Functional kaurene-synthase-like diterpene synthases lacking a gamma domain are widely present in Oryza and related species.

Various diterpene synthases have been functionally identified in cultivated rice (Oryza sativa). These are the homologs of ent-copalyl diphosphate (ent-CDP) synthase and ent-kaurene synthase (KS) that are responsible for the biosynthesis of gibberellins, diterpenoid phytohormones. We isolated a cDNA encoding full-length OsKSL12, a previously uncharacterized KS like (KSL) enzyme that consists of a ß-domain and an α-domain with an active center, but lacks an N-terminal γ-domain. Functional analysis using a bacterial expression system showed that recombinant OsKSL12 converted ent-CDP into ent-manool or ent-13-epi-manool. Comparative genomics revealed that functional OsKSL12 homologs exist in diverse wild species in the Oryzeae-Oryza nivara (Oryza rufipogon), Oryza coarctata, Oryza granulata, Leersia perrieri, and Leersia tisseranti. KSL12 homologs in O. granulata, L. perrieri, and L. tisseranti preferentially reacted with geranylgeranyl diphosphate rather than ent-CDP, resulting in geranyllinalool rather than ent-manool or ent-13-epi-manool as the main product, meaning that KSL12 functionally diversified during evolution in the Oryzeae.

Identification of Chimeric αßγ Diterpene Synthases Possessing both Type†II Terpene Cyclase and Prenyltransferase Activities.

Two unusual diterpene synthases composed of three domains (α, ß, and γ) were identified from fungal Penicillium species. They are the first enzymes found to possess both type II terpene cyclase (TC) and prenyltransferase (PT) activities. These enzymes were characterized by heterologous expression in Aspergillus oryzae and in vitro experiments with wild-type, mutated, and truncated enzymes. The results revealed that the α domain in the C-terminal region of these enzymes was responsible for the PT activity, whereas the ßγ domains in the N-terminal region composed the type II TC, and formed copalyl diphosphate (2). Additionally, between the α and ßγ domains, there is a characteristic linker region, in which minimal secondary structure is predicted. This linker does not exist in the characterized three-domain (αßγ) terpene synthases known as monofunctional type I or type II TCs, or bifunctional type I and type II TC enzymes. Therefore, both the catalytic activities and protein architecture substantially differentiate these new enzymes from the previously characterized terpene synthases.

Identification of reference genes for tissue-specific gene expression in Panax notoginseng using quantitative real-time PCR.

Validated internal controls are prerequisites to accurately normalize gene expression levels. Here, 14 candidate reference genes in Panax notoginseng were characterized. Primer specificity and amplification efficiency were evaluated for each gene. Candidates were subjected to transcript quantification in the root, fibrous root, rhizome, leaf, receptacle, pedicel, and fruit tissues. Expression stability (M value) and normalization factor variation (Vn/Vn+1) were determined by geNorm. 26S-2 and ACT-2 exhibited the highest expression stability among the tissues. Gene expression of dammarenediol synthase was accurately detected after normalization to 26S-2 and ACT-2 was performed. Results were consistent when each or both of 26S-2 and ACT-2 were applied as internal control. Hence, this study provides useful information to normalize gene expression accurately in the tissue-specific transcripts of P. notoginseng.

Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error.

BACKGROUND: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. METHODS: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. RESULTS: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. CONCLUSIONS: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

Identification and characterization of (+)-α-bisabolol and 7-epi-silphiperfol-5-ene synthases from Artemisia abrotanum.

Triquinane is a type of sesquiterpenoid with a unique structure that contains a fused tricyclopentane ring and exhibits a wide range of bioactivities. Like other sesquiterpenoids, the first committed step in triquinane-type sesquiterpenoid biosynthesis is the cyclization of farnesyl pyrophosphate (FPP), a common precursor of all sesquiterpenoids, catalyzed by sesquiterpene synthase. Artemisia abrotanum L. (Asteraceae), a common plant used in the culinary and cosmetics industries, has been reported to accumulate high levels of triquinane silphiperfol-5-en-3-one A. This compound is potentially biosynthesized from the cyclization of FPP into 7-epi-silphiperfol-5-ene followed by a multi-step oxidation to silphiperfol-5-en-3-one A. In this study, we aimed to identify the sesquiterpene synthase responsible for the synthesis of 7-epi-silphiperfol-5-ene. We performed RNA sequencing of A. abrotanum leaves and gene candidates were mined by homology searches using the triquinane α-isocomene synthase of chamomile (MrTPS2) as query. After gene cloning, we obtained five variants of putative sesquiterpene synthase showing greater than 85% amino acid identity to MrTPS2 and greater than 95% amino acid identity to each other. Heterologous expression of these variants in a FPP-high-producing yeast strain revealed the first four variants to be (+)-α-bisabolol synthases (AabrBOS1-4). However, the fifth candidate cyclized FPP into 7-epi-silphiperfol-5-ene and can therefore be defined as a 7-epi-silphiperfol-5-ene synthase (AabrSPS). These findings revealed the first committed enzyme involved in silphiperfol-5-en-3-one A and (+)-α-bisabolol biosyntheses in A. abrotanum. Furthermore, the results of this study will be useful for enhancing the production of these compounds for further applications.

