FEBS Lett, 1998 Nov 27, 440(1-2), 175 - 82 Characterization of the wound-induced metallocarboxypeptidase inhibitor from potato . cDNA sequence, induction of gene expression, subcellular immunolocalization and potential roles of the C-terminal propeptide; Villanueva J et al.; A partial cDNA clone for the potato wound-inducible metallocarboxypeptidase inhibitor (PCI) was isolated from a cDNA library constructed from mRNA of abscisic acid (ABA)-treated potato leaves . The full 5' region of the cDNA was obtained through a RACE-PCR protocol . PCI mRNA encodes a precursor polypeptide which comprises a 29 residue N-terminal signal peptide, a 27 residue N-terminal pro-region, the 39 residue mature PCI protein, and a 7 residue C-terminal extension . Northern blot analysis demonstrates that the PCI gene is transcriptionally activated by wounding, and wound signaling can be induced by ABA and jasmonic acid . Subcellular localization of the protein was investigated by immunocytochemistry and electron microscopy, showing that PCI accumulates within the vacuole . A partial PCI precursor form, comprising the mature protein and the C-terminal extension, has been expressed in Escherichia coli and characterized . Its inability to inhibit carboxypeptidases, and stability to carboxypeptidase digestion, suggest that the C-terminal pro-domain may have, besides a probable vacuolar sorting function, a role in modulation of the inhibitory activity of PCI. FEBS Lett, 1998 Nov 27, 440(1-2), 147 - 52 Expression and characterization of PP7, a novel plant protein Ser/Thr phosphatase distantly related to RdgC/PPEF and PP5; Kutuzov MA et al.; We have recently identified an Arabidopsis thaliana cDNA encoding a putative protein Ser/Thr phosphatase PP7, not closely related to any protein phosphatases in animals or fungi . Here, we describe the characterization of PP7 expressed in a bacterial system . The recombinant protein was inactive unless the longest insert in its catalytic domain was cleaved, suggesting that this insert is an autoinhibitory region . PP7 was resistant to okadaic acid, calyculin and fumonisin B1, and was stimulated by Mn2+ or Fe2+, while Ni2+ and Zn2+ were inhibitory . Polylysine stimulated PP7 activity towards p-nitrophenylphosphate but inhibited activity towards the most efficient protein substrate, myelin basic protein . A tentative model of the control of PP7 activity is proposed. Toxicol Lett, 1998 Nov 12, 99(3), 199 - 205 Cellular inactivation induced by a radiopharmaceutical kit: role of stannous chloride; Assis ML et al.; Stannous chloride (SnCl2) has been used in many sectors of human activities such as food manufacturing and in nuclear medicine to produce radiopharmaceuticals labeled with technetium-99m (99mTc) . Due to its importance and genotoxic potentiality, we decided to evaluate the biological effect induced by a nuclear medicine kit, which includes SnCl2, in association with glucoheptonic acid (GHA) which is employed for brain and renal scintigraphies . These studies were carried out with the Escherichia coli AB1157 strain and the deoxyribonucleic acid (DNA) plasmid pUC 9.1 . The experiments, with different concentrations of SnCl2 and GHA, show an inverse relationship between both agents . When the GHA concentration was increased, the cellular inactivation induced by SnCl2 was reduced, as measured by the number of viable cells . Moreover, GHA protects the DNA molecule against the damage induced by SnCl2. Protein Eng, 1998 Oct, 11(10), 931 - 5 Protein engineering of BamHI restriction endonuclease: replacement of Cys54 by Ala enhances catalytic activity; Mukhopadhyay P et al.; Chemical modification studies of BamHI endonuclease indicated the importance of the cysteine residue in catalysis {Nath, K . (1981) Arch . Biochem . Biophys, 212, 611-617} . Of the three cysteine residues at positions 34, 54 and 64 in the BamHI endonuclease Cys54 and Cys64 are at the DNA-protein interface . The co-crystal structure of the BamHI-DNA complex, however, does not indicate any role of cysteines either in binding or catalysis . In the context of strong biochemical evidence, Cys54 in BamHI was changed to Ala54 to investigate its role in catalysis . The mutation was carried out by PCR overlap extension, the mutant gene was cloned and characterized by sequencing . The mutant BamHI was expressed and purified to homogeneity and the kinetic parameters (K(M) and kcat) of the wild type and the C54A mutant were determined . The mutation results in up to approximately 40% enhancement of kcat and some increase in K(M) . These in vitro results were also supported by in vivo SOS induction assays: the C54A mutant gene under the T7 promoter caused complete lysis in JH139 in absence of T7 RNA polymerase whereas the wild-type gene gave deep blue colonies under the same conditions . The results suggest no direct role of Cys54 in catalysis, but it can influence the catalytic activity through Val57 backbone contact seen in the co-crystal structure. Mutagenesis, 1998 Nov, 13(6), 637 - 41 SOS induction by gamma-radiation in Escherichia coli strains defective in repair and/or recombination mechanisms; Brena-Valle M et al.; Ionizing radiation causes several types of DNA lesions, mainly single- or double-strand breaks and base damage . By means of the chromotest, an assay that allows the level of the SOS response to be monitored via beta-galactosidase enzymatic activity, the roles of several repair (uvrA, recN and oxyR) and recombination (recB, recJ and recO) genes in the response of Escherichia coli to gamma-radiation were studied . The results indicate that all the repair- and recombination-deficient strains were more sensitive to the lethal effects of ionizing radiation . However, the SOS activation pattern was somewhat different . The minimal inducing dose in uvrA and recN mutants was lower than in the wild-type, whereas their SOS response was higher at all doses . Conversely, in the strains lacking an active recB, recJ or recO gene, the doubling dose was almost the same as in the wild-type but the level of induction remained stable over a wide dose range . These findings suggest that neither single- nor double-strand breaks are in themselves direct SOS inducers and that while uvrA, recN and oxyR take part in different repair or protective pathways, apparently recB, recJ and recO participate in damage processing leading to SOS induction, as well as in recombination repair. Mutagenesis, 1998 Nov, 13(6), 625 - 30 Specific mutational spectrum of dimethylnitrosamine in the lacI transgene of Big Blue C57BL/6 mice; Wang X et al.; Dimethylnitrosamine (DMN) produces tumors in mice predominantly in the liver, but also in the kidney and lung . It forms O6-methylguanine adducts in DNA, which induce G:C-->A:T transitions . We have analyzed the spectra of spontaneous and DMN-induced mutations in the lacI transgene of the Big Blue mouse (C57BL/6) . In both cases, mutations in the liver, kidney and lung were predominantly base substitutions, among which G:C-->A:T transitions were the most frequent . In contrast, a high incidence of short deletions (2-23 bp) was only found in the liver of treated mice . The deletions often occurred at direct repeat sequences . Single-base deletion incidence was also higher in the liver than in the kidney and lung . These results imply that accumulation of DNA lesions or their repair in liver is different from other organs . Spontaneous and induced base substitutions and deletions appeared to be randomly distributed in the lacI gene and an apparent hotspot was not observed, except for a 4 bp deletion of a (TGGC)3 sequence at positions 621-632 . The present data demonstrate, for the first time, that DMN induces short deletions especially in the liver, although the mechanism involved needs further investigation. Mutagenesis, 1998 Nov, 13(6), 607 - 12 Mutation studies in lacI transgenic mice after exposure to radiation or cyclophosphamide; Hoyes KP et al.; We have used the Big Blue lacI transgenic mouse reporter system to investigate mutation induction in the testes, spleen and liver after exposure to an internally incorporated radionuclide, 114mIn, whole body irradiation with 60Co gamma-rays and systemically administered cyclophosphamide . Spontaneous mutation frequencies were 6-17x10(-6) . No statistically significant mutation induction was observed in testes or spleen at 35 days after exposure to any test agent, although mutation frequencies tended to be increased (by approximately 1.5-fold) after exposure to 1 Gy gamma-rays . However, liver mutation frequencies were doubled after treatment with 100 mg/kg cyclophosphamide and were elevated by approximately 2.5-fold after systemic administration of 114mIn and 4.5-fold after 1 Gy 60Co gamma-rays . When data from all organs were pooled, mutation frequency was doubled after exposure to 1 Gy gamma-rays, but no other significant increases were observed . These findings support the hypothesis that the lacI transgenic mouse may be relatively inefficient at detecting mutations induced by exposure to ionizing radiation or other agents which produce a spectrum of deletion sizes, including those which are larger than the lacI transgene. Atherosclerosis, 1998 Dec, 141(2), 265 - 71 Ex vivo gene transfer of endothelial nitric oxide synthase to atherosclerotic rabbit aortic rings improves relaxations to acetylcholine; Mozes G et al.; Cholesterol feeding results in impaired endothelium dependent vasorelaxation . The role of nitric oxide in this process is unclear . The aim of this study was to evaluate the role of nitric oxide in cholesterol-induced vasomotor dysfunction by examining the effect of overexpression of eNOS in the hypercholesterolemic rabbit aorta on vascular reactivity . Vascular rings from the thoracic aorta of hypercholesterolemic rabbits were exposed ex vivo either to an adenoviral vector encoding endothelial nitric oxide synthase (AdeNOS) or Escherichia coli beta Galactosidase (AdbetaGal) . Transgene expression was examined by histochemistry for beta galactosidase, immunohistochemistry for eNOS and cyclic GMP measurements and vasomotor studies were performed . Transgene expression was found to localize to the endothelium and adventitia . cGMP levels were significantly greater in AdeNOS compared to AdbetaGal transduced rings . Acetylcholine mediated relaxation was significantly impaired in cholesterol fed rabbits and was markedly improved by overexpression of eNOS . These results suggest that reduced NO bioavailability observed in cholesterol-induced vascular dysfunction can be partially overcome by eNOS gene transfer. Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15519 - 24 Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3'-5' exonuclease proofreading function; Vandewiele D et al.; Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD'2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III) . However, we find that efficient TR can occur in the absence of the Umu proteins if the 3'-5' exonuclease proofreading activity of the pol III epsilon-subunit also is disabled . TR was measured in isogenic uvrA6 DeltaumuDC strains carrying the dominant negative dnaQ allele, mutD5, or DeltadnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T-T dimer . As expected, little TR was observed in the DeltaumuDC dnaQ+ strain . Surprisingly, 26% TR occurred in UV-irradiated DeltaumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain . lexA3 (Ind-) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for approximately 70% of the TR, and another LexA-independent, responsible for the remaining approximately 30% . Curiously, the DeltaumuDC DeltadnaQ spq-2 strain exhibited only the LexA-independent level of TR . The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in DeltadnaQ strains, since introduction of spq-2 into the DeltaumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level . The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon {Palejwala, V . A., Wang, G . E., Murphy, H . S . & Humayun, M . Z . (1995) J . Bacteriol . 177, 6041-6048} . LexA-dependent TR does not result from the induction of pol II, since TR in the DeltaumuDC mutD5 strain is unchanged by introduction of a DeltapolB mutation. Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15275 - 80 Minimal and optimal mechanisms for GroE-mediated protein folding; Ben-Zvi AP et al.; We have analyzed the effects of different components of the GroE chaperonin system on protein folding by using a nonpermissive substrate (i.e., one that has very low spontaneous refolding yield) for which rate data can be acquired . In the absence of GroES and nucleotides, the rate of GroEL-mediated refolding of heat- and DTT-denatured mitochondrial malate dehydrogenase was extremely low, but some three times higher than the spontaneous rate . This GroEL-mediated rate was increased 17-fold by saturating concentrations of ATP, 11-fold by ADP and GroES, and 465-fold by ATP and GroES . Optimal refolding activity was observed when the dissociation of GroES from the chaperonin complex was dramatically reduced . Although GroEL minichaperones were able to bind denatured mitochondrial malate dehydrogenase, they were ineffective in enhancing the refolding rate . The spectrum of mechanisms for GroE-mediated protein folding depends on the nature of the substrate . The minimal mechanism for permissive substrates (i.e., having significant yields of spontaneous refolding), requires only binding to the apical domain of GroEL . Slow folding rates of nonpermissive substrates are limited by the transitions between high- and low-affinity states of GroEL alone . The optimal mechanism, which requires holoGroEL, physiological amounts of GroES, and ATP hydrolysis, is necessary for the chaperonin-mediated folding of nonpermissive substrates at physiologically relevant rates under conditions in which retention of bound GroES prevents the premature release of aggregation-prone folding intermediates from the chaperonin complex . The different mechanisms are described in terms of the structural features of mini- and holo-chaperones. Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15235 - 40 Novel role of phosphorylation in Fe-S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein 1; Brown NM et al.; Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs) . IRPs modulate mRNA utilization by binding to iron-responsive elements (IRE) in the 5' or 3' untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production . IRP1 is also the cytosolic isoform of aconitase . The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its {4Fe-4S} cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase . IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined . To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A) . The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically . Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically . However, when exposed to oxygen, the {4Fe-4S} cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1 . Our findings suggest that stability of the Fe-S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and {4Fe-4S} forms of IRP1 can be modulated independently of cellular iron status . Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression. Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15229 - 34 Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone; Gassler CS et al.; Hsp70 chaperones assist protein folding by ATP-controlled cycles of substrate binding and release . ATP hydrolysis is the rate-limiting step of the ATPase cycle that causes locking in of substrates into the substrate-binding cavity of Hsp70 . This key step is strongly stimulated by DnaJ cochaperones . We show for the Escherichia coli Hsp70 homolog, DnaK, that stimulation by DnaJ requires the linked ATPase and substrate-binding domains of DnaK . Functional interaction with DnaJ is affected by mutations in an exposed channel located in the ATPase domain of DnaK . It is proposed that binding to this channel, possibly involving the J-domain, allows DnaJ to couple substrate binding with ATP hydrolysis by DnaK . Evolutionary conservation of the channel and the J-domain suggests conservation of the mechanism of action of DnaJ proteins. Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15223 - 8 Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ; Suh WC et al.; Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes . ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ . By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding . The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding . A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK . Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70. Biochemistry, 1998 Dec 15, 37(50), 17598 - 609 Porcine recombinant dihydropyrimidine dehydrogenase: comparison of the spectroscopic and catalytic properties of the wild-type and C671A mutant enzymes; Rosenbaum K et al.; Dihydropyrimidine dehydrogenase catalyzes, in the rate-limiting step of the pyrimidine degradation pathway, the NADPH-dependent reduction of uracil and thymine to dihydrouracil and dihydrothymine, respectively . The porcine enzyme is a homodimeric iron-sulfur flavoprotein (2 x 111 kDa) . C671, the residue postulated to be in the uracil binding site and to act as the catalytically essential acidic residue of the enzyme oxidative half-reaction, was replaced by an alanyl residue . The mutant enzyme was overproduced in Escherichia coli DH5alpha cells, purified to homogeneity, and characterized in comparison with the wild-type species . An extinction coefficient of 74 mM-1 cm-1 was determined at 450 nm for the wild-type and mutant enzymes . Chemical analyses of the flavin, iron, and acid-labile sulfur content of the enzyme subunits revealed similar stoichiometries for wild-type and C671A dihydropyrimidine dehydrogenases . One FAD and one FMN per enzyme subunit were found . Approximately 16 iron atoms and 16 acid-labile sulfur atoms were found per wild-type and mutant enzyme subunit . The C671A dihydropyrimidine dehydrogenase mutant exhibited approximately 1% of the activity of the wild-type enzyme, thus preventing its steady-state kinetic analysis . Therefore, the ability of the C671A mutant and, for comparison, of the wild-type enzyme species to interact with reaction substrates, products, or their analogues were studied by absorption spectroscopy . Both enzyme forms did not react with sulfite . The wild-type and mutant enzymes were very similar to each other with respect to the spectral changes induced by binding of the reaction product NADP+ or of its nonreducible analogue 3-aminopyridine dinucleotide phosphate . Uracil also induced qualitatively and quantitatively similar absorbance changes in the visible region of the absorbance spectrum of the two enzyme forms . However, the calculated Kd of the enzyme-uracil complex was significantly higher for the C671A mutant (9.1 +/- 0.7 microM) than for the wild-type dihydropyrimidine dehydrogenase (0.7 +/- 0.09 microM) . In line with these observations, the two enzyme forms behaved in a similar way when titrated anaerobically with a NADPH solution . Addition of an up to 10-fold excess of NADPH to both dihydropyrimidine dehydrogenase forms led to absorbance changes consistent with reduction of approximately 0.5 flavin per subunit, with no indication of reduction of the enzyme iron-sulfur clusters . Absorbance changes consistent with reduction of both enzyme flavins were obtained by removing NADP+ with a NADPH-regenerating system . On the contrary, the two enzyme species differed significantly with respect to their reactivity with dihydrouracil . Addition of dihydrouracil to the wild-type enzyme species, under anaerobic conditions, led to absorbance changes that could be interpreted to result from both partial flavin reduction and the formation of a complex between the enzyme and (dihydro)uracil . In contrast, only spectral changes consistent with formation of a complex between the oxidized enzyme and dihydrouracil were observed when a C671A mutant enzyme solution was titrated with this compound . Furthermore, enzyme-monitored turnover experiments were carried out anaerobically in the presence of a limiting amount of NADPH and excess uracil with the two enzyme forms in a stopped-flow apparatus . These experiments directly demonstrated that the substitution of an alanyl residue for C671 in dihydropyrimidine dehydrogenase specifically prevents enzyme-catalyzed reduction of uracil . Finally, sequence analysis of dihydropyrimidine dehydrogenase revealed that it exhibits a modular structure; the N-terminal region, similar to the beta subunit of bacterial glutamate synthases, is proposed to be responsible for NADPH binding and oxidation with reduction of the FAD cofactor of dihydropyrimidine dehydrogenase . The central region, similar to the FMN subunit of dihydroorotate dehydrogenases, is likely to harbor the site o Biochemistry, 1998 Dec 15, 37(50), 17590 - 7 Conversion of a catalytic into a structural disulfide bond by circular permutation; Hennecke J et al.; The thiol-disulfide oxidoreductase DsbA from Escherichia coli is the strongest oxidant of the enzyme family and required for disulfide bond formation in the bacterial periplasm . The catalytic domain of this 189-residue protein has a thioredoxin-like fold and contains a catalytic disulfide bridge that is located within the sequence Cys30-Pro31-His32-Cys33 at the N-terminus of an alpha-helix . The Cys30-Cys33 disulfide bond destabilizes DsbA by about 16 kJ/mol at pH 7.0, which appears to be caused by the extremely low pKa value of approximately 3.4 of the nucleophilic Cys30 thiol . Here we report the characterization of a circularly permuted variant of DsbA, termed H32-P31, in which the natural termini are connected by a Gly3-Thr-Gly linker and the new termini are located between the active-site cysteines (first residue His32, last residue Pro31) . The disulfide bond in the variant thus connects the second with the penultimate residue . H32-P31 adopts a wild-type-like structure and folds reversibly and cooperatively in both redox forms . However, the permuted variant is catalytically inactive as dithiol oxidase in vivo and in vitro . Both cysteine thiols have pKa values > 8; the variant is 500-fold more reducing than the wild type and more stable in its oxidized form . Thus, the Cys30-Cys33 disulfide in the variant H32-P31 has adopted properties of a structural disulfide bond. Biochemistry, 1998 Dec 15, 37(50), 17554 - 61 Lipid properties and the orientation of aromatic residues in OmpF, influenza M2, and alamethicin systems: molecular dynamics simulations; Tieleman DP et al.; Molecular dynamics simulations allow a direct study of the structure and dynamics of membrane proteins and lipids . We describe the behavior of aromatic residues and lipid properties in POPE and POPC bilayer models with the Escherichia coli OmpF trimer, single alamethicin and Influenza M2 helices, 4-helix M2 bundles, and two alamethicin 6-helix channel models . The total simulation time is over 24 ns, of systems containing solvent, protein, and between 104 and 318 lipids . Various types of adjustment between lipids and proteins occur, depending on the size of the protein and the degree of hydrophobic mismatch between lipid and protein . Single helices cause little measurable effect on nearby lipids whereas the 4-helix bundles, 6-helix channel models, and OmpF cause a significant lowering of order parameters in nearby lipid chains, an increased difference between odd and even chain dihedrals in the magnitude of the trans dihedral fractions and dihedral transition rates, and in most cases a decreased gauche population and a decrease in bilayer thickness . An increased tilt of the lipid chains near the proteins can account for most of the observed decrease in order parameters . The orientation of tryptophans and tyrosines on the outside of the proteins is determined by packing at the protein exterior and non-specific hydrogen bonding with lipids and solvent . The tyrosines in the broad bands that delimit the hydrophobic exterior of OmpF show little change in orientation over one nanosecond . Their rings are oriented predominantly perpendicular to the bilayer plane, with the hydroxyl group pointing toward the lipid-water interface . Phenylalanines in OmpF, alamethicin, and Influenza M2 are more mobile and assume a variety of orientations. Biochemistry, 1998 Dec 15, 37(50), 17448 - 57 Identification of the heme-modified peptides from cumene hydroperoxide-inactivated cytochrome P450 3A4; He K et al.; Cumene hydroperoxide-mediated (CuOOH-mediated) inactivation of cytochromes P450 (CYPs) results in destruction of their prosthetic heme to reactive fragments that irreversibly bind to the protein . We have attempted to characterize this process structurally, using purified, 14C-heme labeled, recombinant human liver P450 3A4 as the target of CuOOH-mediated inactivation, and a battery of protein characterization approaches {chemical (CNBr) and proteolytic (lysylendopeptidase-C) digestion, HPLC-peptide mapping, microEdman sequencing, and mass spectrometric analyses} . The heme-peptide adducts isolated after CNBr/lysylendopeptidase-C digestion of the CuOOH-inactivated P450 3A4 pertain to two distinct P450 3A4 active site domains . One of the peptides isolated corresponds to the proximal helix L/Cys-region peptide 429-450 domain and the others to the K-region (peptide 359-386 domain) . Although the precise residue(s) targeted remain to be identified, we have narrowed down the region of attack to within a 17 amino acid peptide (429-445) stretch of the 55-amino acid proximal helix L/Cys domain . Furthermore, although the exact structures of the heme-modifying fragments and the nature of the adduction remain to be established conclusively, the incremental masses of approximately 302 and 314 Da detected by electrospray mass spectrometric analyses of the heme-modified peptides are consistent with a dipyrrolic heme fragment comprised of either pyrrole ring A-D or B-C, a known soluble product of peroxidative heme degradation, as a modifying species. Biochemistry, 1998 Dec 15, 37(50), 17386 - 401 Coulombic effects of remote subsites on the active site of ribonuclease A; Fisher BM et al.; The active-site cleft of bovine pancreatic ribonuclease A (RNase A) is lined with cationic residues that interact with a bound nucleic acid . Those residues interacting with the phosphoryl groups comprise the P0, P1, and P2 subsites, with the scissile P-O5' bond residing in the P1 subsite . Coulombic interactions between the P0 and P2 subsites and phosphoryl groups of the substrate were characterized previously {Fisher, B . M., Ha, J.