Radiochemical Assay Research Articles

Function and cellular localization of farnesoic acid O-methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis.

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum. FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.

Radiochemical Malonyl-CoA Decarboxylase Assay: Activity and Subcellular Distribution in Heart and Skeletal Muscle

Malonyl-CoA decarboxylase is the main route for the disposal of malonyl-CoA, the key metabolite in the regulation of mitochondrial fatty acid oxidation. We have developed a simple and sensitive radiochemical assay to determine malonyl-CoA decarboxylase activity. The decarboxylation of [2-14C]malonyl-CoA produces [2-14C]acetyl-CoA, which is converted to [2-14C]acetylcarnitine in the presence of excess l-carnitine and carnitine acetyltransferase. The positively charged radiolabeled product, acetylcarnitine, is separated from negatively charged excess radiolabeled substrate and the radioactivity measured by scintillation counting. Measurement of malonyl-CoA decarboxylase activities with this method gives values comparable to those obtained with assays currently in use, but has the advantage of being simpler and less labor intensive. We have applied this assay to rat skeletal muscle of different fiber-type composition and to rat heart. Malonyl-CoA decarboxylase activity (mU/g wet wt) correlates with the oxidative capacity of the muscles, being lowest in type IIb fibers (42.7 ± 3.0) and highest in heart (1071.4 ± 260), with intermediate activity in type IIa fibers (150.7 ± 4.3) and type I fibers (107.8 ± 7.6). Studies on subcellular distribution of malonyl-CoA decarboxylase activity in rat heart and rat skeletal muscle show that approximately 50 and 65% is localized to mitochondria, while 50 and 35% of the activity is extramitochondrial.

Determination of thiopurine S-methyltransferase phenotype using thin-layer chromatography and quantitative scanning.

To develop a non-high-performance liquid chromatography method for the determination of thiopurine- S-methyltransferase (TPMT) phenotype using thin-layer chromatography and quantitative scanning. TPMT reaction was performed using a radiochemical assay. The reaction product [(14)C]-6-methylmercaptopurine was separated using thin-layer chromatography and quantified by means of radioactive scanning. Day-to-day variance was determined to validate results. Determination of TPMT phenotype using thin-layer chromatography and quantitative scanning is reliable (day-to-day variance 8.5+/-1.7%, mean+/-SEM). Mean TPMT activity in 314 randomly selected patient samples was 11.8+/-3.3 units/ml red blood cells (mean+/-SD, range 3.5-25 units/ml). We developed a new assay variant for the determination of TPMT phenotype that is easy to perform, reliable and reduces production of radioactive waste. This may lead to more frequent pretreatment determination of TPMT phenotype and increase drug safety and efficacy by individualising thiopurine doses.

Expression in Escherichia coli, Purification and Kinetic Characterization of Human Heparan Sulfate 3- O-Sulfotransferase-1

Heparan sulfate (HS) glycosaminoglycans are a structurally diverse class of complex biomolecules that modulate many important events at the cell surface and within the extracellular matrix and whose structural heterogeneity derives largely from the sequence-specific N- and O-sulfations catalyzed by an extensive repertoire of sulfating enzymes. We have expressed the human heparan sulfate 3-OST-1 isoform in Escherichia coli and subsequently purified a soluble, active enzyme. To assess its functionality, we determined the kinetic parameters for the recombinant 3-O-sulfotransferase-1 using a radiochemical assay that directly measures the 3-O-sulfation of unlabeled bovine kidney heparan sulfate in vitro using [ 35S]PAPS as the sulfate donor. The apparent K m values measured were in the low micromolar range ( K HS m = 4.3 μM; K PAPS m = 38.6 μM); V max values of 18 and 21 pmol sulfate/min/pmol of enzyme for HS and PAPS, respectively. These values were compared with kinetic parameters likewise measured for recombinant 3-OST-1 purified from baculovirus-infected sf9 cells. The two enzymes appear to modify heparan sulfate in vitro to roughly the same extent and with comparable specificities. The expression of 3-OST-1 in E. coli represents an important step in subsequent structure–function studies.

