Cambrian sea water preserved as inclusions in marine low-magnesium calcite cement.

THE existence of temporal changes in the chemical composition of the oceans, which could provide constraints on the potential variability of the ocean-atmosphere; system, remains an open question. Assessments of the chemistry of ancient oceans have relied largely on analysis of marine precipitates, generally carbonate and evaporite minerals1. These studies suggest that, whereas marine salinity has remained relatively stable over Phanerozoic time1, magnesium, calcium and sulphate concentrations of ancient oceans and CO2 partial pressure of ancient atmospheres may have changed2-4. The ratios of isotopes of carbon, oxygen, sulphur and strontium also appear to have varied3,5,6. Here we present analyses of primary, one-phase fluid inclusions in Cambrian and Ordovician marine cements, which appear to represent aliquots of early Palaeozoic oceans. The cements have trace element, stable isotope and strontium isotope contents that are consistent with their having been precipitated in a Cambrian-Ordovician marine environment, and the fluids have marine salinities. As these (apparently primary) cements are low-magnesium calcite, unlike the predominantly high-magnesium calcite and aragonite of today's carbonate precipitates, the chemistry of the Cambrian ocean-atmosphere system seems to have been different from that of today.

The effect of prostaglandin E2 on cystine uptake and glutathione synthesis by human lung fibroblasts.

Prostaglandin E2 (PGE2) is an inflammatory mediator capable of regulating fibroblast cell proliferation, matrix protein production, and system A amino acid transport. System x-c amino acid transport is regulated by electrophilic agents and oxygen. The effect of PGE2 on the x-c system transport of cystine and the synthesis of glutathione by human lung fibroblasts was examined. Preincubation of fibroblast cultures with PGE2 decreased cystine uptake by 42%. Kinetic studies revealed a 42% decrease in the Vmax of the x-c system transporter in PGE2-treated fibroblasts; however, the apparent Km was not affected. The glutathione content of PGE2-treated fibroblasts was decreased by up to 25% of control. These results demonstrate that system x-c transport of cystine is regulated by PGE2 and suggest that the limited availability of intracellular cysteine inhibited glutathione synthesis.

Retinoic acid-induced inhibition of type I collagen gene expression by human lung fibroblasts.

We examined alpha 1(I) collagen gene expression in all-trans retinoic acid (RA)-treated human lung fibroblast cultures. RA (10(-5)M) decreased steady-state levels for alpha 1(I) collagen mRNA by at least 75% after 24 h. The inhibition was evident within 8 h after addition of RA, was dose dependent and reversible. Treatment with 9-cis-retinoic acid did not affect alpha 1(I) collagen mRNA levels. RA also inhibited the increases in alpha 1(I) mRNA stimulated by transforming growth factor-beta (TGF-beta). The RA-mediated decrease in alpha 1(I) collagen mRNA was blocked by cycloheximide treatment, suggesting that synthesis of a protein intermediate is required for the inhibition. The RA-induced decrease in alpha 1(I) collagen mRNA levels was not mediated by increases in prostaglandin E2 production. RA decreased alpha 1(I) gene transcription as determined by nuclear run-off assays but did not significantly alter the rate of degradation of the alpha 1(I) transcript as determined by actinomycin D treatment. Studies employing cells stably transfected with constructs containing portions of the alpha 1(I) collagen promoter indicate that the DNA sequences which mediate the inhibitory effect are located within 900 bases from the transcription start site.

Discordant regulation of transforming growth factor-beta receptors by prostaglandin E2.

The effect of PGE2 on TGF-beta receptor binding was assessed for both signaling (type I and type II) and non-signaling (type III) TGF-beta receptors. We found in cross-linking studies that PGE2 treatment (24 h) decreased binding of TGF-beta to the type I and type II receptors by approx. 50% and markedly increased binding of TGF-beta to type III receptors nearly 10-fold. Northern analyses indicated that PGE2 decreased type I receptor mRNA levels by approximately 30-50%, decreased type II receptor mRNA levels by approximately 60%, and markedly increased type III receptor mRNA levels. Coincubation with cycloheximide partially blocked the PGE2-induced inhibition of type II receptor mRNA. In contrast, cycloheximide minimally affected PGE2-induced type III receptor mRNA levels. Activation of protein kinase C by phorbol esters had no apparent effect on TGF-beta receptor mRNA levels. Our data suggest that alterations in TGF-beta receptor expression could modify the response of tissues to TGF-beta during injury.

Discordant regulation of human type I collagen genes by prostaglandin E2.

