Identification of putative genes involved in the development of <Emphasis Type="Italic">Tuber borchii</Emphasis> fruit body by mRNA differential display in agarose gel

In order to analyse gene expression during fruit body development of the ectomychorrizal fungus Tuber borchii Vittad., a modified differential display procedure was set up. The procedure used is easier and faster than the traditional one and generates reproducible cDNA banding patterns that can be resolved on a standard ethidium bromide-agarose gel. From 16 cDNA fingerprints, 25 amplicons with apparent differential expression were identified and cloned without a previous reamplification. Fifteen clones showed significant similarity to known proteins that are involved in dikaryosis and fruiting, cell division, transport across membranes, mitochondrial division, intermediary metabolism, biosynthesis of isoprenoid compounds and putative RNA/DNA binding. Northern blot analyses confirmed that seven cDNAs were indeed differentially expressed during fruit body development. The characterisation of these cDNAs represents a starting point in understanding the molecular mechanisms of cellular differentiation leading to the development of the T. borchii fruit body.     

A novel fungal prenyl diphosphate synthase in the dimorphic zygomycete Mucor circinelloides

Two Mucor circinelloides structural genes involved in isoprenoid biosynthesis were isolated and characterised. The isoA gene encodes a typical eukaryotic farnesyl diphosphate synthase (EC 2.5.1.10), whereas the isoB gene deduced amino acid sequence shows similarity to fungal medium-chain prenyl diphosphate synthases. By functional complementation in Escherichia coli, the isoB gene product was shown to be a solanesyl diphosphate synthase (EC 2.5.1.11), which is the first fungal enzyme reported having this specificity. In addition, a M. circinelloides one-marker-per-chromosome map was completed by contour-clamped homogeneous electric field localisation of isoA, isoB and three other isoprenoid biosynthesis genes to individual chromosomes.Abbreviations FPP farnesyl diphosphate (or pyrophosphate) - GGPP geranylgeranyl diphosphate - PrenylPP prenyl diphosphate - DPP decaprenyl diphosphate - HPP hexaprenyl diphosphate - SPP solanesyl diphosphate     

肺炎链球菌HMGS和HMGR有利于寻找抑制先导化合物

 目的 对肺炎链球菌3-羟基-3-甲基戊二酰辅酶A合成酶和还原酶（HMGS和HMGR）进行动力学和功能研究;方法 克隆表达肺炎链球菌HMGS和HMGR,检测洛伐它汀对它们的抑制作用,分别以HMGS酶促反应的产物和3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）作为底物检测几种苗头化合物对HMGR抑制性;结果 洛伐它汀对HMGR有抑制作用,对HMGS无抑制作用,HMGS酶促反应的产物和HMG-CoA在HMGR检测苗头化合物抑制性的筛药反应中效果相似;结论 HMGS酶促反应的产物可以代替HMG-CoA进行苗头化合物初筛,可降低筛选成本利于寻找新型高效抑制先导化合物。     

Sequence comparisons reveal two classes of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Both in eukaryotes and in archaebacteria the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (E.C. 1.1. 1.34) is known to catalyze an early reaction unique to isoprenoid biosynthesis. In humans, the HMG-CoA reductase reaction is rate-limiting for the biosynthesis of cholesterol and therefore constitutes a prime target of drugs that reduce serum cholesterol levels. Recent advances in genome sequencing that permitted comparison of 50 HMG-CoA reductase sequences has revealed two previously unsuspected classes of this enzyme. Based on sequence and phylogenetic considerations, we propose the catalytic domain of the human enzyme and the enzyme from Pseudomonas mevalonii as the canonical sequences for Class I and Class II HMG-CoA reductases, respectively. These sequence comparisons have revealed, in addition, that certain true bacteria, including several human pathogens, probably synthesize isoprenoids by reactions analogous to those of eukaryotes and that there therefore exist two distinct pathways for isoprenoid biogenesis in true bacteria.     

Squalene synthase: a critical enzyme in the cholesterol biosynthesis pathway

High levels of plasma low-density lipoprotein cholesterol (LDL-C) are a significant risk factor for heart disease. Statins (3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors) have been extensively used to treat high-plasma LDL-C levels and are effective in preventing heart disease. However, statins can be associated with adverse side effects in some patients and do not work effectively in others. As an alternative to statins, the development of cholesterol-lowering agents that directly inhibit squalene synthase have shown promise. Clinical studies have shown that squalene synthase inhibitors are effective in lowering plasma levels of total cholesterol and LDL-C. Squalene synthase plays an important role in the cholesterol biosynthesis pathway as it is responsible for the flow of metabolites into either the sterol or the non-sterol branches of the pathway. In addition, variants of the squalene synthase gene appear to modulate plasma cholesterol levels in human populations and therefore may be linked to cardiovascular disease. In this review, we examine squalene synthase and the gene that codes for it (farnesyldiphosphate farnesyltransferase 1). In particular, we investigate their role in the regulation of cellular and plasma cholesterol levels, including data that suggest that squalene synthase may be involved in the etiology of hypercholesterolemia.     