-H., and Raines, R . T . (1998) Biochemistry 37, 12121-12132} . Here, the interactions between these subsites and the active-site residues His12 and His119 are described in detail . A protein variant in which the cationic residues in these subsites (Lys66 in the P0 subsite and Lys7 and Arg10 in the P2 subsite) were replaced with alanine was crystallized, both free and with bound 3'-uridine monophosphate (3'-UMP) . Structures of K7A/R10A/K66A RNase A and the K7A/R10A/K66A RNase A.3'-UMP complex were determined by X-ray diffraction analysis to resolutions of 2.0 and 2.1 A, respectively . There is little observable change between these structures and that of wild-type RNase A, either free or with bound 3'-cytidine monophosphate . K7A/R10A/K66A RNase A was evaluated for its ability to cleave UpA, a dinucleotide substrate that does not span the P0 or the P2 subsites . In comparison to the wild-type enzyme, the value of kcat was decreased by 5-fold and that of kcat/Km was decreased 10-fold, suggesting that these remote subsites interact with the active site . These interactions were characterized by determining the pKa values of His12 and His119 at 0.018 and 0.142 M Na+, both in wild-type RNase A and the K7A/R10A/K66A variant . The side chains of Lys7, Arg10, and Lys66 depress the pKa values of these histidine residues, and this depression is sensitive to the salt concentration . In addition, the P0 and P2 subsites influence the interaction of His12 and His119 with each other, as demonstrated by changes in the cooperativity that gives rise to microscopic pKa values . Finally, the affinity of 3'-UMP for wild-type RNase A and the K7A/R10A/K66A variant at 0.018 and 0.142 M Na+ was determined by isothermal titration calorimetry . 3'-UMP binds to the variant protein with 5-fold weaker affinity at 0.018 M Na+ and 3-fold weaker affinity at 0.142 M Na+ than it binds to wild-type RNase A . Together these data demonstrate that long-range Coulombic interactions are an important feature in catalysis by RNase A. Biochemistry, 1998 Dec 15, 37(50), 17381 - 5 The magnitude of the allosteric conformational transition of aspartate transcarbamylase is altered by mutations; LiCata VJ et al.; Global conformational transitions are of central functional importance for many enzymes and binding proteins . It is not known, however, how much variability can exist in such structural-functional linkages . We have characterized the global magnitude of the T to R conformational transition of Escherichia coli aspartate transcarbamylase (ATCase) by measuring (1) hydration changes using osmotic stress and (2) hydrodynamic changes using high-precision analytical gel chromatography . We find that specific mutations can alter the structural magnitude of the enzyme's conformational transition without abolishing allostery, suggesting that some degree of plasticity exists in the conformational component of allostery. Biochemistry, 1998 Dec 8, 37(49), 17309 - 17 Two stable unfolding intermediates of the disease-causing L68Q variant of human cystatin C; Gerhartz B et al.; In hereditary cystatin C amyloid angiopathy (HCCAA), presence of the Leu68 --> Gln substitution in cystatin C is coupled to a decreased concentration of this major cysteine proteinase inhibitor in cerebrospinal fluid and leads to its amyloid deposition in the brain . We established a high-yield expression system for L68Q cystatin C in Escherichia coli resulting in inclusion body accumulation at a level of 40% of the total cellular protein . Refolding of protein from purified inclusion bodies yielded a pure, almost completely monomeric and active inhibitor . CD and NMR spectroscopy demonstrated that so produced L68Q cystatin C is folded, conformationally homogeneous, and structurally very similar to wild-type cystatin C . Incubation at pH 7.0-5.5 caused the cystatin C variant to dimerize rapidly . The molecular form present at pH 6.0 displayed a slightly increased amount of hydrophobic parts on the surface as measured by 1-anilinonaphthalene-8-sulfonic acid (ANS) binding . NMR results showed that the dimer has a structure similar to that of the wild-type cystatin C dimer formed as a result of slight denaturation . Under more acidic conditions, at pH 4.5, another stable unfolding intermediate of L68Q cystatin C was identified . This molecular form exists in a monomeric state, is characterized by changes in secondary structure according to far UV CD spectroscopy, and shows an altered ANS binding resembling that of a molten globule state . The acidic pH also caused an almost complete monomerization of preformed dimers . The state of denaturation of L68Q cystatin C in vivo is thus a critical factor for the concentration of active cysteine proteinase inhibitor in cerebrospinal fluid and likely also for the development of amyloidosis, in HCCAA patients. Biochemistry, 1998 Dec 8, 37(49), 17280 - 6 Thiol-mediated disassembly and reassembly of {2Fe-2S} clusters in the redox-regulated transcription factor SoxR; Ding H et al.; SoxR, a transcription factor containing {2Fe-2S} clusters, governs the cellular response to oxidative stress in Escherichia coli . The oxidation state of the iron-sulfur clusters regulates the SoxR transcriptional activity . When the reduced iron-sulfur clusters become oxidized ({2Fe-2S}2+ state), SoxR is activated to stimulate transcription of the soxS gene, whose product in turn switches on a group of genes encoding various proteins that defend against oxidative stress and antibiotics . A previous study showed that the oxidized {2Fe-2S} clusters of SoxR are destroyed by a free-radical-dependent process in vitro during aerobic exposure to the biological thiol glutathione . Here, we show that different thiols have differing effects on the SoxR {2Fe-2S} clusters . Like reduced glutathione, N-acetyl-L-cysteine, L-cysteine methyl ester, and L-cysteine ethyl ester disrupted the SoxR {2Fe-2S} clusters in aerobic solution . This disruption was blocked by L-cysteine, which was effective at concentrations 100-fold lower (1-10 microM) than the disrupting thiols (1 mM) . In view of a previous observation that superoxide dismutase and catalase block the disruption process, this result suggests that L-cysteine may quench reactive SoxR or thiol intermediates involved in the cluster disruption reaction, the detailed mechanism of which remains unknown . In contrast, bifunctional thiols such as dithiothreitol or dithioerythritol promoted the aerobic assembly of the functional {2Fe-2S} clusters into apo-SoxR in the presence of Fe2+ and inorganic sulfide . The dithiol protein thioredoxin-A of E . coli acted catalytically in vitro in the presence of thioredoxin reductase and NADPH to promote {2Fe-2S} cluster assembly into apo-SoxR . The regulatory activity of SoxR in vivo, assessed by monitoring the paraquat-mediated induction of a soxS'::lacZ reporter fusion, was significantly lower in a strain lacking both thioredoxin-A and glutathione reductase, which maintains reduced glutaredoxins . Thus, cellular monothiols and dithiol proteins may contribute to SoxR regulation by affecting the disassembly and reassembly of the {2Fe-2S} clusters. Biochemistry, 1998 Dec 8, 37(49), 17163 - 9 Conformational change rate-limits GTP hydrolysis: the mechanism of the ATP sulfurylase-GTPase; Wei J et al.; The fluorescent GTP analogues 3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-(beta, gamma-imidotriphosphate) (mGMPPNP) and 3'-O-(N-methylanthraniloyl)-2'-deoxy-GTP (mGTP) were used to demonstrate that an enzyme isomerization precedes and rate-limits beta,gamma-bond cleavage in the catalytic cycle of the ATP sulfurylase-GTPase, from E . coli K-12 . The binding of mGMPPNP to the E.AMP.PPi complex of ATP sulfurylase is biphasic, indicating that an isomerization occurs in the binding reaction . The isomerization mechanism was assigned based on the results of the enzyme concentration dependence of the observed rate constants, kobs, for both phases of the binding reaction, and sequential-mixing, nucleotide release experiments . The isomerization occurs after, and is driven by, the addition of mGMPPNP . Values were determined for each of the rate constants associated with the two-step kinetic model used in the interpretation of the results . A comparison of the enzyme concentration dependence of kobs for the hydrolysis and binding reactions reveals that the rate constants for the corresponding steps of these two reactions are extremely similar . The virtually identical rate constants for isomerization and beta, gamma-bond scission strongly suggest that isomerization rate-limits bond breaking . The implications of these finding for GTPase/target interactions and the mechanism of energetic linkage in the ATP sulfurylase system are discussed. Biochemistry, 1998 Dec 8, 37(49), 17145 - 56 Reactivity of the human thioltransferase (glutaredoxin) C7S, C25S, C78S, C82S mutant and NMR solution structure of its glutathionyl mixed disulfide intermediate reflect catalytic specificity; Yang Y et al.; Human thioltransferase (TTase) is a 12 kDa thiol-disulfide oxidoreductase that appears to play a critical role in maintaining the redox environment of the cell . TTase acts as a potent and specific reducing agent for protein-S-S-glutathione mixed disulfides (protein-SSG) likely formed during oxidative stress or as redox intermediates in signal transduction pathways . Accordingly, the catalytic cycle of thioltransferase itself involves a covalent glutathionyl enzyme disulfide intermediate (TTase-C22-SSG) . To understand the molecular basis of TTase specificity for the glutathione moiety, we engineered a quadruple Cys to Ser mutant of human TTase (C7S, C25S, C78S, and C82S) which retains only the active site cysteine residue (C22), and we solved its high-resolution NMR solution structure in the mixed disulfide intermediate with glutathione (QM-TTase-SSG) . This mutant which cannot form a C22-S-S-C25 intramolecular disulfide displays the same catalytic efficiency (Vmax/KM) and specificity for glutathionyl mixed disulfide substrates as wild-type TTase, indicating that the Cys-25-SH moiety is not required for catalysis or glutathionyl specificity . The structure of human thioltransferase is characterized by a thioredoxin-like fold which comprises a four-stranded central beta-sheet flanked on each side by alpha-helices . The disulfide-adducted glutathione in the TTase-SSG complex has an extended conformation and is localized in a cleft near the protein surface encompassing the residues from helices-alpha2,alpha3, the active site loop, and the loop connecting helix-alpha3 and strand-beta3 . Numerous van der Waals and electrostatic interactions between the protein and the glutathione moiety are identified as contributing to stabilization of the complex and confering the substrate specificity . Comparison of the human thioltransferase with other thiol-disulfide oxidoreductases reveals structural and functional differences. Biochemistry, 1998 Dec 8, 37(49), 17137 - 44 Functional characterization of the protease of human endogenous retrovirus, K10: can it complement HIV-1 protease? Towler EM, Gulnik SV, Bhat TN, Xie D, Gustschina E, Sumpter TR, Robertson N, Jones C, Sauter M, Mueller-Lantzsch N, Debouck C, Erickson JW. To investigate the biochemical properties of the protease encoded by the human endogenous retrovirus, K10 (HERV-K), 213 amino acids of the 3'-end of the HERV-K protease (PR) open reading frame were expressed in Escherichia coli . Autocatalytic cleavage of the expressed polypeptide resulted in an 18.2 kDa protein which was shown to be proteolytically active against a fluorogenic peptide used as a substrate for HIV-1 protease . On the basis of sequence homology and molecular modeling, the 106 N-terminal amino acids of HERV-K PR were predicted to comprise a retroviral protease core domain . An 11.6 kDa protein corresponding to this region was expressed and shown to be a fully functional enzyme . The 11.6 kDa domain of HERV-K PR is unusually stable over a wide pH range, exhibits optimal catalytic activity between pH 4.0 and 5.0, and exists as a dimer at pH 7.0 with a Kd of 50 microM . Like HIV-1 PR, the HERV-K PR core domain is activated by high salt concentrations and processes HIV-1 matrix-capsid polyprotein at the authentic HIV-1 PR recognition site . However, both the 18.2 and 11.6 kDa forms of HERV-K PR were highly resistant to a number of clinically useful HIV-1 PR inhibitors, including ritonavir, indinavir, and saquinavir . This raises the possibility that HERV-K PR may complement HIV-1 PR during infection, and could have implications for protease inhibitor therapy and drug resistance. Biochim Biophys Acta, 1998 Nov 10, 1388(2), 500 - 5 Limited proteolysis of a trypanosomal hypoxanthine phosphoribosyltransferase yields crystals that diffract X-rays to near atomic resolution; Nieves-Alicea R et al.; Two crystal forms of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi were grown and characterized . Proteolytic modification at the C-terminus of the recombinant enzyme yielded monoclinic crystals that diffract X-rays to higher resolution than the original, trigonal crystal form . Data from the monoclinic crystal form enabled determination of the crystal structure for the trypanosomal HPRT to 1.4 A resolution. Gene, 1998 Nov 26, 223(1-2), 47 - 54 Subdivision of the Escherichia coli K-12 genome for sequencing: manipulation and DNA sequence of transposable elements introducing unique restriction sites; Mahillon J et al.; A transposon-based method of introducing unique restriction sites was used for subdivision of the Escherichia coli genome into a contiguous series of large non-overlapping segments spanning 2.5Mb . The segments, sizes ranging from 150 to 250kb, were isolated from the chromosome using the inserted restriction sites and shotgun cloned into an M13 vector for DNA sequencing . These shotgun sizes proved easily manageable, allowing the genomic sequence of E . coli to be completed more efficiently and rapidly than was possible by previously available methods . The 9bp duplication generated during transposition was used as a tag for accurate splicing of the segments; no further sequence redundancy at the junction sites was needed . The system is applicable to larger genomes even if they are not already well-characterized . We present the technology for segment sequencing, results of applying this method to E . coli, and the sequences of the transposon cassettes. Chem Res Toxicol, 1998 Dec, 11(12), 1468 - 73 Mutagenic specificity of a derivative of 3-nitrobenzanthrone in the supF shuttle vector plasmids; Kawanishi M et al.; 3-Nitrobenzanthrone (NBA) is a powerful bacterial mutagen and a suspected human carcinogen present in diesel exhaust and airborne particulates {Enya, T., et al . (1997) Environ . Sci . Technol . 