Specifically and wash-free labeling of SNAP-tag fused proteins with a hybrid sensor to monitor local micro-viscosity.

Viscosity, as one of the major factors of intracellular microenvironment, influences the function of proteins. To detect local micro-viscosity of a protein, it is a precondition to apply a viscosity sensor for specifically target to proteins. However, all the reported small-molecule probes are just suitable for sensing/imaging of macro-viscosity in biological fluids of entire cells or organelles. To this end, we developed a hybrid sensor BDP-V BG by connecting a viscosity-sensitive boron-dipyrromethene (BODIPY) molecular rotor (BDP-V) to O6-benzylguanine (BG) for specific detection of local micro-viscosity of SNAP-tag fused proteins. We measured and calculated the reaction efficiency between the sensor and SNAP-tag protein in vitro to confirm the high labeling specificity. We also found that the labeling reaction results in a 53-fold fluorescence enhancement for the rotor, which qualifies it as a wash-free sensor with ignorable background fluorescence. The high sensitivity of protein labeled sensor (BDP-V-SNAP) to the changes of local viscosity was evaluated by detecting the enhancement of fluorescence lifetimes. Further, with the sensor BDP-V BG, we achieved high specific labeling of cells expressing two SNAP-tag fused proteins (nuclear histone H2B and mitochondrial COX8A). Two-photon excited fluorescence lifetime imaging revealed that, the micro-viscosities nearby the SNAP-tag fused two proteins are distinct. The different changes of local micro-viscosity of SNAP-tag fused histone protein in apoptosis induced by three nucleus-targeted drugs were also characterized for the first time.

Identification of human dehydrodolichyl diphosphate synthase gene.

We isolated a cDNA encoding human dehydrodolichyl diphosphate (Dedol-PP) synthase and expressed the gene in a yeast mutant strain SNH23-7D, which is deficient in Dedol-PP synthase activity. The identity of the cloned enzyme was confirmed by functional complementation of SNH23-7D strain together with in vitro Dedol-PP synthase activity assay. Northern blot analysis indicated that testis and kidney expressed Dedol-PP synthase mRNA at high levels.

GOT1/AST1 expression status as a prognostic biomarker in pancreatic ductal adenocarcinoma.

Prognostication in pancreatic ductal adenocarcinoma (PDAC) remains a challenge. Recently, a link between mutated KRAS and glutamic-oxaloacetic transaminase (GOT1/AST1) has been described as part of the metabolic reprogramming in PDAC. The clinical relevance of this novel metabolic KRAS-GOT1 link has not been determined in primary human patient samples. Here we studied the GOT1 expression status as a prognostic biomarker in PDAC. We employed three independent PDAC cohorts with clinicopathological- and follow-up data: a) ICGC, comprising 57 patients with whole-exome sequencing and genome-wide expression profiling; b) ULM, composed of 122 surgically-treated patients with tissue-samples and KRAS status; c) a validation cohort of 140 primary diagnostic biopsy samples. GOT1 expression was assessed by RNA level (ICGC) or immunolabeling (ULM/validation cohort). GOT1 expression varied (ICGC) and correlation with the KRAS mutation- and expression status was imperfect (P = 0.2, ICGC; P = 0.8, ULM). Clinicopathological characteristics did not differ when patients were separated based on GOT1 high vs. low (P = 0.08-1.0); however, overall survival was longer in patients with GOT1-expressing tumors (P = 0.093, ICGC; P = 0.049, ULM). Multivariate analysis confirmed GOT1 as an independent prognostic marker (P = 0.009). Assessment in univariate (P = 0.002) and multivariate models in the validation cohort (P = 0.019), containing 66% stage IV patients, confirmed the independency of GOT1. We propose the GOT1 expression status as a simple and reliable prognostic biomarker in pancreatic ductal adenocarcinoma.

