J Appl Physiol, 2001 Jul, 91(1), 130 - 6 Role of nitric oxide in the regulation of glucose kinetics in response to endotoxin in dogs; Moeniralam HS et al.; The purpose of the present in vivo study was to determine the role of nitric oxide (NO) in the regulation of glucose metabolism in response to endotoxin by blocking NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA) . In five dogs, the appearance and disappearance rates of glucose (by infusion of {6,6-(2)H(2)}glucose), plasma glucose concentration, and plasma hormone concentrations were measured on five different occasions: saline infusion, endotoxin alone (E coli, 1.0 microg/kg i.v.), and endotoxin administration plus three different doses of primed, continuous infusion of L-NMMA . Endotoxin increased rate of appearance of glucose from 13.7 +/- 1.6 to 23.6 +/- 3.3 micromol x kg(-1) x min(-1) (P < 0.05), rate of disappearance of glucose from 13.9 +/- 1.1 to 24.8 +/- 3.1 micromol x kg(-1) x min(-1) (P < 0.001), plasma lactate from 0.5 +/- 0.1 to 1.7 +/- 0.1 mmol/l (P < 0.01), and counterregulatory hormone concentrations . L-NMMA did not affect the rise in rate of appearance and disappearance of glucose, plasma lactate, or the counterregulatory hormone response to endoxin . Plasma glucose levels were not affected by endotoxin with or without L-NMMA . In conclusion, in vivo inhibition of NO synthesis by high doses of L-NMMA does not affect glucose metabolism in response to endotoxin, indicating that NO is not a major mediator of glucose metabolism during endotoxemia in dogs. Drug Metab Dispos, 2001 Jul, 29(7), 983 - 9 Screening of organosulfur compounds as inhibitors of human CYP2A6; Fujita K et al.; The capacities to inhibit coumarin 7-hydroxylase activity of human cytochrome P450 2A6 (CYP2A6) by organosulfur compounds were evaluated . Five dialkyl sulfides and five dialkyl disulfides, with alkyl chains from methyl to amyl, were examined . In addition to these chemicals, diallyl sulfide, diallyl disulfide, allyl methyl sulfide, allyl n-propyl sulfide, allyl phenyl sulfide, diphenyl sulfide, diphenyl disulfide, difurfuryl disulfide, phenyl cyclopropyl sulfide, 2,2'-dipyridyl disulfide, 4,4'-dipyridyl sulfide, and 4,4'-dipyridyl disulfide were also examined for their capacity to inhibit CYP2A6 . The membrane fraction of genetically engineered Escherichia coli cells expressing CYP2A6 together with NADPH-cytochrome P450 reductase was used as an enzyme source . Dialkyl disulfides inhibited CYP2A6 more strongly than did dialkyl sulfides . Among dialkyl disulfides examined, di-n-propyl disulfide, contained in onion oil, was the most potent competitive inhibitor of CYP2A6, with a K(i) value of 1.73 microM . Diallyl disulfide, present in garlic oil, inhibited CYP2A6 activity in a competitive/noncompetitive mixed manner, with the K(i) value of 2.13 microM . Among all of the organosulfur compounds tested, 4,4'-dipyridyl disulfide was the most potent inhibitor of CYP2A6, with a K(i) value of 60 nM, followed by 4,4'-dipyridyl sulfide, with a K(i) value of 72 nM . These chemicals inhibited CYP2A6 in a competitive manner . The preincubation time did not affect the inhibitory effects of di-n-propyl disulfide, diallyl disulfide, 4,4'-dipyridyl disulfide, and 4,4'-dipyridyl sulfide on CYP2A6, indicating that these chemicals were not mechanism-based inhibitors of CYP2A6 . 4,4'-Dipyridyl disulfide also inhibited midazolam 1'-hydroxylase activity of CYP3A4 . We discovered 4,4'-dipyridyl disulfide to be a potent and relatively selective inhibitor of CYP2A6. Plasmid, 2001 May, 45(3), 200 - 8 P1 and NR1 plasmid replication during the cell cycle of Escherichia coli; Bogan JA et al.; Replication patterns of the miniP1 plasmid pZC176, the miniNR1 plasmid pRR933, and the high-copy miniNR1 derivative pRR942 were examined during the Escherichia coli cell division cycle and compared to the cycle-specific replication pattern of a minichromosome and the cycle nonspecific pattern of pBR322 . In E . coli cells growing with doubling times of 40 and 60 min, the miniP1 plasmid was found to replicate with a slight periodicity during the division cycle . The periodicity was not nearly as pronounced as that of the minichromosome, was not affected by the presence of a minichromosome, and was not evident in cells growing more rapidly with a doubling time of 25 min . Both miniNR1 plasmids, pRR933 and pRR942, replicated with patterns indistinguishable from that of pBR322 and clearly different from that of the minichromosome . It is concluded that both P1 and NR1 plasmids can replicate at all stages of the cell cycle but that P1 displays a slight periodicity in replication probability in the cycle of slower growing cells . This periodicity does not appear to be coupled to a specific age in the cycle, but could be associated with the achievement of a specific cell mass per plasmid . During temperature shifts of a dnaC(Ts) mutant, the miniP1 plasmid and pBR322 replicated with similar patterns that differed from that of the minichromosome, but were consistent with a brief eclipse between rounds of replication. Mol Ther, 2001 Jun, 3(6), 821 - 30 Specific recognition of protein carboxy-terminal sequences by natural IgM antibodies in normal serum; Sokoloff AV et al.; Our previous study indicated that normal serum contains complement-fixing natural IgM antibodies reacting with a large variety of randomly generated protein carboxy-termini . Here we show that the "carboxy-terminal" IgM (C-IgM) antibodies specifically react with short peptide sequences located immediately at the protein carboxy-terminus . The specificity of C-IgM-peptide interactions is tentatively defined by three to four amino acid residues . All carboxy-terminal peptides in a large peptide library apparently react with C-IgM antibodies . Immobilized synthetic peptides also react with C-IgM antibodies . No interaction of C-IgM antibodies with internal peptide sequences has been observed . C-IgM antibodies are present in germ-free and in athymic adult rats and are absent in newborn rats . The natural ubiquity of protein carboxy-termini in biological structures suggests that C-IgM could play an important role in antigen clearance and presentation to the immune system . From a practical viewpoint, the recognition of carboxy-terminal peptides by complement-fixing C-IgM antibodies has profound implications for the use of peptide- and protein-derivatized delivery vehicles and artificial materials. Fungal Genet Biol, 2001 Jun, 33(1), 15 - 23 Cloning, expression, and characterization of the hxk-1 Gene from the white truffle Tuber borchii vittad.: A first step toward understanding sugar metabolism; Agostini D et al.; Recent biochemical investigations of Tuber borchii Vittad . mycelium have demonstrated the presence of three distinct forms of hexokinase (HK(M1), HK(M2), and HKM3) . In the investigation described here, a gene coding for hexokinase (hxk-1) from T . borchii was isolated and characterized . The hxk-1 gene is characterized by an ORF of 1494 nucleotides and codes for a polypeptide of 497 aa . The gene was overexpressed in Escherichia coli, and the recombinant protein was kinetically characterized . The K(cat) value for fructose is in agreement with the data reported for the hexokinase of Yarrowia lipolytica, the Km for ATP is not dependent on the sugar used, and the enzyme is not inhibited by trehalose 6-phosphate or glucose 6-phosphate . The biochemical characteristics confirm that this enzyme is a hexokinase, as suggested by the Pileup results, and it corresponds to the HKM1 isoform . This work represents the first characterization of the key enzyme of the glycolytic pathway and the related gene in a Tuber species . Cell Biol Int, 2001, 25(6), 557 - 61 Specific invasion of transformed cells by Escherichia coli A2 strain; Efremova T et al.; Bacteria of the spontaneously isolated non-pathogenic strain Escherichia coli A2 producing actin-specific protease ECP 32 (Usmanova and Khaitlina, 1989) were shown to be taken up by transformed cells, whereas finite and immortal cell lines were resistant to the infection . Arthritis Rheum, 2001 Jun, 44(6), 1320 - 30 Plasma levels of nucleosomes and nucleosome-autoantibody complexes in murine lupus: effects of disease progression and lipopolyssacharide administration; Licht R et al.; OBJECTIVE: To evaluate the effect of disease progression and lipopolysaccharide (LPS) administration on the presence of nucleosomes, antinucleosome reactivity, and nucleosome-Ig complexes in the circulation of MRL and control mice . METHODS: Plasma samples from lupus-prone (MRL/lpr and MRL/+) and control (CBA, Swiss, and BALB/c) mice were tested in enzyme-linked immunosorbent assays for the presence of nucleosomes, antinucleosome antibodies, and nucleosome-Ig complexes . Nucleosome kinetics, apoptosis induction, and phagocytosis of apoptotic cells were also analyzed in MRL/lpr, MRL/+, and CBA control mice after a single injection of LPS or phosphate buffered saline . RESULTS: Nucleosomes were found in the circulation of MRL/lpr and MRL/+ mice from week 4 onward . Nucleosomes were also detected in young control mice, but with increasing age, the nucleosomes disappeared . Antinucleosome antibodies, nucleosome-Ig complexes, and albuminuria were found only in the MRL/lpr mice . LPS administration led to a significant increase in circulating nucleosomes (3-8-fold) in all strains tested . In only the MRL/lpr mice was this increase followed by a significant decrease in antinucleosome titers and an increase in nucleosome-Ig complexes . The number of apoptotic cells in the thymus after LPS was significantly higher in the MRL/lpr mice than in the MRL/+ and CBA control mice . LPS caused a profound reduction (50-70%) of the phagocytosis of apoptotic cells by peritoneal macrophages, which was comparable for all strains . CONCLUSION: In MRL lupus-prone mice, nucleosomes are persistently present in the circulation, whereas in control mice, nucleosomes are present only at a young age . The formation of antinucleosome antibodies and nucleosome-Ig complexes is a characteristic feature of MRL/lpr mice . LPS administration increases systemic nucleosome release due to an enhancement of apoptosis and a decrease in the clearance of apoptotic cells. J Chromatogr A, 2001 May 25, 918(2), 311 - 8 Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding; Gu Z et al.; Protein refolding is still a bottleneck for large-scale production of valuable proteins expressed as inclusion bodies in Escherichia coli . Usually biologically active proteins cannot be obtained with high yield at a high concentration after refolding . In order to meet the challenge of protein refolding a urea gradient gel filtration-refolding system was developed in this article . A Superdex 75 column was pre-equilibrated with a linear decreased urea gradient, the denatured protein experienced the gradual decrease in urea concentration as it went through the column . The refolding of denatured lysozyme showed this method could significantly increase the activity recovery of denatured lysozyme at high protein concentration . The activity recovery of 90% was obtained from the initial protein concentration up to 17 mg/ml within 40 min. Adv Microb Physiol, 2001, 44, 1 - 34 Functional versatility in the CRP-FNR superfamily of transcription factors: FNR and FLP; Green J et al.; The cAMP receptor protein (CRP; sometimes known as CAP, the catabolite gene activator protein) and the fumarate and nitrate reduction regulator (FNR) of Escherichia coli are founder members of an expanding superfamily of structurally related transcription factors . The archetypal CRP structural fold provides a very versatile mechanism for transducing environmental and metabolic signals to the transcription machinery . It allows different functional specificities at the sensory, DNA-recognition and RNA-polymerase-interaction levels to be 'mixed and matched' in order to create a diverse range of transcription factors tailored to respond to particular physiological conditions . This versatility is clearly illustrated by comparing the properties of the CRP, FNR and FLP (FNR-like protein) regulators . At the sensory level, the basic structural fold has been adapted in FNR and FLP by the acquisition in the N-terminal region of different combinations of cysteine or other residues; which bestow oxygen/redox sensing mechanisms that are poised according to the oxidative stress thresholds affecting the metabolism of specific bacteria . At the DNA-recognition level, discrimination between distinct but related DNA targets is mediated by amino acid sequence modifications in the conserved core contact between the DNA-recognition helix and target DNA . And, at the level of RNA-polymerase-interaction, different combinations of three discrete regions contacting the polymerase (the activating regions) are used for polymerase recruitment and promoting transcription. Rev Argent Microbiol, 2001 Jan-Mar, 33(1), 52 - 7 {Avian Escherichia coli virulence factors associated with coli septicemia in broiler chickens}; Ramirez Santoyo RM et al.; In order to detect phenotypic characteristics associated with pathogenicity, 25 strains of Escherichia coli, isolated from clinical cases of colisepticemia in broiler chickens, were examined to determine the following properties: colicinogenicity, colicin V production, type 1 fimbriae, hemolysin expression and motility . Colicinogenicity occurred in 72% of the strains, 56% of all strains produced colicin V, 84% were positive for type 1 fimbriae and 80% were positive for motility . None of the strains had hemolytic activity; however, all of them, expressed at least one of the other characteristics studied . These results suggest that the diversity of phenotypes detected partially explain the multifactorial nature of avian colisepticemia. J Commun Dis, 2000 Sep, 32(3), 161 - 8 Incidence of enteroadherence in diarrhoegenic Escherichia coli in infants in Delhi; Varma M et al.; Fifty-six isolates of Escherichia coli including 40 isolates from diarrhoeic infants and 16 from non-diarrhoeic infants were investigated . Twenty-two of the diarrhoeic isolates were typable, the most common serogroup being 086 (33%) . None of the non-diarrheic isolates are typable with EPEC antisera with enteropathogenes . Adherence tests with HEp-2 cell line revealed localized adherence in 23%, diffuse adherence in 14% and aggregative adherence in 5.7% of the 35 isolates tested . Aggregative adherence was not observed in any of the EPEC isolates . None of the isolates in the control group exhibited localized or aggregative adherence . However, 25% of these isolates showed diffuse adherence (DA) which was not significantly different from the incidence of DA (34%) in the test group (p > 0.05) . The importance of serogrouping and studying adherence pattern of E . coli isolates in establishing their pathogenic potential is thus emphasized. Biotechnol Bioeng, 2001 Aug 5, 74(3), 220 - 9 An experimental and theoretical study of the inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides; Cheng B et al.; Previously, we have developed a genetically structured mathematical model to describe the inhibition of Escherichia coli lac operon gene expression by antigene oligos . Our model predicted that antigene oligos targeted to the operator region of the lac operon would have a significant inhibitory effect