We examined the expression of type I collagen genes in prostaglandin E2 (PGE2)-treated human lung fibroblast cultures. Addition of PGE2 to fibroblast cultures inhibited alpha 1(I) mRNA levels by approx. 25% after 6 h and 60% after 24 h. Further studies showed that dibutyryl cAMP did not inhibit alpha 1(I) mRNA levels and that cycloheximide blocked the inhibitory effect of PGE2. In contrast, PGE2 treatment with or without cycloheximide did not affect alpha 2(I) mRNA levels. Moreover, in vitro translation of RNA derived from untreated and PGE2-treated cells yielded similar amounts of alpha 2(I) collagen peptides. Taken together, these results suggest that PGE2 induces a protein which inhibits alpha 1(I) transcription through distinct regulatory elements not under the control of cAMP and provide further evidence that the type I collagen genes can be discordantly regulated.

Structure and expression of the promoter for the R4/ALK5 human type I transforming growth factor-beta receptor: regulation by TGF-beta.

The type I transforming growth factor-beta (TGF-beta) receptors are serine/threonine kinases that are essential for the action of TGF-beta. In this paper, we describe the molecular cloning and expression of the R4/ALK5 human type I TGF-beta receptor promoter. DNA sequence analysis indicates that the promoter lacks a TATA and CAAT box but is highly GC-rich and contains putative Sp1 binding sites. The transcriptional start site is approx. 232 base pairs upstream of the AUG start codon. In human lung fibroblasts, TGF-beta induced a 3-fold increase in steady-state level for type I receptor mRNA. Exposure of cells transfected with a 618 bp promoter fragment to TGF-beta 1 up-regulated transcriptional activity indicating that a TGF-beta response element is contained within this region.

Stimulation of collagen formation by insulin and insulin-like growth factor I in cultures of human lung fibroblasts.

We examined the effects of insulin and insulin-like growth factor I (IGF-I) on the production of collagen by cultures of human embryonic lung fibroblasts. Insulin at 20 ng/ml increased collagen accumulation by 58% and total protein formation by 18%. At 2 micrograms/ml, insulin increased collagen production by 2- to 3-fold and total protein production by 2-fold. The mRNA levels for alpha 1(I) and alpha 1(III) collagen chains were elevated by insulin compared with untreated control values. IGF-I at 10 ng/ml increased collagen production 2-fold. IGF-I at 100 ng/ml maximally increased collagen production 3-fold. A specific antibody to the IGF-I receptor (alpha IR-3) caused a concentration-related decline in insulin-induced collagen formation. The addition of antibody at 1 micrograms/ml, resulted in 80% inhibition of insulin-induced collagen accumulation. Higher levels of antibody were required to inhibit IGF-I mediated collagen formation. The presence of antibody (alpha IR-3) also blocked fibroblast proliferation stimulated by epidermal growth factor plus insulin. These data show that insulin-induced collagen formation is mediated primarily through an interaction with the IGF-I receptor. The modulation of extracellular matrix production by insulin may influence the repair of tissue injury and the development of the accelerated atherosclerosis that accompanies the diabetic state in humans.

Apigenin decreases expression of the myofibroblast phenotype.

We investigated the effect of the dietary flavonoid apigenin on myofibroblast function. We report that in myofibroblasts treated with apigenin, proliferation and basal levels of alpha1(I) collagen and alpha-smooth muscle actin mRNAs were markedly reduced. Apigenin also attenuated the transforming growth factor-beta-stimulated increases of alpha1(I) collagen and alpha-smooth muscle actin mRNAs. Characterization of the apigenin effects indicates that apigenin reduces both the stability of the alpha1(I) collagen mRNA and the rate of transcription of the alpha1(I) collagen gene through a cycloheximide-sensitive pathway. Western blot analyses indicate that Akt activity is reduced in apigenin-treated myofibroblasts.

Potential therapeutic initiatives for fibrogenic lung diseases.

Fibrotic process affecting the lung and other tissues is characterized by stimulation of fibroblast proliferation and connective tissue deposition. Conventional therapy consisting of glucocorticoids or cytotoxic agents is usually ineffective in blocking progression of disease. Potential new therapies have emerged from the use of animal models of pulmonary fibrosis and recent advances in the cellular and molecular biology of inflammatory reactions. Such therapies involve the use of substances directed against the action of certain growth factors, cytokines, or oxidants that are elaborated during the fibrotic reaction. In this article, we review possible therapeutic applications of these advances.

Hypoxia inhibits amino acid uptake in human lung fibroblasts.

