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| Biochem Biophys Res Commun, 1997 Feb 24, 231(3), 645 - 50 Cloning and expression of the adenosine kinase gene from rat and human tissues; McNally T et al.; Adenosine kinase is ubiquitous in eukaryotes and is a key enzyme in the regulation of the intracellular levels of adenosine, an important physiological effector of many cells and tissues . In this paper we report the cloning of cDNAs encoding adenosine kinase from both rat and human tissues . Two distinct forms of adenosine kinase mRNA were identified in human tissues . Sequence variation between the two forms is restricted to the extreme 5'-end of the adenosine kinase mRNA, including a portion of the coding region, and is consistent with differential splicing of a single transcriptional product . We have expressed both forms in E . coli and produced soluble active enzyme which catalyzes the phosphorylation of adenosine with high specific activity in vitro and is susceptible to known adenosine kinase inhibitors. Biochem Pharmacol, 1997 Feb 21, 53(4), 493 - 500 Repression of inducible nitric oxide synthase and cyclooxygenase-2 by prostaglandin E2 and other cyclic AMP stimulants in J774 macrophages; Pang L et al.; The enhanced nitric oxide (NO) and prostaglandin (PG) generation of activated macrophages is controlled by glucocorticoid-sensitive inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively . Negative feedback regulation of iNOS expression by the products of both pathways has been suggested, but their effects on COX-2 expression have not been examined . We hae investigated the effect of E- and l-series prostaglandins that activate adenylate cyclase (AC), forskolin (a direct activator of AC), and other agents that influence the cyclicAMP/cyclicGMP systems on the ability of E . coli endotoxin (lipopolysaccharide, LPS) to induce iNOS and COX-2 in the murine macrophage cell line J774 . After a 2-hr pretreatment before adding endotoxin, PGE2, PGI2, forskolin, IBMX (isobutylmethylxanthine, a cyclicAMP/cyclicGMP phosphodiesterase inhibitor), 8-bromo cyclicAMP, and arachidonic acid itself all inhibited the expression of both iNOS and COX-2 (as shown by Western blotting) and reduced NO release and COX activity, whereas PGF2 alpha and 8-bromo cyclic GMP were only weakly effective . The effects of PGE2, PGI2, and forskolin were enhanced by cotreatment with IBMX . The suppression of LPS-induced iNOS induction by PGE2 was functionally significant, in that it protected against the mild cytotoxicity of the NO generated in response to endotoxin . These results provide the first direct evidence for the feedback regulatory suppression of COX-2 induction by a PG-driven cAMP-mediated process, and show that the modulation of iNOS and COX-2 induction shares common features . They also suggest that such modulation is normally held in check by high phosphodiesterase activity within these cells. Biochim Biophys Acta, 1997 Feb 21, 1324(1), 69 - 75 Fluorescence studies on the interaction of a synthetic signal peptide and its analog with liposomes; Wang Q et al.; The N-terminal signal sequence of glucitol permease of Escherichia coli (Gut22: MIETITPGAVWFIGLFQKGGEC) and its analog (Gut22Ana: MIETITHGAEWFIGLFQKGGEC) were synthesized . The analog had a Pro residue substituted for the His at the 7th position of Gut22 and a Val residue substituted for the Glu at the 10th position . Previous studies indicated that due to its structural rigidity, the interaction of Gut22Ana with lipid bilayer was much weaker than that of Gut22 (Wang, Q.D., Cui, D.F . and Lin, Q.S . (1996) Science in China (Series C) 39, 395-405) . To further probe the location of the tryptophan residues of the peptides in lipid bilayer, the membrane penetration depth of the tryptophan residue of Gut22 was measured using spin-labeled phospholipids, and fluorescence quenching of the peptides by iodide and acrylamide in the presence and absence of phosphatidylserine/phosphatidylcholine liposomes were also studied . Fluorescent labeling of the peptides enabled the study of their association with membrane by fluorospectrophotometry . In the presence of liposomes, the peptides were protected from reaction with chymotrypsin, indicating that the peptide incorporated into the membrane . However, dithionite, which acts external to the membrane, reacted with the peptide, showing that the peptides did not translocate across lipid bilayer spontaneously. J Mol Biol, 1997 Feb 21, 266(2), 381 - 99 Structure and mechanism of a sub-family of enzymes related to N-acetylneuraminate lyase; Lawrence MC et al.; We describe here a sub-family of enzymes related both structurally and functionally to N-acetylneuraminate lyase . Two members of this family (N-acetylneuraminate lyase and dihydrodipicolinate synthase) have known three-dimensional structures and we now proceed to show their structural and functional relationship to two further proteins, trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and D-4-deoxy-5-oxoglucarate dehydratase . These enzymes are all thought to involve intermediate Schiff-base formation with their respective substrates . In order to understand the nature of this intermediate, we have determined the three-dimensional structure of N-acetylneuraminate lyase in complex with hydroxypyruvate (a product analogue) and in complex with one of its products (pyruvate) . From these structures we deduce the presence of a closely similar Schiff-base forming motif in all members of the N-acetylneuraminate lyase sub-family . A fifth protein, MosA, is also confirmed to be a member of the sub-family although the involvement of an intermediate Schiff-base in its proposed reaction is unclear. J Mol Biol, 1997 Feb 21, 266(2), 343 - 56 Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation); Wadzack J et al.; A new technique for neutron scattering, the proton-spin contrast-variation, improves the signal-to-noise ratio more than one order of magnitude as compared to conventional techniques . The improved signal enables small RNA ligands within a large deuterated ribonucleic acid-protein complex to be measured . We used this technique to determine the positions of the two tRNAs within the elongating ribosome before and after translocation . Using a four-sphere model for each of the L-shaped tRNAs, unequivocal solutions were found for the localization of the mass centre of both tRNAs . The centre of gravity is located in the interface cavity separating the ribosomal subunits near the neck of the 30 S subunit . It moves during translocation by 12(+/-4) A towards the head of the 30 S subunit and slightly towards the L1 protuberance of the 50 S subunit. J Mol Biol, 1997 Feb 21, 266(2), 317 - 30 A 29 residue region of the sarcomeric myosin rod is necessary for filament formation; Sohn RL et al.; Myosin is a motor protein whose functional unit in the sarcomere is the thick filament . The myosin molecule is capable of self-assembly into thick filaments through its alpha-helical coiled-coil rod domain . To define more precisely the sequence requirements for this assembly, segments of the human fast IId skeletal myosin rod were expressed in Escherichia coli and examined differential solubility and the formation of ordered paracrystals . We show that both properties appear to require a 29 residue sequence (residues 1874 to 1902) near the C terminus of the rod region . To test further the role of this region in assembly, a protein was constructed which consisted of this assembly competence domain (ACD) fused to the carboxy terminus of an assembly-incompetent myosin rod fragment . This chimeric fragment exhibited myosin's characteristic solubility properties and formed ordered paracrystals . To complement these in vitro experiments, both a full-length myosin heavy chain (MYH) and one from which the 29 residues were deleted were transfected into cultured mammalian cells . While the full-length construct formed the spindle-shaped structures characteristic of arrays of thick filaments, the deleted MYH showed only diffuse staining throughout the cytoplasm by light microscopy . Thus, there appears to be a specific sequence in the C-terminal region of the myosin heavy chain rod which is necessary for ordered paracrystal formation and is sufficient to confer assembly properties to an assembly-incompetent rod fragment. J Mol Biol, 1997 Feb 21, 266(2), 297 - 305 Short-homology-independent illegitimate recombination in Escherichia coli: distinct mechanism from short-homology-dependent illegitimate recombination; Shimizu H et al.; We have shown elsewhere that there is no, or very little, homology at the recombination sites in DNA gyrase-mediated illegitimate recombination in vitro . On the other hand, many reports have indicated that illegitimate recombination takes place between sequences with a short homology . To clarify this contradiction, we analyzed the mechanism of DNA gyrase-mediated illegitimate recombination in vivo, by isolating a temperature-sensitive gyrA mutant (gyrAhr1) that causes spontaneous illegitimate recombination at a higher frequency than that of the wild-type . This mutant also causes spontaneous induction of lambda prophage . It is therefore suggested that the gyrAhr1 mutation induces strand breaks in the chromosome, resulting in the formation of illegitimate recombinants . Analysis of the recombination junctions of lambdabio transducing phages formed spontaneously in the gyrAhr1 mutant revealed that the Escherichia coli bio and lambda recombination sites have an average homologous sequence of only 1.3 base pairs . This is the first indication that homology in vivo is not required for illegitimate recombination . On the other hand, a short homology of 8.4 bp, on average, was found in the junctions of lambdabio transducing phages formed spontaneously in the wild-type bacteria . When the gyrAhr1 mutant was irradiated with UV, short homologies were also detected in the junctions . We concluded that illegitimate recombination, which takes place spontaneously in the gyrAhr1 mutants, is distinguishable from spontaneous recombination in the wild-type and from UV-induced recombination in the mutant with regard to the requirement for short homology . We propose that short-homology-independent illegitimate recombination is mediated by subunit exchange between DNA gyrase, while short-homology-dependent recombination is triggered by double-strand breaks and completed by processing, annealing, and ligation of DNA ends. J Biol Chem, 1997 Feb 21, 272(8), 5335 - 41 NH2-terminal proline acts as a nucleophile in the glycosylase/AP-lyase reaction catalyzed by Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein; Zharkov DO et al.; Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli . The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP lyase), and delta-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA . Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A . P . (1995) J . Biol . Chem . 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride . By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA . This experimental approach may be applicable to the analysis of other protein-DNA complexes. J Biol Chem, 1997 Feb 21, 272(8), 5305 - 12 Lysine 207 as the site of cross-linking between the 3'-end of Escherichia coli initiator tRNA and methionyl-tRNA formyltransferase; Gite S et al.; The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in Escherichia coli . In attempts to identify regions of MTF that come close to the 3'-end of the tRNA, we oxidized 32P-3'-end-labeled E . coli initiator methionine tRNA with sodium metaperiodate and cross-linked it to MTF . The cross-linked MTF was separated from uncross-linked MTF by DEAE-cellulose chromatography, and the tRNA in the cross-linked MTF was hydrolyzed with nuclease P1 and RNase T1, leaving behind an oxidized fragment of {32P}AMP attached to MTF . Trypsin digestion of the cross-linked MTF followed by high pressure liquid chromatography of the digest yielded two peaks of radioactive peptides, I* and II* . These peptides were characterized by N- and/or C-terminal sequencing and by matrix-assisted laser desorption ionization mass spectroscopy . Peptide I* contained amino acids Gln186-Lys210 with Lys207 as the site of the cross-link . Peptide II*, a partial digestion product, contained amino acids Gln186-Arg214 also with Lys207 as the site of the cross-link . The molecular masses of peptides I* and II* indicate that the final product of the cross-linking reaction between the periodate-oxidized AMP moiety of the tRNA and Lys207 is most likely a morpholino derivative rather than a reduced Schiff's base. J Biol Chem, 1997 Feb 21, 272(8), 5141 - 51 Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase, MKP-4; Muda M et al.; Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli . Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues . By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4) . The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX3AYLM (where X is any amino acid) conserved in dual specificity phosphatases . MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2 . When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK . This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK) . Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver . Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells . Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28 . The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression. J Biol Chem, 1997 Feb 21, 272(8), 5082 - 6 Regulation of the soxRS oxidative stress regulon . Reversible oxidation of the Fe-S centers of SoxR in vivo; Gaudu P et al.; SoxR protein, a transcriptional activator of the soxRS (superoxide response) regulon of Escherichia coli, contains autooxidizable {2Fe-2S} centers that are presumed to serve as redox sensors . In vitro transcription experiments previously demonstrated that only the oxidized form is active . Reduced SoxR was detected in overproducing strains by EPR spectroscopy of suspensions of intact cells . Oxidized Fe-S centers were determined by lysing the cells and treating them with the reducing agent sodium dithionite prior to EPR measurements . In uninduced cells, 90% of the SoxR was in the reduced form . Treatment with the redox cycling agents phenazine methosulfate or plumbagin was accompanied by reversible oxidation of the Fe-S centers . Mutant SoxR derivatives that were constitutively activated existed constitutively in an oxidized state . The results indicate the presence of a cellular pathway for countering the autooxidation of SoxR and confirm the hypothesis that induction of the regulon is mediated by a shift in the redox equilibrium of SoxR rather than by assembly of its Fe-S clusters. J Biol Chem, 1997 Feb 21, 272(8), 5000 - 6 Crystal structures of CheY mutants Y106W and T87I/Y106W . CheY activation correlates with movement of residue 106; Zhu X et al.; Position 106 in CheY is highly conserved as an aromatic residue in the response regulator superfamily . In the structure of the wild-type, apo-CheY, Tyr106 is a rotamer whose electron density is observed in both the inside and the outside positions . In the structure of the T87I mutant of CheY, the threonine to isoleucine change at position 87 causes the side chain of Tyr106 to be exclusively restricted to the outside position . In this report we demonstrate that the T87I mutation causes cells to be smooth swimming and non-chemotactic . We also show that another CheY mutant, Y106W, causes cells to be more tumbly than wild-type CheY, and impairs chemotaxis . In the structure of Y106W, the side chain of Trp106 stays exclusively in the inside position . Furthermore, a T87I/Y106W double mutant, which confers the same phenotype as T87I, restricts the side chain of Trp106 to the outside position . The results from these behavioral and structural studies indicate that the rotameric nature of the Tyr106 residue is involved in activation of the CheY molecule . Specifically, CheY's signaling ability correlates with the conformational heterogeneity of the Tyr106 side chain . Our data also suggest that these mutations affect the signal at an event subsequent to phosphorylation. J Biol Chem, 1997 Feb 21, 272(8), 4985 - 92 Mutagenesis studies of the human erythropoietin receptor . Establishment of structure-function relationships; Barbone FP et al.; Mutagenesis of the erythropoietin receptor (EPOR) permits analysis of the contribution that individual amino acid residues make to erythropoietin (EPO) binding . We employed both random and site-specific mutagenesis to determine the function of amino acid residues in the extracellular domain (referred to as EPO binding protein, EBP) of the EPOR . Residues were chosen for site-specific alanine substitution based on the results of the random mutagenesis or on their homology to residues that are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family . Site-specific mutants were expressed in Escherichia coli as soluble EBP and analyzed for EPO binding in several different assay formats . In addition, selected mutant proteins were expressed as full-length EPOR on the surface of COS cells and analyzed for 125I-EPO binding in receptor binding assays . Using these methods, we have identified residues that appear to be involved in EPO binding as well as other residues, most of which are conserved in receptors of the cytokine receptor family, that appear to be necessary for the proper folding and/or stability of the EPOR . We present correlations between these mutagenesis data and the recently solved crystal structure of the EBP with a peptide ligand. J Biol Chem, 1997 Feb 21, 272(8), 4843 - 9 Cooperativity and dimerization of recombinant human estrogen receptor hormone-binding domain; Brandt ME et al.; The estrogen receptor dimerizes and exhibits cooperative ligand binding as part of its normal functioning . Interaction of the estrogen receptor with its ligands is mediated by a C-terminal hormone-binding domain (HBD), and residues within the HBD are thought to contribute to dimerization . To examine dimer interactions in the isolated HBD, a human estrogen receptor HBD fragment was expressed in high yield as a cleavable fusion protein in Escherichia coli . The isolated HBD peptide exhibited affinity for estradiol, ligand discrimination, and cooperative estradiol binding (Hill coefficient approximately 1.6) similar to the full-length protein . Circular dichroism spectroscopy suggests that the HBD contains significant amounts of alpha-helix ( approximately 60%) and some beta-strand ( approximately 7%) and that ligand binding induces little change in secondary structure . HBD dimer dissociation, measured using size exclusion chromatography, exhibited a half-life of approximately 1.2 h, which ligand binding increased approximately 3-fold (estradiol) to approximately 4-fold (4-hydroxytamoxifen) . These results suggest that the isolated estrogen receptor HBD dimerizes and undergoes conformational changes associated with cooperative ligand binding in a manner comparable to the full-length protein, and that one effect of ligand binding is to alter the receptor dimer dissociation kinetics. J Biol Chem, 1997 Feb 21, 272(8), 4820 - 7 Formation of DNA repair intermediates and incision by the ATP-dependent UvrB-UvrC endonuclease; Zou Y et al.; The Escherichia coli UvrB and UvrC proteins play key roles in DNA damage processing and incisions during nucleotide excision repair . To study the DNA structural requirements and protein-DNA intermediates formed during these processes, benzo{a}pyrene diol epoxide-damaged and structure-specific 50-base pair substrates were constructed . DNA fragments containing a preexisting 3' incision were rapidly and efficiently incised 5' to the adduct . Gel mobility shift assays indicated that this substrate supported UvrA dissociation from the UvrB-DNA complex, which led to efficient incision . Experiments with a DNA fragment containing an internal noncomplementary 11-base region surrounding the benzo{a}pyrene diol epoxide adduct indicated that UvrABC nuclease does not require fully duplexed DNA for binding and incision . In the absence of UvrA, UvrB (UvrC) bound to an 11-base noncomplementary region containing a 3' nick (Y substrate), forming a stable protein-DNA complex (Kd approximately 5-10 nM) . Formation of this complex was absolutely dependent upon UvrC . Addition to this complex of ATP, but not adenosine 5'-(beta,gamma-iminotriphosphate) or adenosine 5'-(beta, gamma-methylene)triphosphate, caused incision three or four nucleotides 5' to the double strand-single strand junction . The ATPase activity of native UvrB is activated upon interaction with UvrC and enhanced further by the addition of Y substrate . Incision of this Y structure occurs even without DNA damage . Thus the UvrBC complex is a structure-specific, ATP-dependent endonuclease. J Biol Chem, 1997 Feb 21, 272(8), 4814 - 9 Substrate specificity of hybrid modules from peptide synthetases; Elsner A et al.; Homologous modules from two different peptide synthetases were analyzed for functionally equivalent regions . Hybrids between the coding regions of the phenylalanine-activating module of tyrocidine synthetase and the valine-activating module of surfactin synthetase were constructed by combining the two reading frames at various highly conserved consensus sequences . The resulting DNA fragments were expressed in Escherichia coli as C-terminal fusions to the gene encoding for the maltose-binding protein . The fusion proteins were purified, and the amino acid specificities, the acceptance of different nucleotide analogues, and the substrate binding affinities were analyzed . We found evidence for a large N-terminal domain and a short C-terminal domain of about 19 kDa within the two modules, which are separated by the sequence motif GELCIGG . The two domains could be reciprocally transferred between the two modules, and the constructed hybrid proteins showed amino acid adenylating activity . Hybrid proteins fused at various consensus motifs within the two domains were inactive, indicating that the domains may fold independently and represent complex functional units . The N-terminal domain was found to be responsible for the amino acid specificity of the modules, and it is also involved in the recognition of the ribosyl and the phosphate moieties of the nucleotide substrate . For tyrocidine synthetase I, we could confine the sites for amino acid specificity to a region of 330 residues . The C-terminal domain is essential for the enzymatic activity and has a strong impact on the specific activity of the modules. J Biol Chem, 1997 Feb 21, 272(8), 4770 - 4 Molecular cloning of acetone cyanohydrin lyase from flax (Linum usitatissimum) . Definition of a novel class of hydroxynitrile lyases; Trummler K et al.; Acetone cyanohydrin lyase from Linum usitatissimum is a hydroxynitrile lyase (HNL) which is involved in the catabolism of cyanogenic glycosides in young seedlings of flax . We have isolated a full-length cDNA clone encoding L . usitatissimum HNL (LuHNL) from a cDNA expression library by immunoscreening . LuHNL cDNA was expressed in Escherichia coli and isolated from the respective soluble fraction in an active form which was biochemically indistinguishable from the natural enzyme . An open reading frame of 1266 base pairs encodes for a protein of 45,780 kDa . The derived amino acid sequence shows no overall homologies to the to date cloned HNLs, but has significant similarities to members of the alcohol dehydrogenase (ADH) family of enzymes . In particular, the cysteine and histidine residues responsible for coordination of an active site Zn2+ and a second structurally important Zn2+ in alcohol dehydrogenases are conserved . Nevertheless, we found neither alcohol dehydrogenase activity in LuHNL nor HNL activity in ADH . Moreover, well known inhibitors of ADHs, which interfere with the coordination of the active site Zn2+, fail to affect HNL activity of LuHNL, suggesting principally different mechanisms of cyanohydrin cleavage and alcohol oxidation . Interestingly, LuHNL like ADH and Prunus serotina (PsHNL) possesses an ADP-binding betaalphabeta unit motif, pointing to the possibility that the non-flavoprotein PsHNL and the flavoprotein LuHNL have developed from two independent lines of evolution of a common ancestor with an ADP-binding betaalphabeta unit. J Biol Chem, 1997 Feb 21, 272(8), 4671 - 9 Residues of the Rho family GTPases Rho and Cdc42 that specify sensitivity to Dbl-like guanine nucleotide exchange factors; Li R et al.; The Dbl-like guanine nucleotide exchange factor (GEF) Lbc oncoprotein specifically activates the small GTP-binding protein Rho in mammalian fibroblasts to induce transformation and actin stress fiber formation, whereas another Dbl-related molecule, Cdc24, stimulates guanine nucleotide exchange of the Rho family GTPase Cdc42 to elicit effects on both gene induction and actin-based cytoskeleton change in Saccharomyces cerevisiae . To understand the mechanism of these functional interactions, we have taken a biochemical approach to probe the sites on Rho and Cdc42 that are involved in coupling to their respective GEFs, the Lbc and Cdc24 proteins . Point mutations in the switch II region of the small G-proteins, many of which would affect the interaction with GEF in the case of Ras, or a mutation in the switch I region that was identified as a contact site between Rab3A and Rab GEF had little effect on RhoA or Cdc42Hs with regard to the ability to interact with Lbc or Cdc24, suggesting that there exists a unique mechanism of regulation of the Rho family proteins by their GEFs . Analysis of a panel of chimeras made between RhoA and Cdc42Hs, which all maintained the ability to respond to Dbl, their mutual GEF, and to GTPase-activating protein, revealed that at least two distinct sites in each of the GTPases are required for activation by the respective GEFs . Further site-directed mutagenesis studies showed that the conserved residue Tyr32 in the putative effector region of both GTPases (numbered by Cdc42Hs) is critical for binding of the GEFs and that specific recognition for Lbc or Cdc24 is achieved at least in part through residues Lys27 of Rho and Gln116 of Cdc42 . Moreover, the loss of GEF responsiveness of a RhoA mutation (D76Q) was found to be caused by the impaired GEF catalysis, not by a change in the GEF binding affinity . Together, these results indicate that multiple sites of the Rho GTPases are involved in the regulation by GEFs, contributing to GEF binding or GEF catalysis, and raise the possibility that activation of each Rho family G-protein by a specific GEF may engage in a distinct mechanism. Carbohydr Res, 1997 Feb 20, 298(1-2), 65 - 73 Transglycosylation in a two-phase aqueous-organic system with catalysis by a lipid-coated beta-D-galactosidase; Mori T et al.; A lipid-coated beta-D-galactosidase was prepared in which the enzyme surface is covered with a lipid monolayer and two long alkyl lipophilic tails serve to solubilize the enzyme in organic solvents . In a two-phase aqueous-organic system, a lipid-coated enzyme exists in the organic (2-propyl ether) phase and acts as an efficient transgalactosylation catalyst for various hydrophobic alcohols with lactose in the aqueous buffer solution . When a native beta-D-galactosidase was employed in the two-phase system, neither the transgalactosylation nor the hydrolysis reaction proceeded due to denaturation of the enzyme at the interface . Effects of coating lipid molecules, origins of enzymes, reaction in organic solvents, and chemical structures of acceptor alcohols on the transgalactosylation catalyzed by the lipid-coated enzyme were studied . This system could also be applied in a large-scale synthesis on the 0.1-1 g scale. Mol Gen Genet, 1997 Feb 20, 253(5), 634 - 41 Adducts formed by the food mutagen 2-amino-3-methylimidazo(4,5-f) quinoline induce frameshift mutations at hot spots through an SOS-independent pathway; Maenhaut-Michel G et al.; The potency of 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) adducts to induce -2, -1 and +1 frameshift mutations has been determined on specific target DNA sequences, namely short runs of alternating GpC sequences and short runs of guanines . The genetic control of the mutational processes has been analyzed using different Escherichia coli mutants, affected either in the control or in the mutagenesis pathway of the SOS system . We have shown that IQ adducts induce very efficiently both -1 and -2 frameshift mutations in E . coli . Both types of deletion mutations are induced in bacteria without the need of SOS induction, indicating that no LexA-controlled functions, in particular the UmuDC proteins, are required for mutation fixation . We have also shown that the frequency of IQ-induced -2 frameshift mutations in alternating GC sequences increases with the length of the repetition . The efficiency of IQ adducts to induce -1 and -2 frameshift mutations is similar to that of N-2-acetylaminofluorene (AAF) adducts . Both chemicals are potent carcinogens which form covalent adducts at the C8 position of guanines . We suggest that in both cases the adduct-induced DNA structure allows the replication complex to perform a mutagenic bypass of the lesion by a slippage mechanism . However, in contrast to AAF-induced frameshift mutagenesis, IQ-induced frameshift mutagenesis is SOS-independent. Gene, 1997 Feb 20, 186(1), 55 - 60 A new expression vector for high level protein production, one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins; Zheng CF et al.; Calmodulin-binding peptide (CBP), a peptide of 26 amino acids derived from muscle myosin light chain kinase (MLCK), binds to calmodulin with nanomolar affinity . Proteins fused in frame with CBP can be purified from crude E . coli lysates in a single step using calmodulin affinity chromatography (Stofko-Hahn et al., 1992) . Because the binding between CBP and calmodulin is calcium-dependent, the fusion protein can be eluted from the resin with virtually any buffer containing EGTA (2 mM) and used directly for many applications . To take full advantage of this affinity purification system, we constructed the versatile CBP fusion protein expression vector pCAL-n . The CBP coding sequence was positioned for fusion at the N-terminus, an advantage that ensures consistent high level synthesis of fusion proteins due to the efficient translation of the CBP in E . coli . The production of fusion proteins from pCAL-n is controlled by the tightly regulated T7(lac)O promoter . A versatile multiple cloning site (MCS) was included to facilitate the cloning of genes of interest . The protein coding sequence for the enzyme c-Jun N-terminal kinase (JNK) was inserted into the MCS of pCAL-n, and the resulting fusion protein CBP-JNK synthesized in E . coli cells at 15-20 mg/1 culture . CBP-JNK was purified to near homogeneity in one step with calmodulin affinity resin . Purified CBP-JNK is fully active, and the CBP peptide tag can be removed by cleavage with thrombin . We also show that CBP can be efficiently phosphorylated by cAMP-dependent protein kinase . Hence, the purified fusion proteins can be labeled directly with {gamma-32P}ATP and used to probe protein-protein or protein-nucleic acid interactions. Cell Tissue Res, 1997 Feb 20, 287(3), 507 - 12 Expression and characterization of the "laminin binding protein" in hydra Keppel E, Fenger U, Schaller HC. Recently, a cDNA was isolated from hydra with extensive homology to a mammalian and invertebrate gene which codes for a protein called laminin binding protein (LBP) . In this paper we describe the protein expression of the hydra LBP in Escherichia coli . On SDS gels the recombinant hydra LBP displayed an apparent molecular mass of 43 kDa, although the calculated mass, including six additional histidines, is 33.7 kDa . Polyclonal antibodies were produced against the hydra recombinant LBP . The antiserum reacted with a 42-kDa and a 43-kDa protein from Hydra vulgaris and from a multiheaded mutant of Chlorohydra viridissima, respectively . In hydra, LBP RNA and protein were highly expressed in cells with short cell cycles, such as all cells of the interstitial cell lineage, less in slowly cycling epithelial cells, and at very reduced levels or not at all in differentiated cells . Higher expression in the multiheaded mutant of C . viridissima than in H . vulgaris, the cells of which differ in doubling time, hint at a function in cell proliferation . This is supported by the finding that in vitro hydra LBP is a substrate for the cell-cycle-specific kinase CDC2. Biochemistry, 1997 Feb 18, 36(7), 1943 - 52 {125I}Iberiotoxin-D19Y/Y36F, the first selective, high specific activity radioligand for high-conductance calcium-activated potassium channels; Koschak A et al.; Iberiotoxin (IbTX), a selective peptidyl ligand for high-conductance Ca2(+)-activated K+ (maxi-K) channels cannot be radioiodinated in biologically active form due to the importance of Y36 in interacting with the channel pore . Therefore, an IbTX double mutant (IbTX-D19Y/Y36F) was engineered, expressed in Escherichia coli, purified to homogeneity, and radiolabeled to high specific activity with 125I . IbTX-D19Y/Y36F and {127I}IbTX-D19Y/Y36F block maxi-K channels expressed in Xenopus laevis oocytes with equal potency as wild-type IbTX (Kd approximately 1 nM) . Under low ionic strength conditions, {125I}IbTX-D19Y/Y36F binds with high affinity to smooth muscle sarcolemmal maxi-K channels (Kd of 5 pM as determined by either equilibrium binding or kinetic binding analysis), and with a binding site density of 0.45 pmol/mg of protein . Competition studies with wild-type IbTX, IbTX-D19Y/Y36F or charybdotoxin (ChTX) result in complete inhibition of binding whereas toxins selective for voltage-gated K+ channels (margatoxin (MgTX) or alpha-dendrotoxin (alpha-DaTX) do not have any effect on IbTX binding . Indole diterpene alkaloids, which are selective inhibitors of maxi-K channels, and potassium ions both modulate {125I}IbTX-D19Y/Y36F binding in a complex manner . This pattern is also reflected during covalent incorporation of the radiolabeled toxin into the 31 kDa beta-subunit of the maxi-K channel in the presence of a bifunctional cross-linking reagent . In rat brain membranes, IbTX-D19Y/Y36F does not displace binding of {125I}MgTX or {125I}-alpha-DaTX to sites associated with voltage-gated K+ channels, nor do these latter toxins inhibit {125I}IbTX-D19Y/Y36F binding . Taken together, these results demonstrate that {125I}IbTX-D19Y/Y36F is the first selective radioligand for maxi-K channels with high specific activity. Biochemistry, 1997 Feb 18, 36(7), 1900 - 5 Role of Glu51 for cofactor binding and catalytic activity in pyruvate decarboxylase from yeast studied by site-directed mutagenesis; Killenberg-Jabs M et al.; We investigated the importance of the interaction between the Nl'-atom of the cofactor thiamine diphosphate and glutamic acid residue 51 in pyruvate decarboxylase (EC 4.1 . 1.1) . The yeast wild type gene PDCl and the respective mutant genes (E51Q and E51A) were expressed in Escherichia coli . The three enzymes were purified to homogeneity . They comigrated as a single band during silver-stained SDS/PAGE with a molecular mass of 60 000 Da . A molecular mass of 61 200 +/- 200 Da was determined by mass spectrometry for the subunit . The native enzyme is a homotetramer as demonstrated by gel filtration experiments . Near- and far-UV CD spectra showed no significant differences for the apoenzyme of the wild type and the mutants . Slight differences in the rate of thiamine diphosphate binding to the apoprotein component were observed between the wild type and the E51Q PDC by CD spectroscopy . Compared to the wild type enzyme, thiamine diphosphate binding at the E51A mutant apoprotein is very slow . Only 0.04% of the catalytic activity of the wild type enzyme was observed for the E51Q mutant; the E51A mutant has no detectable catalytic activity . The S0.5 value for the substrate pyruvate is increased 33-fold for the E51Q mutant . Substrate activation was observed for both the wild type and the E51Q mutant . The interaction between the N1'-atom of the coenzyme and glutamic acid 51 strongly influences the catalytic activity but only moderately the binding of the cofactor to the apoenzyme and the substrate activation rate. Biochemistry, 1997 Feb 18, 36(7), 1755 - 65 Miscoding properties of model estrogen-DNA adducts in reactions catalyzed by mammalian and Escherichia coli DNA polymerases; Shibutani S et al.; The miscoding properties of the model estrogen-derived DNA adducts, N2-{3-methoxyestra-1,3,5(10)-trien-6-yl}-2'-deoxyguanosine (dG-N2-3MeE) and N6-{3-methoxyestra-1,3,5(10)-trien-6-yl}-2'- deoxyadenosine (dA-N6-3MeE), have been explored, using an in vitro experimental system to quantify base substitutions and deletions . Site-specifically modified oligodeoxynucleotides containing a single dG-N2-3MeE or dA-N6-3MeE were prepared postsynthetically and used as templates in primer extension reactions catalyzed by Escherichia coli and mammalian DNA polymerases . When the 3'-->5' exonuclease free (exo-) Klenow fragment of DNA polymerase I was used, dG-N2-3MeE promoted mostly one- and two-base deletions, along with small amounts of incorporation of dAMP, dGMP, and dCMP opposite the lesion . dA-N6-3MeE promoted the incorporation of dTMP opposite the lesion as well as two-base deletions, accompanied by the incorporation of dAMP . Using pol alpha, primer extension reactions were blocked at dG-N2-3MeE; however, dA-N6-3MeE promoted preferential incorporation of dTMP opposite the lesion with small amounts of incorporation of dCMP and deletions . Primer extension reactions catalyzed by pol delta were blocked at these lesions . When pol beta was used, dG-N2-3MeE produced small amounts of incorporation of dAMP and deletions . dA-N6-3MeE promoted preferential incorporation of dTMP, along with incorporation of dCMP and two-base deletions . The miscoding specificities and frequencies varied depending on the DNA polymerase used . These results indicate that estrogen-DNA adducts have miscoding potential. Biochemistry, 1997 Feb 18, 36(7), 1748 - 54 Comparative analysis of the interactions of Escherichia coli sigma S and sigma 70 RNA polymerase holoenzyme with the stationary-phase-specific bolAp1 promoter; Nguyen LH et al.; We have investigated the interactions of Escherichia coli sigma 70 and sigma S holoenzyme RNA polymerases (E sigma S and E sigma 70) with the stationary-phase-specific bolAp1 promoter by various footprinting methods in vitro . E sigma S and E sigma 70 have been shown to transcribe the bolApl promoter in vitro . We have determined the effects of salt and holoenzyme concentrations on E sigma S and E sigma 70 open complex formation at the bolAp1 promoter in vitro . We have obtained a high-resolution hydroxyl radical (OH.) footprint of E sigma S and E sigma 70 on the bolApl promoter . The OH . footprinting data show remarkable similarities between the footprints of the heparin-resistant transcription complexes of the two holoenzymes which have the same +1 transcription start site . However, there are distinctive differences in the protection patterns in the region between -20 and -10 of the bolAp1 promoter . KMnO4 reactivity assays reveal that, at 37 degrees C, both holoenzymes produced similar but not identical patterns of reactivities. Biochemistry, 1997 Feb 18, 36(7), 1730 - 9 Dihydrodipicolinate synthase from Escherichia coli: pH dependent changes in the kinetic mechanism and kinetic mechanism of allosteric inhibition by L-lysine; Karsten WE; Dihydrodipicolinate synthase (DHDPS) catalyzes the formation of dihydrodipicolinate from pyruvate and L-aspartate beta-semialdehyde (ASA) . A parallel initial velocity pattern that displays competitive substrate inhibition by ASA and dead-end inhibition patterns obtained at pH 8 are consistent with a ping pong kinetic mechanism for DHDPS . The results suggest that pyruvate binds to free enzyme with subsequent formation of a Schiff base with an enzymic lysine residue followed by binding of ASA to the F enzyme form to initiate the second half-reaction . At low pH (5.7) the initial velocity and dead-end inhibition patterns are consistent with a sequential steady state ordered kinetic mechanism with pyruvate binding to enzyme prior to ASA . The irreversible step in the reaction, leading to the ping pong kinetic mechanism at high pH, is proposed to be loss of a proton from the methyl group of pyruvate in Schiff base with enzyme to form an enamine intermediate . Consistent with this proposal is the change to a sequential steady state ordered kinetic mechanism at low pH at or below the pK of the enamine intermediate . L-Lysine is an allosteric inhibitor of the DHDPS reaction that causes partial inhibition (approximately 90%) at saturating concentrations . Inhibition patterns for L-lysine vs pyruvate and ASA suggest that lysine binds to the F enzyme form at pH 8 with a Ki value of about 0.3 mM . An examination of the effects of different L-lysine concentrations on the kinetic parameters V/Kpyruvate, V/KASA, and V indicate that L-lysine decreases only the values of V/KASA and Vmax, which is consistent with the inhibitory effects of lysine manifested on the second half-reaction . In contrast at low pH the data suggest L-lysine binds to free enzyme with an inhibition constant of about 5 mM. Biochemistry, 1997 Feb 18, 36(7), 1608 - 20 Catalytic mechanism of the quinoenzyme amine oxidase from Escherichia coli: exploring the reductive half-reaction; Wilmot CM et al.; The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has been determined to a resolution of 2.0 A . The inhibitor covalently binds at the 5 position of the quinone ring of the cofactor, 2,4,5-trihydroxyphenylalaninequinone (TPQ) . The inhibitor complex is analogous to the substrate Schiff base formed during the reaction with natural monoamine substrate . A proton is abstracted from a methylene group adjacent to the amine group by a catalytic base during the reaction . The inhibitor, however, has a nitrogen at this position, preventing proton abstraction and trapping the enzyme in a covalent complex . The electron density shows this nitrogen is hydrogen bonded to the side chain of Asp383, a totally conserved residue, identifying it as the probable catalytic base . The positioning of Asp383 is such that the pro-S proton of a substrate would be abstracted, consistent with the stereospecificity of the enzyme determined by 1H NMR spectroscopy . Site-directed mutagenesis and in vivo suppression have been used to substitute Asp383 for 12 other residues . The resulting proteins either lack or, in the case of glutamic acid, have very low enzyme activity consistent with an essential catalytic role for Asp383 . The O4 position on the quinone ring is involved in a short hydrogen bond with the hydroxyl of conserved residue Tyr369 . The distance between the oxygens is less than 2.5 A, consistent with a shared proton, and suggesting ionization at the O4 position of the quinone ring . The Tyr369 residue appears to play an important role in stabilizing the position of the quinone/inhibitor complex . The O2 position on the quinone ring is hydrogen bonded to the apical water ligand of the copper . The basal water ligand, which lies 2.0 A from the copper in the native structure, is at a distance of 3.0 A in the complex . In the native structure, the active site is completely buried, with no obvious route for entry of substrate . In the complex, the tip of the pyridine ring of the bound inhibitor is on the surface of the protein at the edge of the interface between domains 3 and 4, suggesting this as the entry point for the amine substrate. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1556 - 61 Interconversion of red opsin isoforms by the cyclophilin-related chaperone protein Ran-binding protein 2; Ferreira PA et al.; Ran-binding protein 2 (RanBP2) (type II) is a retinal cyclophilin-related protein that binds Ran-GTPase . Type I cyclophilin is a shorter, alternatively spliced isoform of RanBP2 . Recently, we showed that the Ran-binding domain 4 (RBD4)/cyclophilin (CY) supradomain of RanBP2 acts both in vitro and in vivo as a specific chaperone for bovine red/green opsin (R/G opsin) . R/G opsin undergoes a stable modification of its electrophoretic mobility upon binding to RanBP2 . This modification is likely due to cis-trans isomerization of one or more proline residues in the opsin protein . Here, we show that expression of human red opsin in Escherichia coli and COS cells results in the production of still a third electrophoretic variant of this protein . This variant was converted to the RBD4 binding-competent form of opsin through direct interaction with RBD4/CY, both in vivo and in vitro . We suggest that these distinct opsin species may represent kinetically or thermodynamically trapped prolyl conformers that can be interconverted by concerted action of the RBD4 and CY domains of RanBP2 . We also show that the C-terminal half of RBD4 is the binding domain for bovine R/G opsin and that coexpression of human red opsin with type I cyclophilin in vivo enhances the production of functional visual pigment . These observations imply that prolyl isomerization may have importance beyond its role in protein folding, possibly as a molecular switch modulated by cyclophilin for the loading of opsin onto RanBP2 during visual pigment processing in cones. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1304 - 9 High-resolution functional mapping of a cloned gene by genetic footprinting; Singh IR et al.; We describe an efficient method for introducing and analyzing a comprehensive set of mutations in a cloned gene to map its functional organization . The technique, genetic footprinting, uses a retroviral integrase to generate a comprehensive library of mutants, each of which bears a single insertion of a defined oligonucleotide at a random position in the gene of interest . This mutant library is selected for gene function en masse . DNA samples are isolated from the library both before and after selection, and the mutations represented in each sample are then analyzed . The analysis is designed so that a mutation at a particular location gives rise to an electrophoretic band of discrete mobility . For the whole library, this results in a ladder of bands, each band representing a specific mutation . Mutants in which the inserted sequence disrupts a feature that is required for the selected function, ipso facto, fail the selection . The corresponding bands are therefore absent from the ladder of bands obtained from the library after selection, giving rise to a footprint representing features of the gene that are essential for the selected function . Because the sequence of the inserted oligonucleotide is known, and its position can be inferred precisely from the electrophoretic mobility of the corresponding band, the precise location and sequence of mutations that disrupt gene function can be determined without isolating or sequencing individual mutants . This method should be generally applicable for saturation mutagenesis and high-resolution functional mapping of cloned DNA sequences. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1107 - 12 The effect of macromolecular crowding on chaperonin-mediated protein folding; Martin J et al.; The cylindrical chaperonin GroEL and its cofactor GroES mediate ATP-dependent protein folding in Escherichia coli . Recent studies in vitro demonstrated that GroES binding to GroEL causes the displacement of unfolded polypeptide into the central volume of the GroEL cavity for folding in a sequestrated environment . Resulting native protein leaves GroEL upon GroES release, whereas incompletely folded polypeptide can be recaptured for structural rearrangement followed by another folding trial . Additionally, each cycle of GroES binding and dissociation is associated with the release of nonnative polypeptide into the bulk solution . Here we show that this loss of substrate from GroEL is prevented when the folding reaction is carried out in the presence of macromolecular crowding agents, such as Ficoll and dextran, or in a dense cytosolic solution . Thus, the release of nonnative polypeptide is not an essential feature of the productive chaperonin mechanism . Our results argue that conditions of excluded volume, thought to prevail in the bacterial cytosol, increase the capacity of the chaperonin to retain nonnative polypeptide throughout successive reaction cycles . We propose that the leakiness of the chaperonin system under physiological conditions is adjusted such that E . coli proteins are likely to complete folding without partitioning between different GroEL complexes . Polypeptides that are unable to fold on GroEL eventually will be transferred to other chaperones or the degradation machinery. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1096 - 100 Catalysis of protein folding by symmetric chaperone complexes; Sparrer H et al.; The GroE chaperones of Escherichia coli assist protein folding under physiological and heat shock conditions in an ATP-dependent way . Although a number of details of assisted folding have been elucidated, the molecular mechanism of the GroE cycle remains unresolved . Here we present an experimental system that allows the direct analysis of the GroE-mediated folding cycle under stringent conditions . We demonstrate that the GroE proteins efficiently catalyze the folding of kinetically trapped folding intermediates of a mutant of maltose-binding protein (MBP Y283D) in an ATP-dependent way . GroES plays a key role in this reaction cycle, accelerating the folding of the substrate protein MBP Y283D up to 50-fold . Interestingly, catalysis of the folding reaction requires the formation of symmetrical football-shaped GroEL x GroES2 particles and the intermediate release of the nonnative protein from the chaperone complex . Our results show that, in the presence of GroES, the complex architecture of the GroEL toroids allows maintenance of two highly interregulated rings simultaneously active in protein folding. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1069 - 73 Assembly of an active enzyme by the linkage of two protein modules; Nixon AE et al.; The feasibility of creating new enzyme activities from enzymes of known function has precedence in view of protein evolution based on the concepts of molecular recruitment and exon shuffling . The enzymes encoded by the Escherichia coli genes purU and purN, N10-formyltetrahydrofolate hydrolase and glycinamide ribonucleotide (GAR) transformylase, respectively, catalyze similiar yet distinct reactions . N10-formyltetrahydrofolate hydrolase uses water to cleave N10-formyltetrahydrofolate into tetrahydrofolate and formate, whereas GAR transformylase catalyses the transfer of formyl from N10-formyltetrahydrofolate to GAR to yield formyl-GAR and tetrahydrofolate . The two enzymes show significant homology (approximately 60%) in the carboxyl-terminal region which, from the GAR transformylase crystal structure and labeling studies, is known to be the site of N10-formyltetrahydrofolate binding . Hybrid proteins were created by joining varying length segments of the N-terminal region of the PurN gene (GAR binding region) and the C-terminal (N10-formyltetrahydrofolate binding) region of PurU . Active PurN/PurU hybrids were then selected for by their ability to complement an auxotrophic E . coli strain . Hybrids able to complement the auxotrophs were purified to homogeneity and assayed for activity . The specific activity of two hybrid proteins was within 100- to 1000-fold of the native purN GAR transformylase validating the approach of constructing an enzyme active site from functional parts of others. Virology, 1997 Feb 17, 228(2), 200 - 12 Phosphorylated states of vesicular stomatitis virus P protein in vitro and in vivo; Chen JL et al.; We have previously shown that the phosphoprotein (P) of vesicular stomatitis virus (VSV), New Jersey serotype (PNJ) is phosphorylated by casein kinase II, within the N-terminal domain I (P1 form), whereas the C-terminal domain II is phosphorylated by a protein kinase activity associated with the L protein (P2 form) (D . J . Chattopadhyay and A.K . Banerjee, Cell 49, 407, 1987; A.M . Takacs et al., J . Virol . 66, 5842, 1992) . In the present studies, we have mapped the corresponding P1 and P2 phosphorylation sites in the P protein of the well-studied Indiana serotype (PIND) and compared that with the two previously designated NS1 and NS2 forms present in vivo . The PIND expressed in Escherichia coli in an unphosphorylated form (P0) was used as substrate for recombinant casein kinase II (CKII) . By site-directed mutagenesis, the CKII-mediated phosphorylation sites in the P protein were mapped at S60, T62, and S64 within the acidic domain I in vitro . In contrast, using BHK cell extract as the source of CKII or expressing P protein in COS cells labeled with 32PI, the phosphorylation sites were mapped at S60 and S64 with no phosphorylation at T62 residue . We used a peptide mapping technique by which the phosphorylation sites within domain I and domain II were determined . Using this method we demonstrated that the P1 and P2 forms are similar, if not identical, to the previously designated NS1 and NS2 forms, respectively . The domain II phosphorylating kinase activity, associated with the L protein, is shown to be present also in the N-RNA complex, indicating that this activity is of cellular origin . By site-directed mutagenesis, we have shown that S226 and S227 are involved in phosphorylation within domain II . We also demonstrate that the P1 and P2 forms are interconvertible and arise by phosphorylation/dephosphorylation of the phosphate groups in domain II, confirming the precursor-product relationship between the two phosphorylated forms of P protein. EMBO J, 1997 Feb 17, 16(4), 889 - 95 The limited strand-separating activity of the UvrAB protein complex and its role in the recognition of DNA damage; Gordienko I et al.; The recognition by Escherichia coli Uvr nucleotide excision repair proteins of a variety of lesions with diverse chemical structures and the presence of helicase activity in the UvrAB complex which can displace short oligonucleotides annealed to single-stranded DNA led to a model in which this activity moves UvrAB along undamaged DNA to damaged sites where the lesion blocks further translocation and the protein-DNA pre-incision complex is formed . To evaluate this mechanism for damage recognition, we constructed substrates with oligonucleotides of different lengths annealed to single-stranded DNA circles and placed a single 2-(acetylamino)fluorene (AAF) lesion either on the oligonucleotide or on the circle . For the substrates with no lesion, the UvrAB complex effectively displaced a 22-mer but not a 27-mer or longer fragments . The presence of AAF on the oligonucleotide significantly increased the release of the 27-mer but oligomers of 30 or longer were not separated . Placing the lesion on the circular strand did not block the release of the fragments . Instead, the releasing activity of UvrAB was stimulated and also depended on the length of the annealed oligonucleotide . These observations do not agree with the predictions of a damage recognition mechanism that depends on helicase-driven translocation . Most likely, the strand-separating activity of UvrAB is a consequence of local changes occurring during the formation of a DNA-protein pre-incision complex at the damaged site and is not due to translocation of the protein along undamaged DNA to locate a lesion. EMBO J, 1997 Feb 17, 16(4), 880 - 8 UvrAB activity at a damaged DNA site: is unpaired DNA present? Gordienko I, Rupp WD. To study the activity of the Escherichia coli UvrA and UvrB nucleotide excision repair proteins during the formation of the pre-incision complex at a damaged DNA site, we used substrates with modifications around a single 2-(acetylamino)fluorene (AAF) lesion . Based on the release of AAF-containing oligonucleotides from a single-stranded DNA circle, we conclude that during interaction with our substrates UvrAB introduces changes in DNA which are localized at the lesion and are limited to 1-3 bp . Since these changes might include a denaturation of DNA at the lesion site and, consequently, a bubble structure might be present in a pre-incision complex, we studied incision activity of UvrABC excinuclease on substrates with 1-4 unpaired bases next to an AAF adduct . Opening more than one base on either or both sides of the lesion caused a significant decrease in the incision activity of UvrABC, but did not change the position of the incision sites . We conclude that the UvrAB action leading to a pre-incision complex does not include the formation of a bubble intermediate generated by extensive denaturation of base pairs. EMBO J, 1997 Feb 17, 16(4), 826 - 33 Evidence that translation reinitiation abrogates nonsense-mediated mRNA decay in mammalian cells; Zhang J et al.; Nonsense codons upstream of and including position 192 of the human gene for triosephosphate isomerase (TPI) have been found to reduce the abundance of TPI mRNA to approximately 25% of normal . The reduction is due to the decay of newly synthesized TPI mRNA that co-purifies with nuclei . TPI mRNA that co-purifies with cytoplasm is immune to nonsense-mediated decay . Until now, a nonsense codon at position 23 has been the 5'-most nonsense codon that has been analyzed . Here, we provide evidence that a nonsense codon at position 1, 2 or 10 reduces the abundance of nucleus-associated TPI mRNA to an average of only 84% of normal because translation reinitiates at the methionine codon at position 14 . First, converting codon 14 to one for valine increased the effectiveness with which an upstream nonsense codon reduces mRNA abundance . Second, when TPI gene sequences, including codon 14, were fused upstream of and in-frame to the translational reading frame of an Escherichia coli chloramphenicol acetyl transferase (CAT) gene that lacked an initiation codon, a nonsense codon at TPI position 1 or 2 allowed for the production of TPI-CAT that was an estimated 14 amino acids smaller than TPI-CAT produced by a nonsense-free gene, whereas a nonsense codon at TPI position 23 precluded the production of TPI-CAT . These and related findings lend credence to the concept that the nonsense-mediated reduction in the half-life of nucleus-associated TPI mRNA involves cytoplasmic ribosomes. FEBS Lett, 1997 Feb 17, 403(2), 203 - 7 Positively charged lysine at the N-terminus of the signal peptide of the Escherichia coli alkaline phosphatase provides the secretion efficiency and is involved in the interaction with anionic phospholipids; Nesmeyanova MA et al.; Positively charged amino acid residues at the N-terminus of the signal peptide (SP) have been proposed to play a significant role in the initial step of protein secretion in bacteria . To test this hypothesis, Lys(-20) of the Escherichia coli alkaline phosphatase SP was replaced by other amino acid residues, and the effect of these substitutions on protein maturation was studied . The introduction of negatively charged and hydrophobic amino acids resulted in a decrease in secretion efficiency and impaired the SP-APL interaction, whereas His and Tyr had no significant effect . A structural analysis of the SP-APL interaction suggests that the positively charged Lys(-20) determines the stability of the complex. FEBS Lett, 1997 Feb 17, 403(2), 116 - 22 Use of phage display for isolation and characterization of single-chain variable fragments against dihydroflavonol 4-reductase from Petunia hybrida; De Jaeger G et al.; To isolate specific single-chain variable (scFv) fragments against dihydroflavonol 4-reductase (DFR) from Petunia hybrida the phage display technology was used . DFR was overproduced in Escherichia coli, purified and used for immunization . From DFR-immunized mice, a phage display library was made starting from spleen mRNA using an optimized set of primers for V(H) and V(L) amplification . Several rounds of panning against recombinant DFR yielded five different scFv fragments, confirmed by subsequent DNA sequencing . They all specifically bound to recombinant DFR in ELISA and DFR in flower extracts on Western blot . These results show that phage display is a promising technology in plant molecular biology to obtain specific recombinant antibodies not only for ELISA and Western blot but also for in vivo applications in the long run. J Surg Res, 1997 Feb 15, 68(1), 79 - 86 Nitric oxide inhibition decreases neutrophil adhesion at the inflammatory site, while increasing adhesion in remote organs in peritonitis; Fukatsu K et al.; Nitric oxide (NO) is a regulator of leukocyte adhesion in the microcirculation . This study was designed to examine the effects of a NO synthase inhibitor on neutrophil adhesion in the peritoneum, lung, liver, and kidney in a rat peritonitis model using a fluorescence microscopic method . Sprague-Dawley rats were given normal saline (control) or N omega-nitro-L-arginine methyl ester (L-NAME) at dosages of 10 mg/kg (N10) or 100 mg/kg (N100) (n = 66) intraperitoneally . One hour after pretreatment fluorescein-labeled neutrophils were infused without bacterial challenge (0 hr) . Other rats received an injection of 10(7) Escherichia coli into the peritoneal cavity 1 hr after pretreatment . Labeled neutrophils were infused 1 and 5 hr after bacterial challenge . Just 2 min after neutrophil injection, blood samples were obtained and the animals were killed . Five peritoneal samples (omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy . Combined plasma nitrite/nitrate levels were determined . In another set of rats (n = 36), an arterial catheter was inserted after L-NAME treatment and bacterial challenge . At 0, 1, 5, and 12 hr after challenge, blood pressure, heart rate, and arterial blood gas data were measured . One hour after E . coli challenge, the number of neutrophils in the peritoneum was significantly lower in both L-NAME-treated groups than in the control group . In contrast, the number of labeled neutrophils in the lungs was significantly higher in the N100 group than in the control group . Neutrophil accumulation in the lungs and peritoneum at 0 and 5 hr and in the liver and kidney at 0, 1, and 5 hr did not differ among groups, nor did combined plasma nitrite/nitrate levels . L-NAME treatment had no influence on either hemodynamic or blood gas data . In conclusion, administration of L-NAME increases neutrophil adhesion in the lung, while decreasing that in the peritoneum . NO plays an important role in neutrophil adhesion at the inflammatory site, as well as in remote organs, during peritonitis . NO inhibition may be detrimental, due to neutrophil sequestration, in this peritonitis model. Biochem J, 1997 Feb 15, 322 ( Pt 1), 241 - 4 Identification of a putative flexible loop in Arabidopsis glutathione synthetase; Wang CL et al.; Glutathione synthetase catalyses the ATP-dependent ligation of gamma-glutamylcystene with glycine to form glutathione . Amino acid sequence comparisons between the Arabidopsis and the Escherichia coli proteins suggested that a region, identified as a small flexible loop that covers the active site of the E . coli protein, might be conserved in the eukaryotic protein . Three site-directed mutations in the Arabidopsis protein were generated to test this hypothesis . Two mutations within the conserved region (Lys367/ Pro368-->Asn/Ser and Gly374-->Val) inactivated the enzyme in an in vivo assay based on cadmium resistance in S . pombe, and in an in vitro assay of the activity of the enzyme expressed in E . coli . A third mutation outside of this conserved region (Leu363-->Glu) had a smaller effect in both assays . These results are consistent with the idea that this glycine-rich loop in the Arabidopsis and E . coli proteins might serve the same function in covering the active site of the enzyme. Genomics, 1997 Feb 15, 40(1), 41 - 7 PMM (PMM1), the human homologue of SEC53 or yeast phosphomannomutase, is localized on chromosome 22q13; Matthijs G et al.; We have cloned the human homologue of SEC53 or yeast phosphomannomutase (HGMW-approved symbol PMM1) from a liver cDNA library . This cDNA encodes a protein of 262 amino acids with a predicted molecular mass of 29 kDa and 54% identity with yeast phosphomannomutase . Expression of the human cDNA in Escherichia coli yielded an active phosphomannomutase, which was purified to homogeneity . Northern blot analysis of human tissues showed strong expression in liver, heart, brain, and pancreas and a lower expression in skeletal muscle . The gene was assigned to chromosome 22q13.1 by the use of hybrid cell lines and by fluorescence in situ hybridization . Most patients presenting with carbohydrate-deficient glycoprotein syndrome type 1 (CDG1 or Jaeken disease) have a greatly reduced phosphomannomutase activity; the gene encoding this enzyme is a likely candidate for CDG1 . Since the CDG1 locus maps else where in the genome (16p13), mutations in the phosphomannomutase gene encoded by chromosome 22 are not a major cause of CDG1. Nucleic Acids Res, 1997 Feb 15, 25(4), 806 - 13 Role of proofreading and mismatch repair in maintaining the stability of nucleotide repeats in DNA; Strauss BS et al.; The role of the proofreading exonuclease in maintaining the stability of multiply repeated units in DNA was studied in Escherichia coli . Reversion of plasmids in which the beta-galactosidase alpha complementing sequence was moved +2 out of frame by inserts containing (CA)14, (CA)5, (CA)2 or (TA)6 or +1 by creating a run of 8 C was compared in mutS and mutSdnaQ strains . Proofreading corrects at least half of the frameshift errors for all the plasmids and at least 99% of the errors in the (CA)2 plasmid . The (CA)2 plasmid reverts mostly by +1 frameshifts in the restriction sites flanking the insert . With the (CA)14, (TA)6, (CA)5 and 8C plasmids, reversion is mainly by loss of a repeat unit . The data support the hypothesis that the dnaQgene product recognizes frameshifts close to the DNA growing point . Frameshifts distal to the growing point are mainly corrected by mismatch repair.We speculate that mismatches in mononucleotide repeats are susceptible to proofreading because they can either migrate to a point where they are recognized by the exonuclease or, alternatively, because single nucleotide distortions are more readily detected than dinucleotides. Eur J Biochem, 1997 Feb 15, 244(1), 235 - 41 Activity of tethered human immunodeficiency virus 1 protease containing mutations in the flap region of one subunit; Tozser J et al.; The tethered-dimer protease of human immunodeficiency virus 1 (HIV-1) {Cheng Y.-S . E., Yin, F.H., Foundling, S., Blomstrom, D . & Kettner, C . A . (1990) Proc . Natl Acad . Sci . USA 87, 9660-9664} and its mutants containing amino acid substitutions or deletions or both in only one flap region were expressed in Escherichia coli . These mutant enzymes showed various degrees of self-processing and significantly reduced catalytic activity toward oligopeptide substrates compared with the wild type . Kinetic parameters determined for one of the oligopeptide substrates showed a dramatic increase in K(m) and decrease in Kcat values . Unexpectedly, the substrate cleavage was more efficient in low salt concentration for a mutant containing a shortened hydrophilic flap . Assays with oligopeptides representing naturally occurring cleavage sites or oligopeptides containing single amino acid substitutions at the P2 and P2' substrate positions showed only moderate changes in the substrate specificity of the mutant proteases . Predicted structures for the mutants were constructed by molecular modeling and used to interpret the results of kinetic measurements . In general, the data suggest that the mutated part of the flaps does not have a major role in determining substrate specificity; rather, it provides the hydrophobic environment and hydrogen-bond interactions with the conserved water that are necessary for efficient substrate binding and catalysis. Eur J Biochem, 1997 Feb 15, 244(1), 155 - 60 Requirement for the proton-pumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate and its bioenergetic implications; Tran QH et al.; In Escherichia coli the expression of the nuo genes encoding the proton pumping NADH dehydrogenase I is stimulated by the presence of fumarate during anaerobic respiration . The regulatory sites required for the induction by fumarate, nitrate and O2 are located at positions around -309, -277, and downstream of -231 bp, respectively, relative to the transcriptional-start site . The fumarate regulator has to be different from the O2 and nitrate regulators ArcA and NarL . For growth by fumarate respiration, the presence of NADH dehydrogenase I was essential, in contrast to aerobic or nitrate respiration which used preferentially NADH dehydrogenase II . The electron transport from NADH to fumarate strongly decreased in a mutant lacking NADH dehydrogenase I . The mutant used acetyl-CoA instead of fumarate to an increased extent as an electron acceptor for NADH, and excreted ethanol . Therefore, NADH dehydrogenase I is essential for NADH-->fumarate respiration, and is able to use menaquinone as an electron acceptor . NADH-->dimethylsulfoxide respiration is also dependent on NADH dehydrogenase I . The consequences for energy conservation by anaerobic respiration with NADH as a donor are discussed. Arch Biochem Biophys, 1997 Feb 15, 338(2), 157 - 64 Targeted antipeptide antibodies to cytochrome P450 2C18 based on epitope mapping of an inhibitory monoclonal antibody to P450 2C51; Richardson TH et al.; The epitope recognized by the inhibitory monoclonal antibody designated 2F5, which was raised against P450 2C5, was mapped to amino acids 237-260 by immunoblotting using a combination of recombinant antigens and chimeric and partial fusion proteins constructed from rabbit P450s 2C2, 2C4, 2C5, and 2C16, which are recognized by 2F5, and from 2C1 and 2C3, which are not . When the sequence of the epitope for 2F5 (amino acids 237-260) was compared with those of other rabbit 2C P450s, a single lysine residue at position 253 appeared to be a likely determinant of 2F5 immunoreactivity . Substitution of lysine for glutamic acid 253 in P450 2C3 (2C3E253K) conferred immunoreactivity and the ability of 2F5 to inhibit progesterone metabolism catalyzed by P450 2C3E253K . Sequence alignment revealed that this epitope lies in close proximity to the epitope identified for LKM-1 autoantibodies to P450 2D6 . Based on these results, an antipeptide antibody was raised to the corresponding region (amino acids 252-263) of human P450 2C18 . The resulting antipeptide antiserum recognizes P450 2C18 but not P450 2C8, 2C9, or 2C19 . However, the antipeptide 2C18 antiserum did not inhibit 2C18-catalyzed diazepam N-demethylation . Human 2C P450s were also quantitated by immunoblot analysis in a panel of six human liver microsomes using Escherichia coli expressed P450s as standards . Analysis of immunoblots indicated that, if present, P450 2C18 was expressed at very low levels (<2.5 pmol/mg), whereas P450s 2C8, 2C9, and 2C19 were easily detected. Nucleic Acids Res, 1997 Feb 15, 25(4), 781 - 6 Libraries for genomic SELEX; Singer BS et al.; An increasing number of proteins are being identified that regulate gene expression by binding specific nucleic acidsin vivo . A method termed genomic SELEX facilitates the rapid identification of networks of protein-nucleic acid interactions by identifying within the genomic sequences of an organism the highest affinity sites for any protein of the organism . As with its progenitor, SELEX of random-sequence nucleic acids, genomic SELEX involves iterative binding, partitioning, and amplification of nucleic acids . The two methods differ in that the variable region of the nucleic acid library for genomic SELEX is derived from the genome of an organism . We have used a quick and simple method to construct Escherichia coli, Saccharomyces cerevisiae, and human genomic DNA PCR libraries that can be transcribed with T7 RNA polymerase . We present evidence that the libraries contain overlapping inserts starting at most of the positions within the genome, making these libraries suitable for genomic SELEX. Nucleic Acids Res, 1997 Feb 15, 25(4), 764 - 8 Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase; Todo T et al.; Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development . Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts {(6-4)photoproducts} are the two major photoproducts produced in DNA by UV irradiation . Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts {(6-4)photolyase} . (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism . The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family . Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity . Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD . This is the first indication of FAD as the chromophore of (6-4)photolyase. Nucleic Acids Res, 1997 Feb 15, 25(4), 719 - 26 Characterization of a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcus sp . KOD1; Rashid N et al.; The Pk-rec gene, encoding a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcussp . KOD1, was expressed in Escherichia coli . The recombinant Pk-REC was purified to homogeneity and was shown to be in a dimeric form . A striking property of the purified recombinant Pk-REC was the unusual DNase activity on both single- and double-stranded DNAs along with the ATPase activity . The reaction product of this DNase activity was mononucleotides . The optimum temperature and pH for the DNase activity were 60 degrees C and 8-8.5, respectively . In addition, the metal ion requirement for DNase activity was different from that for the ATPase activity . The protein exhibited no DNase activity in the presence of Zn2+ion, which was one of the most preferable divalent cations for ATPase activity . Another unique characteristic of the recombinant protein was that the reaction product of ATPase activity was AMP instead of ADP.Pk-REC may represent a common prototype of the RecA family proteins with high RecA-like activity. Nucleic Acids Res, 1997 Feb 15, 25(4), 713 - 8 DNA flexibility of the UP element is a major determinant for transcriptional activation at the Escherichia coli acetate promoter; Negre D et al.; The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method . Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase . A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter. Mutat Res, 1997 Feb 14, 388(2-3), 239 - 48 Somatic and germ cell mutagenesis in lambda lacZ transgenic mice treated with acrylamide or ethylnitrosourea; Krebs O et al.; The transgenic Muta Mouse in vivo mutagenesis assay was employed to determine the activity of acrylamide and ethylnitrosourea in liver and germ cells after 3, 10 and 100 days following treatment . Each cell of the Muta Mouse carries 80 copies of the lambda gt10 phage including the bacterial lacZ gene, which act as the target gene for the mutagenesis assay . Groups of Muta Mice were given a single intraperitoneal injection of 80 or 160 mg/kg ethylnitrosourea or 50 or 100 mg/kg acrylamide . The tissues were prepared 3, 10 or 100 days post treatment . The liver genomic DNA was extracted with the manufacturer's standard protocol, while the genomic germ cell DNA was extracted with 4 different methods due to problems encountered in DNA yields and packaging efficiency . The mutation analysis of the lacZ gene was carried out by the positive selective assay method {Gossen et al . (1989) Proc . Natl . Acad . Sci . USA, 86, 7971-7975; Dean and Myhr (1994) Mutagenesis, 9, 183-185} . There was a slight increase due to treatment of the observed mutation frequencies in the acrylamide liver group for all three assay times . From the day 3 group to the day 100 group a time dependent decrease in all the absolute mutant frequencies was detectable . The ethylnitrosourea liver group showed a time- and dose-dependent increase in the mutant frequencies from day 3 to day 100 . No meaningful results were obtained for the germ cell tissue assays due to the low amount of genomic DNA extracted which was not packageable in the lambda lacZ assay . At present for the mutagenesis assay of isolated spermatozoa in our laboratory we would be forced to pool tissues from animals to obtain enough DNA for an assay . Since 'jackpot'-animals may exist {Heddle et al . (1992) Mutation Res., 272, 195-203} the individual animals of such a pooled analysis group must be tested before pooling. Mutat Res, 1997 Feb 14, 388(2-3), 229 - 37 Mutations induced in male germ cells after treatment of transgenic mice with ethylnitrosourea; Katoh M et al.; The present study was undertaken to clarify whether the transgenic mouse mutagenesis assay system can be used instead of dominant lethals or specific locus test after treatment of male germ cells in mouse with ethylnitrosourea (ENU) . Male Big Blue transgenic mice (BB) carrying a lacI target gene were given a single intraperitoneal injection of 150 mg/kg ENU . Vasa deferential sperm, caudal epididymal sperm or whole testes were assayed for mutation at 3, 14, 22 and 93 days after treatment with ENU . The average of background lacI- mutant frequencies was 2.05 x 10(-5) . The MF observed in post spermatogonial stage after treatment with ENU were slightly increased over background . On the other hand, ENU induced high MF in the spermatogonial stage . MF detected after treatment of BB male germ cells with ENU were lower than those detected in the mouse visible specific-locus mutations in previous reports . Nevertheless, it is clear that this assay is a practical alternative to the specific locus test for detecting mutations induced in spermatogonial stage. Mutat Res, 1997 Feb 14, 388(2-3), 223 - 8 Germ cell mutagenesis in lacZ transgenic mice treated with methyl methanesulfonate; Itoh S et al.; Mutagenesis induced by methyl methanesulfonate (MMS), a germ cell mutagen, in the testis and the sperm isolated from epididymis and vas deferens have been investigated using lacZ transgenic mice (Muta Mouse) . Male Muta Mice were injected intraperitoneally with MMS at a dose of 80 mg/kg, a potent dominant lethal dose . Animals were killed on days 3 and 7 (Experiment 1) or days 10 and 14 (Experiment 2) after the treatment . Mutant frequencies (MFs) in the testis, sperm and spleen (Experiment 2 only) were analyzed by the positive selection system using E . coli C (GalE-) strain and phenyl beta-D-galactoside . The spontaneous MFs in the testis and sperm were 2.0-3.1 x 10(-5) . No induction of mutation in the testis or sperm of the MMS-treated groups was observed at any sampling point . In the spleen, the spontaneous MF was approximately twice as high as that in the germ cells although the MF at each sampling point was almost the same as the spontaneous MF . MMS is known as a potent clastogen from the results of the dominant lethal assay and the micronucleus assay . The reason for the discrepancy between the results of these assays and the present results may have been insensitivity of the in vitro packaging to large deletion due to the failure to rescue the large deleted gene . It is suggested that the transgenic mouse assay using the in vitro packaging can not replace the dominant lethal assay in the case of MMS. Mutat Res, 1997 Feb 14, 388(2-3), 219 - 22 The detection of gene mutation in the tubular sperm of Muta Mice following a single intraperitoneal treatment with methyl methanesulphonate or ethylnitrosourea; Brooks TM et al.; Transgenic mouse assays, such as Muta Mouse, provide a method to predict the potential target organ carcinogenicity of chemical compounds . As part of a collaborative study, the effects of the direct-acting mutagens, methyl methanesulphonate (MMS) and ethylnitrosourea (ENU), were investigated for gene mutation in the tubular sperm of Muta Mice testes after a single intraperitoneal exposure . Groups of male Muta Mice were dosed intraperitoneally with either 1/15 M phosphate buffer, pH 6.0 (vehicle control), 40 mg/kg methyl methanesulphonate (MMS) or 150 mg/kg ethylnitrosourea (ENU) . The animals were sacrificed 14 days after the single dose . Mutation frequencies were determined in tubular sperm DNA . The results showed a mean mutation frequency (MF) of 2.1 x 10(5) (64 mutants per 3.05 x 10(6) PFU) for the 10 vehicle-treated mice, a mean MF of 2.8 x 10(5) (78 mutants per 2.75 x 10(6) PFU) for the 10 MMS-treated mice and a mean MF of 9.1 x 10(5) (194 mutants per 2.14 x 10(6) PFU) for the 8 ENU-treated mice; this latter value representing a 4.5-fold increase over the vehicle control values. Mutat Res, 1997 Feb 14, 388(2-3), 187 - 95 Evaluation of lacI mutation in germ cells and micronuclei in peripheral blood after treatment of male lacI transgenic mice with ethylnitrosourea, isopropylmethane sulfonate or methylmethane sulfonate; Gorelick NJ et al.; Male C57B1/6 lacI transgenic mice were used to evaluate germ cell mutagenesis in vivo as part of a collaborative study . Groups of 10 mice were administered single intraperitoneal doses of ethylnitrosourea (ENU; 150 mg/kg), isopropyl methanesulfonate (IPMS; 200 mg/kg), methyl methanesulfonate (MMS; 40 mg/kg) or vehicle . Epididymal spermatozoa and testes were recovered 3 days later and DNA isolated subsequently from epididymal spermatozoa and seminiferous tubules were analyzed for lacI mutations . The mutant frequency in seminiferous tubules (average +/- SEM) increased significantly compared with untreated controls (7.2 +/- 0.7 x 10(-5) following treatment with ENU (11.7 +/- 0.8 x 10(-5), p = 0.003) or with IPMS (9.6 +/- 0.5 x 10(-5), p = 0.018) but not following treatment with MMS (8.1 +/- 0.8 x 10(-5), p = 0.213) . Group mutant frequencies were not determined for epididymal spermatozoa from MMS- or IPMS-treated mice because of poor DNA recoveries . As another indicator of the genotoxicity of these alkylating agents, the frequencies of micronuclei were determined in the peripheral blood 48 h after carcinogen administration in the same transgenic mice . The micronuclei frequencies were elevated significantly (p < 0.05) by each treatment (IPMS: 1.0%; MMS: 0.94%) compared to vehicle controls (0.3%) . In a separate experiment, 40 mg/kg ENU was previously found to increase the frequency of micronuclei in peripheral blood of lacI transgenic mice 48 h after treatment (3.2%; Gibson et al., 1995) . These results demonstrate that the lacI transgenic mouse male germ cells are sensitive to some, but not all, mutagens under the conditions used in this experiment . Investigation of other experimental designs would offer additional perspective on the usefulness of this transgenic model for routine mutagenicity testing in germ cells. Mutat Res, 1997 Feb 14, 388(2-3), 155 - 63 Ethyl nitrosourea and methyl methanesulfonate mutagenicity in sperm and testicular germ cells of lacZ transgenic mice (Muta Mouse); Suzuki T et al.; The germ cell mutagens ethyl nitrosourea (ENU) and methyl methanesulfonate (MMS), were tested for their genotoxicity in sperm cells and testicular germ cells using lacZ transgenic mice (Muta Mouse) . Eight- to 10-week-old Muta mice were treated with ENU (150 mg/kg) or MMS (40 mg/kg) by intraperitoneal injection . Three and 14 days after treatment, testes and sperm were collected for lacZ mutation analysis . Sperm were isolated from the epididymis and vas deferens by washing out the minced tissue . Germ cell DNA was isolated from testicular germ cells and sperm with the help of 2-mercaptoethanol, and the target lacZ gene, which is integrated into a lambda shuttle vector, was recovered by in vitro packaging . The resultant phages were allowed to infect to E . coli C (galE), and the lacZ mutant plaques were dominantly selected on a plate containing phenyl-beta-D-galactoside . Spontaneous mutant frequencies (MF) in vehicle-treated control mice were approximately 1 x 10(-5) and 3 x 10(-5) in testicular germ cells and sperm, respectively, at both sampling times . ENU treatment increased the MF in the testicular germ cells to 5 x 10(-5) on days 3 and 14, but did not affect sperm MF . MMS was not mutagenic in either tissue . The peripheral blood micronucleus assay was performed on the same animals 48 h after treatment, and strong inductions of micronucleated reticulocytes (MNRETs) were observed in both ENU- and MMS-treated mice . These data suggest that agents mutagenic to premeiotic germ cells, e.g., ENU, can be detected by transgenic mutation assay system using germ cells isolated from the testis . On the other hand, those mutagenic to postmeiotic cells, e.g., MMS, are insensitive in the assay system. Mutat Res, 1997 Feb 14, 388(2-3), 137 - 43 Evaluation of spontaneous and chemical-induced lacI mutations in germ cells from lambda/lacI transgenic mice; Putman DL et al.; The spontaneous mutant frequency in germ cells isolated from seminiferous tubules of two lambda/lacI transgenic mouse strains, C57BL/6 and B6C3F1 was evaluated . At least 500 000 phage were screened for mutation at lacI for each animal using standardized assay procedures . The germ cell spontaneous lacI mutant frequency was 17.8 +/- 8.1 x 10(-6) in C57BL/6 mice and 17.0 +/- 10.0 x 10(-6) in B6C3F1 mice . The induction of germ cell mutations by three well characterized alkylating agents were also evaluated in C57BL/6 mice on day 3 after a single dose administration . The lacI mutant frequencies were significantly elevated in transgenic mice dosed with ENU at 150 mg/kg (2-fold increase above control) and iPMS at 200 mg/kg (3-fold increase above control) but not in those receiving MMS at 40 mg/kg . These findings suggest that single dose studies using the lambda/lacI transgenic system may be capable of detecting germ mutations induced by chemicals characterized either by point mutations or small, intragenic deletions but not those characterized by a predominance of multi-locus deletions. Mutat Res, 1997 Feb 14, 388(2-3), 129 - 36 Evaluation of the transgenic Lambda/LacI mouse model as a short-term predictor of heritable risk; Provost GS et al.; Transgenic male C57BL/6 lambda/lacI mice were used to assess the mutagenic response in seminiferous tubules and epididymal spermatozoa 3 days after exposure to ethylnitrosourea (ENU), iso-propyl methanesulfonate (iPMS) and methyl methanesulfonate (MMS) . No significant mutagenic response was observed in epididymal spermatozoa for all three compounds, as expected 3 days after treatment . However, ENU and iPMS treated samples demonstrated significant mutagenic inductions relative to controls in seminiferous tubules while MMS treated samples did not . The failure of MMS to induce a mutagenic response in lambda/lacI transgenic mice is likely due to a combination of the low dose used, the short expression time after exposure and the reduced sensitivity to large deletion events in transgenic lambda/lacI shuttle vectors . In addition, ex vivo mutations were measured in control samples and iPMS treated samples, where 33% of mutants from control samples and 35% of mutants from iPMS treated samples were mosaic. J Mol Biol, 1997 Feb 14, 266(1), 122 - 34 The resolving enzyme CCE1 of yeast opens the structure of the four-way DNA junction; White MF et al.; Junction-resolving enzymes exhibit structure-selective binding to DNA, but may also manipulate the DNA structure . CCE1 is a junction-resolving enzyme found in the yeast mitochondrion . To facilitate the analysis of the CCE1-junction interaction, we have exploited the sequence dependence of the cleavage reaction to devise a junction that is refractory to cleavage by this enzyme, even in the presence of magnesium ions . On binding to four-way DNA junctions, pure recombinant CCE1 opens the global structure into a 4-fold symmetrical configuration of arms with an open, chemically reactive centre . The structure of the CCE1-junction complex is independent of the sequence of the junction, and of the presence or absence of magnesium or other ions . This and other functional properties of CCE1 are strikingly similar to those of RuvC resolving enzyme of Escherichia coli. J Mol Biol, 1997 Feb 14, 266(1), 76 - 92 Characterization of the mycobacteriophage L5 attachment site, attP; Pena CE et al.; Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome . Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein . DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange . Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration . We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria. J Mol Biol, 1997 Feb 14, 266(1), 51 - 65 Filamentous phage IKe mRNAs conserve form and function despite divergence in regulatory elements; Stump MD et al.; As a means of determining whether there has been selection to conserve the basic pattern of filamentous phage mRNAs, the major mRNAs representing genes II to VIII have been defined for a phage distantly related to the Ff group specific for Escherichia coli hosts bearing F pili . Phage IKe has a genome with 55% identity with the Ff genome and infects E . coli strains bearing N pili . The results reveal a remarkably similar pattern of overlapping polycistronic mRNAs with a common 3' end and unique 5' ends . The IKe mRNAs, like the Ff phage mRNAs, represent a combination of primary transcripts and processed RNAs . However, examination of the sequences containing the RNA endpoint positions revealed that effectively the only highly conserved regulatory element is the rho-independent terminator that generates the common 3' end . Promoters and processing sites have not been maintained in identical positions, but frequently are placed so as to yield RNAs with similar coding function . By conserving the pattern of transcription and processing despite divergence in the regulatory elements and possibly the requirements for host, endoribonucleases, the results argue that the pattern is not simply fortuitous. J Mol Biol, 1997 Feb 14, 266(1), 40 - 50 Mutations at nucleotides G2251 and U2585 of 23 S rRNA perturb the peptidyl transferase center of the ribosome; Green R et al.; Previous experiments have shown that the phylogenetically conserved G2252 of 23 S rRNA forms a Watson-Crick base-pair with C74 of peptidyl-tRNA . In the studies presented here, site-directed mutations were introduced at two other conserved positions in 23 S rRNA, G2251 and U2585, that were previously implicated in interaction of the CCA acceptor end of tRNA with the 50 S subunit P site . The mutant 23 S rRNAs were characterized by determining (1) the in vivo phenotypes, (2) the ability of mutant ribosomes to bind tRNA oligonucleotide fragments in vitro, using footprinting with allele-specific primer extension and (3) the ability of mutant ribosomes to catalyze peptide bond formation using a chimeric reconstitution approach . Mutations at either position confer a dominant lethal phenotype when the mutant 23 S rRNA is coexpressed with the endogenous wild-type 23 S rRNA . Mutations at 2585 disrupt binding of the wild-type (CCA) tRNA oligonucleotide fragment and cause a modest decrease in the peptidyl transferase activity of reconstituted ribosomes . By contrast, mutations at 2251 abolish both binding of the wild-type (CCA) tRNA fragment and peptidyl transferase activity using the wild-type tRNA fragment . In neither case was the loss of binding or peptidyl transferase activity suppressed by mutations in the tRNA oligonucleotide fragment . Chemical modification analysis revealed that mutations at 2251 perturb the reactivity of bases 2584 to 2586, providing further evidence that the 2250 loop of 23 S rRNA interacts, either directly or indirectly, with the 2585 region in the central loop of domain V of 23 S rRNA. J Biol Chem, 1997 Feb 14, 272(7), 4516 - 21 Identification of a novel gene encoding the yeast mitochondrial dicarboxylate transport protein via overexpression, purification, and characterization of its protein product; Kakhniashvili D et al.; A gene encoding the mitochondrial dicarboxylate transport protein (DTP) has been identified for the first time from any organism . Our strategy involved overexpression of putative mitochondrial transporter genes, selected based on analysis of the yeast genome, followed by purification and functional reconstitution of the resulting protein products . The DTP gene from the yeast Saccharomyces cerevisiae encodes a 298-residue basic protein which, in common with other mitochondrial anion transporters of known sequence and function, displays the mitochondrial transporter signature motif, three homologous 100-amino acid sequence domains, and six predicted membrane-spanning regions . The product of this gene has been abundantly expressed in Escherichia coli where it accumulates in inclusion bodies . Upon solubilization of the overexpressed DTP from isolated inclusion bodies with Sarkosyl, 28 mg of DTP was obtained per liter of E . coli culture at a purity of 75% . The purified, overexpressed DTP was then reconstituted in phospholipid vesicles where both its kinetic properties (i.e . Km = 1 . 