on beta-galactosidase production . In this investigation, the E . coli lac operon gene expression in the presence of antigene oligos was studied experimentally . A 21-mer oligo, which was designed to form a triplex with the operator, was found to be able to specifically inhibit beta-galactosidase production in a dose-dependent manner . In contrast to the 21-mer triplex-forming oligonucleotide (TFO), several control oligos showed no inhibitory effect . The ineffectiveness of the various control oligos, along with the fact that the 21-mer oligo has no homology sequence with lacZYA, and no mRNA is transcribed from the operator, suggests that the 21-mer oligo inhibits target gene expression by an antigene mechanism . To simulate the kinetics of lac operon gene expression in the presence of antigene oligos, a genetically structured kinetic model, which includes transport of oligo into the cell, growth of bacteria cells, and lac operon gene expression, was developed . Predictions of the kinetic model fit the experimental data quite well after adjustment of the value of the oligonucleotide transport rate constant (9.0 x 10(-)(3) min(-)(1)) and oligo binding affinity constant (1.05 x 10(6) M(-)(1)) . Our values for these two adjusted parameters are in the range of reported literature values . Tumour Biol, 2001 Jul-Aug, 22(4), 254 - 61 Analysis of epitopes of mouse monoclonal antibodies against human alpha-fetoprotein; Kang Y et al.; Thirty-six monoclonal antibodies (MAbs) against human alpha-fetoprotein (AFP) were analyzed for the location of their epitopes by reacting them with a set of yeast recombinant AFP proteins using ELISA . Recombinant AFP proteins containing either one, two or all three domains, i.e . domain I, domain III, domain I-II, domain II-III and domain I-II-III, were produced and secreted into the culture medium of yeast cells harboring the expression plasmids . Epitopes of 13 MAbs were localized on domain I and 17 others were on domain III . However, the exact location of the epitopes of the remaining 6 MAbs could not be defined . The epitope of an antibody, namely AFY6, which was located in domain I, was successfully mapped on an octapeptide, C175KAENAVE182, using synthesized overlapping octapeptides . J Biol Chem, 2001 Aug 17, 276(33), 30670 - 7 Epub 2001 Jun 08. The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli; Outten FW et al.; Copper is essential but can be toxic even at low concentrations . Coping with this duality requires multiple pathways to control intracellular copper availability . Three copper-inducible promoters, controlling expression of six copper tolerance genes, were recently identified in Escherichia coli . The cue system employs an inner membrane copper transporter, whereas the cus system includes a tripartite transporter spanning the entire cell envelope . Although cus is not essential for aerobic copper tolerance, we show here that a copper-sensitive phenotype can be observed when cus is inactivated in a cueR background . Furthermore, a clear copper-sensitive phenotype for the cus system is revealed in the absence of O(2) . These results indicate that the cue pathway, which includes a copper exporter, CopA, and a periplasmic oxidase, CueO, is the primary aerobic system for copper tolerance . During anaerobic growth, however, copper toxicity increases, and the independent cus copper exporter is also necessary for full copper tolerance . We conclude that the cytosolic (CueR) and periplasmic (CusRS) sensor systems differentially regulate copper export systems in response to changes in copper and oxygen availability . These results underscore the increased toxicity of copper under anaerobic conditions and the complex adaptation of copper export in E . coli. J Biol Chem, 2001 Aug 10, 276(32), 29979 - 86 Epub 2001 Jun 08. Biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum; Sartori AA et al.; Deamination of cytosine to uracil and 5-methylcytosine to thymine represents a major mutagenic threat particularly at high temperatures . In double-stranded DNA, these spontaneous hydrolytic reactions give rise to G.U and G.T mispairs, respectively, that must be restored to G.C pairs prior to the next round of DNA replication; if left unrepaired, 50% of progeny DNA would acquire G.C --> A.T transition mutations . The genome of the hyperthermophilic archaeon Pyrobaculum aerophilum has been recently shown to encode a protein, Pa-MIG, a member of the endonuclease III family, capable of processing both G.U and G.T mispairs . We now show that this latter activity is undetectable in crude extracts of P . aerophilum . However, uracil residues in G.U mispairs, in A.U pairs, and in single-stranded DNA were efficiently removed in these extracts . These activities were assigned to a approximately 22-kDa polypeptide named Pa-UDG (P . aerophilum uracil-DNA glycosylase) . The recombinant Pa-UDG protein is highly thermostable and displays a considerable degree of homology to the recently described uracil-DNA glycosylases from Archaeoglobus fulgidus and Thermotoga maritima . Interestingly, neither Pa-MIG nor Pa-UDG was inhibited by UGI, a generic inhibitor of the UNG family of uracil glycosylases . Yet a small fraction of the total uracil processing activity present in crude extracts of P . aerophilum was inhibited by this peptide . This implies that the hyperthermophilic archaeon possesses at least a three-pronged defense against the mutagenic threat of hydrolytic deamination of cytosines in its genomic DNA. Teratog Carcinog Mutagen, 2001, 21(4), 275 - 82 Characterization of the mutational specificity of DNA cross-linking mutagens by the Lac+ reversion assay with Escherichia coli; Ohta T et al.; The mutational specificities of DNA cross-linking compounds such as cisplatin, transplatin, carboplatin, mitomycin C, psoralen, and 8-methoxypsoralen were investigated in lacZ reversion assay systems of Escherichia coli . Tester strains were constructed by introducing the six kinds of F' plasmids (lacI-, lacZ461, and proAB+), each of which carries a different base-substitution mutation within the lacZ gene . Each of the six possible base-substitution mutations was assayed by Lac+ reversion . Cisplatin induced G.C-->A.T transitions and G.C-->T.A transversions, with the former predominating . Transplatin induced A.T-->G.C transitions in addition to G.C-->A.T transitions and G.C-->T.A . Carboplatin weakly induced G.C-->A.T transitions . On the other hand, mitomycin C induced only G.C-->T.A transversions, while psoralen and 8-methoxypsoralen reactivated with near-UV irradiation induced A.T-->G.C transitions preferentially . The Lac(+) reversion system was very convenient for rapidly determining mutational spectra . J Biol Chem, 2001 Sep 7, 276(36), 33465 - 70 Epub 2001 Jun 13. Reengineering granulocyte colony-stimulating factor for enhanced stability; Bishop B et al.; Granulocyte colony-stimulating factor is a long-chain cytokine that has both biological and therapeutic applications . It is involved in the production and maturation of neutrophilic progenitor cells and neutrophils and is administered to stimulate the production of white blood cells to reduce the risk of serious infection in immunocompromised patients . We have reengineered granulocyte colony-stimulating factor to improve the thermodynamic stability of the protein, focusing on enhancing the alpha-helical propensity of residues in the antiparallel 4-helix bundle of the protein . These redesigns resulted in proteins with substantially enhanced stability while retaining wild-type levels of biological activity, measured as the ability of the reengineered proteins to stimulate the proliferation of murine myeloid cells transfected