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PubMed1993

An alternative mechanism for the inhibition of cholesterol biosynthesis in HepG2 cells by N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol (MDL 28,815).

Compounds that block hepatic cholesterol biosynthesis and secretion may be useful hypocholesterolemic agents. N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol (MDL 28,815) has been shown to block cholesterol biosynthesis in 3T3 fibroblasts and it causes cellular accumulation of squalene 2,3-epoxide and squalene 2,3:23,24-diepoxide (squalene epoxides), which suggests that it inhibits 2,3-oxidosqualene cyclase. The purpose of the present report was to determine whether MDL 28,815 acts only at the level of 2,3-oxidosqualene cyclase or whether other enzymes in the cholesterol biosynthetic pathway are affected. HepG2 cells, grown in lipoprotein-deficient serum, were incubated with MDL 28,815 and 14C-acetate to radiolabel cholesterol and the intermediates in the cholesterol biosynthetic pathway. Blockade of cholesterol biosynthesis by MDL 28,815 in these cells was associated with the accumulation of two metabolites, one of which was 5 alpha-cholest-8-en-3 beta-ol. The other metabolite was identified by a combination of ultraviolet spectrometry, gas chromatography, mass spectroscopy and analytical high-performance liquid chromatography as 5 alpha-cholest-8,14-dien-3 beta-ol. Maximal blockade of cholesterol biosynthesis was associated with the accumulation of these two metabolites and, in particular, 5 alpha-cholest-8,14-dien-3 beta-ol, rather than with squalene epoxides. These results suggest that MDL 28,815 blocks cholesterol biosynthesis primarily by the inhibition of sterol-delta 14-reductase, and possibly sterol-delta 8-ene isomerase, rather than 2,3-oxidosqualene cyclase.

Animals↗

PubMed1996

Effects of a novel 2,3-oxidosqualene cyclase inhibitor on the regulation of cholesterol biosynthesis in HepG2 cells.

Within the cholesterol biosynthesis cascade, the enzyme 2,3-oxidosqualene cyclase [EC 5.4.99.7] is of special interest due to its dual function: cyclization of 2,3-monoepoxysqualene to lanosterol and 2,3;22,23-diepoxysqualene to oxylanosterol. Further determination of the significance of this enzyme for the intracellular cholesterol homeostasis was done with BIBX 79, a new potent, specific inhibitor of this enzyme. In HepG2 cells the effects of BIBX 79 on cholesterol biosynthesis, 2,3-oxidosqualene cyclase as well as HMG-CoA reductase activities were studied. BIBX 79 is a potent inhibitor of sterol biosynthesis in HepG2 cells (IC50 4 x 10(-9) M). No other enzyme within the cholesterol biosynthesis cascade was significantly inhibited as was evidenced by a radio HPLC detection system. In contrast to simvastatin, no direct interaction with HMG-CoA reductase was observed. When incubating HepG2 cells for 16 h with the HMG-CoA reductase inhibitor simvastatin (10(-6)-10(-10) M) HMG-CoA reductase activity was increased up to 180%. BIBX 79 did also affect HMG-CoA reductase activity under these conditions: in concentrations of BIBX 79 "> or =" 10(-9) "< or =" 10(-7) M, where a partial inhibition of 2,3-oxidosqualene cyclase is observed, HMG-CoA reductase activity was decreased. However, higher concentrations of BIBX 79 that totally blocked 2,3-oxidosqualene cyclase led to an increase in HMG-CoA reductase activity. This effect of BIBX 79 on HMG-CoA reductase is thought to be mainly mediated by oxysterols that are formed by the cyclization of 2,3;22,23-diepoxysqualene. 2,3;22,23-Diepoxysqualene is preferentially cyclized by the 2,3-oxidosqualene cyclase and, consequently, only high inhibitor concentrations will also block 2,3;22,23-diepoxysqualene cyclization. Thus, by partial blockade of this enzyme, both an inhibition of lanosterol and subsequently cholesterol formation as well as a concomitant effect on HMG-CoA reductase can be achieved. Both effects complement each other and lead to an effective control of cholesterol biosynthesis. It is therefore concluded that 2,3-oxidosqualene cyclase plays a crucial role in the regulation of intracellular cholesterol homeostasis. 2,3-Oxidosqualene cyclase inhibitors offer an attractive approach for novel lipid-lowering agents.

