高等植物的3-羟基-3-甲基戊二酰辅酶A还原酶

介绍了植物3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)的结构和调控,并简略讨论了HMGR调控与植物类异戊二烯途径的关系.     

Orthovanadate Suppresses Accumulation of Phenylalanine Ammonia-Lyase mRNA and Chalcone Synthase mRNA in Pea Epicotyls Induced by Elicitor from Mycosphaerella pinodes

Orthovanadate delayed accumulation of mRNAs encoding phenylalanineammonia-lyase and chalcone synthase in pea epicotyls inducedby an elicitor from Mycosphaerella pinodes. However, accumulationof mRNA for a putative P-type ATPase was not affected. The relationshipbetweenthe ATPase and defense responses is discussed. ³Present address: Plant Pathology Laboratory, School of Agriculture,Nagoya University, Chikusa, Nagoya, 464-01 Japan.     

Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway.     

Involvement of de Novo Protein Synthesis,Protein Kinase,Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid Induction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and Isoprenoid Accumulation in Potato (Solanum tuberosum L.)

A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca2+, and lipoxygenase activity in arachidonic acid elicitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum L. cv Kennebec). Gene-specific probes were used to discriminate effects on the expression of two HMGR genes (hmg1 and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. Inhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMGR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid, a Ca2+ chelator, inhibited arachidonate-induction of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmg1. However, staurosporin inhibited arachidonate's suppression of hmg1 gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate's suppression of hmg1 and induction of hmg2. The results indicate that arachidonate's suppression of hmg1 and activation of hmg2 depend on a common intermediate or set of intermediates whose generation is sensitive to the inhibitors tested.     

Phenylalanine Ammonia-Lyase Activity and Anthocyanin Accumulation in Wounded Maize Mesocotyls

Activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) and anthocyanin accumulation were determined in wounded maize (Zea mays L.) mesocotyls. Mesocotyls were wounded with aluminum oxide and were placed in a 15 h light: 9 h dark photoperiod or in the dark. Extractable enzyme activity increased in response to wounding in the photoperiod but not in the dark. Anthocyanin accumulation in mesocotyls placed in the photoperiod decreased in response to wounding. The results are discussed with reference to phenylalanine ammonia-lyase activity in mesocotyl tissue wounded or inoculated with fungal pathogens.     

Ethylene-enhanced Synthesis of Phenylalanine Ammonia-Lyase in Pea Seedlings

The effect of ethylene on the development of phenylalanine ammonia-lyase activity in segments excised from the epicotyl apex of pea seedling was studied. Although there was some increase in phenylalanine ammonia-lyase activity in segments not treated with ethylene, a marked increase in phenylalanine ammonia-lyase activity occurred in ethylene-treated tissues during the incubation. The induction period was estimated to be about 6 hours. The activity reached a maxmum at 30 hours and then declined. On withdrawal of ethylene, the increase was sustained for a short period and then stopped. After retreatment with ethylene, the increase was resumed. Addition of CO₂ reduced the effect of ethylene. Administration of cycloheximide or actinomycin D at an early period almost completely suppressed the increase in phenylalanine ammonia-lyase activity. However, if these inhibitors were administered at a later period, while phenylalanine ammonia-lyase activity was approaching a maximum, they not only failed to reduce but rather stimulated the activity. These results are consistent with the view that there exist both phenylalanine ammonia-lyase-synthesizing and -inactivating systems, and that the development of both systems may involve de novo synthesis of protein.     

Clinical Characteristics of Anti-3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Antibodies in Chinese Patients with Idiopathic Inflammatory Myopathies

Objective

The objective of this study was to detect the prevalence of anti-3-hydroxyl-3- methylglutaryl coenzyme A reductase (anti-HMGCR) antibodies in Chinese patients with idiopathic inflammatory myopathies (IIMs), and to analyze the clinical features of the antibody-positive IIM patients.

Methods

The presence of anti-HMGCR antibodies was detected in 405 patients with IIMs, 90 healthy controls, and 221 patients with other rheumatic diseases by using an ELISA kit. Clinical data from anti-HMGCR antibody-positive and -negative patients were compared. Long-term follow-up of the anti-HMGCR antibody-positive patients was conducted to evaluate the role of anti-HMGCR antibody in IIM disease prognosis.

Results

Of the 405 IIM patients, 22 (5.4%) were found to carry the anti-HMGCR antibody. These IIM patients were predominantly female (73%), and only 3 anti-HMGCR antibody-positive patients with IIM were exposure to statins. Most patients experienced progressive onset, and presented with muscular weakness. Dysphagia was observed in half of the patients (p < 0.01), and 15% of these patients experienced the complication of interstitial lung disease (ILD) (p > 0.05). Mean creatine kinase (CK) levels were higher in antibody-positive patients than in antibody-negative patients (p < 0.05). Muscle biopsies were available from 12 anti-HMGCR antibody-positive patients, eight who experienced myofiber necrosis and showed very little or no evidence of inflammatory cell infiltrates in their muscle biopsies. Of these eleven patients who were followed-up 2.5- to 29-month, 73% experienced improvement after treatment. A cross-sectional study showed that anti-HMGCR antibody levels were significantly associated with CK levels (r = 0.486, p = 0.026) as well as with Myositis Disease Activity Assessment (MYOACT) scores (r = -0.67, p = 0.003) during the initial visit. However, changes in serum anti-HMGCR antibody levels did not correlate with changes in CK levels, Manual Muscle Testing 8 (MMT-8) scores or MYOACT scores in long-term follow-up.