55 mM and Vmax = 3.0 micro;mol/min/mg protein) and its substrate specificity were determined . The intraliposomal substrates malonate, malate, succinate, and phosphate effectively supported {14C}malonate uptake, whereas other anions tested did not . External substrate competition studies revealed a similar specificity profile . Inhibitor studies indicated that the reconstituted transporter was sensitive to inhibition by n-butylmalonate, p-chloromercuribenzoate, mersalyl, and to a lesser extent pyridoxal 5'-phosphate but was insensitive to N-ethylmaleimide and selective inhibitors of other mitochondrial anion transporters . In combination, the above findings indicate that the identified gene encodes a mitochondrial transport protein which upon overexpression and reconstitution displays functional properties that are virtually identical to those of the native mitochondrial dicarboxylate transport system . In conclusion, the present investigation has resulted in identification of a gene encoding the mitochondrial DTP and thus eliminates a major impediment to molecular studies with this metabolically important transporter . Based on both structural and functional considerations, the yeast DTP is assignable to the mitochondrial carrier family . Additionally, the development of a procedure that enables the expression and isolation of large quantities of functional DTP provides the foundation for comprehensive investigations into the structure/function relationships within this transporter via site-directed mutagenesis, as well as for the initiation of crystallization trials. J Biol Chem, 1997 Feb 14, 272(7), 4398 - 403 Mutational analysis of the properties of caveolin-1 . A novel role for the C-terminal domain in mediating homo-typic caveolin-caveolin interactions; Song KS et al.; Caveolin is a principal structural component of caveolae membranes in vivo . Recently, a family of caveolin-related proteins has been identified; caveolin has been retermed caveolin-1 . Caveolin family members share three characteristic properties: (i) detergent insolubility at low temperatures; (ii) self-oligomerization; and (iii) incorporation into low density Triton-insoluble fractions enriched in caveolae membranes . Here, we have used a deletion mutagenesis approach as a first step toward understanding which regions of caveolin-1 contribute to its unusual properties . Two caveolin-1 deletion mutants were created that lack either the C-terminal domain (Cav-1DeltaC) or the N-terminal domain (Cav-1DeltaN); these mutants were compared with the behavior of full-length caveolin-1 (Cav-1FL) expressed in parallel . Our results show that the N-terminal domain and membrane spanning segment are sufficient to form high molecular mass oligomers of caveolin-1 . However, a complete caveolin-1 molecule is required for conveying detergent insolubility and incorporation into low density Triton-insoluble complexes . These data indicate that homo-oligomerization and an intact transmembrane are not sufficient to confer detergent insolubility, suggesting an unknown role for the C-terminal domain in this process . To better understand the role of the C-terminal domain, this region of caveolin-1 (residues 135-178) was expressed as a glutathione S-transferase fusion protein in Escherichia coli . Purified recombinant glutathione S-transferase-C-Cav-1 was found to stably interact with full-length caveolin-1 but not with the two caveolin-1 deletion mutants . These results suggest that the C-terminal domain interacts with both the N-terminal and C-terminal domains of an adjacent caveolin-1 homo-oligomer . This appears to be a specific homo-typic interaction, because the C-terminal domain of caveolin-1 failed to interact with full-length forms of caveolin-2 and caveolin-3 . Homo-typic interaction of the C-terminal domain with an adjacent homo-oligomer could provide a mechanism for clustering caveolin-1 homo-oligomers while excluding other caveolin family members . This type of lateral segregation event could promote caveolae membrane formation and contribute to the detergent insolubility of caveolins-1, -2, and -3. J Biol Chem, 1997 Feb 14, 272(7), 4384 - 90 Cloning and characterization of a wortmannin-sensitive human phosphatidylinositol 4-kinase; Meyers R et al.; Phosphatidylinositol (PtdIns) 4-kinases catalyze the synthesis of PtdIns-4-P, the immediate precursor of PtdIns-4,5-P2 . Here we report the cloning of a novel, ubiquitously expressed PtdIns 4-kinase (PI4Kbeta) . The 2.4-kilobase pair cDNA encodes a putative translation product of 801 amino acids which shows greatest homology to the yeast PIK1 gene . The recombinant protein exhibits lipid kinase activity when expressed in Escherichia coli, and specific antibodies recognize a 110-kDa PtdIns 4-kinase in cell lysates . The biochemical properties of PI4Kbeta are characteristic of a type III enzyme . Interestingly, both recombinant PI4Kbeta and the endogenous protein are inhibited by 150 nM wortmannin, suggesting that we have cloned the previously described PtdIns 4-kinase that is responsible for regulating the synthesis of agonist-sensitive pools of polyphosphoinositides (Nakanishi, S., Catt, J . K., and Balla, T . (1995) Proc . Natl . Acad . Sci . U . S . A . 92, 5317-5321). J Biol Chem, 1997 Feb 14, 272(7), 4281 - 6 Identification and characterization of a novel human matrix metalloproteinase with unique structural characteristics, chromosomal location, and tissue distribution; Pendas AM et al.; We have cloned a novel member of the matrix metalloproteinase (MMP) family of proteins from a human liver cDNA library . The isolated cDNA contains an open reading frame coding for a polypeptide of 508 amino acids, which has been tentatively called MMP-19 . This protein exhibits the domain structure characteristic of previously described MMPs, including a signal sequence, a prodomain with the cysteine residue essential for maintaining the latency of these enzymes, an activation locus with the zinc-binding site, and a COOH-terminal fragment with sequence similarity to hemopexin . However, it lacks a series of structural features distinctive of the diverse MMP subclasses, including the Asp, Tyr, and Gly residues located close to the zinc-binding site in collagenases, the fibronectin-like domain of gelatinases, the transmembrane domain of membrane-type (MT) MMPs, and the furin-activation sequence common to stromelysin-3 and MT-MMPs . In addition, the 9-residue insertion rich in hydrophobic amino acids present at the hinge region in stromelysins is replaced in MMP-19 by a longer insertion very rich in acidic residues . On the basis of these structural characteristics, we propose that MMP-19 does not belong to any of the previously defined MMP-subclasses and may represent the first member of a new MMP subfamily . Chromosomal location of the MMP-19 gene revealed that it maps to chromosome 12q14, which is also a unique location for any MMPs mapped to date . The cDNA encoding a full-length MMP-19 was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade synthetic substrates for MMPs . MMP-19 proteolytic activity was abolished by TIMP-2 and EDTA, thus providing additional evidence that the isolated cDNA codes for an authentic MMP . Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues revealed that MMP-19 is mainly expressed in placenta, lung, pancreas, ovary, spleen, and intestine, suggesting that it may play a specialized role in these tissues. J Biol Chem, 1997 Feb 14, 272(7), 4087 - 93 prl mutations in the Escherichia coli secG gene; Bost S et al.; SecG, an integral membrane component of the Escherichia coli preprotein translocase, contributes to the efficiency of the export process by undergoing cycles of topology inversion in the membrane, coupled with the insertion-deinsertion cycles of SecA . We have previously identified sec alleles of secG that cause a generalized secretion defect . In this study, by screening mutagenized secG libraries for suppressors of a malE signal sequence mutation, we isolated prl alleles of secG . By analogy with secY/prlA, secA/prlD, and secE/prlG, secG could therefore be called secG/prlH . The prlH mutations affect 13 codons distributed along the secG sequence, and none map to the codons affected by sec mutations . prlH suppressors suppress a variety of signal sequence mutations and they allow export of alkaline phosphatase lacking its entire signal sequence . Although secG was not identified in previous selections for prl mutants, several prlH alleles are as strong as the strongest known prlG alleles of secE . Some prlH alleles can also promote the export of alkaline phosphatase fused to predicted cytoplasmic domains of UhpT, an integral membrane protein . These results support the notion that SecG contributes to signal sequence recognition, and suggest that it may also contribute to the topology of integral membrane proteins. J Biol Chem, 1997 Feb 14, 272(7), 3879 - 82 Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase; Tohgo A et al.; CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H . (1993) J . Biol . Chem . 268, 26052-26054) . In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR . To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA . Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129 . We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg . Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR . Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR . These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38. Science, 1997 Feb 14, 275(5302), 945 - 8 Chemical selection for catalysis in combinatorial antibody libraries; Janda KD et al.; For the past decade the immune system has been exploited as a rich source of de novo catalysts . Catalytic antibodies have been shown to have chemoselectivity, enantioselectivity, large rate accelerations, and even an ability to reroute chemical reactions . In many instances catalysts have been made for reactions for which there are no known natural or man-made enzymes . Yet, the full power of this combinatorial system can only be exploited if there was a system that allows for the direct selection of a particular function . A method that allows for the direct chemical selection for catalysis from antibody libraries was so devised, whereby the positive aspects of hybridoma technology were preserved and re-formatted in the filamentous phage system to allow direct selection of catalysis . This methodology is based on a purely chemical selection process, making it more general than biologically based selection systems because it is not limited to reaction products that perturb cellular machinery. Biochem Biophys Res Commun, 1997 Feb 13, 231(2), 452 - 6 The thioesterase I of Escherichia coli has arylesterase activity and shows stereospecificity for protease substrates; Lee YL et al.; A thioesterase I gene was recloned and sequenced from Escherichia coli strain JM109 . The overexpressed, matured enzyme from JM109 was purified to homogeneity . The enzyme showed broad hydrolytic activity toward three kinds of substrates including acyl-CoAs, esters, and amino acid derivatives . The enzyme had a kcat/Km value of 0.363 s-1 microM-1, for a typical thioesterase I substrate, palmitoyl-CoA . The arylesterase activity of the enzyme was observed by its ability to hydrolyze several aromatic esters including alpha-naphthyl acetate, alpha-naphthyl butyrate, phenyl acetate, benzyl acetate, and eight p-nitrophenyl esters . In kinetic studies a chymotrypsin-like substrate (an amino acid derivative), N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (L-NBPNPE), was the best substrate for the enzyme with a catalytic efficiency (kcat/Km) of 4.00 s-1 microM-1, which was 23 times higher than that of the enantiomer D-NBPNPE (0.171 s-1 microM-1) . It was concluded that the thioesterase I of E . coli had arylesterase activity and it possessed stereospecificity for protease substrates. Biochemistry, 1997 Feb 11, 36(6), 1450 - 60 Discrete backbone disorder in the nuclear magnetic resonance structure of apo intestinal fatty acid-binding protein: implications for the mechanism of ligand entry; Hodsdon ME et al.; The three-dimensional structure of the unliganded form of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) has been determined using triple-resonance three-dimensional nuclear magnetic resonance (3D NMR) methods . Sequence-specific 1H, 13C, and 15N resonance assignments were established at pH 7.2 and 33 degrees C and used to determine the consensus 1H/13C chemical shift-derived secondary structure . Subsequently, an eight-stage iterative procedure was used to assign the 3D 13C- and 15N-resolved NOESY spectra, yielding a total of 3335 interproton distance restraints or 26 restraints/residue . The tertiary structures were calculated using a distance geometry/simulated annealing algorithm that employs pairwise Gaussian metrization to achieve improved sampling and convergence . The final ensemble of NMR structures exhibited a backbone conformation generally consistent with the beta-clam motif described for members of the lipid-binding protein family . However, unlike holo-I-FABP, the structure ensemble for apo-I-FABP exhibited variability in a discrete region of the backbone . This variability was evaluated by comparing the apo- and holoproteins with respect to their backbone 1H and 13C chemical shifts, amide 1H exchange rates, and 15N relaxation rates . Together, these results established that the structural variability represented backbone disorder in apo-I-FABP . The disorder was most pronounced in residues K29-L36 and N54-N57, encompassing the distal half of alpha-helix II, the linker between helix II and beta-strand B, and the reverse turn between beta-strands C and D . It was characterized by a destablization of long-range interactions between helix II and the C-D turn and a fraying of the C-terminal half of the helix . Unlike the solution-state NMR structure, the 1.2-A X-ray crystal structure of apo-I-FABP did not exhibit this backbone disorder . In solution, the disordered region may function as a dynamic portal that regulates the entry and exit of fatty acid . We hypothesize that fatty acid binding shifts the order-disorder equilibrium toward the ordered state and closes the portal by stabilizing a series of cooperative interactions resembling a helix capping box . This proposed mechanism has implications for the acquisition, release, and targeting of fatty acids by I-FABP within the cell. Biochemistry, 1997 Feb 11, 36(6), 1281 - 6 Monomer-dimer equilibrium of uncomplemented M15 beta-galactosidase from Escherichia coli; Gallagher CN et al.; A series of gel filtration, native polyacrylamide gel electrophoresis (PAGE) and sucrose density experiments showed that uncomplemented M15 beta-galactosidase is in a monomer-dimer equilibrium and that only under some specific conditions does the equilibrium strongly favor dimerization . The ratio of dimer to monomer increased as a function of the protein concentration, and a very good fit to a theoretical plot of the effect of protein concentration on an associating system of this type was found . The Kdiss (equilibrium constant for dimer dissociation) was 2.5 x 10(-7) M . The addition of 20 mM Mg2+ lowered the Kdiss to 1.5 x 10(-7) M, and the addition of 150 mM NaCl lowered the value to 0.4 x 10(-7) M . Thiol reagents (2-mercaptoethanol and dithiothreitol) caused the equilibrium to shift totally to the dimeric form . The monomer-dimer equilibrium was also found to be dependent upon the pH . The dissociation increased as the pH was raised to 8.5, but there was a reversal of the equilibrium in favor of dimer formation at pH 9.0 . This suggests that one (or more) residues with a pKa value of about 8.0 is involved . Tyr and Lys were eliminated as possible residues involved and it is, therefore, likely that one or more Cys are involved . Further evidence that uncomplemented M15 beta-galactosidase is in a monomer-dimer equilibrium was that the gel-filtration peaks were not totally resolved and that native PAGE bands were diffuse under all conditions except at high thiol concentration. Biochemistry, 1997 Feb 11, 36(6), 1212 - 22 Structural analysis of the H166G site-directed mutant of galactose-1-phosphate uridylyltransferase complexed with either UDP-glucose or UDP-galactose: detailed description of the nucleotide sugar binding site; Thoden JB et al.; Galactose-1-phosphate uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of a uridine 5'-phosphoryl group from UDP-glucose to galactose 1-phosphate . The enzyme from Escherichia coli is composed of two identical subunits . The structures of the enzyme/UDP-glucose and UDP-galactose complexes, in which the catalytic nucleophile His 166 has been replaced with a glycine residue, have been determined and refined to 1.8 A resolution by single crystal X-ray diffraction analysis . Crystals employed in the investigation belonged to the space group P2(1) with unit cell dimensions of a = 68 A, b = 58 A, c = 189 A, and beta = 100 degrees and two dimers in the asymmetric unit . The models for these enzyme/substrate complexes have demonstrated that the active site of the uridylyltransferase is formed by amino acid residues contributed from both subunits in the dimer . Those amino acid residues critically involved in sugar binding include Asn 153 and Gly 159 from the first subunit and Lys 311, Phe 312, Val 314, Tyr 316, Glu 317, and Gln 323 from the second subunit . The uridylyltransferase is able to accommodate both UDP-galactose and UDP-glucose substrates by simple movements of the side chains of Glu 317 and Gln 323 and by a change in the backbone dihedral angles of Val 314 . The removal of the imidazole group at position 166 results in little structural perturbation of the polypeptide chain backbone when compared to the previously determined structure for the wild-type enzyme . Instead, the cavity created by the mutation is partially compensated for by the presence of a potassium ion and its accompanying coordination sphere . As such, the mutant protein structures presented here represent valid models for understanding substrate recognition and binding in the native galactose-1-phosphate uridylyltransferase. Biochemistry, 1997 Feb 11, 36(6), 1199 - 211 Solution structure of the quaternary MutT-M2+-AMPCPP-M2+ complex and mechanism of its pyrophosphohydrolase action; Lin J et al.; The MutT enzyme (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP) by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate . It requires two divalent cations, forming an active E-M2+-NTP-M2+ complex . The solution structure of the free enzyme consists of a five-stranded mixed beta-sheet connected by loop I-alpha-helix I-loop II, by two tight turns, and by loop III and terminated by loop IV-alpha-helix II {Abeygunawardana, C., et al . (1995) Biochemistry 34, 14997-15005} . Assignments of backbone 15N and NH resonances and side chain 15N and NH2 resonances of the quaternary complex were made by 1H-15N HSQC titrations of the free enzyme with MgCl2 followed by equimolar AMPCPP/MgCl2 . H(alpha) assignments were made by 1H-15N 3D TOCSY HSQC, and 1H-13C CT-HSQC spectra and backbone and side chain 1H and 13C assignments were made by 3D HCCH TOCSY experiments . Ligands donated by the protein to the enzyme-bound divalent cation, identified by paramagnetic effects of Co2+ and Mn2+ on CO(C)H spectra, are the carboxylate groups of Glu-56, -57, and -98 and the amide carbonyl of Gly-38 . The solution structure of the complex was computed with XPLOR using a total of 2168 NOE and 83 phi restraints for the protein, 11 intramolecular NOEs for bound Mg2+ AMPCPP, 22 intermolecular NOEs between MutT and AMPCPP, and distances from the enzyme-bound Co2+ to the three phosphorus atoms of Co3+(NH3)4AMPCPP from paramagnetic effects of Co2+ on their T1 values . The fold of the MutT enzyme in the complex is very similar to that of the free enzyme, with minor changes in the metal and substrate binding sites . The adenine ring binds in a hydrophobic cleft, interacting with Leu-4 and Ile-6 on beta-strand A and with Ile-80 on beta-strand D . The 6-NH2 group of adenine approaches the side chain NH2 of Asn-119 . This unfavorable interaction is consistent with the stronger binding by MutT of guanine nucleotides, which have a 6-keto group . The ribose binds with its hydroxyl groups oriented toward the solvent and its hydrophobic face interacting with Leu-4, Ile-6, and the gamma-CH2 of Lys-39 of loop I . The metal-triphosphate moiety appears to bind in the second coordination sphere of the enzyme-bound divalent cation . One of two intervening water ligands is well positioned to attack P(beta) with inversion and to donate a hydrogen bond to the conserved residue, Glu-53, which may deprotonate or orient the attacking water ligand . Lys-39 which is positioned to interact electrostatically with the alpha-phosphoryl group may facilitate the departure of the leaving NMP . On the basis of the structure of the quaternary complex, a mechanism of the MutT reaction is proposed which is qualitatively and quantitatively consistent with kinetic and mutagenesis studies . It is suggested that similar mechanisms may be operative for other enzymes that catalyze substitution at P(beta) of NTP substrates. Biochemistry, 1997 Feb 11, 36(6), 1188 - 93 Asymmetry of catalytic but not of noncatalytic sites on Escherichia coli F1-ATPase in solution as observed using electron spin resonance spectroscopy; Losel RM et al.; We have employed electron spin resonance (ESR) spectroscopy using different spin-labeled nucleotides to probe the environment of nucleotides bound at catalytic and noncatalytic nucleotide binding sites of the Escherichia coli F1-ATPase . We found that nucleotides bound in the noncatalytic binding sites were strongly immobilized and resulted in ESR spectra with one single corresponding spectral component . Nucleotide bound at the catalytic binding sites gave rise to two different signals in the ESR spectra indicative of two distinct conformations of the catalytic sites of the protein . One conformation of the catalytic sites is very tight, resulting in signals identical to those of the noncatalytic sites, while the second type of catalytic sites permitted an unusually high mobility of the bound spin-labeled nucleotide . The findings are compared to the requirements of the binding change mechanism and to the features of the nucleotide binding sites as elucidated from the X-ray structural model of the beef heart mitochondrial enzyme. Hum Gene Ther, 1997 Feb 10, 8(3), 267 - 74 Preclinical studies for cell transplantation: isolation of primate fetal hepatocytes, their cryopreservation, and efficient retroviral transduction; Andreoletti M et al.; Fetal hepatocytes are an attractive target for in utero cellular transplantation . Their use could provide a very efficient way for implanting normal or transduced cells into the livers of affected fetuses . Marking cells with recombinant retroviruses is a powerful tool for evaluating the chimerism of grafted animals . The technique relies on the ex vivo transduction efficiency of the engrafted cells . We have isolated fetal primary hepatocytes from nonhuman primates . The cells were cultured and transduced with a retroviral vector carrying the Escherichia coli beta-galactosidase gene . Optimal gene transfer efficiency was obtained 48-60 hr after plating and was as high as 90% . Cryopreservation had little effect on cell viability and infectivity: The viability of thawed hepatocytes remained high (75-85%) and the infection efficiency was identical to that of freshly isolated cells . Efficient ex vivo retroviral gene transfer into fetal hepatocytes provides an appropriate system for testing allogenic grafting and for modifying immunogenicity of engrafted cells . These results open up new perspectives for cell transplantation through cell banking. FEBS Lett, 1997 Feb 10, 403(1), 5 - 9 Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate,orthophosphate dikinase; Ohta S et al.; Pyruvate,orthophosphate dikinase (PPDK), an enzyme important in C4 photosynthesis, is typically a cold-sensitive enzyme . However, a cold-tolerant form of the enzyme has been isolated from the leaves of Flaveria brownii . Using an E . coli expression system and the PPDK cDNAs from F . brownii (cold-tolerant), F . bidentis (cold-sensitive) and maize (intermediately cold-tolerant), site-directed mutagenesis studies indicated that as few as three amino acids residues (of 880 residues) strongly influence the cold sensitivity of Flaveria PPDK . Gel filtration analysis of the PPDK expressed in E . coli showed that subunit association and cold tolerance are closely linked. J Cell Biol, 1997 Feb 10, 136(3), 609 - 20 A cytosolic serine endopeptidase from Trypanosoma cruzi is required for the generation of Ca2+ signaling in mammalian cells; Burleigh BA et al.; An early event in the Trypanosoma cruzi cell invasion process, the recruitment of host lysosomes, led us to investigate the involvement of signal transduction . Infective trypomastigotes were found to contain a soluble Ca2+-signaling activity for mammalian cells that is sensitive to protease inhibitors . Inhibitor and substrate utilization profiles were used to purify a candidate peptidase for involvement in this process, from which we isolated a full-length cDNA clone . The sequence revealed a novel enzyme, denominated T . cruzi oligopeptidase B, which is homologous to members of the prolyl oligopeptidase family of serine hydrolases, known to participate in the maturation of biologically active peptides . The T . cruzi oligopeptidase B was expressed as a fully active product in Escherichia coli, and antibodies to the recombinant enzyme inhibited both peptidase activity and Ca2+ signaling induced in normal rat kidney cells by trypomastigote extracts . Our data suggest that the T . cruzi oligopeptidase B participates in processing events in the cytoplasm of the parasites, generating a factor with Ca2+-signaling activity for mammalian cells. Biochim Biophys Acta, 1997 Feb 8, 1337(2), 295 - 301 Assembly of nematode cuticle: role of hydrophobic interactions in CUT-2 cross-linking; Parise G et al.; CUT-2 is a component of cuticlin, the highly cross-linked, insoluble residue of the cuticle of the nematode Caenorhabditis elegans . A recombinant fragment of CUT-2, produced in E . coli, can be cross-linked in vitro by horse radish peroxidase via dityrosine formation to give large molecular species {1} . In this paper it is shown that the formation of CUT-2 polymers is greatly favoured over that of CUT-2 oligomers as no low molecular weight intermediates, dimers or trimers can be detected even when the cross-linking reaction is slowed or interrupted before completion . This suggests that recombinant CUT-2 forms large non-covalent complexes that are the only competent substrate for cross-linking . The inhibition of cross-linking by urea and the behavior of recombinant CUT-2 in size-exclusion chromatography under a variety of conditions suggest that hydrophobic interactions are important in the formation and stabilization of these complexes . The complexes are excellent substrates for cross-linking but react poorly with free tyrosine . In contrast, a soluble recombinant CUT-2 is a poor substrate for cross-linking but can efficiently react with free tyrosine. Biochim Biophys Acta, 1997 Feb 8, 1337(2), 248 - 56 Recombinant brain 4-aminobutyrate aminotransferases overexpression, purification, and identification of Lys-330 at the active site; Kim YT et al.; 4-Aminobutyrate aminotransferase (4-aminobutyrate: 2-oxoglutarate aminotransferase EC 2.6.1.19) is a key enzyme of the 4-aminobutyric acid shunt . It catalyzes the conversion of 4-aminobutyrate to succinic semialdehyde . In an effort to clarify the structure-function relationships of 4-aminobutyrate aminotransferase, we analyzed 4-aminobutyrate aminotransferase cDNA from pig brain . The inclusion bodies were formed when recombinant 4-aminobutyrate aminotransferase was overexpressed in Escherichia coli . The unfolded overproduced proteins, were purified by hydroxylapatite chromatography in the presence of urea and refolded by a sequential dialysis method . The renatured protein regained its catalytic activity . The lysyl residue at the 330 position of the amino-acid sequence serves as the anchoring site of the cofactor pyridoxal 5'-P . To verify the catalytic site of 4-aminobutyrate aminotransferase, lysine 330 was mutated to arginine by site-specific mutagenesis . Overexpression and purification of the mutated 4-aminobutyrate aminotransferase (K330R) were performed by the same method used the purification of wild-type 4-aminobutyrate aminotransferase . The purified and renatured K330R protein did not show the catalytic activity of wild type 4-aminobutyrate aminotransferase . Furthermore, the mutated protein did not show any absorption band over the spectral range of 320-460 nm characteristic of pyridoxal 5'-P covalently linked to the protein . From the results presented here, it is concluded that lysine 330 is essential for the catalytic function of the aminotransferase. Biochim Biophys Acta, 1997 Feb 8, 1337(2), 175 - 90 Modulation of human glucokinase intrinsic activity by SH reagents mirrors post-translational regulation of enzyme activity; Tiedge M et al.; The low-affinity glucose phosphorylating enzyme glucokinase plays a key role in the process of glucose recognition in pancreatic B-cells . To evaluate mechanisms of intrinsic regulation of enzyme activity human pancreatic B-cell and liver glucokinase and for comparison rat liver glucokinase were expressed in E . coli bacteria . A one-step purification procedure through metal chelate affinity chromatography revealed 58 kDa proteins with high specific activities in the range of 50 U/mg protein and K(m) values around 8 mM for the substrate D-glucose with a preference for the alpha-anomer . There were no tissue specific differences, no species differences in the electrophoretic mobility, and no differences of the kinetic properties of these well conserved enzymes . The deletion of the 15 tissue-specific NH2-terminal amino acids of the human glucokinase resulted in a catalytically active enzyme whose kinetic properties were not significantly different from those of the wild-type enzymes . The human and rat glucokinase isoforms were non-competitively inhibited by the sulfhydryl group reagents alloxan and ninhydrin with Ki values in the range of 1 microM . The inhibition of glucokinase enzyme activity was reversed by dithiothreitol with an EC50 value of 9 microM for alloxan and of 50 microM for ninhydrin . D-Glucose provided protection against alloxan-induced inhibition of human and rat glucokinase isoenzymes with half-maximal effective concentrations between 11 and 16 mM . The enzyme inhibition by alloxan was accompanied by a change in the electrophoretic mobility with a second lower molecular 49 kDa glucokinase band which can be interpreted as a compact glucokinase molecule locked by disulfide bonds . Quantification of free sulfhydryl groups revealed an average number of 3.6 free sulfhydryl groups per enzyme molecule for the native human glucokinase isoforms . Alloxan decreased the average number of free sulfhydryl groups to 1.9 per enzyme molecule indicating that more than one SH side group is oxidized by this compound . The extraordinary sensitivity of the SH side groups of the glucokinase may be a possible mechanism of enzyme regulation by interconversion of stable (active) and unstable (inactive) conformations of the enzyme . In pancreatic B-cells the glucose-dependent increase of reduced pyridine nucleotides may stabilize the enzyme in the 58 kDa form and provide optimal conditions for glucose recognition and glucose-induced insulin secretion. Gene, 1997 Feb 7, 185(2), 245 - 9 Detection of tetramerization domains in vivo by cooperative DNA binding to tandem lambda operator sites; Zeng X et al.; Chimeric proteins comprising the N-terminal DNA binding domain of lambda repressor fused to a fragment of a foreign protein have been used to detect oligomerization of the latter . Fusions containing dimeric and tetrameric leucine zipper domains can be distinguished based on their in vivo repressor activities on a pair of cat-lacZ reporter strains . Repressor fusions are unable to efficiently repress transcription from a synthetic promoter that overlaps a weak operator site; repression by tetrameric, but not dimeric, fusion proteins is increased by the presence of a strong, upstream operator site . To construct reporters we developed a shuttle system that allows rapid construction of single-copy operon fusions in E . coli, with both cat and lacZ as reporters. J Mol Biol, 1997 Feb 7, 265(5), 519 - 40 RecA protein filaments: end-dependent dissociation from ssDNA and stabilization by RecO and RecR proteins; Shan Q et al.; RecA protein filaments formed on circular (ssDNA) in the presence of ssDNA binding protein (SSB) are generally stable as long as ATP is regenerated . On linear ssDNA, stable RecA filaments are believed to be formed by nucleation at random sites on the DNA followed by filament extension in the 5' to 3' direction . This view must now be enlarged as we demonstrate that RecA filaments formed on linear ssDNA are subject to a previously undetected end-dependent disassembly process . RecA protein slowly dissociates from one filament end and is replaced by SSB . The results are most consistent with disassembly from the filament end nearest the 5' end of the DNA . The bound SSB prevents re-formation of the RecA filaments, rendering the dissociation largely irreversible . The dissociation requires ATP hydrolysis . Disassembly is not observed when the pH is lowered to 6.3 or when dATP replaces ATP . Disassembly is not observed even with ATP when both the RecO and RecR proteins are present in the initial reaction mixture . When the RecO and RecR proteins are added after most of the RecA protein has already dissociated, RecA protein filaments re-form after a short lag . The newly formed filaments contain an amount of RecA protein and exhibit an ATP hydrolysis rate comparable to that observed when the RecO and RecR proteins are included in the initial reaction mixture . The RecO and RecR proteins thereby stabilize RecA filaments even at the 5' ends of ssDNA, a fact which should affect the recombination potential of 5' ends relative to 3' ends . The location and length of RecA filaments involved in recombinational DNA repair is dictated by both the assembly and disassembly processes, as well as by the presence or absence of a variety of other proteins that can modulate either process. J Mol Biol, 1997 Feb 7, 265(5), 507 - 18 Dissection of the switch between genes for replication and transfer of promiscuous plasmid RK2: basis of the dominance of trfAp over trbAp and specificity for KorA in controlling the switch; Jagura-Burdzy G et al.; The trfA and trb operons of broad host range plasmid RK2 are required for replication and conjugative transfer, respectively . Transcription of the trb operon can be initiated at one of two promoters: trbAp or trbBp . trbBp provides a burst of trb transcription on first entry into the cell . trbAp appears to be responsible for steady-state transcription of the trb operon as well as trbA, encoding a repressor which helps to shut down trbBp . The promoters trfAp and trbAp are arranged as face-to-face divergent promoters . trfAp is very strong and shuts off trbAp activity until trfAp is inhibited by KorA, one of the plasmid-encoded global regulators . Although trfAp is also repressed by KorB, a second global regulator encoded along with KorA in the central control operon, trbAp activation only occurs when KorA is present . KorB did not activate trbAp and indeed had a significant inhibitory effect on KorA activation . In vitro trfAp binds RNA polymerease (RNAP) approximately ten times more strongly than trbAp . Comparison of single and multiple rounds of in vitro run-off transcription suggested that the inhibitory effect of trfAp is due to elongating transcription complexes . In vitro studies with purified KorA and KorB on RNAP binding, isomerization and in vitro transcription suggested that both proteins can displace RNAP from trfAp, but that once open complexes have formed at either promoter they have a good chance of generating a transcript even if they encounter an opposing RNAP . In vivo KorB repressed trbAp even when trbAp was derepressed by a trfAp-1 mutation, removing the need for KorA . This suggested that KorB not only fails to derepress but actually represses trbAp despite the KorB operator being located 90 bp downstream of the transcription start point (tsp) . By contrast KorA still activated trbAp when the two promoters were moved further apart or were brought so close that RNAP binding to the two promoters was mutually exclusive . Thus, KorA plays the dominant role in achieving the balance of expression of genes for alternate modes of plasmid propagation but its action is modulated by KorB. Biochim Biophys Acta, 1997 Feb 7, 1350(2), 169 - 82 An3 protein encoded by a localized maternal mRNA in Xenopus laevis is an ATPase with substrate-specific RNA helicase activity; Gururajan R et al.; ATP-dependent RNA helicases from the DEAD box family of proteins are involved in a number of RNA processing and utilization events . An3 protein from Xenopus laevis is an RNA helicase of the DEAD box family of proteins . An3 is synthesized by a mRNA that is localized to one end of Xenopus laevis oocytes . An3 protein is found in the nucleus of ooctes, and more specifically, during the middle stages of oocyte development, with extra nucleoli that contain amplified copies of rRNA genes in the nucleolus . By expressing glutathione-S-transferase:An3 fusion proteins in E . coli, sufficient amounts of An3 protein were isolated to examine its enzymatic activities . ATPase activity, NTP substrate range and RNA helicase activity were tested . An3 protein ATPase activity was evident but not stimulated by any of a variety of RNA tested . An3 protein was able to resolve the duplex formed by an in vitro substrate, in the presence of ATP or dATP . An3 required both 3' and 5' single-stranded regions of RNA flanking the RNA duplex it resolves. Biochim Biophys Acta, 1997 Feb 7, 1350(2), 123 - 7 Cysteine biosynthesis in higher plants: a new member of the Arabidopsis thaliana serine acetyltransferase small gene-family obtained by functional complementation of an Escherichia coli cysteine auxotroph; Howarth JR et al.; A cDNA clone, Sat-52, encoding a novel isoform of serine acetyltransferase (EC 2.3.1.30) was isolated by functional complementation of an Escherichia coli cysE mutant defective in serine acetyltransferase . The 1158 base pair clone contains a full-length open reading frame encoding a deduced protein of 312 amino acids with an M(r) of 32.77 kDa . Northern analysis revealed a single transcript of ca 1.19 kb that did not increase in abundance under sulfate limitation . Genomic Southern hybridization suggests the presence of a single copy of the Sat-52 gene. J Biol Chem, 1997 Feb 7, 272(6), 3683 - 8 Interactions of the subunits of smooth muscle myosin phosphatase; Hirano K et al.; Myosin phosphatase from smooth muscle consists of a catalytic subunit (PP1c) and two non-catalytic subunits, M130 and M20 . Interactions among PP1c, M20, and various mutants of M130 were investigated . Using the yeast two-hybrid system, PP1c was shown to bind to the NH2-terminal sequence of M130, 1-511 . Other interactions were detected, i.e . PP1c to PP1c, M20 to the COOH-terminal fragment of M130, and dimerization of the COOH-terminal fragment of M130 . Mutants of M130 were constructed to localize the PP1c and light chain binding regions . Results from the two-hybrid system indicated two binding sites for PP1c on M130: one site in the NH2-terminal 38 residues and a weaker site(s) in the ankyrin repeats region . Inhibition of PP1c activity with phosphorylase a by the M130 mutants also was consistent with the assignment of these two sites . Overlay assays showed binding of phosphorylated light chain to the ankyrin repeats, probably in the COOH-terminal repeats . Activation of PP1c with phosphorylated light chain required binding sites for PP1c and substrate, plus an additional sequence COOH-terminal to the ankyrin repeats . Thus, activation of phosphatase and binding of PP1c and substrate are properties of the NH2-terminal one-third of M130. J Biol Chem, 1997 Feb 7, 272(6), 3628 - 34 Human methionine synthase . cDNA cloning, gene localization, and expression; Chen LH et al.; Human cDNAs for methionine synthase (5-methyltetrahydrofolate:L-homocysteine S-transmethylase; EC 2.1.1 . 