Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.     

Genetic structure of natural populations of the red-spotted newt,Notophthalmus viridescens

Genetic variation is described at 15 loci in 2 neotenic and 12 nonneotenic populations of red-spotted newts. Though high levels of genetic similarity (I=0.990) were found among all populations, allele frequencies at six of the eight most polymorphic loci show significant heterogeneity across populations. Change in allele frequencies at two of these loci (Pep-2 and Ldh-1) is significantly correlated with latitude. Interspecific homologies are established for newt peptidases based on substrate specificities and lactate dehydrogenases based on tissue distribution, thermal stability, and kinetic properties. Nonneotenic populations are highly variable (H=0.157) and neotenic populations are only slightly, but significantly, less variable (H=0.120). The high levels of heterozygosity detected in nonneotenic populations may result from large effective population size and/or environmental heterogeneity. The unexpectedly high heterozygosity values obtained for the neotenic populations may indicate adult dispersal or the presence of some previously undetected red efts at these localities. In any case, a major change in life history has apparently had little effect on the genetic structure of these populations.This research was supported by grants from the Blakeslee Fund of Smith College.     

Characterization of a 45-kDa polypeptide as the precursor of subunit 1 of cytochrome c oxidase in Neurospora crassa

In a previous paper (Van 't Sant, P., Mak, J.F.C. and Kroon, A.M. (1981) Eur. J. Biochem. 121, 21–26) we showed the existence of three elongated precursor proteins (45, 36 and 25 kDa) of mitochondrial translation products in Neurospora crassa. We presented some indications that the largest precursor could be related to subunit 1 of cytochrome c oxidase. Here we present conclusive evidence that the 45-kDa polypeptide is indeed this precursor by demonstrating that an immunodetectable 45-kDa polypeptide displays the same behaviour as the labeled 45-kDa precursor; both accumulate after long incubation with cycloheximide or by decreasing the temperature and both are not tightly membrane bound. Moreover the antibody against subunit 1 of cytochrome c oxidase also recognizes, in immunoadsorption experiments, besides subunit 1, the 45-kDa polypeptide accumulated by cycloheximide incubation. Furthermore, we developed a small scale purification of antibodies against subunit 1 of cytochrome c oxidase. By means of these purified antibodies it is demonstrated that the 45-kDa polypeptide and subunit 1 have corresponding antigenic determinants. Under the various conditions tested, all three precursors are less firmly membrane-bound than the mature subunits. Finally, it is observed that in short incubations in vivo, chloramphenicol inhibits the processing of the mitochondrially synthesized precursors, under conditions where mitochondrial translation is only partially inhibited.     

Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Metabolism of dodecyldimethylamine by Pseudomonas MA3

Pseudomonas MA3 was isolated from activated sludge on the basis of its capacity to use dodecyldimethylamine as a sole carbon (C) and energy source. Dodecylamine, dodecanal, dodecanoic acid and acetic acid also supported growth of Pseudomonas MA3. Dodecyldimethylamine-grown cells oxidized a wide range of alkylamine derivatives, dodecanal, dodecanoic acid and acetic acid. Degradation of the alkyl chain of dodecyldimethylamine by Pseudomonas MA3 appeared from the stoichiometric liberation of dimethylamine. A dehydrogenase catalysed the cleavage of the C_alkyl-N bond. The first intermediate of the proposed degradation pathway, dodecanal, accumulated in the presence of decanal used as a competitive inhibitor. The second intermediate,dodecanoic acid, was formed in the presence of acrylic acid during the degradation of dodecyldimethylamine. Dodecanal was converted into dodecanoic acid by a dehydrogenase and dodecanoic acid was then degraded via the oxidation pathway.     

Flexible life history responses to flower and rosette bud removal in three perennial herbs

In a garden experiment we investigated the response to continuous removal of either flower buds or rosette buds in three perennial grassland species ( Hypochaeris radicata , Succisa pratensis and Centaurea jacea ), which differ in longevity and flowering type. We distinguished two possible responses: compensation for lost buds by making more buds of the same type, and switching towards development of other life history functions. Both responses were demonstrated in our experiment, but bud removal had significantly different effects in each of the three species. The degree of compensation and the expression of trade-offs between life history functions differed markedly between species and seem related to longevity and developmental constraints. With respect to switching, our results suggest costs of reproduction and a trade-off between life history functions, at least for Hypochaeris and Succisa . For these species weight of new rosettes increased when resource allocation to flowering was inhibited. In Hypochaeris, we see that both compensation for lost flower buds and switching from lost rosette buds increased production of flower buds, underscoring the pivotal role of sexual reproduction in this short-lived species. The most prominent response seen in Centaurea is compensation for lost rosette buds, indicating that this long-lived species with monocarpic rosettes relies on rosette formation. Although Succisa does respond to bud removal, time is an important constraint in this species with long-lived rosettes and preformed flowering stalks. Trade-offs in Succisa seem to operate at a larger time scale, requiring long-lasting experiments to reveal them. We conclude that the response of these species to inflicted damage is likely to be linked to their longevity and developmental constraints.     

Dog-DAT: a direct agglutination test using stabilized, freeze-dried antigen for the serodiagnosis of canine visceral leishmaniasis

Abstract We have evaluated the use of an improved direct agglutination test (DAT) based on stable, freeze-dried antigen for the detection of anti- Leishmania antibodies in canine serum samples. With a cut-off value of 1:640, the sensitivity of the DAT was shown to be 100% and the specificity of the test was 98.8%.     

Removal of enteric viruses from surface water at eight waterworks in The Netherlands.

Eight waterworks in The Netherlands, which use surface water as their raw water source, were sampled repeatedly between November 1978 and June 1981. At five waterworks , 30 of 45 samples of raw water contained viruses. Of 55 samples of partially purified water, 11 were virus positive, including 8 after coagulation, sedimentation, and rapid sand filtration, 2 after storage, coagulation, sedimentation, transport chlorination, and rapid sand filtration, and 1 after storage in open reservoirs for 5 months. No viruses were detected in 100 samples of drinking water of 500 liters each from six waterworks . Most isolated viruses were typed, and a great variety of human enteroviruses were found, reflecting both pollution of raw water sources with sewage and vaccination with oral polio vaccine in neighboring countries.