The effects of acute-phase inducers and dimethyl sulphoxide on delta-aminolaevulinate synthase activity in human HepG2 hepatoma cells.

The effects of acute-phase inducers and dimethyl sulphoxide (Me2SO) on delta-aminolaevulinate (ALA) synthase in HepG2 cells were examined. Treatment of cells with Me2SO resulted in a significant increase in ALA synthase activity. Interleukin-6 increased ALA synthase activity only slightly, but it substantially potentiated the induction of ALA synthase by Me2SO. These data suggest that ALA synthase activity in liver is altered during acute-phase reactions.     

Effects of dimethyl sulphoxide on the synthesis of plasma proteins in the human hepatoma HepG2. Induction of an acute-phase-like reaction.

Effects of dimethyl sulphoxide (Me2SO) on the synthesis of plasma proteins by the human hepatoma cell line HepG2 were examined. Me2SO treatment resulted in decreased synthesis of albumin and alpha-fetoprotein, and in increased synthesis of haptoglobin. Plasma-protein profiles induced by Me2SO treatment were very similar to those seen in acute-phase reactions.     

delta-Aminolaevulinate synthase in human HepG2 hepatoma cells. Repression by haemin and induction by chemicals.

delta-Aminolaevulinate (ALA) synthase, the rate-limiting enzyme in haem biosynthesis in the normal liver, was examined in human HepG2 hepatoma cells. Haemin, up to 100 microM, had no effect on ALA synthase activity in vitro; it did, however, exhibit a dose-dependent inhibitory action when added to cells growing in culture (half-maximal inhibition at 1 microM). The half-life of ALA synthase activity after haemin treatment was 2 h, which was similar to that found after treatment with cycloheximide. Cells treated with actinomycin D showed a longer half-life of the enzyme activity, i.e. 4 h, compared with haemin or cycloheximide treatment. Treatment of cells with succinylacetone markedly inhibited the activity of ALA dehydratase and 59Fe incorporation into haem, but in increased ALA synthase activity. Both the haemin-induced repression and the succinylacetone-mediated de-repression of ALA synthase activity were reversible within 4 h after replacing the medium with fresh medium without the chemical. In addition to succinylacetone, dimethyl sulphoxide and 3-methylcholanthrene induced the enzyme. Induction of ALA synthase by these chemicals was also suppressed by treatment of cells with haemin. These findings indicate that the level of ALA synthase in HepG2 cells is maintained by both synthesis and degradation of the enzyme, and that the synthesis of the enzyme is regulated by the concentration of regulatory free haem in the cell.     

Bradyrhizobium japonicum delta-aminolevulinic acid dehydratase is essential for symbiosis with soybean and contains a novel metal-binding domain.

The Bradyrhizobium japonicum hemA gene product delta-aminolevulinic acid (ALA) synthase is not required for symbiosis of that bacterium with soybean. Hence, the essentiality of the subsequent heme synthesis enzyme, ALA dehydratase, was examined. The B. japonicum ALA dehydratase gene, termed hemB, was isolated and identified on the basis of its ability to confer hemin prototrophy and enzyme activity on an Escherichia coli hemB mutant, and it encoded a protein that was highly homologous to ALA dehydratases from diverse organisms. A novel metal-binding domain in the B. japonicum ALA dehydratase was identified that is a structural composite of the Mg(2+)-binding domain found in plant ALA dehydratases and the Zn(2+)-binding region of nonplant ALA dehydratases. Enzyme activity in dialyzed extracts of cells that overexpressed the hemB gene was reconstituted by the addition of Mg2+ but not by addition of Zn2+, indicating that the B. japonicum ALA dehydratase is similar to the plant enzymes with respect to its metal requirement. Unlike the B. japonicum hemA mutant, the hemB mutant strain KP32 elicited undeveloped nodules on soybean, indicated by the lack of nitrogen fixation activity and plant hemoglobin. We conclude that the hemB gene is required for nodule development and propose that B. japonicum ALA dehydratase is the first essential bacterial enzyme for B. japonicum heme synthesis in soybean root nodules. In addition, we postulate that ALA is the only heme intermediate that can be translocated from the plant to the endosymbiont to support bacterial heme synthesis in nodules.     