Benzamides↗

PubMed1997

Occurrence and biosynthesis of endogenous cannabinoid precursor, N-arachidonoyl phosphatidylethanolamine, in rat brain.

It has been suggested that anandamide (N-arachidonoylethanolamine), an endogenous cannabinoid substance, may be produced through Ca2+-stimulated hydrolysis of the phosphatidylethanolamine (PE) derivative N-arachidonoyl PE. The presence of N-arachidonoyl PE in adult brain tissue and the enzyme pathways that underlie its biosynthesis are, however, still undetermined. We report here that rat brain tissue contains both anandamide (11 +/- 7 pmol/gm wet tissue) and N-arachidonoyl PE (22 +/- 16 pmol/gm), as assessed by gas chromatography/mass spectrometry. We describe a N-acyltransferase activity in brain that catalyzes the biosynthesis of N-arachidonoyl PE by transferring an arachidonate group from the sn-1 carbon of phospholipids to the amino group of PE. We also show that sn-1 arachidonoyl phospholipids are present in brain, where they constitute approximately 0.5% of total phospholipids. N-acyltransferase activity is Ca2+ dependent and is enriched in brain and testis. Within the brain, N-acyltransferase activity is highest in brainstem; intermediate in cortex, striatum, hippocampus, medulla, and cerebellum; and lowest in thalamus, hypothalamus, and olfactory bulb. Pharmacological inhibition of N-acyltransferase activity in primary cultures of cortical neurons prevents Ca2+-stimulated N-arachidonoyl PE biosynthesis. Our results demonstrate, therefore, that rat brain tissue contains the complement of enzymatic activity and lipid substrates necessary for the biosynthesis of the anandamide precursor N-arachidonoyl PE. They also suggest that biosynthesis of N-arachidonoyl PE and formation of anandamide are tightly coupled processes, which may concomitantly be stimulated by elevations in intracellular Ca2+ occurring during neural activity.

Acyltransferases↗

PubMed1997

Lipid biosynthesis pathways as chemotherapeutic targets in kinetoplastid parasites.

Inhibitors of sterol and phospholipid biosynthesis in kinetoplastid parasites such as Trypanosoma cruzi, the causative agent of Chagas' disease, and different species of Leishmania have potent and selective activity as chemotherapeutic agents in vitro and in vivo. Recent work with the sterol C14 alpha-demethylase inhibitor D0870, a bis triazole derivative, showed that this compound is capable of inducing radical parasitological cure in murine models of both acute and chronic Chagas' disease. Other inhibitors of this type, such as SCH 56592, have also shown curative, rather than suppressive, activity against T. cruzi in these models. Leishmania species have different susceptibilities to sterol biosynthesis inhibitors, both in vitro and in vivo. Leishmania braziliensis promastigotes, naturally resistant to C14 alpha-demethylase inhibitors such as ketoconazole and D0870, were susceptible to these drugs when used in combination with the squalene epoxidase inhibitor terbinafine. Inhibitors of delta 24(25) sterol methyl transferase have been shown to act as potent antiproliferative agents against Trypanosoma cruzi, both in vitro and in vivo. New inhibitors of this type which show enhanced activity and novel mechanisms of action have been synthesized. Recent work has also demonstrated that this type of enzyme inhibitors can block sterol biosynthesis and cell proliferation in Pneumocystis carinii, a fungal pathogen which had previously been found resistant to other sterol biosynthesis inhibitors. Ajoene, an antiplatelet compound derived from garlic, was shown to have potent antiproliferative activity against epimastigotes and amastigotes of Trypanosoma cruzi in vitro; this activity was associated with a significant alteration of the phospholipid composition of the cells with no significant effects on the sterol content. In addition, alkyllsophospholipids such as ilmofosine, miltefosine and edelfosine have been shown to block the proliferation of T. cruzi and Leishmania and alter both the phospholipid and sterol composition. These results indicate the potential of lipid biosynthesis inhibitors as useful therapeutic agents in the treatment of leishmaniasis and Chagas' disease.