Hypoxia and amino acid deprivation downregulate expression of extracellular matrix genes in lung fibroblasts. We examined the effect of hypoxia on amino acid uptake and protein formation in human lung fibroblasts. Low O(2) tension (0% O(2)) suppressed incorporation of [(3)H]proline into type I collagen without affecting [(35)S]methionine labeling of other proteins. Initial decreases in intracellular [(3)H]proline incorporation occurred after 2 h of exposure to 0% O(2), with maximal suppression of intracellular [(3)H]proline levels at 6 h of treatment. Hypoxia significantly inhibited the uptake of radiolabeled proline, 2-aminoisobutyric acid (AIB), and 2-(methylamino)isobutyric acid (methyl-AIB) while inducing minor decreases in leucine transport. Neither cycloheximide nor indomethacin abrogated hypoxia-related suppression of methyl-AIB uptake. Efflux studies demonstrated that hypoxia inhibited methyl-AIB transport in a bidirectional fashion. The downregulation of amino acid transport was not due to a toxic effect; function recovered on return to standard O(2) conditions. Kinetic analysis of AIB transport revealed a 10-fold increase in K(m) accompanied by a small increase in maximal transport velocity among cells exposed to 0% O(2). These data indicate that low O(2) tension regulates the system A transporter by decreasing transporter substrate affinity.

Bleomycin-induced lung injury is enhanced by interferon-alpha.

We have evaluated the effect of intraperitoneal (I.P.) injection of human recombinant interferon-2alpha (IFN-alpha) on Bleomycin-induced pulmonary injury in hamsters. Pulmonary injury was induced by a single intratracheal (I.T.) instillation of Bleomycin (Bleo). Six groups of male Syrian hamsters were treated as follows: 1) I.T. Bleo and daily I.P. injections of low-dose interferon-alpha (2 x 10(4) U), 2) I.T. Bleo and daily I.P. injections of high-dose interferon-alpha (10(5) U), 3) I.T. Bleo and I.P. injections of saline, 4) I.T. saline and I.P. low-dose IFN-alpha, 5) I.T. saline and I.P. high-dose IFN-alpha, 6) I.T. saline and I.P. saline. Animals were sacrificed 28 days after I.T. treatment. Lung injury was evaluated histologically and biochemically. Treatment of hamsters with low-dose but not high-dose IFN-alpha significantly augmented the Bleo-induced lung injury, as determined by a semiquantitative morphological index. Lung hydroxyproline measurements were highest in Bleo-low-dose-IFN-alpha followed by Bleo-high-dose-IFN-alpha and Bleo-Sal as compared to Sal-Sal and Sal-IFN-alpha controls. These results suggest that IFN-alpha augments Bleo-induced lung injury but that this effect is complex and does not follow a simple-dose-response pattern.

The effect of suramin on bleomycin-induced lung injury.

Since transforming growth factor beta (TGF-beta) is presumed to play a role in lung fibrosis, we evaluated the effect of suramin (Sur), a substance with an anti-TGF-beta effect, in vivo on bleomycin (Bleo)-induced pulmonary injury in mice and in vitro on human lung fibroblasts. Four groups of C57BL/6 mice each received one of four treatments: (1) intratracheal (i.t.) instillation of Bleo and intraperitoneal (i.p.) injections of Sur, every other day, starting one day before i.t. instillation of Bleo (Bleo-Sur); (2) i.t. Bleo and i.p. injections of saline (Bleo-Sal); (3) i.t. saline and i.p. Sur (Sal-Sur); and (4) i.t. and i.p. saline (Sal-Sal). Animals were sacrificed 14 days after i.t. treatment. Lung injury was evaluated by analysis of bronchoalveolar lavage (BAL) fluid, histologically by the semiquantitative morphological index, and biochemically by analysis of lung hydroxyproline content. In vitro, Sur did not affect TGF-beta induced increase of alpha1 (I) collagen mRNA in human lung fibroblasts. In vivo treatment of mice with Sur did not affect Bleo-induced lung injury. These results indicate that despite its potential anti TGF-beta and lymphocytotoxic effects, Sur is not a therapeutic candidate drug for rescue of lung fibrosis.

Precipitation of low-temperature dolomite from an anaerobic microbial consortium: the role of methanogenic Archaea.