13) have been isolated from fetal and adult liver and HepG2 libraries . The cDNAs span 7.2 kilobases (kb) and consist of a 394-base pair upstream untranslated region, a 3795-base pair open reading frame encoding a 1265-residue 140.3-kDa protein, and about 3 kb of 3' region . The deduced protein sequence shares 53 and 63% identity with the Escherichia coli and the presumptive Caenorhabditis elegans proteins, respectively, and contains all residues implicated in B12 binding to the E . coli protein . Several potential polymorphisms and a cryptic splice deletion were detected in the coding region of the cDNAs . A polymorphism that results in a D919G modification in the protein is fairly common in human DNA samples . Northern analyses of poly(A) mRNA indicated two major species of about 8 and 10 kb in human tissues and some minor, partially spliced species . mRNA levels were highest in the pancreas, skeletal muscle, and heart of the adult and in the kidney in the fetus and were low in adult liver . Genomic clones were isolated and the 5' region was analyzed . Exon 1 is preceded by a number of potential promoter sites, including an E box, CAAT boxes, and a GC box, but this region lacks a TATA element . The human methionine synthase gene was localized to chromosome region 1q42.3-43 by in situ hybridization. J Biol Chem, 1997 Feb 7, 272(6), 3478 - 86 Assembly of human hemoglobin . Studies with Escherichia coli-expressed alpha-globin; Sanna MT et al.; The alpha-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native beta-chains . The functional and structural properties of the expressed alpha-chains were assessed in the isolated state and after assembly into a functional hemoglobin tetramer . The recombinant and native hemoglobins were essentially identical on the basis of sensitivity to effectors (Cl- and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the alpha1beta1 and alpha1beta2 interfaces . However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed alpha-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relative to native HbA . These results indicate that subtle conformational changes in the heme pocket can alter hemoglobin cooperativity in the absence of modifications of quaternary interface contacts or protein dynamics . In addition to incorporation into a HbA tetramer, the alpha-globin refolds and incorporates heme in the absence of the partner beta-chain . Although the CO binding kinetics of recombinant alpha-chains were the same as that of native alpha-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics revealed an additional faster component . These results show that recombinant alpha-chain assumes alternating conformations in the absence of beta-chain and indicate that the isolated alpha-chain exhibits a higher degree of conformational flexibility than the alpha-chain incorporated into the hemoglobin tetramer . These findings demonstrate the utility of the expressed alpha-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships. J Biol Chem, 1997 Feb 7, 272(6), 3179 - 84 Dimerization regulates the enzymatic activity of Escherichia coli outer membrane phospholipase A; Dekker N et al.; The outer membrane phospholipase A (OMPLA) of Escherichia coli is present in a dormant state in the cell envelope . The enzyme is activated by various processes, which have in common that they perturb the outer membrane . Kinetic experiments, chemical cross-linking, and analytical ultracentrifugation were carried out with purified, detergent-solubilized OMPLA to understand the underlying mechanism that results in activation . Under conditions in which the enzyme displayed full activity, OMPLA was dimeric . High detergent concentrations or very dilute protein concentrations resulted in low specific activity of the enzyme, and under those conditions the enzyme was monomeric . The cofactor Ca2+ was required for dimerization . Covalent modification of the active site serine with hexadecylsulfonylfluoride resulted in stabilization of the dimeric form and a loss of the absolute calcium requirement for dimerization . The results of these experiments provide evidence for dimerization as the molecular mechanism by which the enzymatic activity of OMPLA is regulated . This dimerization probably plays a role in vivo as well . Data from chemical cross-linking on whole cells indicate that OMPLA is present in the outer membrane as a monomer and that activation of the enzyme induces dimerization concurrent with the appearance of enzymatic activity. Eur J Pharmacol, 1997 Feb 5, 320(1), 29 - 35 Depression of the inotropic action of isoprenaline by nitric oxide synthase induction in rat isolated hearts; Sun X et al.; The mechanisms involved in myocardial dysfunction during septic shock are not well understood . We have investigated the effects of endotoxin and the role of nitric oxide (NO) in the beta-adrenoceptor responsiveness of rat isolated, ejecting hearts perfused at 60 mmHg of head pressure . In vivo pretreatment with endotoxin (4 mg/kg, i.p., 3 h before heart isolation) significantly attenuated the inotropic response (increase in left ventricular developed pressure, LVP) to isoprenaline (0.15 microgram) after 30 min equilibration and after a further 90 min of perfusion . The peak rate of LVP development (dP/dtmax) in response to isoprenaline was reduced by endotoxin pretreatment, as was the increase of coronary flow . The depression of ventricular contraction was prevented by pretreatment with dexamethasone (1 mg/kg, i.p., 30 min before endotoxin), and was also restored by perfusion with NG-nitro-L-arginine (L-NA, 10 microM) for 60 min, but not by NG-nitro-D-arginine (D-NA, 10 microM) . Mercaptoethylguanidine (MEG, 30 microM), a selective inhibitor of the inducible NO synthase (isoform 2), also reversed the depression of the isoprenaline response caused by endotoxin pretreatment . However, treatment with endotoxin, dexamethasone, L-NA, D-NA or MEG had minimal effects on the baseline parameters of LVP, dP/dtmax and coronary flow, which all tended to decline over the 2 h perfusion period . Western blot analysis using an antibody to NO synthase (isoform 2, but not to isoform 3) revealed the induction of a protein corresponding to NO synthase 2 in the endotoxin-treated hearts but not in control hearts or those treated with dexamethasone or MEG . In summary, these results indicate the endotoxin depresses myocardial contractile function and reduces inotropic responsiveness to beta-adrenoceptor activation . The effect of endotoxin on the inotropic response is mediated, at least in part, by products of an endogenous NO synthase that is suppressed by dexamethasone and a specific inhibitor of NO synthase (isoform 2). Biochemistry, 1997 Feb 4, 36(5), 1173 - 80 Use of a fluorescence spectroscopic readout to characterize the interactions of Cdc42Hs with its target/effector, mPAK-3; Leonard DA et al.; The family of p21-activated kinases (PAKs) has been shown to contain a domain that can independently bind to the Ras-like proteins Cdc42Hs and Rac . We have expressed a 72 amino acid recombinant form of this p21-binding domain (PBD) from mPAK-3 in Escherichia Coli for use in structure-function studies . The protein can be purified on a nickel affinity resin due to a hexa-His tag that is incorporated onto the amino terminus of the domain . PBD binds to Cdc42Hs in a guanine nucleotide-dependent manner as demonstrated by a novel fluorescence assay that takes advantage of the spectroscopic properties of N-methylanthraniloyl (Mant)-guanine nucleotides . Ionic strength has little effect on the affinity of PBD for Cdc42Hs, but alkaline pH values tend to weaken the interaction . We have shown that the inhibition of the GTPase activity of Cdc42Hs, as well as a previously undescribed inhibition of guanine nucleotide dissociation, is mediated by the PBD portion of the mPAK-3 molecule . These findings suggest that PBD binding alters the geometry of the guanine nucleotide binding site on Cdc42Hs, perhaps as an outcome of the target/effector molecule binding in close proximity to the nucleotide domain . We therefore tested if mutations in the effector region of Cdc42Hs (32-40), which in Ras are very close to the guanine nucleotide binding site, had any effect on PBD binding . Changing tyrosine 32 to lysine (Y32K) resulted in a small (5-fold) inhibition of PBD binding, but the very conservative mutation D38E yielded at least a 50-fold decrease in affinity . Finally, the catalytic domain of the GTPase activating protein, Cdc42-GAP, was shown to inhibit PBD binding in a competitive manner, indicating that this target molecule and the negative regulator (GAP) bind to overlapping sites on the Cdc42Hs molecule. Biochemistry, 1997 Feb 4, 36(5), 1148 - 56 Characterization of a recombinant pea 5-aminolevulinic acid dehydratase and comparative inhibition studies with the Escherichia coli dehydratase; Cheung KM et al.; Pea 5-aminolevulinic acid dehydratase (ALAD) was purified 200-fold from a recombinant overproducing strain of Escherichia coli, yielding an octameric enzyme with a specific activity of 280 units mg-1 . Divalent metal ions were essential, Mg2+, Mn2+, and Co2+ ions all supporting activity, whereas Zn2+ ions could not . Equilibrium dialysis and atomic absorption studies revealed two Mg2+ ion binding sites per subunit . Pea ALAD bound the substrate 5-aminolevulinic acid covalently through a Schiff base at the P-site, electrospray mass spectrometry of the reduced enzyme-ALA Schiff base complex showing the presence of one P-site per subunit . The amino acid residue modified by ALA was identified by MALDI-MS and Edman sequencing as Lys-293, analogous to the active site Lys-247 of E . coli ALAD and Lys-252 of mammalian ALAD . Comparative studies of pea ALAD with E . coli ALAD using the inhibitors 3-acetyl-4-oxoheptane-1,7-dioic acid (AOHD) and succinylacetone (SA) indicated similar modes of inhibition, with the formation of a Schiff base complex between the inhibitors and the active site lysine . Studies with the ALA homolog, 4-amino-3-oxobutanoic acid (AOB), revealed that it is specific for the A-site of both the pea and E . coli ALADs . An interesting difference exists between the enzymes, however, pea ALAD being far more susceptible to inhibition with AOB than the E . coli enzyme . AOB bound 10 times better to the A-site of pea ALAD compared to the substrate, ALA . Despite the 2000 times lower Ki of AOB for pea ALAD, no abortive Schiff base intermediate, between enzyme-bound ALA at the P-site and AOB bound at the A-site, could be demonstrated. Biochemistry, 1997 Feb 4, 36(5), 1077 - 84 Preferential interactions of the Escherichia coli LexA repressor with anions and protons are coupled to binding the recA operator; Relan NK et al.; The binding of Escherichia coli LexA repressor to the recA operator was examined as a function of the concentration of NaCl, KCl, NaF, and MgCl2 at pH 7.5, 21 degrees C . The effects of pH at 100 mM NaCl were also examined . Changes both in the qualitative appearance of the binding isotherms and in the magnitude of the apparent binding affinity with changes in solution conditions suggest that binding of anions and protons by LexA repressor is linked to oligomerization and/or operator binding . Binding of LexA repressor to the recA operator in the presence of NaCl ranging from 25 to 400 mM at picomolar DNA concentration showed a broad, apparently noncooperative, binding isotherm . Binding of LexA repressor in NaF at the same {DNA} yielded binding isotherms with a narrow transition, reflecting an apparently cooperative binding process . Also, the apparent binding affinity was weaker in NaF than in NaCl . Furthermore, the binding affinity and also the apparent binding mode, cooperative vs noncooperative, were pH dependent . The binding affinity of LexA repressor for operator was greatest near neutral pH . The apparent binding mode was noncooperative at pH 7-9 but was cooperative at pH 6 or 9.3 . These observations suggest that the specific cation and anion composition and concentrations must be considered in understanding the details of regulation of the SOS system. Proc Natl Acad Sci U S A, 1997 Feb 4, 94(3), 1035 - 40 Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit; Stockinger EJ et al.; Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat/dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit . Here we report the isolation of an Arabidopsis thaliana cDNA that encodes a C-repeat/DRE binding factor, CBF1 (C-repeat/DRE Binding Factor 1) . Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain . CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the Arabidopsis proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function . The transcript levels for CBF1, which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit . Binding of CBF1 to the C-repeat/DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in Escherichia coli . Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat/DRE as an upstream activator sequence but not mutant versions of the DNA element . We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat/DRE DNA regulatory element and, thus, is likely to have a role in cold- and dehydration-regulated gene expression in Arabidopsis. Proc Natl Acad Sci U S A, 1997 Feb 4, 94(3), 1029 - 34 The structure and function of a soybean beta-glucan-elicitor-binding protein; Umemoto N et al.; beta-Glucan elicitor (GE), released from the cell wall of the phytopathogenic fungus Phytophthora megasperma by soybean glucanases, causes defense reactions in soybean . A GE-binding protein (GEBP) was purified from the membrane fraction of soybean root cells, and its cDNA was isolated . Expression of the cDNA clone in tobacco suspension cultured cells and in Escherichia coli conferred GE-binding activity to both . An antibody against the recombinant protein was found to inhibit the GE binding with the soybean cotyledon membrane fraction as well as the resulting accumulation of phytoalexin . Immunolocalization assays indicated that the GEBPs are located in the plasma membrane of root cells . These results suggest that the cDNA encodes a GE receptor and may mediate the signaling of the elicitor. Proc Natl Acad Sci U S A, 1997 Feb 4, 94(3), 946 - 51 Escherichia coli DNA polymerase II catalyzes chromosomal and episomal DNA synthesis in vivo; Rangarajan S et al.; We have investigated a role for Escherichia coli DNA polymerase II (Pol II) in copying chromosomal and episomal DNA in dividing cells in vivo . Forward mutation frequencies and rates were measured at two chromosomal loci, rpoB and gyrA, and base substitution and frameshift mutation frequencies were measured on an F'(lacZ) episome . To amplify any differences in polymerase error rates, methyl-directed mismatch repair was inactivated . When wild-type Pol II (polB+) was replaced on the chromosome by a proofreading-defective Pol II exo- (polBex1), there was a significant increase in mutation frequencies to rifampicin resistance (RifR) (rpoB) and nalidixic acid resistance (NalR) (gyrA) . This increased mutagenesis occurred in the presence of an antimutator allele of E . coli DNA polymerase III (Pol III) (dnaE915), but not in the presence of wild-type Pol III (dnaE+), suggesting that Pol II can compete effectively with DnaE915 but not with DnaE+ . Sequencing the RifR mutants revealed a G --> A hot spot highly specific to Pol II exo- . Pol II exo- caused a significant increase in the frequency of base substitution and frameshift mutations on F' episomes, even in dnaE+ cells, suggesting that Pol II is able to compete with Pol III for DNA synthesis on F episomes. Biochem Biophys Res Commun, 1997 Feb 3, 231(1), 126 - 30 Precise limits of the N-terminal domain of DnaB helicase determined by NMR spectroscopy; Miles CS et al.; Two separate N-terminal fragments of the 470-amino-acid Escherichia coli DnaB helicase, comprising residues 1-142 and 1-161, were expressed in E . coli . The proteins were extracted in a soluble fraction, purified, and characterised physically . In contrast to the full-length protein, which is hexameric, both fragments exist as monomers in solution, as demonstrated by sedimentation equilibrium measurements . CD spectroscopy was used to confirm that the 161-residue fragment is highly structured (mostly alpha-helical) and undergoes reversible thermal denaturation . The structurally well-defined core of the N-terminal domain of the DnaB helicase is composed of residues 24 to 136, as determined by assignment of resonances from flexible residues in NMR spectra . The 1H NMR signals of the flexible residues are located at random coil chemical shifts, and their linewidths are significantly narrower than those of the structured core, indicating complete disorder and increased mobility on the nanosecond time scale . The results support the idea of a flexible hinge region between the N- and C-terminal domains of the native hexameric DnaB protein. Biochem Biophys Res Commun, 1997 Feb 3, 231(1), 68 - 72 Purification and characterization of the periplasmic domain of EnvZ osmosensor in Escherichia coli; Egger LA et al.; The EnvZ-OmpR histidyl-aspartyl phosphorelay system in E . coli responds to osmolarity by differentially modulating the expression of the major outer membrane porins OmpF and OmpC . To date, the natural ligand that activates EnvZ, a transmembrane histidine kinase, has not been identified and the role of the periplasmic domain of EnvZ is unclear . We now report on the purification and characterization of the periplasmic domain of EnvZ (Lys48-Arg162) which has been expressed as a soluble protein in fusion with the maltose-binding protein . Overexpression of the fusion protein did not compete for a signal that activates EnvZ . By amylose affinity chromatography and affinity blotting, interacting proteins could not be detected . The periplasmic domain was released by factor Xa and purified to homogeneity . From circular dichroism analysis, the periplasmic domain was estimated to consist of 35% alpha-helices and 16% beta-sheets. Biochem Biophys Res Commun, 1997 Feb 3, 231(1), 56 - 60 Cloning and expression of human liver rhodanese cDNA; Aita N et al.; cDNA for the human rhodanese (thiosulfate; cyanide sulfurtransferase, EC 2.8.1.1) was cloned from a human fetal liver cDNA library . Sequencing of the cDNA revealed an open reading frame that encodes a 297-residue polypeptide with a calculated mass of 33,427 daltons . When the rhodanese cDNA was transiently expressed in Escherichia coli and Cos7 cells, the rhodanese activity increased 40-fold and 150-fold, respectively . Sequence homology analysis showed that the human rhodanese is 89.6% identical to bovine, 90.2% identical to rat, 91.2% identical to mouse and Chinese hamster, and 71.4% similar to avian counterparts, respectively, and that rhodanese was highly conserved across evolution. FEBS Lett, 1997 Feb 3, 402(2-3), 177 - 80 Insertion of foreign random sequences of 120 amino acid residues into an active enzyme; Doi N et al.; Random sequences of 120-130 amino acid residues were inserted into a surface loop region of Escherichia coli RNase HI . This library was screened and about 10% of the clones were found to retain RNase H activity . Subsequent random mutagenesis led to an increase in RNase H activity and solubility of the protein . The inserted regions were found not to contribute to the secondary structure of the mutant protein . The high frequency of insertion of flexible sequences and the increase in the protein's function by further mutagenesis simulate one of the events in protein evolution. FEBS Lett, 1997 Feb 3, 402(2-3), 116 - 20 19F-NMR studies of retinol transfer between cellular retinol binding proteins and phospholipid vesicles; Rong D et al.; The cellular retinol binding proteins, CRBP and CRBP II, are implicated in the cellular uptake of retinol and intracellular trafficking of retinol between sites of metabolic processing . 19F-NMR studies of retinol transfer between CRBP and CRBP II and phospholipid vesicles, using either fluorine-labeled ligand or protein, demonstrated that there was significantly more transfer of retinol from CRBP II to lipid vesicles than from CRBP . Differences in how readily protein-bound retinol is released to lipid bilayers may lead to differences in how these two proteins modulate intracellular retinol metabolism. EMBO J, 1997 Feb 3, 16(3), 523 - 33 Yeast telomeric sequences function as chromosomal anchorage points in vivo; Mirabella A et al.; Site-specific recombination in Saccharomyces cerevisiae was used to generate non-replicative DNA rings containing yeast telomeric sequences . In topoisomerase mutants expressing Escherichia coli topoisomerase I, the rings adopted a novel DNA topology consistent with the ability of yeast telomeric DNA to block or retard the axial rotation of DNA . DNA fragments bearing portions of the terminal repeat sequence C1-3 A/TG1-3 were both necessary and sufficient to create a barrier to DNA rotation . Synthetic oligonucleotide sequences containing Rap1p binding sites, a well represented motif in naturally occurring C1-3A arrays, also conferred immobilization; mutant Rap1p binding sites and telomeric sequences from other organisms were not sufficient . DNA anchoring was diminished by addition of competing telomeric sequences, implicating a role for an as yet unidentified limiting trans-acting factor . Though Rap1p is a likely protein constituent of the DNA anchor, deletion of the non-essential C-terminal domain did not affect the topology of telomeric DNA rings . Similarly, disruption of SIR2, SIR3 and SIR4, genes which influence a variety of telomere functions in yeast, also had no effect . We propose that telomeric DNA supports the formation of a SIR-independent macromolecular protein-DNA assembly that hinders the motion of DNA because of its linkage to an insoluble nuclear structure . Potential roles for DNA anchoring in telomere biology are discussed. Mol Immunol, 1997 Feb, 34(3), 255 - 61 Determination of the N- and C-terminal sequences required to bind human IgE of the major house dust mite allergen Der f 2 and epitope mapping for monoclonal antibodies; Takai T et al.; B cell epitopes of the major house dust mite allergen Der f 2 from Dermatophagoides farinae were analysed using deletion mutants of Der f 2 expressed as fusion proteins in Escherichia coli . The reactivities of these partial Der f 2 molecules to human anti-mite IgE antibodies in atopic patients and to murine anti-Der f2 monoclonal antibodies (mAbs) were examined by immunoblotting . A C-terminal deletion mutant of Der f 2, 1-123, had almost the same reactivity to human IgE as the whole Der f 2 (1-129) and an N-terminal deletion mutant of Der f 2 (25-129) still had weak reactivity . On the other hand, in two deleted Der f 2 molecules, 1-120 and 30-129, reactivity was lost in spite of long overlapping sequences . These results suggest that the human IgE antibodies to Der f 2 in atopic patient sera recognize the conformational structures dependent on the tertiary structure of Der f 2, including disulfide bond formations, rather than the contiguous sequences of amino acids . The sequences 1-24, 25-29 and 121-123 were revealed as the minimum N- and C- terminal amino acid sequences required for IgE binding . Contrastingly, all three murine mAbs bound to the smaller deletion mutants, 1-90 and 67-129, suggesting that the cores of the epitopes for these mAbs exist in the 24 amino acid sequence of Der f 2, 67-90 overlapping the sequential human IgE epitope on Der p 2, the equivalent allergen from Dermatophagoides pteronyssinus . These findings are important for the understanding of the antigenic structure of Der f 2 and for the manipulation of the allergen for immunotherapy. Mol Immunol, 1997 Feb, 34(3), 201 - 8 Developmental specificity of immunoglobulin heavy chain switch region recombination activities; Li MJ et al.; To understand the regulation of enzymes that carry out immunoglobulin heavy chain class switch recombination, we have assayed recombination of extrachromosomal substrates carrying switch region sequences in cell lines representing different stages of lymphoid cell development . Both pre-B and mature B cell lines supported switch substrate recombination, but B cell lines derived from later stages of cell development did not . Recombination did not occur in an erythroid or a macrophage line . Most recombination junctions in the substrates recovered from transfection of pre-B and B cells mapped to heterogeneous sites within the S mu and Sgamma regions, as do chromosomal switch junctions . Some recombination did occur in T cell lines, but most recombination junctions involved an upstream promoter and did not map preferentially to S regions . Culture of the pre-B cell lines PD31 and 70Z/3 with LPS increased recombination two-fold, to levels approaching those observed in LPS-cultured primary B cells . These results show that the full complement of factors necessary for switch recombination is present only in cells representing a limited spectrum of B cell development and that LPS, which can activate resting splenic B cells to carry out chromosomal recombination, can also stimulate recombination activity in immortalized pre-B cell lines. Comp Immunol Microbiol Infect Dis, 1997 Feb, 20(2), 155 - 62 Comparative effects of bovine cytokines on cattle and bison peripheral blood mononuclear cell proliferation; Stevens MG et al.; Proliferation of peripheral blood mononuclear cells (PBMC) from cattle and bison was measured following stimulation of PBMC with bovine cytokines . Bovine interleukin 1 beta (BoIL-1 beta), interleukin 2 (BoIL-2) or granulocyte-macrophage colony-stimulating factor (BoGM-CSF) at 0.1-100 U/ml were incubated for 48 h with PBMC alone or with PBMC and various mitogens . These included concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) or Escherichia coli 055:B5 lipopolysaccharide (LPS) at 10-0.1 micrograms/ml . BoIL-2 alone, but not BoIL-1 beta and BoGM-CSF alone, induced proliferation of cattle and bison PBMC in the absence of mitogens . In addition, BoIL-1 beta and BoIL-2, but not BoGM-CSF, enhanced proliferation of cattle and bison PBMC induced by mitogens . These results indicate that BoIL-1 beta and BoIL-2 stimulate cattle and bison PBMC proliferation in a similar manner, whereas BoGM-CSF does not appear capable of stimulating either cattle or bison PBMC proliferation. J Infect Dis, 1997 Feb, 175(2), 373 - 81 Receptor specificities of variant Gal(alpha1-4)Gal-binding PapG adhesins of uropathogenic Escherichia coli as assessed by hemagglutination phenotypes; Johnson JR et al.; Receptor specificities of the three recognized variants of PapG, the major adhesin of uropathogenic Escherichia coli, have been deduced in part from the distinctive hemagglutination (HA) patterns these adhesins ostensibly exhibit with different erythrocytes . A comprehensive reevaluation of these HA patterns using sheep, rabbit, and diverse human erythrocytes revealed that the erythrocyte binding ranges of the three PapG variants are broader and more overlapping than generally recognized, suggestive of receptor specificities beyond those posited in the prevailing model . Experiments involving guinea pig erythrocytes artificially labeled with defined Gal(alpha1-4)Gal-containing glycolipids supported this hypothesis and further suggested that HA patterns with naturally occurring erythrocytes cannot be used to determine reliably an adhesin's glycolipid binding specificities . Finally, slide HA assays exhibited considerable interexperiment, interobserver, and intraobserver irreproducibility, limiting their usefulness for assessing either the receptor specificities of PapG variants or the PapG repertoire of wild-type E . coli strains. J Infect Dis, 1997 Feb, 175(2), 352 - 63 Adjuvant activity of the heat-labile enterotoxin from enterotoxigenic Escherichia coli for oral administration of inactivated influenza virus vaccine; Katz JM et al.; Alternative strategies for vaccination against influenza that elicit both systemic antibody and mucosal IgA responses are needed to improve the efficacy in protection against infection . This study demonstrated that oral delivery of inactivated influenza vaccine with the heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli elicited the spectrum of humoral and cell-mediated responses in BALB/c mice critical for the protection and recovery from influenza virus infection . Coadministration of LT with oral influenza vaccine increased antiviral serum IgG and mucosal IgA responses compared with administration of oral influenza vaccine alone . Serum hemagglutination-inhibition and neutralizing antibodies were also augmented by LT . The adjuvant potentiated protection from infection with influenza A H3N2 viruses in mouse lower and upper respiratory tracts, enabling the use of lower doses of oral vaccine . Coadministration of LT with oral inactivated influenza vaccine induced influenza virus-specific proliferative T cells, interleukin-2 production, and major histocompatibility complex class I-restricted cytotoxic T cells. Chem Biol, 1997 Feb, 4(2), 93 - 6 Discriminating among the discriminator bases of tRNAs; Hou YM; Aminoacyl-tRNA synthetases must select their specific tRNAs from the 20 structurally similar tRNAs present in a cell . The discriminator base, at position 73 of the tRNA, is important for this selection but its effects on aminoacylation are variable depending on context . Recent structural studies provide insight into this variability. Biosci Rep, 1997 Feb, 17(1), 33 - 42 Superoxide-driven aconitase FE-S center cycling; Gardner PR; O2- produced by the autoxidation of respiratory chain electron carriers, and other cellular reductants, inactivates bacterial and mammalian iron-sulfur-containing (de)hydratases including the citric acid cycle enzyme aconitase . Release of the solvent-exposed iron atom and oxidation of the {4Fe-4S}2+ cluster accompanies loss of catalytic activity . Rapid reactivation is achieved by iron-sulfur cluster reduction and Fe2+ insertion . Inactivation-reactivation is a dynamic and cyclical process which modulates aconitase and (de)hydratase activities in Escherichia coli and mammalian cells . The balance of inactive and active aconitase provides a sensitive measure of the changes in steady-state O2- levels occurring in living cells and mitochondria under stress conditions . Aconitases are also inactivated by other oxidants including O2, H2O2, NO, and ONOO- which are associated with inflammation, hyperoxia and other pathophysiological conditions . Loss of aconitase activity during oxidant stress may impair energy production, and the liberation of reactive iron may further enhance oxidative damage . Iron-sulfur center cycling may also serve adaptive functions by modulating gene expression or by signaling metabolic quiescence. Genes Cells, 1997 Feb, 2(2), 117 - 28 Chi-activated RecBCD enzyme possesses 5'-->3' nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit; Anderson DG et al.; BACKGROUND: Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme, and is stimulated by DNA elements known as Chi (chi) sites . The RecBCD enzyme is both a helicase and a nuclease . Recognition of chi causes both attenuation of the 3'-->5' exonuclease activity of the RecBCD enzyme, and activation of an exonuclease activity with 5'-->3' polarity, while leaving the helicase activity unaffected . A variety of evidence suggests that chi-recognition by RecBCD enzyme is accompanied by ejection of the RecD subunit . RESULTS: Through examination of RecBCD exonuclease activity under a variety of conditions, we have shown that recognition of chi by the RecBCD enzyme results in a net reduction of nuclease activity . In addition, the exact location of the first cleavage event elicited by chi-activation of the 5'-->3' nuclease is dependent upon the concentration of free magnesium ions . Finally, we have demonstrated that purified RecBC enzyme (i.e . without the RecD subunit) possesses no significant exonuclease activity under conditions where the chi-modified RecBCD enzyme is an active 5'-->3' exonuclease . CONCLUSIONS: We have shown that, despite the activation of a 5'-->3' exonuclease, recognition of chi by the RecBCD enzyme results in a net preservation of DNA . This new chi-activated nucleolytic action shows surprising variability in the exact location of its initial cleavage . We have demonstrated that purified RecBC enzyme is not an exact analogue of the chi-activated RecBCD enzyme, suggesting that the biochemical basis of chi-activation is not simply ejection of the RecD subunit. Biochemistry (Mosc), 1997 Feb, 62(2), 204 - 11 Recognition and conversion of single- and double-stranded oligonucleotide substrates by 8-oxoguanine-DNA glycosylase from Escherichia coli; Ishchenko AA et al.; The substrate specificity of 8-oxoguanine-DNA glycosylase was studied . The data showed for the first time that the enzyme can cleave single-stranded deoxyoligonucleotides containing an 8-oxoG link . The values of K(m) and Vmax for a range of single-stranded and double-stranded oligonucleotides (23 bp) containing 8-oxoG at different positions of one chain . These parameters consistently depended on the position of 8-oxoG in single-stranded or double-stranded substrates . A possible mechanism responsible for substrate recognition by 8-oxoguanine-DNA glycosylase is described. Thromb Haemost, 1997 Feb, 77(2), 350 - 6 Expression and characterization of recombinant porcine plasminogen activator inhibitor-1; Bijnens AP et al.; Porcine models are, among other animal models, very suitable for in vivo investigations in the vascular field especially with respect to the possible relationship between atherosclerosis and thrombosis . In order to use this model to define the in vivo role of PAI-1, the characterization of porcine PAI-1 and its availability for the generation of immunological tools are a prerequisite . Porcine plasminogen activator inhibitor-1 (poPAI-1) cDNA was isolated from a cDNA library prepared from cultured porcine aortic cells and characterized in comparison with PAI-1 cDNA's from other species including human, bovine, rabbit, rat and murine . Subsequently the DNA sequence coding the mature protein was cloned into an appropriate vector for expression in Escherichia coli and recombinant porcine PAI-1 was purified and characterized . On SDS-PAGE the apparent molecular weight was estimated to be 45 kDa, identical to the molecular weight of human PAI-1 . The purified recombinant porcine PAI-1 (rpoPAI-1) had a specific activity of 508,800 +/- 800 U/mg (mean +/- SD, n = 3) towards human tissue-type plasminogen activator (ht-PA) and a functional half-life in vitro of 2.1 +/- 0.8 h (n = 3) . Incubation with a two fold molar excess of ht-PA (n = 3) or human urokinase-type plasminogen activator (hu-PA, n = 2) followed by analysis by SDS-PAGE revealed reaction products corresponding to active (71 +/- 7% resp . 96 +/- 3.6%), latent (12 +/- 0.4% resp . 2.6 +/- 2.4%) and substrate (16.6 +/- 6.8% resp . 1.5 +/- 1.3) forms . Inactivated samples of porcine PAI-1 could be reactivated with guanidinium chloride up to 52% of its original specific activity towards t-PA and u-PA . The second order rate constant of inhibition of ht-PA was 1.64 +/- 0.37 10(7)M-1 s-1 (n = 9) . In gel filtration rpoPAI-1 in buffer eluted at a volume corresponding to 24 kDa, whereas in the presence of porcine plasma, the molecular form containing PAI-1 activity eluted at a volume corresponding to 330 kDa, presumably as a consequence of binding of active PAI-1 to vitronectin . Taken together, these data demonstrate that no obvious functional differences exist between human and porcine PAI-1. Mol Microbiol, 1997 Feb, 23(4), 835 - 45 Transcriptional activation by FNR and CRP: reciprocity of binding-site recognition; Sawers G et al.; Anaerobic expression of the focA pfl operon is dependent on the transcription factors ArcA and FNR and transcription is directed by multiple, anaerobically regulated promoters . A FNR-binding site is centred at -41.5 bp relative to the P6 promoter, inactivation of which severely impairs anaerobic expression of the complete operon . Mutations were introduced into this binding site to create a consensus recognition site for the cAMP-receptor protein, CRP (CC-site), and one that was recognised by both CRP and FNR (CF-site) . Transcription directed by these mutant binding sites in vivo in different promoter constructs was analysed by primer extension and by constructing lacZ operon fusions . With a derivative including only the P6 promoter and the CF-binding site, transcription was shown to be independent of oxygen and was activated by CRP or FNR . In agreement with previous findings, FNR only activated transcription anaerobically . In a construct including the CC-binding site transcription was strong . CRP dependent and initiated at the identical site to the wild-type promoter . Transcription activation from the CC-site was exquisitely sensitive to low cAMP concentration . Surprisingly, in a crp mutant, anaerobically inducible, FNR-dependent transcription directed by the CC-site was detected, indicating that FNR can recognise a consensus CRP-binding site in vivo . A strain unable to synthesise CRP or FNR exhibited no transcription from the P6 promoter . Essentially the same results were observed in a series of constructs that also included the promoter P7 and its regulatory sequences . Evidence is also presented which demonstrates that CRP activates transcription from the natural FNR-binding site of the P6 promoter . In vitro DNA-binding studies showed that CRP specifically interacted with the FNR-binding site, protecting exactly the same sequence as that protected by the FNR protein . Interaction of CRP with the natural FNR-binding site was reduced greater than 50-fold compared to its interaction with the mutant CC-binding site . Although we could not demonstrate that FNR interacted with the CC-binding site in vitro, it did bind to the CF-site giving the same protection as observed with the wild-type FNR-binding site . FNR also activated transcription from the CF-site in vitro, giving further support to the idea that a single functional DNA half-site is sufficient to direct binding and transcription activation by a dimeric transcription factor. Mol Microbiol, 1997 Feb, 23(4), 705 - 17 Integration host factor stimulates both FimB- and FimE-mediated site-specific DNA inversion that controls phase variation of type 1 fimbriae expression in Escherichia coli; Blomfield IC et al.; The site-specific DNA inversion that controls phase variation of type 1 fimbriation in E . coli is catalysed by two recombinases, FimB and FimE . Efficient inversion by either recombinase also requires the leucine-responsive regulatory protein (Lrp) . In addition, FimB recombination is stimulated by the integration host factor (IHF) . The effect of IHF on FimE inversion has not previously been reported . Here it is shown that IHF stimulates FimE recombination; In strain MG1655, mutants containing lesions in either the alpha (ihfA) or beta (ihfB) subunits of IHF show a marked decrease in both FimB- (100-fold) and FimE (15,000-fold)-promoted switching . IHF is shown to bind with high affinity to sites both adjacent to (site I) and within (site II) the fim invertible element . Furthermore, mutations in site I or site II that lower the affinity of IHF binding In vitro were found to lower the frequency of FimE and/ or FimB recombination in vivo . Although site I and site II mutations in combination have an effect on FimB-promoted switching comparable to that of IHF knockout mutations (100-fold), the cis site mutations have a much less marked effect (100-fold) on FimE-promoted switching. Eur J Cell Biol, 1997 Feb, 72(2), 122 - 32 Heterotypic interactions and filament assembly of type I and type II cytokeratins in vitro: viscometry and determinations of relative affinities; Hofmann I et al.; We have determined relative affinities of type I and type II human non-epidermal cytokeratin (CK) polypeptides synthesized in Escherichia coli from cDNA, using the method of surface plasmon resonance, and have compared the influence of ionic strength and ion quality on the assembly to intermediate filaments (IFs) by viscometry and electron microscopy . By surface plasmon resonance (total internal reflection) we determined the real-time relative binding of various type I CKs to the type II CK8 . Surprisingly, the various type I CKs examined (i.e., CKs 13, 18, 19 and 20) differed markedly in their relative binding rate: For example, CK18 and CK13 displayed much higher resonance signals than CK19 and CK20 . In addition, soluble complexes of type II CK8 with various type I cytokeratins were reconstituted at slightly alkaline pH and low ionic strength . Subsequent IF assembly was induced by simultaneous shifting to neutral pH and increasing the ionic strength . Both mono- and divalent ions as well as tris (hydroxymethyl)-aminomethane (Tris) alone were able to induce IF assembly . When we compared the time course of viscosity increase of CK8 in combination with either CK13, CK18, CK19 or CK20, we found that the cytokeratin pairs 8:18 and 8:20 attained the highest viscosity values, whereas the cytokeratin pair 8:19 displayed a relatively low value . Significantly lower values of specific viscosity were also observed in competition experiments when CK18 was gradually replaced by CK19 . The observed differences in relative affinities and assembly kinetics of certain CK combinations allow quantitations of the interactions between different CKs and are discussed in relation to IF stability and cell differentiation. Pediatr Surg Int, 1997 Feb, 12(2-3), 198 - 9 Cholelithiasis in early infancy; Wilcox DT et al.; This report describes three children, age range 7 weeks to 5 months, who presented with obstructive jaundice secondary to gallstones . Previous Escherichia coli septicaemia and frusemide therapy were predisposing risk factors in two of the patients . All three were successfully treated with cholecystectomy and exploration of the common bile duct. Neuropediatrics, 1997 Feb, 28(1), 33 - 6 Expression studies of CLN3 protein; Kaczmarski W et al.; Expression of the gene for Batten disease (CLN3) was studied in Escherichia coli and in a cell-free rabbit reticulocyte expression systems . A full-length recombinant fusion CLN3 protein was not produced in the bacterial systems used . However, both N-terminal fragment encompassing 246 amino acids and short C-terminal fragment containing 428-438 amino acids of the CLN3 protein were successfully overexpressed in bacteria . Further studies showed that the C-terminal sequence of the CLN3 protein corresponding to the 356-438 amino acid residues was responsible for inhibition of protein synthesis in bacteria . The full-length CLN3 gene product was readily synthesized in vitro in the cell-free rabbit reticulocyte expression system . The product obtained, corresponding to core CLN3 protein, showed an approximate molecular weight of 43 kDa . Immunoprecipitation of this product with pAb to 4-19 amino acids of the CLN3 protein allows us to suggest that CLN3 protein translation starts at Met-1. J Immunoassay, 1997 Feb, 18(1), 49 - 65 An immunoligand assay for quantitation of process specific Escherichia coli host cell contaminant proteins in a recombinant bovine somatotropin; Whitmire ML et al.; We have developed a Threshold System immunoligand assay for the quantitation of residual, process-specific, Escherichia coli host cell contaminant proteins (HCP) in somavubove (a normal sequence recombinant bovine somatotropin) . The assay has a dynamic range of 2 to 160 ng/mL, with a limit of quantitation of 2 ng/mL . Daily analytical precision (CV) for six replicates of the designated somavubove laboratory standard is 3.0% . Cumulative analytical precision for multiple assay runs of this standard (a total of 69 laboratory standard replicates) is 8.4% . A conservative alert limit of 100 ng/mL has been assigned in order to assure analytical precision for each assay sample under conditions of stoichiometric antibody excess . Although the assay was designed for use as a profile-release specification assay, it has also been used to validate removal of HCP by the proprietary somavubove purification process . This use is consistent with regulatory guidelines related to "well characterized" recombinant biopharmaceutical proteins. Immunology, 1997 Feb, 90(2), 286 - 93 Effects of 1 alpha,25-dihydroxyvitamin D3 and cytokines on the expression of MHC antigens, complement receptors and other antigens on human blood monocytes and U937 cells: role in cell differentiation, activation and phagocytosis; Spittler A et al.; The effect of calcitriol/1 alpha,25-dihydroxyvitamin D3, alone and in combination with cytokines, on the expression of various antigens (Ag) on human peripheral blood monocytes and U937 cells was studied by flow cytometry . Both constitutive and interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and tumour necrosis factor-alpha (TNF-alpha)-induced human leucocyte antigen (HLA)-DR, HLA-DP and HLA-DQ Ag expression on monocytes was significantly down-regulated by calcitriol, IL-10 and transforming growth factor-beta (TGF-beta) . The effects of calcitriol were concentration dependent and reached maximal inhibitory levels after 3-5 days . Modulation of HLA-DR by calcitriol and IFN-gamma at the protein level correlated with the amount of mRNA specific for the HLA-DR alpha-chain, as judged by Northern blot analysis . The basal as well as IL-4, IL-6, IFN-gamma, TNF-alpha and TGF-beta-driven levels of HLA-ABC Ag were significantly diminished by calcitriol . On U937 cells calcitriol markedly induced CD11a and CD11b expression and weakly up-regulated CD11c whereas on monocytes, constitutive CD11a, CD11b and CD11c expression was significantly down-regulated by calcitriol . The expression of CD14 Ag was strongly induced on U937 cells but only modestly on monocytes . Both the basal level of CD71 and IL-4, IFN-gamma or TNF-alpha-driven expression was diminished on calcitriol-treated U937 cells . In addition, calcitriol suppressed the expression of CD71 Ag on monocytes . The ability of monocytes to phagocytize opsonized Escherichia coli was diminished by calcitriol . Our results demonstrate that calcitriol, alone or in combination with cytokines, modulates expression of MHC, CD11b, CD11c, CD14 and CD71 Ag on both monocytes and U937 cells, and impairs the phagocytic property of monocytes. Immunology, 1997 Feb, 90(2), 176 - 82 Relationship between a low toxicity of the mutant A subunit of enterotoxigenic Escherichia coli enterotoxin and its strong adjuvant action; Tsuji T et al.; In the work described here it was determined if and how unnicking in the A subunit of Escherichia coli enterotoxin at Arg192 or nearby residues affected biological activities of the toxin . The mutant toxin was constructed to lack the nick site in the A subunit by deleting the tripeptide Arg192-Thr193-Ile194, which is essential for toxicity . The mutant toxin did not exhibit agmatine ADP-ribosyltransferase activity in the presence or absence of the ADP-ribosylation factor and had less diarrhoeal activity and lower induction of cyclic AMP than did LT . The mutant toxin exhibited a much stronger adjuvant action on antibody responses to measles virus, keyhole limpt haemocyanin, bovine immunoglobulin and ovalbumin compared with LT . The altered toxicity of the mutant toxin might be closely related to the potent adjuvant action on antibody responses to antigens . The relationship between two activities is discussed. Arch Microbiol, 1997 Feb-Mar, 167(2-3), 143 - 50 The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase; Miksch G et al.; Heterologous gene products produced by Escherichia coli cells can be exported into the culture medium by the action of the kil gene of the ColE1 plasmid, which encodes a bacterial release protein . The kil gene was fused with the stationary-phase promoter of the fic gene of E . coli, and a secretion cassette (Kil-Km cassette) containing the regulated kil gene, the Km-resistance gene, and multiple cloning sites for the integration of target genes was constructed . Using the gene for beta-glucanase (bgl) as a target gene, it was shown that the protein produced was only secreted into the medium during the stationary phase . Quasi-lysis and lethality were not observed . The primary effect of the induction of the kil gene was the overproduction of beta-glucanase . The total amount produced per milliliter of bacterial culture was almost threefold higher than that of the corresponding Kil- control . The protein pattern of periplasm and culture medium was analyzed before and after induction of the kil gene expression, indicating that the release of periplasmic proteins is semiselective . This secretion system is the first to use a growth-phase-regulated promoter for the expression of the kil gene. Arch Microbiol, 1997 Feb-Mar, 167(2-3), 126 - 36 Requirement of a large K+-uptake capacity and of extracytoplasmic protease activity for protamine resistance of Escherichia coli; Stumpe S et al.; The effect of protamine on growing cells of Escherichia coli K-12 strains containing different K+-uptake systems was investigated . Immediately after the addition of the toxic peptide, growth ceased and all strains lost most of their K+ . In addition, these cells released a significant amount of their ATP into the medium, and the cytoplasmic volume of these cells decreased by 70% . Whereas cells without rapid K+-uptake systems did not recover, cells containing either the Trk systems or the overproduced Kup system slowly reversed the effects of protamine, and growth resumed after the cells had reached their original volume . Experiments with a set of strains carrying mutations in the K+-uptake gene trkA showed a reasonably satisfactory correlation between inhibition of net K+ uptake and the lag time for resumption of growth after addition of protamine . Cells carrying mutations in three extracytoplasmic proteases were hypersusceptible to protamine, suggesting that the toxic peptide is degraded by these proteases . Data on the effect of a second addition of protamine suggest that protamine degradation activity is inducible . These data are interpreted to mean that reaccumulation of K+ by protamine-treated cells triggers recovery of the cells, thereby allowing induction of extracytoplasmic proteases . These, in turn, degrade protamine, leading to complete recovery of the cells and resumption of growth . Cells that cannot take up K+ rapidly remain metabolically compromised to such an extent that extracytoplasmic protease activity is not induced, leading to a prolonged susceptibility of the cells to the toxic peptide. Cell Mol Neurobiol, 1997 Feb, 17(1), 119 - 27 The use of Epstein-Barr virus-based shuttle vectors in rat PC12 cells; Lindenboim L et al.; 1 . The rat pheochromocytoma PC12 cell line has been a commonly used model for studies of neuronal development, function, and death . Thus the ability to transfect PC12 cells in an efficient manner and to manipulate their gene expression would enhance the usefulness of these cells . 