J Biochem (Tokyo), 2000 Aug, 128(2), 195 - 200 Molecular construction of a multidrug exporter system, AcrAB: molecular interaction between AcrA and AcrB, and cleavage of the N-terminal signal sequence of AcrA; Kawabe T et al.; The AcrAB system of Escherichia coli is an intrinsic efflux protein with a broad substrate specificity . AcrA was thought to be localized in the periplasmic space, and to be linked to AcrB and TolC . The AcrAB-TolC system directly exports diverse substrates from the cell interior to the medium . In this study, we have determined the cellular localization of AcrA . By using the osmotic shock method, sucrose density gradient centrifugation, urea washing and Western blotting analysis, we reveal that AcrA is a peripheral inner membrane protein . A mutant plasmid encoding both the AcrA-TetBCt fusion protein and the AcrB-His fusion protein was constructed . Membrane vesicles prepared from cells expressing these fusion proteins were solubilized and AcrB-His was immunoprecipitated with an anti-polyhistidine antibody . After SDS-PAGE, Western blotting was performed with anti-TetBCt antiserum, resulting in the appearance of a 40 kDa band, indicating that AcrA co-precipitated with AcrB . Next we performed site-directed chemical labeling of Cys-introduced mutants of AcrA with {(14)C}N-ethylmaleimide . As judged from the labeling pattern and the molecular mass shift, the N-terminus of AcrA was removed and the mature protein is on the periplasmic surface . On the other hand, C25A mutants retained the N-terminal signal sequence on the cytoplasmic side of the membrane . We conclude that AcrA exists as a complex with AcrB on the periplasmic surface of the inner membrane after removal of the signal sequence. J Biochem (Tokyo), 2000 Aug, 128(2), 161 - 6 Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds; Kouzuma Y et al.; Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds {Kouzuma et al . J . Biochem . 119 (1996) 1106-1113} . A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe . The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues . The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca . The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined . SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor . Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins. Proc Natl Acad Sci U S A, 2000 Aug 15, 97(17), 9425 - 30 Autocatalytic proteolysis of the transcription factor-coactivator C1 (HCF): a potential role for proteolytic regulation of coactivator function; Vogel JL et al.; Site-specific proteolysis is an important biological mechanism for the regulation of cellular processes such as gene expression, cell signaling, development, and apoptosis . In transcriptional regulation, specific proteolysis regulates the localization and activity of many regulatory factors . The C1 factor (HCF), a cellular transcription factor and coactivator, undergoes site-specific proteolytic processing at a series of unusual amino acid reiterations to generate a family of amino- and carboxyl-terminal polypeptides that remain tightly associated . Expression and purification of bacterially expressed domains of the C1 factor identifies an autocatalytic activity that is responsible for the specific cleavage of the reiterations . In addition, coexpression of the autocatalytic domain with a heterologous protein containing a target cleavage site demonstrates that the C1 protease may also function in trans. Biophys J, 2000 Aug, 79(2), 1146 - 54 Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin; Simplaceanu V et al.; Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions . However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra . We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A) . This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin . Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect . In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A . In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer . The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A . These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa. Biophys J, 2000 Aug, 79(2), 602 - 13 Replacement of glycine 232 by aspartic acid in the KdpA subunit broadens the ion specificity of the K(+)-translocating KdpFABC complex; Schrader M et al.; Replacement of glycine residue 232 with aspartate in the KdpA subunit of the K(+)-translocating KdpFABC complex of Escherichia coli leads to a transport complex that has reduced affinity for K(+) and has lost the ability to discriminate Rb(+) ions (, J . Biol . Chem . 270:6678-6685) . This glycine residue is the first in a highly conserved GGG motif that was aligned with the GYG sequence of the selectivity filter (P- or H5-loop) of K(+) channels (, Nature . 371:119-122) . Investigations with the purified and reconstituted KdpFABC complex using the potential sensitive fluorescent dye DiSC(3)(5) and the "caged-ATP/planar bilayer method" confirm the altered ion specificity observed in uptake measurements with whole cells . In the absence of cations a transient current was observed in the planar bilayer measurements, a phenomenon that was previously observed with the wild-type enzyme and with another kdpA mutant (A:Q116R) and most likely represents the movement of a protein-fixed charge during a conformational transition . After addition of K(+) or Rb(+), a stationary current could be observed, representing the continuous pumping activity of the KdpFABC complex . In addition, DiSC(3)(5) and planar bilayer measurements indicate that the A:G232D Kdp-ATPase also transports Na(+), Li(+), and H(+) with a reduced rate . Similarities to mutations in the GYG motif of K(+) channels are discussed. Biophys J, 2000 Aug, 79(2), 788 - 801 A Grand Canonical Monte Carlo-Brownian dynamics algorithm for simulating ion channels; Im W et al.; A computational algorithm based on Grand Canonical Monte Carlo (GCMC) and Brownian Dynamics (BD) is described to simulate the movement of ions in membrane channels . The proposed algorithm, GCMC/BD, allows the simulation of ion channels with a realistic implementation of boundary conditions of concentration and transmembrane potential . The method is consistent with a statistical mechanical formulation of the equilibrium properties of ion channels (; Biophys . J . 77:139-153) . The GCMC/BD algorithm is illustrated with simulations of simple test systems and of the OmpF porin of Escherichia coli . The approach provides a framework for simulating ion permeation in the context of detailed microscopic models. Biophys J, 2000 Aug, 79(2), 747 - 55 Secondary structure components and properties of the melibiose permease from Escherichia coli: a fourier transform infrared spectroscopy analysis; Dave N et al.; The structure of the melibiose permease from Escherichia coli has been investigated by Fourier transform infrared spectroscopy, using the purified transporter either in the solubilized state or reconstituted in E . coli lipids . In both instances, the spectra suggest that the permease secondary structure is dominated by alpha-helical components (up to 50%) and contains beta-structure (20%) and additional components assigned to turns, 3(10) helix, and nonordered structures (30%) . Two distinct and strong absorption bands are recorded at 1660 and 1653 cm(-1), i.e., in the usual range of absorption of helices of membrane proteins . Moreover, conditions that preserve the transporter functionality (reconstitution in liposomes or solubilization with dodecyl maltoside) make possible the detection of two separate alpha-helical bands of comparable intensity . In contrast, a single intense band, centered at approximately 1656 cm(-1), is recorded from the inactive permease in Triton X-100, or a merged and broader signal is recorded after the solubilized protein is heated in dodecyl maltoside . It is suggested that in the functional permease, distinct signals at 1660 and 1653 cm(-1) arise from two different populations of alpha-helical domains . Furthermore, the sodium- and/or melibiose-induced changes in amide I line shape, and in particular, in the relative amplitudes of the 1660 and 1653 cm(-1) bands, indicate that the secondary structure is modified during the early step of sugar transport . Finally, the observation that approximately 80% of the backbone amide protons can be exchanged suggests high conformational flexibility and/or a large accessibility of the membrane domains to the aqueous solvent. Mutat Res, 2000 Jun 30, 451(1-2), 151 - 67 Mismatch repair proteins and mitotic genome stability; Harfe BD et al.; Mismatch repair (MMR) proteins play a critical role in maintaining the mitotic stability of eukaryotic genomes . MMR proteins repair errors made during DNA replication and in their absence, mutations accumulate at elevated rates . In addition, MMR proteins inhibit recombination between non-identical DNA sequences, and hence prevent genome rearrangements resulting from interactions between repetitive elements . This review provides an overview of the anti-mutator and anti-recombination functions of MMR proteins in the yeast Saccharomyces cerevisiae. J Immunol Methods, 2000 Jul 31, 241(1-2), 147 - 58 Phage display of peptide/major histocompatibility complex; Le Doussal J et al.; To date, there is no direct way to determine the antigenic specificity of T-cells . While B-cell epitopes can be selected from phage-displayed libraries of peptides, the corresponding molecular tool for identifying T-cell epitopes does not yet exist . The natural ligands of the T-cell antigen-receptor (TCR) are essentially antigenic peptides (P) associated with the products of the major histocompatibility complex (MHC) . Here, we report phages displaying P-MHC complexes . Single-chain P-MHC class I molecules, produced in E . coli periplasm, stimulate T-cells in a peptide-specific fashion . The same P-MHC, fused at the tip of filamentous phage, directed their binding to a recombinant TCR restricted to the displayed MHC haplotype (H-2K(d)) . Importantly, the binding of P-K(d)-fd to a K(d)-restricted TCR, and also to K(d)-restricted T-cell hybridomas, was modulated by the displayed peptide . Therefore, we suggest phage display of P-MHC as a direct molecular tool for probing T-cell specificity, and for selecting TCR ligands from genetic libraries encoding randomized or natural peptides. J Biol Chem, 2000 Oct 13, 275(41), 31689 - 94 From malate dehydrogenase to phenyllactate dehydrogenase . Incorporation of unnatural amino acids to generate an improved enzyme-catalyzed activity; Wright SK et al.; Malate dehydrogenase (MDH) from Escherichia coli is highly specific for its keto acid substrate . The placement of the active site-binding groups in MDH effectively discriminates against both the shorter and the longer keto dicarboxylic acids that could potentially serve as alternative substrates . A notable exception to this specificity is the alternative substrate phenylpyruvate . This aromatic keto acid can be reduced by MDH, albeit at a somewhat slower rate and with greatly diminished affinity, despite the presence of several substrate-binding arginyl residues and the absence of a hydrophobic pocket in the active site . The specificity of MDH for phenylpyruvate has now been enhanced, and that for the physiological substrate oxaloacetate has been diminished, through the replacement of one of the binding arginyl residues with several unnatural alkyl and aryl amino acid analogs . This approach, called site-specific modulation, incorporates systematic structural variations at a site of interest . Molecular modeling studies have suggested a structural basis for the affinity of native MDH for phenylpyruvate and a rationale for the improved catalytic activity that is observed with these new, modified phenyllactate dehydrogenases. J Biol Chem, 2000 Nov 17, 275(46), 35868 - 75 Flavohemoglobin Hmp affords inducible protection for Escherichia coli respiration, catalyzed by cytochromes bo' or bd, from nitric oxide; Stevanin TM et al.; Respiration of Escherichia coli catalyzed either by cytochrome bo' or bd is sensitive to micromolar extracellular NO; extensive, transient inhibition of respiration increases as dissolved oxygen tension in the medium decreases . At low oxygen concentrations (25-33 microm), the duration of inhibition of respiration by 9 microm NO is increased by mutation of either oxidase . Respiration of an hmp mutant defective in flavohemoglobin (Hmp) synthesis is extremely NO-sensitive (I(50) about 0.8 microm); conversely, cells pre-grown with sodium nitroprusside or overexpressing plasmid-borne hmp(+) are insensitive to 60 microm NO and have elevated levels of immunologically detectable Hmp . Purified Hmp consumes O(2) at a rate that is instantaneously and extensively (>10-fold) stimulated by NO due to NO oxygenase activity but, in the absence of NO, Hmp does not contribute measurably to cell oxygen consumption . Cyanide binds to Hmp (K(d) 3 microm) . Concentrations of KCN (100 microm) that do not significantly inhibit cell respiration markedly suppress the protection of respiration from NO afforded by Hmp and abolish NO oxygenase activity of purified Hmp . The results demonstrate the role of Hmp in protecting respiration from NO stress and are discussed in relation to the energy metabolism of E . coli in natural O(2)-depleted environments. Exp Neurol, 2000 Aug, 164(2), 292 - 302 Schwann cells transplanted into normal and X-irradiated adult white matter do not migrate extensively and show poor long-term survival; Iwashita Y et al.; Although Schwann cells are able to enter the central nervous system (CNS) when the integrity of the glia limitans is disrupted, their ability to migrate through intact CNS remains unclear . We have addressed this issue by transplanting lacZ-labeled Schwann cells into normal adult spinal cord white matter, and into X-irradiated spinal cord (an environment that, unlike normal spinal cord, permits the migration of transplanted oligodendrocyte progenitors) . Schwann cell cultures, obtained from neonatal rat sciatic nerve and expanded using bovine pituitary extract and forskolin, were transfected by repeated exposure to retroviral vectors encoding the Escherichia coli lacZ gene . The normal behavior of the transduced cells was confirmed by transplantation into a nonrepairing area of demyelination in the spinal cord, where they formed myelin sheaths around demyelinated axons . A single microliter containing 4 x 10(4) cells was then transplanted into unlesioned normal and X-irradiated white matter of the spinal cord of adult syngeneic rats . One hour after injection, blue cells were observed as a discrete mass within the dorsal funiculus with a longitudinal distribution of 2-3 mm, indicating the extent of passive spread of the injected cells . At subsequent survival times (1, 2, and 4 weeks posttransplantation) blue cells had a distribution that was no more extensive than that seen 1 h after transplantation . However, the number of Schwann cells declined with time following transplantation such that at 4 weeks there were few surviving Schwann cells in both X-irradiated and nonirradiated spinal cord . These results indicate that transplanted Schwann cells do not migrate extensively and show poor long-term survival when introduced into a normal CNS environment . J Infect Dis, 2000 Aug, 182(2), 526 - 33 Epub 2000 Jul 24. In vivo neutrophil dysfunction in cirrhotic patients with advanced liver disease; Fiuza C et al.; Bacterial infections are frequent, life-threatening complications in cirrhotic patients . This study investigated in vivo neutrophil migration and phagocytic activity in cirrhotic patients with advanced liver disease, in liver transplant recipients, and in healthy volunteers, by use of the skin window technique . Complement receptor type III (CR3) expression was also measured in blood and elicited neutrophils . Neutrophil migration to skin windows and neutrophil in vivo phagocytosis of heat-killed Escherichia coli were significantly decreased in cirrhotic patients compared with healthy controls . Neutrophil migration and phagocytosis were decreased in cirrhotic patients with previous episodes of bacterial infection compared with noninfected patients . Expression of CR3 in circulating neutrophils was significantly higher in cirrhotic patients, whereas it was significantly reduced in elicited neutrophils of cirrhotic patients with previous bacterial infection . These data suggest that deficient neutrophil recruitment to the infection site and impaired phagocytic activity may contribute to bacterial infections in cirrhotic patients with advanced liver disease. Clin Cancer Res, 2000 Jul, 6(7), 2835 - 43 Immunotoxins with increased activity against epidermal growth factor receptor vIII-expressing cells produced by antibody phage display; Beers R et al.; Recombinant immunotoxins are fusion proteins composed of Fv regions of antibodies and bacterial or plant toxins that are being developed for the targeted therapy of cancer . MR1(Fv)-PE38 is a single-chain recombinant immunotoxin that targets a mutant form of the epidermal growth factor receptor (EGFR), EGFRvIII, that is frequently overexpressed in malignant glioblastomas . We have used random complementarity determining region (CDR) mutagenesis to obtain mutants of MR1(Fv) with an increased affinity for EGFRvIII and an increased activity when converted to a recombinant immunotoxin . Initially, nine residues of heavy chain CDR3 were randomly mutagenized, and several mutants with increased binding affinity were isolated . All mutations were located at amino acids 98 and 99, which correspond to a DNA hot spot, a DNA sequence that mutates at high frequency during natural antibody maturation . A specific region of variable region of antibody light chain CDR3 was mutagenized that corresponded to a hot spot and a mutant (MR1-1) with an additional increase in affinity, and cytotoxic activity was isolated . These studies show that targeting hot spots in the CDRs of Fvs is an effective approach to obtaining Fvs with increased affinity . The increased affinity of MR1-1(Fv) makes it an attractive candidate for the targeted therapy of glioblastomas. Can J Microbiol, 2000 Jun, 46(6), 577 - 83 Eight UCA codons differentially affect the expression of the lacZ gene in the divE42 mutant of Escherichia coli; Kubo T et al.; In the divE mutant, which has a temperature-sensitive mutation in the tRNA1(Ser) gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature . In Escherichia coli, the UCA codon is only recognized by tRNA1(Ser) . Several genes containing UCA codons are normally expressed at 42 degrees C in the divE mutant . Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1(Ser) . In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant . We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA . It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462 . A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant . We propose a model to explain these results. Can J Microbiol, 2000 Jun, 46(6), 532 - 41 Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter; Donovan RS et al.; The expression of a monoclonal antibody Fab fragment in Escherichia coli strain RB791/pComb3, induced with either lactose or isopropyl-beta-D-thiogalactoside (IPTG), was compared to determine if lactose might provide an inexpensive alternative to induction with IPTG . Induction of Fab expression imposed a metabolic load on the recombinant cells, resulting in lower final cell yields compared to the non-induced controls . An IPTG concentration of 0.05 mM was sufficient to achieve maximal expression of soluble Fab protein when inducing in the early-, mid-, or late-log phases of batch cultures grown using either glucose or glycerol as a carbon source . The largest overall yield of Fab fragments when using 0.05 mM IPTG was achieved by increasing the final yield of cells through glycerol feeding following induction in late-log phase . Lactose was as effective as IPTG for inducing Fab expression in E . coli RB791/pComb3 . The greatest overall level of Fab expression was found when cells grown on glycerol were induced with 2 g/L lactose in late-log phase . Since the cost of 0.05 mM of IPTG is significantly greater than the cost of 2 g/L lactose, lactose provides an inexpensive alternative to IPTG for inducing the expression of Fab fragments, and possibly other recombinant proteins, from the E . coli lac promoter. FEMS Microbiol Lett, 2000 Aug 1, 189(1), 1 - 8 Antibody engineering and its applications in tumor targeting and intracellular immunization; Chames P et al.; During the last decade, recombinant antibody engineering has emerged as one of the most promising approaches for the design, selection and production of molecules for basic research, medicine and the pharmaceutical industry . This MiniReview describes the major findings that have led to the development of this powerful technique, with an emphasis on the use of Escherichia coli and filamentous phage as a tool allowing powerful selection procedures from large libraries as well as the use of intracellular expression of antibody fragments as a new class of neutralizing molecules with a potential use in therapy . The future of these rapidly evolving technologies is discussed. FEMS Microbiol Lett, 2000 Jul 15, 188(2), 197 - 202 Frame-shift mutations in NAD(P)H flavin oxidoreductase encoding gene (frxA) from metronidazole resistant Helicobacter pylori ATCC43504 and its involvement in metronidazole resistance; Kwon DH et al.; Metronidazole is a critical ingredient for combination therapies of Helicobacter pylori infection, the major cause of peptic ulcer and gastric cancer . It has been recently reported that metronidazole resistance from H . pylori ATCC43504 is caused by the insertion of a mini-IS605 sequence and deletion of sequences in an oxygen insensitive NAD(P)H nitroreductase encoding gene (rdxA) . We also found that an additional gene (frxA) encoding NAD(P)H flavin oxidoreductase in the same strain was truncated by frame-shift mutations . To assess whether the frxA truncation is also involved in metronidazole resistance, metronidazole sensitive H . pylori strains ATCC43629 and SS1 were transformed by the truncated frxA gene cloned from strain ATCC43504 . All transformed cells grew on agar plates containing 16 microg ml(-1) of metronidazole . The involvement of the frxA gene in metronidazole resistance was also confirmed by insertion inactivation of frxA and/or rdxA genes from strain ATCC43629 and one metronidazole sensitive clinical isolate H . pylori 2600 . In addition, the frxA gene cloned from the H . pylori 2600 showed metronidazole nitroreductase activity in Escherichia coli and rendered ordinary metronidazole resistant E . coli to metronidazole sensitive cell . These results indicate that the frxA gene may also be involved in metronidazole resistance among clinical H . pylori isolates. FEMS Microbiol Lett, 2000 Jul 15, 188(2), 141 - 6 Analysis of the dormancy-inducible narK2 promoter in Mycobacterium bovis BCG; Hutter B et al.; Upon depletion of oxygen, the obligate aerobe mycobacteria switch from growth to a state of non-replicating persistence or dormancy . Here, we report the first functional analysis of a dormancy-dependent mycobacterial promoter in Mycobacterium bovis BCG . Promoter probing using a 'lacZ reporter detected a dormancy-inducible promoter activity upstream of the coding sequence for the putative nitrite extrusion protein NarK2 . Primer extension analysis mapped a transcriptional start point 47 bp upstream of the narK2 start codon . Deletion analysis revealed that the sequence -222 to -133 bp upstream from the transcriptional start point was required for basal and dormancy-inducible reporter expression . The sequence +1 to +47 downstream of the transcriptional start point had a strong inhibitory effect on the level of dormancy-induced beta-galactosidase activity . The identification of apparent activating and inhibiting regions suggests that the narK2 promoter is at least under dual control. FEBS Lett, 2000 Jul 7, 476(3), 266 - 71 Functional Fv fragment of an antibody specific for CD28: Fv-mediated co-stimulation of T cells; Takemura S et al.; The most predominant co-stimulation pathway, which is critical for T cell activation and proliferation, is the CD28-B7 pathway . The anti-CD28 monoclonal antibody (mAb) also provides a co-stimulatory signal to T cells . In order to construct a functional Fv fragment (complex of VH and VL domains) of anti-CD28 antibody using a bacterial expression system, cDNA encoding the variable regions of immunoglobulin from 15E8 hybridoma cells was cloned and expressed in Escherichia coli . The Fv fragment was obtained as a soluble protein from the periplasmic fraction and showed a binding pattern similar to parental IgG . The Fv fragment induced proliferation of peripheral blood mononuclear cells in the presence of anti-CD3 or anti-CD2 mAb and enhanced anti-tumor activity of anti-MUC1x(anti)-CD3 bispecific antibody when tested with lymphokine-activated killer cells with T cell phenotype . Thus, the anti-CD28 Fv fragment will be promising not only for the study of co-stimulation, but also for cancer immunotherapy. FEBS Lett, 2000 Jul 7, 476(3), 229 - 33 Nascent Lep inserts into the Escherichia coli inner membrane in the vicinity of YidC, SecY and SecA; Houben EN et al.; Targeting and assembly of the Escherichia coli inner membrane protein leader peptidase (Lep) was studied using a homologous in vitro targeting/translocation assay . Assembly of full-length Lep was efficient in the co-translational presence of membrane vesicles and hardly occurred when membranes were added post-translationally . This is consistent with the signal recognition particle-dependent targeting of Lep . Crosslinking experiments showed that the hydrophilic region P1 of nascent membrane-inserted Lep 100-mer was in the vicinity of SecA and SecY, whereas the first transmembrane domain H1 was in the vicinity of YidC . These results suggested that YidC, together with the Sec translocase, functions in the assembly of Lep . YidC might be a more generic component in the assembly of inner membrane proteins. FEBS Lett, 2000 Jul 7, 476(3), 174 - 8 Effect of DNA topology on plasmid DNA repair in vivo; Park JY et al.; Escherichia coli nucleotide excision repair (NER) is responsible for removing bulky DNA adducts by dual incisions of the UvrABC endonuclease . Although the activity of the UvrAB complex which can induce DNA conformational change is employed in NER, the involvement of DNA topology and DNA topoisomerases remains unclear . We examined the effect of topoisomerase inhibitions on a NER in vivo system . The repair analysis of intracellular plasmid revealed that the DNA damage on positive supercoils generated by gyrase inhibition remained unrepaired, whereas the DNA damage was repaired in topoisomerase I mutants . These results suggest that DNA topology affects the NER process and the removal of positive supercoils by gyrase is vital for the efficiency of the E . coli NER system. FEBS Lett, 2000 Jul 7, 476(3), 113 - 7 Kinetic model of ATP synthase: pH dependence of the rate of ATP synthesis; Jain S et al.; Recently, a novel molecular mechanism of torque generation in the F(0) portion of ATP synthase was proposed {Rohatgi, Saha and Nath (1998) Curr . Sci . 