31, 2772-2776} . In the accompanying paper {Enya, T., et al . (1998) Chem . Res . Toxcol . 11, 1460-1467}, N-acetoxy-N-acetyl-3-aminobenzanthrone (N-Aco-N-Ac-ABA) was synthesized to yield the DNA adducts of NBA . In this work, to investigate the mutagenic specificity of NBA in human cells, we analyzed mutations induced by N-Aco-N-Ac-ABA using the supF shuttle vector plasmids . Base sequence analysis of 110 and 100 plasmids with mutations in the supF gene propagated in normal cells {WI38-VA13} and nucleotide excision repair deficient cells {XP2OS(SV)}, respectively, revealed that the majority of the mutations were base substitutions (85 and 90%) and the rest were deletions and insertions (10 and 15%) in both cell lines . About half of the mutant plasmids had a single base substitution . Of the base substitutions, the most frequent mutation was G.C to T.A transversion (41 and 51%), followed by G.C to A.T transitions (18 and 24%) in either cell . The mutations were distributed not randomly but located at several hot spots, and almost all (nine of ten) hot spots were at the sites of G.C base pairs . The polymerase stop assay in the supF gene revealed that N-Aco-N-Ac-ABA preferentially bound to guanine residues, and mutation sites were generally consistent with the sites where the guanine adducts were formed. Vet Immunol Immunopathol, 1998 Nov 24, 66(2), 127 - 41 Interferon induction in turkeys by oral administration of the imidazoquinolinamine S-28828 and modulation of the pathogenesis of Escherichia coli; Rautenschlein S et al.; A synthetic imidazoquinolinamine, S-28828, has been shown to be an effective antiviral and antitumor agent in mammals . This immune modifier induces a number of cytokines such as interferons, tumor necrosis factor-alpha, interleukins and granulocyte-macrophage colony-stimulating factors in mammals . We showed that when turkeys were given S-28828 orally, high serum titers of IFN were induced in a dose-dependent manner . Turkeys, once stimulated by S-28828, became refractory to IFN production by repeated stimulation . S-28828 induced spleen, bone marrow and peripheral leukocytes to produce IFN in vitro . Splenic adherent cells were the main producers of IFN after in vitro stimulation . S-28828-induced IFN was identified as type I IFN that was pH-resistant but heat-labile . We examined the effect of a high dose (100 mg kg(-1) body weight) of S-28828 on the pathogenesis of E . coli in turkeys . Treatment with S-28828 increased mortality in infected birds and impaired E . coli clearance from the liver . The enhancement of the pathogenicity of E . coli by S-28828 may have been due to the massive release of cytokines inducing a shock-like syndrome in infected turkeys. Vet Immunol Immunopathol, 1998 Nov 24, 66(2), 99 - 112 Production and characterisation of monoclonal antibodies specific for bovine interleukin-4; Weynants V et al.; Genetic immunisation is a simple method for producing polyclonal antibodies in mice . By this method, we produced antibodies against bovine interleukin-4 (BoIL-4) . After a final injection with a recombinant BoIL-4 protein, nine stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine . Specific binding of each of the MAbs to recombinant BoIL-4 produced by Escherichia coli, baculovirus, and Trypanosoma brucei was demonstrated in an indirect ELISA and/or in Western blotting . These MAbs recognise the same antigenic region localised in the first 47 amino acids of the mature protein . None of them was able to neutralise the biological activity of the BoIL-4 under the conditions tested but one allowed the detection of BoIL-4 by flow cytometry. Anticancer Res, 1998 Sep-Oct, 18(5A), 3475 - 9 Inhibition by Porphyromonas gingivalis LPS of apoptosis induction in human peripheral blood polymorphonuclear leukocytes; Hiroi M et al.; Many factors, such as cytokines and bacterial products, are known to affect the life-span of polymorphonuclear leukocytes (PMNs) . We investigated here whether Porphyromonas gingivalis (P.gingivalis) LPS slows down the apoptotic process of PMNs . During incubation in regular culture medium, the viability of PMNs progressively declined, producing apoptotic cells, characterized by chromatin condensation, internucleosomal DNA fragmentation, cell shrinkage and blebbing of plasma membrane . P.gingivalis LPS significantly increased the viability of PMNs and reduced the production of fragmented DNA, as efficiently as E.coli LPS . The present study suggests that the retardation of PMNs apoptosis by P.gingivalis LPS may modulate the restoration of acute inflammation in the periodontal tissues, where activated PMNs accumulate. Anticancer Res, 1998 Sep-Oct, 18(5A), 3369 - 73 Production and characterisation of a recombinant single-chain anti ErbB2-clavin immunotoxin; D'Alatri L et al.; We generated a recombinant immunotoxin, named scFv(MGR6)-Cla, composed of the Fv region of an anti ErbB2 monoclonal antibody (MGR6) fused to clavin, a type 1 ribosome-inactivating protein (RIP) from Aspergillus clavatus . ErbB2 is a tyrosine kinase receptor which is overexpressed in most adenocarcinomas; clavin is a 17 kDa ribonuclease which inhibits protein synthesis by inactivating ribosomes . A recombinant DNA construct containing the cDNA of the single chain Fv fragment (scFv) of the MGR6 antibody fused to the clavin cDNA, was expressed at high levels in Escherichia coli as an insoluble fusion protein containing an N-terminal affinity tag of six consecutive histidine residues . Inclusion bodies were denatured and the recombinant fusion protein was purified under denaturing conditions by single-step purification using immobilised metal ion affinity chromatography (IMAC) . The purified immunotoxin was renatured at high yield and histidine tag removed by digestion with enterokinase . The purity of the immunotoxin obtained after refolding was confirmed by SDS-PAGE, RP-HPLC, GPC-HPLC and N-terminal sequence analysis . Cell-free protein synthesis inhibition and binding assays showed that both clavin and scFv(MGR6) maintained their properties after refolding. Biochim Biophys Acta, 1998 Nov 10, 1388(2), 489 - 99 The function of Glu338 in the catalytic triad of the carbamoyl phosphate synthetase amidotransferase domain; Hewagama A et al.; The synthesis of carbamoyl phosphate by the mammalian multifunctional protein, CAD, involves the concerted action of the 40 kDa amidotransferase domain (GLN), that hydrolyzes glutamine and the 120 kDa synthetase (CPS) domain that uses the ammonia, thus produced, ATP and bicarbonate to make carbamoyl phosphate . The separately cloned GLN domain has very low activity due to a reduction in kcat and an increase in Km but forms a hybrid complex with the isolated Escherichia coli CPS subunit . The hybrid has full glutamine-dependent catalytic activity and a functional interdomain linkage . The mammalian-E . coli hybrid was used to investigate the functional consequence of replacing His336 and Glu338, two residues postulated to participate in catalysis as part of a catalytic triad . The mutant mammalian GLN domains formed stable complexes with the E . coli CPS subunit, but the catalytic activity was severely impaired . While the His336Asn mutant does not form measurable amounts of the gamma-glutamyl thioester, the steady state concentration of the intermediate with the Glu338Gly mutant was comparable to the wild type hybrid because both the rate of formation and breakdown of the thioester are reduced . This result is consistent with the postulated role of Glu338 in maintaining His336 in the optimal orientation for catalysis and suggests a mechanism for the GLN CPS functional linkage. Biochim Biophys Acta, 1998 Nov 10, 1388(2), 315 - 24 DNA binding properties and processive proofreading of herpes simplex virus type 1 DNA polymerase; Strick R et al.; The DNA binding properties of herpes simplex virus type 1 DNA polymerase (HSV pol), an alpha-like DNA polymerase, were investigated using an optimized band-shift assay . With linear double-stranded DNA (dsDNA), HSV pol formed two complexes . The favored DNA template was dsDNA with protruding 5'-phosphoryl termini . Stable binding of HSV pol was observed with a DNA hairpin containing a primer region of 9 bp of dsDNA, a 6-base loop and a 12-base 5'-terminal single-stranded extension . For the polymerization activity of HSV pol on poly(dT) an optimal primer length of 8 to 10 nucleotides was determined . The DNA binding event could be clearly separated from the enzymatic activities by its unique response to divalent cations and salt . Under ionic strength conditions where HSV pol exerts optimal polymerization activity in vitro, novel polymerase-DNA complexes were detected by band-shift analysis . These new complexes were similar while either in DNA polymerase or 3',5' exonuclease mode . Using a polymerase trap method and high-resolution polyacrylamide gel electrophoresis, HSV pol demonstrated internal switching from 3',5' exonuclease to polymerase-active mode during one DNA binding event . These results support the role of HSV pol as a true replicase, which proofreads without dissociating from the DNA template. Gene, 1998 Nov 26, 223(1-2), 235 - 45 GalR-mediated repression and activation of hybrid lacUV5 promoter: differential contacts with RNA polymerase; Ryu S et al.; The GalR repressor regulates expression of genes of the gal regulon in Escherichia coli . We studied the regulatory effect of GalR in vitro on a heterologous promoter, lacUV5, by placing the GalR-binding site, OE, at different locations upstream of this promoter . Despite the fact that the lacUV5 promoter is transcribed efficiently by RNA polymerase (RNP) alone, GalR modulated transcription from many of the PlacUV5 variants . Depending on the location of OE and the neighboring DNA sequence, GalR repressed, activated or had no effect on the promoter . Both repression and activation involved formation of GalR-RNP-DNA ternary complexes and required an intact c-domain of the alpha subunit of the holoenzyme . These results support the differential contact model of a regulator action, in which a regulator differentially binds to, and lowers the energy of, intermediates of transcription initiation either to hinder or to facilitate a step of initiation . The nature of the contacts depends upon the context, i.e . the geometry of the ternary complex . The observed repression and activation effect of GalR on a heterologous promoter also underscores the point that a regulator is not a dedicated protein for repression or for activation. Gene, 1998 Nov 26, 223(1-2), 221 - 31 Using chromosomal lacIQ1 to control expression of genes on high-copy-number plasmids in Escherichia coli; Glascock CB et al.; Transcription of the lac and the hybrid tac promoters is repressed by the lac repressor and induced by the non-metabolizable substrate IPTG . The degree of repression depends upon the ratio of LacI molecules in a cell to the DNA operator sites . In the absence of an inducer, repression of Ptac on a high-copy-number (hcn) plasmid was equivalent in strains containing lacIQ1 on the chromosome, or lacI+ on the plasmid, but not from strains with lacI+ or lacIQ only on the chromosome . Induction of Ptac on hcn plasmids in strains in which expression was controlled by lacIQ1 occurred at very low inducer concentrations (3-10microM IPTG) and reached levels significantly higher than in strains with lacI+ on the plasmid . Greater than 300-fold induction of a beta-LacZ fusion was observed, and >600-fold induction was estimated from recombinant hemoglobin synthesis . Transcription from PlacIQ1 initiated in the same point as PlacI+, but was 170-fold stronger, consistent with the lac repressor levels required to control LacI-regulated genes on hcn plasmids . The DNA sequence upstream of lacI was used to develop a simple PCR test to identify lacIQ1 by a characteristic 15-bp deletion . This deletion created a consensus -35 hexamer, responsible for the increased lacI transcription, and was easily detectable in a variety of strains . Using lacIQ1 hosts eliminates the requirement to maintain lacI on the plasmid to regulate gene expression on hcn expression plasmids. Gene, 1998 Nov 26, 223(1-2), 205 - 11 TrfA dimers play a role in copy-number control of RK2 replication; Toukdarian AE et al.; Copy-number regulation of the broad-host-range plasmid RK2 is dependent on the plasmid-encoded initiator protein, TrfA, and the RK2 origin of replication . The handcuffing model for copy-number control proposes that TrfA-bound oris reversibly couple to prevent the further initiation of plasmid replication when the copy number in vivo is at or above the replicon-specific copy number . TrfA mutants have been isolated which allow for oriV replication at elevated copy numbers . To better understand the mechanism of 'handcuffing', the copy-up TrfA(G254D/S267L) mutant was characterized further . In the present study we show by size exclusion chromatography and native gel electrophoresis that unlike wt TrfA which is largely dimeric, purified His6-TrfA(G254D/S267L) is primarily monomeric . In vivo, TrfA33(G254D/S267L) supports replication of an RK2 ori plasmid in trans at a greatly elevated copy number, while in cis the plasmid exhibits runaway replication . However, expression of either of two previously isolated DNA-binding defective TrfA mutants, TrfA33(P151S) or TrfA33(S257F), in a cell transformed with a mini-RK2 replicon encoding TrfA33(G254D/S267L) results in suppression of