Here we report precipitation of dolomite at low temperature (30 degrees C) mediated by a mixed anaerobic microbial consortium composed of dissimilatory iron-reducing bacteria (DIRB), fermenters, and methanogens. Initial solution geochemistry is controlled by DIRB, but after 90 days shifts to a system dominated by methanogens. In live experiments conditions are initially saturated with respect to dolomite (Omega(dol) = 19.40) and increase by two orders of magnitude (Omega(dol) = 2 330.77) only after the onset of methanogenesis, as judged by the increasing [CH(4)] and the detection of methanogenic micro-organisms. We identify ordered dolomite in live microcosms after 90 days via powder X-ray diffraction, while sterile controls precipitate only calcite. Scanning electron microscopy and transmitted electron microscopy demonstrate that the precipitated dolomite is closely associated with cell walls and putative extra-cellular polysaccharides. Headspace gas measurements and denaturing gradient gel electrophoresis confirm the presence of both autotrophic and acetoclastic methanogens and exclude the presence of DIRB and sulfate-reducing bacteria after dolomite begins forming. Furthermore, the absence of dolomite in the controls and prior to methanogenesis confirm that methanogenic Archaea are necessary for the low-temperature precipitation of dolomite under the experimental conditions tested.

Control of type I collagen formation in the lung.

Type I collagen is a major structural protein in the lung, the accumulation of which is stimulated during certain inflammatory reactions in the lung. Accumulating evidence suggests that type I collagen formation parallels changes in steady-state mRNA levels. Specific inflammatory substances modulate transcription of collagen genes and stabilization of collagen mRNA in vitro. However, the precise role for any particular mediator during fibrotic processes is difficult to identify because of the complex nature of the inflammatory reaction and potential interaction among mediators. The signal transduction mechanisms that regulate collagen accumulation remain to be defined. This review focuses on the regulation of collagen accumulation in the lung by specific inflammatory substances.

Response of the aging hamster lung to elastase injury.

The effect of age on the induction of emphysema in hamsters was investigated by administering porcine pancreatic elastase endotracheally to old (18 months of age) and young (4 months of age) hamsters; age-matched untreated animals served as controls. Lung volumes, quasi-static deflation volume pressure relations, and mean linear intercept, an index of alveolar size, were measured. Elastase-treated young hamsters demonstrated an increased volume of air in the lungs at a transpulmonary pressure of 25 cm H2O and an increased mean linear intercept. The lungs of old elastase-treated animals showed less pronounced changes than those of young hamsters in lung volume at transpulmonary pressure of 25 cm H2O and in mean linear intercept. Untreated old hamsters had a larger lung volume at a transpulmonary pressure of 25 cm H2O and an 85% increase in total lung collagen. Age-related alterations in connective tissues may account for the increased resistance of old lungs to injury by pancreatic elastase.

Regulation of sulfated glycosaminoglycan production by prostaglandin E2 in cultured lung fibroblasts.

Prostaglandin E2 (PGE2) has been shown to increase the synthesis of hyaluronic acid in cultured fibroblasts by increasing the activity of hyaluronate synthetase, a group of plasma membrane-bound synthetic enzymes. We examined whether PGE2 also increased the activity of those enzyme systems involved in the synthesis of sulfated glycosaminoglycan in the human embryonic lung fibroblast. Exposure of cells to PGE2 resulted in dose-dependent increases in glucosamine incorporation into all sulfated glycosaminoglycan subtypes. PGE2 at 10(-7) mol/L increased total glycosaminoglycan per dish to 21.6 +/- 3.1 micrograms versus 12.0 +/- 2.5 micrograms in control untreated cultures. Stimulation of endogenous PGE2 production by bradykinin had a similar effect on glycosaminoglycan synthesis. To examine whether PGE2 affected sulfated glycosaminoglycan protein core production, cells were labeled with tritiated glucosamine in the presence of cycloheximide. Under these conditions, incorporation of radiolabel into all glycosaminoglycan subtypes was reduced. However, when exogenous sulfated glycosaminoglycan chain initiator (p-nitrophenyl beta-D-xyloside) was added, incorporation of tritiated glucosamine into sulfated glycosaminoglycan increased but not to levels found in control cultures. Application of PGE2 to cultures treated with cycloheximide alone, or to cultures treated with cycloheximide plus xyloside, increased tritiated glucosamine incorporation into chondroitin, dermatan sulfate, and to a lesser extent into heparan sulfate. We conclude that PGE2 stimulates synthesis of all sulfated glycosaminoglycan even in the absence of new protein core production, probably by increasing activities of sulfated glycosaminoglycan synthetase enzymes. PGE2 stimulation of heparan sulfate synthesis is partially dependent on the availability of heparan sulfate-specific protein core.