75, 716-718} . In this mechanism, rotation of the c-subunit was conceived to take place in 12 discrete steps of 30 degrees each due to the binding and unbinding of protons to/from the leading and trailing Asp-61 residues of the c-subunit, respectively . Based on this molecular mechanism, a kinetic scheme has been developed in this work . The scheme considers proton transport driven by a concentration gradient of protons across the proton half-channels, and the rotation of the c-subunit by changes in the electrical potential only . This kinetic scheme has been analyzed mathematically and an expression has been obtained to explain the pH dependence of the rate of ATP synthesis by ATP synthase under steady state operating conditions . For a single set of three enzymological kinetic parameters, this expression predicts the rates of ATP synthesis which agree well with the experimental data over a wide range of pH(in) and pH(out) . A logical consequence of our analysis is that DeltapH and Deltapsi are kinetically inequivalent driving forces for ATP synthesis. Biochemistry, 2000 Aug 1, 39(30), 9018 - 29 Porphobilinogen synthase from pea: expression from an artificial gene, kinetic characterization, and novel implications for subunit interactions; Kervinen J et al.; Porphobilinogen synthase (PBGS) is present in all organisms that synthesize tetrapyrroles such as heme, chlorophyll, and vitamin B(12) . The homooctameric metalloenzyme catalyzes the condensation of two 5-aminolevulinic acid molecules to form the tetrapyrrole precursor porphobilinogen . An artificial gene encoding PBGS of pea (Pisum sativum L.) was designed to overcome previous problems during bacterial expression caused by suboptimal codon usage and was constructed by recursive polymerase chain reaction from synthetic oligonucleotides . The recombinant 330 residue enzyme without a putative chloroplast transit peptide was expressed in Escherichia coli and purified in 100-mg quantities . The specific activity is protein concentration dependent, which indicates that a maximally active octamer can dissociate into less active smaller units . The enzyme is most active at slightly alkaline pH; it shows two pK(a) values of 7.4 and 9.7 . Atomic absorption spectroscopy shows maximal binding of three Mg(II) per subunit; kinetic data support two functionally distinct types of Mg(II) and the third appears to be nonphysiologic and inhibitory . Analysis of the protein concentration dependence of the specific activity suggests that the minimal functional unit is a tetramer . A model of octameric pea PBGS was built to predict the location of intermolecular disulfide linkages that were revealed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis . As verified by site-specific mutagenesis, disulfide linkages can form between four cysteines per octamer, each located five amino acids from the C-terminus . These data are consistent with the protein undergoing conformational changes and the idea that whole-body motion can occur between subunits. Biochemistry, 2000 Aug 1, 39(30), 8878 - 87 The N-terminal fragment of human parathyroid hormone receptor 1 constitutes a hormone binding domain and reveals a distinct disulfide pattern; Grauschopf U et al.; The N-terminal extracellular parts of human G-protein coupled receptor class B, for example, receptors for secretin, glucagon, or parathyroid hormone, are involved in ligand binding . To obtain structural and functional information on the N-terminal receptor fragment of human parathyroid hormone receptor 1 (PTHR1), the truncated receptor was expressed in the cytosol of Escherichia coli in the form of inclusion bodies . Oxidative refolding of inclusion body material resulted in stable, soluble, monomeric protein . Ligand binding was proved by surface plasmon resonance spectroscopy and isothermal titration calorimetry . Refolded receptor fragment was able to bind parathyroid hormone with an apparent dissociation constant of 3-5 microM . Far-UV circular dichroism spectra showed that the refolded polypeptide contained approximately 25% alpha-helical and 23% beta-sheet secondary structures . Analysis of the disulfide bond pattern of the refolded receptor fragment revealed disulfide bonds between Cys170 and Cys131, Cys148 and Cys108, and Cys117 and Cys48 . These results demonstrate that the extracellular N-terminal domain of the parathyroid hormone receptor (PTHR1) possesses a well-defined, stable conformation, which shows a significant ligand binding activity. Biochemistry, 2000 Aug 1, 39(30), 8791 - 802 Molecular structure of Escherichia coli PurT-encoded glycinamide ribonucleotide transformylase; Thoden JB et al.; In Escherichia coli, the PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, catalyzes an alternative formylation of glycinamide ribonucleotide (GAR) in the de novo pathway for purine biosynthesis . On the basis of amino acid sequence analyses, it is known that the PurT transformylase belongs to the ATP-grasp superfamily of proteins . The common theme among members of this superfamily is a catalytic reaction mechanism that requires ATP and proceeds through an acyl phosphate intermediate . All of the enzymes belonging to the ATP-grasp superfamily are composed of three structural motifs, termed the A-, B-, and C-domains, and in each case, the ATP is wedged between the B- and C-domains . Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E . coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR . The latter structure is of special significance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in ribonucleotide binding . Specifically in PurT transformylase, the GAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363 . Key amino acid side chains involved in binding the AMPPNP to the enzyme include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267 . Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 A from one of the gamma-phosphoryl oxygens of the AMPPNP. Biochemistry, 2000 Aug 1, 39(30), 8719 - 27 Determination of the structure of Escherichia coli glyoxalase I suggests a structural basis for differential metal activation; He MM et al.; The metalloenzyme glyoxalase I (GlxI) converts the nonenzymatically produced hemimercaptal of cytotoxic methylglyoxal and glutathione to nontoxic S-D-lactoylglutathione . Human GlxI, for which the structure is known, is active in the presence of Zn(2+) . Unexpectedly, the Escherichia coli enzyme is inactive in the presence of Zn(2+) and is maximally active with Ni(2+) . To understand this difference in metal activation and also to obtain a representative of the bacterial enzymes, the structure of E . coli Ni(2+)-GlxI has been determined . Structures have also been determined for the apo enzyme as well as complexes with Co(2+), Cd(2+), and Zn(2+) . It is found that each of the protein-metal complexes that is catalytically active has octahedral geometry . This includes the complexes of the E . coli enzyme with Ni(2+), Co(2+), and Cd(2+), as well as the structures reported for the human Zn(2+) enzyme . Conversely, the complex of the E . coli enzyme with Zn(2+) has trigonal bipyramidal coordination and is inactive . This mode of coordination includes four protein ligands plus a single water molecule . In contrast, the coordination in the active forms of the enzyme includes two water molecules bound to the metal ion, suggesting that this may be a key feature of the catalytic mechanism . A comparison of the human and E . coli enzymes suggests that there are differences between the active sites that might be exploited for therapeutic use. Biochemistry, 2000 Jul 25, 39(29), 8632 - 7 Colicin pore-forming domains bind to Escherichia coli trimeric porins; Dover LG et al.; Colicin N kills sensitive Escherichia coli cells by first binding to its trimeric receptor (OmpF) via its receptor binding domain . It then uses OmpF to translocate across the outer membrane and in the process it also needs domains II and III of the protein TolA . Recent studies have demonstrated sodium dodecyl sulfate- (SDS) dependent complex formation between trimeric porins and TolA-II . Here we demonstrate that colicin N forms similar complexes with the same trimeric porins and that this association is unexpectedly solely dependent upon the pore-forming domain (P-domain) . No binding was seen with the monomeric porin OmpA . In mixtures of P-domain and TolA with OmpF porin, only binary and no ternary complexes were observed, suggesting that binding of these proteins to the porin is mutually exclusive . Pull-down assays in solution show that porin-P-domain complexes also form in the presence of outer membrane lipopolysaccharide . This indicates that an additional colicin-porin interaction may occur within the outer membrane, one that involves the colicin pore domain rather than the receptor-binding domain . This may help to explain the role of porins and TolA-II in the later stages of colicin translocation. Biochemistry, 2000 Jul 25, 39(29), 8603 - 8 GDP-mannose mannosyl hydrolase catalyzes nucleophilic substitution at carbon, unlike all other Nudix hydrolases; Legler PM et al.; GDP-mannose mannosyl hydrolase (GDPMH) from Escherichia coli is a 36 . 8 kDa homodimer which, in the presence of Mg(2+), catalyzes the hydrolysis of GDP-alpha-D-mannose or GDP-alpha-D-glucose to yield sugar and GDP . On the basis of its amino acid sequence, GDPMH is a member of the Nudix family of enzymes which catalyze the hydrolysis of nucleoside diphosphate derivatives by nucleophilic substitution at phosphorus . However, GDPMH has a sequence rearrangement (RE to ER) in the conserved Nudix motif and is missing a Glu residue characteristic of the Nudix signature sequence . By (1)H NMR, the initial hydrolysis product of GDP-alpha-D-glucose is beta-D-glucose, indicating nucleophilic substitution with inversion at C1' of glucose . Substitution at carbon was confirmed by two-dimensional (1)H-(13)C HSQC spectra of the products of hydrolysis in 48.4% (18)O-labeled water which showed an additional C1' resonance of beta-D-glucose with a typical upfield (18)O isotope shift of 18 ppb and an intensity of 47.6% of the total signal . No (18)O isotope-shifted resonances (<4%) were found in the (31)P NMR spectrum of the GDP product . Thus, unlike all other Nudix enzymes studied so far, GDPMH catalyzes nucleophilic substitution at carbon rather than at phosphorus . A small solvent kinetic deuterium isotope effect on k(cat) of 1.76 +/- 0.25, independent of pH over the range of 6.0-9.3, suggests that the deprotonation of water may be part of the rate-limiting step. Biochemistry, 2000 Jul 25, 39(29), 8448 - 59 Restricted motion of the lipoyl-lysine swinging arm in the pyruvate dehydrogenase complex of Escherichia coli; Jones DD et al.; The three lipoyl (E2plip) domains of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase (PDH) complex of Escherichia coli house the lipoyl-lysine side chain essential for active-site coupling and substrate channelling within the complex . The structure of the unlipoylated form of the innermost domain (E2plip(apo)) was determined by multidimensional NMR spectroscopy and found to resemble closely that of a nonfunctional hybrid domain determined previously {Green et al . (1995) J . Mol . Biol . 