Iron mobilization by succinylacetone methyl ester in rats. A model study for hereditary tyrosinemia and porphyrias characterized by 5-Aminolevulinic acid overload

Accumulation of 5-aminolevulinic acid (ALA) is an event characteristic of porphyrias that may contribute to their pathological manifestations. To investigate effects of ALA independent of porphyrin accumulation we treated rats with the methyl ester of succinylacetone, an inhibitor of 5-aminolevulinic acid dehydratase that accumulates in the porphyric-like syndrome hereditary tyrosinemia. Acute 2-day treatment of fasted rats with succinylacetone methyl ester (SAME) promoted a 27% increase in plasma ALA. This increase in plasma ALA was accompanied by augmentation of the level of total nonheme iron in liver (37%) and brain (20%). Mobilization of iron was also indicated by 49% increase in plasma iron and a 77% increase in plasma transferrin saturation. Liver responded with a mild (12%) increase in ferritin. Under these acute conditions, some indications of oxidative stress were evident: a 15% increase in liver reactive protein carbonyls, and a 42% increase in brain subcellular membrane TBARS. Brain also showed a 44% increase in CuZnSOD activity, consistent with observations in treatment with ALA. Overall, the data indicate that SAME promotes ALA-driven changes in iron metabolism that could lead to increased production of free radicals. The findings support other evidence that accumulation of ALA in porphyrias and hereditary tyrosinemia may induce iron-dependent biological damage that contributes to neuropathy and hepatoma.     

Iron mobilization by succinylacetone methyl ester in rats. A model study for hereditary tyrosinemia and porphyrias characterized by 5-aminolevulinic acid overload

Accumulation of 5-aminolevulinic acid (ALA) is an event characteristic of porphyrias that may contribute to their pathological manifestations. To investigate effects of ALA independent of porphyrin accumulation we treated rats with the methyl ester of succinylacetone, an inhibitor of 5-aminolevulinic acid dehydratase that accumulates in the porphyric-like syndrome hereditary tyrosinemia. Acute 2-day treatment of fasted rats with succinylacetone methyl ester (SAME) promoted a 27% increase in plasma ALA. This increase in plasma ALA was accompanied by augmentation of the level of total nonheme iron in liver (37%) and brain (20%). Mobilization of iron was also indicated by 49% increase in plasma iron and a 77% increase in plasma transferrin saturation. Liver responded with a mild (12%) increase in ferritin. Under these acute conditions, some indications of oxidative stress were evident: a 15% increase in liver reactive protein carbonyls, and a 42% increase in brain subcellular membrane TBARS. Brain also showed a 44% increase in CuZnSOD activity, consistent with observations in treatment with ALA. Overall, the data indicate that SAME promotes ALA-driven changes in iron metabolism that could lead to increased production of free radicals. The findings support other evidence that accumulation of ALA in porphyrias and hereditary tyrosinemia may induce iron-dependent biological damage that contributes to neuropathy and hepatoma.     

Effects of modulators of protein phosphorylation on heme metabolism in human hepatic cells: induction of delta-aminolevulinic synthase mRNA and protein by okadaic acid

Effects of modulators of protein phosphorylation on delta-aminolevulinic acid (ALA) synthase and heme oxygenase-1 mRNA were analyzed in the human hepatic cell lines Huh-7 and HepG2 using a quantitative RNase protection assay. Okadaic acid was found to induce ALA synthase mRNA in a concentration-dependent fashion in both Huh-7 and HepG2 cells. The EC(50) for induction of ALA synthase mRNA in Huh-7 cells was 13.5 nM, with maximum increases occurring at okadaic acid concentrations of 25-50 nM. The EC(50) for induction of ALA synthase mRNA in HepG2 cells was 35.5 nM, with maximum increases occurring at okadaic acid concentrations of 50 nM. Concentration-dependent induction of ALA synthase mRNA paralleled the increase in ALA synthase protein. Maximum induction of ALA synthase was observed between 5 and 10 h post-treatment in both cell lines. Induction of ALA synthase mRNA in Huh-7 cells, but not HepG2 cells, was associated with an increase in ALA synthase mRNA stability. Okadaic acid also induced heme oxygenase-1 mRNA in both cell lines, but the magnitude of induction was only twofold, and was rapid and transient. Okadaic acid and phorbol 12-myristate 13-acetate significantly decreased heme-mediated induction of heme oxygenase-1 mRNA in both Huh-7 and HepG2 cells. Wortmannin diminished the heme-mediated induction of heme oxygenase-1 mRNA in HepG2 cells, but not Huh-7 cells. These results report a novel property of okadaic acid to affect heme metabolism in human cell lines.     