the runaway phenotype . His6-TrfA(P151S) and His6-TrfA(S257F) purify as dimers, and when expressed in vivo are incapable of supporting RK2 plasmid replication . In contrast, combination of the trfA(P151S) or trfA(S257F) mutation with the trfA(G254D/S267L) mutations results in the expression of mutant TrfA proteins which are mainly monomers and which can no longer restore copy control to replication directed by TrfA33(G254D/S267L) in vivo . On the basis of these findings a handcuffing model is proposed, whereby oriV-bound TrfA monomers are coupled by dimeric TrfA molecules. Biochim Biophys Acta, 1998 Dec 9, 1415(1), 114 - 24 The turgor sensor KdpD of Escherichia coli is a homodimer; Heermann R et al.; Escherichia coli responds to K+-limitation or high osmolarity by induction of the kdpFABC operon coding for the high affinity K+-translocating KdpFABC complex . Expression of the corresponding operon is controlled by the membrane-bound sensor kinase KdpD and the cytoplasmic response regulator KdpE . Here, we examine the oligomeric state of KdpD . KdpD-His673-->Gln and KdpD-Asn788-->Asp are kinase inactive . When the corresponding genes are coexpressed, the resulting KdpD protein regains kinase activity in vitro, suggesting that the functional state of KdpD is at least a dimer and that the kinase reaction is a result of a trans-phosphorylation between two monomers . Furthermore, coexpression of kdpD-6His and kdpD-(Delta128-391) leads to stable heterooligomers that can bind to Ni-NTA agarose and that are coeluted . Purified and solubilized KdpD-6His has been electrophoresed in blue native polyacrylamide gels (BN-PAGE), and unphosphorylated and phosphorylated KdpD resulted in the same band pattern suggesting that the oligomeric state of KdpD does not change upon phosphorylation . In addition, determination of the molecular masses of KdpD-6His and KdpD-6His approximately 32P by gel filtration reveals a value of 245 kDa for both forms of the protein . The Stokes radius is determined to be 5.4 nm . Sucrose gradient sedimentation analysis of KdpD-6His results in a molecular mass of 289 kDa . The calculated molecular mass of a KdpD-6His monomer is 99.6 kDa . Considering the detergent bound to KdpD the obtained data reveal that KdpD is a homodimer and there is no change in the oligomeric state upon activation . Crosslinking experiments with single Cys KdpD molecules indicate that there is a close contact between the monomers in the transmitter as well as in transmembrane domain 1 . BN-PAGE of solubilized and purified KdpD-6His devoid of Cys residues demonstrates that Cys residues do not contribute to the stabilization of the dimer. Gene, 1998 Nov 26, 223(1-2), 105 - 13 Arrangement and functional identification of genes in the regulatory region of lambdoid phage H-19B, a carrier of a Shiga-like toxin; Neely MN et al.; H-19B is a lambdoid phage that carries the genes (stx-I) encoding the two toxin subunits of a Shiga-like toxin; Escherichia coli lysogens of H-19B are converted to toxin producers . Based on the determination of a 17-kb region of the H-19B genome and functional studies, we have identified the early regulatory region and associated genes of H-19B, as well as the location of the late regulatory region and the toxin and lysis genes . A comparative analysis of the sequence of the H-19B genome reveals the presence of ORFs and genes found in analogous positions on the genomes of a number of other lambdoid phages . A cloned genomic fragment that confers immunity to an infecting H-19B phage contains an ORF of an analogous size and genomic location for a repressor gene, adjacent to a putative operator region . The lambda replication genes, O and P, are conserved in H-19B except for a 39-bp insert in the O gene creating two new O protein-binding sites in the origin of replication (ori), giving H-19B six binding sites as opposed to the four sites found in lambda . We identify ORFs and sequences involved in transcriptional regulation encoding N-like antitermination systems like those found in other lambdoid phages and nearly identical to sequences found in phage HK97 . Our functional studies show that these sequences support antitermination even though they contain significant differences from those of other lambdoid phages . We also identify ORFs and sequences analogous to the Q-p'R late antiterminators-promoters found in other lambdoid phages . The Shiga-like stx-I genes are located directly downstream of the promoter, p'R, for the late genes, and upstream of the lysis genes. Gene, 1998 Nov 26, 223(1-2), 83 - 91 Genomic DNA sequencing by SPEL-6 primer walking using hexamer ligation; Kaczorowski T et al.; DNA sequencing by SPEL-6 (Sequential Primer Elongation by Ligation of 6-mers) primer walking is based on the rapid assembly of true primers by ligation of several (three to 10) contiguous hexamers complementary to a DNA template saturated with Escherichia coli single-stranded DNA-binding protein . To prove the usefulness and to check the reliability of this method, a 3-kb DNA fragment carrying the genes encoding the EcoVIII restriction-modification (RM) system was sequenced with low redundancy (2.8) . The use of both single-stranded (ss) and double-stranded (ds) DNA templates was compared . For this project, 27 primers were assembled by hexamer ligation to form 18-30-nt strings of three to five hexamers . Each primer was designed based on nucleotide sequence determined in a previous run, and was produced in a matter of minutes . The overall length of the easily readable sequencing ladders was about 300-450nt . We found that strong secondary structures in the ss DNA tend to interfere with its template function for the primer assembly by hexamer ligation, especially when they overlap the 3'-end of such a primer . This was easily overcome either by avoiding such hairpin regions or by using longer strings of hexamers, since we show that their ligation is highly cooperative, and ligation efficiency increases with the length of the string () . Some general rules for successful primer assembly and prospects for using the SPEL-6 method for large-scale, fully automated fluorescent sequencing of large genomes are discussed. Biochim Biophys Acta, 1998 Dec 9, 1415(1), 77 - 84 Assembly of the Kdp complex, the multi-subunit K+-transport ATPase of Escherichia coli; Gassel M et al.; Kdp, the high affinity ATP-driven K+-transport system of Escherichia coli, is a complex of the membrane-bound subunits KdpA, KdpB, KdpC and the small peptide KdpF . The assembly of this complex was studied by the analysis of mutants that expressed two of the three large subunits and inserted them into the cytoplasmic membrane . In the strains that do not express KdpC or KdpA the other two subunits did not copurify on dye-ligand affinity columns after solubilization with non-ionic detergent . In the mutant lacking KdpB the other two subunits copurified under the same conditions . It is concluded that KdpC forms strong interactions with the KdpA subunit, serving to assemble and stabilise the Kdp complex . A structure in which KdpC could be one of the connecting links between the energy-delivering subunit KdpB and the K+-transporting subunit KdpA is suggested by these data. Mutat Res, 1999 Jan 2, 438(1), 53 - 62 Sunlight mutagenesis: changes in mutational specificity during the irradiation of phage M13mp2; Yang SJ et al.; We reported previously that the mutations in phage M13mp2, a single-stranded DNA phage, induced by sunlight exposure are predominated by G-to-C transversions . We have now made an unexpected observation that an exposure to sunlight for a short period of time results in induction mainly of C-to-T transitions while a longer exposure results in the induction of G-to-C transversions . This peculiar phenomenon suggests that DNA damage formed by initial sunlight exposure can be transformed during an elongated exposure . 7, 8-Dihydro-8-oxoguanine (8-oxoG) in DNA might be involved in the shift of the mutational specificity, as 8-oxoG was formed in the phage DNA upon the sunlight exposure . We also compared the mutagenic activity of UVB irradiation with that of sunlight exposure . The results demonstrate that the genotoxic properties of sunlight and UVB in phage M13mp2 mutagenesis are different . The shift in the mutational specificity associated with the dose of the sunlight may call for general cautions in the studies of agent-induced mutagenesis . Mol Cell Biol, 1999 Jan, 19(1), 556 - 66 The nuclease activity of Mre11 is required for meiosis but not for mating type switching, end joining, or telomere maintenance; Moreau S et al.; The Saccharomyces cerevisiae MRE11 gene is required for the repair of ionizing radiation-induced DNA damage and for the initiation of meiotic recombination . Sequence analysis has revealed homology between Mre11 and SbcD, the catalytic subunit of an Escherichia coli enzyme with endo- and exonuclease activity, SbcCD . In this study, the purified Mre11 protein was found to have single-stranded endonuclease activity . This activity was absent from mutant proteins containing single amino acid substitutions in either one of two sequence motifs that are shared by Mre11 and SbcD . Mutants with allele mre11-D56N or mre11-H125N were partially sensitive to ionizing radiation but lacked the other mitotic phenotypes of poor vegetative growth, hyperrecombination, defective nonhomologous end joining, and shortened telomeres that are characteristic of the mre11 null mutant . Diploids homozygous for the mre11-H125N mutation failed to sporulate and accumulated unresected double-strand breaks (DSB) during meiosis . We propose that in mitotic cells DSBs can be processed by other nucleases that are partially redundant with Mre11, but these activities are unable to process Spo11-bound DSBs in meiotic cells. Mol Cell Biol, 1999 Jan, 19(1), 173 - 81 The beta subunit of eukaryotic translation initiation factor 2 binds mRNA through the lysine repeats and a region comprising the C2-C2 motif; Laurino JP et al.; Eukaryotic translation initiation factor 2 (eIF2) has been implicated in the selection of the AUG codon as the start site for eukaryotic translation initiation, since mutations in its three subunits in yeast that allow the recognition of a UUG codon by the anticodon of the initiator Met-tRNAMet have been identified . All such mutations in the beta subunit of eIF2 (eIF2beta) mapped to a region containing a putative zinc finger structure of the C2-C2 type, indicating that these sequences could be involved in RNA recognition . Another feature of eIF2beta that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three repeats of seven lysine residues which are highly conserved in other species . We show here the ability of eIF2beta, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro . Through a deletion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C2-C2 motif . Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run . We further show that only one run of lysine residues is sufficient for the in vivo function of eIF2beta, probably through charge interaction, since its replacement by arginines did not impair cell viability, whereas substitution for alanines resulted in inviable cells . mRNA binding, but not GTP-dependent initiator Met-tRNAMet binding, by the eIF2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit. J Exp Med, 1998 Dec 21, 188(12), 2277 - 88 Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses; Ermak TH et al.; We examined the roles of cell- and antibody-mediated immunity in urease vaccine-induced protection against Helicobacter pylori infection . Normal and knockout mice deficient in major histocompatibility complex (MHC) class I, MHC class II, or B cell responses were mucosally immunized with urease plus Escherichia coli heat-labile enterotoxin (LT), or parenterally immunized with urease plus aluminum hydroxide or a glycolipid adjuvant, challenged with H . pylori strain X47-2AL, and H . pylori organisms and leukocyte infiltration in the gastric mucosa quantified . In an adjuvant/route study in normal mice, there was a direct correlation between the level of protection and the density of T cells recruited to the gastric mucosa . In knockout studies, oral immunization with urease plus LT protected MHC class I knockout mice {beta2-microglobulin (-/-)} but not MHC class II knockout mice {I-Ab (-/-)} . In B cell knockout mice {microMT (-/-)}, vaccine-induced protection was equivalent to that observed in immunized wild-type (+/+) mice; no IgA+ cells were detected in the stomach, but levels of CD4(+) cells equivalent to those in the wild-type strain (+/+) were seen . These studies indicate that protection of mice against H . pylori infection by immunization with the urease antigen is dependent on MHC class II-restricted, cell-mediated mechanisms, and antibody responses to urease are not required for protection. Br J Haematol, 1998 Dec, 103(3), 839 - 41 R411C mutation of the ALAS2 gene encodes a pyridoxine-responsive enzyme with low activity; Furuyama K et al.; A R411C missense mutation of the erythroid-specific delta-aminolaevulinate synthase (ALAS2) gene was identified in a pedigree with X-linked pyridoxine-responsive sideroblastic anaemia (XLSA) . The normal and the mutant cDNAs were expressed in E . coli, and the enzyme protein was purified . ALAS activity of the mutant enzyme was 12% and 25%, when incubated in the absence and the presence of pyridoxal 5'-phosphate, respectively, compared with that of the wild-type enzyme . These findings suggest that the R411C mutation accounts for low ALAS activity and a partial pyridoxine-responsiveness of the disease in the patient. Virus Genes, 1998, 17(2), 107 - 15 Orf virus encodes a homolog of the vaccinia virus