activity was up to 8-fold higher in SH-SY5Y cells overexpressing CREB. These results suggest that SgII expression is a marker for neuronal differentiation in human neuroblastoma cell lines and is dependent on the level of CREB expression.

A culture apparatus for maintaining H2 at sub-nanomolar concentrations.

We devised a microbial culture apparatus capable of maintaining sub-nanomolar H2 concentrations. This apparatus provides a method for study of interspecies hydrogen transfer by externally fulfilling the thermodynamic requirement for low H2 concentrations, thereby obviating the need for use of cocultures to study some forms of metabolism. The culture vessel is constructed of glass and operates by sparging a liquid culture with purified gases, thereby removing H2 as it is produced. We used the culture apparatus to decouple a syntrophic association in an ethanol-consuming, methanogenic enrichment culture, allowing ethanol oxidation to dominate methane production. We also used the culture apparatus to grow pure cultures of the ethanol-oxidizing, proton-reducing Pelobacter acetylenicus (WoAcy 1), and to study the bioenergetics of growth.

Climatically driven emissions of hydrocarbons from marine sediments during deglaciation.

Marine hydrocarbon seepage emits oil and gas, including methane ( approximately 30 Tg of CH(4) per year), to the ocean and atmosphere. Sediments from the California margin contain preserved tar, primarily formed through hydrocarbon weathering at the sea surface. We present a record of variation in the abundance of tar in sediments for the past 32,000 years, providing evidence for increases in hydrocarbon emissions before and during Termination IA [16,000 years ago (16 ka) to 14 ka] and again over Termination IB (11-10 ka). Our study provides direct evidence for increased hydrocarbon seepage associated with deglacial warming through tar abundance in marine sediments, independent of previous geochemical proxies. Climate-sensitive gas hydrates may modulate thermogenic hydrocarbon seepage during deglaciation.

New perspectives on anaerobic methane oxidation.

Anaerobic methane oxidation is a globally important but poorly understood process. Four lines of evidence have recently improved our understanding of this process. First, studies of recent marine sediments indicate that a consortium of methanogens and sulphate-reducing bacteria are responsible for anaerobic methane oxidation; a mechanism of 'reverse methanogenesis' was proposed, based on the principle of interspecies hydrogen transfer. Second, studies of known methanogens under low hydrogen and high methane conditions were unable to induce methane oxidation, indicating that 'reverse methanogenesis' is not a widespread process in methanogens. Third, lipid biomarker studies detected isotopically depleted archaeal and bacterial biomarkers from marine methane vents, and indicate that Archaea are the primary consumers of methane. Finally, phylogenetic studies indicate that only specific groups of Archaea and SRB are involved in methane oxidation. This review integrates results from these recent studies to constrain the responsible mechanisms.

Induction of secretogranin II mRNA by protein synthesis inhibitors in GH4C1 cells.

The effect of cycloheximide (CHX) on the expression of secretogranin II (SgII), a member of the granin family of secretory proteins, was investigated in rat pituitary GH4C1 (GH) cells. The administration of CHX resulted in a dose- and time-dependent increase in SgII mRNA expression, the greatest effect (3.8-fold above control) being achieved with 1 microgram/ml CHX, which resulted in > 90% inhibition of protein synthesis. Emetine (1 microgram/ml), pactamycin (0.6 microgram/ml), anisomycin (2.5 micrograms/ml), and puromycin (100 micrograms/ml), protein synthesis inhibitors structurally and mechanistically unrelated to CHX, also increased the level of SgII mRNA expression. Treatment with forskolin (10 microM) alone had no effect on SgII mRNA levels but potentiated the effect of CHX. Neither 100 nM phorbol 12-myristate 13-acetate nor 45 mM KCl affected SgII mRNA levels in the absence or presence of 1 microgram/ml CHX. The effect of CHX was blocked by the transcription inhibitors actinomycin D (5 micrograms/ml) and 5,6-dichlorobenzimidazole riboside (20 micrograms/ml) but not by the coadministration of the polysome destabilizer pactamycin (0.6 microgram/ml), suggesting that the effect of CHX was transcriptional. These studies show that the expression of SgII mRNA is induced by protein synthesis inhibitors in GH cells, suggesting the presence of a labile repressor, which not only controls the basal expression of the SgII gene but also completely inhibits the stimulatory effect of forskolin.

Hydrogen production by methanogens under low-hydrogen conditions.

Hydrogen production was studied in four species of methanogens (Methanothermobacter marburgensis, Methanosaeta thermophila, Methanosarcina barkeri, and Methanosaeta concilii) under conditions of low (sub-nanomolar) ambient hydrogen concentration using a specially designed culture apparatus. Transient hydrogen production was observed and quantified for each species studied. Methane was excluded as the electron source, as was all organic material added during growth of the cultures (acetate, yeast extract, peptone). Hydrogen production showed a strong temperature dependence, and production ceased at temperatures below the growth range of the organisms. Addition of polysulfides to the cultures greatly decreased hydrogen production. The addition of bromoethanesulfonic acid had little influence on hydrogen production. These experiments demonstrate that some methanogens produce excess reducing equivalents during growth and convert them to hydrogen when the ambient hydrogen concentration becomes low. The lack of sustained hydrogen production by the cultures in the presence of methane provides evidence against "reverse methanogenesis" as the mechanism for anaerobic methane oxidation.