Aminolevulinic acid dehydratase in pea (Pisum sativum L.). Identification of an unusual metal-binding domain in the plant enzyme

Aminolevulinic acid dehydratase (ALA dehydratase) catalyzes the second step of tetrapyrrole synthesis leading to the formation of heme and chlorophyll in higher plant cells. Antibodies elicited against spinach leaf ALA dehydratase were used to immunoscreen lambda gt11 cDNA libraries constructed from etiolated pea (Pisum sativum L.) leaf poly(A)+ RNAs. A set of overlapping cDNAs was characterized that encode the pea enzyme. The predicted amino acid sequence of the pea ALA dehydratase is similar to those reported for other eukaryotic and prokaryotic ALA dehydratases. The pea enzyme has an active site domain centered on lysine that is highly conserved in comparison to other known ALA dehydratases. Consistent with the previously reported requirement of Mg2+ for catalytic activity by plant ALA dehydratases, the pea enzyme lacks the characteristic Zn(2+)-binding domain present in other eukaryotic ALA dehydratases, but contains a distinctive metal ligand-binding domain based upon aspartate. Northern blot analyses demonstrated that ALA dehydratase mRNA is present in leaves, stems, and to a lesser extent in roots. Steady state levels of mRNA encoding ALA dehydratase exhibit little or no change during light-induced greening.     

Light-dependent Synthesis of δ-aminolaevulinic Acid Dehydratase During the Developmental Period of Trapella Seedlings

Mature seeds of Trapella sinensis, after removal of receptacles, were germinated for 10 days at 23°C in the dark and then were illuminated. After illumination, the activity of δ-aminolaevulinic acid (ALA) dehydratase was determined in relation to chlorophyll synthesis, and increases in dry weight of the whole or of parts of a developing seedling. Internal programming of development was related to changes in ALA dehydratase activity on the basis of porphobilinogen synthesis. Exogenously added ALA as well as an extract of Chlorella cells promoted this activity. There was an enhancement of chlorophyll formation resulting in an enhancement of the photosynthetic rate and in an increase of growth promotion by addition of Chlorella extract.     

Plant delta-aminolevulinic acid dehydratase. Expression in soybean root nodules and evidence for a bacterial lineage of the Alad gene.

We isolated a soybean (Glycine max) cDNA encoding the heme and chlorophyll synthesis enzyme delta-aminolevulinic acid (ALA) dehydratase by functional complementation of an Escherichia coli hemB mutant, and we designated the gene Alad. ALA dehydratase was strongly expressed in nodules but not in uninfected roots, although Alad mRNA was only 2- to 3-fold greater in the symbiotic tissue. Light was not essential for expression of Alad in leaves of dark-grown etiolated plantlets as discerned by mRNA, protein, and enzyme activity levels; hence, its expression in subterranean nodules was not unique in that regard. The data show that soybean can metabolize the ALA it synthesizes in nodules, which argues in favor of tetrapyrrole formation by the plant host in that organ. Molecular phylogenetic analysis of ALA dehydratases from 11 organisms indicated that plant and bacterial enzymes have a common lineage not shared by animals and yeast. We suggest that plant ALA dehydratase is descended from the bacterial endosymbiont ancestor of chloroplasts and that the Alad gene was transferred to the nucleus during plant evolution.     