248, 328-343} . The domain comprises two four-stranded beta-sheets, with the target lysine residue residing at the tip of a type-I beta-turn in one of the sheets; the N- and C-termini lie close together at the opposite end of the molecule in the other beta-sheet . Measurement of (15)N NMR relaxation parameters and backbone hydrogen/deuterium (H/D) exchange rates reveals that the residues in and surrounding the lipoyl-lysine beta-turn in the E2plip(apo) form of the domain become less flexible after lipoylation of the lysine residue . This implies that the lipoyl-lysine side chain may not sample the full range of conformational space once thought . Moreover, reductive acetylation of the lipoylated domain (E2plip(holo) --> E2plip(redac)) was accompanied by large changes in chemical shift between the two forms, and multiple resonances were observed for several residues . This implies a change in conformation and the existence of multiple conformations of the domain on reductive acetylation, which may be important in stabilizing this catalytic intermediate. Biochemistry, 2000 Jul 25, 39(29), 8418 - 25 Active site of lysyl-tRNA synthetase: structural studies of the adenylation reaction; Desogus G et al.; Aminoacyl-tRNA synthetases play a key role in protein biosynthesis by catalyzing the specific aminoacylation of tRNA . The energy required for the formation of the ester bond between the amino acid carboxylate group and the tRNA acceptor stem is supplied by coupling the reaction to the hydrolysis of ATP . Lysyl-tRNA synthetase from Escherichia coli belongs to the family of class II synthetases and carries out a two-step reaction, in which lysine is activated by being attached to the alpha-phosphate of AMP before being transferred to the cognate tRNA . Crystals of the thermo-inducible E . coli lysyl-tRNA synthetase LysU which diffract to 2.1 A resolution have been used to determine crystal structures of the enzyme in the presence of lysine, the lysyl-adenylate intermediate, and the nonhydrolyzable ATP analogue AMP-PCP . Additional data have been obtained from crystals soaked in a solution containing ATP and Mn(2+) . The refined crystal structures give "snapshots" of the active site corresponding to key steps in the aminoacylation reaction and provide the structural framework for understanding the mechanism of lysine activation . The active site of LysU is shaped to position the substrates for the nucleophilic attack of the lysine carboxylate on the ATP alpha-phosphate . No residues are directly involved in catalysis, but a number of highly conserved amino acids and three metal ions coordinate the substrates and stabilize the pentavalent transition state . A loop close to the catalytic pocket, disordered in the lysine-bound structure, becomes ordered upon adenine binding. J Biol Chem, 2000 Oct 27, 275(43), 33297 - 301 Identification of subunits a, b, and c1 from Acetobacterium woodii Na+-F1F0-ATPase . Subunits c1, c2, AND c3 constitute a mixed c-oligomer; Aufurth S et al.; The Na(+)-F(1)F(0)-ATPase operon of Acetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunit a and b, a gene encoding a 16-kDa proteolipid (subunit c(1)), and two genes encoding 8-kDa proteolipids (subunits c(2) and c(3)) . Because subunits a, b, and c(1) were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme . The genes were overproduced, and specific antibodies were raised . Western blots revealed that subunits a, b, and c(1) are produced and localized in the cytoplasmic membrane . Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis . N-terminal sequence analyses revealed the presence of subunits a, c(2), c(3), b, delta, alpha, gamma, beta, and epsilon . Biochemical and immunological analyses revealed that subunits c(1), c(2), and c(3) are all part of the c-oligomer, the first of a F(1)F(0)-ATPase that contains 8- and 16-kDa proteolipids. J Biol Chem, 2000 Oct 13, 275(41), 32277 - 80 MioC is an FMN-binding protein that is essential for Escherichia coli biotin synthase activity in vitro; Birch OM et al.; Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro . We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase . We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization . Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide . The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein . The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin. J Biol Chem, 2000 Oct 13, 275(41), 31594 - 600 Changing the donor cofactor of bovine alpha 1, 3-galactosyltransferase by fusion with UDP-galactose 4-epimerase . More efficient biocatalysis for synthesis of alpha-Gal epitopes; Chen X et al.; Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC ) and alpha1, 3-galactosyltransferase (EC ) with an N-terminal His(6) tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3' terminus (f1) or to the 5' terminus (f2) of a truncated bovine alpha1, 3-galactosyltransferase gene, respectively . Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins . Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes . Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of alpha1, 3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galalpha1,3Gal-terminated oligosaccharides by more than 40-fold . For enzymatic synthesis of Galalpha1,3Galbeta1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase . These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity . The kinetic parameters suggested a random mechanism for the substrate binding of the alpha1, 3-galactosyltransferase . This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one. J Biol Chem, 2000 Oct 6, 275(40), 30970 - 6 Homophilic interaction of junctional adhesion molecule; Bazzoni G et al.; Junctional adhesion molecule (JAM) is an integral membrane protein that belongs to the immunoglobulin superfamily, localizes at tight junctions, and regulates both paracellular permeability and leukocyte transmigration . To investigate molecular determinants of JAM function, the extracellular domain of murine JAM was produced as a recombinant soluble protein (rsJAM) in insect cells . rsJAM consisted in large part of noncovalent homodimers, as assessed by analytical ultracentrifugation . JAM dimers were also detected at the surface of Chinese hamster ovary cells transfected with murine JAM, as evaluated by cross-linking and immunoprecipitation . Furthermore, fluid-phase rsJAM bound dose-dependently solid-phase rsJAM, and such homophilic binding was inhibited by anti-JAM Fab BV11, but not by Fab BV12 . Interestingly, Fab BV11 exclusively bound rsJAM dimers (but not monomers) in solution, whereas Fab BV12 bound both dimers and monomers . Finally, we mapped the BV11 and BV12 epitopes to a largely overlapping sequence in proximity of the extracellular amino terminus of JAM . We hypothesize that rsJAM dimerization induces a BV11-positive conformation which in turn is critical for rsJAM homophilic interactions . Dimerization and homophilic binding may contribute to both adhesive function and junctional organization of JAM. J Biol Chem, 2000 Oct 20, 275(42), 33110 - 5 Analytical sedimentation of the IIAChb and IIBChb proteins of the Escherichia coli N,N'-diacetylchitobiose phosphotransferase system . Demonstration of a model phosphotransfer transition state complex; Keyhani N et al.; The phosphoenolpyruvate:glycose transferase system (PTS) is a prototypic signaling system responsible for the vectorial uptake and phosphorylation of carbohydrate substrates . The accompanying papers describe the proteins and product of the Escherichia coli N, N-diacetylchitobiose ((GlcNAc)(2)) PTS-mediated permease . Unlike most PTS transporters, the Chb system is composed of two soluble proteins, IIA(Chb) and IIB(Chb), and one transmembrane receptor (IIC(Chb)) . The oligomeric states of PTS permease proteins and phosphoproteins have been difficult to determine . Using analytical ultracentrifugation, both dephospho and phosphorylated IIA(Chb) are shown to exist as stable dimers, whereas IIB(Chb), phospho-IIB(Chb) and the mutant Cys10SerIIB(Chb) are monomers . The mutant protein Cys10SerIIB(Chb) is unable to accept phosphate from phospho-IIA(Chb) but forms a stable higher order complex with phospho-IIA(Chb) (but not with dephospho-IIA(Chb)) . The stoichiometry of proteins in the purified complex was determined to be 1:1, indicating that two molecules of Cys10SerIIB(Chb) are associated with one phospho-IIA(Chb) dimer in the complex . The complex appears to be a transition state analogue in the phosphotransfer reaction between the proteins . A model is presented that describes the concerted assembly and disassembly of IIA(Chb)-IIB(Chb) complexes contingent on phosphorylation-dependent conformational changes, especially of IIA(Chb). J Biol Chem, 2000 Oct 20, 275(42), 33102 - 9 The transport/phosphorylation of N,N'-diacetylchitobiose in Escherichia coli . Characterization of phospho-IIB(Chb) and of a potential transition state analogue in the phosphotransfer reaction between the proteins IIA(Chb) AND IIB(Chb); Keyhani NO et al.; Enzyme II permeases of the phosphoenolpyruvate:glycose phosphotransferase system comprise one to five separately encoded polypeptides, but most contain similar domains (IIA, IIB, and IIC) . The phosphoryl group is transferred from one domain to another, with histidine as the phosphoryl acceptor in IIA and cysteine as the acceptor in certain IIB domains . IIB(Chb) is a phosphocarrier in the uptake/phosphorylation of the chitin disaccharide, (GlcNAc)(2) by Escherichia coli and is unusual because it is separately encoded and soluble . Both the crystal and solution structures of a IIB(Chb) mutant (C10S) have been reported . In the present studies, homogeneous phospho-IIB(Chb) was isolated, and the phosphoryl-Cys linkage was established by (31)P NMR spectroscopy . Rate constants for the hydrolysis of phospho-IIB(Chb) plotted versus pH gave the same shape peak reported for the model compound, butyl thiophosphate, but was shifted about 4 pH units . Evidence is presented for a stable complex between homogeneous Cys10SerIIB(Chb) (which cannot be phosphorylated) and phospho-IIA(Chb), but not with IIA(Chb) . The complex (a tetramer (3)) contains equimolar quantities of the two proteins and has been chemically cross-linked . It appears to be an analogue of the transition state complex in the reaction: phospho-IIA(Chb) + IIB(Chb) <--> IIA(Chb) + phospho-IIB(Chb) . This is apparently the first report of the isolation of a transition state analogue in a protein-protein phosphotransfer reaction. J Biol Chem, 2000 Oct 20, 275(42), 33091 - 101 Isolation and characterization of IIAChb, a soluble protein of the enzyme II complex required for the transport/phosphorylation of N, N'-diacetylchitobiose in Escherichia coli; Keyhani NO et al.; N,N'-Diacetylchitobiose is transported/phosphorylated in Escherichia coli by the (GlcNAc)(2)-specific Enzyme II permease of the phosphoenolpyruvate:glycose phosphotransferase system . IIA(Chb), one protein of the Enzyme II complex, was cloned and purified to homogeneity . IIA(Chb) and phospho-IIA(Chb) form stable homodimers () . Phospho-IIA(Chb) behaves as a typical epsilon2-N (i.e . N-3) phospho-His protein . However, the rate constants for hydrolysis of phospho-IIA(Chb) at pH 8.0 unexpectedly increased 7-fold between 25 and 37 degrees C and increased approximately 4-fold with decreasing protein concentration at 37 degrees C (but not 25 degrees C) . The data were explained by thermal denaturation studies using CD spectroscopy . IIA(Chb) and phospho-IIA(Chb) exhibit virtually identical spectra at 25 degrees C (approximately 80% alpha-helix), but phospho-IIA(Chb) loses about 30% of its helicity at 37 degrees C, whereas IIA(Chb) shows only a slight change . Furthermore, the T(m) for thermal denaturation of IIA(Chb) was 54 degrees C, only slightly affected by concentration, whereas the T(m) for phospho-IIA(Chb) was much lower, ranging from 40 to 46 degrees C, depending on concentration . In addition, divalent cations (Mg(2+), Cu(2+), and Ni(2+)) have a dramatic and differential effect on the structure, depending on the state of phosphorylation of the protein . Thus, phosphorylation destabilizes IIA(Chb) at 37 degrees C, potentially affecting the monomer/dimer transition, which correlates with its chemical instability at this temperature . The physiological consequences of this phenomenon are briefly considered. J Biol Chem, 2000 Oct 20, 275(42), 33084 - 90 The chitin disaccharide, N,N'-diacetylchitobiose, is catabolized by Escherichia coli and is transported/phosphorylated by the phosphoenolpyruvate:glycose phosphotransferase system; Keyhani NO et al.; We have previously reported that wild type strains of Escherichia coli grow on the chitin disaccharide N,N'-diacetylchitobiose, (GlcNAc)(2), as the sole source of carbon (Keyhani, N . O., and Roseman, S . (1997) Proc . Natl . Acad . Sci., U . S . A . 94, 14367-14371) . A nonhydrolyzable analogue of (GlcNAc)(2,) methyl beta-N, N'-{(3)H}diacetylthiochitobioside ({(3)H}Me-TCB), was used to characterize the disaccharide transport process, which was found to be mediated by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) . Here and in the accompanying papers (Keyhani, N . O., Boudker, O., and Roseman, S . (2000) J . Biol . Chem . 275, 33091-33101; Keyhani, N . O., Bacia, K., and Roseman, S . (2000) J . Biol . Chem . 275, 33102-33109; Keyhani, N . O., Rodgers, M., Demeler, B., Hansen, J., and Roseman, S . (2000) J . Biol . Chem . 