Production of the carotenoids lycopene, beta-carotene, and astaxanthin in the food yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, beta-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promotes and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, beta-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.     

The mode of action of AKD-2C, an antifungal antibiotic from Streptomyces sp. OCU-42815

The mechanism of the antifungal action of AKD-2C was studied by using Torulaspora delbrueckii IFO 1621 as a model. AKD-2C slightly inhibited the incorporation of radioactive precursors into protein, RNA and lipid, but not into DNA. On the other hand, AKD-2C greatly enhanced the leakage of K+ ions from treated cells and showed a potent effect on liposomal glucose leakage. Using electron microscopic studies, though drastic morphological changes were not observed, an increase in cell membrane irregularities and swelling of the mitochondria caused by AKD-2C were demonstrated. These results suggest that the antifungal action of AKD-2C is due to effects on the yeast cell membrane.     

Inhibition of Sindbis virus replication by cyclopentenone prostaglandins: a cell-mediated event associated with heat-shock protein synthesis

Cyclopentenone prostaglandins (PGs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report on the effect of prostaglandin A1 (PGA1) on the multiplication of a positive strand RNA virus, Sindbis virus, in Vero cells under one-step multiplication conditions. PGA1 was found to inhibit Sindbis virus production dose-dependently, and virus yield was reduced by more than 90% at the concentration of 8 micrograms/ml, which was non-toxic to the cells and did not inhibit DNA, RNA or protein synthesis in Vero cells. The cyclopentenone prostaglandin delta 12-PGJ2 was also shown to be a potent inhibitor of Sindbis virus replication. Virus-induced reduction of [3H]uridine uptake by cells was partially prevented by PGA1 treatment, which also caused a 1 h delay in the peak of virus RNA synthesis. SDS-PAGE analysis of [35S]methionine-labeled proteins showed that PGA1 moderately inhibited the synthesis of the viral structural proteins E1, E2 and C, and induced the synthesis of a 72 kDa M(r) protein, identified as a heat-shock protein related to the HSP70 group, in both virus-infected and uninfected cells. Actinomycin D treatment completely prevented PGA1-antiviral activity, indicating that a cellular product is responsible for this action. PGA1-induced HSP70 is a good candidate for this role.     

The growth of Rhodotorula rubra yeasts and their synthesis of ergosterol on media with lovastatin

The effect of lovastatin, a competing inhibitor of 3-hydroxy-3-methylglutaryl-CoA-reductase (HMG-CoA-reductase) of the fungal origin on the growth of and ergosterol biosynthesis by Rh. rubra VKPM Y 1337 was studied. It was shown that the yeast growth was inhibited by lovastatin in concentrations of 0.25 to 5.0 micrograms/ml when the inhibitor was added to the cultivation medium either with the inoculum or at later periods of the yeast cultivation. In concentrations of 2.0 to 5.0 micrograms/ml lovastatin almost completely inhibited the yeast growth. In the concentrations tested the inhibitor did not decrease the ergosterol level in the yeast cells below 40 per cent of the control. When lovastatin was added in concentrations of 1.0 to 5.0 micrograms/ml the maximum inhibition of the ergosterol biosynthesis was observed at the end of the growth log phase. Further cultivation of the yeast in the presence of the inhibitor resulted in an increase of the ergosterol biosynthesis. It is believed that the increase in the quantity of ergosterol may be due either to the HMG-CoA-reductase induction under the action of the inhibitor or to its inactivation as a result of the hydroxylation.     

Biochemical nature and cellular distribution of the paired immunoglobulin-like receptors, PIR-A and PIR-B

Feline immunodeficiency virus (FIV) multiplication is totally blocked by incubation of infected cells at 41 degrees. This inhibition does not take place with a thermoresistant strain of FIV, designated m41, indicating the role played by the viral genome in temperature sensitivity. We have investigated the steps in the life cycle of wild-type FIV that are thermosensitive and found that they depend on the host cells infected. In CrFK cells, FIV replication was inhibited after the penetration step at 41 degrees. Synthesis of viral RNA and DNA was barely detectable and no viral antigen appeared in the extracellular medium. Nevertheless, viral multiplication resumed on incubation at 37 degrees, suggesting a state of latency at the elevated temperature. In peripheral blood mononuclear cells (PBMCs), the FIV cycle was inhibited at 41 degrees after the synthesis of viral DNA. Several viral mRNAs failed to appear as fully spliced products and no viral antigen was found in the extracellular medium. As in CrFK cells, viral multiplication occurred in PBMCs after a shift to the permissive temperature. These results suggest that at least two steps in the viral life cycle are sensitive to 41 degrees and that two different viral functions of the thermoresistant mutant m41 are modified to overcome temperature sensitivity.     