with the granulocyte colony-stimulating factor receptor. J Biol Chem, 2001 Aug 17, 276(33), 31067 - 73 Epub 2001 Jun 13. Heterogeneous RNA-binding protein M4 is a receptor for carcinoembryonic antigen in Kupffer cells; Bajenova OV et al.; Here we report the isolation of the recombinant cDNA clone from rat macrophages, Kupffer cells (KC) that encodes a protein interacting with carcinoembryonic antigen (CEA) . To isolate and identify the CEA receptor gene we used two approaches: screening of a KC cDNA library with a specific antibody and the yeast two-hybrid system for protein interaction using as a bait the N-terminal part of the CEA encoding the binding site . Both techniques resulted in the identification of the rat heterogeneous RNA-binding protein (hnRNP) M4 gene . The rat ortholog cDNA sequence has not been previously described . The open reading frame for this gene contains a 2351-base pair sequence with the polyadenylation signal AATAAA and a termination poly(A) tail . The mRNA shows ubiquitous tissue expression as a 2.4-kilobase transcript . The deduced amino acid sequence comprised a 78-kDa membrane protein with 3 putative RNA-binding domains, arginine/methionine/glutamine-rich C terminus and 3 potential membrane spanning regions . When hnRNP M4 protein is expressed in pGEX4T-3 vector system in Escherichia coli it binds (125)I-labeled CEA in a Ca(2+)-dependent fashion . Transfection of rat hnRNP M4 cDNA into a non-CEA binding mouse macrophage cell line p388D1 resulted in CEA binding . These data provide evidence for a new function of hnRNP M4 protein as a CEA-binding protein in Kupffer cells. J Biotechnol, 2001 Jun 15, 88(2), 95 - 105 Some observations in freeze-drying of recombinant bioluminescent Escherichia coli for toxicity monitoring; Gu MB et al.; A recombinant bioluminescent bacteria, containing a fabA::luxCDABE fusion gene, has been used to characterize freeze-drying methods, which may be conveniently used as a tool for the development of a portable biosensor . Through residual water, viability, biosensing activity and scanning electron microscopy analyses, the characteristics that four cryoprotectants, trehalose, sucrose, sorbitol, and mannitol, conferred on freeze-dried samples were elucidated, including the morphology, water content and activity of the cells . It was found that trehalose showed the best freeze-drying efficiency among the tested cryoprotectants and it might have a specific capacity limitation in protection of the cells during the freeze step . Humidity might result in damage to the cells, according to the viability, when exposed to air during storage, while the water remaining post freeze-drying showed good correlation with damage to the freeze-dried cells when under air-tight storage conditions . The results with other recombinant bioluminescent bacteria indicated that these findings might be general features of the freeze-drying processes. Int J Radiat Biol, 2001 Jun, 77(6), 645 - 54 Radiosensitivity of DNA in a specific protein-DNA complex: the lac repressor-lac operator complex; Begusova M et al.; PURPOSE: To calculate the probability of radiation-induced frank strand breakage (FSB) at each nucleotide in the Escherichia coli lac repressor-lac operator system using a simulation procedure . To compare calculated and experimental results . To asses the contribution of DNA conformational changes and of the masking by the protein to DNA protection by the repressor . MATERIALS AND METHODS: Two structures of the complex were extracted from the PDB databank: crystallography- and NMR-based structures . Calculations were made of the accessibility of the atoms mainly involved in strand breakage (H4' and H5') to O&Hdot; and of the FSB probabilities, along: (1) DNA in the complex; (2) DNA in the complex depleted of the repressor; and (3) a linear DNA having the same sequence . An 80bp fragment bearing the operator was irradiated alone or in presence of the repressor . The relative probabilities of FSB at each nucleotide were determined using sequencing gel electrophoresis . RESULTS: Calculations predict modulation of the accessibility of H4' and H5' atoms and of the probabilities of FSB along the DNA fragments of complexes . This is due to the protein-induced conformational change and to masking by bound protein . The best agreement with the experimental FSB was observed for calculations that use the crystallography-based structure . CONCLUSIONS: For specific DNA-protein complexes, our calculations can predict the protein radiolytic footprints on DNA . They show the significant contribution of the protein-induced DNA conformational change to DNA protection. Parasitol Res, 2001 May, 87(5), 390 - 5 Heterologous expression and functional characterization of thioredoxin from Fasciola hepatica; Salazar-Calderon M et al.; The full thioredoxin coding sequence from Fasciola hepatica has been cloned into the pGEX-2T expression vector and produced in Escherichia coli as a fusion protein . The recombinant protein proved to be biologically active, using an insulin reduction assay, and was also able to activate thioredoxin peroxidase from F . hepatica . These observations suggest that this protein could participate in a redox cascade involved in the maintenance of cell homeostasis as well as in parasite protection against reactive oxygen species produced by the host. Arch Virol, 2001, 146(4), 801 - 6 Group C rotavirus NSP4 induces diarrhea in neonatal mice; Sasaki S et al.; Nonstructural glycoprotein NSP4 of group A rotavirus induces diarrhea in neonatal mice by functioning as an enterotoxin . Previously, our laboratory reported that the structural features of group A and group C rotavirus NSP4 proteins are well conserved despite a lack of sequence homology between group A and group C rotavirus NSP4 proteins {Horie Y, et al., Arch Virol (1997) 142: 1865-1872} . To test whether group C rotavirus NSP4 has an enterotoxigenic activity, we expressed in Escherichia coli the carboxy two-thirds (corresponding to amino acid residues 55-150) of the NSP4 protein derived from group C rotavirus strain Ehime 9301 . This truncated NSP4 protein was able to induce diarrhea in 5-day-old CD-1 mice when administered intraperitoneally . Thus, group C rotavirus NSP4 acts as an enterotoxin like group A rotavirus NSP4. Oncogene, 2001 Apr 26, 20(18), 2318 - 24 The 'wildtype' conformation of p53: epitope mapping using hybrid proteins; Wang PL et al.; The function of p53 correlates with its 'wildtype' conformation, specifically recognized by antibodies PAb1620 and PAb246, and many cancer-associated mutations cause loss of this conformation . The epitopes of these antibodies were identified using hybrid p53 proteins created by a new method . Plasmids carrying homologous genes cut at appropriate sites recombined efficiently when transformed into RecE(+) E . coli . PAb1620 and PAb246 recognize mouse but not chicken p53; we created mouse-chicken hybrids of the p53 core domain and tested antibody reactivity . PAb246 binding mapped to residues 201-212, while PAb1620 required both residues 145-157 and 201-212 (human p53 numbering used throughout) . An alanine-scan showed that the key residues for PAb246 and PAb1620 are completely distinct: PAb246 recognizes residues 202-204 (Tyr-Pro-Glu) while PAb1620 recognizes residues Arg156, Leu206, Arg209, and Gln/Asn210, the last two residues being essential . Both antibody epitopes are far from the p53 interface with DNA, but near the epitope of the 'mutant' conformation antibody PAb240 . These epitope locations may help in dissecting the interactions of p53, including those with E6/E6-AP and in its DNA-bound state. Plant Physiol, 2001 Jun, 126(2), 601 - 12 The