Conclusion

The major clinical features of anti-HMGCR antibody-positive Chinese IIM patients were muscle weakness and dysphagia, which were seen in patients with and without statin exposure. This subtype of patients were responsive to immunosuppressive treatment and received good prognoses after treatment, but serum levels of the anti-HMGCR antibody do not correlate with disease activity.     

Enhanced Production of a Plant Monoterpene by Overexpression of the 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Catalytic Domain in Saccharomyces cerevisiae

Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T₇₃ was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount). The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a haploid laboratory strain, engineering of this key step also improved linalool yield.Monoterpenes are a class of isoprenoids of increasing industrial and clinical interest usually produced by plants. They are used as aromatic additives in the food and cosmetics industries and are also important components in wine aroma. Moreover, certain monoterpenes display antimicrobial, antiparasitic, and antiviral properties as well as a plethora of promising health benefits (for recent reviews, see references 2, 7, 15, 28, and 30 and references cited therein). To date, many studies have focused on plant metabolic engineering of monoterpene production (for selected reviews, see references 1, 14, 19, 29, and 35 and references cited therein), and few studies have been carried out on microorganisms (9, 21, 22, 34, 38). Efficient microbial production of these metabolites could provide an alternative to the current methods of chemical synthesis or extraction from natural sources. In this regard, a considerable number of studies have shown the utility of Saccharomyces cerevisiae as a valuable platform for sesquiterpene, diterpene, triterpene, and carotene production (references 5, 10, 23, 26, 30, 31, 32, and 33 and references cited therein). However, all the efforts dedicated to the improvement of isoprenoid yields in S. cerevisiae have been performed using conventional laboratory strains, and there are no studies concerning natural or industrially relevant isolates.In recent years, many genes that encode plant monoterpene synthases (MTS), a family of enzymes which specifically catalyze the conversion of the ubiquitous C₁₀ intermediate of isoprenoid biosynthesis geranyl pyrophosphate (GPP) to monoterpenes, have been characterized. Such is the case with the LIS gene (codes for S-linalool synthase) of Clarkia breweri, the first MTS-encoding gene to be isolated (13). In contrast to plants, S. cerevisiae cannot produce monoterpenes efficiently, mainly due to the lack of specific pathways involving MTS. However, GPP is formed as a transitory intermediate in the two-step synthesis of farnesyl pyrophosphate (FPP), catalyzed by FPP synthase (FPPS) (Fig. ​(Fig.1),1), and some natural S. cerevisiae strains have been shown to possess the ability to produce small amounts of monoterpenes (8). Whether this occurs through unspecific dephosphorylation of a more available endogenous pool of GPP and subsequent bioconversions is not known. In addition, it has recently been established that S. cerevisiae has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions (22, 34).Open in a separate windowFIG. 1.Simplified isoprenoid pathway in S. cerevisiae, including the branch point to linalool. Dotted arrows indicate that more than one reaction is required to convert the substrate to the product indicated. Dashed arrows indicate the engineered steps. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase.Here we present the process for selecting and optimizing yeast strains for foreign monoterpene production. We have chosen the C. breweri LIS gene as a prototype because, when heterologously expressed in S. cerevisiae, it specifically results in the production of linalool (3,7-dimethyl-1,6-octadien-3-ol; a floral scent and bioactive acyclic monoterpene identified in numerous fruits and flowers) and no other by-products (22). Two S. cerevisiae strains of different origins have been selected and their endogenous mevalonate (MVA) pathways engineered to enhance the production of linalool. These strategies might be employed to produce any other recombinant monoterpene in S. cerevisiae by expressing the appropriate monoterpene synthase.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production

Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.     

Protein Kinase Inhibitors Inhibit Stimulation of Inositol Phospholipid Turnover and Induction of Phenylalanine Ammonia-Lyase in Fungal Elicitor-Treated Tobacco Suspension Culture Cells

Elicitor prepared from Phytophthora nicotianae stimulated inositolphospholipid turnover and induced phenylalanine ammonia-lyaseactivity in tobacco suspension culture cells [Kamada and Muto(1994) Plant Cell Physiol. 35: 397]. Protein kinase inhibitors,K252a and staurosporine inhibited both responses. These resultssuggest that inositol phospholipid turnover plays an importantrole in PAL induction through protein kinases. In addition,their mode of inhibition were different, proposing that severaltypes of protein kinases are involved in these elicitor-inducedresponses. ¹Present address: The Johns Hopkins University School of Hygieneand Public Health, 615 N. Wolfe St., Baltimore, Maryland 21205,U.S.A. ²Present address: Nagoya University BioScience Center and GraduateSchool of Agricultural Sciences, Nagoya University, Chikusa-ku,Nagoya, 464-01 Japan.     

抗癌新药K－AM的合成

本文以鼠李糖和对氨基苯甲酸为原料合成了抗癌新药Ｋ－ＡＭ，并作了红外鉴定。     

抗癌新药K－AM的合成

本文以鼠李糖和对氨基苯甲酸为原料合成了抗癌新药Ｋ－ＡＭ,并作了红外鉴定。