Enzyme activity and liver tissue PCB concentrations of chicks of northern fulmars (F. glacialis) and black-legged kittiwakes (R. tridactyla) from Kongsfjorden

Arctic seabirds are exposed to a wide range of halogenated organic contaminants (HOCs). Exposure occurs mainly through food intake, and many pollutants accumulate in lipid-rich tissues. Little is known about how HOCs are biotransformed in arctic seabirds. In this study, we characterized biotransformation enzymes in chicks of northern fulmars (Fulmarus glacialis) and black-legged kittiwakes (Rissa tridactyla) from Kongsfjorden (Svalbard, Norway). Phase I and II enzymes were analyzed at the transcriptional, translational and activity levels. For gene expression patterns, quantitative polymerase chain reactions (qPCR), using gene-sequence primers, were performed. Protein levels were analyzed using immunochemical assays of western blot with commercially available antibodies. Liver samples were analyzed for phase I and II enzyme activities using a variety of substrates including ethoxyresorufin (cytochrome (CYP)1A1/1A2), pentoxyresorufin (CYP2B), methoxyresorufin (CYP1A), benzyloxyresorufin (CYP3A), testosterone (CYP3A/CYP2B), 1-chloro-2,4-nitrobenzene (CDNB) (glutathione S-transferase (GST)) and 4-nitrophenol (uridine diphosphate glucuronyltransferase (UDPGT)). In addition, the hydroxylated (OH-) polychlorinated biphenyls (PCBs) were analyzed in the blood, liver and brain tissue, whereas the methylsulfone (MeSO2-) PCBs were analyzed in liver tissue. Results indicated the presence of phase I (CYP1A4/CYP1A5, CYP2B, and CYP3A) and phase II (GST and UDPGT) enzymes at the activity, protein and/or mRNA level in both species. Northern fulmar chicks had higher enzyme activity than black-legged kittiwake chicks. This in combination with the higher XOH-PCB to parent PCB ratios suggests that northern fulmar chicks have a different biotransformation capacity than black-legged kittiwake chicks.

(Table 1) Polychlorinated biphenyl (PCB) and PCB metabolite concentrations in ringed seals (Phoca hispida) from Svalbard and the Baltic Sea

Polychlorinated biphenyls (PCBs) may induce activity of hepatic enzymes, mainly Phase I monooxygenases and conjugating Phase II enzymes, that catalyze the metabolism of PCBs leading to formation of metabolites and to potential adverse health effects. The present study investigates the concentration and pattern of PCBs, the induction of hepatic phase I and II enzymes, and the formation of hydroxy (OH) and methylsulfonyl (CH3SO2=MeSO2) PCB metabolites in two ringed seal (Phoca hispida) populations, which are contrasted by the degree of contamination exposure, that is, highly contaminated Baltic Sea (n = 31) and less contaminated Svalbard (n = 21). Phase I enzymes were measured as ethoxyresorufin-O-deethylation (EROD), benzyloxyresorufin-O-dealkylation (BROD), methoxyresorufin-O-demethylation (MROD), and pentoxyresorufin-O-dealkylation (PROD) activities, and phase II enzymes were measured as uridine diphosphophate glucuronosyl transferase (UDPGT) and glutathione-S-transferase (GST). Geographical comparison, multivariate, and correlation analysis indicated that sum-PCB had a positive impact on Phase I enzyme and GST activities leading to biotransformation of group III (vicinal ortho-meta-H atoms and <=1 ortho-chlorine (Cl)) and IV PCBs (vicinal meta-para-H atoms and <=2 ortho-Cl). The potential precursors for the main OH-PCBs detected in plasma in the Baltic seals were group III PCBs. MeSO2-PCBs detected in liver were mainly products of group IV PCB metabolism. Both CYP1A- and CYP2B-like enzymes are suggested to be involved in the PCB biotransformation in ringed seals.