Cytotoxicity of captafol in mammalian cells

The cytotoxicity of captafol, a phthalimide-derived fungicide, was evaluated in IB-RS-2 cells. Captafol at 0.12-1.0 microgram/ml blocks the cell multiplication. This effect is concentration-dependent, only partially reversible and the degree of inhibition increases with time. The synthesis of DNA and RNA is inhibited in parallel by increasing concentrations of the chemical.     

Polyamine evaluation in serum and erythrocytes of pediatric patients with neoplasms

A major allergen/antigen, Asp fl, secreted by Aspergillus fumigatus exhibits cytotoxicity towards eukaryotic cell lines. Asp fl inhibited protein synthesis in RAW cells with an IC50 of 4.5 nM and also degraded ribosomal RNA of RAW cells at a similar concentration. Ribosomal inactivation by Asp fl may be the probable mechanism for protein synthesis inhibition. Specific ribonuclease activity of Asp fl was observed to be 100,000 U/mg. Presence of strong RNase activity in Asp fl was further confirmed by agar gels containing yeast RNA. Electrophoretic run on agarose gels showed that Asp fl degrades all species of naked RNA. Modification of histidine residues of Asp fl with diethyl pyrocarbonate and alkylation of cysteines with iodoacetamide resulted in loss of ribonuclease activity and cytotoxicity of Asp fl. The current study establishes the ribonuclease activity of a purified major allergen of A. fumigatus that inhibits protein synthesis and kills the eukaryotic cells.     

Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP-1 macrophages

The free cholesterol content of cells can be monitored by the intensity of fluorescence emissions from the polyene antibiotic filipin. In a previous study (Hassall: Cytometry 13:381-388, 1992) using THP-1 macrophages, a decrease in filipin fluorescence in response to increasing concentrations of modified lipoprotein was observed, suggesting a reduction in the free cholesterol content of the cells. In this study, THP-1 macrophages were treated with a number of agents known to modulate cholesterol biosynthesis and cholesterol esterification. Changes in filipin fluorescence emissions were measured by flow cytometry, and correlated with changes in cholesterol biosynthesis measured by incorporation of [14C]acetate into cholesterol. A correlation between decreases in filipin fluorescence and reductions in cholesterol biosynthesis was apparent, even when cholesterol esterification was inhibited. These results suggest that the decreases in filipin fluorescence observed may be due, at least in part, to reduction in cholesterol biosynthesis.     

Sequences within a small yeast RNA required for inhibition of internal initiation of translation: interaction with La and other cellular proteins influences its inhibitory activity

We recently reported purification, determination of the nucleotide sequence, and cloning of a 60-nucleotide RNA (I-RNA) from the yeast Saccharomyces cerevisiae which preferentially blocked cap-independent, internal ribosome entry site (IRES)-mediated translation programmed by the poliovirus (PV) 5' untranslated region (UTR). The I-RNA appeared to inhibit IRES-mediated translation by virtue of its ability to bind a 52-kDa polypeptide which interacts with the 5' UTR of viral RNA. We demonstrate here that the HeLa 52-kDa I-RNA-binding protein is immunologically identical to human La autoantigen. Moreover, I-RNA-mediated purified La protein. By using I-RNAs with defined deletions, we have identified sequences of I-RNA required for inhibition of internal initiation of translation. Two smaller fragments of I-RNA (16 and 25 nucleotides) inhibited PV UTR-mediated translation from both monocistronic and bicistronic RNAs. When transfected into HeLa cells, these derivatives of I-RNA inhibited translation of PV RNA. A comparison of protein binding by active and inactive I-RNA mutants demonstrates that in addition to the La protein, three other polypeptides with apparent molecular masses of 80, 70, and 37 kDa may influence the translation-inhibitory activity of I-RNA.     

Effects of hormones on protein and amino acid metabolism in mammary-gland explants of mice

The effects of insulin, cortisol and prolactin on amino acid uptake and protein biosynthesis were determined in mammary-gland explants from mid-pregnant mice. Insulin stimulated [3H]leucine incorporation into protein within 15 min of adding insulin to the incubation medium. Insulin also had a rapid stimulatory effect on the rate of aminoiso[14C]butyric acid uptake, but it had no effect on the intracellular accumulation of [3H]leucine. Cortisol inhibited the rate of [3H]leucine incorporation into protein during the initial 4h of incubation, but it had no effect at subsequent times. [3H]Leucine uptake was unaffected by cortisol, but amino[14C]isobutyric acid uptake was inhibited after a 4h exposure period to this hormone. Prolactin stimulated the rate of [3H]leucine incorporation into protein when tissues were exposed to this hormone for 4h or more; up to 4h, however, no effect of prolactin was detected. At all times tested, prolactin had no effect on the uptake of either amino[14C]isobutyric acid or [3H]leucine. Incubation with actinomycin D abolished the prolactin stimulation of protein biosynthesis, but this antibiotic did not affect the insulin response. A distinct difference in the mechanism of action of these hormones on protein biosynthesis in the mammary gland is thus apparent.     