Animals↗

PubMed1998

Bacteria inhibit biosynthesis of bone matrix proteins in human osteoblasts.

The effect of extracts from Staphylococcus aureus and Staphylococcus epidermidis on bone matrix production were assessed by analyzing the biosynthesis of osteocalcin and Type I collagen in a human osteoblastic osteosarcoma cell line (MG-63). In MG-63 cells, extracts from Staphylococcus aureus and Staphylococcus epidermidis decreased 1,25(OH)2-vitamin D3 stimulated osteocalcin biosynthesis, and insulin-like growth factor I induced production of Type I collagen in a concentration dependent manner. The basal rate of osteocalcin and Type I collagen formation was unaffected by the bacterial extracts. The inhibitory effect of the bacteria on osteocalcin biosynthesis was seen after 24 hours of treatment and was maintained for at least 96 hours. The extracts of Staphylococcus aureus and Staphylococcus epidermidis enhanced prostaglandin E2 formation in the MG-63 cells. Abolition of the prostaglandin E2 response by treatment with indomethacin and flurbiprofen did not affect bacteria induced inhibition of osteocalcin production. Stimulation of osteocalcin biosynthesis by 1,25(OH)2-vitamin D3 was associated with a decreased rate of cell proliferation. The inhibitory action of the bacterial extracts was not linked to any inhibition of [3H]-thymidine incorporation into deoxyribonucleic acid. These data show that extracts of Staphylococcus aureus and Staphylococcus epidermidis have the ability to inhibit the biosynthesis of bone matrix proteins by a nonprostaglandin and noncytotoxic dependent mechanism and suggest that bone loss in inflammatory processes containing Staphylococcus aureus or Staphylococcus epidermidis may not be caused only by enhanced bone resorption but also by decreased bone formation.

Calcitriol↗

PubMedDate not supplied

Doxorubicin-induced inhibition of prolidase activity in human skin fibroblasts and its implication to impaired collagen biosynthesis.

One of the recognized side effects accompanying doxorubicin administration is poor wound healing resulting from impairement of collagen biosynthesis. However, the precise mechanism of doxorubicin-induced inhibition of collagen synthesis has not been established. We considered prolidase, an enzyme involved in collagen metabolism as a possible target for doxorubicin-induced inhibition of synthesis of this protein. Prolidase [E.C.3.4.13.9] cleaves imidodipeptides containing C-terminal proline, providing large amount of proline for collagen resynthesis. Therefore, we compared the effect of doxorubicin on prolidase activity and collagen biosynthesis in cultured human skin fibroblasts. We have found that doxorubicin induces coordinately inhibition of prolidase activity (IC50 approximately 10 +/- 3 microM) and collagen biosynthesis (IC50 approximately 15 +/- 3 microM) in cultured human skin fibroblasts. The inhibitory effect of doxorubicin on prolidase activity and collagen biosynthesis was not due to cytotoxicity of this drug as shown by cell viability tetrazoline test. The decrease in prolidase activity in fibroblasts treated with doxorubicin was not accompanied by differences in the amount of the enzyme protein recovered from these cells as shown by western immunoblot analysis. It may suggest that the inhibition is a posttranslational event. The data presented here rise possibility that doxorubicin-induced decrease in collagen biosynthesis is mostly due to the inhibition of prolidase activity.

Antibiotics, Antineoplastic↗

PubMed1995

Negative feedback regulation of wild-type p53 biosynthesis.