Developmental disorder associated with increased cellular nucleotidase activity

Four unrelated patients are described with a syndrome that included developmental delay, seizures, ataxia, recurrent infections, severe language deficit, and an unusual behavioral phenotype characterized by hyperactivity, short attention span, and poor social interaction. These manifestations appeared within the first few years of life. Each patient displayed abnormalities on EEG. No unusual metabolites were found in plasma or urine, and metabolic testing was normal except for persistent hypouricosuria. Investigation of purine and pyrimidine metabolism in cultured fibroblasts derived from these patients showed normal incorporation of purine bases into nucleotides but decreased incorporation of uridine. De novo synthesis of purines and cellular phosphoribosyl pyrophosphate content also were moderately decreased. The distribution of incorporated purines and pyrimidines did not reveal a pattern suggestive of a deficient enzyme activity. Assay of individual enzymes in fibroblast lysates showed no deficiencies. However, the activity of cytosolic 5′-nucleotidase was elevated 6- to 10-fold. Based on the possibility that the observed increased catabolic activity and decreased pyrimidine salvage might be causing a deficiency of pyrimidine nucleotides, the patients were treated with oral pyrimidine nucleoside or nucleotide compounds. All patients showed remarkable improvement in speech and behavior as well as decreased seizure activity and frequency of infections. A double-blind placebo trial was undertaken to ascertain the efficacy of this supplementation regimen. Upon replacement of the supplements with placebo, all patients showed rapid regression to their pretreatment states. These observations suggest that increased nucleotide catabolism is related to the symptoms of these patients, and that the effects of this increased catabolism are reversed by administration of uridine.

The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA

tRNA pseudouridine synthase I (ΨSI) catalyzes the conversion of uridine to Ψ at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. ΨSI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNAPhe containing FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-tRNA. The covalent adduct between FUra-tRNA and ΨSI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluorouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for ΨS.

Coiled Bodies and U2 snRNA Genes Adjacent to Coiled Bodies Are Enriched in Factors Required for snRNA Transcription

A significant percentage of the gene clusters that contain the human genes for U1 small nuclear RNA (snRNA) or for U2 snRNA have been found associated with small nuclear domains, known as coiled bodies. We show here, by immunofluorescent labeling of human cells, that coiled bodies are enriched in factors required for the transcription of these snRNA genes. The 45-kDa γ-subunit of the transcription factor, proximal element sequence-binding transcription factor (PTF), which is specific for the snRNA genes, was found in high concentrations in coiled bodies, along with the general transcription factor TATA-box binding protein and a subset of RNA polymerase II. We show that the transcription factors and RNA polymerase II are concentrated in irregularly shaped domains that not only overlap with coiled bodies but also extend to their immediate surroundings. Fluorescent in situ hybridization showed that these domains can overlap with U2 snRNA genes adjacent to coiled bodies. In addition, we found the domains to contain newly synthesized RNA, visualized by 5-bromo-uridine triphosphate labeling. Our data suggest that coiled bodies are involved in the expression of snRNA genes, which leads us to propose the model that coiled bodies are associated with snRNA genes to facilitate and regulate their transcription. These findings point to a general principle of higher order organization of gene expression in the nucleus.

Numbers and Organization of RNA Polymerases, Nascent Transcripts, and Transcription Units in HeLa Nuclei

Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) the number of transcription sites, and 3) the number of polymerases associated with one transcription unit. To count engaged polymerases, cells were encapsulated in agarose, permeabilized, treated with ribonuclease, and the now-truncated transcripts extended in [32P]uridine triphosphate; then, the number of growing transcripts was calculated from the total number of nucleotides incorporated and the average increment in length of the transcripts. Approximately 15,000 transcripts were elongated by polymerase I, and ∼75,000 were elongated by polymerases II and III. Transcription sites were detected after the cells were grown in bromouridine for <2.5 min, after which the resulting bromo-RNA was labeled with gold particles; electron microscopy showed that most extranucleolar transcripts were concentrated in ∼2400 sites with diameters of ∼80 nm. The number of polymerases associated with a transcription unit was counted after templates were spread over a large area; most extranucleolar units were associated with one elongating complex. These results suggest that many templates are attached in a “cloud” of loops around a site; each site, or transcription “factory,” would contain ∼30 active polymerases and associated transcripts.

