Toxin-induced blood vessel inclusions caused by the chronic administration of aluminum and sodium fluoride and their implications for dementia.
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Aluminum Compounds↗
Biomedical subjects
K F Jensen
Publications and source records attributed to K F Jensen.
NusA is required for ribosomal antitermination and for modulation of the transcription elongation rate of both antiterminated RNA and mRNA.
Ribosomal RNA (rRNA) is elongated twice as fast as mRNA in vivo due to the presence of antitermination sequences in the 5' part of the rRNA transcripts. A number of Nus factors bind to RNA polymerase at the antitermination sites and help confer resistance to Rho-dependent termination of transcription. In this paper, the effects of the nusAcs10 allele on the elongation rate of both mRNA and antiterminated RNA were investigated. The results indicate that NusA is required to achieve a high elongation rate of RNA chains carrying the ribosomal antitermination boxA and that antitermination is defective when the rate of transcription elongation is decreased by the nusAcs10 allele. Furthermore, the nusAcs10 allele had no significant effects on the elongation rate of normal lacZ mRNA during steady state growth, but it abolished the inhibition of lacZ mRNA elongation by guanosine 3',5'-bis(diphosphate) (ppGpp). These results suggest that NusA is the component of the transcription elongation complex required for inhibition of mRNA elongation by ppGpp.
Alleles↗
The crystal structure of the flavin containing enzyme dihydroorotate dehydrogenase A from Lactococcus lactis.
BACKGROUND: . Dihydroorotate dehydrogenase (DHOD) is a flavin mononucleotide containing enzyme, which catalyzes the oxidation of (S)-dihydroorotate to orotate, the fourth step in the de novo biosynthesis of pyrimidine nucleotides. Lactococcus lactis contains two genes encoding different functional DHODs whose sequences are only 30% identical. One of these enzymes, DHODA, is a highly efficient dimer, while the other, DHODB, shows optimal activity only in the presence of an iron-sulphur cluster containing protein with which it forms a complex tetramer. Sequence alignments have identified three different families among the DHODs: the two L. lactis enzymes belong to two of the families, whereas the enzyme from E. coli is a representative of the third. As no three-dimensional structures of DHODs are currently available, we set out to determine the crystal structure of DHODA from L. lactis. The differences between the two L. lactis enzymes make them particularly interesting for studying flavoprotein redox reactions and for identifying the differences between the enzyme families. RESULTS: . The crystal structure of DHODA has been determined to 2.0 resolution. The enzyme is a dimer of two crystallographically independent molecules related by a non-crystallographic twofold axis. The protein folds into and alpha/beta barrel with the flavin molecule sitting between the top of the barrel and a subdomain formed by several barrel inserts. Above the flavin isoalloxazine ring there is a small water filled cavity, completely buried beneath the protein surface and surrounded by many conserved residues. This cavity is proposed as the substrate-binding site. CONCLUSIONS: . The crystal structure has allowed the function of many of the conserved residues in DHODs to be identified: many of these are associated with binding the flavin group. Important differences were identified in some of the active-site residues which vary across the distinct DHOD families, implying significant mechanistic differences. The substrate cavity, although buried, is located beneath a highly conserved loop which is much less ordered than the rest of the protein and may be important in giving access to the cavity. The location of the conserved residues surrounding this cavity suggests the potential orientation of the substrate.
Amino Acid Sequence↗
The B form of dihydroorotate dehydrogenase from Lactococcus lactis consists of two different subunits, encoded by the pyrDb and pyrK genes, and contains FMN, FAD, and [FeS] redox centers.