interferon-resistance gene E3L; McInnes CJ et al.; A homolog of the vaccinia virus (VAC) interferon resistance gene E3L has been discovered in orf virus strain NZ-2, a parapoxvirus that infects sheep, goats and humans . The gene is located 20 kb from the left terminus of the orf virus genome and is transcribed towards this terminus . RNase protection studies have been used to define the limits of the gene and Northern analysis revealed that it is expressed early in infection . The predicted amino acid sequence of the orf virus protein shares 31% identity (57% similarity) with the VAC E3L protein . Four of the six residues identified as being essential to dsRNA binding in the vaccinia virus protein are conserved in the orf virus protein whilst the other two amino acid changes are conservative substitutions . The orf virus gene has been sequenced in two other orf virus strains which vary markedly in their ability to produce experimental lesions in vivo . Their predicted protein sequences vary by less than 3% from the NZ-2 protein . The recombinant orf virus protein, expressed as a fusion protein in E . coli, bound double-stranded (ds)RNA but not dsDNA, single-stranded (ss)DNA or ssRNA . This is the first demonstration of a VAC E3L-like gene encoded by a parapoxvirus. J Clin Immunol, 1998 Nov, 18(6), 421 - 9 Presence of activated B-1 cells in chronic inflamed gingival tissue; Aramaki M et al.; B-1 cells are physically and functionally unique B cells that produce polyreactive natural antibody . This study examined the activation of B-1 cells in inflamed gingival tissue . Serum IgG antibodies to phosphorylcholine, E . coli LPS, DNA, and some commensal bacteria were examined in adult periodontitis patients and healthy subjects . In addition, the proportion of B-1a (CD20+CD5+) cells and the amount of IL-6 and IL-10 in the inflamed gingival tissues were examined . The serum levels of IgG antibodies to phosphorylcholine, E . coli LPS, and commensal bacteria were significantly higher in the adult periodontitis patients than the healthy subjects . The proportion of B-1a cells and the amount of IL-6 and IL-10 were significantly higher in the inflamed gingival tissues than in peripheral blood from the healthy subjects . These results suggest the activation of B-1 cells in the inflamed gingival tissue of adult periodontitis patients, and that B-1 cells may serve as the first line of defense by producing polyreactive antibodies to phosphorylcholine, LPS, and commensal bacteria. J Biol Chem, 1998 Dec 25, 273(52), 35307 - 18 Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains; Foster KA et al.; Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis . We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli . Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP . Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules . The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively . The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release . Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex . These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate. J Biol Chem, 1998 Dec 25, 273(52), 35282 - 90 Cloning and characterization of a novel mammalian PP2C isozyme; Tong Y et al.; PP2C is a structurally diversified protein phosphatase family with a wide range of functions in cellular signal transduction . A novel PP2C subtype, designated PP2Cdelta, was identified from a rat cDNA clone, which encodes a protein of 392 amino acid residues . While PP2Cdelta shares approximately 30% sequence identity in its catalytic domain with the mammalian PP2C, it lacks a 90-residue carboxyl-terminal sequence conserved in mammalian PP2C . Northern blot analysis showed that PP2Cdelta is widely expressed in rat tissues . The transcription of the PP2Cdelta gene was activated in response to stress, such as the addition of ethanol to the culture medium or UV irradiation of cells . Recombinant PP2Cdelta purified from bacteria exhibited a potent Mn2+-dependent serine/threonine phosphatase activity . Unlike other members of the PP2C family, the activity of PP2Cdelta was inhibited, rather than stimulated, by Mg2+ . Transfection with PP2Cdelta resulted in inhibition of cell growth, precluding generation of stable 293 or CHO transfectants . Using a modified tetracycline-regulated PP2Cdelta-GFP dicistronic expression cassette, it was revealed that overexpression of PP2Cdelta blocked cell cycle progression and arrested cells at early S phase, resulting in inhibition of DNA synthesis and leading to cell death . These results suggest that PP2Cdelta plays a role in regulation of cell cycle progression via dephosphorylation of its substrates whose appropriate phosphorylation states might be crucial for cell proliferation. J Biol Chem, 1998 Dec 25, 273(52), 35245 - 9 Structure-function studies of human leptin; Imagawa K et al.; To elucidate the structural requirement of human leptin for its functions, the wild-type, mutant-type, C-terminal deletion, and N-terminal deletion were expressed in Escherichia coli and purified in soluble forms . These leptin analogs were intracerebroventrically injected into C57BL/6J ob/ob mice, and their in vivo biological activities were evaluated . The mutant-type leptin lacking a C-terminal disulfide bond reduced food intake at doses of more than 15 pmol/mouse, which was as effective as the wild-type leptin . C-terminal deletion without the loop structure, also significantly, but to a lesser extent, reduced food intake at doses of more than 90 pmol/mouse . However, N-terminal deletions showed no effect on food intake . We also evaluated the effects of the leptin analogs on radiolabeled leptin binding to its receptor in the choroid plexus using autoradiography . An excess of unlabeled mutant-type leptin as well as wild-type leptin led to complete inhibition of binding . C-terminal deletions led to weak inhibitory activity, whereas N-terminal deletions caused no inhibitory activity . These results clearly demonstrate that the N-terminal region of leptin is essential for both its biological and receptor binding activities . The amino acid sequence of the C-terminal loop structure is also important for enhancing these actions, whereas the C-terminal disulfide bond is not needed. J Biol Chem, 1998 Dec 25, 273(52), 35126 - 31 Identification of a potent DNase activity associated with RNase T of Escherichia coli; Viswanathan M et al.; RNase T was first identified as an enzyme responsible for end turnover of tRNA in Escherichia coli . Its activity, specific for tRNA-C-C-A, catalyzes the release of tRNA-C-C and AMP . RNase T, along with several other RNases, plays a role in maturation of several other RNA species by a similar limited nuclease activity . In previous work, we identified the gene for RNase T, rnt, as a high copy suppressor of the UV sensitivity conferred by deficiency in three single-strand DNA-specific exonucleases, RecJ, exonuclease I, and exonuclease VII . This suggested that RNase T may process DNA substrates as well . In this work, we show that purified RNase T possesses a potent 3' to 5' single-strand DNA-specific exonucleolytic activity . Its Km for single-strand DNA substrates is many orders of magnitude lower than that for tRNA, suggesting that single-strand DNA may be a natural biological substrate for RNase T . We suggest that the DNase activity of RNase T may play a role in end trimming reactions during DNA recombination and/or DNA repair. J Biol Chem, 1998 Dec 25, 273(52), 35109 - 17 Identification of multiple Caenorhabditis elegans caspases and their potential roles in proteolytic cascades; Shaham S; Proteases of the caspase family play a central role in the execution of programmed cell death in all metazoans examined . The Caenorhabditis elegans caspase CED-3 is essential for programmed cell death in this organism . Three additional C . elegans caspase-related genes, csp-1 (caspase homolog-1), which encodes the csp-1A, csp-1B, and csp-1C RNA species; csp-2, which encodes the csp-2A and csp-2B RNA species; and csp-3 are identified . CSP-1A, CSP-1B, CSP-2A, and CSP-2B proteins are similar in sequence to caspase proproteins . CSP-1C is similar only to large caspase subunits, and CSP-3 is similar only to small caspase subunits . CSP-1B can be activated to become a cysteine protease by processing at internal aspartate residues . Activated CSP-1B can cleave the CSP-1B, CED-3, and CSP-2B proproteins, and activated CED-3 can cleave the CED-3 and CSP-2B proproteins . Inhibitor and synthetic substrate studies further suggest that activated CSP-1B and activated CED-3 have different substrate specificities . These results suggest that C . elegans encodes several caspases that might act in proteolytic cascades to regulate processes such as programmed cell death. J Biol Chem, 1998 Dec 25, 273(52), 34976 - 82 The rub family of ubiquitin-like proteins . Crystal structure of Arabidopsis rub1 and expression of multiple rubs in Arabidopsis; Rao-Naik C et al.; Several proteins with significant identity to ubiquitin have been characterized recently . In contrast to ubiquitin's main role in targeting proteins for degradation, a described function of one family of ubiquitin-related proteins, the Rub family, is to serve as a stable post-translational modification of a complex involved in the G1-to-S cell cycle transition . Rub proteins have been found in animals, plants, and fungi and consist of 76 residues with 52-63% identity to ubiquitin . In this study three different RUB proteins within the plant Arabidopsis are identified; two differ by only 1 amino acid, while the third is only 77.6% identical to the other two . Genes encoding all three are expressed in multiple organs . In addition, we report the crystal structure of higher plant RUB1 at 1.7-A resolution to help elucidate the functional differences between Rub and ubiquitin . RUB1 contains a single globular domain with a flexible COOH-terminal extension . The overall RUB1 structure is very similar to ubiquitin . The majority of the amino acid differences between RUB1 and ubiquitin map to the surface . These changes alter the electrostatic surface potential in two regions and likely confer specificity between ubiquitin and RUB1 and their ubiquitin-activating enzyme (E1) or E1-like activating enzymes. J Biol Chem, 1998 Dec 25, 273(52), 34920 - 5 Biochemical characterization of tomato annexin p35 . Independence of calcium binding and phosphatase activities; Lim EK et al.; Tomato annexin p35 has been cloned and used in a site-directed mutagenesis study to explore the phospholipid binding and catalytic properties of the protein in detail . Analysis of the cDNA sequence of p35 reveals that the annexin has only two typical endonexin folds, corresponding to repeats I and IV . Expression of recombinant p35 in Escherichia coli confirmed both phospholipid binding and a nucleotide phosphatase activity that could be inhibited on interaction of the recombinant annexin with phospholipids . Site-directed mutagenesis in which the acidic residues Glu-68 (repeat I), and Asp-297 (repeat IV) were changed to Asn, generated two mutant forms, E68N and D297N, respectively . Both mutant forms of the annexin continued to express catalytic activity . Changing repeat I had little effect on phospholipid binding, whereas the change to repeat IV abolished this property . These data show that, in this plant annexin, repeat IV plays a more critical role in calcium-dependent phospholipid binding than repeat I, and that the catalytic and phospholipid binding activity of the protein can be separated experimentally. J Biol Chem, 1998 Dec 25, 273(52), 34896 - 903 Characterization of the Escherichia coli damage-independent UvrBC endonuclease activity; Moolenaar GF et al.; Incision of damaged DNA templates by UvrBC in Escherichia coli depends on UvrA, which loads UvrB on the site of the damage . A 50-base pair 3' prenicked DNA substrate containing a cholesterol lesion is incised by UvrABC at two positions 5' to the lesion, the first incision at the eighth and the second at the 15th phosphodiester bond . Analysis of a 5' prenicked cholesterol substrate revealed that the second 5' incision is efficiently produced by UvrBC independent of UvrA . This UvrBC incision was also found on the same substrate without a lesion and, with an even higher efficiency, on a DNA substrate containing a 5' single strand overhang . Incision occurred in the presence of ATP or ADP but not in the absence of cofactor . We could show an interaction between UvrB and UvrC in solution and subsequent binding of this complex to the substrate with a 5' single strand overhang . Analysis of mutant UvrB and UvrC proteins revealed that the damage-independent nuclease activity requires the protein-protein interaction domains, which are exclusively needed for the 3' incision on damaged substrates . However, the UvrBC incision uses the catalytic site in UvrC which makes the 5' incision on damaged DNA substrates. J Biol Chem, 1998 Dec 25, 273(52), 34799 - 805 The C331A mutant of neuronal nitric-oxide synthase is defective in arginine binding; Martasek P et al.; It has been proposed that Cys99 of human endothelial nitric oxide synthase (eNOS) is responsible for tetrahydrobiopterin (BH4) binding . To examine this possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escherichia coli, with the homologous Cys331 to Ala mutation, and characterized structural and functional attributes of the purified, mutated enzyme . C331A-nNOS, as isolated, was catalytically incompetent . Upon prolonged incubation with L-arginine (L-Arg), not only BH4 binding but also catalytic activity could be restored . In contrast to wild-type nNOS (WT-nNOS), which exhibits an absorbance maximum at 407 nm that shifts immediately