2 . We demonstrate that EBV-based vectors provide a useful expression system for gene manipulation in rat PC12 cells . 3 . The EBV-based vectors replicate episomally in PC12 cells for at least 2 months, as evidence by their recovery from the transfected cells and by the digestion of the episomal plasmid with the isoschizomer MboI and DpnI restriction enzymes . 3 . PC12 cells are efficiently transfected by EBV-based vectors both transiently and stably . 4 . Transfection of PC12 cells with an EBV-based vector containing tau cDNA in the antisense orientation resulted in a decrease in the level of tau protein in the transfected cells . 5 . The results demonstrate that EBV-based vectors can be a useful expression system for gene manipulation in PC12 cells. Protein Expr Purif, 1997 Feb, 9(1), 91 - 9 High-level expression and purification of MyoD, myogenin, and E12; Maleki SJ et al.; The myogenic regulatory factors (MRFs), MyoD, myogenin, Myf-5, and MRF4, function as transcriptional activators of muscle-specific gene expression by forming heterodimers with the ubiquitously expressed products of the E2A gene, E12 and E47 . To enable quantitative biochemical and biophysical analyses of the wild-type proteins, as well as mutants designed to reveal structure-function relationships, we developed protocols for the high-level expression and rapid purification of milligram quantities of MyoD, myogenin, and E12 using conventional biochemical techniques . T7 expression systems were used to direct expression of cDNA encoded proteins in Escherichia coli . Whereas MyoD and E12 were expressed well without alteration, high-level expression of myogenin required changing several rare arginine codons by in vitro mutagenesis to a commonly used E . coli arginine codon . Presumably, inefficient translation of the rare arginine codons inhibited high-level expression of myogenin in the original expression plasmid . Purification protocols are described which involve a simple strategy of cell lysis, ammonium sulfate precipitation, and ion-exchange chromatography . Using this approach, 20 to 50 mg of MyoD, myogenin, or E12 can be purified to 90-95% homogeneity from induced cell pellets in 1 day's time. Protein Expr Purif, 1997 Feb, 9(1), 53 - 60 Expression of functional eIF-4Ehuman: purification, detailed characterization, and its use in isolating eIF-4E binding proteins; Hagedorn CH et al.; Protein-mRNA cap interactions represent a critical point for regulating gene expression in vivo . For example, a rapid stimulation of gene expression at the mRNA level is mediated by insulin regulating the availability of functional cap-binding protein (eIF-4E) . In addition, several viruses modify cap binding proteins to regulate host vs viral gene expression . However, little is known about the molecular details of eIF-4E interactions with m7GTP mRNA caps, with regulatory proteins (e.g., eIF-4E binding proteins), and with proteins within the eIF-4F complex . To study these protein-mRNA and protein-protein interactions in mammalian systems we have constructed a T7 polymerase-driven expression vector containing the coding sequence for human eIF-4E . Recombinant eIF-4Ehuman was purified in a functional state by m7GTP affinity chromatography and Mono Q FPLC . This recombinant protein has biological and physical characteristics that are similar or identical to native eIF-4E . Fluorescence titration studies determined the equilibrium constant for recombinant eIF-4E/m7GTP binding to be 10.1 +/- 0.3 x 10(5) M(-1) . To isolate eIF-4E binding proteins, recombinant eIF-4E was linked to agarose beads and incubated with cell lysates . Several proteins were isolated, including a 220-kDa protein that was confirmed to be the p220 subunit of eIF-4F by its proteolysis during incubation with lysates of poliovirus-infected cells . We conclude that recombinant eIF-4E produced in Escherichia coli provides a useful tool for studying eIF-4E/protein and eIF-4E/mRNA cap interactions and their role in regulating mammalian gene expression. Protein Expr Purif, 1997 Feb, 9(1), 33 - 9 High-level expression and purification of the major birch pollen allergen, Bet v 1; Hoffmann-Sommergruber K et al.; Bet v 1, the single major allergen from birch pollen, shares IgE epitopes with all major tree pollen allergens from closely related species such as alder, hazel, hornbeam, beech, and European chestnut . Because of high sequence homologies among these allergens and the well-studied cross-reactivities on B cell epitopes, Bet v 1 is a representative model protein which can be used for in vitro studies . The cDNA coding for Bet v 1, the single major allergen from birch pollen, was cloned into the T7-based Escherichia coli expression system pMW 175/BL21(DE3) and synthesized as a nonfusion protein . In contrast to other E . coli systems (e.g., pKK233-2/JM105), this system produces high levels of readily extractable proteins corresponding to 5-10% of E . coli total protein, the percentage varying with culture conditions . The overall yield was 8-10 mg of purified recombinant protein per liter of culture medium . The recombinant allergen was purified by several steps, including ion-exchange and hydrophobic interaction chromatography . The purified recombinant allergen showed identical immunological properties with the respective natural counterpart . The use of recombinant allergens of high purity is expected to result in more accurate diagnostic procedures, but possibly also in a superior immunotherapy of Type I allergic diseases when compared with methods using crude allergen extracts containing various amounts of allergen concentrations. Protein Expr Purif, 1997 Feb, 9(1), 15 - 26 Purification and biochemical characterization of the 19-kDa signal recognition particle RNA-binding protein expressed as a hexahistidine-tagged polypeptide in Escherichia coli; Henry KA et al.; The signal recognition particle (SRP) is a ribonucleoprotein complex that mediates translocation of proteins into the endoplasmic reticulum . Protein SRP19 is an essential structural component of SRP and is believed to promote assembly of the particle . In order to have a convenient source for the purification of milligram amounts of SRP19, we expressed in Escherichia coli a human SRP19 cDNA with an amino-terminal addition of six histidine residues . Expression at 25 degrees C eliminated formation of insoluble SRP19 and resulted in accumulation of soluble hexahistidine-SRP19 to 68% of total cell protein after 24 h . Metal chelation chromatography yielded 40 mg of hexahistidine-SRP19 per liter of culture, with a purity slightly greater than 97% . To examine protein function, the RNA-binding properties of the purified protein were determined by RNA electromobility shift assays . The histidine-tagged SRP19 bound specifically to a 150-nucleotide RNA derived from SRP RNA, with an apparent Kd of 1 nM, and bound, with greatly reduced affinity, to a mutagenized form of the SRP RNA derivative that contained an altered helix 6 tetranucleotide loop . The purified protein was also photochemically crosslinked to the 150-nucleotide SRP RNA fragment, providing the means to potentially identify portions of hexahistidine-SRP19 which are in close proximity to the RNA molecule. Protein Expr Purif, 1997 Feb, 9(1), 115 - 24 High-yield expression and purification of recombinant human macrophage migration inhibitory factor; Mozetic-Francky B et al.; We have expressed the human macrophage migration inhibitory factor (MIF) in Escherichia coli using the pKP 1500 expression plasmid, which contains the tac promoter and a temperature-sensitive origin of replication, to ensure a high plasmid copy number at elevated temperatures . The recombinant protein accumulated intracellularly in soluble form . We have designed a simple two-step procedure for protein purification by gel filtration on Sephadex G-50 and cation exchange chromatography on CM cellulose columns . This results in significantly improved yields . One gram of recombinant human MIF was isolated from 50 g of E . coli cells (wet weight) . The 12.5-kDa protein was shown to be pure by SDS-PAGE, IEF, and HPLC . The identity of the purified protein was verified by N-terminal amino acid sequencing . The purified protein exhibits MIF activity . The near-UV CD and the 1H NMR spectra confirmed its highly ordered, native-like structure . The far-UV CD spectrum revealed that recombinant human MIF contains well-defined secondary structure. Protein Expr Purif, 1997 Feb, 9(1), 1 - 9 Expression, purification, and characterization of the two human primase subunits and truncated complexes from Escherichia coli; Copeland WC; Eukaryotic DNA replication is primed by small RNA primers synthesized by the two-subunit primase complex, p58 and p49, where the p49 subunit contains the catalytic activity . The cDNA's for these two human DNA primase subunits were amplified, sequenced, and overexpressed in Escherichia coli . Specific assays for initiation revealed that although the smaller subunit contains catalytic function, initiation requires the presence of the larger subunit . A two-plasmid system was developed for the coexpression of both subunits in E . coli . This system was exploited to express and study truncations of the larger, human p58 subunit in order to investigate its role in primer formation . Analysis of the complexes formed between the truncated human p58 subunits and the native human p49 subunit revealed that protein-protein contacts between these two subunits occur over several regions of the human p58 subunit . Of four primase complexes containing different truncated p58 subunits only one complex supported initiation as measured by the formation of dinucleotides . All complexes supported the extension of oligoA-primed polydT, suggesting that the intrinsic RNA polymerase activity residing in the smaller subunit was not affected . These results suggest that several regions of the human p58 subunit are in contact with the human p49 subunit during the initiation of primer synthesis. J Belge Radiol, 1997 Feb, 80(1), 9 - 10 Delayed remote abscess formation after spillage of infected gallstone during laparoscopic cholecystectomy: CT and US findings; Van Hover P et al.; We report on a patient who presented an abdominal wall abscess two years after percutaneous cholecystectomy . The diagnosis of wall abscess caused by migrating gallstones could be made sonographically, whereas thin-section spiral CT failed to show the cause of the abscess: a non-calcified gallstone. J Biomol NMR, 1997 Feb, 9(2), 167 - 80 1H, 13C, and 15N NMR backbone assignments and chemical-shift-derived secondary structure of glutamine-binding protein of Escherichia coli; Yu J et al.; 1H, 13C, and 15N NMR assignments of the backbone atoms and beta-carbons have been made for liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with 226 amino acid residues and a molecular weight of 24935 Da . GlnBP is a periplasmic binding protein which plays an essential role in the active transport of L-glutamine through the cytoplasmic membrane . The assignments have been obtained from three-dimensional triple-resonance NMR experiments on a 13C, 15N uniformly labeled sample as well as specifically labeled samples . Results from the 3D triple-resonance experiments, HNCO, HN(CO)CA, HN(COCA)HA, HNCA, HN(CA)HA, HN(CA)CO, and CBCA(CO)NH, are the main sources used to make the resonance assignments . Other 3D experiments, such as HNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirm the resonance assignments and to extend connections where resonance peaks are missing in some of the experiments mentioned above . We have assigned more than 95% of the polypeptide backbone resonances of GlnBP . The result of the standard manual assignment is in agreement with that predicted by an automated probailistic method developed in our laboratory . A solution secondary structure of the GlnBP-Gln complex has been proposed based on chemical shift deviations from random coil values . Eight alpha-helices and 10 beta-strands are derived using the Chemical Shift Index method. J Biomol NMR, 1997 Feb, 9(2), 151 - 66 Automated probabilistic method for assigning backbone resonances of (13C,15N)-labeled proteins; Lukin JA et al.; We present a computer algorithm for the automated assignment of polypeptide backbone and 13C beta resonances of a protein of known primary sequence . Input to the algorithm consists of cross peaks from several 3D NMR experiments: HNCA, HN(CA)CO, HN(CA)HA, HNCACB, COCAH, HCA(CO)N, HNCO, HN(CO)CA, HN(COCA)HA, and CBCA(CO)NH . Data from these experiments performed on glutamine-binding protein are analyzed statistically using Bayes' theorem to yield objective probability scoring functions for matching chemical shifts . Such scoring is used in the first state of the algorithm to combine cross peaks from the first five experiments to form intraresidue segments of chemical shifts (Ni,HiN,Ci alpha, Ci beta, Ci'), while the latter five are combined into interresidue segments (Ci alpha,Ci beta,Ci',Ni + 1,Hi + 1N) . Given a tentative assignment of segments, the second stage of the procedure calculates probability scores based on the likelihood of matching the chemical shifts of each segment with (i) overlapping segments; and (ii) chemical shift distributions of the underlying amino acid type (and secondary structure, if known) . This joint probability is maximized by rearranging segments using a simulated annealing program, optimized for efficiency . The automated assignment program was tested using CBCANH and CBCA(CO)NH cross peaks of the two previously assigned proteins, calmodulin and CheA . The agreement between the results of our method and the published assignments was excellent . Our algorithm was also applied to the observed cross peaks of glutamine-binding protein of Escherichia coli, yielding an assignment in excellent agreement with that obtained by time-consuming, manual methods . The chemical shift assignment procedure described here should be most useful for NMR studies of large proteins, which are now feasible with the use of pulsed-field gradients and random partial deuteration of samples. J Biomol NMR, 1997 Feb, 9(2), 113 - 26 Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution; Hinds MG et al.; The chemical shift assignments and secondary structure of a murine-human chimera, MH35, of leukaemia inhibitory factor (LIF), a 180-residue protein of molecular mass 20 kDa, have been determined from multidimensional heteronuclear NMR spectra acquired on a uniformly 13C, 15N-labelled sample . Secondary structure elements were defined on the basis of chemical shifts, NH-C alpha H coupling constants; medium-range NOEs and the location of slowly exchanging amide protons . The protein contains four alpha-helices, the relative orientations of which were determined on the basis of long-range, interhelical NOEs . The four helices are arranged in an up-up-down-down orientation, as found in other four-helical bundle cytokines . The overall topology of MH35-LIF is similar to that of the X-ray crystallographic structure for murine LIF {Robinson et al . (1994) Cell, 77, 1101-1116} . Differences between the X-ray structure and the solution structure are evident in the N-terminal tail, where the solution structure has a trans-Pro17 compared with the cis-Pro17 found in the crystal structure and the small antiparallel beta-sheet encompassing residues in the N-terminus and CD loop in the crystal structure is less stable. Semin Liver Dis, 1997 Feb, 17(1), 71 - 8 Autoantibodies against "nuclear dots" in primary biliary cirrhosis; Szostecki C et al.; Autoantibodies against nuclear proteins are not always but rather frequently present in sera of patients with primary biliary cirrhosis (PBC) . The specificity and diagnostic value of these autoantibodies for PBC have only recently become clear through cloning of the cDNA of some of the corresponding autoantigens which allowed the establishment of immunological assays with recombinant autoantigens expressed in E . coli and eukaryotic cells . In this report we summarize primarily the knowledge on the structure and putative function of two nuclear autoantigens, the Sp100 and PML proteins, which are present in so-called nuclear dots (NDs) and against which autoantibodies are present in a subpopulation of PBC patients . Furthermore, the type of autoimmune response (epitope specificity and immunoglobulin class) against both the Sp100 and PML proteins and the specificity of the anti-Sp100 and anti-PML autoantibodies for PBC patients and patients with other autoimmune diseases is reviewed . Current knowledge clearly indicates that determination of anti-Sp100 and anti-PML autoantibodies substantially improves diagnosis of PBC as these autoantibodies are highly specific for this disease even when autoantibodies against mitochondrial antigens, a hallmark of most PBC patients, are not found . The type of autoimmune response against the Sp100 and PML proteins also provides some clues about possible mechanisms which lead to autoantigenicity of both proteins. Protein Eng, 1997 Feb, 10(2), 175 - 80 Maltodextrin-binding protein hybrids carrying epitopes from the preS2 region of hepatitis B virus: expression, antibody-binding and preliminary crystallographic studies; Vulliez-le Normand B et al.; Five hybrid constructions of maltodextrin-binding protein (MBP), each containing an inserted epitope(s) from the preS2 region of the envelope proteins of hepatitis B virus (HBV), have been expressed . The anti-preS2 monoclonal antibody S2.3 was shown to cross-react with the MBP hybrid constructions, demonstrating that the epitopes presented by these recombinant proteins mimic the antigenic behaviour of the native viral antigen . In addition, all five hybrid proteins have been crystallized . Preliminary structural solutions obtained by molecular replacement indicate that the native conformation of MBP is preserved in the hybrid constructions despite the significant length of the epitope insertions. J Biochem (Tokyo), 1997 Feb, 121(2), 370 - 5 Dimerization of granulocyte-colony stimulating factor receptor: the Ig plus CRH construct of granulocyte-colony stimulating factor receptor forms a 2:2 complex with a ligand; Horan TP et al.; We have previously shown that the extracellular domain of granulocyte-colony stimulating factor receptor (soluble G-CSFR), prepared from CHO cell conditioned media, dimerizes upon binding its ligand, G-CSF . The most stable ligand-receptor complex occurs at a 2:2 stoichiometry, unlike the growth hormone and erythropoietin systems . In the latter cases, each ligand uses two sites to bring two receptors together . In this study, we have generated a truncated G-CSF receptor, known to be sufficient for high affinity ligand binding, which consists of an Ig-like domain and a cytokine receptor homology module . With an affinity purified receptor, sedimentation equilibrium experiments clearly demonstrated that this truncated form of the receptor behaves very similarly to the entire extracellular domain . The sedimentation equilibrium data are consistent with the model that the truncated receptor has a weak tendency to self-associate into a dimer in the absence of a ligand, this receptor-receptor interaction is enhanced by ligand binding, and the most stable complex occurs at a 2:2 stoichiometry . These results are very different from those described by others for various murine G-CSF receptor constructs from either Escherichia coli or insect expression systems. J Biochem (Tokyo), 1997 Feb, 121(2), 331 - 7 GroE assists refolding of recombinant human pro-urokinase; Xu Z et al.; GroE, one of the molecular chaperones, facilitates correct protein folding both in vitro and in vivo . The refolding of recombinant human pro-urokinase, a protein with a high content of disulfide bonds, was used as a model system to illustrate the mechanism of action of GroE . Aggregation of this protein predominates during its in vitro refolding, as indicated by a strong, concentration-dependent increase in light scattering . The addition of GroE and Mg-ATP significantly increases the yield of the active protein . GroE specifically inhibits the aggregation reaction that competes with correct folding, as shown by a strong decrease in the intensity of light scattering . GroEL rapidly binds to unfolded or partially folded pro-urokinase molecules and thus protects them from the aggregation reaction . Interaction with GroES and ATP hydrolysis are required for the release of the polypeptide chain from GroEL and further acquisition of the completely folded, native conformation. J Biochem (Tokyo), 1997 Feb, 121(2), 270 - 7 The hydrophobic region of signal peptides is a determinant for SRP recognition and protein translocation across the ER membrane; Hatsuzawa K et al.; Newly recognized mammalian secretory proteins such as preprolactin are translocated across the endoplasmic reticulum (ER) in a signal recognition particle (SRP)-dependent manner . Recent studies revealed that there are two recognition steps for signal peptides during this translocation . The first step is recognition by SRP, which results in elongation arrest, and the second step is interaction between signal peptides and the translocation channel embedded in the ER membrane . To determine the roles of the hydrophobic region of signal peptides in the recognition by SRP and the membrane-embedded translocation machinery, we constructed chimeric proteins consisting of the mature region of preprolactin and signal peptides containing different numbers of leucine residues . The translocation of these chimeric proteins was completely dependent on SRP, and the efficiency increased as the number of leucine residues increased up to 10 and then decreased . Although the efficiency of elongation arrest also increased as the number of leucine residues increased up to 10, it only slightly decreased as the number increased up to 20 . Similar results were obtained when the hydrophobic region was replaced by alternate leucine and alanine residues, except that the most efficient translocation occurred when the number was 14 . Taken together, the present results suggests that the total hydrophobicity of the hydrophobic region of signal peptides is a determinant for recognition by both SRP and the membrane-embedded translocation machinery, although the specificities of the two signal recognition steps are slightly different from each other. Int J Parasitol, 1997 Feb, 27(2), 203 - 13 Molecular and biochemical studies on the hypoxanthine-guanine phosphoribosyltransferases of the pathogenic haemoflagellates; Ullman B et al.; All genera of protozoan parasites are auxotrophic for purines, and thus, purine acquisition from the host is a nutritional necessity for the survival and growth of these pathogens . Many of these parasites, including Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., access host purines by phosphoribosylating purine bases via purine phosphoribosyltransferase (PRT) enzymes . The trypanosomatid hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme has been implicated as a critical enzyme of purine salvage in members of the Trypanosomatidae family . Moreover, the HGPRT enzymes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp . can also initiate the metabolism of certain cytotoxic purine base analogs that have little effect on the mammalian host . This implies that either inhibitors or substrates of HGPRT might serve as efficacious and selective agents for the treatment of diseases for which trypanosomatids are the etiologic agent . The hgprt genes from Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani have all been cloned, sequenced and overexpressed in E . coli, and the recombinant proteins have all been purified to homogeneity and characterized with respect to kinetic parameters and physicochemical properties . This paper presents an overview of recent molecular and biochemical studies on trypanosomatid HGPRT proteins and future efforts to validate HGPRT as a rational target for the chemotherapeutic manipulation of African sleeping sickness, Chagas disease and leishmaniasis. Biol Chem, 1997 Feb, 378(2), 71 - 6 Activation of progelatinase A and progelatinase A/TIMP-2 complex by membrane type 2-matrix metalloproteinase; Kolkenbrock H et al.; C-terminal truncated membrane-type 2 matrix metalloproteinase (MT2-MMP1-269), comprising prodomain and catalytic domain, was expressed as a soluble protein in Escherichia coli . Unlike the corresponding form of MT1-MMP, which can be isolated as a 31 kDa protein, MT2-MMP1-269 proved to be comparatively instable, and already the freshly isolated preparation displayed several proteins in SDS-PAGE representing MT2-MMP1-269 (33 kDa) and four N-truncated forms with N-termini methionine32 (30 kDa), isoleucine37 (30 kDa), leucine84 (24 kDa), and leucine93 (22 kDa), the catalytic domain . After thawing of frozen preparations the 33 and the 30 kDa proforms were no longer detectable in SDS-PAGE, and only the 24 and 22 kDa forms remained . The catalytic domain of MT2-MMP activated progelatinase A as well as the progelatinase A/TMP-2 complex by cleaving the 72 kDa progelatinase A to yield 67 kDa gelatinase A, which is then transformed into 62 kDa gelatinase A . The 62 kDa form is about twice as active as the 67 kDa form towards the synthetic substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg . No significant difference in activity was found between free and complexed gelatinase A forms . the activation of the progelatinase A/TIMP-2 complex proceeds in two steps: At first MT2-MMP is inhibited by the progelatinase A/TIMP-2/MT2-MMP, complex, whereby a ternary complex, progelatinase A/TIMP-2/ MT-2MMP is generated . This ternary complex is then activated by excess MT2-MMP . Our results suggest a mechanism for spatially regulated extracellular gelatinase A activity mediated by activation with membrane-type MMPs; Free gelatinase A is released into the extracellular space, while gelatinase A/TIMP-2 bound to MT-MMP remains anchored on the cell surface. Rev Rhum Engl Ed, 1997 Feb, 64(2), 82 - 8 Cross-reactivity of anti-nDNA antibodies with nuclear envelope proteins . Isolation of a cDNA encoding the 70 kDa annular protein recognized by autoantibodies from patients with systemic lupus erythematosus; Herrera-Diosdado R et al.; OBJECTIVES: To determine whether annular rDNA is complexed with the nuclear envelope proteins . METHODS: From a batch of lupus sera with anti-nDNA, we selected a lupus serum containing annular anti-nDNA autoantibodies resistant to DNase digestion and used it to isolate several cDNA clones from a lambda gt11 HeLa cell library . RESULTS: The cloned fusion protein immunoadsorbed the annular anti-nDNA autoantibodies, and the immunoaffinity autoantibodies eluted from the recombinant filters produced an annular pattern around the nucleus in fluorescent assays on HEp-2 cells; by Western blot, they also recognized a 70 kDa protein from HEp-2 cell extracts . Annular-lambda gt11 lysogens generated in E . coli Y1089 produced a fusion protein that recognized annular anti-nDNA autoantibody-containing lupus sera by Western blot . The recombinant filters and annular fusion protein were also recognized by a prototype anti-lamin serum . To determine whether the annular recombinant protein bound DNA, an interaction assay was performed in vitro using DNA minicircles and DNA from HEp-2 cells; this assay resulted in a slowing of the electrophoretic mobility of the DNA . CONCLUSIONS: 1) The annular DNA in eukaryotic cells is complexed with nuclear envelope proteins . 2) Annular anti-nDNA autoantibodies from lupus patients cross-react with perinuclear proteins . 3) Perinuclear proteins recognized by anti-nDNA are lamins . 4) An interaction between DNA and the 70 kDa protein is inducible in vitro . Whether this interaction affects cell function is still unknown. Hybridoma, 1997 Feb, 16(1), 47 - 52 Production of a single-chain fragment of the murine anti-idiotypic antibody ACA125 as phage-displayed and soluble antibody by recombinant phage antibody technique; Schlebusch H et al.; The F(ab')2 fragment of the murine monoclonal anti-idiotypic antibody ACA125 mimicking the tumor-associated antigen CA125 is used as a vaccine for the induction of an anti-tumoral immunity in patients with ovarian carcinoma . We tried to generate a single-chain fragment (ScFv) composed of ACA125 heavy- and light-chain variable domains connected by a polypeptide linker as an alternative to the corresponding F(ab')2 fragment . Heavy- and light-chain genes of antibody-producing mouse hybridoma cell line were amplified separately and assembled into a ScFv gene with linker DNA by the polymerase chain reaction (PCR) . The ScFv gene was ligated into the phagemid vector pCANTAB5E, which allows the production of both phage-displayed and soluble ScFv . Transformed Escherichia coli TG1 cells were infected with M13K07 helper phage to yield recombinant phage, which display ScFv fragments as a g3p fusion protein on the surface of the filamentous phage M13 . Recombinant phages could be selected by binding to the idiotypic antibody OC125 after one round of panning and directly used to reinfect E . coli TG1 cells . The E . coli nonsuppressor strain HB2151 was infected with an antigen-positive phage clone, previously screened by enzyme-linked immunosorbent assay (ELISA), to express soluble ScFv fragments . Functional soluble ScFv binding to the idiotypic antibody OC125 F(ab')2 could be detected in the bacterial periplasm by Western blot and ELISA . The variable heavy- and light-chain genes of the ACA125 ScFv fragment were further sequenced and compared with known antibody sequences. Mol Biochem Parasitol, 1997 Feb, 84(2), 203 - 14 Molecular characterisation of an expressed sequence tag locus of Toxoplasma gondii encoding the micronemal protein MIC2; Wan KL et al.; The expressed sequence tag (EST) dataset of Toxoplasma gondii provides a wealth of information towards gene discovery . The complete cDNA and genomic sequence of EST tgc050 locus shows that it contains five copies of the conserved thrombospondin (TSP)-like motif present in a number of molecules with adhesive properties . A conserved region implicated with the adhesive characteristic of another group of proteins including several integrins, is also present in this molecule . The protein encoded by this sequence (rc50) is strongly recognised by monoclonal antibodies to MIC2 . Affinity purified anti-rc50 antisera specifically reacted with a single protein of identical molecular mass as MIC2 and exclusively labeled the micronemes of T . gondii by cryo-immunoelectron microscopy . These results demonstrate that c50 encodes for MIC2, a previously characterised microneme protein of T . gondii . The extensive sequence similarity across multiple protein domains provides evidence that the protein encoded by this locus is the homologue to the Etp100 microneme protein of Eimeria tenella. J Mol Med, 1997 Feb, 75(2), 145 - 52 Detection of antibodies against viral capsid proteins of human herpesvirus 8 in AIDS-associated Kaposi's sarcoma; Andre S et al.; Sequences of a new herpesvirus with homology to gammaherpesvirinae were recently identified in AIDS-associated Kaposi's sarcoma (KS) . Subsequently this novel virus, called KS-associated virus (KSHV) or human herpesvirus (HHV) 8 was detected in classical KS and AIDS-associated body cavity based lymphomas by polymerase chain reaction . In this report major and minor capsid proteins of HHV-8 were molecularly cloned and produced as recombinant proteins in Escherichia coli . Sera from 69 HIV-1 infected patients with KS, 30 HIV-1 infected patients without KS and 106 control individuals were tested by enzyme-linked immunosorbent assay for anti-HHV-8 capsid IgM and IgG antibodies . Sera from four patients were tested over periods ranging from 18 months to 6 years . IgG antibodies directed against HHV-8 capsid antigens were detected in patients with AIDS-associated KS and in some AIDS patients without KS . Seroconversion with IgM and IgG antibodies directed against HHV-8 capsid proteins occurred more than 1 year prior to diagnosis of KS . In a considerable portion of KS patients no IgM or IgG antibodies against HHV-8 capsid proteins were detected . In these patients there was an inverse relationship between antibodies against HHV-8orf26 and the CD4/CD8 ratio, suggesting that the inconsistency of anti-HHV-8orf26 antibodies is due at least partly to an impaired immune response . No reactivity against HHV-8 capsid antigens was detected in the vast majority of sera from HIV-negative control individuals . Our findings indicate that a specific humoral immune response against capsid proteins is raised in HHV-8 infected individuals, and that anti-capsid antibodies can be used to diagnose HHV-8 infection . The correlation between occurrence of anti-HHV-8 antibodies and KS supports the hypothesis of a causative role of HHV-8. Curr Biol, 1997 Feb 1, 7(2), R97 - 9 Identification of a prime suspect; Buc H; Recent findings help to define the multiple functions of the sigma subunit of bacterial RNA polymerase, from promoter recognition to the release of pausing during initial RNA elongation; these functions can now be confronted with a crystal structure of an essential domain of the sigma subunit. Curr Biol, 1997 Feb 1, 7(2), R100 - 3 The Tom and Tim machine; Pfanner N et al.; Translocation of precursor proteins into mitochondria depends on loosely assembled protein complexes in the outer and inner membranes . Recent studies indicate that dynamic interactions of subcomplexes and cooperation with molecular chaperones drive key steps in protein import. Toxicon, 1997 Feb, 35(2), 185 - 93 Leukocyte response induced by Bothrops jararaca crude venom: in vivo and in vitro studies; Farsky SH et al.; The effect of Bothrops jararaca crude venom (BjV) on the cellular component of inflammatory responses was investigated in vivo and in vitro . In vivo leukocyte accumulation and release of eicosanoids (thromboxane A2, TXA2, and leukotriene B4, LTB4) at the site of injection of the venom were assessed using the air pouch method in rats . Administration of BjV caused a significant cell accumulation, maximal values being obtained after 6-8 hr . Neutrophils were the predominant cell type in the inflammatory exudate . High concentrations of LTB4 were detected 1-4 hr after the injection of the venom . TXA2 concentrations were significantly increased only at the early stages of the response to the venom . In vitro chemotaxis assays were performed and showed that the venom per se was not able to induce oriented neutrophil migration because varying concentrations of the venom dissolved in Hanks' balanced salt solution (HBSS) evoked a response equivalent to that of HBSS alone . Furthermore, the venom did not affect cellular intrinsic mechanisms involved with neutrophil locomotion because previous incubation of the cells with BjV produced no effect . However, high concentrations of the venom were able to generate serum chemotactic factor(s) . Incubation of serum with the venom evoked a neutrophil migration similar to that observed with serum activated by lipopolysaccharide from Escherichia coli . Participation of chemotactic factors derived from the complement system is suggested by data showing loss of this activity when serum was heated (56 degrees C) before the addition of BjV . The present results suggest that leukocyte accumulation in the locality of a lesion induced by BjV is dependent on secretion or activation of endogenous components responsible for several steps in leukocyte recruitment instead of a direct effect of the venom on leukocytes. Cancer Genet Cytogenet, 1997 Feb, 93(2), 160 - 6 Cytogenetic studies in seventy-six cases of B-chronic lymphoproliferative disorders; Sole F et al.; The results of cytogenetic studies are reported in 76 patients with B-chronic lymphoproliferative disorders (B-CLPD): 60 patients with chronic lymphocytic leukemia (CLL), six with follicular lymphoma in leukemic phase (FLLP), five with splenic B-cell lymphoma with villous lymphocytes (SLVL), two with chronic prolymphocytic leukemia (CPL), two with hairy cell leukemia (HCL), and one with plasma cell leukemia (PCL) . PHA (phytohemagglutinin), PWM (pokeweed mitogen), LPS (lipopolysaccharide from Escherichia Coli), TPA (phorbol 12-myristate acetate), IL6 (interleukin 6), and DxS (dextran sulfate) were used as mitogens . Mitoses were obtained in 75 cases . Clonal aberrations could be demonstrated in 34 cases (44%) . In CLL, classical type, chromosomes 6, 11, and 13 were more frequently involved, whereas trisomy 12 was frequently found in CLL mixed-cell type, in FLLP, and CPL . In SLVL the deletion del(7)(q32) is noteworthy and miscellaneous chromosome abnormalities in the remaining patients were observed . Regarding the efficiency of mitogens, PHA turned to be the most effective in obtaining metaphases and in detecting clonal chromosomal aberrations. Kansenshogaku Zasshi, 1997 Feb, 71(2), 162 - 8 {Isolation of verotoxin-producing Escherichia coli from cattle and characterization of the isolated strains}; Takesue K et al.; Attempts to isolate Verotoxin-producing Escherichia coli (VTEC) from 53 dead baby cattle were made during a period from January 1989 to July 1991 . From 6 cattle of 5 farms, VTECs were isolated . Further examination of cattle from 2 farms with dead baby cattle and from 1 farm with an outbreak of diarrhea among baby cattle, from 8 of 26 cattle with diarrhea and from 9 of 58 healthy cattle, VTECs were isolated . Several serotypes which caused food poisoning in Japan as well as in the US and Europe were included in the isolated strains . VTECs were also isolated from several cattle imported from the US suggesting that these VTECs were from outside the country together with the imported cattle. Kansenshogaku Zasshi, 1997 Feb, 71(2), 153 - 61 {Efficacy of nasal influenza vaccine combined with Escherichia coli heat-labile enterotoxin B subunit containing a trace amount of the holotoxin in healthy volunteers}; Hashigucci K et al.; We conducted a field trial to evaluate the efficacy of nasal influenza vaccine combined with Escherichia coli heat-labile enterotoxin B subunit (LTB) containing a trace amount of the holotoxin (LT) in preventing or attenuating influenza among volunteers during the winter season of 1994-1995 . A trivalent inactivated influenza vaccine, composed of A/Yamagata/32/89 (H1N1), A/Kitakyusyu/159/93 (H2N2) and B/Mie/1/93 influenza virus strains, was administered intranasally together with recombinant LTB containing 1% recombinant LT (LTB*) . Vaccination was done twice 4 weeks apart . Salivary secretory IgA and serum HI antibodies were measured before and 8 weeks after the primary vaccination . Thirty-two volunteers were enrolled in this study; 18 volunteers (mean age 37.7 +/- 11.3) were given LTB*-combined vaccine and 14 volunteers (mean age 44.1 +/- 11.3) given placebo . Outbreaks of H3N2 subtype and B type virus were observed during this study period . Six (42.9%) of the 14 volunteers in the placebo group and 3 (16.7%) of the 18 receiving the LTB*-combined vaccine contracted influenza . There was no statistically significant difference between the two groups, because the number of subjects was small . Higher percentage of positive IgA and HI antibody responses among vaccines given vaccine with LTB* were observed as compared with those in the placebo group . Positive IgA antibody response to all vaccine strains were observed in 46.7% (7/15) of the vaccine group . On the other hand, none of the placebo group showed positive IgA antibody response to all vaccine strains . These results suggest that nasal influenza vaccine with LTB* appears to be effective in preventing influenza. Plant J, 1997 Feb, 11(2), 251 - 62 Cysteine synthesis in plants: protein-protein interactions of serine acetyltransferase from Arabidopsis thaliana; Bogdanova N et al.; The biosynthesis of cysteine represents the final step of sulfate assimilation in bacteria and plants . It is catalyzed by the sequential action of serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which form a cysteine synthase (CS) complex in vitro . SAT and OAS-TL from Arabidopsis thaliana have previously been cloned, and now the first evidence is presented for the CS complex and SAT self-interaction in vivo employing the yeast two-hybrid system . Application of this method proved to be an efficient tool for the analysis of protein-protein interactions within a plant metabolic protein complex . Mapping of SAT domain structure revealed two new, independent domains with specific functions in protein-protein interaction . Analysis using truncated proteins proved the C-terminus of SAT to be sufficient for association with OAS-TL and to correlate with the putative transferase activity domain . SAT/SAT interaction was localized in the central region of the protein and occurred also between SAT isoforms . Both protein interaction domains coincided with distinct alpha-helical and beta-sheet clusters and together correlated with the minimal protein structure required for SAT catalysis as shown by functional complementation of an Escherichia coli mutant . The homo- and hetero-oligomerization properties are discussed with respect to the assumed function of the CS complex in metabolic channeling and activation of SAT by interaction with OAS-TL. Plant J, 1997 Feb, 11(2), 227 - 36 Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato; Moehs CP et al.; A cDNA encoding solanidine glucosyltransferase (SGT) was isolated from potato . The cDNA was selected from a yeast expression library using a positive selection based on the higher toxicity of steroidal alkaloid aglycons relative to their associated glycosylated forms . The cDNA contained an open reading frame encoding a 56 kDa polypeptide with regions of similarity to previously characterized UDP-glucosyltransferases . The enzyme activity and reaction products of recombinant SGT in yeast were consistent with those observed for the endogenous enzyme from potato . SGT mRNA and protein accumulated in tubers in response to wounding . The time course for SGT mRNA accumulation paralleled that of 3-hydroxy-3-methylglutaryl-coenzymeA isoform 1 (hmg1) mRNA . Steady-state SGT mRNA levels also increased transiently upon wounding of leaves. Plant J, 1997 Feb, 11(2), 213 - 26 The ubiquitin-activating enzyme (E1) gene family in Arabidopsis thaliana; Hatfield PM et al.; Conjugation of multiple ubiquitins