The B form of dihydroorotate dehydrogenase from Lactococcus lactis (DHOdehase B) is encoded by the pyrDb gene. However, recent genetic evidence has revealed that a co-transcribed gene, pyrK, is needed to achieve the proper physiological function of the enzyme. We have purified DHOdehase B from two strains of Escherichia coli, which harbored either the pyrDb gene or both the pyrDb and the pyrK genes of L. lactis on multicopy plasmids. The enzyme encoded by pyrDb alone (herein called the delta-enzyme) was a bright yellow, dimeric protein that contained one molecule of tightly bound FMN per subunit. The delta-enzyme exhibited dihydroorotate dehydrogenase activity with dichloroindophenol, potassium hexacyanoferrate(III), and molecular oxygen as electron acceptors but could not use NAD+. The DHOdehase B purified from the E. coli strain that carried both the pyrDb and pyrK genes on a multicopy plasmid (herein called the deltakappa-enzyme) was quite different, since it was formed as a complex of equal amounts of the two polypeptides, i.e. two PyrDB and two PyrK subunits. The deltakappa-enzyme was orange-brown and contained 2 mol of FAD, 2 mol of FMN, and 2 mol of [2Fe-2S] redox clusters per mol of native protein as tightly bound prosthetic groups. The deltakappa-enzyme was able to use NAD+ as well as dichloroindophenol, potassium hexacyanoferrate(III), and to some extent molecular oxygen as electron acceptors for the conversion of dihydroorotate to orotate, and it was a considerably more efficient catalyst than the purified delta-enzyme. Based on these results and on analysis of published sequences, we propose that the architecture of the deltakappa-enzyme is representative for the dihydroorotate dehydrogenases from Gram-positive bacteria.
Amino Acid Sequence↗
Different oligomeric states are involved in the allosteric behavior of uracil phosphoribosyltransferase from Escherichia coli.
Uracil phosphoribosyltransferase, catalyzing the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl-alpha-1-diphosphate (PPRibP), was purified from an overproducing strain of Escherichia coli. GTP was shown to activate the enzyme by reducing K(m) for PPRibP by about fivefold without affecting Vmax. When started by addition of enzyme, the reactions accelerated over an extended period of time, while enzyme solutions incubated first with GTP and PPRibP displayed constant velocities. This indicated that PPRibP and GTP influenced the structure of the enzyme. Gel-filtration and sedimentation analyses showed that the apparent oligomeric state of uracil phosphoribosyltransferase is defined by a dynamic equilibrium between a slowly sedimenting form (dimeric or trimeric) that has only a little activity, and a more highly aggregated form (pentameric or hexameric), which is more active. It appears that the smaller form predominates in the absence of substrates, while the larger form predominates in the presence of GTP and PPRibP. Guanosine-3',5'-bis(diphosphate) was found to activate the enzyme much like GTP.
Allosteric Regulation↗
Uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP.
Uracil phosphoribosyltransferase, which catalyses the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl alpha-1-pyrophosphate (PRPP), was partly purified from the extreme thermophilic archaebacterium Sulfolobus shibatae. The enzyme required divalent metal ions for activity and it showed the highest activity at pH 6.4. The specific activity of the enzyme was 50-times higher at 95 degrees C than at 37 degrees C, but the functional half-life was short at 95 degrees C. The activity of uracil phosphoribosyltransferase was strongly activated by GTP, which increased Vmax of the reaction by approximately 20-fold without much effect on K(m) for the substrates. The concentration of GTP required for half-maximal activation was about 80 microM. CTP was a strong inhibitor and acted by raising the concentration of GTP needed for half-maximal activation of the enzyme. We conclude that uracil phosphoribosyltransferase from S. shibatae is an allosteric enzyme which is activated by a purine nucleotide and inhibited by a pyrimidine nucleotide as seen for several enzymes in the pyrimidine nucleotide biosynthetic pathway of Escherichia coli, but not observed before for any phosphoribosyltransferase.
Adenosine Triphosphate↗
A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase.