When growth-arrested mouse fibroblasts re-entered the cell-cycle, the rise in tumour suppressor p53 mRNA level markedly preceded the rise in expression of the p53 protein. Furthermore, gamma-irradiation of such cells led to a rapid increase in p53 protein biosynthesis even in the presence of the transcription inhibitor actinomycin D. Both findings strongly suggest that p53 biosynthesis in these cells is regulated at the translational level. We present evidence for an autoregulatory control of p53 expression by a negative feed-back loop: p53 mRNA has a predicted tendency to form a stable stem-loop structure that involves the 5'-untranslated region (5'-UTR) plus some 280 nucleotides of the coding sequence. p53 binds tightly to the 5'-UTR region and inhibits the translation of its own mRNA, most likely mediated by the p53-intrinsic RNA re-annealing activity. The inhibition of p53 biosynthesis requires wild-type p53, as it is not observed with MethA mutant p53, p53-catalysed translational inhibition is selective; it might be restricted to p53 mRNA and a few other mRNAs that are able to form extensive stem-loop structures. Release from negative feed-back regulation of p53 biosynthesis, e.g. after damage-induced nuclear transport of p53, might provide a means for rapidly increasing p53 protein levels when p53 is required to act as a cell-cycle checkpoint determinant after DNA damage.

3T3 Cells↗

PubMed1978

Methionine metabolism in BHK cells: preliminary characterization of the physiological effects of cycloleucine, an inhibitor of s-adenosylmethionine biosynthesis.

Cycloleucine is in vitro a competitive inhibitor of methionine adenosyltransferase, an enzyme involved in S-adenosylmethionine biosynthesis. The physiological effects of this drug on baby hamster kidney cells have been studied. When cells are grown in a medium containing 10 micron methionine, cycloleucine is an inhibitor of cell proliferation; high concentrations of methionine are able to withdraw this inhibition suggesting that cycloleucine toxicity is related to methionine metabolism. The drug does not primarily affect methionine uptake and its subsequent use for protein biosynthesis. Cycloleucine toxicity is correlated with a block of SAM biosynthesis and nucleic acids methylations. The actions of cycloleucine on progression in the cell cycle and DNA, RNA and protein biosynthesis are studied. The implications of these results are discussed.

Amino Acids↗

PubMed1986

The role of chalcone synthase in the regulation of flavonoid biosynthesis in developing oat primary leaves.

The role of chalcone synthase in the regulation of flavonoid biosynthesis during organogenesis of oat primary leaves has been investigated at the level of enzyme activity and mRNA translation in vitro. Chalcone synthase was purified about 500-fold. The apparent Km values were 1.5 and 6.3 microM for 4-coumaroyl-CoA and malonyl-CoA, respectively. The end products of oat flavonoid biosynthesis, three C-glucosylflavones, did not inhibit the reaction at concentrations as measured up to 60 microM each. Apigenin (4',5,7-trihydroxyflavone), a stable structural analog of the reaction product, 2',4,4',6'-tetrahydroxychalcone, was found to be a strong competitive inhibitor of 4-coumaroyl-CoA binding and a strong noncompetitive inhibitor of malonyl-CoA binding. Although apigenin is not supposed to be an intermediate of C-glucosylflavone biosynthesis, this compound might be a valuable tool for future kinetic studies. To date, there is no indication of chalcone synthase regulation by feedback or similar mechanisms which modulate enzyme activity. Mathematical correlation of chalcone synthase activity and flavonoid accumulation during leaf development, however, indicates that chalcone synthase is the rate-limiting enzyme of the pathway. By in vitro translation studies using preparations of total RNA from different leaf stages, we could demonstrate for the first time that the translational activity of chalcone synthase mRNA undergoes marked daily changes. The high values found at the end of the dark phase suggest that light does not exert direct influence on flavonoid biosynthesis but probably functions by controlling the basic diurnal rhythm.

Acyltransferases↗

PubMed1988

Rate of basement membrane biosynthesis as an index to angiogenesis.

A method was developed for assessing collagenous protein biosynthesis from [U-14C]proline in relation to angiogenesis in the chick chorioallantoic membrane (CAM). The rate of collagenous protein biosynthesis both in vitro and in vivo was maximum between days 8 and 11 of chick embryo development. This was the stage of maximum angiogenesis as shown by morphological evaluation of the vascular density. At day 10 the rate of collagenous protein biosynthesis was 11-fold higher than that of day 15, when angiogenesis had reached a plateau. The collagenous protein formed by CAM co-elutes on SDS-agarose chromatography with the collagenous component of [3H]-acetylated-basement membrane (BM) from bovine lens capsule. 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide (GPA1734), which was shown previously to be a specific inhibitor of BM collagen biosynthesis, caused about 80% reduction in collagenous protein synthesis by CAM. These results indicate that most of the collagenous protein synthesized by CAM was BM collagen and this can be used as a biochemical index of angiogenesis.