Frontal Affinity Chromatography Coupled to Mass Spectrometry for Screening Mixtures of Enzyme Inhibitors

Frontal affinity chromatography coupled online to mass spectrometry (FAC/MS) has previously been used to estimate binding constants for individual protein ligands present in mixtures of compounds. In this study FAC/MS is used to determine enzyme substrate kinetic parameters and binding constants for enzyme inhibitors. Recombinant human N-acetylglucosaminyltransferase V was biotinylated and adsorbed onto immobilized streptavidin in a microcolumn (20 μL). The enzyme was shown to be catalytically competent transferring GlcNAc from the donor UDP-GlcNAc to β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-β-d-Glcp-OR acceptor giving β-d-GlcpNAc-(1→2)-[β-d-GlcpNAc-(1→6)]-α-d-Manp-(1→6)-β-d-Glcp-OR as the reaction product. The kinetic parameters Km and Vmax for the immobilized enzyme could be determined by FAC/MS and were comparable to those measured in solution. Analysis of a mixture of eight trisaccharide analogs in a single run yielded Kd values for each of the eight compounds ranging from 0.3 to 36 μM. These Kd values were 2 to 10 times lower than the inhibition constants, KI's, determined in solution using a standard radiochemical assay. However, the ranking order of Kd's was the same as the ranking of KI values. FAC/MS assays can therefore be employed for the rapid estimation of inhibitor Kd values making it a valuable tool for enzyme inhibitor evaluations.

The radiochemical assay for juvenile hormone biosynthesis in insects: problems and solutions

Measurement of juvenile hormone (JH) production using the radiochemical assay (RCA) for JH biosynthesis and release is usually a reliable and precise technique. However problems with radiolabeled precursors and misunderstanding of the data, the techniques and the calculations have contributed towards uncertainty with respect to published experimental results. Problems with the purity of [ methyl− 3 H] -methionine or determination of its specific radioactivity have had detrimental effects on the reliability of results using the RCA. Proper control procedures and the use of 14 C/ 3 H -double-label RCA can be useful in detecting irregularities in the experimental results, and in determining contributing factors to any problems. The use of [ methyl− 14 C] -methionine and an awareness of normally expected RCA values can also assist the researcher in checking the validity of results. The radiolabeled methyl moiety of methionine is incorporated into JH without discrimination relative to unlabeled methyl methionine, by the o-methyl transferase. However unexpected preferential incorporation of the [ methyl− 14 C] - vs. [ methyl− 3 H] -moiety into JH occurs, but is only evident at concentrations of radiolabeled methionine outside the normal range of the RCA. Changes in radioactive precursor formulation have no effect on the RCA.

Phenotype Determination of Thiopurine Methyltransferase in Erythrocytes by HPLC

Thiopurine methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S -methylation of thiopurine drugs, which are used in cancer chemotherapy and as immunosuppressive agents (1). TPMT activity is controlled by a common genetic polymorphism that contributes to interindividual variability in drug response and, consequently, to implications for thiopurine therapeutic efficacy and toxicity (2). Severe myelosuppression has been reported for TPMT-deficient patients treated with standard doses of thiopurines (3)(4)(5), and high TPMT activity has been associated with the rejection of transplanted organs (6). Because of the clinical significance of the TPMT genetic polymorphism, determination of the TPMT phenotype in red blood cells is routinely performed to optimize and individualize thiopurine treatment (5). Variant alleles of the TPMT gene have been characterized and associated with low TPMT activity (7)(8). Recently, Spire-Vayron de la Moureyre et al. (9) reported that genotypic analysis of TPMT allows the correct determination of metabolic capacity for 87% of individuals. A lower correlation was found for individuals with TPMT activity that was close to the antimode value. Thus, phenotypic analysis may be useful and could be performed concomitantly with genotyping tests. TPMT activity has classically been measured using a radiochemical assay (10). Few HPLC methods using nonradiolabeled calibrators with liquid-liquid extraction (11)(12) or solid-phase extraction (13) have been reported. Here, we report a reversed-phase HPLC method that uses a simple and rapid treatment procedure for the determination …

Oral colonization and cariogenicity of Streptococcus gordonii in specific pathogen-free TAN:SPFOM(OM)BR rats consuming starch or sucrose diets