Pitavastatin decreases tau levels via the inactivation of Rho/ROCK

Epidemiological studies have shown that long-term treatment with statins decreases the risk of developing Alzheimer's disease. Statins have pleiotropic effects by lowering the concentration of isoprenoid intermediates. Although several studies have shown that statins may reduce amyloid beta protein levels, there have been few reports on the interaction between statins and tau. We report here that pitavastatin reduces total and phosphorylated tau levels in a cellular model of tauopathy, and in primary neuronal cultures. The decrease caused by pitavastatin is reversed by the addition of mevalonate, or geranylgeranyl pyrophosphate. The maturation of small G proteins, including RhoA was disrupted by pitavastatin, as was the activity of glycogen synthase kinase 3β (GSK3β), a major tau kinase. Toxin A, inhibitor of glycosylation of small G proteins, and Rho kinase (ROCK) inhibitor decreased phosphorylated tau levels. Rho kinase inhibitor also inactivated glycogen synthase kinase 3β. Although the mechanisms responsible for the reduction in tau protein by pitavastatin require further examination, this report sheds light on possible therapeutic approaches to tauopathy.     

Vγ9Vδ2 TCR‐activation by phosphorylated antigens requires butyrophilin 3 A1 (BTN3A1) and additional genes on human chromosome 6

Pyrophosphorylated metabolites of isoprenoid‐biosynthesis (phosphoantigens, PAgs) activate Vγ9Vδ2 T cells during infections and trigger antitumor activity. This activation depends on expression of butyrophilin 3 A1 (BTN3A1) by antigen‐presenting cells. This report defines the minimal genetic requirements for activation of Vγ9Vδ2 T cells by PAgs and mAb 20.1. We compared PAg‐presentation by BTN3A1‐transduced CHO hamster cells with that of CHO cells containing the complete human chromosome 6 (Chr6). BTN3A1 expression alone was sufficient for activation of Vγ9Vδ2 T‐cell receptor transductants by mAb 20.1., while activation by PAgs also required the presence of Chr6. We take this finding as evidence that gene(s) on Chr6 in addition to BTN3A1 are mandatory for PAg‐mediated activation of Vγ9Vδ2 T cells. This observation is important for the design of animal models for PAg‐mediated immune responses and provokes speculations about the analogy between genes controlling PAg presentation and MHC‐localized genes controlling peptide‐antigen presentation.     

Upregulation of sterol C14-demethylase expression in Trypanosoma cruzi treated with sterol biosynthesis inhibitors

Infection with the protozoan, Trypanosoma cruzi, is the cause of Chagas disease that occurs widely throughout Latin America. T. cruzi contains sterol biosynthesis enzymes, and produces sterol products similar to those found in fungi. Antifungal drugs that inhibit ergosterol biosynthesis have potent anti-T. cruzi activity in vitro and in animal models. In this report, we describe the effects of sterol biosynthesis inhibitors (simvistatin, zaragosic acid, terbinafine, a lanosterol synthase inhibitor, ketoconazole, and tridemorph) on the regulation of two sterol biosynthesis genes and their protein products. Culturing T. cruzi in the presence of the lanosterol synthase inhibitor, terbinafine, or ketoconazole increased mRNA levels of the sterol C14-demethylase gene approximately 7-12-fold. The sterol C14-demethylase protein levels were also elevated. The effects of the sterol biosynthesis inhibitors on hydroxymethylglutaryl-CoA reductase expression were minimal. Control of the upregulation of sterol C14-demethylase appears to be mediated through the 3'-untranslated region of the gene. The findings demonstrate that T. cruzi can specifically regulate gene expression in response to derangements in its cellular functions.     

Polygenic association with total homocysteine in the post-folic acid fortification era: The CARDIA study

Elevated plasma concentration of total homocysteine (tHcy) has been linked with many diseases. tHcy is associated with a variety of factors, including polymorphisms in genes involved in homocysteine metabolism. It is not clear whether US-mandated fortification of grain products with folic acid has affected the association of genetic variants with tHcy levels. We determined tHcy concentrations in sera from 997 Caucasians and 692 African Americans participants in the Coronary Artery Risk Development in Young Adults (CARDIA) study before and after folic acid fortification. DNA was genotyped for variants present in four genes involved in homocysteine metabolism: cystathionine β-synthase (CBS) 844ins68, methionine synthase (MS) 2756A>G; methionine synthase reductase (MTRR) 66A>G and methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C. A greater number of African Americans were homozygous for the MS 2756GG, MTRR 66GG and CBS 844ins68 genotypes compared to Caucasians, while prevalence of MTHFR 677TT and 1298CC genotypes was substantially lower in African Americans compared to Caucasians. The overall variance in tHcy levels at y 0, 7 and 15 that can be explained by the combined presence of all five variants increased slightly over time in Caucasians (17%, y 0; 21%, y 7; and 26%, y 15) and in African Americans (13%, y 0; 17% y 7; and 18% y 15) largely due to decrease in tHcy variance.     