Immunological analyses of alkyl-dihydroxyacetone-phosphate synthase in human peroxisomal disorders.

Alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) is a peroxisomal enzyme involved in the biosynthesis of ether phospholipids. To localize the enzyme in human peroxisomal disorders, indirect immunofluorescence and immunoblot analysis was performed. In Zellweger syndrome and rhizomelic chondrodysplasia punctata fibroblast cell lines, alkyl-DHAP synthase protein levels on immunoblots were strongly decreased and residual immunofluorescence was diffusely localized throughout the cytoplasm. In a particular neonatal adrenoleukodystrophy cell line, characterized by the absence of a functional peroxisomal targeting signal 1 receptor, the precursor form of the enzyme was detected in Western blots at levels comparable to that of the mature enzyme in control fibroblasts. Similarly, fibroblasts from patients with a single deficiency in the activity of either alkyl-DHAP synthase or DHAP-acyltransferase showed normal levels of the mature alkyl-DHAP synthase protein on immunoblots. Immunofluorescence experiments revealed a peroxisomal localization of both the precursor and the mature form of the enzyme. Collectively, these results visualize the peroxisomal localization of alkyl-DHAP synthase, indicate that the enzyme is unstable outside its target organelle and explain that normal enzyme protein levels found in some peroxisomal disorders result from protection against cytoplasmic degradation through import into peroxisomes. Additionally, alkyl-DHAP synthase could be detected in rat mesangial cells and murine NIH-3R3 fibroblasts by immunofluorescence as well as immunoblot analysis. Immunoelectron microscopy showed that the enzyme is predominantly located on the lumenal side of the peroxisomal membrane in rat and guinea pig liver.

Biotechnology and the soybean.

Glyphosate-tolerant soybeans (GTSs), the first biotechnologically improved soybeans to be marketed, became available commercially in 1996. The safety of GTSs was assessed in 2 ways: study of the introduced protein and of the soybean seed and selected processing fractions. Because soybeans are a major source of protein in most farm animals' diets, animal feeding studies in wholesomeness were done to complement the analyses. Analysis of the expressed protein in GTSs [3-phosphoshikimate 1-carboxyvinyltransferase (EC 2.5.1.19)] showed it to be readily digestible and to possess no allergenic concerns. In addition, comparison of the composition of seeds and selected processing fractions from 2 GTS lines with the parental line showed that the lines are equivalent. Feeding studies in various animal species confirmed that the feeding value of GTS lines is comparable with that of the parental line. These studies support the conclusion that GTSs are as safe and nutritious as traditional soybeans marketed currently and can be incorporated safely into feed and food products in the near future.

Novel DNA repair alkyltransferase from Caenorhabditis elegans.

O6-alkylguanine DNA-alkyltransferase (AGT) is a widely distributed DNA repair protein that protects living organisms from endogenous and exogenous alkylation damage to DNA at the O6-position of guanine. The search of the C. elegans genome database for an AGT protein revealed the presence of a protein (cAGT-2) with some similarity to known AGTs in addition to the easily recognized cAGT-1 protein. The predicted protein sequence of cAGT-2 contains the amino acid sequence -ProCysHisPro- at the presumed active site of the protein, whereas all other known AGTs have -ProCysHisArg-. A truncated version of the cAGT-2 protein was expressed in E. coli. This purified recombinant protein was able to repair O6-methylguanine and O4-methylthymine adducts in DNA in vitro and also reacted with the bulky benzyl adduct in O6-benzylguanine. This fragment of cAGT-2 (104 amino acids) is the smallest protein possessing AGT activity yet described. The full-length cAGT-2 protein (274 amino acids) totally lacks the N-terminal domain present in all other known AGTs but has a long C-terminal extension that has significant homology to histone 1C. Expression of cAGT-2 in an E. coli strain lacking endogenous AGT activity provided modest but statistically significant resistance to the toxicity of N-methyl-N'-nitro-N-nitrosoguanidine, confirming that cAGT-2 is an alkyltransferase.

Effect of pulp and paper mill effluent (BKME) on physiological parameters of roach (Rutilus rutilus) infected by the digenean Rhipidocotyle fennica.