275, 33110-33115), we report that transport of {(3)H}Me-TCB and (GlcNAc)(2) involves a specific PTS Enzyme II complex, requires Enzyme I and HPr of the PTS, and results in the accumulation of the sugar derivative as a phosphate ester . The phosphoryl group is linked to the C-6 position of the GlcNAc residue at the nonreducing end of the disaccharide . The {(3)H}Me-TCB uptake system was induced only by (GlcNAc)(n), n = 2 or 3 . The apparent K(m) of transport was 50-100 micrometer, and effective inhibitors of uptake included (GlcNAc)(n), n = 2 or 3, cellobiose, and other PTS sugars, i.e . glucose and GlcNAc . Presumably the PTS sugars inhibit by competing for PTS components . Kinetic properties of the transport system are described. J Bacteriol, 2000 Aug, 182(16), 4658 - 60 Identification, expression, and characterization of Escherichia coli guanine deaminase; Maynes JT et al.; Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein . The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically . The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a K(m) of 15 microM with guanine and a k(cat) of 3.2 s(-1) . The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG{FL}VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein . The E . coli guanine deaminase locus is 3' from an open reading frame which shows homology to a bacterial purine base permease. J Bacteriol, 2000 Aug, 182(16), 4654 - 7 3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase of Legionella pneumophila transfers two kdo residues to a structurally different lipid A precursor of Escherichia coli; Brabetz W et al.; The 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase gene of Legionella pneumophila was cloned and sequenced . Despite remarkable structural differences in lipid A, the gene complemented a corresponding Escherichia coli mutant and was shown to encode a bifunctional enzyme which transferred 2 Kdo residues to a lipid A acceptor of E . coli. J Bacteriol, 2000 Aug, 182(16), 4640 - 3 Developmental regulation of the cell division protein FtsZ in Anabaena sp . strain PCC 7120, a cyanobacterium capable of terminal differentiation; Kuhn I et al.; Heterocysts are terminally differentiated cells devoted to nitrogen fixation in the filamentous cyanobacterium Anabaena sp . strain PCC 7120 . We show here that the cell division protein FtsZ is present in vegetative cells but undetectable in heterocysts . These results provide a first rational explanation for the inability of mature heterocysts to undergo cell division. J Bacteriol, 2000 Aug, 182(16), 4628 - 31 A carboxy-terminal 16-amino-acid region of sigma(38) of Escherichia coli is important for transcription under high-salt conditions and sigma activities in vivo; Ohnuma M et al.; sigma(38) (or sigma(S), the rpoS gene product) is a sigma subunit of RNA polymerase in Escherichia coli and directs transcription from a number of stationary-phase promoters as well as osmotically inducible promoters . In this study, we analyzed the function of the carboxy-terminal 16-amino-acid region of sigma(38) (residues 315 to 330), which is well conserved among the rpoS gene products of enteric bacterial species . Truncation of this region was shown to result in the loss of sigma activity in vivo using promoter-lacZ fusion constructs, but the mutant sigma(38) retained the binding activity in vivo to the core enzyme . The in vitro transcription analysis revealed that the transcription activity of sigma(38) holoenzyme under high potassium glutamate concentrations was significantly decreased by the truncation of the carboxy-terminal tail element. J Bacteriol, 2000 Aug, 182(16), 4625 - 7 Role of the yiaR and yiaS genes of Escherichia coli in metabolism of endogenously formed L-xylulose; Ibanez E et al.; Genes yiaP and yiaR of the yiaKLMNOPQRS cluster of Escherichia coli are required for the metabolism of the endogenously formed L-xylulose, whereas yiaS is required for this metabolism only in araD mutants . Like AraD, YiaS was shown to have L-ribulose-5-phosphate 4-epimerase activity . Similarity of YiaR to several 3-epimerases suggested that this protein could catalyze the conversion of L-xylulose-5-phosphate into L-ribulose-5-phosphate, thus completing the pathway between L-xylulose and the general metabolism. J Bacteriol, 2000 Aug, 182(16), 4617 - 24 Regulation of expression of the yiaKLMNOPQRS operon for carbohydrate utilization in Escherichia coli: involvement of the main transcriptional factors; Ibanez E et al.; The yiaKLMNOPQRS (yiaK-S) gene cluster of Escherichia coli is believed to be involved in the utilization of a hitherto unknown carbohydrate which generates the intermediate L-xylulose . Transcription of yiaK-S as a single message from the unique promoter found upstream of yiaK is proven in this study . The 5' end has been located at 60 bp upstream from the ATG . Expression of the yiaK-S operon is controlled in the wild-type strain by a repressor encoded by yiaJ . No inducer molecule of the yiaK-S operon has been identified among over 80 carbohydrate or derivative compounds tested, the system being expressed only in a mutant strain lacking the YiaJ repressor . The lacZ transcriptional fusions in the genetic background of the mutant strain revealed that yiaK-S is modulated by the integration host factor and by the cyclic AMP (cAMP)-cAMP receptor protein (Crp) activator complex . A twofold increase in the induction was observed during anaerobic growth, which was independent of ArcA or Fnr . Gel mobility shift assays showed that the YiaJ repressor binds to a promoter fragment extending from -50 to +121 . These studies also showed that the cAMP-Crp complex can bind to two different sites . The lacZ transcriptional fusions of different fragments of the promoter demonstrated that binding of cAMP-Crp to the Crp site 1, centered at -106, is essential for yiaK-S expression . The 5' end of the yiaJ gene was determined, and its promoter region was found to overlap with the divergent yiaK-S promoter . Expression of yiaJ is autogenously regulated and reduced by the binding of Crp-cAMP to the Crp site 1 of the yiaK-S promoter. J Bacteriol, 2000 Aug, 182(16), 4606 - 16 sigma(BldN), an extracytoplasmic function RNA polymerase sigma factor required for aerial mycelium formation in Streptomyces coelicolor A3(2); Bibb MJ et al.; Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci . whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N'-nitro-N-nitrosoguanidine)-induced whi strains (N . J . Ryding et al., J . Bacteriol . 181:5419-5425, 1999), was defined by two mutants, R112 and R650 . R650 produced frequent spores that were longer than those of the wild type . In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement . Subcloning and sequencing showed that whiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors . A constructed whiN null mutant failed to form aerial mycelium (the "bald" phenotype) and, as a consequence, whiN was renamed bldN . This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145 . The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S . coelicolor bld mutants . Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficient bld strains . bldN was transcribed from a single promoter, bldNp . bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC, bldF, bldK, or bldJ or on bldN itself . Transcription from the p1 promoter of the response-regulator gene bldM depended on bldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for sigma(BldN) holoenzyme in vitro. J Bacteriol, 2000 Aug, 182(16), 4587 - 95 Escherichia coli DNA polymerase IV mutator activity: genetic requirements and mutational specificity; Wagner J et al.; The dinB gene of Escherichia coli is known to be involved in the untargeted mutagenesis of lambda phage . Recently, we have demonstrated that this damage-inducible and SOS-controlled gene encodes a novel DNA polymerase, DNA Pol IV, which is able to dramatically increase the untargeted mutagenesis of F' plasmid . At the amino acid level, DNA Pol IV shares sequence homologies with E . coli UmuC (DNA Pol V), Rev1p, and Rad30p (DNA polymerase eta) of Saccharomyces cerevisiae and human Rad30A (XPV) proteins, all of which are involved in translesion DNA synthesis . To better characterize the Pol IV-dependent untargeted mutagenesis, i.e., the DNA Pol IV mutator activity, we analyzed the genetic requirements of this activity and determined the forward mutation spectrum generated by this protein within the cII gene of lambda phage . The results indicated that the DNA Pol IV mutator activity is independent of polA, polB, recA, umuDC, uvrA, and mutS functions . The analysis of more than 300 independent mutations obtained in the wild-type or mutS background revealed that the mutator activity clearly promotes single-nucleotide substitutions as well as one-base deletions in the ratio of about 1:2 . The base changes were strikingly biased for substitutions toward G:C base pairs, and about 70% of them occurred in 5'-GX-3' sequences, where X represents the base (T, A, or C) that is mutated to G . These results are discussed with respect to the recently described biochemical characteristics of DNA Pol IV. J Bacteriol, 2000 Aug, 182(16), 4564 - 71 Identification and characterization of genes required for early Myxococcus xanthus developmental gene expression; Guo D et al.; Starvation and cell density regulate the developmental expression of Myxococcus xanthus gene 4521 . Three classes of mutants allow expression of this developmental gene during growth on nutrient agar, such that colonies of strains containing a Tn5 lac Omega4521 fusion are Lac(+) . One class of these mutants inactivates SasN, a negative regulator of 4521 expression; another class activates SasS, a sensor kinase-positive regulator of 4521 expression; and a third class blocks lipopolysaccharide (LPS) O-antigen biosynthesis . To identify additional positive regulators of 4521 expression, 11 Lac(-) TnV.AS transposon insertion mutants were isolated from a screen of 18,000 Lac(+) LPS O-antigen mutants containing Tn5 lac Omega4521 (Tc(r)) . Ten mutations identified genes that could encode positive regulators of 4521 developmental expression based on their ability to abolish 4521 expression during development in the absence of LPS O antigen and in an otherwise wild-type background . Eight of these mutations mapped to the sasB locus, which encodes the known 4521 regulators SasS and SasN . One mapped to sasS, whereas seven identified new genes . Three mutations mapped to a gene encoding an NtrC-like response regulator homologue, designated sasR, and four others mapped to a gene designated sasP . One mutation, designated ssp10, specifically suppressed the LPS O-antigen defect; the ssp10 mutation had no effect on 4521 expression in an otherwise wild-type background but reduced 4521 developmental expression in the absence of LPS O antigen to a level close to that of the parent strain . All of the mutations except those in sasP conferred defects during growth and development . These data indicate that a number of elements are required for 4521 developmental expression and that most of these are necessary for normal growth and fruiting body development. J Bacteriol, 2000 Aug, 182(16), 4443 - 52 Glucose transporter mutants of Escherichia coli K-12 with changes in substrate recognition of IICB(Glc) and induction behavior of the ptsG gene; Zeppenfeld T et al.; In Escherichia coli K-12, the major glucose transporter with a central role in carbon catabolite repression and in inducer exclusion is the phosphoenolpyruvate-dependent glucose:phosphotransferase system (PTS) . Its membrane-bound subunit, IICB(Glc), is encoded by the gene ptsG; its soluble domain, IIA(Glc), is encoded by crr, which is a member of the pts operon . The system is inducible by D-glucose and, to a lesser degree, by L-sorbose . The regulation of ptsG transcription was analyzed by testing the induction of IICB(Glc) transporter activity and of a single-copy Phi(ptsGop-lacZ) fusion . Among mutations found to affect directly ptsG expression were those altering the activity of adenylate cyclase (cyaA), the repressor DgsA (dgsA; also called Mlc), the general PTS proteins enzyme I (ptsI) and histidine carrier protein HPr (ptsH), and the IIA(Glc) and IIB(Glc) domains, as well as several authentic and newly isolated UmgC mutations . The latter, originally thought to map in the repressor gene umgC outside the ptsG locus, were found to represent ptsG alleles . These affected invariably the substrate specificity of the IICB(Glc) domain, thus allowing efficient transport and phosphorylation of substrates normally transported very poorly or not at all by this PTS . Simultaneously, all of these substrates became inducers for ptsG . From the analysis of the mutants, from cis-trans dominance tests, and from the identification of the amino acid residues mutated in the UmgC mutants, a new regulatory mechanism involved in ptsG induction is postulated . According to this model, the phosphorylation state of IIB(Glc) modulates IIC(Glc) which, directly or indirectly, controls the repressor DgsA and hence ptsG expression . By the same mechanism, glucose uptake and phosphorylation also control the expression of the pts operon and probably of all operons controlled by the repressor DgsA. J Bacteriol, 2000 Aug, 182(16), 4437 - 42 Substrate specificity and signal transduction pathways in the glucose-specific enzyme II (EII(Glc)) component of the Escherichia coli phosphotransferase system; Notley-McRobb L et al.; Escherichia coli adapted to glucose-limited chemostats contained mutations in ptsG resulting in V12G, V12F, and G13C substitutions in glucose-specific enzyme II (EII(Glc)) and resulting in increased transport of glucose and methyl-alpha-glucoside . The mutations also resulted in faster growth on mannose and glucosamine in a PtsG-dependent manner . By use of enhanced growth on glucosamine for selection, four further sites were identified where substitutions caused broadened substrate specificity (G176D, A288V, G320S, and P384R) . The altered amino acids include residues previously identified as changing the uptake of ribose, fructose, and mannitol . The mutations belonged to two classes . First, at two sites, changes affected transmembrane residues (A288V and G320S), probably altering sugar selectivity directly . More remarkably, the five other specificity mutations affected residues unlikely to be in transmembrane