The significance of Streptococcus gordonii in dental caries is undefined, as is that of other α-amylase-binding bacteria (ABB) commonly found in the mouth. To clarify the ecological and cariological roles of S. gordonii our specific pathogen-free Osborne–Mendel rats, TAN:SPFOM(OM)BR, were fed either diet 2000 (containing 56% confectioner's sugar, most of which is sucrose) or diet 2000CS (containing 56% cornstarch, in lieu of confectioner's sugar) and inoculated with S. gordonii strains. Uninoculated rats were free of both indigenous mutans streptococci (MS) and ABB, including S. gordonii, as shown by culture on mitis salivarius and blood agars of swabs and sonicates of dentitions after weanlings had consumed these diets for 26 days. ABB were detected by radiochemical assay using [ 125I]-amylase reactive to α-amylase-binding protein characteristic of the surface of S. gordonii and other ABB. No ABB were detected (detection limit <1 colony-forming units in 10 6 colony-forming units). Thus the TAN:SPFOM(OM)BR colony presents a ‘clean animal model’ for subsequent study. Consequently, S. gordonii strains Challis or G9B were used to inoculate weanling rat groups consuming either the high-sucrose diet 2000 or the cornstarch diet 2000CS. Two additional groups fed each of these diets remained unioculated. Recoveries of inoculants were tested 12 and 26 days later by oral swabs and sonication of the molars of one hemimandible of each animal, respectively. Uninoculated animals were reconfirmed to be free of ABB and mutans streptococci, but inoculated ones eating diet 2000CS had S. gordonii recoveries of 1–10% or, if eating diet 2000, 10–30% of total colony-farming units in sonicates. There were no statistically significant differences among the inoculated and uninoculated animal groups’ caries scores when they ate the cornstarch diet. Lesion scores for sucrose-eating rats were, however, from 2.4–5.1-fold higher than for cornstarch-eating rats, P<0.001, and were still higher if animals had been inoculated with either Challis (1.41-fold) or G9B (1.64-fold), than if uninoculated, both P<0.001, so long as the rats ate the sucrose diet. Therefore, TAN:SPFOM(OM)BR rats do not harbour ABB or S. gordonii but can be colonized by S. gordonii. Colonization levels of S. gordonii on the teeth are higher in the presence of high sucrose than with high starch-containing diets. Caries scores are augmented by sucrose compared with starch, and are further augmented by S gordonii colonization. S. gordonii is thus cariologically significant in the presence of sucrose, at least in this rat.

Genotype-phenotype correlation for thiopurine S-methyltransferase in healthy Italian subjects.

The aim of the present study was to estimate the concordance rate between erythrocyte thiopurine methyltransferase (TPMT) activity and genotype at the TPMT locus in an Italian population sample. The TPMT phenotype and genotype were determined in an unrelated population of 103 Italian healthy blood donors. Erythrocyte TPMT activity was measured with a radiochemical assay using 12.5 microM S-adenosyl-L-(methyl-14C)-methionine and 4 mM 6-mercaptopurine. The genotyping assay was based on restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) methods. All subjects had detectable TPMT activity. The activity of TPMT varied 2.8-fold between the 5th and 95th percentile. This variation was neither age (P = 0.63) nor gender (P = 0.44) regulated and the frequency distribution of TPMT activity is compatible with a polymorphic distribution. The presence of the four most common defective alleles, i.e. TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C, was examined through the entire phenotyped population. Ninety-two subjects did not carry any of the tested mutations. Eleven individuals were heterozygous for one of the mutant alleles and had a TPMT activity lower than 30 pmol/min/mg. Eight subjects were TPMT*1/TPMT*3A, two TPMT*1/TPMT*3C and one was TPMT*1/TPMT*2. The TPMT*3B allele was not detected in the samples analysed. There was a concordance of 97% between genotype and phenotype. All the heterozygotes had an intermediate phenotype. However, the wide variation range in TPMT activity detected in the wild-type homozygotes indicates that other genetic or epigenetic factors influence the TPMT phenotype.