2H-Benzo(a)quinolizin-2-ol, 2-Ethyl-1,3,4,6,7,11b-↗

PubMed1977

Regulation of cholesterol biosynthesis by normal and leukemic (L2C) guinea pig lymphocytes.

The cholesterol production of guinea pig leukemic (L2C) lymphocytes preceeds at greater than 30 times the rate found in normal cells. Fatty acid biosynthesis is also enhanced in L2C cells. Exposure of L2C cells to cholesterol/lecithin liposomes does not depress their sterol biosynthesis, in contrast to the behavior of normal lymphocytes [Philippot, J.R., Cooper, A.G. & Wallach, D. F. H. (1975) Biochim. Biophys. Acta 406, 161-166]. However, 25-hydroxycholesterol, an inhibitor of hydroxymethylglutaryl-CoA reductase (NADPH) [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], the rate limiting enzyme in cholesterogenesis, and 25-hydroxycholecalciferol, a biologically potent form of vitamin D3, block sterol biosynthesis of both normal and L2C lymphocytes [Philippot, j.r., cooper, A.G. & Wallach, D.F.H. (1976) Biochem. Biophys. Res. Commun. 72, 1035-1041]. Moreover, both cell types exchange cholesterol equivalently with cholesterol/lecithin liposomes. The only difference in sterol biosynthesis observed between the two cell types is in the temperature response of the enzyme. Arrhenius plots of this enzyme activity exhibit a prominent discontinuity at about 24 degrees in the case of normal cells, but none in the case of L2C. The activation energies for L2C cells and normal cells, above the normal cell transition temperature, were not significantly different. All of the data suggest that the regulatory defect in L2C lymphocytes arises from a deficiency in these cells' internal membranes.

Androstanes↗

PubMed2003

Glucose-induced translational control of proinsulin biosynthesis is proportional to preproinsulin mRNA levels in islet beta-cells but not regulated via a positive feedback of secreted insulin.

Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (</=2h) this is due to increases in the translation of pre-existing mRNA. However, prolonging glucose stimulation (24 h) also increases preproinsulin mRNA levels. It has been proposed that secreted insulin from the pancreatic beta-cell regulates its own synthesis through a positive autocrine feedback mechanism. Here the comparative contributions of translation and mRNA levels on the levels of proinsulin biosynthesis were examined in isolated pancreatic islets. Also, the autocrine role of insulin upon four beta-cell functions (insulin secretion, proinsulin translation, preproinsulin mRNA levels, and total protein synthesis) was investigated in parallel. The results showed that proinsulin biosynthesis is regulated, in the short term (1 h), solely at the level of translation, through an approximately 6-fold increase in response to glucose (2.8 mm versus 16.7 mm glucose). In the longer term, when preproinsulin mRNA levels have increased approximately 2-fold, a corresponding increase was observed in the fold response of proinsulin translation to a stimulatory glucose concentration (>/=10-fold). Importantly, neither exogenously added nor secreted insulin were found to play any role in regulating insulin secretion, proinsulin translation, preproinsulin mRNA levels, or total protein synthesis. The results presented here indicate that long term nutritional state sets the preproinsulin mRNA level in the beta-cell at which translation control regulates short term changes in rates of proinsulin biosynthesis in response to glucose, but this is not mediated by any autocrine effect of insulin.

Animals↗

PubMed1984

Molecular mechanism for feedback regulation of C4 biosynthesis in guinea pig peritoneal macrophage.