Physiological parameters were measured after experimental infection of roach (Rutilus rutilus L.) with Rhipidocotyle fennica Gibson, Valtonen et Taskinen, 1992 (Digenea) cercariae. The fish were caught from two lakes: a eutrophic bleached kraft mill effluent (BKME)-contaminated lake and an oligotrophic unpolluted lake. The intensity of infection was followed up to 10 days post infection (p.i.) and physiological parameters indicating non-specific stress responses and the condition of fish were examined simultaneously. The mean abundance, the number of parasites per fish, of R. fennica was significantly higher in the fish from the contaminated water during the first two days p.i., probably reflecting the decreased resistance of these fish to infection. The decrease of leukocrit, as well as the increase of the activity of transaminases (GOT and GPT) in infected fish of both groups are suggestive of pathological processes caused by cercariae penetrating the fish. A significantly lower leukocrit value, as well as higher alkaline phosphatase activity and plasma chloride levels were noted in fish originating from the contaminated lake compared to those from the unpolluted lake. No significant differences were noted in haematocrit, plasma protein and calcium values between the fish from the uncontaminated and contaminated lakes, or between the infected and uninfected control fish.

Evaluation of analgesic, antipyretic activity and toxicity study of Bryonia laciniosa in mice and rats.

Analgesic, antipyretic activity and toxicity study of the leaves of Bryonia laciniosa Linn. (Family: Cucurbitaceae) was evaluated in the standard animal models. The methanol extract of Bryonia laciniosa (MEBL) was evaluated by hot plate and acetic acid-induced writhing methods to assess analgesic activity. The antipyretic activity of the extract was also evaluated by normal body temperature and yeast-induced hyperpyrexia. The extract showed significant analgesic and antipyretic activity. The MEBL was further evaluated for toxicity at the doses of 125 and 250 mg/kg administered orally for 14 days in rats. At the end of experiments, the blood, liver function and kidney metabolism were observed. The hematological profile and different biochemical parameters such as SGOT, SGPT and ALP were estimated. The present study revealed that MEBL exhibited significant analgesic and antipyretic activity in the tested experimental animal models. The toxicity study indicates that the extract is not toxic at the tested doses.

Determination of enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase by LC/MS.

A liquid chromatography-mass spectrometry (LC/MS) method for determining the enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), an enzyme of the shikimate pathway, was developed. EPSP synthase catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S-3-P) and phosphoenolpyruvate (PEP) in microorganisms and plants. The enzymatic activity of EPSP synthase was assessed by the determination of EPSP after a 30-min incubation with S-3-P and PEP using the LC/MS system. EPSP synthase activity is given in terms of the produced EPSP (pmol/min/mg protein). Glyphosate (N-phosphonomethyl glycine)-tolerant EPSP synthase from the Agrobacterium sp. strain CP4 (CP4-EPSP synthase) in genetically modified soybeans (GM-soybeans) was found to have an enzymatic activity of 736 EPSP pmol/min/mg protein in the presence of 3 nmol of S-3-P. In contrast, the enzyme activity of non-GM-soybeans was 21 EPSP pmol/min/mg protein. The EPSP synthase activity was markedly decreased in the non-GM-soybeans by the addition of glyphosate, but the enzyme activity of the GM-soybeans was only slightly decreased with this treatment. This LC/MS system could also be applicable to the measurement of EPSP synthase activity in different plant species and the detection of herbicide-tolerant EPSP synthase in GM foods.

Trypanosoma brucei solanesyl-diphosphate synthase localizes to the mitochondrion.

Polyprenyl-diphosphate synthase is a key enzyme in the biosynthesis of ubiquinone, a molecule considered essential for a typical eukaryotic cell. Its orthologue in the American stercorarian flagellate Trypanosoma cruzi, solanesyl diphosphate synthase, has been previously localized into the glycosomes. We wondered whether this unique cellular localization is shared by other trypanosome species. Using digitonin permeabilization, immunofluorescence and in situ tagging techniques, we show that in Trypanosoma brucei, the African salivarian flagellate, the enzyme localizes to the mitochondrion.

Rab-geranylgeranyl transferase regulates glucose-stimulated insulin secretion from pancreatic ß cells.