Fatty acid synthesis in developing leaves of Brassica napus in relation to leaf growth and changes in activity of 3-oxoacyl-ACP reductase

In young expanding leaves of Brassica napus, the demand for fatty acids is met by de novo biosynthesis of fatty acid synthase components, as demonstrated by 3-oxoacyl-ACP reductase. Using a novel radio-chemical assay for 3-oxoacyl-ACP reductase and specific antibodies, we have demonstrated a direct relationship between the increase in activity and synthesis of polypeptide. The maximum rate of fatty acid synthesis was between 4 and 7 days post-emergence, but slowed after this point even though 3-oxoacyl-ACP reductase activity was high. Leaf area continued to expand in a linear fashion after reductions in both enzyme activity and the rate of fatty acid synthesis.

A generic medium throughput activity assay procedure for serine–threonine protein phosphatases using 14C-labelled N-acetyl-Arg-Arg-Ala-Thr(P)-Val-Ala

A new and sensitive activity assay for the Ser-Thr protein phosphatases PP1 and PP2A based upon the hydrolysis of the synthetic radiolabelled phosphopeptide substrate N-acetyl-Arg-Arg-Ala-Thr(P)-Val-Ala, is described. The protocol is also applicable to the assay of and PP2C activity. The radiolabelled phosphopeptide is stable and can be stored for prolonged periods without deterioration or loss of radioactivity offering advantages over the use of 32P-labelled substrates. The assay method involves the separation of phosphopeptide substrate from the peptide alcohol product by anion exchange chromatography. The separation protocol is not sensitive to the presence of inorganic phosphate anion or metal cations. Using PP1 as the enzyme the radiochemical assay procedure afforded a Vmax value of 17 (±2) μM s−1 [28 (±3) μM s−1 μg−1] and a KM value of 3.7 (±0.9) mM for the substrate Ac-Arg-Arg-Ala-Thr(P)-Val-Ala with significantly greater accuracy than for a Malachite Green based assay. Inorganic phosphate was shown to be a competitive product inhibitor (Ki = 1.6 mM) and nodularin was found to be a potent competitive inhibitor (Ki = 0.19 nM) for PP1 with respect to the phosphopeptide substrate. This assay procedure was employed to determine the mode and magnitude of the inhibition of PP1 by the nodularin analogue described in the previous article [M. E. O'Donnell, J. Sanvoisin and D. Gani, J. Chem. Soc., Perkin Trans. 1, 2001 (DOI:10.1039/b100402f)]. The enzyme PP2A afforded a Vmax value of 2.8 (±0.2) μM s−1 [45 (±2) μM s−1μg−1] and a KM value of 4.1 (±0.7) mM for the radiolabelled substrate.

The critical period for caste determination in Bombus terrestris and its juvenile hormone correlates.

The critical period for caste determination and its juvenile hormone (JH III) correlates were studied in Bombus terrestris. Larvae of known age and instar were taken from young colonies, in which they would have been reared as workers, and placed into groups of queenless workers. Under these conditions the critical age for caste determination was 5 days, during the second instar. Endocrine correlates of caste determination were obtained by determining profiles of juvenile hormone titer and juvenile hormone biosynthesis, measured by chiral-specific radioimmunoassay and the in vitro radiochemical assay, respectively. By the middle of the second instar prospective queen larvae had significantly higher rates of juvenile hormone biosynthesis and juvenile hormone titer than prospective worker larvae. Based on the coincidence of timing of both the critical period and the appearance of caste-specific juvenile hormone titer, we suggest that juvenile hormone plays a role in the mechanisms that control caste determination in B. terrestris.

Injection of Dip-allatostatin or Dip-allatostatin pseudopeptides into mated female Diploptera punctata inhibits endogenous rates of JH biosynthesis and basal oocyte growth

Studies on the catabolism of allatostatins (ASTs) provided the rationale for the design of a series of Dip-allatostatin-derived pseudopeptide mimetic analogues. In vitro, the Dip-ASTs and pseudopeptides show varying degrees of resistance to catabolism and all show significant inhibition of juvenile hormone (JH) biosynthesis. This study was undertaken to determine whether potent Dip-ASTs and/or their pseudopeptide mimetic counterparts caused ‘allatostatic’ effects in vivo following injection into mated female Diploptera punctata. Animals injected with aqueous solvent or Dip-AST 7(1–7) N-terminal fragment, which excludes the active core region of the ASTs, were used as controls. An in vitro radiochemical assay revealed that injection of Dip-AST 5, 7 or pseudopeptide analogues 397-2 or AST(b)φ2 significantly inhibited the biosynthesis of JH ( P<0.05). The results also indicate that basal oocyte growth was significantly inhibited by injection of these same compounds, with the exception of Dip-AST 7 ( P<0.05). Analogues 396-1 and 419 did not significantly inhibit rates of JH biosynthesis but did significantly inhibit the growth of basal oocytes. Analyses of feeding, excretion and food absorption/utilization patterns of these same animals suggested that these compounds are not toxic to the insect; rather they directly inhibit the biosynthesis of JH by the corpora allata, and reduce the rate of growth of basal oocytes. Disruption of critical reproductive and/or developmental processes by pseudopeptide analogues of the ASTs could provide novel and selective strategies for future insect pest management.