Orotate phosphoribosyltransferase (OPRTase) is involved in the biosynthesis of pyrimidine nucleotides. Alpha-D-ribosyldiphosphate 5-phosphate (PRPP) and orotate are utilized to form pyrophosphate and orotidine 5'-monophosphate (OMP) in the presence of divalent cations, preferably Mg2+. OMP is thereafter converted to uridine 5'-monophosphate by OMP decarboxylase. We have determined the 2.4 angstrom structure of Escherichia coli OPRTase, ligated with sulfate, by molecular replacement and refined the structure to an R-factor of 18.3% for all data. In the structure of the E. coli enzyme we have determined the fold of a flexible loop region with a highly conserved amino acid sequence among OPRTases, a region known to take part in catalysis. The structure of this region was not determined in the model used for molecular replacement, and it involves interactions at the dimer interface through a bound sulfate ion. Crystalline E. coli OPRTase is a homodimer, with sulfate ions inhibiting enzyme activity bound in the dimer interface close to the flexible loop region. Although this loop is very close in space to the sulfate binding site, and sulfate is found in both interfaces of the homodimer, the loop structure is only traceable in one monomer. We expect that the mobility of this loop is important for catalysis, and, on the basis of the reported structure and the structure of Salmonella typhimurium OPRTase.OMP, we propose that the movement of this loop in association with the movement of OMP is vital to catalysis. Apart from the flexible loop region and a solvent-exposed loop (residues 158-164), the most significant differences in structure between S. typhimurium OPRTase.OMP and E. coli OPRTase are found in the substrate binding regions: the 5'-phosphate binding region (residues 120-131), the binding region for the orotate part of OMP (residues 25-27), and the pyrophosphate binding region (residues 71-73).
Amino Acid Sequence↗
Purification and characterization of dihydroorotate dehydrogenase A from Lactococcus lactis, crystallization and preliminary X-ray diffraction studies of the enzyme.
Lactococcus lactis is the only organism known to contain two dihydroorotate dehydrogenases, i.e., the A- and B-forms. In this paper, we report the overproduction, purification, and crystallization of dihydroorotate dehydrogenase A. In solution, the enzyme is bright yellow. It is a dimer of subunits (34 kDa) that contain one molecule of flavin mononucleotide each. The enzyme shows optimal function in the pH range 7.5-9.0. It is specific for L-dihydroorotate as substrate and can use dichlorophenolindophenol, potassium hexacyanoferrate (III), and, to a lower extent, also molecular oxygen as acceptors of the reducing equivalents, whereas the pyridine nucleotide coenzymes (NAD+, NADP+) and the respiratory quinones (i.e., vitamins Q6, Q10 and K2) were inactive. The enzyme has been crystallized from solutions of 30% polyethylene glycol, 0.2 M sodium acetate, and 0.1 M Tris-HCl, pH 8.5. The resulting yellow crystals diffracted well and showed little sign of radiation damage during diffraction experiments. The crystals are monoclinic, space group P21 with unit cell dimensions a = 54.19 A, b = 109.23 A, c = 67.17 A, and beta = 104.5 degrees. A native data set has been collected with a completeness of 99.3% to 2.0 A and an Rsym value of 5.2%. Analysis of the solvent content and the self-rotation function indicates that the two subunits in the asymmetric unit are related by a noncrystallographic twofold axis perpendicular to the crystallographic b and c axes.
Bacterial Proteins↗
Increased neurotoxicity following concurrent exposure to pyridostigmine bromide, DEET, and chlorpyrifos.