A growing body of evidence implicates essential roles for small molecular weight G-proteins (e.g., Cdc42, Rac1, Arf6 and Rab3A and Rab27A) in islet ß-cell function including glucose-stimulated insulin secretion (GSIS). One of the known mechanisms for optimal activation of small G-proteins involves post-translational prenylation, which is mediated by farnesyltransferase (FTase) and geranylgeranyl transferases (GGTases I and II). The FTase catalyzes incorporation of a 15-carbon farnesyl group while the GGTase mediates incorporation of a 20-carbon geranylgeranyl group into the C-terminal cysteines of G-proteins. The FTase, GGTase I and GGTase II prenylate Ras, Cdc42/Rac1, and Rab G-proteins, respectively. While considerable evidence exists on FTase/GGTase I-mediated regulation of GSIS, very little is known about GGTase II (also referred to as Rab GGTase; RGGT) and its regulatory proteins in the cascade of events leading to GSIS. Herein, we provide the first immunological evidence to suggest expression of α- and ß-subunits of RGGT in clonal INS 832/13 ß-cells, normal rat islets and human islets. Furthermore, Rab escort protein1 (REP1), which has been shown to be critical for prenylation of Rab G-proteins, is also expressed in these cells. Furthermore, evidence is presented to suggest that siRNA-mediated knockdown of α- or ß-subunits of RGGT and REP1 markedly attenuates GSIS in INS 832/13 cells. These findings provide the first evidence in support of key roles for RGGT and its regulatory proteins in GSIS.

Immunofluorescence localization of levopimaradiene/abietadiene synthase in methyl jasmonate treated stems of Sitka spruce (Picea sitchensis) shows activation of diterpenoid biosynthesis in cortical and developing traumatic resin ducts.

Conifers produce terpenoid-rich oleoresin in specialized resin ducts as a main line of defence against pests and pathogens. In spruce species (Picea spp.), axial resin ducts are either present constitutively in the cortex tissue (cortical resin ducts, CRDs) or are formed de novo as traumatic resin ducts (TRDs) in the cambial zone upon attack by insects, fungi or treatment with methyl jasmonate (MeJA). Using immunofluorescence localization we tested if previously formed CRDs respond to MeJA treatment with increased capacity for diterpenoid biosynthesis. We also tested the dynamics of diterpene synthase localization in the cambial zone. Immunofluorescence localization was performed using an antibody against a diterpene synthase, levopimaradiene/abietadiene synthase (LAS), in stem cross-sections of untreated and 0.1% MeJA-treated 4-year old Sitka spruce (P. sitchensis) trees. No fluorescence signal was observed in untreated stem cross-sections; however, signal was present 2 days after treatment with MeJA exclusively in the epithelial cells of CRDs. Fluorescence steadily increased in the CRD epithelial cells 4 and 8 days after treatment. At 8days, additional fluorescence was observed in developing epithelial cells of traumatic resin ducts TRDs in the cambial zone. These results confirm that resin duct epithelial cells are the main site of diterpene biosynthesis in Sitka spruce, diterpenoid biosynthesis is induced in CRD epithelial cells early upon treatment with MeJA, and immature developing TRD epithelial cells produce diterpene synthase enzyme. Overall, the results of this work improve our understanding of spatial and temporal patterns of induced diterpene resin acid biosynthesis in conifers.

A splicing mutation in the gene encoding phytoene synthase causes orange coloration in Habanero pepper fruits.

Peppers (Capsicum spp.) display a variety of fruit colors that are reflected by the composition and amount of diverse carotenoid pigments accumulated in the pericarp. Three independent loci, c1, c2, and y, are known to determine the mature color of pepper fruits by their allelic combinations. We examined the inheritance of fruit color in recombinant inbred lines (RILs) derived from an interspecific cross between C. annuum cv. TF68 (red) and C. chinense cv. Habanero (orange). The c2 gene encodes phytoene synthase (PSY), a rate-limiting enzyme in the carotenoid biosynthesis pathway. TF68 has a dominant c2+ allele whereas Habanero is homozygous for the recessive c2 allele, which determined RIL fruit color. Here we report that the recessive c2 allele has a point mutation in the PSY gene that occurs at a splice acceptor site of the fifth intron leading to both a frame shift and premature translational termination, suggesting that impaired activity of PSY is responsible for orange fruit color. During ripening, PSY is expressed at a significantly high level in orange colored fruits compared to red ones. Interestingly, the PSY gene of red Habanero has a conserved splice acceptor dinucleotide AG. Further analysis suggests that red Habanero is a wild type revertant of the PSY mutant orange Habanero.

An assay for thiaminase I in complex biological samples.

An alternative method for measuring thiaminase I activity in complex samples is described. This assay is based on the selective consumption of the highly chromophoric 4-nitrothiophenolate by thiaminase I, resulting in a large decrease in absorbance at 411nm. This new assay is simple and sensitive, and it requires only readily available chemicals and a visible region spectrophotometer. In addition, the assay is optimized for high-throughput analysis in a 96-well format with complex biological samples.