serves as a committed step in the degradation of a variety of intracellular eukaryotic proteins by the 26S proteasome . Conjugates are formed via a three-enzyme cascade; the initial step requires ubiquitin-activating enzyme (E1), which couples ubiquitin activation to ATP hydrolysis . Previously, we showed that many higher plants contain multiple E1 proteins and described several E1 genes from wheat . To facilitate understanding of the roles of the different plant E1s, we characterized the E1 gene and protein family from Arabidopsis thaliana . Arabidopsis E1s are encoded by two genes (AtUBA1 and AtUBA2) that synthesize approximately 123-kDa proteins with 81% amino acid sequence identity to each other and 44-75% sequence identity with confirmed E1s from other organisms . Like other E1 proteins, AtUBA1 and 2 contain a cysteine residue in the putative active site for forming the ubiquitin thiol-ester intermediate . Enzymatic analysis of the corresponding proteins expressed in Escherichia coli demonstrated that both proteins activate ubiquitin in an ATP-dependent reaction and transfer the activated ubiquitin to a variety of Arabidopsis E2s with near equal specificity . Expression studies by quantitative RT-PCR and histochemistry with transgenic plants containing AtUBA promoter-beta-glucuronidase-coding region fusions showed that the AtUBA1 and 2 genes are co-expressed in most, if not all, Arabidopsis tissues and cells . Collectively, the data indicate that E1 proteins, and presumably the rest of the ubiquitin pathway, are present throughout Arabidopsis . They also show that the AtUBA1 and 2 genes are not differentially expressed nor do they encode E1s with dramatically distinct enzymatic properties. Plant J, 1997 Feb, 11(2), 191 - 201 Cloning and expression in Escherichia coli of the obtusifoliol 14 alpha-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14 alpha-demethylases (CYP51) from fungi and mammals; Bak S et al.; Obtusifoliol 14 alpha-demethylase from Sorghum bicolor (L.) Moench has been cloned using a gene-specific probe generated using PCR primers designed from an internal 14 amino acid sequence . The sequence identifies sorghum obtusifoliol 14 alpha-demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14 alpha-demethylases from fungi and mammals . The presence of highly conserved regions in the amino acid sequences, analogous substrates and the same metabolic role demonstrate that the sterol 14 alpha-demethylases are orthologous enzymes . The sterol 14 alpha-demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14 alpha-methyl group in all known functional sterols . A functional sorghum obtusifoliol 14 alpha-demethylase was expressed at high levels in Escherichia coli and purified using an efficient method based on temperature-induced Triton X-114 phase partitioning . The recombinant purified enzyme produced a type I spectrum with obtusifoliol as substrate . Reconstitution of purified recombinant enzyme with sorghum NADPH-cytochrome P450 reductase in dilaurylphosphatidylcholine micelles confirms that obtusifoliol 14 alpha-demethylase catalyses the 14 alpha-demethylation of obtusifoliol to 4 alpha-methyl-5 alpha-ergosta-8, 14,24(28)-trien-3 beta-ol as evidenced by GC-MS . The isolation of a cDNA clone encoding the plant sterol 14 alpha-demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14 alpha-demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14 alpha-demethylases. Cell Mol Biol (Noisy-le-grand), 1997 Feb, 43(1), 67 - 73 Characteristics of human protoporphyrinogen oxidase in controls and variegate porphyrias; Dailey HA et al.; Protoporphyrinogen oxidase (E.C.1.3.3.4) (PPO) catalyzes the penultimate step in the heme biosynthetic pathway . Deficiency in activity of this enzyme results in the human genetic disease variegate porphyria . Herein we detail the cloning, expression, purification and characterization of the normal and variegate porphyria forms of human PPO . The cDNA sequence for human ppo is approximately 1.8 kb in length and codes for a protein of 477 amino acids . This protein, which does not contain a typical cleavable mitochondrial targeting sequence, is approximately 51 kDa and contains a putative dinucleotide binding motif near the amino terminus . The active enzyme is a homodimer and contains an FAD . Attachment of a six his amino terminal tag allows for the rapid and efficient purification of approximately 10 mg of enzyme from one liter of E . coli culture . Three variegate porphyria mutant PPO enzymes were expressed and characterized . These mutations, R59W, R168C and A433P, result in decreased enzyme activity by causing a decrease in kcat without a significant change in Km for the substrate protoporphyrinogen IX . Purified R59W lacks the FAD cofactor which may be explained by the fact that this mutation resides within the dinucleotide binding motif of PPO. Cell Mol Biol (Noisy-le-grand), 1997 Feb, 43(1), 59 - 66 Molecular defects of the coproporphyrinogen oxidase gene in hereditary coproporphyria; Sassa S et al.; Hereditary coproporphyria (HCP) is an acute hepatic porphyria, and is an autosomal dominant disorder but with a variable degree of clinical expression . Molecular cloning, sequencing and expression of the defective gene for coproporphyrinogen oxidase (CPO) in a patient with HCP were carried out . Enzyme assays revealed that CPO activity in EBV-transformed lymphoblastoid cells from the proband and one of her sisters was approximately 50% of normal . Nucleotide sequence analysis of CPO cDNAs isolated from the proband's cells demonstrated 3 base substitutions which accompanied 3 different amino acid substitutions . An A514-->C transition causing an Asn172-->His substitution occurred in one allele, while two other transitions, G265-->A and G580-->A, caused Gly89-->Ser and Val194-->Ile substitutions, respectively, in the other allele . The A514-->C and the G580-->A transitions were shown to be genetic polymorphisms . Transfection of CPO cDNA into E . coli demonstrated that cDNA with the G265-->A transition produced a protein with less than 5% of normal enzyme activity . These findings indicate that the G265-->A transition, involving the highly conserved glycine residue at the 89th position, is responsible for the CPO defect in the patient and accounts for the partial deficiency of CPO activity in this pedigree . This mutation is different from three other mutations reported in patients with HCP . Molecular defects in the porphyrias including HCP are highly heterogeneous. J Endocrinol, 1997 Feb, 152(2), 317 - 27 Large-scale preparation and characterization of recombinant ovine placental lactogen; Sakal E et al.; To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep placental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5' and 3' primers containing, respectively, NcoI and PstI sites . The oPL cDNA fragment amplified between these two primers extended from A(-1) to the natural stop codon . The PCR product was gel-purified and subcloned into a Puc vector and the insert was sequenced on both strands, revealing several differences relative to the published sequence: S19N, S69N, D129E and R165Q . We assume that these differences can be accounted for by the high level of individual polymorphism, which has been described in detail for PLs of different species . The insert was subcloned into NcoI/ PstI-digested pTrc99A procaryotic expression plasmid and protein expression was induced by isopropyl-1-thio-beta-D-galactopyranoside . Because of low expression, oPL's cDNA was further subcloned into pET8 procaryotic expression plasmid . Its expression in E . coli strain BL21 transformed with this vector yielded 30-40 mg/l . The expressed protein, found in the inclusion bodies, was refolded into a monomer and purified on a Q-Sepharose column to homogeneity . Structural analysis using circular dichroism revealed a spectrum similar to that of human GH (hGH) thereby indicating proper refolding . Gel filtration and binding experiments, including real-time kinetic measurements using the surface plasmon resonance method revealed that oPL forms transient homodimeric complexes with extracellular domains of prolactin receptors from rabbit, rat and bovine and with hGH receptor . The purified oPL was biologically active in an Nb2-11C cell proliferation bioassay, in its ability to stimulate beta-casein synthesis in explants of ovine and rabbit mammary gland and fat synthesis in explants of bovine mammary gland, and in a proliferation assay using FDC-P1 cells transfected with rabbit or hGH receptors. Cytokine, 1997 Feb, 9(2), 119 - 25 Inhibition of tumour necrosis factor production in endotoxin-stimulated human mononuclear leukocytes by the prostacyclin analogue iloprost: cellular mechanisms; Jorres A et al.; The prostacyclin analogue iloprost has been shown to inhibit effectively TNF-alpha production in human peripheral blood mononuclear leukocytes (PBMC) stimulated with bacterial lipopolysaccharide (LPS) . The current paper set out to analyse further the possible mechanisms involved in the regulation of TNF-alpha synthesis by iloprost . Healthy human PBMC were challenged with Escherichia coli LPS and assessed for TNF-alpha gene transcription, mRNA stability and protein secretion . Iloprost reduced both steady-state TNF-alpha mRNA expression and protein release as assessed by Northern blot analysis, polymerase chain reaction and enzyme immunoassay . This effect was related both to a reduction of TNF-alpha transcriptional activity (as evaluated by nuclear run-on transcription analysis) and a decrease in TNF-alpha mRNA stability (as assessed by serial Northern blot analysis of TNF-alpha mRNA content in PBMC blocked with actinomycin D) . When collectively assessed, these data demonstrate that iloprost regulates TNF-alpha synthesis at both transcriptional and post-transcriptional level. Cytokine, 1997 Feb, 9(2), 112 - 8 A monoclonal antibody based elisa for quantitation of human leukaemia inhibitory factor; Taupin JL et al.; The authors report on the development of a new sandwich enzyme-linked immunoabsorbent assay (ELISA) for the quantitation of the human cytokine leukaemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) with high accuracy and sensitivity (23 pg/ml), in less than 5 h and in various biological fluids . The antibodies used in this assay were raised against recombinant glycosylated LIF expressed in vivo following inoculation of recombinant vaccinia viruses, and screened with the biologically active cytokine in a flow cytometry assay using cells expressing a membrane-bound form of LIF . Furthermore, this home-made assay was compared with two commercially available ELISA kits . The results led to the conclusion that these three assays are far from being equivalent between each other, in terms of sensitivity towards non-glycosylated vs glycosylated LIF . Two major parameters must be incriminated: the glycosylation status of the LIF molecule used as the calibrator, and the binding characteristics of the monoclonal antibodies used to set up these assays toward LIF derived from Escherichia coli or from eukaryotic cells . This points out the importance of these parameters for the design of ELISAs meant for the quantitation of glycosylated cytokines in biological fluids. Arch Environ Contam Toxicol, 1997 Feb, 32(2), 146 - 53 Use of stress-inducible transgenic nematodes as biomarkers of heavy metal pollution in water samples from an english river system; Mutwakil MH et al.; Transgenic strains of the nematode Caenorhabditiselegans, which carry stress-inducible lacZ reporter genes, aremeasurably stressed by exposure to heavy metals in aqueous solution . Thisstress response can be quantified, using enzymatic assays for the reportergene-product (Escherichia coli beta-galactosidase), or estimatedapproximately by in situ staining for beta-galactosidase in exposedworms . Stress responses to heavy metals have been demonstrated both inlaboratory tests using Cd2+ or Hg2+, and also in watersamples taken from a metal-polluted river system in southwest England . TheRiver Carnon flows through an area with an ancient mining history,principally for Sn, but also for Cu and other metals; As, Cd, Al, Mn, and Zn,as well as large amounts of Fe, are all present in these ore bodies . Foursites in the Carnon river basin were compared with respect to theirmacroinvertebrate diversity, physical and chemical characteristics (includingthe concentrations of As, Cd, Al, Cu, Mn, Zn, and Fe) . Transgenic worms wereexposed to water samples from these four sites, and also to a 0.33%(v/v) dilution of metal-laden minewater from the principal local mine (WhealJane) . Transgene expression was induced in all five cases, though markedlyless so for the least polluted of the sites (which also supported a richermacroinvertebrate fauna) . Two different transgenic strains were tested inthis study; strain PC72 (using a homologous hsp16 promoter) isslightly more sensitive to most metal-containing water samples than strainCB4027 (using a heterologous Drosophila hsp70 promoter) . Bothtransgenic strains and two different assay methods gave essentially similarresults . These findings demonstrate that transgenic nematodes could provide arapid and simple assessment of aquatic pollution, in that the transgeneresponse is inducible by mixtures of dissolved metals at concentrationsactually encountered in metal-polluted watercourses. J Bioenerg Biomembr, 1997 Feb, 29(1), 55 - 9 Expression of bacteriorhodopsin in Sf9 and COS-1 cells; Heymann J et al.; We report studies on the expression of the archaebacterial membrane protein bacteriorhodopsin in Sf9 insect cells and in COS-1 mammalian cells . In both cell systems, the apoprotein bacterio-opsin was expressed at levels of approximately 1 microgram/10(6) cells . Immunofluorescence studies showed that the expressed protein was accumulated in the endoplasmic reticulum . However, upon addition of all-trans retinal to membranes isolated from either Sf9 or COS-1 cells expressing bacterio-opsin, the characteristic bacteriorhodopsin chromophore (lambda max at approximately 560 nm) was rapidly generated . This is in contrast to bacterio-opsin expressed in E . coli, which cannot be functionally reconstituted with retinal unless it is first denatured, and then renatured in vitro . These studies demonstrate that the bacterio-opsin expressed is correctly folded and show that localization of a heterologously expressed membrane protein in the endoplasmic reticulum does not necessarily imply that it is misfolded. Photochem Photobiol, 1997 Feb, 65(2), 323 - 9 Ultraviolet irradiation produces novel endonuclease III-sensitive cytosine photoproducts at dipyrimidine sites; Jen J et al.; Ultraviolet light irradiation of DNA in vitro and in vivo induces cyclobutane dimers, (6-4) pyrimidine-pyrimidone photoproducts and a variety of minor products . Using a defined DNA fragment, we have identified two classes of sites that can be cleaved by Escherichia coli endonuclease III: single cytosines whose heat lability corresponds to that of cytosine hydrates and more heat-stable dipyrimidines containing cytosine . The dipyrimidine products are induced at sites suggestive of (6-4) photoproducts but are not recognized as (6-4) photoproducts by radioimmunoassay . Use of oligonucleotides containing a single cyclobutane thymine dimer, a (6-4) photoproduct or the Dewar photoisomer of the (6-4) photoproduct also indicated that these products are not substrates for endonuclease III . We have therefore identified a minor UV photoproduct that has the same sequence specificity as the two major dipyrimidine photoproducts; it may be a minor isomer, a unique derivative or an oxidative lesion confined to dipyrimidine sites . Its biological significance is not yet known but may be masked by the preponderance of major products at the same sites . Its occurrence at the particular site in dipyrimidine sequences involved in the mutagenic action of UV photoproducts suggests that it may play a role in generating C to T transitions that are common UV-induced mutations. Vaccine, 1997 Feb, 15(2), 225 - 9 Suppression of delayed-type hypersensitivity and IgE antibody responses to ovalbumin by intranasal administration of Escherichia coli heat-labile enterotoxin B subunit-conjugated ovalbumin; Tamura S et al.; Oral administration of a small amount of antigen conjugated cholera toxin B subunit is known to induce tolerance to the antigen . In the present experiments, whether nasal administration of allergen conjugated to Escherichia coli heat-labile toxin B subunit (LTB) induced tolerance was examined in BALB/c mice . A single administration of a small amount of LTB-coupled ovalbumin (OVA) suppressed the induction of delayed-type hypersensitivity and IgE antibody responses to OVA which was administered parenterally after nasal administration of LTB-coupled OVA . The antigen-specific suppression was abrogated by the addition of the holotoxin to LTB-coupled OVA . The suppression, induced by nasal administration with a small amount of allergen conjugated to a mucosa-binding molecule, may be applicable for preventing the development of allergy. Vaccine, 1997 Feb, 15(2), 111 - 20 An Escherichia coli CS31A fibrillum chimera capable of inducing memory antibodies in outbred mice following booster immunization with the entero-pathogenic coronavirus transmissible gastroenteritis virus; Der Vartanian M et al.; CS31A fibrillae are thin, flexible, heteropolymeric proteinaceous appendages exposed as a capsule-like material around the cell surface of certain Escherichia coli strains . Two antigenic peptides of the S spike glycoprotein (TGEV-S) amino acids (aa) 363-371 and 521-531 of the transmissible gastroenteritis virus (TGEV) were tandemly introduced in the loop-structured, variable region aa 202-218 of the major ClpG subunit protein composing the bulk of CS31A . The resulting hybrid fibrillae with a 25 aa heterologous peptide were produced at the cell surface . Using a monoclonal antibody (Mab) specific for the TGEV epitopes, purified hybrid fibrillae were analysed in Western blotting under native conditions, which showed that the two viral epitopes were recognized immunologically as an integral part of the hybrid fibrillae, and therefore that they were antigenically active . The immunogenicity of the fusion construct was evaluated with live recombinant bacteria, purified hybrid ClpG monomers, and purified chimeric CS31A polymers . Whatever the form of hybrid used as antigen, intraperitoneally immunized outbred mice elicited serum anti-TGEV peptides antibodies (Abs) with significant titres and capable of recognizing native TGEV particles, indicating that the epitopes are exposed in an immunogenic conformation in all cases . However, virus neutralization titres were only obtained after immunization with either purified polymers or monomers . Furthermore, 4 months after an ultimate immunization with 20 micrograms of hybrid fibrillae mice developed a strong anamnestic Ab response against the two TGEV peptides following booster inoculation with virions . We conclude that CS31A fibrillae carrying a combination of TGEV epitopes as insert can induce an immunological memory in outbred animals infected with TGEV, and therefore that hybrid CS31A fibrillae may prove efficient as components of a subunit vaccine. Planta Med, 1997 Feb, 63(1), 15 - 7 Cytotoxicity and immunosuppressive activity of withanolides from Discopodium penninervium; Habtemariam S; The cytotoxicity and immunosuppressive activities of jaborosalactone-L and three 16 alpha-oxygenated withanolides from the leaves of Discopodium penninervium were studied . Jaborosalactone-L showed cytotoxicity only to the murine macrophage cell line, RAW 264.7, but the 16 alpha-oxygenated withanolides exhibited cytotoxicity to both human (COR-L23 and ECV 304) and murine (L929 and RAW 264.7) carcinoma cell lines with IC50 values ranging from 1.2-150 microM . The non-toxic concentrations of these withanolides were also found to reduce the proliferative response of both inactive and E . coli lipopolysaccharide and concanavalin A activated rat spleen cells in vitro. J Dent Res, 1997 Feb, 76(2), 641 - 7 Cloning, characterization, and heterologous expression of exon-4-containing amelogenin mRNAs; Hu CC et al.; The formation of dental enamel is dependent upon amelogenins, a family of proteins constituting most of the developing enamel matrix . Depending upon the species, these enamel proteins are expressed from either one or two copies of the amelogenin gene . Each gene directs the synthesis of a variety of amelogenin isoforms through alternative splicing of their pre-mRNA transcript(s) . Before the role of amelogenins in dental enamel formation can be better understood, one must know the isoforms that are secreted and their biochemical properties . Previously, we cloned and characterized 7 mouse amelogenin RNA messages generated by alternative splicing . The largest amelogenin cDNA encoded a 194-residue amelogenin isoform which was the only clone to contain the 42-nucleotide exon 4 segment . Anti-peptide antibodies raised against the derived translation of this exon revealed an unexpectedly diverse assortment of murine amelogenins, suggesting that additional splicing variants could contain the exon 4 coding region . Using exon-4-specific oligonucleotide primers, we have amplified, cloned, and characterized three different amelogenin RNA messages . These messages encode amelogenin polypeptides (exclusive of signal peptides) 194, 170, and 73 amino acids in length . The isotope-averaged molecular weights for the deduced, single-phosphorylated, proteins are 21,897.1, 19,113.9, and 8176.5 Daltons, respectively . Splice-site selection for the generation of these mRNAs was identical to that of the previously characterized messages for the M180, M156, and M59 except for the inclusion of exon 4 . The exon-4-containing amelogenin isoforms were heterologously expressed in E . coli by means of the pET11 expression system (Novagen, Madison, WI). Vet Res Commun, 1997 Feb, 21(2), 101 - 15 Leukocyte and cytokine accumulation in the ovine teat and udder during endotoxin-induced inflammation; Waller KP et al.; The accumulation of leukocytes, ovine serum albumin and the cytokines interleukin-1 beta(IL-1 beta), tumour necrosis factor-alpha(TNF-alpha), interleukin-8 (IL-8), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma(IFN-gamma) was studied during endotoxin-induced inflammation in lactating and dry ovine udders, and in the teat cisterns of dry ewes after surgical closure of the passage between the teat and udder cisterns . Samples were taken before infusion and hourly up to 10 h after infusion of 0.1, 1 or 10 micrograms of endotoxin, or infusion of pyrogen-free saline (PFS) as a control . Rectal temperatures were measured . A significant dose- and time-dependent accumulation of leukocytes, mainly neutrophils, was observed in the lactating udders and in the teat cisterns . In the dry udders, the leukocyte accumulation was significant for time but not for dose . Peak numbers of cells were reached at 3-4 h in the dry udders and in the teat cisterns, but not until 10 h after infusion in the lactating udders . The changes in the ovine serum albumin concentrations mostly paralleled changes in leukocyte numbers . A role was indicated for TNF-alpha, IL-8 and GM-CSF, but not for IL-1 beta and IFN-gamma, during endotoxin-induced inflammation in the ovine udder . Release of TNF-alpha, IL-8 and GM-CSF was most prominent in lactating udders, peaking at 2 or 3 h after infusion, but was also detected in dry udders and teat cisterns . Detectable levels of IL-1 beta and IFN-gamma were occasionally found in all three groups. Proteins, 1997 Feb, 27(2), 322 - 4 Purification, crystallization, and preliminary x-ray studies of a bifunctional 5,10-methenyl/methylene-tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli; Cheung E et al.; A bifunctional enzyme that catalyzes the conversion of formyltetrahydrofolate to methylene-tetrahydrofolate (5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylene tetrahydrofolate dehydrogenase), has been subcloned from a cDNA library, purified to homogeneity, and crystallized . The crystals belong to space group I222, with unit cell dimensions of a = 64.5 A, b = 84.9 A, c = 146.1 A . The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme monomer, and a specific volume of the unit cell of 3.2 A3/Da . The crystals diffract to at least 2.8 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector . A 2.56 A resolution native data set has been collected at beamline X12-C at the NSLS. Proteins, 1997 Feb, 27(2), 204 - 9 Rab7: NMR and kinetics analysis of intact and C-terminal truncated constructs; Neu M et al.; Rab proteins are a family of approximately 25kD ras-related GTPases which are associated with distinct intracellular membranes where they control vesicle traffic between intracellular compartments . The late-endosomal rab protein rab7(1-207), (lacking only the C-terminal lipids of the native molecule) and three C-terminal truncated constructs rab7(1-202), rab7(1-197) and rab7(1-182) were purified using an E . coli expression system . The C-terminal tail region of rab proteins is of special interest because it is thought to target rab proteins to particular intracellular membranes . A comparison of TOCSY-NMR spectra from intact rab7(1-207) and the tail-less construct rab7(1-182) suggested that much of the C-terminal tail is flexible in solution . The GTP hydrolysis, and GDP association and dissociation rates for all the truncated and intact constructs were similar, showing that the tail region of rab7(1-207) has little influence on the hydrolysis and exchange rates of the nucleotide. Bioorg Med Chem, 1997 Feb, 5(2), 323 - 34 New aromatic inhibitors of EPSP synthase incorporating hydroxymalonates as novel 3-phosphate replacements; Shah A et al.; A new, aromatic analogue of the EPSP synthase enzyme reaction intermediate 1 has been identified, which contains a 3-hydroxymalonate moiety in place of the usual 3-phosphate group . This simplified inhibitor was readily prepared in five steps from ethyl 3,4-dihydroxybenzoate . The resulting tetrahedral intermediate mimic 9 is an effective, competitive inhibitor versus S3P with an apparent Ki of 0.57 +/- 0.06 microM . This result demonstrates that 3-hydroxymalonates exhibit potencies comparable to aromatic inhibitors containing the previously identified 3-malonate ether replacements and can thus function as suitable 3-phosphate mimics in this system . These new compounds provide another example in which a simple benzene ring can be used effectively in place of the more complex shikimate ring in the design of EPSP synthase inhibitors . Furthermore, the greater potency of 9 versus the glycolate derivative 10 and the 5-deoxy-analog 11, again confirms the requirement for multiple anionic charges at the dihydroxybenzoate 5-position in order to attain effective inhibition of this enzyme. J Med Microbiol, 1997 Feb, 46(2), 139 - 44 TH1 pattern of cytokine secretion by splenic cells from pyelonephritic mice after in-vitro stimulation with hsp-65 of Escherichia coli; Chopra U et al.; Splenic lymphocytes and peritoneal macrophages from BALB/c mice with Escherichia coli pyelonephritis were obtained at various intervals after infection . These cells were stimulated in vitro with different antigens and cytokine release was assayed in the supernate of the cultured cells . It was observed that both specific antigens such as outer-membrane proteins (OMPs), porins and heat-shock protein-65 (hsp-65), as well as non-specific mitogens such as phytohaemagglutinin (PHA), were able to induce cytokine production by splenic cells from infected mice . Of all these antigens, hsp-65 was found to be the best inducer of cytokine release . In the acute stage of pyelonephritis, the release of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) was found to increase with time; both reached their peak values on the seventh day after infection . The TH1 pattern of cytokine secretion by splenic cells was observed, i.e., IL-2 and IFN-gamma, whereas there was complete absence of IL-4 secretion . In the chronic stage of pyelonephritis, i.e., 150 days after infection, a decrease in the level of IL-2 and IFN-gamma was observed . Peritoneal macrophages released IL-1 on stimulation with hsp-65, which increased with the progression of disease . The possible implications of this study for the disease process are discussed. Biosci Biotechnol Biochem, 1997 Feb, 61(2), 324 - 31 Gypsophilin, a new type 1 ribosome-inactivating protein from Gypsophila elegans: purification, enzymatic characterization, and subcellular localization; Yoshinari S et al.; Gypsophila elegans contains a new type 1 ribosome-inactivating protein, which we named gypsophilin . The protein was purified to apparent homogeneity by (NH4)2SO4 fractionation, ion-exchange chromatography, and adsorption chromatography . The protein was found to have a molecular mass of 28.0 kDa and a pI of about 10.1 . It does not contain glycosidic linkages . The sequence of the N-terminal 22 amino acids of the protein shows a close relationship to other RIPs . The enzyme strongly inhibits protein synthesis in a rabbit reticulocyte lysate and depurinates 28S rRNA in rat liver ribosomes in a manner identical to that of ricin A-chain and other RIPs . Using a direct method for the measurement of the RNA N-glycosidase activity, the substrate specificity of gypsophilin was identified . EC50 of the protein for ribosomes of rat liver, wheat germ, and E . coli was 39.8 pM, 0.24 nM, and 0.82 microM, respectively . Gypsophilin may be one of the most active RNA N-glycosidases among the RIPs known to date . Immunoelectron microscopic localization of gypsophilin in the leaves shows that the protein is accumulated densely in the intercellular spaces and is also distributed within vacuoles in the cytoplasm. Eur J Biochem, 1997 Feb 1, 243(3), 739 - 47 Cloning, sequencing and functional expression of dihydrolipoamide dehydrogenase from the human pathogen Trypanosoma cruzi; Schoneck R et al.; This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis) . The full-length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues . LipDH is a homodimeric protein with FAD as prosthetic group . The calculated molecular mass of the subunit of the mature protein with bound FAD is 50,066 . Comparison of the deduced amino acid sequence of LipDH from T . cruzi with that of Trypanosoma brucei and man shows identities of 81% and 50%, respectively . An N-terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids . The gene lpd1 of T . cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH . For this purpose an ATG codon was introduced directly upstream the codon for Asn10 which represents the N-terminus of the mature protein . This system allowed the synthesis of 1000 U T . cruzi LipDH/1 bacterial cell culture . The recombinant protein was purified to homogeneity by (NH4)2SO4-precipitation and affinity chromatography on 5' AMP-Sepharose . The K(m) values for NAD+, NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite . LipDH is present in all major developmental stages of T . cruzi as shown by northern and western blot analyses . This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite . In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redoxcycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox . For that reason T . cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs . The bacterial expression system for the parasite enzyme will now allow the study of the role of T . cruzi LipDH in drug activation and the crystallization of the protein. Eur J Biochem, 1997 Feb 1, 243(3), 684 - 9 Inert chromium and cobalt complexes as probes of magnesium-dependent enzymes . Evaluation of the mechanistic role of the essential metal cofactor in Escherichia coli exonuclease III; Black CB et al.; An investigation of the metal ion dependence of Escherichia coli exonuclease III, 3'-5'-exonuclease and exoribonuclease H activities is reported . Catalytic activation of E . coli exonuclease III has been examined for a series of inert chromium complexes Cr(NH3)6-x(H2O)x3+ (x = 0-6) that bear water and ammine ligands in well defined inner-sphere geometries . The importance of hydrogen bonding and electrostatic stabilization for catalysis of this reaction were quantitatively evaluated . Catalytic activation by the essential metal cofactor appears to be mediated through transition-state stabilization by outer-sphere complex formation with substrate . Hydrogen bonding to metal-bound water molecules is the dominant stabilizing interaction. Eur J Biochem, 1997 Feb 1, 243(3), 670 - 7 Methanol:coenzyme M methyltransferase from Methanosarcina barkeri . Purification, properties and encoding genes of the corrinoid protein MT1; Sauer K et al.; In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methylcoenzyme M from methanol and coenzyme M . This methyl transfer reaction is catalyzed by two enzymes, designated MT1 and MT2 . Transferase MT1 is a corrinoid protein . The purification, catalytic properties and encoding genes of MT2 (MtaA) have been described previously {Harms, U . and Thauer, R.K . (1996) Eur . J . Biochem . 235, 653-659} . We report here on the corresponding analysis of MT1 . The corrinoid protein MT1 was purified to apparent homogeneity and showed a specific activity of 750 mumol min-1 mg-1 . The enzyme catalyzed the methylation of its bound corrinoid in the cob(I)amide oxidation state by methanol . In addition to this automethylation, the purified enzyme was found to catalyze the methylation of free cob(I)alamin to methylcob(III)alamin . It was composed of two different subunits designated MtaB and MtaC, with apparent molecular masses of 49 kDa and 24 kDa, respectively . The subunit MtaC was shown to harbour the corrinoid prosthetic group . The genes mtaB and mtaC were cloned and sequenced . They were found to be juxtapositioned and to form a transcription unit mtaCB . The corrinoid-harbouring subunit MtaC exhibits 35% sequence similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli. Eur J Biochem, 1997 Feb 1, 243(3), 660 - 9 Stability and kinetics of unfolding and refolding of cAMP receptor protein from Escherichia coli; Malecki J et al.; cAMP receptor protein (CRP) is involved in regulation of expression of several genes in Escherichia coli . The protein is a homodimer and each monomer is folded into two distinct structural domains . The mechanism of the biological activity of the protein may involve the interaction between the subunits and domains . In order to determine the interaction between the subunits or domains of CRP, we have studied the reversible denaturation of the protein by guanidine hydrochloride . The unfolding and refolding kinetics of CRP was monitored using stopped-flow fluorescence spectroscopy at 20 degrees C and pH 7.9 . The results of CRP denaturation indicate that the transition can be described by a three-state model: (CRP native)2<=> 2 (CRP native)<=>2 (CRP denatured) . The faster process, characterized by the relaxation time tau 2 = 80 +/- 3 ms, corresponds to the dissociation of CRP dimer into monomers . The slower process has the relaxation time tau t = 1.9 +/- 0.1 s and corresponds to the cooperative unfolding of CRP monomer . The free energy change in the absence of denaturant upon CRP dissociation is delta G dis degrees = 46.9 +/- 2.5 kJ/mol and for monomer unfolding delta G unf degrees = 30.9 +/- 1.3 kJ/mol . The thermal unfolding of CRP was studied by circular dichroism and fluorescence spectroscopy at various guanidine hydrochloride concentrations . It has been found that the native protein is maximally stable at about 21 +/- 0.3 degrees C and is denatured upon heating and cooling from this temperature . The apparent free energy change for CRP unfolding at 21 degrees C is equal to 30.5 +/- 0.4 kJ/mol and the apparent specific heat change is equal to delta Cp, app = 10.7 +/- 0.7 kJ mol-1 K-1 . The predicted values of cold denaturation midpoint is equal to tau G = -18.8 +/- 1.5 degrees C and for high-temperature transition tau G = 63.1 +/- 1.5 degrees C . The predicted midpoint of high-temperature unfolding transition is about the same as determined experimentally. Vet Microbiol, 1997 Feb, 54(2), 145 - 53 Virulence properties of Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups isolated from calves with diarrhea; Saridakis HO et al.; Nineteen Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups were isolated from calves with diarrhea in Parana State . Brazil, and studied for virulence markers associated with EPEC or enterohemorrhagic E . coli (EHEC) . The 19 isolates belonged to 12 serotypes with isolates of O26:H11, O119:H25 and O114:H- being the most prevalent Localized adherence (LA) was demonstrated for 37% of the isolates, consisting of all four O26:H11, both O114:H- and one O114:H40 isolates . All the LA strains were positive in the fluorescent-actin staining (FAS) test and possessed attaching-effacing E . coli (eae) sequences, but only O114 strains hybridized with the EPEC adherence factor (EAF) probe . None of the strains produced Shiga-like toxins (Verotoxin) . Only the O26:H11 strains hybridized with the EHEC plasmid specific (CVD419) probe and were enterohemolytic, properties associated with EHEC strains . This investigation demonstrates that among the bovine strains isolated only those of serogroup O114 behaved as typical EPEC. Vet Microbiol, 1997 Feb, 54(2), 123 - 32 Distribution of K88 Escherichia coli-adhesive and nonadhesive phenotypes among pigs of four breeds; Baker DR et al.; Enterotoxigenic Escherichia coli expressing K88 fimbrial adhesins often cause diarrhea in young pigs . However, some pigs are inherently resistant to colibacillosis, because they lack receptors on their epithelial cell brush borders to which the fimbriae bind . Phenotypic diversity with respect to the binding of E . coli expressing K88 of the three variant types (K88ab, K88ac, and K88ad) was reported by Bijlsma et al . (1982), and binding specificities for each phenotype were described: A (adhesive to all three variants), B (adhesive to K88ab and K88ac), C (adhesive to K88ab and K88ad), D (adhesive to K88ad) and E (nonadhesive) . Because brush border adhesiveness has been correlated with disease susceptibility, swine K88 adhesive phenotypes are of significance in the control of enteric disease . To determine the prevalence of the various K88 adhesive phenotypes in the swine population in the Midwestern United States, we tested epithelial cell brush borders of 24 purebred pigs from each of four breeds (Chester White, Duroc, Hampshire and Yorkshire) for adhesiveness to each of the K88 variants . Four, 4-week-old pigs (the largest and smallest healthy female littermates from two litters) were collected from each of 24 farms . Brush border vesicles from the pigs were tested for ability to bind E . coli expressing each K88 variant . The five brush border adherence patterns described for phenotypes A-E were observed . In addition, brush borders from some pigs only bound K88ab + bacteria . Nearly three quarters of the pigs whose brush borders tested, were found to be phenotype A (43%) or phenotype E (28%) . These were the most common phenotypes in each breed, except Hampshire, in which case phenotypes C (17%) and D (25%) were more common than E (8%) . There appeared to be no relationship between the phenotype of a pig and its weight relative to its littermate. Poult Sci, 1997 Feb, 76(2), 244 - 7 Dietary effects on immune response of fast-growing chicks to inoculation of sheep erythrocytes and Escherichia coli; Praharaj NK et al.; Responses to SRBC and Escherichia coli inoculations were measured during the early posthatch period in broiler cockerels fed diets differing in nutrient density . Diet A consisted of 20% protein and 2,685 kcal ME/kg and Diet B consisted of 24% protein and 3,146 kcal ME/kg . There was no effect of diet on antibody response of chicks inoculated at 10 d of age with 0.25, 2.50, 5.00, or 25.00% suspensions of SRBC . A significantly larger proportion of chicks, however, produced antibody at the 25.00% than at the 0.25% dosage of SRBC . When inoculated at 15 d of age with 10(-6), 10(-4), or 10(-2) dosage of E . coli, there were no significant diet by dosage interactions for lesion scores, or relative change in BW 24, 48, and 120 h after inoculation . There were differences among E . coli dosages for severity of lesions and mortality, with rankings being 10(-2) > 10(-4) > 10(-6) . Lesion scores and mortality were higher for the chicks fed Diet B than those fed Diet A . Also, deleterious effects of E . coli on BW 24, 48, and 120 h after inoculation were greater for chicks fed Diet B than for chicks fed Diet A . Responses to inoculations of SRBC and E . coli of broilers fed a diet with a lower nutrient density were equal or superior to those of broilers fed a diet with a higher nutrient density. Dig Dis Sci, 1997 Feb, 42(2), 242 - 50 Role of central interleukin-1 beta in gastrointestinal motor disturbances induced by lipopolysaccharide in sheep; Plaza MA et al.; Cytokines are involved in the symptoms of the acute phase response induced by infectious diseases in humans as well as in animals, and interleukin-1 beta (IL-1 beta) has a pivotal role in these changes . The role of central IL-1 beta in the gastrointestinal hypomotility and fever evoked by intravenous administration of lipopolysaccharide (LPS) and the mechanisms involved, were investigated in sheep as an experimental model . LPS (0.1 microgram/kg, intravenously) induced gastrointestinal hypomotility and fever that were significantly reduced by prior intracerebroventricular administration of IL-1 receptor antagonist protein (IL-1ra, 2 micrograms/kg) . The effects of LPS were mimicked by intracerebroventricular IL-1 beta (50 ng/kg), whereas IL-1 beta injected intravenously at the same dose only caused a slight and transient fever without modifying the gastrointestinal motility . Prior intracerebroventricular administration of the cyclooxygenase inhibitor indomethacin (100 micrograms/kg) but not the corticotropin-releasing factor (CRF) receptor antagonist alpha-helical CRF9-41 (5 micrograms/kg) blocked all effects by both LPS and IL-1 beta . These results suggest that in sheep, LPS induces digestive motor disturbances through a central release of IL-1 beta and prostaglandins. J Anim Sci, 1997 Feb, 75(2), 409 - 16 Immune response and growth of stressed weanling pigs fed diets supplemented with organic or inorganic forms of chromium; van Heugten EV et al.; A 2 x 4 factorial arrangement of treatments was used in a randomized complete block designed study to determine the effects of chromium level and source on growth and immune response of stressed and non-stressed 3-wk-old crossbred weanling pigs (BW was 6.35 kg) . Factors included 1) immune stress or control and 2) no supplemental Cr or .2 ppm of supplemental Cr from either CrCl3, Cr-picolinate, or Cr-nicotinic acid complex . The basal diet was a corn-soybean meal-whey diet containing 1.2% lysine . Escherichia coli lipopolysaccharide (LPS) was the stress-inducing agent and was injected on d 7, 10, and 13 of the experiment . Immune challenge with LPS resulted in reduced gain (P < .05) and feed intake (P < .10) . Supplementation with Cr was not effective in alleviating the depression in growth due to LPS . However, supplementation of control pigs with Cr tended to improve (P < .10) gain and feed intake . In vitro cellular immune response as measured by a lymphocyte blastogenesis assay was increased (P < .10) in pigs fed supplemental Cr from CrCl3, or Cr-picolinate . Antibody response to sheep red blood cells tended to be increased (P < .10) in pigs supplemented with Cr-nicotinic acid, but antibody response to ovalbumin was decreased (P < .05) in pigs supplemented with organic forms of Cr . At the end of the study, effects of Cr supplementation on lymphocyte proliferative response were investigated before and after ACTH administration . Injections of ACTH resulted in increased (P < .001) serum cortisol levels and increased lymphocyte proliferation . Supplementation of Cr did not affect lymphocyte blastogenic response before or after ACTH injection (P > .10) . These data suggest that Cr supplementation was not beneficial during immune stress in pigs. J Vet Pharmacol Ther, 1997 Feb, 20(1), 61 - 8 Effects of pentoxifylline and polymyxin B on the acute-phase-response to Escherichia coli endotoxin in dwarf goats; van Miert AS et al.; The purpose of this study was to assess whether polymyxin B together with pentoxifylline, had beneficial effects on the acute-phase-response to E . coli endotoxin in the dwarf goat (n = 6) . Polymyxin B partly neutralizes E . coli endotoxin by forming inactive polymyxin B-lipopolysaccharide (LPS) complexes; pentoxifylline has been reported to suppress the LPS-induced production of tumour necrosis factor (TNF-alpha) . E . coli LPS (0.0067 microgram/kg/min over 30 min) induced fever, tachycardia, inhibition of rumen motility, a decline in WBC, lymphopenia, and decreases in plasma zinc and iron concentrations . Most of the haematological, blood biochemical and clinical effects of E . coli LPS were significantly reduced by polymyxin B pretreatment (0.1 mg/kg/min over 30 min, i.v.) . Pentoxifylline (0.3 mg/kg/min over 30 min, i.v.) did not reduce the clinical and blood biochemical effects of E . coli LPS, however, it modulated the number of circulating neutrophils . No synergistic effects were observed after i.v . infusion of polymyxin B with pentoxifylline . The lack of synergy may be due to the production and release of pro-inflammatory cytokines other than TNF-alpha. Chem Res Toxicol, 1997 Feb, 10(2), 156 - 64 Substrate specificity for the epoxidation of terpenoids and active site topology of house fly cytochrome P450 6A1; Andersen JF et al.; Heterologous expression in Escherichia coli, purification, and reconstitution of house fly P450 6A1 and NADPH-cytochrome P450 reductase were used to study the metabolism of terpenoids . In addition to the epoxidation of cyclodiene insecticides demonstrated previously {Andersen et al . (1994) Biochemistry 33, 2171-2177}, this cytochrome P450 was shown to epoxidize a variety of terpenoids such as farnesyl, geranyl, and neryl methyl esters, juvenile hormones I and III, and farnesal but not farnesol or farnesoic acid . P450 6A1 reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine did not metabolize alpha-pinene, limonene, of the insect growth regulators hydroprene and methoprene . The four geometric isomers of methyl farnesoate were metabolized predominantly to the 10,11-epoxides, but also the 6,7-epoxides and to the diepoxides . The 10,11-epoxide of methyl (2E,6E)-farnesoate was produced in a 3:1 ratio of the (10S) and (10R) enantiomers . Monoepoxides of methyl farnesoate were metabolized efficiently to the diepoxides . Methyl farnesoate epoxidation was strongly inhibited by a bulky substituted imidazole . The active site topology of P450 6A1 was studied by the reaction of the enzyme with phenyldiazene to form a phenyl-iron complex . Ferricyanide-induced in situ migration of the phenyl group showed formation of the N-phenylprotopor-phyrinporphyrin IX adducts in a 17:25:33:24 ratio of the NB:NA:NC:ND isomers . These experiments suggest that metabolism of xenobiotics by this P450, constitutively overexpressed in insecticide-resistant strains of the house fly, is not severely limited by stereochemically constrained access to the active site. Plant Mol Biol, 1997 Feb, 33(3), 565 - 70 Functional activity of sporamin from sweet potato (Ipomoea batatas Lam.): a tuber storage protein with trypsin inhibitory activity; Yeh KW et al.; Sporamin accounts for about 60% to 80% of total soluble protein in sweet potato tubers, and the predicted protein sequence of sporamin shares significant amino acid sequence identity with some Kunitz-type trypsin inhibitors . We constructed three recombinant plasmids with cDNAs that encode preprosporamin, prosporamin, and sporamin, and these three were expressed in Escherichia coli cells as fusion proteins . All three forms of sporamin expressed in E . coli were shown to have strong inhibitory activity to trypsin in vitro, suggesting that post-translational modifications are not essential for trypsin inhibitory activity . Northern blot analysis showed that sporamin transcripts could be systemically induced in leaf tissue of sweet potato by wounding . Therefore, sporamin may have a defense role as a protease inhibitor, in addition to its role as a storage protein. Plant Mol Biol, 1997 Feb, 33(3), 457 - 66 Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts; Krishna P et al.; A polyclonal antibody, R2, was raised against a fusion protein consisting of a portion of plant hsp90 fused to the trpE protein of Escherichia coli . This antibody was found to be specific towards plant hsp90, showing little or no cross-reactivity with mouse and human hsp90 proteins . The R2 antibody identified an 83 kDa protein as the hsp90 homologue in cytosolic extracts of several dicot and monocot plants . Two-dimensional gel electrophoresis indicated that at least two different isoforms of hsp90 are expressed in Brassica napus seedlings . An examination of the native state of hsp90 by non-denaturing gel electrophoresis showed that this protein exists as a monomer, dimer and as a high-molecular-mass complex of ca . 