mitochondrial isovaleryl-coenzyme a dehydrogenase of arabidopsis oxidizes intermediates of leucine and valine catabolism; Daschner K et al.; We recently identified a cDNA encoding a putative isovaleryl-coenzyme A (CoA) dehydrogenase in Arabidopsis (AtIVD) . In animals, this homotetrameric enzyme is located in mitochondria and catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA as an intermediate step in the leucine (Leu) catabolic pathway . Expression of AtIVD:smGFP4 fusion proteins in tobacco (Nicotiana tabacum) protoplasts and biochemical studies now demonstrate the in vivo import of the plant isovaleryl-CoA dehydrogenase (IVD) into mitochondria and the enzyme in the matrix of these organelles . Two-dimensional separation of mitochondrial proteins by blue native and SDS-PAGE and size determination of the native and overexpressed proteins suggest homodimers to be the dominant form of the plant IVD . Northern-blot hybridization and studies in transgenic Arabidopsis plants expressing Ativd promoter:gus constructs reveal strong expression of this gene in seedlings and young plants grown in the absence of sucrose, whereas promoter activity in almost all tissues is strongly inhibited by exogeneously added sucrose . Substrate specificity tests with AtIVD expressed in Escherichia coli indicate a strong preference toward isovaleryl-CoA but surprisingly also show considerable activity with isobutyryl-CoA . This strongly indicates a commitment of the enzyme in Leu catabolism, but the activity observed with isobutyryl-CoA also suggests a parallel involvement of the enzyme in the dehydrogenation of intermediates of the valine degradation pathway . Such a dual activity has not been observed with the animal IVD and may suggest a novel connection of the Leu and valine catabolism in plants. J Biol Chem, 2001 Aug 17, 276(33), 31394 - 401 Epub 2001 Jun 11. Recombinant forms of tetanus toxin engineered for examining and exploiting neuronal trafficking pathways; Li Y et al.; Tetanus toxin is a fascinating, multifunctional protein that binds to peripheral neurons, undergoes retrograde transport and trans-synaptic transfer to central inhibitory neurons where it blocks transmitter release, thereby, causing spastic paralysis . As a pre-requisite for exploiting its unique trafficking properties, a novel recombinant single chain was expressed at a high level in Escherichia coli as a soluble, easily purifiable protein . It could be activated with enterokinase to produce a dichain that matched native toxin in terms of proteolytic and neuroinhibitory activities, as well as induction of spastic paralysis in mice . Importantly, nicking was not essential for protease activity . Substitution of Glu(234) by Ala created a protease-deficient atoxic form, which blocked the neuroparalytic action of tetanus toxin in vitro, with equal potency to its heavy chain; but, the mutant proved >30-fold more potent in preventing tetanus in mice . This observation unveils differences between the intoxication processes resulting from retrograde transport of toxin in vivo and its local uptake into peripheral or central nerves in vitro, dispelling a popularly held belief that the heavy chain is the sole determinant for efficient trafficking . Thus, this innocuous mutant may be a useful vehicle, superior to the heavy chain, for drug delivery to central neurons. Infect Immun, 2001 Jul, 69(7), 4678 - 80 Dr operon-associated invasiveness of Escherichia coli from pregnant patients with pyelonephritis; Goluszko P et al.; We used a gentamicin protection assay to assess the ability of gestational pyelonephritis isolates of Escherichia coli to invade HeLa cells . The ability to enter HeLa cells was strongly associated with the presence of Dr operons coding for Dr adhesins . In contrast, the nonivasive isolates predominantly expressed papG, coding for P fimbriae. Infect Immun, 2001 Jul, 69(7), 4580 - 9 Decreased apoptosis in the ileum and ileal Peyer's patches: a feature after infection with rabbit enteropathogenic Escherichia coli O103; Heczko U et al.; Significant changes occur in intestinal epithelial cells after infection with enteropathogenic Escherichia coli (EPEC) . However, it is unclear whether this pathogen alters rates of apoptosis . By using a naturally occurring weaned rabbit infection model, we determined physiological levels of apoptosis in rabbit ileum and ileal Peyer's patches (PP) and compared them to those found after infection with adherent rabbit EPEC (REPEC O103) . Various REPEC O103 strains were first tested in vitro for characteristic virulence features . Rabbits were then inoculated with the REPEC O103 strains that infected cultured cells the most efficiently . After experimental infection, intestinal samples were examined by light and electron microscopy . Simultaneously, ileal apoptosis was assessed by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase 3 assays and by apoptotic cell counts based on morphology (hematoxylin-and-eosin staining) . The highest physiological apoptotic indices were measured in PP germinal centers (median = 14.7%), followed by PP domed villi (8.1%), tips of absorptive villi (3.8%), and ileal crypt regions (0.5%) . Severe infection with REPEC O103 resulted in a significant decrease in apoptosis in PP germinal centers (determined by TUNEL assay; P = 0.01), in the tips of ileal absorptive villi (determined by H&E staining; P = 0.04), and in whole ileal cell lysates (determined by caspase 3 assay; P = 0.001) . We concluded that REPEC O103 does not promote apoptosis . Furthermore, we cannot rule out the possibility that REPEC O103, in fact, decreases apoptotic levels in the rabbit ileum. Infect Immun, 2001 Jul, 69(7), 4465 - 72 Cloning and expression of two novel hemin binding protein genes from Treponema denticola; Xu X et al.; Treponema denticola does not appear to produce siderophores, so it must acquire iron by other pathways . Indeed, T . denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability . To characterize the HbpA protein, its gene was cloned from genomic DNA libraries of T . denticola . Sequence analysis of the hbpA open reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide . HbpA has no significant homology to any proteins in the databases . Southern blot analysis demonstrated that hbpA is present in several T . denticola ATCC strains and clinical isolates, but not in Treponema pectinovorum, Treponema socranskii, or Escherichia coli . HbpA, expressed as a recombinant protein in E . coli and purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining . Northern blot analysis showed that there were two hbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced . Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA . This downstream gene, called hbpB, was cloned and sequenced and its product was expressed as a fusion protein in E . coli . The hbpB gene product is 49% identical to HbpA and binds hemin . Thus, T . denticola has two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway. Infect Immun, 2001 Jul, 69(7), 4430 - 7 Cloning, expression, and characterization of a neuraminidase gene from Arcanobacterium pyogenes; Jost BH et al.; Arcanobacterium pyogenes is an opportunistic pathogen, associated with suppurative infections in domestic animals . In addition to pyolysin, a pore-forming, cholesterol-binding toxin, A . pyogenes expresses a number of putative virulence factors, including several proteases and neuraminidase activity . A 3,009-bp gene, nanH, was cloned and sequenced and conferred neuraminidase activity on an Escherichia coli host strain . The predicted 107-kDa NanH protein displayed similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases . Recombinant His-tagged NanH was found to have pH and temperature optima of 5.5 to 6.0 and 55 degrees C, respectively . Insertional deletion of the nanH gene resulted in the reduction, but not absence, of neuraminidase activity, indicating the presence of a second neuraminidase gene in A . pyogenes . NanH was localized to the A . pyogenes cell wall . A . pyogenes adhered to HeLa, CHO, and MDBK cells in a washing-resistant manner . However, the nanH mutant was not defective for adherence to epithelial cells . The role of NanH in host epithelial cell adherence may be masked by the presence of a second neuraminidase in A . pyogenes. Infect Immun, 2001 Jul, 69(7), 4390 - 7 Naturally acquired antibody responses to Plasmodium falciparum merozoite surface protein 4 in a population living in an area of endemicity in Vietnam; Wang L et al.; Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylphosphatidylinositol-anchored integral membrane protein that is being developed as a component of a subunit vaccine against malaria . We report here the measurement of naturally acquired antibodies to MSP4 in a population of individuals living in the Khanh-Hoa region of Vietnam, an area where malaria is highly endemic . Antibodies to MSP4 were detected in 94% of the study population at titers of 1:5,000 or greater . Two forms of recombinant MSP4 produced in either Escherichia coli or Saccharomyces cerevisiae were compared as substrates in the enzyme-linked immunosorbent assay . There was an excellent correlation between reactivity measured to either, although the yeast substrate was recognized by a higher percentage of sera . Four different regions of MSP4 were recognized by human antibodies, demonstrating that there are at least four distinct epitopes in this protein . In the carboxyl terminus, where the single epidermal growth factor-like domain is located, the reactive epitope(s) was shown to be conformation dependent, as disruption of the disulfide bonds almost completely abolished reactivity with human antibodies . The anti-MSP4 antibodies were mainly of the immunoglobulin G1 (IgG1) and IgG3 subclasses, suggesting that such antibodies may play a role in opsonization and complement-mediated lysis of free merozoites . Individuals in the study population were drug-cured and followed up for 6 months; no significant correlation was observed between the anti-MSP4 antibodies and the absence of parasitemia during the surveillance period . As a comparison, antibodies to MSP1(19), a leading vaccine candidate, were measured, and no correlation with protection was observed in these individuals . The anti-MSP1(19) antibodies were predominantly of the IgG1 isotype, in contrast to the IgG3 predominance noted for MSP4. Infect Immun, 2001 Jul, 69(7), 4382 - 9 Type I Helicobacter pylori lipopolysaccharide stimulates toll-like receptor 4 and activates mitogen oxidase 1 in gastric pit cells; Kawahara T et al.; Guinea pig gastric pit cells express an isozyme of gp91-phox, mitogen oxidase 1 (Mox1), and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phox) . Helicobacter pylori lipopolysaccharide (LPS) and Escherichia coli LPS have been shown to function as potent activators for the Mox1 oxidase . These cells spontaneously secreted about 10 nmol of superoxide anion (O(2)(-))/mg of protein/h under LPS-free conditions . They expressed the mRNA and protein of Toll-like receptor 4 (TLR4) but not those of TLR2 . LPS from type I H . pylori at 2.1 endotoxin units/ml or higher stimulated TLR4-mediated phosphorylations of transforming growth factor beta-activated kinase 1 and its binding protein 1 induced TLR4 and p67-phox and up-regulated O(2)(-) production 10-fold . In contrast, none of these events occurred with H . pylori LPS from complete or partial deletion mutants of the cag pathogenicity island . Lipid A was confirmed to be a bioactive component for the priming effects, while removal of bisphosphates from lipid A completely eliminated the effects, suggesting the importance of the phosphorylation pattern besides the acylation pattern for the bioactivity . H . pylori LPS is generally accepted as having low toxicity; however, our results suggest that type I H . pylori lipid A may be a potent stimulator for innate immune responses of gastric mucosa by stimulating the TLR4 cascade and Mox1 oxidase in pit cells. Infect Immun, 2001 Jul, 69(7), 4373 - 81 Establishing a direct role for the Bartonella bacilliformis invasion-associated locus B (IalB) protein in human erythrocyte parasitism; Coleman SA et al.; The invasion-associated locus A and B genes (ialAB) of Bartonella bacilliformis were previously shown to confer an erythrocyte-invasive phenotype upon Escherichia coli, indirectly implicating their role in virulence . We report the first direct demonstration of a role for ialB as a virulence factor in B . bacilliformis . The presence of a secretory signal sequence and amino acid sequence similarity to two known outer membrane proteins involved in virulence suggested that IalB was an outer membrane protein . To develop an antiserum for protein localization, the ialB gene was cloned in frame into an expression vector with a six-histidine tag and under control of the lacZ promoter . The IalB fusion protein was purified by nickel affinity chromatography and used to raise polyclonal antibodies . IalB was initially localized to the bacterial membrane fraction . To further localize IalB, B . bacilliformis inner and outer membranes were fractionated by sucrose density gradient centrifugation and identified by appearance, buoyant density (rho), and cytochrome b content . Inner and outer membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and IalB was positively identified by Western blot . Contrary to expectations, IalB was localized to the inner membrane of the pathogen . To directly demonstrate a role for IalB in erythrocyte parasitism, the B . bacilliformis ialB gene was disrupted by insertional mutagenesis . The resulting ialB mutant strain was complemented in trans with a replicative plasmid encoding the full-length ialB gene . PCR and high-stringency DNA hybridization confirmed mutagenesis and transcomplementation events . Abrogation and restoration of ialB expression was verified by SDS-PAGE and immunoblotting . In vitro virulence assays showed that mutagenesis of ialB decreased bacterial association and invasion of human erythrocytes by 47 to 53% relative to controls . Transcomplementation of ialB restored erythrocyte association and invasion rates to levels observed in the parental strain . These data provide direct evidence for IalB's role in erythrocyte parasitism and represent the first demonstration of molecular Koch's postulates for a Bartonella species. Am J Respir Crit Care Med, 2001 Jun, 163(7), 1591 - 8 Local inflammatory responses following bronchial endotoxin instillation in humans; O'Grady NP et al.; To study local lung inflammation, 34 subjects had endotoxin (1-4 ng/kg) instilled into a lung segment and saline instilled into a contralateral segment followed by bronchoalveolar lavage (BAL) at 2 h, 6 h, 24 h, or 48 h . Endotoxin instillation resulted in a focal inflammatory response with a distinct time course . An early phase (2 h to 6 h) revealed an increase in neutrophils (p = 0.0001) with elevated cytokines (tumor necrosis factor {TNF}-alpha, TNF receptors {TNFR}, interleukin {IL}-1beta, IL-1 receptor antagonist, IL-6, granulocyte-colony-stimulating factor {G-CSF}, all p < or = 0.002, but no change in IL-10) and chemokines (IL-8, epithelial neutrophil activating protein-78, monocyte chemotactic protein-1, macrophage inflammatory protein {MIP}-1alpha, MIP-1beta, all p < or = 0.001, but no change in growth-regulated