Previous reports have shown that regulation of local extrahepatic production of complement may not reflect the regulation of plasma concentrations of the corresponding proteins and, further, that alteration of the tissue microenvironment can affect local macrophage protein synthesis. This report describes the molecular basis for control of the biosynthesis and secretion of a class III major histocompatibility complex gene product, the fourth component of complement (C4), from guinea pig macrophages by extracellular native C4 protein. The effect is specific for C4 synthesis, since production of C2 and total secreted protein was unaffected by fluid phase C4. C4 synthesis by extracellular C4 is regulated at a pretranslational level, without an effect on posttranslational proteolytic cleavage, glycosylation, or secretion. Specific C4 and factor B cDNA probes were used to demonstrate, by dot hybridization and Northern blot analysis, a decrease in messenger RNA coding for C4 that paralleled the inhibition of C4 biosynthesis, while the amount of total RNA and mRNA specific for factor B remained constant. Inhibition of C4 biosynthesis and the disappearance of mRNA encoding C4 occurred between 4 and 6 h after exposure of the macrophages to biologically active or methylamine-inactivated C4 protein. These data demonstrate that regulation of C4 biosynthesis by guinea pig macrophages serves as a model for the study of the molecular mechanisms of macrophage activation as well as the control of production of a component of the inflammatory response.

Animals↗

PubMed1980

Transcriptional and translational control of glucose-stimulated (pro)insulin biosynthesis.

Islets of Langerhans were isolated from the pancreata of fed or 48-h-fasted Wistar rats. The islets were incubated with either [3H]leucine of [3H]uridine. Inhibition of RNA synthesis by actinomycin D or by alpha-amanitin for 4 h had no influence on the (pro)insulin biosynthesis of isolated islets of fed rats. The (pro)insulin biosynthesis was not inhibited after two days incubation of islets of fed rats with alpha-amanitin either. Incorporation of labelled uridine into total RNA for 3 h was stimulated by glucose in islets of fasted, but not of fed rats. Therefore, it was concluded that transcriptional control does not participate, even for longer periods than believed previously, in acute regulation of (pro)insulin biosynthesis of islets isolated fed rats. Despite the strong and preferential stimulation of (pro)insulin biosynthesis of islets of fed rats by glucose the radioactivity of the [3H]uridine-labelled polysomes active in proinsulin synthesis remained unchanged. To interprete these experimental data we suggest that glucose triggers the transformation of a translationally inactive form of pre-proinsulin mRNA to a translationally active form.

Amanitins↗

PubMed1984

Subcellular site of biosynthesis of the catecholamine biosynthetic enzymes in bovine adrenal medulla.

The subcellular site of biosynthesis of the catecholamine biosynthetic enzymes was examined. Free and membrane-bound polysomes were prepared from bovine adrenal medulla and mRNA was isolated from these polysomes. Both were active in directing cell-free translations. Immunoprecipitation of cell-free products with specific antisera localized the biosynthesis of the subunits of tyrosine hydroxylase (TH) (apparent Mr = 61,000) and of phenylethanolamine N-methyltransferase (PNMT) (apparent Mr = 32,000) on free polysomes, compared with biosynthesis of subunits of dopamine beta-hydroxylase (DBH) (apparent Mr = 67,000) on membrane-bound polysomes. Cross-reactivity between translation products was observed. Antibodies for DBH recognized a polypeptide with electrophoretic mobility identical to newly synthesized PNMT. However increasing concentrations of antibodies to DBH recognized at most 1/20 of the PNMT formed. The results of this study show the subcellular distribution of the catecholamine synthesizing enzymes is determined by their site of biosynthesis.

Adrenal Medulla↗

PubMed2026

Light regulates capsaicinoid biosynthesis via the CaHY5-CaBBX2-CaACS8 module in pepper.