The crystal structure of the Rev binding element of HIV-1 reveals novel base pairing and conformational variability

The crystal and molecular structure of an RNA duplex corresponding to the high affinity Rev protein binding element (RBE) has been determined at 2.1-Å resolution. Four unique duplexes are present in the crystal, comprising two structural variants. In each duplex, the RNA double helix consists of an annealed 12-mer and 14-mer that form an asymmetric internal loop consisting of G-G and G-A noncanonical base pairs and a flipped-out uridine. The 12-mer strand has an A-form conformation, whereas the 14-mer strand is distorted to accommodate the bulges and noncanonical base pairing. In contrast to the NMR model of the unbound RBE, an asymmetric G-G pair with N2-N7 and N1-O6 hydrogen bonding, is formed in each helix. The G-A base pairing agrees with the NMR structure in one structural variant, but forms a novel water-mediated pair in the other. A backbone flip and reorientation of the G-G base pair is required to assume the RBE conformation present in the NMR model of the complex between the RBE and the Rev peptide.

Identification of 10 novel snoRNA gene clusters from Arabidopsis thaliana

Ten novel small nucleolar RNA (snoRNA) gene clusters, consisting of two or three snoRNA genes, respectively, were identified from Arabidopsis thaliana. Twelve of the 25 snoRNA genes in these clusters are homologous to those of yeast and mammals according to the conserved antisense sequences that guide 2′-O-ribose methylation of rRNA. The remaining 13 snoRNA genes, including two 5.8S rRNA methylation guides, are new genes identified from A.thaliana. Interestingly, seven methylated nucleotides, predicted by novel snoRNAs Z41a–Z46, are methylated neither in yeast nor in vertebrates. Using primer extension at low dNTP concentration the six methylation sites were determined as expected. These snoRNAs were recognized as specific guides for 2′-O-ribose methylation of plant rRNAs. Z42, however, did not guide the expected methylation of 25S rRNA in our assay. Thus, its function remains to be elucidated. The intergenic spacers of the gene clusters are rich in uridine (up to 40%) and most of them range in size from 35 to 100 nt. Lack of a conserved promoter element in each spacer and the determination of polycistronic transcription from a cluster by RT–PCR assay suggest that the snoRNAs encoded in the clusters are transcribed as a polycistron under an upstream promoter, and individual snoRNAs are released after processing of the precursor. Numerous snoRNA gene clusters identified from A.thaliana and other organisms suggest that the snoRNA gene cluster is an ancient gene organization existing abundantly in plants.

Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. donovani (TUBA5) that is deficient in LdNT1 transport and consequently resistant to tubercidin. In TUBA5 cells, the LdNT1.2 genes had the same sequence as wild-type cells. However, because LdNT1.2 mRNA is not detectable in either wild-type or TUBA5 promastigotes, LdNT1.2 does not contribute to nucleoside transport in this stage of the life cycle. In contrast, the TUBA5 cells were compound heterozygotes at the LdNT1.1 locus containing two mutant alleles that encompassed distinct point mutations, each of which impaired transport function. One of the mutant LdNT1.1 alleles encoded a G183D substitution in predicted TM 5, and the other allele contained a C337Y change in predicted TM 7. Whereas G183D and C337Y mutants had only slightly elevated adenosine Km values, the severe impairment in transport resulted from drastically (≈20-fold) reduced Vmax values. Because these transporters were correctly targeted to the plasma membrane, the reduction in Vmax apparently resulted from a defect in translocation. Strikingly, G183 was essential for pyrimidine nucleoside but not adenosine transport. A mutant transporter with a G183A substitution had an altered substrate specificity, exhibiting robust adenosine transport but undetectable uridine uptake. These results suggest that TM 5 is likely to form part of the nucleoside translocation pathway in LdNT1.1

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase1

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [Km] = 29 μm when UMP is the other substrate and Km = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (Km = 153 μm) and CMP (Km = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P1, P5-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.