segments and were additionally associated with increased ptsG transcription in the absence of glucose . Increased expression of wild-type EII(Glc) was not by itself sufficient for growth with other sugars . A model is proposed in which the protein conformation determining sugar accessibility is linked to transcriptional signal transduction in EII(Glc) . The conformation of EII(Glc) elicited by either glucose transport in the wild-type protein or permanently altered conformation in the second category of mutants results in altered signal transduction and interaction with a regulator, probably Mlc, controlling the transcription of pts genes. J Bacteriol, 2000 Aug, 182(16), 4430 - 6 Effect of altered spacing between uhpT promoter elements on transcription activation; Chen Q et al.; Many bacterial promoters possess multiple sites for binding of transcriptional activator proteins . The uhpT promoter, which controls expression of the sugar phosphate transport system in Escherichia coli, possesses multiple sites for its specific activator protein, UhpA, and a single site for binding of the global regulator, the catabolite gene activator protein (CAP) . The binding of UhpA to the uhpT promoter was determined by DNase protection assays; UhpA displayed different affinities for the target sites . The upstream or strong sites, between positions -80 and -50, exhibited a higher affinity for UhpA than did the downstream or weak sites, between positions -50 and -32, adjoining the RNA polymerase-binding site . Phosphorylation of UhpA strongly increased its affinity for both sites . To examine the possible roles of the two sets of UhpA-binding sites, a series of insertion and deletion mutations were introduced at the boundary between them, as suggested from the positions that were protected by UhpA against hydroxyl radical cleavage . Deletions extended in the direction of the weak sites . The insertion or deletion of one helical turn of DNA resulted in the loss of promoter activity and of occupancy by UhpA of the remaining weak-site sequences but was accompanied by normal occupancy of the strong site and no change in the gel retardation behavior of the promoter fragments . However, the deletion of two helical turns of DNA, i.e., 20, 21, or 22 bp, resulted in the novel appearance of UhpA-independent expression and in an additional level of expression that was dependent on UhpA but independent of an inducing signal . The UhpA-independent promoter activity was shown to result from activation by CAP at its more proximal position . UhpA-dependent activity under noninducing conditions appears to result from the binding of unphosphorylated UhpA to the strong sites, which are now in the position normally occupied by the weak sites . Thus, regulated phosphorylation of the response regulator UhpA enhances its occupancy of the weak sites where favorable contacts can allow the binding of RNA polymerase to the promoter. Am J Physiol Endocrinol Metab, 2000 Aug, 279(2), E235 - 43 Hyperinsulinemia compensates for infection-induced impairment in net hepatic glucose uptake during TPN; Donmoyer CM et al.; In animals receiving total parenteral nutrition (TPN), infection impairs net hepatic glucose uptake (NHGU) by 40% and induces mild hyperinsulinemia . In the normal animal, the majority of the glucose taken up by the liver is diverted to lactate, but in the infected state, lactate release is curtailed . Because of the hyperinsulinemia and reduced NHGU, more glucose is utilized by peripheral tissues . Our aims were to determine the role of infection-induced hyperinsulinemia in 1) limiting the fall in NHGU and hepatic lactate release and 2) increasing the proportion of glucose disposed of by peripheral tissues . Chronically catheterized dogs received TPN for 5 days via the inferior vena cava . On day 3, a fibrin clot with a nonlethal dose of E . coli was placed into the peritoneal cavity; sham dogs received a sterile clot . On day 5, somatostatin was infused to prevent endogenous pancreatic hormone secretion, and insulin and glucagon were replaced at rates matching incoming hormone concentrations observed previously in sham or infected dogs . The TPN-derived glucose infusion was adjusted to maintain a constant arterial plasma glucose level of approximately 120 mg/dl . after a basal blood sampling period, the insulin infusion rate was either maintained constant (infected time control, Hi-Ins, n = 6; sham time control, Sham, n = 6) or decreased (infected + reduced insulin, Lo-Ins; n = 6) for 180 min to levels seen in noninfected dogs (from 23 +/- 2 to 12 +/- 1 microU/ml) . Reduction of insulin to noninfected levels decreased NHGU by 1.4 +/- 0.5 mg x kg(-1) x min(-1) (P < 0.05) and nonhepatic glucose utilization by 4.8 +/- 0.8 mg x kg(-1) x min(-1) (P < 0.01) . The fall in NHGU was caused by a decline in HGU (Delta-0.6 +/- 0.4 mg x kg(-1) x min(-1)) and a concomitant increase in hepatic glucose production (HGP, Delta0.8 +/- 0.5 mg x kg(-1) x min(-1)); net hepatic lactate release was not altered . Hyperinsulinemia that accompanies infection 1) primarily diverts glucose carbon to peripheral tissues, 2) limits the fall in NHGU by enhancing HGU and suppressing HGP, and 3) does not enhance hepatic lactate release, thus favoring hepatic glucose storage . Compensatory hyperinsulinemia plays a critical role in facilitating hepatic and peripheral glucose disposal during an infection. Eur J Gastroenterol Hepatol, 2000 Jun, 12(6), 703 - 5 Infected hepatic cyst in a patient with multiple hepatic cysts: report of a case diagnosed by change of ultrasonographic findings; Shoji F et al.; Liver cysts are commonly observed, but infection of a liver cyst is a rare complication . Although patients have clinical symptoms, such as a high-grade fever and abdominal pain, diagnosing an infected cyst by abdominal ultrasonography, computed tomography (CT) with contrast medium and magnetic resonance imagings (MRI) is not always easy . We experienced an unusual case who had only clinical symptoms, such as high-grade fever and a right quadrant abdominal pain, but no imaging findings when admitted . Careful observation using ultrasonography once a week revealed signs of an infected cyst containing echogenic fluid 32 days after admission . We performed percutaneous transhepatic cystic drainage . When a patient has liver cysts and complains of high-grade fever and abdominal pain, liver cysts should be considered as a focus of sepsis, and we recommend repeat use of ultrasonography or CT, even if no typical findings occur the first time. Eur J Gastroenterol Hepatol, 2000 Jun, 12(6), 641 - 4 Helicobacter pylori increases gastrin release from cultured canine antral G-cells; Beales IL et al.; OBJECTIVE: To investigate the mechanisms underlying the hypergastrinaemia of Helicobacter pylori by examining the effects of H . pylori on basal and stimulated gastrin release from cultured canine G-cells . METHODS: Canine antral G-cells were prepared by collagenase-EDTA digestion and cultured for 40 h . G-cells were then cultured for a further 24 h with two different H . pylori sonicates before basal and bombesin-stimulated gastrin release were measured by radioimmunoassay . RESULTS: Treatment of G-cells with both H . pylori sonicates significantly enhanced basal gastrin release (by 17-27%) and bombesin-stimulated gastrin release (by 115-133%) . This effect was independent of cagA and vacuolating cytotoxin status . Control treatment with Escherichia coli sonicate had no effect on gastrin release . There was no change in the cellular content of gastrin . CONCLUSIONS: Incubation of antral G-cells with H . pylori constituents enhances subsequent basal and bombesin-stimulated gastrin release . Direct contact between H . pylori and G-cells in the gastric antrum may be responsible for the hypergastrinaemia seen with the infection. Mol Cell, 2000 Jun, 5(6), 1025 - 34 Unexpected structural diversity in DNA recombination: the restriction endonuclease connection; Hickman AB et al.; Transposition requires a coordinated series of DNA breakage and joining reactions . The Tn7 transposase contains two proteins: TnsA, which carries out DNA breakage at the 5' ends of the transposon, and TnsB, which carries out breakage and joining at the 3' ends of the transposon . TnsB is a member of the retroviral integrase superfamily whose hallmark is a conserved DDE motif . We report here the structure of TnsA at 2.4 A resolution . Surprisingly, the TnsA fold is that of a type II restriction endonuclease . Thus, Tn7 transposition involves a collaboration between polypeptides, one containing a DDE motif and one that does not . This result indicates that the range of biological processes that utilize restriction enzyme-like folds also includes DNA transposition. Carcinogenesis, 2000 Aug, 21(8), 1559 - 66 Identification of the human liver microsomal cytochrome P450s involved in the metabolism of N-nitrosodi-n-propylamine; Teiber JF et al.; The ability of human liver cytochrome P450s to metabolize the environmental carcinogen N-nitrosodi-n-propylamine (NDPA) was investigated . The maximum rate of NDPA depropylation in seven human liver microsomal samples was 1.15 nmol/min/mg (range 0.53-2.60) . Troleandomycin, a P450 3A4/5 inhibitor, inhibited depropylation modestly (10-60%) in three of seven samples . Diethyldithiocarbamic acid, a potent 2E1 inhibitor, and a 2E1 inhibitory monoclonal antibody (mAb) inhibited the reaction in all samples (23 to almost 100%) . No significant inhibition was observed with the 2C9 inhibitor sulfaphenazole or with mAbs to 3A4, 2A6 and 2D6 . The 2C8/9/18/19 mAb inhibited depropylation in one sample by approximately 25% and approximately 25% of the activity in another sample could not be accounted for by the inhibitors . Denitrosation of NDPA by three of the microsomal samples exhibited low K(m) values (51-86 microM) while two of these also had high K(m) values (2.6 and 4.6 mM) . Purified human P450 2B6 and 3A4 and human P450 2A6, 2C8, 2C9 and 2D6 membranes had high K(m) values relative to their maximum turnover rates and are unlikely to participate in NDPA metabolism at micromolar concentrations . Conversely, purified rabbit 2E1 exhibited K(m) and V(max) values for depropylation of 52 microM and 13.4 nmol propionaldehyde/min/nmol P450, respectively . Values for denitrosation were 66 microM and 1.44 nmol nitrite/min/nmol P450, respectively . The toxicity of NDPA in transfected human liver epithelial cells expressing 2E1 was dose dependent down to 50 microM . No toxicity was observed in control cells or those expressing 2A6 . These results indicate that 2E1 is the major human liver microsomal isoform responsible for NDPA metabolism at low micromolar concentrations . We also show that purified P450s catalyze the denitrosation of NDPA at approximately 10-20% of the rate of depropylation and K(m) values for both reactions are the same for each isozyme . This is consistent with the formation of an initial intermediate common to both pathways, presumably an alpha-nitrosamino radical. Protein Expr Purif, 2000 Aug, 19(3), 419 - 24 Use of modified BL21(DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage; Kleber-Janke T et al.; We previously cloned a panel of peanut allergens by phage display technology . Examination of the codons used in these sequences indicated that most of the cDNAs contain an excess of the least used codons in Escherichia coli, namely AGG/AGA, that correspond to a minor tRNA, the product of the dnaY gene . To achieve high-level expression of the peanut allergens, the cDNAs were subcloned into an expression vector of the pET series (Novagen) in order to produce (His)(10)-tagged fusion proteins in conventional E . coli BL21(DE3) cells . The peanut allergens Ara h 1, Ara h 2, and Ara h 6 with an AGG/AGA codon content of 8-10% were only marginally expressed, whereas the peanut profilin Ara h 5, with an AGG/AGA codon content of only 0.8%, was efficiently expressed in these cells . Hence, by using modified BL21(DE3) E . coli cells, namely BL21-CodonPlus(DE3)-RIL cells (Stratagene) with extra copies of E . coli argU, ileY, and leuW tRNA genes, it was possible to attain high-level expression of the proteins affected by rare codon usage . IPTG-induced expression of several recombinant peanut allergens, such as Ara h 1, Ara h 2, and Ara h 6, was greatly increased in these special cells compared to the expression yield achieved by conventional E . coli hosts . The purification of the soluble and the insoluble fraction of Ara h 2 was performed by metal-affinity chromatography and yielded a total of about 30 mg (His)(10)-tagged recombinant protein per liter of culture of transformed BL21(DE3)CodonPlus-RIL cells . This is over 100 times more than achieved by production of Ara h 2 in conventional BL21(DE3) cells . Protein Expr Purif, 2000 Aug, 19(3), 411 - 8 Overexpression in Escherichia coli and characterization of the chloroplast fructose-1,6-bisphosphatase from wheat; Tang GL et al.; An important Calvin cycle enzyme, chloroplast fructose-1, 6-bisphosphatase (FBPase) from wheat, has been cloned and expressed up to 15% of the total cell protein using a pPLc expression vector in Escherichia coli by replacing the codons in the 5'-terminal encoding sequence with optimal and A/T-rich ones . The overexpressed