Emopamil-binding protein, a mammalian protein that binds a series of structurally diverse neuroprotective agents, exhibits delta8-delta7 sterol isomerase activity in yeast

Delta8-delta7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of delta8-sterols to their corresponding delta7-isomers. No sequence data for high eukaryote sterol isomerase being available so far, we have cloned a murine sterol isomerase-encoding cDNA by functional complementation of the corresponding deficiency in the yeast Saccharomyces cerevisiae. The amino acid sequence deduced from the cDNA open reading frame is highly similar to human emopamil-binding protein (EBP), a protein of unknown function that constitutes a molecular target for neuroprotective drugs. A yeast strain in which the sterol isomerase coding sequence has been replaced by that of human EBP or its murine homologue recovers the ability to convert delta8-sterol into delta7-sterol, both in vivo and in vitro. In these recombinant strains, both cell proliferation and the sterol isomerization reaction are inhibited by the high affinity EBP ligand trifluoperazine, as is the case in mammalian cells but not in wild type yeast cell. In contrast, the recombinant strains are much less susceptible to the sterol inhibition effect of haloperidol and fenpropimorph, as compared with wild type yeast strains. Our results strongly suggest that EBP and delta8-delta7 sterol isomerase are identical proteins in mammals.     

8-OH-DPAT influences extracellular levels of serotonin and dopamine in the medial preoptic area of male rats

The effect of phospholipase C treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with phosphatidylcholine-specific Clostridium welchii phospholipase C reduced the pool size of phosphatidylcholine compared with controls whereas the pool size of cardiolipin and phosphatidylglycerol were unaffected. Pulse labeling experiments with [1,3-3H]glycerol and pulse-chase labeling experiments with [1,3-3H]glycerol were performed in cells incubated or pre-incubated in the absence or presence of phospholipase C. In all experiments, radioactivity incorporated into cardiolipin and phosphatidylglycerol were reduced in phospholipase C-treated cells with time compared with controls indicating attenuated de novo biosynthesis of these phospholipids. Addition of 1,2-dioctanoyl-sn-glycerol, a cell permeable 1,2-diacyl-sn-glycerol analog, to cells mimicked the inhibitory effect of phospholipase C on cardiolipin and phosphatidylglycerol biosynthesis from [1,3-3H]glycerol indicating the involvement of 1,2-diacyl-sn glycerol. The mechanism for the reduction in cardiolipin and phosphatidylglycerol biosynthesis in phospholipase C-treated cells appeared to be a decrease in the activities of phosphatidic acid:cytidine-5'triphosphate cytidylyltransferase and phosphatidylglycerolphosphate synthase, mediated by elevated 1,2-diacylsn-glycerol levels. Upon removal of phospholipase C from the incubation medium, phosphatidylcholine biosynthesis from [methyl-3H]choline was markedly stimulated. These data suggest that de novo phosphatidylglycerol and cardiolipin biosynthesis may be regulated by 1,2-diacyl-sn-glycerol and support the notion that phosphatidylglycerol and cardiolipin biosynthesis may be coordinated with phosphatidylcholine biosynthesis in H9c2 cardiac myoblast cells.     

Improvement in nerve regeneration by monoclonal antibodies to ICAM-1 and LFA-1 in allogeneic mice

A mixed membrane fraction isolated from C. albicans yeast cells catalyzed the transfer of glucose from UDP-Glc into three classes of endogenous acceptors: glucolipid, glycoprotein and lipid-linked oligosaccharides. About 80% of the total radioactivity transferred into these products corresponded to the glucolipid which was identified as dolichol phosphate glucose by several criteria. The remainder was detected in about equal proportions in the other two fractions. Conditions that stimulated or inhibited glucolipid synthesis did not affect the extent of glycoprotein labeling. The synthesis of dolichol phosphate glucose exhibited a K(m) of 104 microM UDP-Glc and was stimulated by Mg2+ but not by Mn2+ or Ca2+. The latter cations were, however, better stimulators of glycoprotein labeling than Mg2+. Most nucleotides strongly inhibited the synthesis of dolichol phosphate glucose, UMP being a competitive inhibitor with a Ki of 100 microM. The dolichol phosphate glucose synthase reaction was reversed about 57% by 0.62 mM UDP but not by UMP.     

Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of both acute and chronic inflammatory responses in many diseases. Tristetraprolin (TTP), the prototype of a class of Cys-Cys-Cys-His (CCCH) zinc finger proteins, inhibited TNF-alpha production from macrophages by destabilizing its messenger RNA. This effect appeared to result from direct TTP binding to the AU-rich element of the TNF-alpha messenger RNA. TTP is a cytosolic protein in these cells, and its biosynthesis was induced by the same agents that stimulate TNF-alpha production, including TNF-alpha itself. These findings identify TTP as a component of a negative feedback loop that interferes with TNF-alpha production by destabilizing its messenger RNA. This pathway represents a potential target for anti-TNF-alpha therapies.     