680 kDa in cell extracts of spinach cotyledons and leaves, B . napus seedlings and wheat germ . Native gel analysis and cross-linking studies of purified hsp90 showed that plant hsp90 exists predominantly as a monomer . When 35S-labelled B . napus cytosolic extracts were immunoprecipitated with the R2 antiserum, hsp90 and two additional proteins with approximate molecular masses of 49 and 45 kDa were detected in the immunoprecipitates . These results are consistent with the idea that hsp90:protein heterocomplexes exist in plant cells. Trends Biochem Sci, 1997 Feb, 22(2), 59 - 63 Protein misfolding in the cell envelope of Escherichia coli: new signaling pathways; Missiakas D et al.; Depending on their cellular localization, misfolded proteins in Escherichia coli trigger two different heat-shock responses . Cytoplasmic proteins induce the 'classical' heat-shock regulon transcribed by the E sigma 32 polymerase . By contrast, misfolding of proteins in the cell envelope induces the newly described E sigma E-dependent regulon . This implies that there is an inducible transduction machinery in the inner membrane . The response to protein misfolding in the cell envelope is a finely tuned system regulated by a cascade of phosphorylation and dephosphorylation reactions. Mol Microbiol, 1997 Feb, 23(3), 515 - 24 Characterization of the ModE DNA-binding sites in the control regions of modABCD and moaABCDE of Escherichia coli; McNicholas PM et al.; The Escherichia coli molybdate transporter, encoded by the modABCD operon, is negatively regulated by the modE gene product in response to the intracellular molybdate concentration . Utilizing an in vivo titration assay, we localized the ModE-binding site to the start of modA transcription . This localization was further characterized using in vitro gel-shift assays and DNase I footprinting . ModE bound the wild-type modA promoter with an apparent dissociation constant (Kd) of 45 nM, and addition of molybdate, in physiologically relevant amounts, significantly increased DNA binding . Consistent with these data, modA promoter fragments containing mutations that reduced ModE repression in vivo displayed proportionately higher apparent Kd values in vitro . DNase I footprinting of the modA promoter revealed a single protected region that overlapped the start site of transcription and extended from position -18 to +10, relative to the transcript start site . Gel-shifting assays, employing the promoter regions from the tor, nrf, moa and moe operons, revealed that ModE bound only the moa promoter region, with an apparent Kd of 24nM . Footprint analysis of the moaA promoter revealed a single protected region located immediately upstream of the putative -35 consensus sequence and extending from position -202 to -174, relative to the start of translation . In vivo expression of a moaA-lacZ operon fusion was stimulated twofold by ModE . However, relative to modA, binding of ModE to the moaA promoter appeared to be largely molybdate independent both in vitro and in vivo . These findings demonstrate that ModE acts both as a repressor and activator of the mod and moa operons, respectively, depending on the properties of the binding site. Mol Microbiol, 1997 Feb, 23(3), 459 - 71 Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus; Hanlon WA et al.; The Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/Thr protein kinases . The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M . xanthus life cycle . Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development . It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M . xanthus . This region also shows 29, 25 and 29% identify with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively . A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane . The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function . When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues . Disruption of the pkn9 kinase catalytic subdomains I-III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal . Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time . Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced . Two-dimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4) . These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production. Microbiology, 1997 Feb, 143 ( Pt 2), 675 - 88 The Mycoplasma hominis P120 membrane protein contains a 216 amino acid hypervariable domain that is recognized by the human humoral immune response; Nyvold C et al.; In the antigenically heterogeneous species Mycoplasma hominis a monoclonal antibody, mAb 26.7D, was previously found to recognize a 120 kDa polypeptide from M . hominis 7488 . This antibody did not react with the type strain PG21 . The homologous gene from M . hominis PG21 was cloned and sequenced and found to have a sequence identity of 91% with the gene of strain 7488 . One hypervariable and two semivariable regions were detected . The epitope for mAb 26.7D was mapped to the hypervariable domain by expression of various parts of this domain in Escherichia coli using expression vector systems . A polyclonal antiserum (pAb 121) generated against the hypervariable region of P120 from PG21 identified the P120 homologue in M . hominis PG21 . Fusion proteins of the hypervariable and constant parts of the proteins were constructed and tested for reactivity with 21 human sera . Twelve sera reacted with the 7488 hypervariable fusion protein, but only four reacted with PG21 hypervariable fusion protein . No reactivity was seen with a fusion protein containing part of the constant region of P120 . Gene fragments amplified from 18 M . hominis isolates by PCR confirmed the heterogeneity of the hypervariable domain . Based on restriction endonuclease cleavage patterns of the hypervariable domain the 18 isolates could be divided into four cases . Reactivity with both mAb 26.7D and pAb 121 confirmed these classes . The hypervariable, but not the constant, part of P120 was recognized by the human humoral immune response . Such a variable domain may be important in evasion of the host's immune response, and thus aid survival of the micro-organism. Microbiology, 1997 Feb, 143 ( Pt 2), 585 - 94 Missense mutations in the 3' end of the Escherichia coli dnaG gene do not abolish primase activity but do confer the chromosome-segregation-defective (par) phenotype; Versalovic J et al.; Isogenic dnaG strains of Escherichia coli with the parB and dnaG2903 alleles in the MG1655 chromosomal background displayed the classic par phenotype at the nonpermissive temperature of 42 degrees C . These strains synthesized DNA at 42 degrees C, but remained chromosome segregation defective as determined by cytology . A strain with the dnaG2903 allele was tested for its ability to support DNA replication of a primase-dependent G4ori(c)-containing M13 phage derivative by quantitative competitive PCR (QC-PCR) . The dnaG2903 strain converted the single-stranded DNA into double-stranded replicative form DNA at 42 degrees C . These results indicate that DnaG2903 retains primase activity at the restrictive temperature . Nucleoids remained unsegregated in the central region of cell filaments at 42 degrees C . The observed suppression of cell filamentation in dnaG sfiA or dnaG lexA double mutants suggests that the SOS response is induced at the restrictive temperature in parB and dnaG2903 strains but fails to account entirely for the cell filamentation phenotype . ParB and DnaG2903 presumably can synthesize primer RNA for DNA replication, but may be defective in their interactions with DNA replication proteins, cell cycle regulatory factors, or the chromosome segregation apparatus itself. Microbiology, 1997 Feb, 143 ( Pt 2), 547 - 52 IS900 targets translation initiation signals in Mycobacterium avium subsp . paratuberculosis to facilitate expression of its hed gene; Doran T et al.; The Mycobacterium avium subsp . paratuberculosis (formerly Mycobacterium paratuberculosis) atypical insertion sequence, IS900, encodes a novel gene on the complementary strand to the putative transposase, p43 . This gene requires a promoter, ribosome binding site (RBS) and termination codon to be acquired upon insertion into the M . avium subsp . paratuberculosis genome and hence is designated the hed (host expression-dependent) gene of IS900 . Analysis of IS900 insertion sites suggests that this element targets translation initiation signals in M . avium subsp . paratuberculosis, specifically inserting between the RBS and start codon of a putative gene sequence . This aligns the hed initiation codon adjacent to a functional RBS and possibly downstream of an active promoter, driving expression of Hed protein . We have confirmed this unique targeting process by detecting expression of hed in M . avium subsp . paratuberculosis at the level of transcription by reverse transcription-PCR . Further, two Hed-specific antibodies detected Hed translation products in Western blots of protein extracts from M . avium subsp . paratuberculosis . A recombinant form of Hed expressed and purified from Escherichia coli will facilitate studies of IS900 transposition and will also be assessed as a diagnostic antigen for M . avium subsp . paratuberculosis disease . Implications of IS900 insertion in M . avium subsp . paratuberculosis pathogenicity are discussed. Microbiology, 1997 Feb, 143 ( Pt 2), 457 - 66 Enzymological and physiological consequences of restructuring the lipoyl domain content of the pyruvate dehydrogenase complex of Escherichia coli; Guest JR et al.; The core-forming lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli contain three tandemly repeated lipoyl domains although one lipoyl domain is apparently sufficient for full catalytic activity in vitro . Plasmids containing IPTG-inducible aceEF-IpdA operons which express multilip-PDH complexes bearing one N-terminal lipoyl domain and up to seven unlipoylated (mutant) domains per E2p chain, were constructed . Each plasmid restored the nutritional lesion of a strain lacking the PDH complex and expressed a sedimentable PDH complex, although the catalytic activities declined significantly as the number of unlipoylated domains increased above four per E2p chain . It was concluded that the extra domains protrude from the 24-meric E2p core without affecting assembly of the E1p and E3 subunits, and that the lipoyl cofactor bound to the outermost domain can participate successfully at each of the three types of active site in the assembled complex . Physiological studies with two series of isogenic strains expressing multilip-PDH complexes from modified chromosomal pdh operons (pdhR-aceEF-IpdA) showed that three lipoyl domains per E2p chain is optimal and that only the outermost domain need be lipoylated for optimal activity . It is concluded that the reason for retaining three lipoyl domains is to extend the reach of the outermost lipoyl cofactor rather than to provide extra cofactors for catalysis. J Trauma, 1997 Feb, 42(2), 183 - 90 Alanylglutamine-enriched total parenteral nutrition improves protein metabolism more than branched chain amino acid-enriched total parenteral nutrition in protracted peritonitis; Naka S et al.; Branched chain amino acids (BCAAs) and glutamine are both recommended in catabolic states . The object of this study was to compare the efficacies of alanylglutamine (Ala-Gln)-enriched and BCAA-enriched total parenteral nutrition (TPN) on the protein kinetics in peritonitis . Rats were divided into Ala-Gln and BCAA groups after intraperitoneal implantation of an osmotic pump, delivering a continuous infusion of Escherichia coli . Glutamine composed 30.0% (w/v) of the total amino acids in the Ala-Gln group, and BCAA composed 30.5% (w/v) of the total amino acids in the BCAA group . The two solutions were isocaloric and isonitrogenous . Whole body protein turnover and organ fractional protein synthetic rates (FSR) were measured on days 3 and 5 . Serum amino acid levels and mucosal morphology were determined . Ala-Gln group had higher rates of whole body protein turnover, and hepatic FSR on both days . Serum glutamine levels correlated with hepatic and muscle FSR . Ala-Gln TPN group had greater mucosal thickness, numbers of mitoses per crypt, and FSR in distal intestine . Ala-Gln-enriched TPN may be a useful nutritional treatment modality in sepsis. Epidemiol Infect, 1997 Feb, 118(1), 51 - 61 Molecular epidemiology of recent outbreaks of swine vesicular disease: two genetically and antigenically distinct variants in Europe, 1987-94; Brocchi E et al.; Viruses from the recent epidemic of swine vesicular disease (SVD) in Europe have been isolated and characterized by antigenic and genetic methods to examine the likely epidemiological origins of the disease . Antigenic analysis was performed on 77 SVD viruses (SVDV) isolated in Europe between 1966 and 1994 using two panels of monoclonal antibodies (MAb) in a trapping ELISA . Genetic analysis of 33 of the SVD viruses by reverse transcription-polymerase chain-reaction (RT-PCR) amplification and nucleotide sequencing of the ID (VP1) coding region was also performed . Comparison of the nucleotide sequences with each other and with three other previously published SVDV sequences revealed four distinct groups which correlated exactly with the results of the pattern of reactivity with MAbs . The first group consisted solely of the earliest SVD virus isolated (ITL/1/66) while the second group comprised viruses present in Europe and Japan between 1972 and 1981 . The third group consisted of viruses isolated from outbreaks of SVD in Italy between December 1988 and June 1992 . Viruses isolated between 1987 and 1994 from Romania, the Netherlands, Italy and Spain formed a fourth group . The genetic and antigenic similarity of the most recent virus isolates from Western Europe to a virus isolated in Romania 5 years previously suggests that the possible origin of the recent epidemic of swine vesicular disease in Western Europe was in Eastern Europe. Protein Sci, 1997 Feb, 6(2), 444 - 9 Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: a model for studying phosphagen kinases; Zhou G et al.; Phosphagen kinases catalyze the reversible transfer of a phosphoryl group between guanidino phosphate compounds and ADP, thereby regenerating ATP during bursts of cellular activity . Large quantities of highly pure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in arthropods, have been isolated from E . coli, into which the cDNA for the horseshoe crab enzyme had been cloned . Purification involves size exclusion and anion exchange chromatographies applied in the denatured and refolded states . The recombinant enzyme has been crystallized as a transition state analog complex . Near complete native diffraction data have been collected to 1.86 A resolution . Substitution of a recombinant source for a natural one, improvement in the purification, and data collection at cryo temperatures have all yielded significant improvements in diffraction. Protein Sci, 1997 Feb, 6(2), 438 - 43 The role of helix VIII in the lactose permease of Escherichia coli: II . Site-directed sulfhydryl modification; Frillingos S et al.; Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S . Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport . In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) . Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with {14C}NEM is attenuated by TDG . In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue . Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent . Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys . The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change. Protein Sci, 1997 Feb, 6(2), 431 - 7 The role of helix VIII in the lactose permease of Escherichia coli: I . Cys-scanning mutagenesis; Frillingos S et al.; Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys . Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less) . As expected (Ujwal ML, Sahin-Toth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport . Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels . The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner . Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys . Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys) . The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism . More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S . Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ. Protein Sci, 1997 Feb, 6(2), 383 - 90 Complete 1H, 13C, and 15N NMR resonance assignments and secondary structure of human glutaredoxin in the fully reduced form; Sun C et al.; Human glutaredoxin is a member of the glutaredoxin family, which is characterized by a glutathione binding site and a redox-active dithiol/disulfide in the active site . Unlike Escherichia coli glutaredoxin-1, this protein has additional cysteine residues that have been suggested to play a regulatory role in its activity . Human glutaredoxin (106 amino acid residues, M(r) = 12,000) has been purified from a pET expression vector with both uniform 15N labeling and 13C/15N double labeling . The combination of three-dimensional 15N-edited TOCSY, 15N-edited NOESY, HNCA, HN(CO)CA, and gradient sensitivity-enhanced HNCACB and HNCO spectra were used to obtain sequential assignments for residues 2-106 of the protein . The gradient-enhanced version of the HCCH-TOCSY pulse sequence and HCCH-COSY were used to obtain side chain 1H and 13C assignments . The secondary structural elements in the reduced protein were identified based on NOE information, amide proton exchange data, and chemical shift index data . Human glutaredoxin contains five helices extending approximately from residues 4-10, 24-36, 53-64, 83-92, and 94-104 . The secondary structure also shows four beta-strands comprised of residues 15-19, 43-48, 71-75, 78-80, which form a beta-sheet almost identical to that found in E . coli glutaredoxin-1 . Complete 1H, 13C, and 15N assignments and the secondary structure of fully reduced human glutaredoxin are presented . Comparison to the structures of other glutaredoxins is presented and differences in the secondary structure elements are discussed. Protein Sci, 1997 Feb, 6(2), 340 - 6 Introduction of a {4Fe-4S (S-cys)4}+1,+2 iron-sulfur center into a four-alpha helix protein using design parameters from the domain of the Fx cluster in the Photosystem I reaction center; Scott MP et al.; We describe the insertion of an iron-sulfur center into a designed four alpha-helix model protein . The model protein was re-engineered by introducing four cysteine ligands required for the coordination of the mulinucleate cluster into positions in the main-chain directly analogous to the domain predicted to ligand the interpeptide {4Fe-4S (S-cys)4} cluster, Fx, from PsaA and PsaB of the Photosystem I reaction center . This was achieved by inserting the sequence, CDGPGRGGTC, which is conserved in PsaA and PsaB, into interhelical loops 1 and 3 of the four alpha-helix model . The holoprotein was characterized spectroscopically after insertion of the iron-sulfur center in vitro . EPR spectra confirmed the cluster is a {4Fe-4S} type, indicating that the cysteine thiolate ligands were positioned as designed . The midpoint potential of the iron-sulfur center in the model holoprotein was determined via redox titration and shown to be -422 mV (pH 8.3, n = 1) . The results support proposals advanced for the structure of the domain of the {4Fe-4S} Fx cluster in Photosystem I based upon sequence predictions and molecular modeling . We suggest that the lower potential of the Fx cluster is most likely due to factors in the protein environment of Fx rather than the identity of the residues proximal to the coordinating ligands. Protein Sci, 1997 Feb, 6(2), 331 - 9 The active site histidines of creatine kinase . A critical role of His 61 situated on a flexible loop; Forstner M et al.; A histidine residue with a pKa of 7 has been inferred to act as a general acid-base catalyst for the reaction of creatine kinase (CK), catalyzing the reversible phosphorylation of creatine by ATP . The chicken sarcomeric muscle mitochondrial isoenzyme Mib-CK contains several histidine residues that are conserved throughout the family of creatine kinases . By X-ray crystal structure analysis, three of them (His 61, His 92, and His 186) were recently shown to be located close to the active site of the enzyme . These residues were exchanged against alanine or aspartate by in vitro mutagenesis, and the six mutant proteins were expressed in E . coli and purified . Structural integrity of the mutant proteins was checked by small-angle X-ray scattering . Kinetic analysis showed the mutant His 61 Asp to be completely inactive in the direction of ATP consumption while exhibiting a residual activity of 1.7% of the wild-type (wt) activity in the reverse direction . The respective His to Ala mutant of residue 61 showed approximately 1% wt activity in the forward and 10% wt activity in the reverse reaction . All other mutants showed near wt activities . Changes in the kinetic parameters K(m) or Vmax, as well as a significant loss of synergism in substrate binding, could be observed with all active mutants . These effects were most pronounced for the binding of creatine and phosphocreatine, whereas ATP or ADP binding were less severely affected . Based on our results, we assume that His 92 and His 186 are involved in the binding of creatine and ATP in the active site, whereas His 61 is of importance for the catalytic reaction but does not serve as an acid-base catalyst in the transphosphorylation of creatine and ATP . In addition, our data support the idea that the flexible loop bearing His 61 is able to move towards the active site and to participate in catalysis. Protein Sci, 1997 Feb, 6(2), 304 - 14 The NMR side-chain assignments and solution structure of enzyme IIBcellobiose of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli; Ab E et al.; The assignment of the side-chain NMR resonances and the determination of the three-dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli are presented . The side-chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH-TOCSY, HCCH-COSY, and COCCH-TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments . In order to obtain the three-dimensional structure, NOE data were collected from 15N-NOESY-HSQC, 13C-HSQC-NOESY, and 2D NOE experiments . The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols . In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated . The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 A on C alpha atoms . IIBcel consists of a four stranded parallel beta-sheet, in the order 2134 . The sheet is flanked with two and three alpha-helices on either side . Residue 10, a cysteine in the wild-type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first beta-strand . A loop that is proposed to be involved in the binding of the phosphoryl-group follows the cysteine . The loop appears to be disordered in the unphosphorylated state. Am J Gastroenterol, 1997 Feb, 92(2), 271 - 4 Solitary and multiple pyogenic liver abscesses: characteristics of the patients and efficacy of percutaneous drainage; Tazawa J et al.; OBJECTIVES: Although percutaneous drainage has emerged as one of the first line of therapies for pyogenic liver abscesses, the presence of multiple abscesses may warrant surgical drainage, which remains controversial in the literature . We studied whether the multiplicity of the lesions influences the outcome of the treatment . METHODS: Ultrasonography-guided percutaneous drainage was carried out in 48 patients with pyogenic liver abscesses . The abscesses were solitary in 38 patients and multiple (two to seven lesions) in 10 patients . Clinical characteristics and the efficacy of the treatment were compared between these two groups . RESULTS: Biliary diseases and malignancies were more frequently observed in the solitary cases than multiple cases . A past history of surgery for cholelithiasis was seen exclusively in the multiple cases . E . coli was more frequently cultured from the abscesses in the multiple cases . Three of the multiple cases required more than a single catheter . All of the multiple cases and 36 of the 38 solitary cases were successfully treated . Two patients died of biliary peritonitis as a complication of the procedure, and three died of other underlining diseases . CONCLUSION: Ultrasonography-guided percutaneous drainage is effective even in patients with multiple pyogenic liver abscesses by adding catheters to obtain sufficient drainage. FEMS Microbiol Lett, 1997 Feb 1, 147(1), 147 - 9 Organization of the nar genes at the chlZ locus; Bonnefoy V et al.; The two membrane-bound respiratory nitrate reductases of Escherichia coli are encoded by distinct operons at two different loci, chlC and chlZ, on the chromosome . The chlZ locus includes a narK homologue, narU, encoding a nitrite extrusion protein, and narZYWV encoding nitrate reductase Z . No apparent homologue to the narXL operon has been found . Homology between narU and narK on the one hand and narZYWV and narGHJI on the other hand is limited to the coding regions. FEMS Microbiol Lett, 1997 Feb 1, 147(1), 139 - 45 A novel bend of DNA CIT: changeable bending-center sites of an intrinsic curvature under temperature conditions; Agrawal GK et al.; We found a novel DNA curvature, which has changeable bending-center sites of an intrinsic curvature under temperature conditions (CIT) in the cyanobacterium strain Microcystis aeruginosa K-81 . Circular permutation analyses (CPA) for CIT under different temperature conditions (4-50 degrees C) revealed that the changeable bending-center sites are located in the 5'-upstream region (-141 to -184) of the psbA2 gene, encoding the D1 protein homolog for photosynthesis . The nucleotide sequence around the bending center contains several dT (deoxy thymine) tracts, which seem to be a pivotal determinant for CIT. FEMS Microbiol Lett, 1997 Feb 1, 147(1), 89 - 95 Comparison of the nucleotide sequence of enteroaggregative Escherichia coli heat-stable enterotoxin 1 genes among diarrhea-associated Escherichia coli; Yamamoto T et al.; The presence of the enteroaggregative Escherichia coli (EAggEC) heat-stable enterotoxin 1 (EAST1) gene was investigated in 15 strains each of EAggEC, enteropathogenic E . coli (EPEC), EPEC-related strains of non-EPEC serotypes, diffusely adhering E . coli (type 1 DAEC) that carries F1845 adhesive pili (or a related adhesin), and enteroinvasive E . coli (EIEC) by PCR and colony hybridization . The EAST1 gene or its homologue was present in 53.3% of EAggEC, 20% of EPEC, 13.3% of the EPEC-related strains, and 6.7% of type 1 DAEC, EIEC and E . coli unrelated with diarrhea had no gene with sequence similarity to the EAST1 gene . Comparison of the EAST1 gene sequences analyzed in this study as well as those reported previously showed that EAggEC (including strain O42, which was shown to be pathogenic in volunteer experiments), EPEC, type 1 DAEC, type 2 DAEC (which carries the 57-kDa outer membrane protein as an adhesin), and enterotoxigenic E . coli shared a common sequence . A variant type of the EAST1 gene sequence was present in the EAggEC strain 17-2 (initially characterized for the EAST1 gene) and in an EPEC-related strain of a non-EPEC serotype . These data suggest that the EAST1 gene or its variant is a virulence gene widely distributed among diarrhea-associated E . coli. Shock, 1997 Feb, 7(2), 139 - 46 Interleukin-6 and tumor necrosis factor production in an enterocyte cell model (Caco-2) during exposure to Escherichia coli; Michalsky MP et al.; Data linking interactions between bacteria and the intestine with elevated serum cytokine levels has led to the concept of the gut as a cytokine-producing organ . An in vitro cell culture model was used to investigate the potential role of intestinal mucosa within this paradigm . Polarized monolayers of human enterocytes (Caco-2) were grown in a two compartment system where the apical and basal aspects of the membrane could be studied . Supernatant was collected at 0, 1, 3, 6, and 24 h after the monolayer was exposed (apically or basally) to 10(2), 10(5), or 10(8) colony-forming units of Escherichia coli C25/mL and saved for interleukin (IL)-6 and tumor necrosis factor (TNF) bioassay analysis . Caco-2 cells (not bacterially challenged) secreted significant amounts of constitutive IL-6, but not TNF, into the apical and basal chambers . Both cytokines levels were increased in a dose-dependent fashion (p < .05) after the E . coli challenge . This stimulated cytokine response was polar, in that the highest cytokine levels were at the side of the bacterial challenge and were most notable at the highest dose (10(8) colony-forming units/mL) of E . coli C25 tested . Caco-2 cells produce IL-6 and TNF in a dose-dependent fashion in response to E . coli C25 and the magnitude of this response is maximal on the side of the bacterial challenge . This data supports the hypothesis that bacterially challenged human enterocytes may be important producers of cytokines. Shock, 1997 Feb, 7(2), 105 - 10 Endothelial structural integrity is maintained during endotoxic shock in an interleukin-1 type 1 receptor knockout mouse; Sutton ET et al.; The derangement of arterial endothelial cell morphology is a good indicator of a severe shock state . Because interleukin (IL)-1 has been implicated in this process, we examined the structural integrity of aortic endothelial cells in conjunction with serum IL-6 concentrations and nitric oxide levels, which are known to increase during endotoxemia in animals genetically devoid of the type 1 IL-1 receptor . Endotoxin (10 mg/kg Escherichia coli, injected intraperitoneally) (LD100) or saline vehicle was administered to adult male C57BL/129J wild-type control mice and C57BL/129J knockout mice possessing a homozygous deletion of the type 1 IL-1 receptor . The integrity of the aortic endothelium was determined by comparisons of ultrastructure . Mice injected with sterile vehicle showed normal endothelial ultrastructure with intact membranes . Wild-type and knockout control animals receiving saline vehicle showed a complete aortic endothelium (29.11 +/- .27 and 30.85 +/- .21 intact endothelial cells per millimeter of internal elastic lamina (IEL), respectively, p = N.S.) . Endotoxin-treated wild-type animals showed extensive endothelial damage with most sections showing only denuded IEL on the luminal surface (1.83 +/- .38 cells/mm IEL, p < .001 vs . control) . Knockout animals treated with endotoxin showed complete maintenance of endothelial structural integrity (34.08 +/- .57 cells/mm IEL, p < .001 vs . endotoxin-treated wild type) with ultrastructural morphology appearing identical to those given saline vehicle . Also, no apparent correlation was observed between serum IL-6 concentrations or serum nitric oxide levels and aortic endothelial damage . The maintenance of endothelial integrity in animals devoid of the IL-1 receptor confirms earlier observations of endothelial cell protection with IL-1 receptor antagonism and suggests that IL-1 contributes significantly to sepsis-induced endothelial damage. Pediatr Nephrol, 1997 Feb, 11(1), 36 - 9 Possible person-to-person transmission of Escherichia coli O111--associated hemolytic uremic syndrome; Boudailliez B et al.; Over a 3-month period, ten children (aged 1-13 years) from a 15-km radius in southern Picardy developed typical D+ hemolytic uremic syndrome (HUS) . Polymerase chain reaction, using two pairs of verocytotoxin 1-(VT1) and VT2-specific oligonucleotide primers and an internal control was used to detect VT genes directly from stools samples . VT2 gene was detected in seven of nine patients' stools and in 5 of 14 contacts' stool samples . A VT2-producing Escherichia coli (VTEC) O111 was isolated from five of nine children's stools and in 3 adults' stools of the 14 tested . A retrospective case-control study was performed which showed a higher rate of absence in school A, where the first four cases were detected, compared with a control school . The odds ratio for the whole school was 2.77 (confidence interval 1.46-5.26), and 15 (confidence interval 2.54-115.6) if only the nursery classes were considered . A culture of all food samples from households was always negative for VTEC . A retrospective cohort study performed in 89% of children attending school A showed no linkage between food or drink and gastroenteritis . These findings emphasize the potential for person-to-person transmission of VT2-producing E . coli O111, since the only salient risk factor was close contact. Nat Struct Biol, 1997 Feb, 4(2), 133 - 9 The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome; Rohrwild M et al.; HslVU is a new two-component protease in Escherichia coli composed of the proteasome-related peptidase HslIV and the ATPase HsIU . We have used electron microscopy and image analysis to examine the structural organization of HslV and HslU homo-oligomers and the active HslVU enzyme . Electron micrographs of HslV reveal ring-shaped particles, and averaging of top views reveal six-fold rotational symmetry, in contrast to other beta-type proteasome subunits, which form rings with seven-fold symmetry . Side views of HslV show two rings stacked together, thus, HslV behaves as dodecamer . The ATPase HslU forms ring-shaped particles in the presence of ATP, AMP-PNP or ADP, suggesting that nucleotide binding, but not hydrolysis, is required for oligomerization . Subunit crosslinking, STEM mass estimation, and analysis of HslU top views indicate that HslU exists both as hexameric and heptameric rings . With AMP-PNP present, maximal proteolytic activity is observed with a molar ratio of HslU to HslV subunits of 1:1, and negative staining electron microscopy shows that HslV and HsIU form cylindrical four-ring structures in which the HsIV dodecamer is flanked at each end by a HslU ring. Nat Struct Biol, 1997 Feb, 4(2), 112 - 4 Phosphorylation destabilizes alpha-helices; Szilak L et al.; Phosphorylation of threonine destabilizes the leucine zipper of a bZIP protein by 4.6 kcal mol-1 dimer-1, which reduces DNA binding 100-fold . This decrease in stability reflects the low alpha-helix forming propensity of a phosphorylated threonine. Nat Struct Biol, 1997 Feb, 4(2), 101 - 4 The RecA hexamer is a structural homologue of ring helicases; Yu X et al.; The RecA protein forms a hexameric ring that is similar to the core of the F1-ATPase . Several lines of evidence suggest that this hexamer may be a structural homologue of ring helicases. Biochem J, 1997 Feb 1, 321 ( Pt 3), 623 - 7 Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Bertrand L et al.; In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site . Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences . In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine . Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity . Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000- and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold . In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase . CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins . The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis . Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity . These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase. Biochem J, 1997 Feb 1, 321 ( Pt 3), 615 - 21 Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase; Bertrand L et al.; Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain . The sequence of the kinase domain was also compared with proteins of known three-dimensional structure . No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase . This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed {Bazan, Fletterick and Pilkis (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 9642-9646} . However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase . A structural model of the 2-kinase domain based on adenylate kinase is proposed . It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain . It also allows residues potentially involved in catalysis and/or substrate binding to be predicted. Biochem J, 1997 Feb 1, 321 ( Pt 3), 583 - 5 Analysis of glycosylation sites on gp91phox, the flavocytochrome of the NADPH oxidase, by site-directed mutagenesis and translation in vitro; Wallach TM et al.; Flavocytochrome b558 of the NADPH oxidase which generates superoxide in phagocytic cells, is a alpha1 beta1 heterodimer of gp91phox and p22phox, which together form a membrane-spanning electron-transport chain that transfers electrons from NADPH in the cytosol to oxygen . The C-terminal portion of gp91phox is a member of the ferredoxin-NADP+ reductase family of reductases . Little is known of the organization of the N-terminal section of this molecule, which is associated with the two haem structures . It is N-glycosylated, and site-directed mutagenesis has been used to eliminate the five potential N-linked glycosylation consensus sites . Mutated cDNAs were expressed in vitro . This approach provided evidence for glycosylation of residues Asn131, Asn148 and Asn239, but not of Asn96 and Asn429. Am J Respir Crit Care Med, 1997 Feb, 155(2), 513 - 9 Impact of arachidonic versus eicosapentaenoic acid on exotonin-induced lung vascular leakage: relation to 4-series versus 5-series leukotriene generation; Grimminger F et al.; Escherichia coli hemolysin (HlyA) is a proteinaceous pore-forming exotoxin that is implicated as a significant pathogenicity factor in extraintestinal E . coli infections including sepsis . In perfused rabbit lungs, subcytolytic concentrations of the toxin evoke thromboxane-mediated vasoconstriction and prostanoid-independent protracted vascular permeability increase (11) . In the present study, the influence of submicromolar concentrations of free arachidonic acid (AA) and eicosapentaenoic acid (EPA) on the HlyA-induced leakage response was investigated . HlyA at concentration from 0.02 to 0.06 hemolytic units/ml provoked a dose-dependent, severalfold increase in the capillary filtration coefficient (Kfc), accompanied by the release of leukotriene(LT)B4, LTC4, and LTE4 into the recirculating buffer fluid . Simultaneous application of 100 nmol/L AA markedly augmented the HlyA-elicited leakage response, concomitant with an amplification of LTB4 release and a change in the kinetics of cysteinyl-LT generation . In contrast, 50 to 200 nmol/L EPA suppressed in a dose-dependent manner the HlyA-induced increase in Kfc values . This was accompanied by a blockage of 4-series LT generation and a dose-dependent appearance of LTB5, LTC5, and LTE5 . In addition, EPA fully antagonized the AA-induced amplification of the HlyA-provoked Kfc increase, again accompanied by a shift from 4-series to 5-series LT generation . We conclude that the vascular leakage provoked by HlyA in rabbit lungs is differentially influenced by free AA versus free EPA, related to the generation of 4- versus 5-series leukotrienes . The composition of lipid emulsions used for parenteral nutrition may thus influence inflammatory capillary leakage. Curr Opin Struct Biol, 1997 Feb, 7(1), 41 - 52 Chaperone-assisted protein folding; Martin J et al.; Molecular chaperones of the Hsp70 and chaperonin families are basic constituents of the cellular machinery that mediates protein folding . Recent functional and structural studies corroborate existing models for the mechanism of these components . Highlights of the past year include the X-ray crystallographic analysis of the peptide-binding domain of the Escherichia coli Hsp70 homolog, DnaK, the direct demonstration of protein folding in the central cavity of the chaperonin GroEL, and the visualization of conformational changes in GroEL during the chaperonin folding cycle. Curr Opin Struct Biol, 1997 Feb, 7(1), 76 - 85 Lac repressor-operator complex; Kercher MA et al.; For many years the lac operon of Escherichia coli has been the paradigm for gene regulation . Recently, the structures of the lac repressor core bound to isopropyl-beta-D-1-thiogalactoside (IPTG), the intact apo lac repressor, the intact lac repressor complexes with IPTG and a 21-base-pair symmetric operator, and the refined headpiece of the repressor have been determined . These structures have provided a framework for understanding a wealth of biochemical and genetic information . An analysis of these structures, as well as a description of their function and a comparison to homologous proteins, is now possible. Br J Pharmacol, 1997 Feb, 120(3), 357 - 66 The inhibitory effects of mercaptoalkylguanidines on cyclo-oxygenase activity; Zingarelli B et al.; 1 . It has been proposed that in inflammatory conditions, in which both the inducible isoforms of nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) are induced, inhibition of NOS also results in inhibition of arachidonic acid metabolism . In the present study we have investigated whether mercaptoalkylguanidines, a novel class of selective iNOS inhibitors, may also influence the activity of cyclo-oxygenase (COX) . Therefore, the effect of mercaptoethylguanidine (MEG) and related compounds on the activity of the constitutive (COX-1) and the inducible COX (COX-2) was investigated in cells and in purified enzymes . Aminoguanidine, NG-methyl-L-arginine (L-NMA) and NG-nitro-L-arginine methyl ester (L-NAME) were also studied for comparative purposes . 