Overexpression of the FK506-binding immunophilin FKBP51 is the common cause of glucocorticoid resistance in three New World primates.

Many New World primates have high circulating levels of cortisol to compensate for the expression of glucocorticoid receptors (GRs) with low activity. Recent work in squirrel monkeys has suggested that this may be due to either the expression of GRs that are transcriptionally incompetent or the expression of an FK506-binding immunophilin that inhibits GR binding. The goal of this study was to resolve this controversy by determining the molecular basis of glucocorticoid resistance not only in species of squirrel monkeys but also in other glucocorticoid-resistant New World primates. First, the transcriptional activity of the GR from the Bolivian squirrel monkey was compared to that of the human GR. Incubation of COS-7 cells transfected with the squirrel monkey GR with 10 nM dexamethasone resulted in a robust stimulation of MMTV-luciferase activity (up to 260-fold), which was similar in magnitude to that achieved with the human GR. Second, the effect of FK506 on GR binding was determined in cytosol from cells from two species of squirrel monkeys as well as glucocorticoid-resistant cotton-top tamarins and owl monkeys. Incubation with 10 microM FK506 increased GR binding by at least 4-fold in cytosol from cells of each of the New World primates but had no effect on GR binding in cytosol from human WI-38 VA13 cells. Third, Western blots showed elevated expression of FKBP51 in New World primate cells and liver samples from two squirrel monkey species. On the other hand, the levels of FKBP52 were significantly lower in cells and liver from New World primates. The sequences of FKBP51 from the cotton-top tamarin, owl monkey and squirrel monkey are closely related and share differences from the human, rhesus monkey, mouse, and lemur FKBP51 sequences in the same 18 positions. Fourth, the relative activities of FKBP51 from the cotton-top tamarin, owl monkey and squirrel monkey were determined in cytosol mixing and GR transactivation studies and showed that FKBP51 from each of these primates was a potent inhibitor of GR activity. These results indicate that the elevated expression of FKBP51 contributes to glucocorticoid resistance in three New World primate genera.

Epitope mapping of monoclonal antibodies to bovine prolactin.

The epitopes recognized by three monoclonal antibodies generated to sheep prolactin were determined by evaluating their cross-reactivities by immunodot analysis with 14 mutants of bovine prolactin, in which individual amino acids had been deleted or substituted. Mutations were made throughout the molecule and included disruption of the amino-terminal, carboxyl-terminal, and central disulfide loops. Lack of immunoreactivity was taken as an indication that the site of mutation was part of the epitope. Antibody 6F11 reacted with all bovine prolactin mutants tested, except those in which the carboxyl-terminal cysteine (position 199) was substituted by a serine. Antibodies 5G2 and 4C10 reacted with all of the bovine prolactin mutants, except those in which the amino-terminal cysteine (position 4) was substituted by a serine. Western blot analysis of sheep, squirrel monkey, and rat prolactins with the monoclonal antibodies revealed that 5G2 and 4C10 were specific for sheep prolactin, whereas antibody 6F11 cross-reacted with prolactins from all three species. The mitogenic activity of sheep or rat prolactin in the Nb2 bioassay was determined in the presence of the antibodies to determine whether the epitopes were part of the functional domains of these prolactins. The bioactivity of sheep prolactin (0.4 ng/ml) was unaffected by the monoclonal antibodies [0.01-1 microgram immunoglobulin G (IgG)/ml], whereas the bioactivity of rat prolactin (1.25 ng/ml) was inhibited by 6F11 with an apparent 50% inhibitory concentration of 0.25 microgram IgG/ml. These results indicate that monoclonal antibodies 5G2 and 4C10 cross-react with a species-specific region of the amino-terminal disulfide loop of bovine prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional regulation of secretogranin II and chromogranin B by cyclic AMP in a rat pheochromocytoma cell line.