wheat FBPase is soluble, fully active, and heat stable . It can be purified by chromatography in turn on DEAE-Sepharose and Sephacryl S-200, and around 15 mg of purified enzymes (>95%) is obtained from 1 liter of cultured bacteria . Its special activity is 8.8 u/mg, K(cat) is 22.9/S, K(m) is 121 microM, and V(max) is 128 micromol/min . mg . The recombinant FBPase can be activated by DTT, Na(+), or low concentrations of Li(+), Ca(2+), Zn(2+), GuHCl, and urea, while it can be inhibited by K(+) or NH(+)(4) . Protein Expr Purif, 2000 Aug, 19(3), 375 - 83 Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli; Richter W et al.; A 5'-truncated PDE4A-cDNA corresponding to the amino acid positions 200-886 of the "full-length" sequence (Accession No . L20965) was generated from human leukocyte mRNA by RT-PCR . Several PDE4A constructs containing the catalytic region and differing in their degree of N- and/or C-terminal truncation (amino acid positions 200-886, 200-704, 342-886, and 342-704) were expressed in Escherichia coli to investigate the effect of truncations on purification characteristics, long-term stability, and aggregation . All peptides accumulated as inclusion bodies, necessitating refolding prior to purification by dye and metal chelate affinity chromatography . The constructs differed in long-term stability due to variable levels of protease contamination . The position of the His-tag also influenced the purification results . The best results were obtained with the N- and C-truncated form C-terminally His-tagged, appropriate quantities of which were obtained in pure form and was found to be stable against proteolysis at 4 degrees C for at least 6 weeks . The comparison of the molecular mass of the investigated PDE4A constructs obtained by SDS electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation indicated that C-terminal truncated PDE4A forms dimers whereas PDE4A constructs with a complete C-terminus tend to form larger aggregates . Protein Expr Purif, 2000 Aug, 19(3), 355 - 61 Purification of the Caenorhabditis elegans transposase Tc1A refolded during gel filtration chromatography; Garcia-Saez I et al.; Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form . The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration . This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein . This step is very sensitive to the concentration of protein . Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein . The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography . Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking . This new Tc1A purification procedure gives a yield of 12-16 mg/liter E . coli culture, in a form suitable for crystallization studies . Protein Expr Purif, 2000 Aug, 19(3), 321 - 8 Gag-derived proteins of HIV-1 isolates from Indian patients: cloning, expression, and purification of p24 of B- and C-subtypes; Gupta S et al.; A simple and efficient method for hyperexpression in Escherichia coli and purification of capsid protein, p24, of human immunodeficiency virus type 1 (HIV-1) of both B- and C-subtypes is described . DNA-encoding p24 of C-subtype was cloned from C-subtype gag sequence which was obtained by PCR amplification using DNA extracted from peripheral blood mononuclear lymphocytes (PBMLs) of an HIV-1-infected patient from India . DNA-encoding p24B protein was amplified directly by two-step PCR using genomic DNA obtained from PBMLs of an HIV-infected individual . A T7 promoter-based expression system was optimized for hyperexpression of p24 in the soluble form . Both p24 (B- and C-subtype) were purified to near homogeneity using conventional chromatographic techniques . Purification of p24 (C subtype) was described for the first time with yield of 53 mg from 1 liter of culture . The yield of p24 (B-subtype) was 67 mg from 1 liter of culture, which was severalfold better than reported earlier . The immunoreactivity of both types of p24 to sera from HIV-infected individuals was comparable . This report describes a simple, highly efficient, and reproducible method for obtaining large quantities of highly pure p24 of both B- and C-subtype, which can be used for structural, biochemical, and immunological characterization and, eventually, for diagnostic and prognostic applications . Exp Parasitol, 2000 Jun, 95(2), 113 - 21 Plasmodium falciparum: cloned and expressed CIDR domains of PfEMP1 bind to chondroitin sulfate A; Degen R et al.; Adherence of erythrocytes infected with mature asexual Plasmodium falciparum parasites (iRBC) to microvascular endothelial cells contributes to the pathology of P . falciparum malaria . It has been shown that the variant P . falciparum erythrocyte membrane protein 1 (PfEMP1) confers adhesion to a wide range of cell surface receptors . Previously, the cysteine-rich interdomain region (CIDR) of PfEMP1 has been identified as binding site to CD36 . We provide evidence that the same region can also mediate binding to chondroitin sulfate A (CSA) . CIDR domains of two different parasite strains were expressed in Escherichia coli as a 6xHis-tagged protein . Purified recombinant protein bound to Chinese hamster ovary (CHO) cells which naturally express chondroitin sulfate A . Treatment of wild-type CHO cells with chondroitinase ABC reduced binding up to 94.4% . Competitive binding using soluble CSA inhibited binding to CHO cells by up to 100% at 2 mg/ml and by 62.4% at 0.5 mg/ml, whereas 1 mg/ml heparan sulfate had only a little effect (18.1%) . In contrast, a recombinant 6xHis-tagged DBL1 domain showed no binding to wild-type CHO cells . Such an approach of analyzing various domains of PfEMP1 as recombinant proteins may elucidate their functions and may lead to novel anti-adherence therapeutics, especially for maternal malaria infections . J Bacteriol, 2000 Aug, 182(16), 4670 - 2 H-NS-Dependent regulation of flagellar synthesis is mediated by a LysR family protein; Ko M et al.; H-NS regulates the flagellar master operon (flhDC) and thus is necessary for flagellation of Escherichia coli . However, the molecular mechanism of its regulation has remained unknown . Genetic screening of a transposon insertion abolishing the H-NS effect revealed a previously unidentified gene, named hdfR, encoding a LysR family protein . Binding of purified HdfR to the flhDC promoter was demonstrated by a DNA mobility shift assay, indicating that HdfR is a transcriptional regulator for the flagellar master operon . Furthermore, the expression of the hdfR gene was shown to be negatively regulated by H-NS. Cancer Res, 2000 Jul 1, 60(13), 3440 - 4 Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity; Hanna IH et al.; Activation of 17beta-estradiol (E2) through the formation of catechol estrogen metabolites, 2-OH-E2 and 4-OH-E2, and the C-16alpha hydroxylation product, 16alpha-OH-E2, has been postulated to be a factor in mammary carcinogenesis . Cytochrome P450 1B1 (CYP1B1) exceeds other P450 enzymes in both estrogen hydroxylation activity and expression level in breast tissue . To determine whether inherited variants of CYP1B1 differ from wild-type CYP1B1 in estrogen hydroxylase activity, we expressed recombinant wild-type and five polymorphic variants of CYP1B1: variant 1 (codon 48Arg-->Gly), variant 2 (codon 119Ala-->Ser), variant 3 (codon 432Val-->Leu), variant 4 (codon453Asn-->Ser), variant 5 (48Gly, 119Ser, 432Leu, 453Ser) . The His-tagged proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography and analyzed by electrophoresis and spectrophotometry . We performed assays of E2 hydroxylation activity and quantitated production of 2-OH-E2, 4-OH-E2, and 16alpha-OH-E2 by gas chromatography/mass spectrometry . Wild-type CYP1B1 formed 4-OH-E2 as main product (Km, 40+/-8 microM; k(cat) 4.4+/-0.4, min(-1); k(cat)/Km, 110 mM(-1) min(-1)), followed by 2-OH-E2 (Km, 34+/-4 microM; k(cat), 1.9+/-0.1 min(-1); k(cat)/Km, 55 mM(-1)min(-1)) and 16alpha-OH-E2 (Km, 39+/-5.7 microM; k(cat), 0.30+/-0.02 min(-1); k(cat)/Km, 7.6 mM(-1)min(-1)) . The CYP1B1 variants also formed 4-OH-E2 as the main product but displayed 2.4- to 3.4-fold higher catalytic efficiencies k(cat)/Km than the wild-type enzyme, ranging from 270 mM(-1)min(-1) for variant 4, to 370 mM(-1)min(-1) for variant 2 . The variant enzymes also exceeded wild-type CYP1B1 with respect to 2- and 16alpha-hydroxylation activity . Thus, inherited alterations in CYP1B1 estrogen hydroxylation activity may be associated with significant changes in estrogen metabolism and, thereby, may possibly explain interindividual differences in breast cancer risk associated with estrogen-mediated carcinogenicity. Shock, 2000 Jul, 14(1), 68 - 72 Endotoxemia alters splanchnic capacitance; Ujhelyi MR et al.; The splanchnic circulation constitutes a major portion of the total capacitance vasculature and may affect venous return and subsequently cardiac output during low output states . This study assessed the effects of rapid (10 microg/kg over 5 min) and slow (10 microg/kg over 60 min) induction of endotoxin (Escherichia coli) shock on splanchnic blood volume in 8 farm swine . Blood volume was measured by using Tc99m-labeled erythrocytes and radionuclide imaging . Baseline arterial pressure (MAP), central venous pressure (CVP), and liver, splenic, mesenteric and total splanchnic volumes were stable during the 30-min baseline . Approximately 30 min after the rapid endotoxin infusion, splenic volume decreased by 45%, whereas liver volume increased by 40% and MAP decreased by 60% (P < 0.01) . The reduction in splenic volume occurred within 10 min of the endotoxin infusion, whereas liver volume changes occurred after MAP reduction . The slow endotoxin infusion also reduced splenic volume by approximately 50% (P = 0.05), whereas MAP declined by 30% (P < 0.05) . However, the slow endotoxin infusion lowered liver volume (P < 0.05) . Mesenteric volume was unaffected by the fast or slow endotoxin infusion . Total splanchnic volume was unaffected by the fast infusion but decreased by 37% in the slow infusion group (P < 0.05) . In summary, E . coli endotoxin reduces splenic blood volume and increases liver blood volume after acute hypotension ensues . Endotoxin does not increase total splanchnic blood volume and may actually decrease total splanchnic volume in the absence of circulatory collapse . This endotoxin shock model is not associated with blood volume pooling in the splanchnic capacitance circulation. J Biomol NMR, 2000 May, 17(1), 33 - 42 Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium; Hochuli M et al.; Isotope effects on the central carbon metabolism due to the addition of variable amounts of D2O (0 to 70%) were investigated with biosynthetically directed fractional 13C-labeling for Escherichia coli BL21(DE3) cells during exponential growth on a M9 minimal medium containing a mixture of 70% unlabeled and 30% uniformly 13C-labeled glucose as the sole carbon source . The resulting 13C-labeling patterns in the amino acids were analysed by two-dimensional {13C,1H}-correlation spectroscopy . With the aforementioned growth conditions, higher D2O contents resulted in an increase of the anaplerotic supply of the tricarboxylic acid cycle via carboxylation of phosphoenolpyruvate when compared to the influx of acetyl-CoA . Furthermore, the addition of D2O affected the C1 metabolic pathways that involve Ser and Gly . Otherwise the E . coli cells showed identical topologies of the active biosynthetic pathways in H2O and at elevated D2O contents, and the metabolic flux ratios characterizing glycolysis and the pentose phosphate pathway were not measurably affected by the addition of D2O . Cells that had been adapted for growth in D2O exhibited the same response to the presence of D2O in the nutrient medium as non-adapted cells . Implications of these data for the preparation of recombinant deuterated proteins for NMR studies are discussed. FEBS Lett, 2000 Jul 21, 477(3), 278 - 82 ATPase cycle controls the conformation of an archaeal chaperonin as visualized by cryo-electron microscopy; Gutsche I et al.; Chaperonins are double-ring protein folding machines fueled by ATP binding and hydrolysis . Conformational rearrangements upon ATPase cycling of the group I chaperonins, typified by the Escherichia coli GroEL/GroES system, have been thoroughly investigated by cryo-electron microscopy and X-ray crystallography . For archaeal group II chaperonins, however, these methods have so far failed to provide a correlation between the structural and the functional states . Here, we show that the conformation of the native alphabeta-thermosome of Thermoplasma acidophilum in vitrified ice is strictly regulated by adenine nucleotides. FEBS Lett, 2000 Jul 21, 477(3), 224 - 9 The HslU ATPase acts as a molecular chaperone in prevention of aggregation of SulA, an inhibitor of cell division in Escherichia coli; Seong IS et al.; HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase . SulA, which is an inhibitor of cell division and has high tendency of aggregation, is degraded by HslVU protease . Here we show that HslU plays a role not only as a regulatory component for the HslV-mediated proteolysis but also as a molecular chaperone . Purified HslU prevented aggregation of SulA in a concentration-dependent fashion . This chaperone activity required oligomerization of HslU subunits, which could be achieved by ATP-binding or in the presence of high HslU protein concentrations . hsl mutation reduced the SulA-mediated inhibition of cell growth and this effect could be reversed upon overproduction of HslU, suggesting that HslU promotes the ability of SulA to block cell growth through its chaperone function . Thus, HslU appears to have two antagonistic functions: one as a chaperone for promotion of the ability of SulA in cell growth inhibition by preventing SulA aggregation and the other as the regulatory component for elimination of SulA by supporting the HslV-mediated degradation. Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 8932 - 7 Crystal structure of Escherichia coli CyaY protein reveals a previously unidentified fold for the evolutionarily conserved frataxin family; Cho SJ et al.; Friedreich ataxia is an autosomal recessive neurodegenerative disease caused by defects in the FRDA gene, which encodes a mitochondrial protein called frataxin . Frataxin is evolutionarily conserved, with homologs identified in mammals, worms, yeast, and bacteria . The CyaY proteins of gamma-purple bacteria are believed to be closely related to the ancestor of frataxin . In this study, we have determined the crystal structure of the CyaY protein from Escherichia coli at 1.4-A resolution . It reveals a protein fold consisting of a six-stranded antiparallel beta-sheet flanked on one side by two alpha-helices . This fold is likely to be shared by all members of the conserved frataxin family . This study also provides a framework for the interpretation of disease-associated mutations in frataxin and for understanding the possible functions of this protein family. Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 9009 - 14 The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli; Schwartz CJ et al.; The cysteine desulfurase, IscS, provides sulfur for Fe-S cluster synthesis in vitro, but a role for IscS in in vivo Fe-S cluster formation has yet to be established . To study the in vivo function of IscS in Escherichia coli, a strain lacking IscS was constructed and characterized . Using this iscS deletion strain, we have observed decreased specific activities for proteins containing {4Fe-4S} clusters from soluble (aconitase B, 6-phosphogluconate dehydratase, glutamate synthase, fumarase A, and FNR) and membrane-bound proteins (NADH dehydrogenase I and succinate dehydrogenase) . A specific role for IscS in in vivo Fe-S cluster assembly was demonstrated by showing that an Fe-S cluster independent mutant of FNR is unaffected by the lack of IscS . These data support the conclusion that, via its cysteine desulfurase activity, IscS provides the sulfur that subsequently becomes incorporated during in vivo Fe-S cluster synthesis . We also have characterized a growth phenotype associated with the loss of IscS . Under aerobic conditions the deletion of IscS caused an auxotrophy for thiamine and nicotinic acid, whereas under anaerobic conditions, only nicotinic acid was required . The lack of IscS also had a general effect on the growth of E . coli because the iscS deletion strain grew at half the rate of wild type in many types of media even when the auxotrophies were satisfied. Mol Pharmacol, 2000 Aug, 58(2), 288 - 99 Characterization of naturally occurring and recombinant human N-acetyltransferase variants encoded by NAT1; de Leon JH et al.; The genotype at the NAT1* locus of an interethnic population of 38 unrelated subjects was determined by direct sequencing of 1.6-kb fragments amplified by PCR . The coding exon alone and together with the 3' noncoding exon of the wild-type (NAT1*4) and the three mutant alleles (NAT1*10, *11, and *16) detected was expressed in Escherichia coli and COS-1 cells, respectively, and the cytosolic fraction of mononuclear leukocytes from NAT1*4/*4 and NAT1*10/*10 homozygotes was also isolated . Recombinant and leukocyte cytosolic preparations were thoroughly characterized by N-acetylation activity with several NAT1-specific and -selective substrates, as well as by steady-state kinetics with varying amounts of the substrate (fixed acetyl CoA) and acetyl CoA (fixed substrate), thermodynamics, stability, and protein immunoreactivity with a polyclonal human anti-NAT1 . The polyadenylation signal mutation in the 3' noncoding sequence of NAT1*10 affected none of the aforementioned parameters evaluated both with recombinant NAT1*10 and with the naturally occurring allele . Function was also unaffected by the coding and 3' noncoding exon mutations in NAT1*11 . In contrast, the three extra adenosines located immediately after the sixth position of the polyadenylation signal in the 3' untranslated region of NAT1*16 ostensibly caused disruption of the predicted secondary structure of the pre-mRNA for NAT1 16, culminating in parallel 2-fold decreases in the amount and catalytic activity of NAT1 16 in COS-1 cell cytosol . This novel finding in N-acetylation pharmacogenetics clearly demonstrates a direct link between reduced catalytic activity and structural alteration in the 3' untranslated region of an NAT variant (NAT1*16) brought about by mutation. Lab Invest, 2000 Jul, 80(7), 1079 - 87 Down-regulation of renal endothelial nitric oxide synthase expression in experimental glomerular thrombotic microangiopathy; Zhou XJ et al.; Infection with certain strains of Escherichia coli and endotoxemia results in renal glomerular thrombotic microangiopathy (TMA) characterized by endothelial swelling and prominent glomerular microthrombus formation . Nitric oxide (NO) is an endogenous biologic modulator with diverse physiologic functions including vasodilation and inhibition of platelet adhesion and aggregation . NO is synthesized from conversion of L-arginine to L-citrulline by a family of NO synthases (NOS), which include constitutive and inducible isoforms . Indirect evidence supports the hypothesis that TMA is associated with depressed intrarenal NO production . However, the effect of TMA on renal tissue NOS expression has not been fully elucidated . We studied rats with TMA induced by iv bolus injection of high dose (20 mg/kg) E . coli endotoxin . Subgroups of six animals each were sacrificed before or at 30, 90, 180, 360, and 720 minutes after the administration of endotoxin . Renal histology and tissue expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) were examined . Additionally, we examined the effect of endotoxin on glomerular NO production, and eNOS and iNOS protein expression in vitro . Glomerular capillary thrombosis developed by 180 minutes after endotoxin administration in approximately half of the animals . The glomeruli without thrombotic lesions apparent by light microscopy disclosed early signs of TMA characterized by endothelial swelling, platelet accumulation/adhesion, and patchy fibrinogen deposition . These morphologic changes were associated with a marked reduction of renal tissue eNOS expression beyond 180 minutes after the endotoxin administration . The fall in eNOS expression was coupled with a significant rise in iNOS protein abundance, which was expressed largely by glomerular circulating neutrophils and endothelial cells, peritubular vascular endothelium, and collecting ducts of cortex and medulla . In vitro incubation of isolated glomeruli with endotoxin also resulted in a marked reduction in eNOS expression and a significant rise in iNOS content . Administration of E . coli endotoxin leads to a sustained fall in renal eNOS expression both in vivo and in vitro . The associated decline in intrarenal endothelial NO production/availability may result in renal vasoconstriction and a hypercoagulative state, which may contribute to the pathogenesis of endotoxin-induced TMA. Lipids, 2000 May, 35(5), 481 - 6 Metabolism of 1-acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in castor oil biosynthesis; Lin JT et al.; We have examined the role of 2-oleoyl-PE (phosphatidylethanolamine) in the biosynthesis of triacylglycerols (TAG) by castor microsomes . In castor microsomal incubation, the label from 14C-oleate of 1-palmitoyl-2-{1-(14)C}oleoyl-sn-glycero-3-phosphoethanolamine is incorporated into TAG containing ricinoleate . The enzyme characteristics, such as optimal pH, and the effect of incubation components of the oleoyl-12-hydroxylase using 2-oleoyl-PE as incubation substrate are similar to those for 2-oleoyl-PC (phosphatidylcholine) . However, compared to 2-oleoyl-PC, 2-oleoyl-PE is a less efficient incubation substrate of oleoyl-12-hydroxylase in castor microsomes . Unlike 2-oleoyl-PC, 2-oleoyl-PE is not hydroxylated to 2-ricinoleoyl-PE by oleoyl-12-hydroxylase and is not desaturated to 2-linoleoyl-PE by oleoyl-12-desaturase . We have demonstrated the conversion of 2-oleoyl-PE to 2-oleoyl-PC and vice versa . The incorporation of label from 2-{14C}oleoyl-PE into TAG occurs after its conversion to 2-oleoyl-PC, which can then be hydroxylated or desaturated . We detected neither PE-N-monomethyl nor PE-N,N-dimethyl, the intermediates from PE to PC by N-methylation . The conversion of 2-oleoyl-PE to 2-oleoyl-PC likely occurs via hydrolysis to 1,2-diacyl-sn-glycerol by phospholipase C and then by cholinephosphotransferase . This conversion does not appear to play a key role in driving ricinoleate into TAG. J Biol Inorg Chem, 2000 Jun, 5(3), 402 - 8 Recombinant superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilium; Whittaker MM et al.; Superoxide dismutase (SOD) from the hyperthermophilic archaeon Pyrobaculum aerophilum (a facultative aerobe) has been cloned and expressed in a mesophilic host (Escherichia coli) as a soluble tetrameric apoprotein . The purified apoprotein can be reconstituted with either Mn or Fe by heating the protein with the appropriate metal salt at an elevated temperature (95 degrees C) . Both Mn- and Fe-reconstituted P . aerophilum SOD exhibit superoxide dismutase activity, with the Mn-containing enzyme having the higher activity . P . aerophilum SOD is extremely thermostable and the reconstitution with Mn(II) can be performed in an autoclave (122 degrees C, 18 psi) . The Mn(III) optical absorption spectrum of Mn-reconstituted P . aerophilum SOD is distinct from that of most other MnSODs and is unchanged upon addition of NaN3 . The optical absorption spectrum of Fe-reconstituted P . aerophilum SOD is typical of Fe-substituted MnSODs and authentic FeSOD and exhibits a pH-dependent transition with an effective pKa value higher than that found for Fe-substituted MnSOD from either E . coli or Thermus spp . Amino acid sequence analysis shows that the P . aerophilum SOD is closely related to SODs from other hyperthermophilic archaea (Aeropyrum pernix and Sulfolobus spp.), forming a family of enzymes distinct from the hyperthermophilic bacterial SOD from Aquifex pyrophilus and from mesophilic SODs. J Vet Med Sci, 2000 Jun, 62(6), 661 - 4 ELISA for bovine interleukin-1 receptor antagonist and its application to mastitic sera and whey; Yamanaka H et al.; A sandwich ELISA for the bovine IL-1 receptor antagonist (bIL-1ra) was developed using recombinant (r) bIL-1ra produced by Escherichia coli, anti-rbIL-1ra rabbit IgG, its biotinylated one and avidin-peroxidase . This ELISA system enabled detection of rbIL-1ra at a concentration of more than 2 ng/ml . This ELISA was applied to quantitation of bIL-1ra in sera and whey of mastitic and healthy cows . The results indicate that although IL-1ra levels in healthy and mastitic sera and whey were comparable, serum IL-1ra/IL-1beta ratio of euthanized cows was significantly lower than that of the recovered. J Vet Med Sci, 2000 Jun, 62(6), 621 - 6 Involvement of apoptosis in the endotoxemic lesions of the liver and kidneys of piglets; Nakajima Y et al.; The involvement of apoptosis was evaluated in lesions of endotoxemic piglets . A single injection with E . coli O111:B4 lipopolysaccharide (LPS) induced foci of coagulative necrosis in the liver and kidneys . No significant change was observed in these organs at 1.5 hr after LPS injection, but at 6 hr, epithelial cells with chromatin condensation or fragmentation and apoptotic bodies were visible . Foci of coagulative necrosis were formed within 24 hr after LPS inoculation . In and adjacent to the necrotic foci, dead hepatocytes with nuclear condensation or fragmentation were scattered . These dead cells were positively stained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) methods . Electronmicroscopy revealed apoptotic cells with condensed or fragmented homogeneous nuclear chromatin, and necrotic cells with irregularly destroyed nuclei and cytoplasmic membranes . Apoptotic cell death were also observed in parietal cells of the stomach and lymphocytes in the lymphatic system . DNA ladders with approximately 200-bp multimers were observed in hepatic, renal and thymic samples prepared after 6 and 24 hr of LPS injection by agarose gel electrophoresis . These results suggest that apoptosis is involved in the pathology of swine endotoxemia. Radiats Biol Radioecol, 2000 May-Jun, 40(3), 319 - 22 {The effect of magnetic fields on the growth and division of the lon mutant of Escherichia coli K-12}; Stepanian RS et al.; It was shown that the static magnetic field (SMF) and electromagnetic field (EMF) caused inhibition of the cell division in Escherichia coli K-12 lon mu