Identification and Partial Characterization of the Pectin Methyltransferase “Homogalacturonan-Methyltransferase” from Membranes of Tobacco Cell Suspensions1

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg−1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.

An AU-box motif upstream of the SD sequence of light-dependent psbA transcripts confers mRNA instability in darkness in cyanobacteria

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. The expression of the psbA2 transcript has been shown to be light-dependent as assessed under light and dark (12/12 h) cycling conditions. We aligned the 5′-untranslated leader regions (UTRs) of psbAs from different photosynthetic organisms and identified a conserved sequence, UAAAUAAA or the ‘AU-box’, just upstream of the SD sequences. To clarify the role of 5′-upstream cis-elements containing the AU-box for light-dependent expression of psbA2, a series of deletion and point mutations in the region were introduced into the genome of heterologous cyanobacterium Synechococcus sp. strain PCC 7942, and psbA2 expression was examined. A clear pattern of light-dependent expression was observed in recombinant cyanobacteria carrying the K-81 psbA2 –38/+36 region (which includes the minimal promoter element and a light-dependent cis-element with the AU-box), +1 indicating the transcription start site. A constitutive pattern of expression, in which the transcripts remained almost stable under dark conditions, was obtained in cells harboring the –38/+14 region (the minimal element), indicating that the +14/+36 region with the AU-box is important for the observed light-dependent expression. Point mutations analyses within the AU-box also revealed that changes in number, direction and identity (as assayed by adenine/uridine nucleotide substitutions) influenced the light-dependent pattern of expression. The level of psbA2 transcripts increased markedly in CG- or deletion-box mutants in the dark, strongly indicating that the AU- (AT-) box acts as a negative cis-element. Furthermore, characterization of transcript accumulation in cells treated with rifampicin suggests that psbA2 5′-mRNA is unstable in the dark, supporting the view that the light-dependent expression is controlled at the post-transcriptional level. We discuss various mechanisms that may lead to altered mRNA stability such as the binding of factor(s) or ribosomes to the 5′-UTR and possible roles of the AU-box motif and the SD sequence.

Increased glycogen accumulation in transgenic mice overexpressing glycogen synthase in skeletal muscle.

To investigate the role of glycogen synthase in controlling glycogen accumulation, we generated three lines of transgenic mice in which the enzyme was overexpressed in skeletal muscle by using promoter-enhancer elements derived from the mouse muscle creatine kinase gene. In all three lines, expression was highest in muscles composed primarily of fast-twitch fibers, such as the gastrocnemius and anterior tibialis. In these muscles, glycogen synthase activity was increased by as much as 10-fold, with concomitant increases (up to 5-fold) in the glycogen content. The uridine diphosphoglucose concentrations were markedly decreased, consistent with the increase in glycogen synthase activity. Levels of glycogen phosphorylase in these muscles increased (up to 3-fold), whereas the amount of the insulin-sensitive glucose transporter 4 either remained unchanged or decreased. The observation that increasing glycogen synthase enhances glycogen accumulation supports the conclusion that the activation of glycogen synthase, as well as glucose transport, contributes to the accumulation of glycogen in response to insulin in skeletal muscle.

Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.

We have previously shown that specific nuclear pre-mRNA transcripts and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are packaged in large nuclear ribonucleoprotein (InRNP) particles that sediment at the 200S region in sucrose gradients. The InRNP particles contain all uridine-rich small nuclear ribonucleoprotein complexes required for pre-mRNA splicing, as well as protein splicing factors. In this paper we show that all of the phosphorylated, mAb 104 detectable, Ser/Arg-rich essential splicing factors (SR proteins) in the nucleoplasm are integral components of the InRNP particles, whereas only part of the essential splicing factor U2AF65 (U2 snRNP auxiliary factor) and the polypyrimidine tract binding protein (PTB) are associated with these particles. This finding suggests a limiting role for SR proteins in the assembly of the InRNP particles. We further show that the structural integrity of InRNP particles is sensitive to variations in the phosphorylation levels of the SR proteins.