The operating environment of the service personnel during the Persian Gulf War involved psychological, biological, and chemical elements including exposure to pesticides such as the insect repellent DEET (N,N-diethyl-m-toluamide) and the insecticide chlorpyrifos (O,O-diethyl O-3,5,6-trichloropyridinyl phosphorothioate) and to pyridostigmine bromide (PB,3-dimethylaminocarbonyloxy-N-methylpyridinium bromide) that was administered as a prophylactic agent against possible nerve gas attack. The present study was designed to determine the toxicity produced by individual or coexposure of hens 5 days/week for 2 months to 5 mg PB/kg/day in water, by gavage; 500 mg DEET/kg/day, neat, sc; and 10 mg chlorpyrifos kg/day in corn oil, sc. Coexposure to various binary treatments produced greater neurotoxicity than that caused by individual exposures and was characterized by severe neurologic deficit and neuropathological alterations. Also, neurotoxicity was further enhanced following concurrent administration of the three chemicals. Severe inhibition of plasma butyrylcholinesterase (BuChE) activity was produced in hens treated with PB (activity 17% of control) compared to those treated with chlorpyrifos (activity 51% of control) or DEET (activity 83% of control). BuChE inhibition was further increased in binary and tertiary treatment groups compared to individual treatment groups. In contrast, a significant inhibition of brain acetylcholinesterase (AChE) was produced in hens administered chlorpyrifos alone (activity 67% of control), while those given chlorpyrifos in combination with other compounds exhibited a significant inhibition of brain AChE activity ranging from 43 to 76%. Brain neurotoxicity target esterase (NTE) was not inhibited in any of the individual treatment groups or PB/DEET, but was significantly inhibited and had activity expressed as a percentage of control in groups administered combined chlorpyrifos with PB of 73% or DEET of 74% and in the tertiary treatment group of 71%. We hypothesize that test compounds may compete for xenobiotic metabolizing enzymes in the liver and blood and may also compromise the integrity of the blood-brain barrier, leading to an increase in their "effective concentrations" in the nervous system to levels equivalent to the toxic doses of individual compounds. This is consistent with the present observation of increases in (1) the inhibition of brain AChE and NTE, (2) the extent of neurologic dysfunction, and (3) the severity and frequency of neuropathologic lesions in the combined treatment groups compared to those administered individual compounds.
Animals↗
Neurotoxicity resulting from coexposure to pyridostigmine bromide, deet, and permethrin: implications of Gulf War chemical exposures.
Of the three-quarters of a million service personnel involved in the Persian Gulf War, approximately 30,000 have complained of neurological symptoms of unknown etiology. One contributing factor to the emergence of such symptoms may be the simultaneous exposure to multiple agents used to protect the health of service personnel, in particular, the anti-nerve agent pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methylpyridinium bromide), the insect repellent DEET (N,N-diethyl-m-toluamide), and the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxyphenyl)methyl ester). This study investigated neurotoxicity produced in hens by individual or simultaneous exposure to these agents (5 d/wk for 2 months to 5 mg/kg/d PB in water, po; 500 mg/kg/d DEET, neat, sc; and 500 mg/kg/d permethrin in corn oil, sc). At these dosages, exposure to single compounds resulted in minimal toxicity. Combinations of two agents produced greater neurotoxicity than that caused by individual agents. Neurotoxicity was further enhanced following concurrent administration of all three agents. We hypothesize that competition for liver and plasma esterases by these compounds leads to their decreased breakdown and increased transport of the parent compound to nervous tissues. Thus, carbamylation of peripheral esterases by PB reduces the hydrolysis of DEET and permethrin and increases their availability to the nervous system. In effect, PB "pumps" more DEET and permethrin into the central nervous system. Consistent with this hypothesis, hens exposed to the combination of the three agents exhibited neuropathological lesions with several characteristics similar to those previously reported in studies of near-lethal doses of DEET and permethrin. If this hypothesis is correct, then blood and liver esterases play an important "buffering" role in protecting against neurotoxicity in the population at large. It also suggests that individuals with low plasma esterase activity may be predisposed to neurologic deficits produced by exposure to certain chemical mixtures.
Administration, Oral↗
Chemienzymatic synthesis of uridine nucleotides labeled with [15N] and [13C].