upon L-arginine addition to a high spin form, the C331A-nNOS mutant, as isolated, exhibited an absorbance maximum at 420 nm . C331A-nNOS, as isolated, did not bind detectable levels of either {3H}Nomega-nitro-L-arginine or {3H}BH4, but {3H}BH4 binding was reinstated after extended incubation with excess L-arginine . On the other hand, C331A-nNOS and WT-NOS were identical with regard to imidazole binding affinity, CaM binding affinity, and rates of cytochrome c and 2, 6-dichlorophenolindophenol reduction . EPR spectroscopy revealed conversion of low to high spin heme after extended incubation with high concentrations of L-arginine (0.1-10 mM) . The estimated Kd for L-arginine binding to C331A-nNOS was two orders of magnitude greater than WT-nNOS (>100 microM versus 2-3 microM) . Here we propose that Cys331 plays an important role in stabilizing L-arginine binding to nNOS . Our findings suggest that the primary dysfunction in the C331A mutant of nNOS, as isolated, is disruption of the BH4-substrate binding interactions as broadcast from this mutated cysteine residue . Prolonged incubation with L-arginine appears to cause remodeling of the mutant protein to a form similar to that of WT-nNOS, allowing for normalized BH4 binding and nitric oxide synthetic activity. J Biol Chem, 1998 Dec 25, 273(52), 34753 - 9 A mutational approach shows similar mechanisms of recognition for the isolated and integrated versions of a protein epitope; Rondard P et al.; Antibody mAb164 is directed against the native form of the TrpB2 subunit of Escherichia coli tryptophan synthase . It recognizes a synthetic peptide, P11, constituted of residues 273-283 of TrpB, with high affinity . We introduced 16 single and 3 double mutations in each of the two contexts, TrpB2 and P11, and used them as local probes to study the cross-reactivity of mAb164 toward these two antigens . The equilibrium constant, KD, of dissociation from mAb164 was measured for each of the mutant derivatives of TrpB2 and P11 by a competition enzyme-linked immunosorbent assay and compared with the wild type one . The variation of the free energy of interaction, DeltaDeltaG, covered nearly 8 kcal/mol for the different mutations . The values of DeltaDeltaG for the mutant derivatives of TrpB2 and for those of P11 were close and the two sets of values were strongly correlated (r = 0.96) . This correlation showed that mAb164 recognized the integrated and isolated versions of residues 273-283 with very similar mechanisms . A few significant differences between the recognitions of TrpB2 and P11 by mAb164 suggested some adaptability of the interaction . The results were compatible with a recognition of residues 273-283 of TrpB in a loop conformation, close to their structure in the crystals of the complete tryptophan synthase, TrpA2TrpB2. Mol Cells, 1998 Oct 31, 8(5), 623 - 8 A site in the dinucleotide-fold domain contributes to the accuracy of tRNA selection by Escherichia coli methionyl-tRNA synthetase; Kim HY et al.; Interactions of specific amino acid residues of the carboxyl-terminal domain of MetRS with the CAU anticodon of tRNAMet assure accurate and efficient aminoacylation . The substitution of one such residue, Trp461 by Phe, impairs the binding of cognate tRNA, but enhances the binding of noncognate tRNAs, particularly those containing G at the wobble position . However, the enhanced binding of noncognate tRNAs is not accompanied by the increased aminoacylation of these tRNAs . A genetic screening procedure was designed to isolate methionyl-tRNA synthetase mutants which were able to aminoacylate a GGU (threonine) anticodon derivative of tRNAfMet . One such mutant, obtained from W461F MetRS, had an Ile29 to Thr substitution in helix A located in the amino-terminal dinucleotide-fold domain that forms the site for amino acid activation . Analysis of the catalytic properties of the I29T/W461F enzyme indicates that the mutation in helix A of the dinucleotide-fold domain affects kcat for aminoacylation of tRNAs having a GGU threonine anticodon . Interactions with cognate tRNAfMet (CAU), as well as with methionine and ATP were not affected by the Ile29 to Thr substitution . We conclude that the I29T substitution leads to a slight adjustment of the alignment of the CCA stem of noncognate tRNAs (GGU) in the catalytic domain of the enzyme, reflected in the increase in kcat, which also allows mischarging in vivo . A function of Ile29 is therefore to minimize the mischarging of tRNAThr (GGU) by methionyl-tRNA synthetase . The methods described here provide useful tools for examining the mechanisms of tRNA selection by aminoacyl-tRNA synthetases. Mol Cells, 1998 Oct 31, 8(5), 614 - 7 CRP.cAMP-dependent transcription activation of the Escherichia coli pts Po promoter by the heat shock RNA polymerase (Esigma32) in vitro; Ryu S; The P0 promoter of the Escherichia coli pts operon that is activated during growth in the presence of glucose contains a recognition sequence for the RNA polymerase holoenzyme containing sigma 70 (Esigma70) which is the RNA polymerase holoenzyme responsible for the transcription of heat shock genes, and a putative recognition element for Esigma32, the Esigma32 was found to be able to transcribe from the pts P0 promoter with in vitro transcription assays using purified RNA polymerase holoenzymes . Esigma32 and Esigma70 used the same transcriptional start site and both RNA polymerases were activated in the presence of a cAMP receptor protein (CRP) and cAMP complex (CRP.cAMP) . These results suggest the possibility that the contact between CRP.cAMP and the alpha subunit of the RNA polymerase can be made regardless of the kind of sigma subunit that is associated with the core RNA polymerase. Mol Cells, 1998 Oct 31, 8(5), 578 - 84 Cloning and characterization of novel disintegrins from Agkistrodon halys venom; Park D et al.; Snake venom disintegrins act as potent inhibitors of platelet aggregation . In this report, we isolated genes encoding novel members of disintegrins through the screening of Agkistrodon halys venom gland cDNA library . Subsequent characterization of positives revealed the presence of distinct disintegrins named salmosinl, 2, and 3, each containing a characteristic RGD/KGD sequence essential for the binding to integrins . Whereas salmosinl was identical to previously described salmosin purified from A . halys venom, salmosin2 and salmosin3 were predicted to be a novel, 73 amino acid protein with a KGD sequence, and an 80 amino acid protein with an additional 7th disulfide bond, respectively . Taken together, this is the first report describing 3 unique disintegrins, namely, salmosinl with RGD, salmosin2 with KGD and salmosin3 with 7 disulfide bonds are found in a single species of venom . Subsequently, to compare the platelet aggregation inhibitory potential of the recombinant protein with that of natural protein, salmosinl was expressed in E . coli and purified to homogeneity . Recombinant and natural salmosin1 inhibited the binding of alphaIIbbeta3 to fibrinogen with an almost identical IC50 value of 2.2 nM and 4.5 nM respectively . Moreover, recombinant salmosinl displayed an IC50 value approximately 5-fold lower than flavoridin, which was previously described as the most potent venom disintegrin so far . In conclusion, we identified 3 disintegrins with distinct properties through the molecular cloning approach and found that the recombinant salmosinl retained one of the most potent alphaIIbbeta3 antagonist activity. Arch Virol, 1998, 143(11), 2133 - 58 Ultrastructural localization of nonstructural and coat proteins of 19 potyviruses using antisera to bacterially expressed proteins of plum pox potyvirus; Riedel D et al.; Antisera to the bacterially expressed nonstructural proteins (NSP) HC-Pro, CI, NIa, and NIb and the coat protein (CP) of plum pox potyvirus (PPV) were used for analysing the composition of virus-induced cytoplasmic and nuclear inclusions by electron microscopy . The antisera reacted with NSP and CP of PPV on immunogold-labelled ultrathin sections . Antiserum to CP reacted with virions of seven out of 18 other potyviruses . CP was distributed throughout the cytoplasm of infected cells . Antisera to PPV NSP specifically reacted with virus-specific cytoplasmic and/or nuclear inclusions induced by 17 different potyviruses . NSP were furthermore localized in confined cytoplasmic areas in between complex accumulations of virus-specific inclusions . Cylindrical inclusions induced by the potyviruses were proven to consist of CI protein . Most other cytoplasmic or nuclear inclusions were shown to be composed of two or more NSP . An unexpected composition of virus-induced inclusions was observed for the crystalline nuclear inclusions of tobacco etch virus . Here, in addition to the expected presence of NIa and NIb, HC-Pro could be demonstrated . Furthermore, amorphous cytoplasmic inclusions induced by papaya ringspot virus contained the expected HC-Pro but additionally NIa, NIb and CI . Beet mosaic virus-induced nuclear inclusions ('satellite bodies') contained in their electron-dense matrix NIa, NIb, Hc-Pro and CI and in their lacunae CP in bundles of virion-like filaments . The results indicate that all cytoplasmic or nuclear inclusions of potyviruses have to be regarded as deposition sites of excessively produced viral NSP. Carcinogenesis, 1998 Nov, 19(11), 1879 - 87 Molecular analysis of lacI mutants from transgenic fibroblasts exposed to 1,2-epoxybutene; Saranko CJ et al.; 1,3-Butadiene (BD) is a genotoxic carcinogen that is bioactivated to at least two mutagenic metabolites: 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB) . We reported previously that lacI transgenic mice exposed to BD had an increased frequency of specific base substitution mutations in the bone marrow and spleen relative to unexposed controls . In the experiments described here, we determined the mutagenicity and mutational spectrum of EB in Rat2 lacI transgenic fibroblasts as a means of assessing the contribution of this metabolite to the lacI mutational spectrum of BD . Rat2 cells were exposed to 0, 0.4, 0.6, 0.8 or 1.0 mM EB for 24 h, resulting in a range of cell survival from 100 to 15%, respectively . Mutagenicity was assessed at 0, 0.6 and 1.0 mM EB . Unexposed controls had a background mutant frequency of 6 +/- 1 +/- 10(-5), while the mutant frequency in cells exposed to 0.6 and 1.0 mM EB was increased 2- and 3-fold, respectively . DNA sequence analysis of 154 lacI mutants recovered in these experiments revealed an increase in the frequency of specific base substitution mutations in cells exposed to 1.0 mM EB compared with controls . These included G:C-->A:T transitions at non-CpG sites, G:C-->T:A transversions and A:T-->T:A transversions, which have all been observed in lacI mutants isolated from transgenic mice exposed to BD . These results suggest that EB causes mutation primarily by base substitution and that the spectrum of these mutations closely resembles that of BD . These data, along with previous findings from our laboratory, suggest that EB is more likely than DEB to be primarily responsible for the lacI mutational spectrum observed in lacI transgenic mice exposed to BD. Comp Biochem Physiol B Biochem Mol Biol, 1998 Aug, 120(4), 665 - 73 Role of isozyme group-specific sequence 4 in the isozyme-specific properties of human aldolase C; Kusakabe T et al.; To assess which regions of the aldolase C molecule are required for exhibiting isozyme-specific kinetic properties, we have constructed nine chimeric enzymes of human aldolases A and C . Kinetic studies of these chimeric enzymes revealed that aldolase C absolutely required its own isozyme group-specific sequences (IGS), particularly IGS-4, for exhibiting the characteristics of aldolase C which differ significantly from those of isozymes A and B (Kusakabe T, Motoki K, Hori K . Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli . J Biochem (Tokyo) 1994;115:1172-7) . Whereas human aldolases A and B required their own isozyme group-specific sequences-1 and -4 (IGS-1 and -4) as the main determinants of isozyme-specific kinetic properties (Motoki K, Kitajima Y, Hori K . Isozyme-specific modules on human aldolase A molecule . J Biol Chem 1993;268:1677-83; Kusakabe T, Motoki K, Sugimoto Y, Takasaki Y, Hori K . Human aldolase B: liver-specific properties of the isoenzyme depend on type B isozyme group-specific sequence . Prot . Eng . 1994;7:1387-93), the present studies indicate that the IGS-1 is principally substitutable between aldolases A and C . The kinetic data also suggests that the connector-2 (amino acid residues 243-306) may modulate the interaction of IGS units with the alpha/beta barrel of the aldolase molecule. Comp Biochem Physiol B Biochem Mol Biol, 1998 Aug, 120(4), 657 - 63 Production of a biologically active novel goldfish growth hormone in Escherichia coli; Mahmoud SS et al.; Goldfish pituitary contains two types of growth hormones . One with five cysteine residues (type-I) similar to other Cyprinid GHs, and the other with four Cys residues (type-II) similar to those of other fish and tertapod species . Recombinant goldfish type II GH (gfGH-II) was produced in Escherichia coli using the pRSETB expression vector . The gfGH-II was produced fused to a leader sequence, which sequestered into inclusion bodies after expression . The inclusion bodies were solubilized using sodium hydroxide and the fusion protein purified by chelating affinity chromatography . Subsequently, gfGH-II was cleaved and analyzed by Western blotting, using a specific antiserum . For comparison we also produced recombinant common carp GH (cGH) which has 95% similarity to gfGH-II, and tested their growth promoting activity in goldfish . Both forms of GH significantly increased the growth rate of goldfish (P < 0.05), although cGH was found to have a somewhat higher potency than gfGH-II. Biochem J, 1999 Jan 1, 337 ( Pt 1), 141 - 51 Gene structure, expression and chromosomal localization of murine theta class glutathione transferase mGSTT1-1; Whittington A et al.; We have isolated and characterized a cDNA and partial gene encoding a murine subfamily 1 Theta class glutathione transferase (GST) . The cDNA derived from mouse GSTT1 has an open reading frame of 720 bp encoding a peptide of 240 amino acids with a calculated molecular mass of 27356 Da . The encoded protein shares only 51% deduced amino acid sequence identity with mouse GSTT2, but greater than 80% deduced amino acid sequence identity with rat GSTT1 and human GSTT1 . Mouse GSTT1-1 was expressed in Escherichia coli as an N-terminal 6x histidine-tagged protein and purified using immobilized-metal affinity chromatography on nickel-agarose . The yield of the purified recombinant protein from E . coli cultures was approx . 