peptide-alpha) . A later phase (24 h to 48 h) showed increased neutrophils, macrophages, monocytes, and lymphocytes (all p < or = 0.02), and a return to basal levels of most mediators . Elevated levels of inflammatory markers (TNFR(1), TNFR(2), L-selectin, lactoferrin, and myeloperoxidase) persisted in the BAL at 48 h (p < or = 0.001) . Increased permeability to albumin occurred throughout both phases (p = 0.001) . Blood C-reactive protein, serum amyloid A, IL-6, IL-1ra, G-CSF, but not TNF-alpha increased by 8 h (all p < or = 0.008) . The local pulmonary inflammatory response to endotoxin has a unique qualitative and temporal profile of inflammation compared with previous reports of intravenous endotoxin challenges . This model provides a means to investigate factors that initiate, amplify, and resolve local lung inflammation. Am J Respir Crit Care Med, 2001 Jun, 163(7), 1578 - 83 Defective natural killer and phagocytic activities in chronic obstructive pulmonary disease are restored by glycophosphopeptical (inmunoferón); Prieto A et al.; We have investigated both modifications in natural (innate) immunity caused by chronic obstructive pulmonary disease (COPD) and the effects of a glycophosphopeptical immunomodulator (Inmunoferon) treatment on COPD-associated immunoalterations . In a double-blinded clinical trial, 60 patients with COPD received glycophosphopeptical or placebo during 90 consecutive days at oral doses of 3 g/d . Fifty-six sex- and age-matched healthy control subjects were included as a reference group for immunologic parameters . Peripheral blood natural killer (PBNK) cell cytotoxic activity and phagocytic activity of peripheral monocytes/macrophages (Mo/Ma) and polymorphonuclear (PMN) cells were assessed at baseline and then again at the end of treatments . We found both PBNK activity and phagocytic activity to be significantly decreased in patients with COPD compared with levels in healthy volunteers . The treatment with glycophosphopeptical provoked significant stimulatory effects on PBNK cytotoxic activity . This stimulation was not mediated by an increase in CD3(-)CD56(+) NK cells . Further, glycophosphopeptical significantly increased the percentage of monocytes and PMNs that phagocytize Escherichia coli in vitro, as well as increased phagocytic indices . We conclude that peripheral blood cells of patients with COPD show clear defects in natural immunity that are partially rescued by glycophosphopeptical. Mol Microbiol, 2001 Jun, 40(5), 1155 - 64 Poly(A) polymerase activity and RNA polyadenylation in Streptomyces coelicolor A3(2); Bralley P et al.; The Streptomyces coelicolor genome sequence was searched for open reading frames (ORFs) similar to Escherichia coli poly(A) polymerase I, revealing an ORF with 36% amino acid sequence identity to that protein . Mycelial extracts prepared from S . coelicolor cultures incorporated radioactive ATP into an acid-insoluble form, and some of the products of this incorporation had the properties expected of poly(A) . {3H}-uridine and {3H}-adenosine were used to label the RNA in S . coelicolor cultures of different ages, and total RNA was fractionated by oligo dT cellulose chromatography . Approximately 3% of the total uridine-labelled RNA and 11% of the adenosine-labelled RNA were retained by the oligo dT cellulose columns . Enzymatic digestion of the retained RNA supported the conclusion that a significant fraction of the adenosine label was present in 3'-poly(A) chains . Measurement of poly(A) tail lengths by end labelling of total RNA and RNase digestion revealed a maximum length of approximately 18 residues . Radioactive cDNA prepared from the RNA fraction retained by oligo dT cellulose hybridized to the 16S and 23S genes from a streptomycete ribosomal RNA operon but not to the 5S gene . Reverse transcription-polymerase chain reaction (RT-PCR) revealed the presence of mRNAs in the RNA fraction retained by oligo dT cellulose. Mol Microbiol, 2001 Jun, 40(5), 1141 - 54 Systematic mutagenesis of the DNA binding sites for SoxS in the Escherichia coli zwf and fpr promoters: identifying nucleotides required for DNA binding and transcription activation; Griffith KL et al.; SoxS is the direct transcriptional activator of at least 15 genes of the Escherichia coli superoxide regulon . SoxS is small (107 amino acids), binds DNA as a monomer and recognizes a highly degenerate DNA binding site, termed 'soxbox' . Like other members of the AraC/XylS family, SoxS has two putative helix-turn-helix (HTH) DNA-binding motifs, and it has been proposed that each HTH motif recognizes a highly conserved recognition element of the soxbox . To determine which nucleotides are important for SoxS binding, we conducted a systematic mutagenesis of the DNA binding sites for SoxS in the zwf and fpr promoters and determined the effect of the soxbox mutations on SoxS DNA binding and transcription activation in vivo by measuring beta-galactosidase activity in strains with fusions to lacZ . We found that the sequences GCAC and CAAA, termed recognition elements 1 and 2 (RE 1 and RE 2), respectively, are critical for SoxS binding, as mutations within these elements severely hinder or eliminate SoxS-dependent transcription activation; substitutions within RE 2 (CAAA), however, are tolerated better than changes within RE 1 (GCAC) . Although substitutions at the seven positions separating the two REs had only a modest effect on SoxS binding, AT basepairs were favoured within this 'spacer' region, presumably because, by facilitating DNA bending, they help bring the two recognition elements into proper juxtaposition . We also found that the 'invariant A' present at position 1 of 14/15 functional soxboxes identified thus far is important for SoxS binding, as a change to any other nucleotide at this position reduced SoxS-dependent transcription by approximately 50% . In addition, positions surrounding the REs seem to show a context effect, in that certain substitutions there have little or no effect when the RE has the optimal binding sequence, but produce a pronounced effect when the RE has a suboptimal sequence . We propose that these nucleotides play an important role in effecting differential expression from the various promoters . Lastly, we used gel retardation assays to show that alterations in transcription activation in vivo are caused by effects on DNA binding . Based on this exhaustive mutagenesis, we propose the following optimal sequence for SoxS binding: AnVGCACWWWnKRHCAAAHn (n = A, C, G, T; V = A, C, G; W = A, T; K = G, T; R = A, G; H = A, C, T). Mol Microbiol, 2001 May, 40(4), 932 - 40 Experimental genome evolution: large-scale genome rearrangements associated with resistance to replacement of a chromosomal restriction-modification gene complex; Handa N et al.; Type II restriction enzymes are paired with modification enzymes that protect type II restriction sites from cleavage by methylating them . A plasmid carrying a type II restriction-modification gene complex is not easily replaced by an incompatible plasmid because loss of the former leads to cell death through chromosome cleavage . In the present work, we looked to see whether a chromosomally located restriction-modification gene complex could be replaced by a homologous stretch of DNA . We tried to replace the PaeR7I gene complex on the Escherichia coli chromosome by transducing a homologous stretch of PaeR7I-modified DNA . The replacement efficiency of the restriction-modification complex was lower than expected . Some of the resulting recombinant clones retained the recipient restriction-modification gene complex as well as the homologous DNA (donor allele), and slowly lost the donor allele in the absence