UTP, labeled with 15N and 13C (at all carbon atoms of the ribose moiety), was obtained enzymatically from [15N]uracil and [13C6]glucose. Eleven enzymes and suitable substrates reconstituted a metabolic pathway in which glucose was first transformed to 5-phosphoribosyl-1-pyrophosphate. The latter compound plus uracil yielded UMP in a second step by the reaction catalyzed by uracil phosphoribosyltransferase. UMP was subsequently phosphorylated to the corresponding di- and triphosphate. ATP, required for five phosphorylation reactions, was regenerated from creatine phosphate, whereas NADP+ necessary for the oxidation of glucose 6-phosphate and 6-phosphogluconate was recycled by glutamate dehydrogenase and excess of ammonia and alpha-oxoglutarate. Despite the number and complexity of the enzymatic steps, the synthesis of [15N, 13C]UTP is straightforward with an overall yield exceeding 60%. This method, extended and diversified to the synthesis of all natural ribonucleotides, is a more economical alternative for obtaining nucleic acids for structural analysis by heteronuclear NMR spectroscopy.
Adenosine Triphosphate↗
Effects of the antiterminator BoxA on transcription elongation kinetics and ppGpp inhibition of transcription elongation in Escherichia coli.
It has been shown previously that two different mRNA chains (lacZ and infB) are elongated at a rate of approximately 40 nucleotides (nt)/s during steady state growth on minimal medium and that the rate of mRNA chain elongation is inhibited by ppGpp in vivo. On the other hand, it was found that a truncated ribosomal RNA chain was elongated at a rate of approximately 80 nt/s, independent of growth condition (Vogel, U., and Jensen, K. F. (1994) J. Biol. Chem. 269, 16236-16241). We reasoned that the different transcriptional behavior of mRNA genes and rRNA operons might be caused by the antiterminator sequences present in the rRNA operons. To test this possibility, we have (a) inserted the minimal antiterminator boxA sequence between the promoter and the lacZ and infB genes and (b) deleted the antiterminator sequences from the rRNA transcription unit and measured transcription elongation rates in vivo on the resulting hybrid genes. We found that insertion of boxA in front of the coding region of lacZ increased the transcription elongation rate from 42 nt/s to 69 nt/s during steady state growth and that it eliminated the ppGpp-dependent decrease in the transcription elongation rate during the stringent response. On the other hand, deletion of the antiterminator sequences from the rRNA operon resulted in a reduced transcription elongation rate, but the elongation rate was still insensitive to changes in the ppGpp pool. These results are consistent with the hypothesis that the antiterminator boxA is a primary determinant of the rate of transcription elongation rate.
Base Sequence↗
Quantitative aspects of drug and toxicant-induced astrogliosis.
A universal cellular reaction to damage of the CNS is hypertrophy of astrocytes. The hallmark of this response, often termed 'reactive gliosis', is the enhanced expression of the major intermediate filament protein of astrocytes, glial fibrillary acidic protein (GFAP). This latter observation suggests that increased synthesis of GFAP would occur in response to diverse neurotoxic insults. To investigate this possibility, prototype neurotoxicants were administered to experimental animals and the effects of these agents on the tissue content of GFAP was determined by immunoassay. Assays of GFAP were found to reveal dose-, time- and region-dependent patterns of neurotoxicity at toxicant dosages below those that cause light microscopic evidence of cell loss or damage. Moreover, the temporal and regional increments in GFAP correspond to the temporal and regional patterns of argyrophilia, as revealed by the cupric silver degeneration stain of de Olmos. Our findings indicate that assays of GFAP represent a sensitive, simple and quantitative approach for evaluation of nervous system damage. Combining this indirect yet quantitative indicator of neurotoxicity with more traditional neuroanatomical endpoints, should augment the armamentarium of techniques useful for detection and characterization of neurotoxicity.
Animals↗
Crystallization and preliminary X-ray diffraction studies on the Apo form of orotate phosphoribosyltransferase from Escherichia coli.
Three different crystal forms of the apoenzyme orotate phosphoribosyltransferase, with M(r) = 23,552 from Escherichia coli have been grown. The crystals, which are all suitable for X-ray diffraction analysis, have been grown by the hanging drop vapour diffusion method. The first form crystallizes in the orthorhombic space group P2(1)2(1)2, with cell dimensions: a = 136.34 A, b = 75.98 A and c = 40.32 A; the second form in the monoclinic space group P2, with unit cell dimensions: a = 101.61 A, b = 40.49 A, c = 79.05 A and beta = 87.33 degrees; and the third form in P2(1)2(1)2(1), the cell dimensions being a = 70.27 A, b = 103.53 A, c = 53.83 A.
