Determination of GDC-0449, a small-molecule inhibitor of the Hedgehog signaling pathway, in human plasma by solid phase extraction-liquid chromatographic-tandem mass spectrometry.

To support clinical development, a solid phase extraction (SPE) liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0449 concentrations in human plasma has been developed and validated. Samples (200 microl) were extracted using an Oasis MCX 10 mg 96-well SPE plate and the resulting extracts were analyzed using reverse-phase chromatography coupled with a turbo-ionspray interface. The method was validated over calibration curve range 5-5000 ng/mL. Quadratic regression and 1/x(2) weighing were used. Within-run relative standard deviation (%RSD) was within 10.1% and accuracy ranged from 88.6% to 109.0% of nominal. Between-run %RSD was within 8.6% and accuracy ranged from 92.4% to 105.3% of nominal. Extraction recovery of GDC-0449 was between 88.3% and 91.2% as assessed using quality control sample concentrations. GDC-0449 was stable in plasma for 315 days when stored at -70 degrees C and stable in reconstituted sample extracts for 117 h when stored at room temperature. Quantitative matrix effect/ion suppression experiment was performed and no significant matrix ion suppression was observed. This assay allows for the determination of GDC-0449 plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.

Guidelines for 18F-FDG PET and PET-CT imaging in paediatric oncology.

OBJECTIVE: The purpose of these guidelines is to offer to the nuclear medicine team a framework that could prove helpful in daily practice. These guidelines contain information related to the indications, acquisition, processing and interpretation of (18)F-fluorodeoxyglucose positron emission tomography ((18)F-FDG PET) in paediatric oncology. The Oncology Committee of the European Association of Nuclear Medicine (EANM) has published excellent procedure guidelines on tumour imaging with (18)F-FDG PET (Bombardieri et al., Eur J Nucl Med Mol Imaging 30:BP115-24, 2003). These guidelines, published by the EANM Paediatric Committee, do not intend to compete with the existing guidelines, but rather aim at providing additional information on issues particularly relevant to PET imaging of children with cancer. CONCLUSION: The guidelines summarize the views of the Paediatric Committee of the European Association of Nuclear Medicine. They should be taken in the context of "good practice" of nuclear medicine and of any national rules, which may apply to nuclear medicine examinations. The recommendations of these guidelines cannot be applied to all patients in all practice settings. The guidelines should not be deemed inclusive of all proper procedures or exclusive of other procedures reasonably directed to obtaining the same results.

FDG-PET in 10 children with non-Hodgkin's lymphoma: initial experience in staging and follow-up.

BACKGROUND: The use of FDG-PET was evaluated for initial staging and therapy efficacy in paediatric patients with non-Hodgkin's lymphoma (NHL) and compared to the established conventional imaging modalities (CIM). The results of this retrospective analysis are presented in conjunction with a critical review of the current literature. PATIENTS AND METHODS: Ten paediatric patients with NHL were examined using whole-body FDG-PET initially (n = 6), during therapy (n = 5) and after completion of therapy (n = 5), respectively. FDG-PET findings were compared to CIM performed according to the protocol of the German NHL-BFM 95 study. The results were evaluated for their impact on disease classification and therapy decision (St. Jude, REAL) in correspondence to a clinical follow-up of at least 24 months. RESULTS: Concerning initial staging, all lymphoma manifestations detected by conventional imaging were also detected by FDG-PET (15 nodal, 2 extranodal). Furthermore, an additional nodal lesion was detected by FDG-PET in three patients. This resulted in an upstaging followed by an intensified poly-chemotherapy in one patient. In five patients showing unclear residual masses on conventional imaging during therapy, FDG-PET indicated viable residual tumours in one case. This patient showed a relapse during follow-up while the four FDG-PET negative patients did not. After completion of initial therapy, FDG-PET revealed in one out of five patients persistent tumour metabolism in the primary lesions and also detected new manifestations. The patient died shortly after restaging due to disease progression. CONCLUSIONS: These first results on the use of FDG-PET in paediatric non-Hodgkin lymphoma indicate a high potential to improve the therapeutic management.

DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity.

Acyl CoA:diacylgycerol acyltransferase (EC; DGAT) catalyzes the final step in the production of triacylglycerol. Two polypeptides, which co-purified with DGAT activity, were isolated from the lipid bodies of the oleaginous fungus Mortierella ramanniana with a procedure consisting of dye affinity, hydroxyapatite affinity, and heparin chromatography. The two enzymes had molecular masses of 36 and 36.5 kDa, as estimated by gel electrophoresis, and showed a broad activity maximum between pH 6 and 8. Based on partial peptide sequence information, polymerase chain reaction techniques were used to obtain full-length cDNA sequences encoding the purified proteins. Expression of the cDNAs in insect cells conferred high levels of DGAT activity on the membranes isolated from these cells. The two proteins share 54% homology with each other but are unrelated to the previously identified DGAT gene family (designated DGAT1), which is related to the acyl CoA:cholesterol acyltransferase gene family, or to any other gene family with ascribed function. This report identifies a new gene family, including members in fungi, plants and animals, which encode enzymes with DGAT function. To distinguish the two unrelated families we designate this new class DGAT2 and refer to the M. ramanniana genes as MrDGAT2A and MrDGAT2B.

Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members.

Studies involving the cloning and disruption of the gene for acyl-CoA:diacylglycerol acyltransferase (DGAT) have shown that alternative mechanisms exist for triglyceride synthesis. In this study, we cloned and characterized a second mammalian DGAT, DGAT2, which was identified by its homology to a DGAT in the fungus Mortierella rammaniana. DGAT2 is a member of a gene family that has no homology with DGAT1 and includes several mouse and human homologues that are candidates for additional DGAT genes. The expression of DGAT2 in insect cells stimulated triglyceride synthesis 6-fold in assays with cellular membranes, and DGAT2 activity was dependent on the presence of fatty acyl-CoA and diacylglycerol, indicating that this protein is a DGAT. Activity was not observed for acyl acceptors other than diacylglycerol. DGAT2 activity was inhibited by a high concentration (100 mm) of MgCl(2) in an in vitro assay, a characteristic that distinguishes DGAT2 from DGAT1. DGAT2 is expressed in many tissues with high expression levels in the liver and white adipose tissue, suggesting that it may play a significant role in mammalian triglyceride metabolism.

Accumulation of palmitate in Arabidopsis mediated by the acyl-acyl carrier protein thioesterase FATB1.

The acyl-acyl carrier protein thioesterase B1 from Arabidopsis (AtFATB1) was previously shown to exhibit in vitro hydrolytic activity for long chain acyl-acyl carrier proteins (P. Dörmann, T.A. Voelker, J.B. Ohlrogge [1995] Arch Biochem Biophys 316: 612-618). In this study, we address the question of which role in fatty acid biosynthesis this enzyme plays within the plant. Over-expression of the AtFATB1 cDNA under a seed-specific promoter resulted in accumulation of high amounts of palmitate (16:0) in seeds. RNA and protein-blot analysis in Arabidopsis and rapeseed (Brassica napus) showed that the endogenous AtFATB1 expression was highest in flowers and lower in leaves. All floral tissues of wild-type plants contained elevated amounts of 16:0, and in the polar lipid fraction of flowers close to 50 mol % of the fatty acids were 16:0. Therefore, flowers contain polar lipids with an unusually high amount of saturated fatty acids as compared to all other plant tissues. Antisense expression of the AtFATB1 cDNA under the cauliflower mosaic virus 35S promoter resulted in a reduction of seed and flower 16:0 content, but no changes in leaf fatty acids. We conclude that the AtFATB1 thioesterase contributes to 16:0 production particularly in flowers, but that additional factors are involved in leaves.

Broad-range and binary-range acyl-acyl-carrier protein thioesterases suggest an alternative mechanism for medium-chain production in seeds.

In the current model of medium-chain (C8-14) fatty acid biosynthesis in seeds, specialized FatB acyl-acyl-carrier-protein (ACP) thioesterases are responsible for the production of medium chains. We have isolated and characterized FatB cDNAs from the maturing seeds of elm (Ulmus americana) and nutmeg (Myristica fragrans), which accumulate predominantly caprate (10:0)- and myristate (14:0)-containing oils, respectively. In neither species were we able to find cDNAs encoding enzymes specialized for these chain lengths. Nutmeg FatB hydrolyses C14-18 substrates in vitro and expression in Brassica napus seeds leads to an oil enriched in C14-18 saturates. Elm FatB1 displays a binary specificity: one activity is centered on 10:0-ACP, and a second is centered on palmitate (16:0)-ACP. After expression in B. napus seeds the oil is enriched in C10-18 saturates, predominantly 16:0, 14:0, and 10:0. The composition of free fatty acids produced by elm FatB1 in Escherichia coli shifts from C14-16 to mostly C8-10 by increasing the rate of chain termination by this enzyme. These results suggest the existence of an alternative mechanism used in the evolution of medium-chain production, a model of which is presented.

Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana.

