Cloning and Expression Analysis on 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase from Aquilaria sinensis

Objective To clone the full-length cDNA of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGR) from Aquilaria sinensis(AsHMGR1) and to analyze its expression profile in different tissues and in response to different treatments.HMGR is the first rate-limiting enzyme for sesquiterpene synthesis in the mevalonate pathway.Methods RT-PCR and RACE were used to clone the full-length cDNA of HMGR from A.sinensis based on the conserved HMGR gene fragments.The bioinformatic analysis was performed on its nucleic acid and protein sequence.The expression profile of AsHMGR1 in different tissues and in response to different treatments was analyzed by quantitative RT-PCR.Results The full-length AsHMGR1 cDNA was 2026 bp,containing a 1719 bp open reading frame which encoded a protein of 572 amino acids.Amino acid sequence homology alignment and phylogenetic analysis demonstrated that AsHMGR1 belonged to the HMGR gene family.The detection of tissue expression patterns showed that AsHMGR1 was mainly expressed in the stem,followed by roots and branches.AsHMGR1 could be stimulated by methyl jasmonate and H2O2to varying degrees in a time-dependent manner.Conclusion These data will provide a foundation for further investigation on AsHMGR1 functions and regulatory mechanisms in sesquiterpene synthesis in A.sinensis.

Identification of a Highly Expressed 3-Hydroxy-3-Methylglutaryl-CoA Reductase Gene in the Root Tissue of <i>Taraxacum kok-saghyz</i>

Kazakh dandelion (Taraxacum kok-saghyz, Tk) is a rubber-producing plant currently being investigated as a source of natural rubber for industrial applications. Like many other isoprenoids, rubber is a downstream product of the mevalonate pathway. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, a key regulatory step in the MVA pathway. Such regulated steps provide targets for increases in isoprenoid and rubber contents via genetic engineering to increase enzyme activities. In this study, we identify a TkHMGR1 gene that is highly expressed in the roots of Kazakh dandelion, the main tissue where rubber is synthesized and stored. This finding paves the way for further molecular and genetic studies of the TkHMGR1 gene, and its role in rubber biosynthesis in Tk and other rubber-producing plants.

Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis

The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway,the HMGR gene from Glycyrrhiza uralensis Fisch.(GuHMGR)was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC.The results showed that all the recombinant P.pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains.However,as the copy number increased,the content of ergosterol showed an increasing–decreasing–increasing pattern.This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G.uralensis.

Molecular cloning and functional identification of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Tripterygium wilfordii

The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase(HDR) is the last step key enzyme of the methylerythritol phosphate(MEP) pathway,synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate,which is important for regulation of isoprenoid biosynthesis.Here the full-length cDNA of HDR,designated TwHDR(GenBank Accession No.KJ933412.1),was isolated from Tripterygium wilfordii for the first time.TwHDR has an open reading frame(ORF) of 1386 bp encoding461 amino acids.TwHDR exhibits high homology with HDRs of other plants,with an N-terminal conserved domain and three conserved cysteine residues.TwHDR cDNA was cloned into an expression vector and transformed into an Escherichia coli hdr mutant.Since loss-of-function E.coli hdr mutant is lethal,the result showed that transformation of TwHDR cDNA rescued the E.coli hdr mutant.This complementation assay suggests that the TwHDR cDNA encodes a functional HDR enzyme.The expression of TwHDR was induced by methyl-jasmonate(MJ) in T.wilfordii suspension cells.The expression of TwHDR reached the highest level after 1 h of MJ treatment.These results indicate that we have identified a functional TwHDR enzyme,which may play a pivotal role in the biosynthesis of diterpenoid triptolide in T.wilfordii.

Immune-mediated necrotizing myopathy:Report of two cases

BACKGROUND Immune-mediated necrotizing myopathy is a rare autoimmune myopathy characterized by muscle weakness and elevated serum creatine kinase,with unique skeletal muscle pathology and magnetic resonance imaging features.CASE SUMMARY In this paper,two patients are reported:One was positive for anti-signal recognition particle antibody,and the other was positive for anti-3-hydroxy-3-methylglutaryl coenzyme A reductase antibody.CONCLUSION The clinical characteristics and treatment of the two patients were analysed,and the literature was reviewed to improve the recognition,diagnosis,and treatment of this disease.