Innate immune functions of human gammadelta T cells

gammadelta T cells expressing the Vgamma9Vdelta2 T cell receptor (TCR) account for 1-10% of CD3(+) peripheral blood T lymphocytes. Vgamma9Vdelta2 T cells use their TCR as a pattern recognition receptor to sense the presence of infection through specific recognition of intermediates of the microbial non-mevalonate pathway of isoprenoid biosynthesis. Such phosphoantigens rapidly and selectively activate human gammadelta T cells to produce proinflammatory cytokines, notably interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). In addition, human gammadelta T cells express certain Toll-like receptors (TLR) and directly respond to the corresponding ligands. We have demonstrated expression of TLR3 in Vgamma9Vdelta2 T cells and striking costimulatory effects of the ligand polyinosinic-polycytidylic acid (polyI:C) on TCR-stimulated IFN-gamma production. Gene expression studies by microarray analysis identified additional genes that were up-regulated by combined TCR- and TLR3 stimulation. We discuss these findings in the context of the suspected role of human Vgamma9Vdelta2 T cells as a link between innate and adaptive immune responses.     

Stimulation of Vgamma9/Vdelta2 T-lymphocyte proliferation by the isoprenoid precursor, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate

(E)-1-Hydroxy-2-methyl-but-2-enyl 4-diphosphate, a recently discovered intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis, has been shown to act as a potent immunomodulator. In cultures of human peripheral blood mononuclear cells from eight non-related donors, the compound stimulated the proliferation of Vgamma9/Vdelta2 T lymphocytes with a median EC(50) of 70 pM when 10 U/ml of IL-2 was used as costimulant. Isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP) and some structural analogs of (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate also stimulated Vgamma9/Vdelta2 T-cell proliferation, albeit at much higher concentrations. The Vgamma9/Vdelta2 T-cell proliferation is highly dependent on the seeding density used in culture. All phosphoantigens tested elicited the proliferation of two T-lymphocyte populations with different apparent ratios between the expression level of Vdelta2 and Vgamma9 chains.     

Age-related changes of cholesterol and dolichol biosynthesis in rat liver

Ageing has been defined as a gradually decreased ability to maintain homeostatic potential and increased risk to die, associated with a tissue accumulation of altered proteins and lipids. Among other, increased concentration of an isoprenoid compound, dolichol (Dol), in mammalian tissues during ageing has been reported and it has been considered as a new biomarker of ageing. However, the mechanism and the role of this accumulation is still unknown. Aim of this work was to study the mechanism of age-dependent Dol accumulation in the liver analysing the activity of the hepatic rate-limiting enzyme of isoprenoid biosynthesis, the 3-hydroxy 3-methylglutaryl CoA reductase (HMGCoA reductase), the Dol synthesis by mevalonate (MVA), the Dol level in the plasma, and the cholesterol (Chol) synthesis and content of ageing rat fed ad libitum (AL) or subjected to the effect of food restriction. Since the caloric restrictions are the most reproducible way to slow ageing and to extend life span, animals on these nutritional regimens were used to study ageing related mechanisms. The data show that during ageing the hepatic Dol accumulation is associated with an increase of HMGCoA reductase activity, which is affected by diet restriction, and with an increase of MVA incorporation in Dol and Chol, which is not. In addition, the liver of aged rats maintains the capability to regulate its Chol content and to modify Chol delivery into the blood.     

A novel polyketide biosynthesis gene cluster is involved in fruiting body morphogenesis in the filamentous fungi <Emphasis Type="Italic">Sordaria macrospora</Emphasis> and <Emphasis Type="Italic">Neurospora crassa</Emphasis>

During fungal fruiting body development, hyphae aggregate to form multicellular structures that protect and disperse the sexual spores. Analysis of microarray data revealed a gene cluster strongly upregulated during fruiting body development in the ascomycete Sordaria macrospora. Real time PCR analysis showed that the genes from the orthologous cluster in Neurospora crassa are also upregulated during development. The cluster encodes putative polyketide biosynthesis enzymes, including a reducing polyketide synthase. Analysis of knockout strains of a predicted dehydrogenase gene from the cluster showed that mutants in N. crassa and S. macrospora are delayed in fruiting body formation. In addition to the upregulated cluster, the N. crassa genome comprises another cluster containing a polyketide synthase gene, and five additional reducing polyketide synthase (rpks) genes that are not part of clusters. To study the role of these genes in sexual development, expression of the predicted rpks genes in S. macrospora (five genes) and N. crassa (six genes) was analyzed; all but one are upregulated during sexual development. Analysis of knockout strains for the N. crassa rpks genes showed that one of them is essential for fruiting body formation. These data indicate that polyketides produced by RPKSs are involved in sexual development in filamentous ascomycetes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.