Activation of the progesterone-signaling pathway by methyl-beta-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa Galphas

Treatment of Xenopus laevis oocytes with cholesterol-depleting methyl-β-cyclodextrin (MeβCD) stimulates phosphorylation of mitogen-activated protein kinase (MAPK) and oocyte maturation, as reported previously [Sadler, S.E., Jacobs, N.D., 2004. Stimulation of Xenopus laevis oocyte maturation by methyl-β-cyclodextrin. Biol. Reprod. 70, 1685-1692.]. Here we report that treatment of oocytes with MeβCD increased levels of immunodetectable 39-kDa mos protein. The protein synthesis inhibitor, cycloheximide, blocked the appearance of Mos, blocked MeβCD-stimulated phosphorylation of MAPK, and inhibited MeβCD-induced oocyte maturation. These observations suggest that MeβCD activates the progesterone-signaling pathway. Chemical inhibition of steroid synthesis and mechanical removal of follicle cells were used to verify that MeβCD acts at the level of the oocyte and does not require production of steroid by surrounding follicle cells. Cortical Gα_s is contained in low-density membrane; and treatment of oocytes with progesterone or MeβCD reduced immunodetectable levels of Gα_s protein in cortices and increased internal levels of 45-kDa Gα_s in cortical-free extracts. Dose-dependent increases in internal Gα_s after treatment of oocytes with progesterone correlated with the steroid-induced maturation response, and the increase in internal Gα_s after hormone treatment was comparable to the decrease in cortical Gα_s. These results are consistent with a model in which release of Gα_s from the plasma membrane is involved in activation of the progesterone-signaling pathway that leads to amphibian oocyte maturation.     

A correlation between phorbol diester-induced protein phosphorylation and superoxide anion generation in HL-60 cells during granulocytic maturation

As HL-60 cells matured along the granulocytic pathway, phorbol diester-induced superoxide anion production was compared to phorbol diester-induced protein phosphorylation using an in vitro phosphorylation technique. Maturation was induced by 0, 2, 4, or 6 days incubation with dimethyl sulfoxide (Me2SO). In 0 day Me2SO HL-60 cells, phorbol 12-myristate 13-acetate induced phosphorylation of protein pp29 (Mr = 28,600) and to a lesser extent protein pp76 (Mr = 76,300). With increased time of Me2SO incubation, phorbol 12-myristate 13-acetate induced phosphorylation of pp212 (Mr = 211,800), pp134 (Mr = 134,200), and pp76, whereas the phosphorylation of pp29 did not change appreciably. In close agreement with this increase in protein phosphorylation was the observed increase in phorbol diester-induced superoxide anion formation. Morphological characterization of cells during Me2SO-induced differentiation reveals that these increases in phorbol diester responses are probably attributable to the proportional rise in metamyelocytes, band, and segmented neutrophils. A variety of phorbol diesters increased superoxide anion generation in HL-60 cells differentiated into granulocyte-like cells by 6-day incubation with Me2SO. The structure-activity relationship of these phorbol diester derivatives for protein phosphorylation was strongly correlated to their ability to increase superoxide anion generation. Thus, we propose that phorbol diester-induced phosphorylation of pp212, pp134, and pp76, but not pp29 may play a role in mediating the functional response of phorbol diester-induced superoxide anion generation in HL-60 cells differentiated into mature granulocyte-like cells.     

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemin control of heme biosynthesis in mouse Friend virus-transformed erythroleukemia cells in culture.

Hemin treatment of mouse Friend virus-transformed cells in cultured caused a dose-dependent increase in hemoglobin synthesis. By the addition of radioactively labeled hemin and by the analysis of the radioactive heme in hemoglobin, only 60 to 70% of heme in the newly synthesized hemoglobin was accounted for by the exogenously added hemin. In keeping with this finding, hemin treatment increased the activity of two enzymes in the heme biosynthetic activity, i.e. delta-aminolevulinate (ALA) dehydratase and uroporphyrinogen-I (URO) synthase in these cells. Incorporation of [2(-14C)]glycine, [14C]ALA, and 59Fe into heme was also significantly increased in the cells treated with hemin, suggesting that essentially all enzyme activities in the heme biosynethetic pathway were increased after hemin treatment. These results indicate that heme in the newly synthesized hemoglobin in hemin-treated Friend cells derives both from hemin added to the culture and from heme synthesized intracellularly. In addition, these results suggest that the stimulation of heme biosynthesis by hemin in Friend virus-transformed cells is in contrast to the hemin repression of heme biosynthesis in liver cells.     

Protoporphyrin IX fluorescence kinetics in C6 glioblastoma cells after delta-aminolevulinic acid incubation: effect of a protoporphyrinogen oxidase inhibitor.