Hepatitis B virus induces expression of cholesterol metabolism-related genes via TLR2 in HepG2 cells

AIM:To investigate whether hepatitis B virus(HBV) exacerbates hepatic cholesterol accumulation,and explore the underlying mechanisms.METHODS:HepG2 cells were infected with adenovirus(Ad) containing 1.3-fold overlength HBV genome.Realtime polymerase chain reaction and Western blotting were used to measure mRNA and protein expression of target genes.Cholesterol accumulation was measured by fluorescence microscopy.Cell toxicity due to Ad-HBV treatment was determined by the mitochondrial tetrazolium assay.The protein levels of toll-like receptors(TLRs) were determined by Western blotting.RESULTS:Ad-HBV increased hepatic cholesterol accumulation and enhanced the mRNA and protein levels oflow-density lipoprotein receptor(LDLR) and 3-hydroxy3-methylglutharyl-coenzyme A reductase(HMGCoAr) mRNA and protein expression in HepG2 cells.In addition,these inductive effects were partly offset by suppressing TLR2 expression levels by small interfering RNA in HepG2 cells.CONCLUSION:Ad-HBV increases LDLR and HMGCoAr expression,resulting in exacerbated cholesterol accumulation in HepG2 cells,which was mediated via the TLR2 pathway.

Statins and the risk of colorectal cancer: An updated systematic review and meta-analysis of 40 studies

AIM:To investigate the association between statin use and colorectal cancer risk,we conducted an updated meta-analysis of published studies.METHODS:We performed a comprehensive search for studies published up to July 2013.Eligible studies for this meta-analysis were either randomized controlled trials(RCTs)or observational studies(casecontrol or cohort)evaluating any exposure to statins and the risk of colorectal cancer.Two reviewers selected studies based on predefined inclusion criteria,and abstracted the data.Pooled relative risk(RR)estimates with their 95%CI were calculated using fixedand random-effects models.Then,we assessed the potential presence of publication bias and betweenstudies heterogeneity.To evaluate the results,we also performed a"leave-one-out"sensitivity analysis.RESULTS:A total of 40 studies,involving more than eight million subjects,contributed to the analysis.They were grouped on the basis of study design and,consequently,three separate meta-analyses were conducted.A similar modest reduction in the risk of colorectal cancer with statin use was observed,which was not statistically significant among RCTs(RR=0.89,95%CI:0.74-1.07;n=8),but reached statistical significance among cohort studies(RR=0.91,95%CI:0.83-1.00;n=13)and case-control studies(RR=0.92,95%CI:0.87-0.98;n=19).While we did not find significant evidence of selective outcome reporting or publication bias,substantial heterogeneity was detected,mainly among the observational studies.The sensitivity analysis confirmed the stability of our results.CONCLUSION:A modest reduction in risk of colorectal cancer among statin users cannot be disproved.Further targeted research is warranted.2014 Baishideng Publishing Group Co.,Limited.All

Regulation and deregulation of cholesterol homeostasis: The liver as a metabolic "power station"

Cholesterol plays several structural and metabolic roles that are vital for human biology. It spreads along the entire plasma membrane of the cell, modulating fluidity and concentrating in specialized sphingolipid-rich domains called rafts and caveolae. Cholesterol is also a substrate for steroid hormones. However, too much cholesterol can lead to pathological pictures such as atherosclerosis, which is a consequence of the accumu- lation of cholesterol into the cells of the artery wall. The liver is considered to be the metabolic power station of mammalians, where cholesterol homeostasis relies on an intricate network of cellular processes whose deregulations can lead to several life-threatening pathologies, such as familial and age-related hypercholesterolemia. Cholesterol homeostasis maintenance is carried out by: biosynthesis, via 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity; uptake, through low density lipoprotein receptors (LDLr); lipoprotein release in the blood; storage by esterification; and degradation and conversion into bile acids. Both HMGR and LDLr are transcribed as a function of cellular sterol amount by a family of transcription factors called sterol regulatory element binding proteins that are responsible for the maintenance of cholesterol homeostasis through an intricate mechanism of regulation. Cholesterol obtained by hepatic de novo synthesis can be esterified and incorporated into apolipoprotein B-100-containing very low density lipoproteins, which are then secreted into the bloodstream for transport to peripheral tissues. Moreover, dietary cholesterol is transferred from the intestine to the liver by high density lipoproteins (HDLs); all HDL particles are internalized in the liver, interacting with the hepatic scavenger receptor (SR-B1). Here we provide an updated overview of liver cholesterol metabolism regulation and deregulation and the causes of cholesterol metabolism-related diseases. Moreover, current pharmacological treatment and novel hypocho-lesterolemic strategies will also be introduced.