14 mg/l . Recombinant mouse GSTT1-1 was catalytically active towards 1, 2-epoxy-3-(p-nitrophenoxy)propane, 4-nitrobenzyl chloride and dichloromethane . Low activity towards 1-menaphthyl sulphate and 1-chloro-2,4-dinitrobenzene was detected, whereas mouse GSTT1-1 was inactive towards ethacrynic acid . Recombinant mouse GSTT1-1 exhibited glutathione peroxidase activity towards cumene hydroperoxide and t-butyl hydroperoxide, but was inactive towards a range of secondary lipid-peroxidation products, such as the trans-alk-2-enals and trans,trans-alka-2,4-dienals . Mouse GSTT1 mRNA is most abundant in mouse liver and kidney, with some expression in intestinal mucosa . Mouse GSTT1 mRNA is induced in liver by phenobarbital, but not by butylated hydroxyanisole, beta-napthoflavone or isosafrole . The structure of mouse GSTT1 is conserved with that of the subfamily 2 Theta class GST genes mouse GSTT2 and rat GSTT2, comprising five exons interrupted by four introns . The mouse GSTT1 gene was found, by in situ hybridization, to be clustered with mouse GSTT2 on chromosome 10 at bands B5-C1 . This region is syntenic with the location of the human Theta class GSTs clustered on chromosome 22q11.2 . Similarity searches of a mouse-expressed sequence tag database suggest that there may be two additional members of the Theta class that share 70% and 88% protein sequence identity with mouse GSTT1, but less than 55% sequence identity with mouse GSTT2. Nature, 1998 Dec 3, 396(6710), 486 - 91 Crystal structure of the ligand-binding domain of the receptor tyrosine kinase EphB2; Himanen JP et al.; The Eph receptors, which bind a group of cell-membrane-anchored ligands known as ephrins, represent the largest subfamily of receptor tyrosine kinases (RTKs) . They are predominantly expressed in the developing and adult nervous system and are important in contact-mediated axon guidance, axon fasciculation and cell migration . Eph receptors are unique among other RTKs in that they fall into two subclasses with distinct ligand specificities, and in that they can themselves function as ligands to activate bidirectional cell-cell signalling . We report here the crystal structure at 2.9 A resolution of the amino-terminal ligand-binding domain of the EphB2 receptor (also known as Nuk) . The domain folds into a compact jellyroll beta-sandwich composed of 11 antiparallel beta-strands . Using structure-based mutagenesis, we have identified an extended loop that is important for ligand binding and class specificity . This loop, which is conserved within but not between Eph RTK subclasses, packs against the concave beta-sandwich surface near positions at which missense mutations cause signalling defects, localizing the ligand-binding region on the surface of the receptor. Nature, 1998 Dec 3, 396(6710), 482 - 6 The receptor Msn5 exports the phosphorylated transcription factor Pho4 out of the nucleus; Kaffman A et al.; The movement of many transcription factors, kinases and replication factors between the nucleus and cytoplasm is important in regulating their activity . In some cases, phosphorylation of a protein regulates its entry into the nucleus; in others, it causes the protein to be exported to the cytoplasm . The mechanism by which phosphorylation promotes protein export from the nucleus is poorly understood . Here we investigate how the export of the yeast transcription factor Pho4 is regulated in response to changes in phosphate availability . We show that phosphorylation of Pho4 by a nuclear complex of a cyclin with a cyclin-dependent kinase, Pho80-Pho85, triggers its export from the nucleus . We also find that the shuttling receptor used by Pho4 for nuclear export is the importin-beta-family member Msn5, which is required for nuclear export of Pho4 in vivo and binds only to phosphorylated Pho4 in the presence of the GTP-bound form of yeast Ran in vitro . Our results reveal a simple mechanism by which phosphorylation can control the nuclear export of a protein. Proteins, 1998 Dec 1, 33(4), 558 - 66 Homology modeling of an RNP domain from a human RNA-binding protein: Homology-constrained energy optimization provides a criterion for distinguishing potential sequence alignments; Sahasrabudhe PV et al.; We have recently described an automated approach for homology modeling using restrained molecular dynamics and simulated annealing procedures (Li et al, Protein Sci., 6:956-970,1997) . We have employed this approach for constructing a homology model of the putative RNA-binding domain of the human RNA-binding protein with multiple splice sites (RBP-MS) . The regions of RBP-MS which are homologous to the template protein snRNP U1A were constrained by "homology distance constraints," while the conformation of the non-homologous regions were defined only by a potential energy function . A full energy function without explicit solvent was employed to ensure that the calculated structures have good conformational energies and are physically reasonable . The effects of mis-alignment of the unknown and the template sequences were also explored in order to determine the feasibility of this homology modeling method for distinguishing possible sequence alignments based on considerations of the resulting conformational energies of modeled structures . Differences in the alignments of the unknown and the template sequences result in significant differences in the conformational energies of the calculated homology models . These results suggest that conformational energies and residual constraint violations in these homology-constrained simulated annealing calculations can be used as criteria to distinguish between correct and incorrect sequence alignments and chain folds. Yeast, 1998 Nov, 14(15), 1399 - 406 Isolation and sequence analysis of the orotidine-5'-phosphate decarboxylase gene (URA3) of Candida utilis . Comparison with the OMP decarboxylase gene family; Rodriguez L et al.; The URA3 gene of Candida utilis encoding orotidine-5'-phosphate decarboxylase enzyme was isolated by complementation in Escherichia coli pyrF mutation . The deduced amino-acid sequence is highly similar to that of the Ura3 proteins from other yeast and fungal species . An extensive analysis of the family of orotidine-5'-phosphate decarboxylase is shown . The URA3 gene of C . utilis was able to complement functionally the ura3 mutation of Saccharomyces cerevisiae. Am J Respir Crit Care Med, 1998 Dec, 158(6), 1883 - 9 Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury; Kristof AS et al.; The role of nitric oxide (NO) in lung injury remains unclear . Both beneficial and detrimental roles have been proposed . In this study, we used mutant mice lacking the inducible nitric oxide synthase (iNOS) to assess the role of this isoform in sepsis-associated lung injury . Wild-type and iNOS knockout mice were injected with either saline or Escherichia coli endotoxin (LPS) 25 mg/kg and killed 6, 12, and 24 h later . Lung injury was evaluated by measuring lactate dehydrogenase activity in the bronchoalveolar lavage, pulmonary wet/dry ratio, and immunostaining for nitrotyrosine formation . In the wild-type mice, LPS injection elicited more than a 3-fold rise in lactate dehydrogenase activity, a significant rise in lung wet/dry ratio and extensive nitrotyrosine staining in large airway and alveolar epithelium, macrophages, and pulmonary vascular cells . This was accompanied by induction of iNOS protein and increased lung nitric oxide synthase activity . By comparison, LPS injection in iNOS knockout mice elicited no iNOS induction and no significant changes in lung NOS activity, lactate dehydrogenase activity, lung wet/dry ratio, or pulmonary nitrotyrosine staining . These results indicate that mice deficient in iNOS gene are more resistant to LPS-induced acute lung injury than are wild-type mice. J Protein Chem, 1998 Oct, 17(7), 579 - 90 Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis; Funane K et al.; Incubation of maize branching enzyme, mBEI and mBEII, with 100 microM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes . Treatment of the DEPC-inactivated enzymes with 100500 mM hydroxylamine restored the enzyme activities . Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification . The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding . In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508) . His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis . Expression of these mutants in E . coli showed a significant decrease of the activity and the mutant enzymes had Km values 10 times higher than the wild type . Therefore, residues His320 and His508 do play an important role in substrate binding. Hum Gene Ther, 1998 Nov 20, 9(17), 2577 - 83 Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method; Mizuguchi H et al.; An efficient method for constructing a recombinant adenovirus (Ad) vector, based on an in vitro ligation, has been developed . To insert the foreign gene into an adenoviral DNA, we introduced three unique restriction sites, I-CeuI, SwaI, and PI-SceI, into the E1 deletion site of the vector plasmid, which contains a complete E1, E3-deleted adenovirus type 5 genome . I-CeuI and PI-SceI are intron-encoded endonucleases with a sequence specificity of at least 9-10 and 11 bp, respectively . A shuttle plasmid, pHM3, containing multiple cloning sites between the I-CeuI and PI-SceI sites, was constructed . After the gene of interest was inserted into this shuttle plasmid, the plasmid for E1-deleted adenovirus vector could be easily prepared by in vitro ligation using the I-CeuI and PI-SceI sites . SwaI digestion of the ligation products prevented the production of a plasmid containing a parental adenovirus genome (null vector) . After transformation into E . coli, more than 90% of the transformants had the correct insert . To make the vector, a PacI-digested, linearized plasmid was transfected into 293 cells, resulting in a homogeneous population of recombinant virus . The large number and strategic location of the unique restriction sites will not only increase the rapidity of production of new first-generation vectors for gene transfer but will allow for rapid further improvements in the vector DNA backbone. Biol Pharm Bull, 1998 Nov, 21(11), 1139 - 41 Identification of proteins whose amounts are altered by mutation in the pgsA gene of Escherichia coli; Ohba A et al.; We identified proteins whose amounts were altered in an Escherichia coli pgsA3 mutant lacking the potential to synthesize phosphatidylglycerolphosphate, a precursor of phosphatidylglycerol . Proteins whose amounts were increased in the mutant were protease Do, periplasmic oligopeptide-binding protein, tryptophanase, and an unidentified protein, while the decreased one was flagellin . Transformation of the mutant with a plasmid containing the wild type pgsA gene complemented the phenotype, indicating that the pgsA3 mutation is responsible for the phenotype. Biol Pharm Bull, 1998 Nov, 21(11), 1128 - 33 pH-dependent growth retardation of Escherichia coli caused by overproduction of Na+/H+ antiporter; Inoue H et al.; The overproduction of Na+/H+ antiporter NhaA in Escherichia coli caused growth retardation . Quantities and the activity of the antiporter were studied for their causative roles in terms of this retardation . We constructed a series of nhaA-expression plasmids differing in their transcriptional and translational efficiencies . Low-level nhaA expression complemented the defect of an nhaA mutant and allowed the mutant to survive on a high-NaCl or high-LiCl medium . However, when the production of NhaA was strongly induced by the combination of a stronger promoter, an efficient translational initiation signal and a high copy number plasmid, the growth of the cells carrying the plasmid was severely retarded . This growth retardation correlated well with the amount of NhaA protein produced from the plasmids . Surprisingly, the growth retardation caused by overproduction of NhaA was enhanced more extensively at an alkaline pH than at a neutral pH, in which the antiporter activity was stimulated . However, these retardations were also observed for mutant NhaA antiporters without the activity . These results indicated that the growth retardation was due to an overproduction of the antiporter rather than its increased antiporter activity, and also affected by a pH-dependent change in NhaA, possibly its structural change. Cancer J Sci Am, 1998 Nov-Dec, 4(6), 364 - 9 Selective radiosensitization of 9L glioma in the brain transduced with double suicide fusion gene; Kim JH et al.; PURPOSE: Suicide gene therapy has proved to be successful in enhancing the therapeutic index by sensitizing genetically modified tumor cells to prodru