Molecular Cloning of a Taxa-4(20),11(12)-dien-5α-ol-O-Acetyl Transferase cDNA from Taxus and Functional Expression in Escherichia coli

The taxa-4(20),11(12)-dien-5α-ol-O-acetyl transferase which catalyzes the third step of Taxol biosynthesis has been isolated from methyl jasmonate-induced Taxus cells, and partially purified and characterized (K. Walker, R. E. B. Ketchum, M. Hezari, D. Gatfield, M. Golenowski, A. Barthol, and R. Croteau, Arch. Biochem. Biophys. 364, 273–279 1999). A revised purification method allowed internal amino acid microsequencing of the enzyme, from which primers were designed and employed to amplify a transacetylase gene-specific fragment. This radiolabeled, 900-bp amplicon was used as a hybridization probe to screen a cDNA library constructed from poly(A)+ RNA isolated from induced Taxus cells, from which a full-length transacetylase sequence was obtained. Expression of this clone from pCWori+ in Escherichia coli JM109 cells yielded the functional enzyme, as determined by radiochemical assay and combined capillary gas chromatographic-mass spectrometric verification of the acetylated product. The full-length DNA has an open-reading frame of 1317 nucleotides corresponding to a deduced amino acid sequence of 439 residues that exhibits high sequence identity to the proteolytic fragments of the native enzyme, which the recombinant transacetylase resembles in properties. Consistent with the size of the operationally soluble native enzyme, the DNA appears to encode a monomeric protein of molecular weight 49,079 that bears no N-terminal organellar targeting information. Sequence comparison of the taxadien-5α-ol-O-acetyl transferase with the few other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (64–67%). The efficient conversion of taxadien-5α-yl acetate to further hydroxylated intermediates of the Taxol pathway confirms the significance of this acylation step and suggests this taxadienol transacetylase to be an important target for genetic manipulation to improve Taxol production.

Mammalian 5′(3′)-Deoxyribonucleotidase, cDNA Cloning, and Overexpression of the Enzyme in Escherichia coli and Mammalian Cells

5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known. It was earlier purified to homogeneity from human placenta. We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa. The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells. The recombinant enzyme was purified to homogeneity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme. Several polymerase chain reaction-based approaches to find this sequence met with no success. A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme.

Responsiveness of honey bee ( Apis mellifera L.) corpora allata to allatoregulatory peptides from four insect species

Five neuropeptides with known allatotropic or allatostatic activity in other insect species were examined for their effects on honey bee corpora allata. Using an in vitro radiochemical assay, we assessed the ability of these peptides to affect the biosynthesis of juvenile hormone III and its immediate precursor methyl farnesoate, as well as their effects on the conversion of methyl farnesoate into juvenile hormone. None of the allatostatins tested affected JH biosynthesis during the last larval instar of honey bee workers. Manduca sexta allatotropin, however, stimulated JH biosynthesis in a stage-specific and dose-dependent manner. Analysis of intraglandular contents of juvenile hormone and its precursor revealed that the allatotropin significantly increased JH precursor but did not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for early fifth-instar worker larvae. Studies also indicated that the allatotropic effect was reversible at the level of methyl farnesoate production.

Time-Resolved Fluorescence Energy Transfer DNA Helicase Assays for High Throughput Screening.