The Mexican shrub Cuphea hookeriana accumulates up to 75% caprylate (8:0) and caprate (10:0) in its seed oil. An acyl-ACP thioesterase cDNA from C. hookeriana, designated Ch FatB2, has been identified, which, when expressed in Escherichia coli, provides thioesterase activity specific for 8:0- and 10:0-ACP substrates. Expression of this clone in seeds of transgenic canola, an oilseed crop that normally does not accumulate any 8:0 and 10:0, resulted in a dramatic increase in the levels of these two fatty acids accompanied by a preferential decrease in the levels of linoleate (18:2) and linolenate (18:3). The Ch FatB2 differs from Ch FatB1, another Cuphea hookeriana thioesterase reported recently, in both substrate specificity and expression pattern. The Ch FatB1 has a broad substrate specificity with strong preference for 16:0-ACP and is expressed throughout the plant; whereas Ch FatB2 is specific for 8:0/10:0-ACP and its expression is confined to the seed. It is proposed that the amplified expression of Ch FatB2 in the embryo provides the hydrolytic enzyme specificity determining the fatty acyl composition of Cuphea hookeriana seed oil.

Crystallization of glycosylated and nonglycosylated phytohemagglutinin-L.

In the seeds of legume plants a class of sugar-binding proteins can be found, generally called legume lectins. In this paper we present the crystallization of phytohemagglutinin-L (PHA-L), a glycosylated lectin from the seeds of the common bean (Phaseolus vulgaris). Single PHA-L crystals were grown by vapor diffusion, using PEG as precipitant. The protein crystallizes in the monoclinic space group C2, and diffracts to a resolution of 2.7 angstroms. The unit cell parameters are a=106.3 angstroms, 121.2 angstroms, c=90.8 angstroms, and beta=93.7 degrees. The asymmetric unit probably contains one PHA-L tetramer. Crystals of a recombinant nonglycosylated form of PHA-L, grown under identical conditions, and crystals of the native PHA-L, grown in the presence of isopropanol, did not survive the mounting process.

Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering.

The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant. These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired. The ability to modify TE specificity should allow the production of additional "designer oils" in genetically engineered plants.

Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase.

Alteration of acyl-acyl carrier protein pools and acetyl-CoA carboxylase expression in Escherichia coli by a plant medium chain acyl-acyl carrier protein thioesterase.

Expression of a plant lauroyl-acyl carrier protein (ACP) thioesterase in an Escherichia coli strain deficient in beta oxidation results in the accumulation of free fatty acids in the culture. Overall fatty acid production by the cultures is increased severalfold, particularly in the late log and stationary stages of growth. In control E. coli cells, malonyl-ACP levels and rates of fatty acid synthesis are highest during rapid logarithmic growth and decline to undetectable levels in stationary stage. In contrast, in cells expressing plant acyl-ACP thioesterase, malonyl-ACP levels remain high in late log and stationary stage in association with the continued fatty acid production. In addition, the biotin carboxyl carrier protein component of acetyl-CoA carboxylase is expressed at higher levels in cultures expressing the acyl-ACP thioesterase. The data presented indicate that removal of the acyl-ACP products of fatty acid synthesis results in increased production of both malonyl-ACP and fatty acids, which may in turn result from higher activity and/or expression of acetyl-CoA carboxylase.

Cloning and expression in Escherichia coli of a novel thioesterase from Arabidopsis thaliana specific for long-chain acyl-acyl carrier proteins.

An Arabidopsis thaliana partial cDNA was previously identified with a sequence similar to the lauroyl-acyl carrier protein (ACP) thioesterase from Umbellularia california (Grellet et al., 1993, Plant Physiol. Biochem. 31, 599-602). Using this DNA fragment, we isolated a 1.8-kb cDNA coding for a 412-amino-acid preprotein. The deduced amino acid sequence is 51% identical to the lauroyl-ACP thioesterase but only 39% identical to safflower oleoyl-ACP thioesterase. The cDNA was expressed in Escherichia coli and the gene product showed thioesterase activity for long-chain acyl-ACPs (14:0, 16:0, 18:0, 18:1 delta 9cis). When expressed in beta-oxidation mutants of E. coli, lipid analysis revealed that cells transformed with the thioesterase produced high amounts of free fatty acids that mostly consisted of 16:0 and some 14:0, 16:1 delta 9cis, and 18:1 delta 11cis. Antibodies were raised to the recombinant protein and used to determine tissue-specific and developmental expression in A. thaliana and Brassica napus. A 40-kDa protein was detected by immunoblots in A. thaliana siliques, leaves, and roots. A maximal expression of the B. napus protein between 18 and 31 days after flowering was found, which correlates with the rapid accumulation of triacylglycerols in the seeds. Based upon these results, we suggest that this long-chain acyl-ACP thioesterase may be a ubiquitous enzyme in plants which is involved in the synthesis of long-chain fatty acids.

Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase.