When PC-12 cells were treated with 10 microM forskolin, the expression of two members of the granin family, secretogranin II (SgII) and chromogranin B (CgB), were differentially regulated. SgII mRNA levels declined progressively after forskolin treatment to reach a level of 22 +/- 1% of control after 48 hr, whereas CgB mRNA levels increased more rapidly, reaching a maximum of 3-fold above control after 24 hr. The dependence of these changes on an increase in cellular cAMP levels, activation of cAMP-dependent protein kinase (PKA), protein synthesis, and changes in the rate of transcription was investigated. The effects of forskolin on SgII and CgB mRNAs were reproduced by 1 mM 8-bromo-cAMP but not by 10 microM 1,9-dideoxyforskolin, an inactive analog of forskolin. The actions of forskolin on SgII and CgB mRNAs were blocked by treatment with 60 microM H-89, a selective PKA inhibitor, and were blunted in PKA-deficient PC-12 cell clones. To examine whether forskolin action was dependent on ongoing protein synthesis, PC-12 cells were treated with 1 microgram/ml cycloheximide before the addition of forskolin. The reduction in SgII mRNA levels by forskolin was not evident in PC-12 cells treated with cycloheximide. Rather, in the presence of cycloheximide, forskolin stimulated SgII mRNA levels 3.6 +/- 0.7-fold above control. The induction of CgB mRNA by forskolin was not affected by cycloheximide treatment. The superinduction of SgII mRNA by cycloheximide and forskolin was related to the extent of protein synthesis inhibition, was observed in cells treated with forskolin and other protein synthesis inhibitors, and was blunted in PKA-deficient PC-12 cells, suggesting that this effect was dependent on inhibition of protein synthesis and activation of PKA. To determine whether changes in SgII and CgB mRNA levels resulted from changes in the rate of transcription, nuclear run-on assays were performed in nuclei isolated from PC-12 cells that had been treated for 2 hr with cycloheximide, forskolin, or the two combined. Transcription of the SgII gene was not significantly affected by treatment with either forskolin or cycloheximide alone but was increased 12.9 +/- 1.0-fold above control in nuclei from cells treated with cycloheximide and forskolin together. Forskolin caused a 3.8 +/- 0.8-fold induction of CgB transcription.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in bioactive prolactin-like activity in plasma and its relationship to incubation behavior in breeding ring doves.

Previous radioimmunoassay (RIA) data indicate that plasma prolactin (PRL) is elevated during the late incubation and the early posthatching periods of the ring dove breeding cycle. Although these changes are temporally associated with changes in PRL-dependent crop sac growth, the precise relationship between immunoreactive and bioactive PRL has not been directly examined. To investigate this question and to further explore the relationship between sitting behavior and PRL secretion, we used rat Nb2 lymphoma cell proliferation to estimate the concentration of bioactive PRL-like activity (PLA) in the plasma of breeding ring doves. Serial dilutions of dove pituitary homogenate and dove plasma stimulated mitogenic responses that were parallel to those observed with purified ovine PRL. Changes in plasma PLA during the breeding cycle closely resembled changes in PRL that have been previously reported by RIA, although the relative changes in PLA were more pronounced. In both sexes, PLA remained at basal levels prior to egg laying and during early incubation (Day 4-5) but then abruptly increased to reach peak values near the time of hatching (Day 14-15). Activity remained high for 3-4 days after hatching, declined gradually thereafter, and returned to baseline values by Posthatching Days 14-17. Plasma PLA levels of birds sampled at the end of incubation were correlated with those of their breeding partners. In the majority of pairs, females had higher PLA levels than their mates at this stage even though no significant overall sex differences in PLA levels were observed. Plasma PLA declined precipitously in birds that were nest deprived on the last day of the incubation period. Nevertheless, plasma PLA levels of normally breeding birds at the end of incubation were not correlated with the average time spent in the nest during the incubation period. However, day-to-day variability in time spent in the nest correlated negatively with plasma PLA in incubating males, and females exhibited a similar trend that approached significance. These data suggest (1) that published RIA estimates of PRL are reasonably accurate reflections of changes in bioactive PLA in dove plasma and (2) that while sitting duration itself is not strongly related to plasma PLA, large day-to-day fluctuations in nest occupation time are associated with reduced PLA levels in incubating doves.

Glucocorticoid-resistant B-lymphoblast cell line derived from the Bolivian squirrel monkey (Saimiri boliviensis boliviensis).

The goal of the study reported here was to develop a continuous cell line from the squirrel monkey that expresses the species-specific phenotype of impaired sensitivity to glucocorticoids. Thirty milliliters of blood from a male Bolivian squirrel monkey (Saimiri boliviensis boliviensis) was fractionated, and the buffy coat was obtained and incubated in the presence of B95-8 cell-conditioned medium, an abundant source of Epstein-Barr virus (EBV), and 2 micrograms of cyclosporin A/ml. Cell growth was detected within 8 weeks, after which the cells were cloned by use of the limiting dilution method. One clone (4D8) was characterized in detail. The chromosomal count and G-banding pattern confirmed that the cells were of Bolivian squirrel monkey origin. The B-cell origin of these cells was indicated by electron microscopic analysis and was confirmed by expression of CD20. The cells stained strongly for LMP1, a marker of latent EBV infection, and occasionally for the lytic infection marker ZEBRA (BZLF1). The responsiveness of clone 4D8 cells to glucocorticoids was determined by comparing the effects of dexamethasone on cell growth and the induction of a glucocorticoid-inducible mRNA in 4D8 cells with the effects on a human EBV-transformed B-lymphoblast cell line (HL). Dexamethasone inhibited the growth of HL cells, with IC50 of approximately 9 nM, but had no effect on the growth of 4D8 cells. The induction of FK506-binding protein FKBP51 mRNA by dexamethasone was also significantly blunted in 4D8 cells. Thus, we have developed and characterized a squirrel monkey lymphoblastic cell line derived by transformation of B-lymphocytes with EBV; the cell line has diminished growth and transcriptional responses to glucocorticoids.