Capsaicinoids are a class of unique alkaloids that confer the pungent taste to pepper fruits. However, it remains largely unknown how light regulates the biosynthesis of capsaicinoids. We conducted a metabolic analysis on light- and dark-adapted pepper fruits. The results showed that dark-adapted pepper fruits had lower capsaicinoid contents and correspondingly downregulated transcription of capsaicinoid biosynthetic genes (CBGs), indicating that light plays a crucial role in capsaicinoid biosynthesis. Furthermore, silencing of CaHY5, a pivotal transcription factor gene in the light signaling pathway, decreased the content of capsaicinoid and suppressed the expression of CBGs, whereas transient overexpression of CaHY5 generated exactly opposite results. CaHY5 can bind to the G-box motif in the promoters of CaBBX2 and CaACS8, thereby enhancing their transcriptional levels. The activated CaBBX2 then binds to the T/G-box in the CaACS8 promoter to stimulate its expression. CaBBX2 or CaACS8 silencing led to decreased levels of capsaicinoids, while their transient overexpression produced increased capsaicinoid contents. Collectively, our results indicated that the light-activated CaHY5-CaBBX2-CaACS8 regulatory module plays a pivotal role in capsaicinoid biosynthesis. These findings provide new insights into the influence of light on capsaicinoid biosynthesis and potential targets for activation of this biosynthetic pathway in pepper.

Capsicum↗

PubMed2025

Ethylene response factors ERF.B2 and ERF.B5 synergically regulate ascorbic acid biosynthesis at multiple sites in tomato.

Ascorbic acid (AsA) is an important growth regulator and antioxidant in plants. It is acknowledged as a quality indicator in tomato (Solanum lycopersicum). Although the AsA biosynthetic pathway has been elucidated, its regulatory mechanisms remain largely unknown. In the present study, two members of the ethylene response factor (ERF) family, SlERF.B2 and SlERF.B5, were found to be co-expressed with SlGGP1, a pivotal gene in AsA biosynthesis. These two transcription factors were biochemically confirmed to bind to the DRE motif (GCCGAC/GTCGGC) of the SlGGP1 promoter. Notably, the SlERF.B2 and SlERF.B5 functioned as a dimer to regulate SlGGP1 expression and AsA biosynthesis. Overexpression of SlERF.B2 and SlERF.B5 enhanced the AsA levels up to 149 and 140%, respectively, whereas knockout of either of them could significantly decrease the AsA levels by up to 27%. DNA affinity purification sequencing (DAP-seq) indicated that SlERF.B2 synergistically regulates AsA biosynthesis at multiple sites by targeting the promoters of SlGPI and SlDHAR1. Overexpression of SlERF.B2 or SlERF.B5 in tomato conferred a high capacity for scavenging reactive oxygen species and enhanced tolerance to oxidation and salt stress, potentially by elevating the AsA content. This study unravels novel regulators of AsA biosynthesis and elucidates a molecular network that should facilitate the improvement of this nutrient in tomato and enhance stress tolerance in plants.

Solanum lycopersicum↗

PubMed2006

Delayed biosynthesis of varicella-zoster virus glycoprotein C: upregulation by hexamethylene bisacetamide and retinoic acid treatment of infected cells.

In the course of examining the trafficking pathways of varicella-zoster virus (VZV) glycoproteins gE, gI, gH, and gB, we discovered that all four are synthesized within 4 to 6 h postinfection (hpi) in cultured cells. Thereafter, they travel via the trans-Golgi network to the outer cell membrane. When we carried out a similar analysis on VZV gC, we observed little gC biosynthesis in the first 72 hpi. Further examination disclosed that gC was present in the inocula of infected cells, but no new gC biosynthesis occurred during the first 24 to 48 h thereafter, during which time new synthesis of gE, gH, and major capsid protein was easily detectable. Similarly, delayed gC biosynthesis was confirmed with three different VZV strains and two different cell lines. Bioinformatics analyses disclosed the presence of PBX/HOX consensus binding domains in the promoter/enhancer regions of the genes for VZV gC and ORF4 protein (whose orthologs transactivate gC in other herpesviruses). Bioinformatics analysis also identified two HOXA9 activation regions on ORF4 protein. Treatment of infected cultures with chemicals known to induce the production of PBX/HOX transcription proteins, namely, hexamethylene bisacetamide (HMBA) and retinoic acid, led to more rapid gC biosynthesis. Immunoblotting demonstrated a fivefold increase in the HOXA9 protein after HMBA treatment. In summary, these results documented that gC was not produced during early VZV replication cycles, presumably related to a deficiency in the PBX/HOX transcription factors. Furthermore, these results explain the apparent spontaneous loss of VZV gC in some passaged viruses, as well as other anomalous gC results.

Acetamides↗