2 . Western blot analysis demonstrated a significant COX-1 activity in unstimulated J774 macrophages and in unstimulated human umbilical vein endothelial cells (HUVEC) . Immunostimulation of the J774 macrophages by endotoxin (lipopolysaccharide of E . coli, LPS 10 micrograms ml-1) and interferon gamma (IFN gamma, 100 u ml-1) for 6 h resulted in a significant induction of COX-2, and a down-regulation of COX-1 . No COX-2 immunoreactivity was detected in unstimulated HUVEC or unstimulated J774 cells . Therefore, in subsequent studies, the effect of mercaptoalkylguanidines on COX-1 activity was studied in HUVEC stimulated with arachidonic acid for 6 h, and in J774 cells stimulated with arachidonic acid for 30 min . The effect of mercaptoalkylguanidines on COX-2 activity was studied in immunostimulated J774 macrophages, both on prostaglandin production by endogenous sources, and on prostaglandin production in response to exogenous arachidonic acid stimulation . In addition, the effect of mercaptoalkylguanidines on purified COX-1 and COX-2 activities was also studied . 3 . In experiments designed to measure COX-1 activity in HUVEC, the cells were stimulated by arachidonic acid (15 microM) for 6 h . This treatment induced a significant production of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha, the stable metabolite of prostacyclin), while nitrite production was undetectable by the Griess reaction . MEG (1 microM to 3 mM) caused a dose-dependent inhibition of the accumulation of 6-keto-PGF1 alpha, with an IC50 of 20 microM . However, aminoguanidine, L-NAME or L-NMA (up to 3 mM) did not affect the production of 6-keto-PGF1 alpha in this experimental system . In experiments designed to measure COX-1 activity in J774.2 macrophages, the cells were stimulated by arachidonic acid (15 microM) for 30 min; this also induced a significant production of 6-keto-PGF1 alpha and MEG (1 microM to 3 mM), aminoguanidine (at 1 and 3 mM), but neither L-NAME nor L-NMA inhibited the production of prostaglandins . 4 . In experiments designed to measure prostaglandin production by COX-2 with endogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h in the absence or presence of various inhibitors . In experiments designed to measure prostaglandin production by COX-2 with exogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h, followed by a replacement of the culture medium with fresh medium containing arachidonic acid and various inhibitors . Both of these treatments induced a significant production of 6-keto-PGF1 alpha . Nitrite production, an indicator of NOS activity, was moderately increased after immunostimulation . MEG (1 microM to 3 mM) caused a dose-dependent inhibition of the accumulation of COX metabolites . Similar inhibition of LPS-stimulated 6-keto PGF1 alpha production was shown by other mercaptoalkylguanidines (such as N-methyl-mercaptoethylguanidine, N,N'-dimethyl-mercaptoethylguanidine, S-methyl-mercaptoethylguanidine and guanidino-ethyldisulphide), with IC50 values ranging between 34-55 microM . However, aminoguanidine, L-NAME and L-NMA (up to 3 mM) did not affect the production of prostaglandins.(ABSTRACT TRUNCATED) Brain Res Mol Brain Res, 1997 Feb, 44(1), 125 - 33 Long-term histological follow-up of genetically modified myoblasts grafted into the brain; Lisovoski F et al.; Although primary muscle cells have been used as intracerebral vehicles for transgene expression in the past, data concerning their long-term survival after grafting into the brain, and the reaction of the host tissue to their implantation are lacking . In order to study these aspects, we have implanted, into the brain, primary muscle cells infected ex vivo with recombinant retroviruses carrying the E . coli LacZ gene . The muscle cells were delivered stereotaxically into different areas of the brain of adult rats and the grafts were analyzed up to 105 days after implantation . Intraventricular implantations did not lead to surviving grafts . In contrast, myoblasts developed when they were grafted into gray or white matter regions . They appeared numerous during the first weeks, but decreased dramatically in number over time . Over months, the grafts appeared to fill up with collagen . Astrocytes elaborated a continuous glia limitans surrounding the implant . Blood vessels coming from the host tissue were found within the grafts . The blood-brain barrier was permanently disrupted within the transplants . beta-Galactosidase activity was abundant during the first weeks, but decreased to a very low level subsequently . This decrease paralleled that of the number of muscle cells . In conclusion, myoblasts transplanted into the adult brain survived only temporarily, which implies a transient transgene expression . In addition, before being eliminated, muscle cells were surrounded by a glia limitans, which may limit exchanges with the host tissue . Altogether, these results suggest that intracerebral transplantation of myoblasts may possibly provide a relevant vehicle only for short-term delivery of a gene product. Genes Dev, 1997 Feb 1, 11(3), 371 - 82 Disruption of the terminal base pairs of retroviral DNA during integration; Scottoline BP et al.; Integrase catalyzes two essential steps in the integration of the retroviral genome--end processing and strand transfer--both of which require the interaction of integrase with viral att sites located at the ends of viral genomic DNA . These two different polynucleotidyl transfer reactions are apparently carried out by a single active site . The end product of these reactions, the integrated provirus, does not undergo transposition and remains a stable part of the host cell genome . A central question in understanding the mechanism of integration is how a single active site accomplishes two distinct polynucleotidyl transfer reactions . We propose that integrase distorts DNA substrates to accommodate both reactions within the active site . Evidence is provided for disruption of base-pairing at the terminus of viral DNA during end processing . Furthermore, we show that this end fraying is a required step in end processing and that it appears to occur after initial binding of the viral DNA end . This requirement for base-pair disruption may account for the inability of integrase to use internal sites on DNA molecules as viral att sites . The specificity of integrase for DNA ends solves a problem posed by the long terminal repeat structure of the viral genome, and may help to prevent transposition of integrated proviruses. Genes Dev, 1997 Feb 1, 11(3), 358 - 70 RLF2, a subunit of yeast chromatin assembly factor-I, is required for telomeric chromatin function in vivo; Enomoto S et al.; In the yeast Saccharomyces cerevisiae, telomere repeat DNA is assembled into a specialized heterochromatin-like complex that silences the transcription of adjacent genes . The general DNA-binding protein Rap1p binds telomere DNA repeats, contributes to telomere length control and to telomeric silencing, and is a major component of telomeric chromatin . We identified Rap1p localization factor 2 (RLF2) in a screen for genes that alleviate antagonism between telomere and centromere sequences on plasmids . In rlf2 mutants, telomeric chromatin is perturbed: Telomeric silencing is reduced and Rap1p localization is altered . In wild-type cells, Rap1p and telomeres localize to bright perinuclear foci . In rlf2 strains, the number of Rap1p foci is increased, Rap1p staining is more diffuse throughout the nucleus, Rap1p foci are distributed in a much broader perinuclear domain, and nuclear volume is 50% larger . Despite the altered distribution of Rap1p in rlf2 mutant cells, fluorescence in situ hybridization to subtelomeric repeats shows that the distribution of telomeric DNA is similar in wild-type and mutant cells . Thus in rlf2 mutant cells, the distribution of Rap1p does not reflect the distribution of telomeric DNA . RLF2 encodes a highly charged coiled-coil protein that has significant similarity to the p150 subunit of human chromatin assembly factor-I(hCAF-I), a complex that is required for the DNA replication-dependent assembly of nucleosomes from newly synthesized histones in vitro . Furthermore, RLF2 is identical to CAC1, a subunit of yeast chromatin assembly factor-I (yCAF-I) which assembles nucleosomes in vitro . In wild-type cells, epitope-tagged Rlf2p expressed from the GAL10 promoter localizes to the nucleus with a pattern distinct from that of Rap1p, suggesting that Rlf2p is not a component of telomeric chromatin . This study provides evidence that yCAF-I is required for the function and organization of telomeric chromatin in vivo . We propose that Rlf2p facilitates the efficient and timely assembly of histones into telomeric chromatin. Genes Dev, 1997 Feb 1, 11(3), 334 - 44 Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing; Xiao SH et al.; ASF/SF2 is a member of a conserved family of splicing factors known as SR proteins . These proteins, which are necessary for splicing in vitro, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repeating Arg-Ser dipeptides (RS domains) . Previous studies have suggested that RS domains participate in protein-protein interactions with other RS domain-containing proteins . Here we provide evidence that the RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not sufficient, for binding to the U1 snRNP-specific 70-kD protein (70K) in vitro . An apparent interaction of the isolated RS domain with 70K was observed if contaminating RNA was not removed, suggesting a nonspecific bridging between the basic RS domain, RNA, and 70K . In vitro phosphorylation of recombinant ASF/SF2 both significantly enhanced binding to 70K and also eliminated the RS domain-RNA interaction . Providing evidence that these interactions are relevant to splicing, ASF/SF2 can bind selectively to U1 snRNP in an RS domain-dependent, phosphorylation-enhanced manner . We also describe conditions that reveal for the first time a phosphorylation requirement for ASF/SF2 splicing activity in vitro. Exp Parasitol, 1997 Feb, 85(2), 121 - 34 Plasmodium falciparum: an epitope within a highly conserved region of the 47-kDa amino-terminal domain of the serine repeat antigen is a target of parasite-inhibitory antibodies; Fox BA et al.; Previously, the Plasmodium falciparum serine repeat antigen has been shown to be protective in primate models of malaria immunity and also to be a target of in vitro parasite-inhibitory antibodies . To further define parasite-inhibitory epitopes a series of deletions from the amino-terminal 47-kDa domain of the serine repeat antigen (SERA) were constructed as glutathione-S-transferase fusion proteins . Several GST-SERA fusion proteins were used to vaccinate mice with Freund's adjuvant and the resulting immune sera were used to assay for the inhibition of P . falciparum invasion of erythrocytes in vitro . The minimal epitope shown to be the target of invasion-blocking antibodies was SERA amino acids 17-165 . Additional GST-SERA deletion constructs of the 47-kDa domain were developed and evaluated for reactivity, by Western immunoblot analysis, with a parasite-inhibitory murine monoclonal antibody (mAb 43E5), a parasite-inhibitory pooled goat polyclonal sera, and a pooled human Nigerian immune serum . The parasite-inhibitory epitope defined by mAb 43E5 was mapped to SERA amino acids 17-110 and, at least, part of the epitope was defined to include amino acids in the region of amino acids 59-72 . The parasite-inhibitory epitope recognized by mAb 43E5 appears to be well conserved between diverse geographical isolates of P . falciparum . The results have relevance for malaria vaccine development and suggest that an appropriately designed recombinant SERA antigen produced from a synthetic gene in Escherichia coli may be an effective component of a candidate malaria vaccine. J Virol Methods, 1997 Feb, 64(1), 103 - 10 Simple hand-held devices for the efficient infection of plants with viral-encoding constructs by particle bombardment; Gal-On A et al.; An efficient method for infection of plants with a cloned potyvirus by particle bombardment has been described (Gal-On et al., 1995) . A simplified method is described now whereby a vaccuum chamber and helium propulsive gas are not required to achieve a high efficiency of infection . The new device-the 'HandGun'--is hand-held, and easily constructed from readily available materials . With this technique it is possible to bombard soft plants and seedings that do not survive particle bombardment by other devices . bombardment of C . pepo plants with a full length clone of zucchini yellow mosaic potyvirus results in approximately 100% infection at 100 pg cDNA per plant using air or helium to propel the microprojectiles . The HandGun is 10(5)-fold more efficient than mechanical inoculation . Tungsten and gold were found to be the most efficient materials tested for use as microprojectiles . Crude extracts of plasmids from E . coli were found to be effective, as well as column-purified cDNA . A functional, simple version of the HandGun--'the Blowpipe'--was also constructed, which does not require an electrically controlled valve . Plants can be inoculated with plant viruses from sap with the HandGun. Drug Metab Dispos, 1997 Feb, 25(2), 133 - 9 Diazepam metabolism by cDNA-expressed human 2C P450s: identification of P4502C18 and P4502C19 as low K(M) diazepam N-demethylases; Jung F et al.; The present study provides a detailed kinetic analysis of diazepam metabolism by all four known members of the human P4502C subfamily expressed from their cDNAs in Escherichia coli . Both P4502C18 and P4502C19 were found to be low K(M) diazepam N-demethylases with apparent K(M) values of 24 +/- 4 microM and 21 +/- 3 microM, respectively . These values closely resemble the low K(M) component of diazepam N-demethylase activity exhibited by human liver microsomes . In addition, P4502C19 also catalyzed diazepam 3-hydroxylation with a K(M) value of 21 +/- 9 microM . Although P4502C8 was essentially inactive in catalyzing diazepam metabolism, P4502C9 catalyzed the N-demethylation with a relatively high K(M) of 80 +/- 15 microM and an overall 3- to 6-fold lower catalytic efficiency, compared with P4502C18 and P4502C19, respectively . At a substrate concentration of 10 microM, diazepam N-demethylation in a panel of human liver microsomes was inhibited 42 +/- 12% (mean +/- SD, N = 6) by a polyclonal anti-CYP2C antibody . In the same experiment, 3-hydroxylation remained unaffected (<10% inhibition) . 1 microM of the CYP3A inhibitor ketoconazole inhibited 37 +/- 19% of the N-demethylation and 86 +/- 5% of 3-hydroxylation . Estimates of relative contributions to diazepam N-demethylation of P4502C9 (8 +/- 4%), P4502C18 (<2%), and P4502C19 (33 +/- 14%) and to diazepam 3-hydroxylation of P4502C19 (9 +/- 3%) based on the kinetic parameters of the recombinant enzymes and on specific contents of the individual 2C P450s determined in immunoblots are consistent with the inhibition data . In conclusion, these data confirm that both P4502C19 and P4503A are major contributors to human liver microsomal diazepam N-demethylation at low substrate concentrations, whereas P4503A is the major enzyme responsible for 3-hydroxylation. J Cell Biochem, 1997 Feb, 64(2), 217 - 24 Isolation, characterization, and chromosomal mapping of a novel cDNA clone encoding human selenium binding protein; Chang PW et al.; We have isolated the full-length human 56 kDa selenium binding protein (hSP56) cDNA clone, which is the human homolog of mouse 56 kDa selenium binding protein . The cDNA is 1,668 bp long and has an open reading frame encoding 472 amino acids . The calculated molecular weight is 52.25 kDa and the estimated isoelectric point is 6.13 . Using Northern blot hybridization, we found that this 56 kDa selenium binding protein is expressed in mouse heart with an intermediate level between those found in liver/lung/kidney and intestine . We have also successfully expressed hSP56 in Escherichia coli using the expression vector-pAED4 . The hSP56 gene is located at human chromosome 1q21-22). Anat Rec, 1997 Feb, 247(2), 214 - 24 Ultrastructural and immunocytochemical study of the pulmonary intravascular macrophages of Escherichia coli lipopolysaccharide-treated sheep; Singh B et al.; BACKGROUND: Pulmonary intravascular macrophages (PIMs) of sheep, cattle, goats, and horses have a novel heparin-sensitive chain of globules, called a surface coat, on their plasma membrane . The globules are arranged at a distance of 32-39 nm from the plasma membrane of PIMs . Intravascular nonbiological tracer particles complex with these globules prior to their endocytosis by the PIMs . METHODS: We conducted a preliminary in vivo time-course study in sheep to investigate responses of the coat globules to a single dose of Escherichia coli lipopolysaccharide (E . coli LPS) . Six sheep (6-9 months of age) were used in this study, and five of them were intravenously injected with E . coli (1 microgram/kg body weight) and euthanised at 3, 8, 10, 30, and 180 min (n = 1 each) after treatment . One sheep injected with saline solution served as the control . Acid phosphatase (AcPase) cytochemistry and immunocytochemistry using a polyclonal antibody were employed to localize secretory activity and E . coli LPS respectively in the PIMs . RESULTS: The surface coat of PIMs disappeared rapidly following the LPS administration . Escherichia coli LPS micelles and coat globules were colocalized as a complex in the endosomes of PIMs . At 8-10 min following the treatment, endosomal and the other membranes were disrupted, and the LPS was identified in cytoplasm and nuclear matrix of PIMs simultaneously with the development of pulmonary interstitial edema . Progression of AcPase reactivity along the nucleus-Golgi complex axis coupled with intense buildup of coated transport vesicles within 30 min of the LPS injection suggested enhanced biosynthetic activity in the PIMs . CONCLUSIONS: This study provides initial data on the sensitivity of the coat globules and their possible role in the endocytosis of E . coli LPS by the PIMs . Rapid biosynthetic activation of PIMs concurrent with loss of the coat and treatment with the LPS probably results in the secretion of inflammatory substances and contributes to the enhanced susceptibility of sheep to endotoxin-induced lung pathology. Anal Biochem, 1997 Feb 1, 245(1), 85 - 92 Sequestration stabilizes lac repressor-DNA complexes during gel electrophoresis; Vossen KM et al.; The gel electrophoresis mobility shift assay is widely used for both qualitative and quantitative characterization of protein-nucleic acid interactions . Often it is found that protein-nucleic acid complexes persist within gels for much longer than would be expected on the basis of their free solution lifetimes . Excluded volume and matrix-interaction mechanisms have been proposed to account for the enhanced stabilities of complexes within gels . To test these mechanisms, we have investigated the influences of gel composition and concentration on the pseudo first-order dissociation kinetics of complexes containing the Escherichia coli lactose (lac) repressor protein and lactose promoter DNA . In both polyacrylamide and agarose gels, dissociation rates were slower than those in free solution and decreased with increasing gel concentration . This result is inconsistent with mechanisms of stabilization that require specific interactions with the gel matrix . Under standard reaction conditions, free solution values of kdiss were proportional to {DNA}0.83 +/- 0.11, while in 10% polyacrylamide gels kdiss values were proportional to {DNA}0.48 +/- 0.09 . These results suggest that the lifetimes of lac repressor-DNA complexes in free solution are limited by their encounter frequency with molecules of DNA or with protein-DNA complexes; some or all of the stabilization observed in gels may be due to a reduction in this frequency. Curr Opin Genet Dev, 1997 Feb, 7(1), 105 - 13 MutS homologs in mammalian cells; Fishel R et al.; Alterations of the human mismatch repair genes have been linked to hereditary non-polyposis colon cancer (HNPCC) as well as to sporadic cancers that exhibit microsatellite instability . The human mismatch repair genes are highly conserved homologs of the Escherichia coli MutHLS system . Six MutS homologs have been identified in Saccharomyces cerevisiae and four MutS homologs have been identified in human cells . At least three of these eukaryotic MutS homologs are involved in the recognition/binding of mispaired nucleotides and nucleotide lesions . MSH2 plays a fundamental role in mispair recognition whereas MSH3 and MSH6 appear to modify the specificity of this recognition . The redundant functions of MSH3 and MSH6 explain the greater prevalence of hmsh2 mutations in HNPCC families. Gastroenterology, 1997 Feb, 112(2), 437 - 43 Guanosine 3',5'-cyclic monophosphate-dependent protein kinase II mediates heat-stable enterotoxin-provoked chloride secretion in rat intestine; Vaandrager AB et al.; BACKGROUND & AIMS: Escherichia coli heat-stable enterotoxins (STa) provoke electrogenic Cl- secretion in the intestine through a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent signal transduction pathway . The cGMP receptor involved in the activation of the Cl- channel is not known with certainty but may comprise either adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (cAK) or cGMP-dependent protein kinase (cGK) type II . The aim of this study was to discriminate between these possibilities using specific kinase inhibitors . METHODS: Intestinal electrogenic Cl- secretion was determined by measuring short-circuit current (Isc) in a Ussing chamber . RESULTS: The general protein kinase inhibitors staurosporine and H-8 inhibited rat cGK II activity in vitro with 50% inhibitory concentration values of 4 nmol/L and 3 mumol/L, respectively, which are lower than those reported for cAK . Both staurosporine and H-8, when added to rat proximal colon at concentrations that did not affect the Isc response to 8-bromo-cAMPS, inhibited the STa- and 8-bromo-cGMP-provoked Isc response for more than 80% . Furthermore, the relative specific cGK inhibitor Rp isomer of 8-(chlorophenylthio)-cGMP, but not the cAK inhibitor RP isomer of (Rp) 8-bromo-cAMPS, inhibited the Isc response to submaximal levels of STa in rat proximal colon . CONCLUSIONS: These data provide further evidence for an important role of cGK II in STa-mediated Cl- secretion in native rat intestinal epithelium. Appl Environ Microbiol, 1997 Feb, 63(2), 785 - 9 In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp . strain KOD1; Yan Z et al.; The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp . strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli . The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da . The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH) . Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH . CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess . In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo . These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit. Appl Environ Microbiol, 1997 Feb, 63(2), 771 - 4 Limitations of highly sensitive enzymatic presence-absence tests for detection of waterborne coliforms and Escherichia coli; Van Poucke SO et al.; This study presents evidence for the unfeasibility of enzymatic presence-absence tests to detect one total coliform or one Escherichia coli organism in 100 ml of drinking water within a working day . The results of field trials with prototype chemiluminometric procedures indicated that the sensitivity-boosting measures that are essential to achieve the required speed compromise the specificity of the tests. Appl Environ Microbiol, 1997 Feb, 63(2), 761 - 2 Mutational analysis of the feedback sites of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli; Kikuchi Y et al.; In Escherichia coli, aroF, aroG, and aroH encode 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isozymes that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively . In vitro chemical mutagenesis of the cloned aroG gene was used to identify residues and regions of the polypeptide essential for phenylalanine feedback inhibition. Appl Environ Microbiol, 1997 Feb, 63(2), 703 - 9 Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains; Kuhnert P et al.; We describe a rational approach to simultaneously test Escherichia coli strains for the presence of known virulence genes in a reverse dot blot procedure . Specific segments of virulence genes of E . coli designed to have similar hybridization parameters were subcloned on plasmids and subsequently amplified by PCR as unlabeled probes in amounts sufficient to be bound to nylon membranes . Various pathogenic isolates and laboratory strains of E . coli were probed for the presence of virulence genes by labeling the genomic DNA of these strains with digoxigenin and then hybridizing them to the prepared nylon membranes . These hybridization results demonstrated that besides the E . coli K-12 safety strain derivatives, E . coli B and C strains are also devoid of genes encoding any of the investigated virulence factors . In contrast, pathogenic E . coli control strains, used to evaluate the method, showed typical hybridization patterns . The described probes and their easy application on a single filter were shown to provide a useful tool for the safety assessment of E . coli strains to be used as hosts in biotechnological processes . This approach might also be used for the identification and characterization of clinically significant E . coli isolates from human and animal species. J Bacteriol, 1997 Feb, 179(4), 1413 - 6 Role of His243 in the phosphatase activity of EnvZ in Escherichia coli; Skarphol K et al.; EnvZ undergoes autophosphorylation at His243 and subsequently transfers the phosphate group to OmpR . EnvZ also possesses an OmpR-phosphate phosphatase activity . We examined the role of His243 in the phosphatase function by replacing His with either Val, Tyr, Ser, Asp, or Asn . EnvZH243V and EnvZH243Y were both shown to possess phosphatase activity in vitro . In addition, the mutant proteins were able to reduce the high level of OmpR-phosphate present in the envZ473 strain . These results indicate that His243 of EnvZ is not essential for stimulating the dephosphorylation of OmpR-phosphate. J Bacteriol, 1997 Feb, 179(4), 1400 - 3 Evidence that KpsT, the ATP-binding component of an ATP-binding cassette transporter, is exposed to the periplasm and associates with polymer during translocation of the polysialic acid capsule of Escherichia coli K1; Bliss JM et al.; KpsT utilizes ATP to effect translocation of the polysialic acid capsule of Escherichia coli K1 . We have previously proposed a mechanistic model for the action of this protein . Here, we provide evidence to support two predictions of the model: that KpsT associates with polymer and that KpsT is accessible from the periplasmic surface of the inner membrane. J Bacteriol, 1997 Feb, 179(4), 1393 - 9 Replication and segregation of a miniF plasmid during the division cycle of Escherichia coli; Helmstetter CE et al.; Replication of the miniF plasmid pML31 was examined during the division cycle of Escherichia coli growing with doubling times between 40 and 90 min at 37 degrees C and compared to the replication of plasmid pBR322 and the minichromosome pAL70 . The replication pattern of pML31 was indistinguishable from that of pBR322 at all growth rates and very different from the cell-cycle-specific replication of the minichromosome . It is concluded that both pML31 and pBR322 plasmids can replicate at all stages of the division cycle, with a probability of replication that increases gradually, but perhaps not exponentially, during the cycle . In contrast, the modes of segregation of pML31 and pBR322 plasmids into daughter cells at division appeared to differ, raising the possibility that pML31 may segregate in a nonrandom fashion similar to that of chromosomes and minichromosomes. J Bacteriol, 1997 Feb, 179(4), 1337 - 43 MalFGK complex assembly and transport and regulatory characteristics of MalK insertion mutants; Lippincott J et al.; MalK is a peripheral cytoplasmic membrane protein that has multiple activities in Escherichia coli . It associates with integral cytoplasmic membrane proteins MalF and MalG to form the maltose transport complex (MalFGK), a member of the ATP-binding cassette (ABC) superfamily of proteins . In addition, MalK participates in two different regulatory pathways which modulate mal gene expression and MalFGK transport activity . We have created a set of malK mutations for analysis of the protein's structure and folding . These mutations, distributed throughout malK, are all similar insertions of 31 codons . The ability of each mutant to function in maltose transport and MalK-dependent regulation was characterized . Furthermore, we have exploited a sensitive biochemical assay to classify our MalK insertion mutants into two additional categories: MalFGK complex assembly proficient and complex assembly defective . The regions containing the insertions in the assembly-proficient class should correspond to areas within MalK that are surface exposed within the MalFGK complex . Affected regions in assembly-deficient mutants may be involved in critical structural contacts within the complex . One mutant apparently blocks assembly at an intermediate stage prior to oligomerization of the final MalFGK complex . This work contributes to the analysis of ABC transport proteins and to the study of the assembly process for hetero-oligomeric membrane proteins. J Bacteriol, 1997 Feb, 179(4), 1317 - 23 Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae; Hsu T et al.; Colonization of the swine respiratory tract by Mycoplasma hyopneumoniae is accomplished by specific binding to the cilia of the mucosal epithelial cells . Previous studies have implicated a 97-kDa outer membrane-associated protein, P97, that appeared to mediate this interaction . In order to further define the role of P97 in adherence to porcine cilia, the structural gene was cloned and sequenced, and the recombinant products were analyzed . Monoclonal antibodies were used to identify recombinant clones in a genomic library expressed in an opal suppressor host because of alternate codon usage by mycoplasmas . The gene coding for P97 was then identified by Tn1000 mutagenesis of recombinant clones . DNA sequence analysis revealed an open reading frame coding for a 124.9-kDa protein with a hydrophobic transmembrane spanning domain . The N-terminal sequence of purified P97 mapped at amino acid position 195 of the translated sequence, indicating that a processing event had occurred in M . hyopneumoniae . Both recombinant P97 protein expressed in an Escherichia coli opal suppressor host and M . hyopneumoniae bound specifically to swine cilia, and the binding was inhibited by heparin and fucoidan, thus supporting the hypothesis that P97 was actively involved in binding to swine cilia in vivo. J Bacteriol, 1997 Feb, 179(4), 1298 - 306 Molecular characterization of glucokinase from Escherichia coli K-12; Meyer D et al.; glk, the structural gene for glucokinase of Escherichia coli, was cloned and sequenced . Overexpression of glk resulted in the synthesis of a cytoplasmic protein with a molecular weight of 35,000 . The enzyme was purified, and its kinetic parameters were determined . Its Km values for glucose and ATP were 0.78 and 3.76 mM, respectively . Its Vmax was 158 U/mg of protein . A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression . Under all conditions tested, only growth on glucose reduced the expression of glk by about 50% . A fruR mutation slightly increased the expression of glk-lacZ, whereas the overexpression of plasmid-encoded fruR+ weakly decreased expression . A FruR consensus binding motif was found 123 bp upstream of the potential transcriptional start site of glk . Overexpression of glk interfered with the expression of the maltose system . Repression was strongest in strains that exhibited constitutive mal gene expression due to endogenous induction and, in the absence of a functional MalK protein, the ATP-hydrolyzing subunit of the maltose transport system . It was least effective in wild-type strains growing on maltose or in strains constitutive for the maltose system due to a mutation in malT rendering the mal gene expression independent of inducer . This demonstrates that free internal glucose plays an essential role in the formation of the endogenous inducer of the maltose system. J Bacteriol, 1997 Feb, 179(4), 1239 - 45 DNA-binding determinants of sigma 54 as deduced from libraries of mutations; Guo Y et al.; PCR mutagenesis was used to obtain libraries of mutations in the region between amino acids 300 and 400 in the DNA-binding domain of Escherichia coli sigma 54 . Two hundred changes that did not alter function were identified . These were compared with a somewhat smaller number of changes that did alter function . Several important regions were identified . Single point mutations in two of these, near amino acids 363 and 383, destroyed the ability of sigma to bind DNA, as assayed by band shift analysis . A third segment from amino acids 327 to 347 is also a candidate for contributing to DNA binding . Comparison with data in the literature leads to testable proposals for the complex mode of DNA binding that is associated with sigma 54. J Bacteriol, 1997 Feb, 179(4), 1230 - 8 Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp . pallidum rare outer membrane protein (Tromp2); Champion CI et al.; In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp . pallidum rare outer membrane protein (TROMP), designated Tromp2 . The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala . The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3 . The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins . Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector . Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane . Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations . Following Triton X-114 extraction and phase separation of T . pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase . Alkali and high-salt treatment of purified outer membrane from T . pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein . Whole-mount immunoelectron microscopy of E . coli cells expressing rTromp2 showed specific surface antibody binding . These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T . pallidum. J Bacteriol, 1997 Feb, 179(4), 1174 - 9 Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp . strain 7; Suzuki T et al.; The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp . strain 7 was cloned, sequenced, and expressed in Escherichia coli . The recombinant Sulfolobus sp . enzyme was extremely stable to heat . The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts . Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric . Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources . These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family. J Bacteriol, 1997 Feb, 179(4), 1135 - 42 A conserved glutamate residue, Glu-257, is important for substrate binding and transport by the Escherichia coli mannitol permease; Saraceni-Richards CA et al.; The mannitol permease, or D-mannitol-specific enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) of Escherichia coli, both transports and phosphorylates its substrate . Previous analyses of the amino acid sequences of PTS permeases specific for various carbohydrates in different species of bacteria revealed several regions of similarity . The most highly conserved region includes a GIXE motif, in which the glutamate residue is completely conserved among the permeases that contain this motif . The corresponding residue in the E . coli mannitol permease is Glu-257, which is located in a large putative cytoplasmic loop of the transmembrane domain of the protein . We used site-directed mutagenesis to investigate the role of Glu-257 . The properties of proteins with mutations at position 257 suggest that a carboxylate side chain at this position is essential for mannitol binding . E257A and E257Q mutant proteins did not bind mannitol detectably, while the E257D mutant could still bind this substrate . Kinetic studies with the E257D mutant protein also showed that a glutamate residue at position 257 of this permease is specifically required for efficient mannitol transport . While the E257D permease phosphorylated mannitol with kinetic parameters similar to those of the wild-type protein, the Vmax for mannitol uptake by this mutant protein is less than 5% that of the wild type . These results suggest that Glu-257 of the mannitol permease and the corresponding glutamate residues of other PTS permeases play important roles both in binding the substrate and in transporting it through the membrane. J Bacteriol, 1997 Feb, 179(4), 1117 - 25 Specific DNA cleavage mediated by the integrase of conjugative transposon Tn916; Taylor KL et al.; The conjugative transposon Tn916 encodes a protein called INT(Tn916) which, based on DNA sequence comparisons, is a member of the integrase family of site-specific recombinases . Integrase proteins such as INT(lambda), FLP, and XERC/D that promote site-specific recombination use characteristic, conserved amino acid residues to catalyze the cleavage and ligation of DNA substrates during recombination . The reaction proceeds by a two-step transesterification reaction requiring the formation of a covalent protein-DNA intermediate . Different requirements for homology between recombining DNA sites during integrase-mediated site-specific recombination and Tn916 transposition suggest that INT(Tn916) may use a reaction mechanism different from that used by other integrase recombinases . We show that purified INT(Tn916) mediates specific cleavage of duplex DNA substrates containing the Tn916 transposon ends and adjacent bacterial sequences . Staggered cleavages occur at both ends of the transposon, resulting in 5' hydroxyl protruding ends containing coupling sequences . These are sequences that are transferred with the transposon from donor to recipient during conjugative transposition . The nature of the cleavage products suggests that a covalent protein-DNA linkage occurs via a residue of INT(Tn916) and the 3'-phosphate group of the DNA . INT(Tn916) alone is capable of executing the strand cleavage step required for recombination during Tn916 transposition, and this reaction probably occurs by a mechanism similar to that of other integrase family site-specific recombinases. J Bacteriol, 1997 Feb, 179(4), 1082 - 9 Conservation of msp, the gene encoding the major outer membrane protein of oral Treponema spp; Fenno JC et al.; The major surface protein (Msp) of Treponema denticola has been implicated as a mediator of the interaction between the spirochete and the gingival epithelium in periodontal diseases . Previous studies showed that the Msp of T . denticola ATCC 35405 had porin activity, depolarized epithelial cell membranes, bound to extracellular matrix components of epithelial cells, and formed a regular hexagonal surface array in the treponemal outer membrane . The gene encoding Msp in ATCC 35405 was recently cloned, sequenced, and expressed in Escherichia coli (J . C . Fenno, K.-H . Muller, and B . C . McBride, J . Bacteriol . 178:2489-2496, 1996) . In the present study, we identified genes encoding Msp-like proteins in several oral spirochetes . A prominent heat-modifiable Msp-like protein having an apparent molecular mass of between 43 and 64 kDa was present in all oral spirochete strains tested . Antibodies raised against the ATCC 35405 Msp reacted strongly with the Msp proteins of T . denticola ATCC 35404 and T . vincentii, reacted very weakly with the Msp protein of T . denticola ATCC 33520, and did not react with T . denticola OTK, T . socranskii, and T . pectinovorum . The msp loci of the T . denticola strains and T . vincentii were identified in analyses using PCR with oligonucleotide primers derived from the DNA sequence flanking msp in ATCC 35405 . Southern blot analysis showed at least three groups of related msp DNA sequences . Comparison of DNA sequences of the 5' and 3' ends of the msp genes showed high sequence homology in the flanking regions and signal peptide coding regions, while the homologies between regions encoding the mature peptide were as low as 50% . The entire msp DNA sequences of T . denticola ATCC 33520 and OTK were determined, and the deduced Msp amino acid sequences were compared to the sequence of the previously reported Msp of ATCC 35405 . The results show that the msp locus is conserved in oral treponemes but that there are significant differences between the mature Msp peptides of different strains . Further studies of the antigenic domains, functional domains, and physical structures of Msp proteins, based on these results, will enhance understanding of the role of Msp in the cytopathology associated with oral spirochetes. J Bacteriol, 1997 Feb, 179(4), 1077 - 81 Carbon source-dependent synthesis of SecB, a cytosolic chaperone involved in protein translocation across Escherichia coli membranes; Seoh HK et al.; SecB is a cytosolic chaperone involved in protein translocation across cytoplasmic membranes in Escherichia coli . It has been shown to be required for efficient translocation of a subset of precursor proteins but is not essential for cell viability . This study investigated whether synthesis of SecB is growth rate dependent . Interestingly, the total amount of SecB synthesized in the cells was relatively small . Moreover, the levels of SecB were found to be carbon source dependent since more SecB was produced in cells grown in glycerol media than in cells grown in glucose media, regardless of the growth rate . This is in contrast to the other Sec proteins, whose synthesis is growth rate dependent and not related to glucose as a carbon source . In addition, cyclic AMP (cAMP) partially relieves the lower levels of SecB observed in glucose medium, a compensatory effect that depends on the presence of both cya and crp gene products . Thus, the glucose-dependent synthesis of SecB may be related to the cAMP-cAMP receptor protein complex-mediated activation. J Bacteriol, 1997 Feb, 179(4), 1051 - 8 Critical base pairs and amino acid residues for protein-DNA interaction between the TyrR protein and tyrP operator of Escherichia coli; Hwang JS et al.; In Escherichia coli K-12, the repression of tyrP requires the binding of the TyrR protein to the operator in the presence of coeffectors, tyrosine and ATP . This operator contains two 22-bp palindromic sequences which are termed TyrR boxes . Methylation, uracil, and ethylation interference experiments were used to identify the important sites in the TyrR boxes that make contacts with the TyrR protein . Methylation interference studies demonstrated that guanines at positions +8, -5, and -8 of the strong TyrR box and positions +8, -4, and -8 of the weak box are close to the TyrR protein . Uracil interference revealed that strong van der Waals contacts are made by the thymines at position -7 and +5 of the top strands of both strong and weak boxes and that weaker contacts are made by the thymines at positions +7 (strong box) and -5 and +7 (weak box) of the bottom strand . In addition, ethylation interference suggested that the phosphate backbone contacts are located at the end and central regions of the palindrome . These findings are supported by our results derived from studies of symmetrical mutations of the tyrP strong box . Overall, the results confirm the critical importance of the invariant (G x C)(C x G)8 base pairs for TyrR recognition and also indicate that interactions with (T x A)(A x T)7 are of major importance . In contrast, mutations in other positions result in weaker effects on the binding affinity of TyrR protein, indicating that these positions play a lesser role in TyrR protein recognition . Alanine scanning of both helices of the putative helix-turn-helix DNA-binding motif of TyrR protein has identified those amino acids whose side chains play an essential role in protein structure and DNA binding. J Bacteriol, 1997 Feb, 179(4), 1007 - 12 Survival during exposure to the electrophilic reagent N-ethylmaleimide in Escherichia coli: role of KefB and KefC potassium channels; Ferguson GP et al.; The role of the KefB and KefC potassium efflux systems in protecting Escherichia coli cells against the toxic effects of the electrophile N-ethylmaleimide has been investigated . Activation of KefB and KefC aids the survival of cells exposed to high concentrations (> 100 microM) of NEM . High potassium concentrations reduce the protection afforded by activation of KefB and KefC, but the possession of these systems is still important under these conditions . The Kdp system, which confers sensitivity to the electrophile methylglyoxal, did not affect the survival of cells exposed to NEM . Survival is correlated with the reduction of the cytoplasmic pH upon activation of the channels . In particular, the kinetics of the intracellular pH (pHi) change are crucial to the retention of viability of cells exposed to NEM; slow acidification does not protect cells as effectively as rapid lowering of pHi . Cells treated with low levels of NEM (10 microM) recover faster if they activate KefB and KefC, and this correlates with changes in pHi . The pHi does not significantly alter the rate of NEM metabolism . The possible mechanisms by which protection against the electrophile is mediated are discussed.
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