DNA helicases are responsible for the unwinding of double-stranded DNA, facilitated by the binding and hydrolysis of 5'-nucleoside triphosphates. These enzymes represent an important class of targets for the development of novel anti-infective agents particularly because opportunity exists for synergy with existing therapies targeted at other enzymes involved in DNA replication. Unwinding reactions are conventionally monitored by low throughput, gel-based radiochemical assays; to overcome the limitations of low throughput to achieve comprehensive characterization of adenosine triphosphate (ATP)-dependent unwinding by viral and bacterial helicases and the screening for unwinding inhibitors, we have developed and validated homogeneous time-resolved fluorescence energy transfer (TRET) assays. Rapid characterization and screening of DNA helicase has been performed in 96- and 384-well plate densities, and the ability to assay in 1536-well format also demonstrated. We have successfully validated and are running full high throughput runs using 384-well TRET helicase assays, culminating in the identification of a range of chemically diverse inhibitors of viral and bacterial helicases. For screening in mixtures, we used a combination of quench correction routines and confirmatory scintillation proximity (SP) assays to eliminate false-positives due to the relatively high levels of compound quenching (unlike other Ln(3+)-based assays). This strategy was successful yet emphasised the need for further improvements in helicase assays.

A Radiochemical Assay for β-Ureidopropionase Using Radiolabeled N-Carbamyl-β-alanine Obtained via Hydrolysis of [2-14C]5,6-Dihydrouracil

A radiochemical assay was developed to measure the activity of β-ureidopropionase in human liver homogenates which is based on the detection of the reaction product 14CO2 by liquid scintillation counting. Radiolabeled N-carbamyl-β-alanine was prepared within 15 min by a simple hydrolysis of [2-14C]5,6-dihydrouracil under alkaline conditions at 37°C. The enzymatic reaction proved to be linear with time up to at least 3.5 h and protein concentrations up to at least 1 mg/ml. Human β-ureidopropionase obeyed Michaelis–Menten kinetics with an apparent Km for N-carbamyl-β-alanine of 15.5 ± 1.9 μM. The assay proved to be very accurate and sensitive with an intraassay coefficient of variation of 2% and a detection limit of 28 pmol for the product CO2.

Radiochemical assay for determination of dihydropyrimidinase activity using reversed-phase high-performance liquid chromatography

A radiochemical assay was developed to measure the activity of dihydropyrimidinase (DHP) in human liver homogenates. The method is based on the separation of radiolabeled dihydrouracil from N-carbamyl-β-alanine by HPLC with on-line detection of radioactivity combined with detection of 14CO 2 by liquid scintillation counting. The assay was linear with time and protein concentration. The minimum amount of radiolabeled products which could be determined proved to be 12 pmol using a purified stock solution of [2- 14C]-5,6-dihydrouracil. This highly sensitive assay is especially suitable to identify patients with a dihydropyrimidinase deficiency.

Juvenile hormone biosynthesis in diapausing and nondiapausing Culex pipiens (Diptera: Culicidae).

The synthesis of juvenile hormone in vitro by diapausing and nondiapausing Culex pipiens L. was measured by radiochemical assay. Paired corpora allata from diapausing females synthesized < 18 fmols of juvenile hormone per hour during the first 3 wk after emergence. In contrast, juvenile hormone synthesis in nondiapausing females increased rapidly reaching a peak of 87.3 +/- 21 (mean +/- SE) fmol/h 3 d after emergence. By 3 wk, juvenile hormone synthesis had decreased in both groups of females, but corpora allata from nondiapausing mosquitoes still were 3 times more active than those from diapausing mosquitoes. By 16 wk after diapause induction, females maintained at 8:16 (L:D) h and 15 degrees C produced levels of juvenile hormone similar to 3-wk-old nondiapausing females. When females were held in diapause conditions for up to 22 wk, follicles gradually grew longer and by 15 wk were significantly longer than in the previous 14 wk. Blood feeding also increased in older females, indicating that over time, juvenile hormone synthesis gradually stimulated blood-feeding behavior. When 21-d-old diapausing mosquitoes were moved to a long-day photoperiod of 16:8 (L:D) h and 26 degrees C, juvenile hormone synthesis increased rapidly and peaked 5 d later while the ovarian follicles grew to the resting stage. Allatectomy of young diapausing females prevented follicle growth and blood feeding when diapause was terminated prematurely, demonstrating that the physiological events associated with diapause termination were associated with juvenile hormone biosynthesis.