The expression of a plant (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (BTE) cDNA in Escherichia coli results in a very high level of extractable medium-chain-specific hydrolytic activity but causes only a minor accumulation of medium-chain fatty acids. BTE's full impact on the bacterial fatty acid synthase is apparent only after expression in a strain deficient in fatty acid degradation, in which BTE increases the total fatty acid output of the bacterial cultures fourfold. Laurate (12:0), normally a minor fatty acid component of E. coli, becomes predominant, is secreted into the medium, and can accumulate to a level comparable to the total dry weight of the bacteria. Also, large quantities of 12:1, 14:0, and 14:1 are made. At the end of exponential growth, the pathway of saturated fatty acids is almost 100% diverted by BTE to the production of free medium-chain fatty acids, starving the cells for saturated acyl-ACP substrates for lipid biosynthesis. This results in drastic changes in membrane lipid composition from predominantly 16:0 to 18:1. The continued hydrolysis of medium-chain ACPs by the BTE causes the bacterial fatty acid synthase to produce fatty acids even when membrane production has ceased in stationary phase, which shows that the fatty acid synthesis rate can be uncoupled from phospholipid biosynthesis and suggests that acyl-ACP intermediates might normally act as feedback inhibitors for fatty acid synthase. As the fatty acid synthesis is increasingly diverted to medium chains with the onset of stationary phase, the rate of C12 production increases relative to C14 production. This observation is consistent with activity of the BTE on free acyl-ACP pools, as opposed to its interaction with fatty acid synthase-bound substrates.

Effects of Elevated Sucrose-Phosphate Synthase Activity on Photosynthesis, Assimilate Partitioning, and Growth in Tomato (Lycopersicon esculentum var UC82B).

The expression of a sucrose-phosphate synthase (SPS) gene from maize (Zea mays, a monocotyledon) in tomato (Lycopersicon esculentum, a dicotyledon) resulted in marked increases in extractable SPS activity in the light and the dark. Diurnal modulation of the native tomato SPS activity was found. However, when the maize enzyme was present the tomato leaf cells were unable to regulate its activation state. No detrimental effects were observed and total dry matter production was unchanged. However, carbon allocation within the plants was modified such that in shoots it increased, whereas in roots it decreased. There was, therefore, a change in the shoot:root dry weight ratio favoring the shoot. This was positively correlated with increased SPS activity in leaves. SPS was a major determinant of the amount of starch in leaves as well as sucrose. There was a strong positive correlation between the ratio of sucrose to starch and SPS activity in leaves. Therefore, SPS activity is a major determinant of the partitioning of photosynthetically fixed carbon in the leaf and in the whole plant. The photosynthetic rate in air was not significantly increased as a result of elevated leaf SPS activity. However, the light- and CO2-saturated rate of photosynthesis was increased by about 20% in leaves expressing high SPS. In addition, the temporary enhancement of the photosynthetic rate following brief exposures to low light was increased in the high SPS plants relative to controls. We conclude that the level of SPS in the leaves plays a pivotal role in carbon partitioning. Furthermore, high SPS levels have the potential to boost photosynthetic rates under favorable conditions.

Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants.

Medium-chain fatty acids (FAs), found in storage lipids of certain plants, are an important renewable resource. Seeds of undomesticated California bay accumulate laurate (12:0), and a 12:0-acyl-carrier protein thioesterase (BTE) has been purified from this tissue. Sequencing of BTE enabled the cloning of a complementary DNA coding for a plastid-targeted preprotein. Expression of the complementary DNA in the seeds of Arabidopsis thaliana resulted in BTE activity, and medium chains accumulated at the expense of long-chain (greater than or equal to 16) FAs. Laurate became the most abundant FA species and was deposited in the storage triacylglycerols. These results demonstrate a mechanism for medium-chain FA synthesis in plants.

Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning.

We isolated a complementary DNA sequence for the enzyme sucrose phosphate synthase (SPS) from maize utilizing a limited amino acid sequence. The 3509-bp cDNA encodes a 1068-amino acid polypeptide. The identity of the cDNA was confirmed by the ability of the cloned sequence to direct sucrose phosphate synthesis in Escherichia coli. Because no plant-specific factors were necessary for enzymatic activity, we can conclude that SPS enzyme activity is conferred by a single gene product. Sequence comparisons showed that SPS is distantly related to the enzyme sucrose synthase. When expressed from a ribulose bisphosphate carboxylase small subunit promoter in transgenic tomatoes, total SPS activity was boosted up to sixfold in leaves and appeared to be physiologically uncoupled from the tomato regulation mechanism. The elevated SPS activity caused a reduction of starch and increase of sucrose in the tomato leaves. This result clearly demonstrates that SPS is involved in the regulation of carbon partitioning in the leaves.