PpIX synthesis after incubation with delta-aminolevulinic acid (ALA) is highly variable from one cell to another within a single cell population and in human glioblastomas in vivo. To improve PpIX synthesis, we attempted to modify the PpIX synthesis pathway in a C6 glioma cell model. To perform this experiment we used confocal microspectrofluorometry to analyse the effects of a highly purified form of sulfentrazone (FP846) on the kinetics of PpIX synthesis after ALA administration to living C6 cells. Our results show that PpIX fluorescence was maximal (seven-fold higher than basal values) 3 to 4 hrs. after the beginning of incubation with ALA. FP846 depressed this increase in fluorescence nearly to basal levels not only in C6 cells but also in HT29 and HepG2 cells. Fluorescence spectra shape were not affected by FP846, except for intensity. ALA/PpIX-induced photocytoxicity was perfectly correlated with fluorescence intensity recorded in cell cytoplasm. ALA alone (100 microg/ml) did not induce a significant decrease in cell survival, but irradiation of 25 J/cm2 leading to an overall cell death of 60%. FP846 added together with ALA suppressed ALA/PpIX-induced phototoxicity. The fact that the FP846-induced decrease in PpIX synthesis was not the same in animal and plant cells suggests that the porphyrin metabolic pathway differs due to the relative amounts of substrate or the effect of inhibitor and that another chemical would be needed alone or in combination with FP846 to improve PpIX synthesis.     

Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration.

A recently developed sandwich culture system, in which hepatocytes are sandwiched between two layers of collagen, has been shown to be capable of maintaining long-term expression of hepatocellular function (J. C. Y. Dunn et al., Biotechnol. Prog. 7, 237-245, 1991). The development of an adequate technique for the cryopreservation of hepatocytes in such a stable culture configuration would ensure a ready supply of hepatocytes for use in bioreactors or bioartificial liver support devices. This report describes the effects of exposing hepatocytes in sandwich culture to different concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) at 22 degrees C on Day 7 of culture. Cell function, morphology, and cytoskeletal organization were followed for 14 days after exposure. Hepatocellular morphology and albumin secretion remained normal when cultures were exposed for up to 120 min to predicted final Me2SO concentrations up to 1.33 M. Exposure for less than 60 min to equilibrium concentrations of up to 3.33 M Me2SO did not adversely affect cell morphology or albumin secretion rate, but at the highest concentration (3.33 M), increase of the exposure time to 60 or 120 min resulted in dramatic, irreversible cell damage and loss of function. Actin filament organization was shown to be undisturbed when the cells were exposed to 1.33 M Me2SO for 60 min, but was irreversibly disrupted by exposure to 3.33 M for 120 min. Based on these results, a simple and safe procedure is suggested for the addition of Me2SO to hepatocytes in a sandwich culture configuration and its subsequent removal, which will be valuable for studies on hepatocyte cryopreservation.     

The role of Ca2+ in dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells

Cultured Friend cells can be induced by dimethyl sulfoxide (Me2SO) and several other agents to mature along the erythroid pathway. Evidence has been presented that an increase in Ca2+ influx is an early and necessary prelude to the commitment to maturation by these cells (Levenson, R., Housman, D., and Cantley, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5948-5952). The simplest hypothesis supporting all the available data is that Me2SO and other inducers elevate the cytosolic Ca2+ concentration. We have now measured cytosolic Ca2+ using the fluorescent indicator quin-2, and find, contrary to expectation, a small decrease upon treatment of cells with Me2SO. Cytosolic Ca2+ was increased by raising the Ca2+ in the medium, but was not dramatically altered by addition of ouabain or monensin or by incubation in Na+-free medium. Measurement of total cell Ca2+ by a triple-labeling technique using 3H2O and 125I-albumin to determine cell water and extracellular space, respectively, revealed no significant change upon treatment with Me2SO for up to 40 h. A decrease in the initial rate of 45Ca2+ influx was observed in Me2SO-treated cells, when measured at 4 degrees C. These data do not support the hypothesis that an increase in cell Ca2+ is necessary for the induction of Friend cell differentiation or that Na+/Ca2+ exchange is a significant regulator of cytosolic Ca2+ in Friend cells.