Inhibition of steroidogenesis in rat adrenal cells by 18-ethynyldeoxycorticosterone: evidence for an alternative pathway of aldosterone biosynthesis

The effect of the mechanism-based inhibitor 18-ethynyldeoxycorticosterone (18-E-DOC) on the late steps of the aldosterone biosynthetic pathway was examined in freshly isolated cells of the zona glomerulosa (ZG) and fasciculata (ZF) from rat adrenal glands. ZG synthesis of aldosterone was inhibited by 18-E-DOC in a time- and concentration-dependent manner with a Ki of approximately 0.05 microM. The maximal degree of inhibition of ZG production of aldosterone and 18-hydroxycorticosterone (18-OH-B) was approximately 80%. ZF cells, perhaps surprisingly, were found to secrete 18-OH-B at levels approximately one-third to one-fourth those of ZG cells and the Ki of 18-E-DOC inhibition of 18-OH-B secretion was approximately 10 microM for ZF cells, 200-fold higher than for ZG cells. The inhibitor had no effect on the secretion of corticosterone by either ZG or ZF, and the secretion of 18-hydroxydeoxycorticosterone (18-OH-DOC) by both the ZG and ZF was inhibited only to a minor degree. 18-E-DOC inhibited the biosynthesis of aldosterone by ZG cells incubated with 10 microM added DOC or 18-OH-DOC by approximately 75%, similar to the degree of inhibition of aldosterone biosynthesis from endogenous substrate, whereas ZF biosynthesis of 18-OH-B from either substrate was inhibited by less than 40%. ZF cells do not express aldosterone synthase, the only enzyme known to convert 18-OH-DOC into 18-OH-B. Incubation of MA-10 cells stably transfected with the cDNA of the rat aldosterone synthase with 18-E-DOC resulted in a complete inhibition of the conversion of DOC to aldosterone with a Ki of approximately 0.02 microM. In addition, transfected cells expressing 11beta-hydroxylase convert DOC to 18-OH-B in very small quantities only and cannot convert 18-OH-DOC to 18-OH-B. These data suggest that neither 11beta-hydroxylase nor aldosterone synthase are responsible for the biosynthesis of 18-OH-B by ZF cells from DOC or 18-OH-DOC, that 20% of aldosterone synthesis appears not to be attributable to the actions of aldosterone synthase and that an unknown CYP11B enzyme is also involved in the biosynthesis of 18-OH-B.     

Arabidopsis ent-kaurene oxidase catalyzes three steps of gibberellin biosynthesis

The Arabidopsis GA3 cDNA was expressed in yeast (Saccharomyces cerevisiae) and the ability of the transformed yeast cells to metabolize ent-kaurene was tested. We show by full-scan gas chromatography-mass spectrometry that the transformed cells produce ent-kaurenoic acid, and demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid.     

Chemical constituents of original plants of Cimicifugae rhizoma in Chinese medicine

Cimicifugae Rhizoma have been used as an anti-inflammatory, analgesic and antipyretic remedy in the traditional Chinese medicines. Many 9,19-cyclolanostane glycosides have been isolated from Cimicifuga and related genera. Two biogenetically key compounds, acetylshengmanol xyloside and cimicifugoside H-1, were isolated and their chemical structures were elucidated by our group. The former compound seems to be the parent component of the other glycosides such as cimigenol xyloside from C. dahurica, C. iaponica and C. acerina. The latter glycoside, cimicifugoside H-1 was isolated together with cimicifugosides H-2-H-6 from commercial Cimicifugae Rhizoma. They are novel glycosides having a hydroxyl group ay C-11, and cimicifugosides H-3, H-4 and H-6 were trinor-triterpenol glycosides. Cimicifugoside H-1 changed into H-2, H-3 and H-4 under acidic or alkaline conditions. In this review, the structure elucidation of the above glycosides and their chemical transformation into other Cimicifuga glycosides are described.     

Nucleolar localization of early tRNA processing

There is little information as to the location of early tRNA biosynthesis. Using fluorescent in situ hybridization in the budding yeast, Saccharomyces cerevisiae, examples of nuclear pre-tRNAs are shown to reside primarily in the nucleoli. We also probed the RNA subunit of RNase P. The majority of the signal from RNase P probes was nucleolar, with less intense signals in the nucleoplasm. These results demonstrate that a major portion of the tRNA processing pathway is compartmentalized in nucleoli with rRNA synthesis and ribosomal assembly. The spatial juxtaposition suggests the possibility of direct coordination between tRNA and ribosome biosynthesis.