Mycorrhizas Affect Polyphyllin Accumulation of Paris polyphylla var.yunnanensis through Promoting PpSE Expression

Paris polyphylla var.yunnanensis is a traditional Chinese medicinal plant,in which polyphyllin as the main medicinal component is an important secondary metabolite with bioactivity.Arbuscular mycorrhizal fungi(AMF)have multiple positive effects on plants,while it is not clear whether AMF increase the content of medicinal components in medicinal plants.In this study,a total of nine AMF treatments were laid to analyze the mycorrhizal effect on polyphyllin accumulation and PpHMGR and PpSE expression of P.polyphylla var.yunnanensis.AMF increased the content of polyphyllin in the cultivated variety with low relation to the increase of inoculation intensity.Polyphyllin I,II,and VII were identified and partly improved by AMF inoculation,dependent on AMF treatments and culture environments.Similarly,the PpHMGR and PpSE expression was induced by mycorrhization,dependent on AMF species,whilst the induction was more obvious in PpSE than in PpHMGR after mycorrhization.It concluded that the symbiotic relationship between P.polyphylla var.yunnanensis and AMF increased polyphyllin content level in the plant,which was associated with the up-regulation of PpSE transcripts.

Cholesterol synthesis inhibition or depletion in axon regeneration

Cholesterol is biosynthesized by all animal cells. Beyond its metabolic role in steroidogenesis, it is enriched in the plasma membrane where it has key structural and regulatory functions. Cholesterol is thus presumably important for post-injury axon regrowth, and this notion is supported by studies showing that impairment of local cholesterol reutilization impeded regeneration. However, several studies have also shown that statins, inhibitors of 3-hydroxy-3-methylglutaryl-Co A reductase, are enhancers of axon regeneration, presumably acting through an attenuation of the mevalonate isoprenoid pathway and consequent reduction in protein prenylation. Several recent reports have now shown that cholesterol depletion, as well as inhibition of cholesterol synthesis per se, enhances axon regeneration. Here, I discussed these findings and propose some possible underlying mechanisms. The latter would include possible disruptions to axon growth inhibitor signaling by lipid raft-localized receptors, as well as other yet unclear neuronal survival signaling process enhanced by cholesterol lowering or depletion.

Statins in risk-reduction and treatment of cancer

Statins,which are competitive inhibitors of 3-hydroxy-3-methyl-glutarylcoenzyme A reductase,reduce cholesterol blood levels and the risk of developing cardiovascular diseases and their related complications.In addition to this main activity,statins show pleiotropic effects such as antioxidant,anti-inflammatory and antiproliferative properties,with applications in many pathologies.Based on their antiproliferative properties,in vitro and in vivo studies have investigated their effects on various types of cancer(i.e.,breast cancer,prostate cancer,colorectal cancer,ovarian cancer,lung cancer)with different genetic and molecular characteristics.Many positive results were obtained,but they were highly dependent on the physiochemical properties of the statins,their dose and treatment period.Combined therapies of statins and cytotoxic drugs have also been tested,and synergistic or additive effects were observed.Moreover,observational studies performed on patients who used statins for different pathologies,revealed that statins reduced the risk of developing various cancers,and improved the outcomes for cancer patients.Currently,there are many ongoing clinical trials aimed at exploring the potential of statins to lower the mortality and the disease-recurrence risk.All these results are the foundation of new treatment directions in cancer therapy.

Expression of <i>HMGR</i>in Lilu cattle tissues

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR ) is an essential enzyme in cholesterol biosynthesis. To study the expression of HMGR in adipose and muscle tissues, and some performance indexes of four age stages, twelve Lilu cattle were selected. The results indicated that the Lilu beef cattle have good production and slaughter performance. HMGR mRNA expression level in adipose was higher than in muscle, but the trend in adipose was the same as in muscle. HMGR mRNA expression is difference in adipose and muscle tissues suggesting this gene is expressed in a tissue-dependent manner in cattle. Understanding the causes of variation in HMGR gene expression may provide crucial information about cholesterol biosynthesis in Lilu beef cattle.

Phenotypical statin-associated immune-mediated necrotizing myositis with histological features of inclusion body myositis

Introduction:Statin-associated immune-mediated necrotizing myositis(IMNM)is a rare but distinct idiopathic inflammatory myopathy(IIM)that requires early recognition and intervention to prevent irreversible muscle damage.It is typically characterized by active statin use,elevated creatinine kinase(CK)levels,proximal muscle weakness,and at times,a positive 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase(HMGCR)antibody.Treatment includes immediate discontinuation of the statin and may include corticosteroids,intravenous immunoglobulin(IVIG),and/or immunosuppressive therapy.Inclusion body myositis(IBM),another distinct IIM,also presents with elevated CK levels but with insidious onset of distal upper and proximal lower extremity weakness and is typically refractory to treatment.Case Description:A 64-year-old female patient presented with proximal muscle weakness,elevated CK levels,and a positive HMGCR antibody in the setting of statin use with muscle pathology suggestive of both statinassociated IMNM and IBM.She responded to subcutaneous methotrexate and a slow prednisone taper over several months,however,will require close monitoring for symptoms associated with either disease.Conclusion:In conclusion,we report a case of muscle weakness with muscle pathology demonstrating both statin-associated IMNM and IBM.This case highlights the importance of understanding the clinical and pathological features of statin-associated IMNM and IBM.