Crystallization↗
Effects of guanosine 3',5'-bisdiphosphate (ppGpp) on rate of transcription elongation in isoleucine-starved Escherichia coli.
We measured the transcription elongation rate on two mRNA genes, i.e. infB and lacZ, and on a part of the rrnB gene under conditions when wild type (rel+) Escherichia coli and relaxed (relA) mutants were exposed to isoleucine starvation. The RNA chain growth rates were calculated from the time lag between induction of transcription and the appearance of specific hybridization to probes complementary to the 3' ends of the genes, i.e. from the transcription time. The rate of mRNA chain elongation responded differently in rel+ and relA strains exposed to isoleucine starvation as it decreased (approximately 50%) in rel+ strains that accumulated high concentrations of guanosine 3',5'-bisdiphosphate (ppGpp) and increased (approximately 15%) the relA mutant whose ppGpp pool decayed during starvation. These results show that ppGpp inhibits mRNA chain elongation in vivo. However, stable RNA chain elongation appeared unaffected by ppGpp pool size and was twice as fast as mRNA chain elongation in exponentially growing cells.
Base Sequence↗
High concentrations of ppGpp decrease the RNA chain growth rate. Implications for protein synthesis and translational fidelity during amino acid starvation in Escherichia coli.
We show that the RNA chain growth rate on lacZ is reduced by an elevated ppGpp level even in the absence of starvation. Under these conditions the polypeptide chain elongation rate is affected little, if at all. These results lead us to re-examine the role of ppGpp in the reduction of protein synthesis and translational fidelity during amino acid starvation. We find that ppGpp has little or no direct effect on translation rate or fidelity. Rather, the effects of ppGpp on translation are indirectly caused by the fact that ppGpp inhibits mRNA synthesis, making mRNA limiting for translation during amino acid starvation. The reduced level of mRNA thereby reduces the severity of the aminoacyl-tRNA limitation and, in turn, decreases mistranslation. Mistranslation in the starved relA strain therefore results from an increased severity of aminoacyl-tRNA limitation due to the failure of this strain to reduce mRNA levels by increasing the level of ppGpp. Finally, the initial rise of the ppGpp level in the starved stringent strain, followed by a characteristic reduction to a steady poststarved level, can now be explained by the initially high, and then decreasing number of "hungry" codons adjusted through the mRNA pool.
Amino Acids↗
The RNA chain elongation rate in Escherichia coli depends on the growth rate.
We determined the rates of mRNA and protein chain elongation on the lacZ gene during exponential growth on different carbon sources. The RNA chain elongation rate was calculated from measurements of the time elapsing between induction of lacZ expression and detection of specific hybridization with a probe near the 3' end of the mRNA. The elongation rate for the transcripts decreased 40% when the growth rate decreased by a factor of 4, and it always correlated with the rate of translation elongation. A similar growth rate dependency was seen for transcription on the infB gene and on a part of the rrnB gene fused to a synthetic, inducible promoter. However, the untranslated RNA chain specified by the rrnB gene was elongated nearly twice as fast as the two mRNA species encoded by infB and lacZ.
Cell Division↗
The DNA damage-inducible dinD gene of Escherichia coli is equivalent to orfY upstream of pyrE.
The DNA damage-inducible gene dinD, originally identified by Kenyon and Walker (C. J. Kenyon and G. C. Walker, Proc. Natl. Acad. Sci. USA 77:2819-2823, 1980) by selection of the dinD::MudI (Ap lac) fusion, is shown here to be equivalent to the open reading frame orfY near pyrE. The evidence for identity between the two genes includes results from P1 transduction, Southern hybridization, and cloning and sequencing of the dinD fusion. No data were obtained that reveal any hints about the function of the dinD gene.
Bacterial Proteins↗