of selection . Analysis of their genome-wide rearrangements by Southern hybridization, inverse polymerase chain reaction (iPCR) and sequence determination demonstrated the occurrence of unequal homologous recombination between copies of the transposon IS3 . It was strongly suggested that multiple rounds of unequal IS3-IS3 recombination caused large-scale duplication and inversion of the chromosome, and that only one of the duplicated copies of the recipient PaeR7I was replaced. Mol Microbiol, 2001 May, 40(4), 909 - 16 Transcription of essential cell division genes is linked to chromosome replication in Escherichia coli; Liu G et al.; Cell division normally follows the completion of each round of chromosome replication in Escherichia coli . Transcription of the essential cell division genes clustered at the mra region is shown here to depend on continuing chromosomal DNA replication . After chromosome replication was blocked by either nalidixic acid treatment or thymine starvation, the transcription of these cell division genes was repressed significantly . This suggests a way in which cell division is controlled by chromosome replication. Mol Microbiol, 2001 May, 40(4), 835 - 45 Different localization of SeqA-bound nascent DNA clusters and MukF-MukE-MukB complex in Escherichia coli cells; Ohsumi K et al.; MukF, MukE and MukB proteins form a complex that may participate in the organization of folded sister chromosomes in Escherichia coli . We have found that a MukB-GFPuv4 fusion protein is observed as discrete fluorescent foci, which are localized within cellular spaces occupied by nucleoids, but not at the constriction site of cell division in living cells . In contrast, MukB-GFPuv4 is distributed throughout the whole cell when either MukF or MukE is absent . Statistical analysis revealed that most newborn cells have two foci of mukB-gfpUV4 at one-quarter and three-quarter positions in the cell length and one focus of SeqA-bound nascent DNA at or near the middle of the cell . Subsequently, the single SeqA focus divides into two foci, and then these migrate to the one-quarter and three-quarter positions . Before cell division, most long cells have two SeqA foci and four MukB-GFPuv4 foci . In early stationary phase, SeqA foci disappear, but one or two foci of MukB-GFPuv4 remain . We discuss the reorganization and proper arrangement of folded sister chromosome in the cell quarter positions, which are performed after release from the long-time cohesion of sister chromosomes. Mol Microbiol, 2001 May, 40(4), 824 - 34 Signalling substitutions in the periplasmic domain of chemoreceptor Trg induce or reduce helical sliding in the transmembrane domain; Beel BD et al.; We used in vivo oxidative cross-linking of engineered cysteine pairs to assess conformational changes in the four-helix transmembrane domain of chemoreceptor Trg . Extending previous work, we searched for and found a fourth cross-linking pair that spanned the intrasubunit interface between transmembrane helix 1 (TM1) and its partner TM2 . We determined the effects of ligand occupancy on cross-linking rate constants for all four TM1-TM2 diagnostic pairs in conditions that allowed the formation of receptor-kinase complexes for the entire cellular complement of Trg . Occupancy altered all four rates in a pattern that implicated sliding of TM2 relative to TM1 towards the cytoplasm as the transmembrane signalling movement in receptor-kinase complexes . Transmembrane signalling can be reduced or induced by single amino acid substitutions in the ligand-binding region of the periplasmic domain of Trg . We determined the effects of these substitutions on conformation in the transmembrane domain and on ligand-induced changes using the diagnostic TM1-TM2 cysteine pairs . Effects on rates of in vivo cross-linking showed that induced signalling substitutions altered the relative positions of TM1 and TM2 in the same way as ligand binding, and reduced signalling substitutions blocked or attenuated the ligand-induced shift . These results provide strong support for the helical sliding model of transmembrane signalling. Mol Microbiol, 2001 May, 40(4), 779 - 85 Co-ordinate regulation of the Escherichia coli cell cycle or The cloud of unknowing; Donachie WD; A discussion of some aspects of the regulation of chromosome replication, segregation and cell division in Escherichia coli. Biochemistry, 2001 Jun 19, 40(24), 7334 - 41 Regulatory properties of tropomyosin effects of length, isoform, and N-terminal sequence; Maytum R et al.; The regulatory properties of naturally occurring tropomyosins (Tms) of differing lengths have been examined . These Tms span from 4 to 7 actin subunits . Native proteins have been used to study the common 7 actin-spanning skeletal and smooth muscle variants and expressed recombinant proteins to study the shorter fibroblast 5a, 5b, yeast Tm1 and yeast Tm2 Tms (6, 6, 5, and 4 actin-spanning variants, respectively) . The yTm2 has been overexpressed in Escherichia coli with N-terminal constructs equivalent to those previously used for yTm1 {Maytum, R., et al . (2000) Biochemistry 39, 11913} . The regulation of myosin subfragment 1 (S1) binding to actin by Tm has been assessed using a sensitive S1 binding titration . The equilibrium between closed and open (C to M states, KT = 0.1-0.14) was similar for all vertebrate Tms . Apart from skTm where the apparent cooperative unit size (n) is the same as the structural size (n = 7 actin sites), the other vertebrate Tms that were studied exhibited large n values (n = 12-14) . The yeast Tms also exhibited large values of n (6-9) in comparison to their structural sizes (4-5) . The determined value of KT depended on the N-terminal sequence (KT = 0.15-1) . These results are compared with the effect of S1 upon Tm's affinity for actin . The yeast Tms have regulatory parameters similar to those of skTm, but unlike skTm, S1 has little effect upon their actin affinity . This shows that an actin state with a high affinity for S1 and Tm is not necessary for regulation, and the higher affinity of S1 for actin in the presence of vertebrate Tms is probably the result of a direct interaction of S1 with Tm. Biochemistry, 2001 Jun 19, 40(24), 7324 - 33 Requirements for osmosensing and osmotic activation of transporter ProP from Escherichia coli; Racher KI et al.; Transporter ProP of Escherichia coli, a solute-H+ symporter, can sense and respond to osmotic upshifts imposed on cells, on membrane vesicles, or on proteoliposomes that incorporate purified ProP-(His)6 . In this study, proline uptake catalyzed by ProP was used as a measure of its osmotic activation, and the requirements for osmosensing were defined using the proteoliposome system . The initial rate of proline uptake increased with decreasing external pH and increasing DeltaPsi, lumen negative . Osmotic upshifts increased DeltaPsi by concentrating lumenal K+, but osmotic activation of ProP could be distinguished from this effect . Osmotic activation of ProP resulted from changes in Vmax, though osmotic shifts also increased the KM for proline . Osmotic activation could be described as a reversible, osmotic upshift-dependent transition linking (at least) two transporter protein conformations . No correlation was observed between ProP activation and the position of the anions of activating sodium salts within the Hofmeister series of solutes . Both the magnitude of the osmotic upshift required to activate ProP and the ProP activity attained were similar for membrane-impermeant osmolytes, including NaCl, glucose, and PEG 600 . The membrane-permeant osmolytes glycerol, urea, PEG 62, and PEG 106 failed to activate ProP . Two poly(ethylene glycol)s, PEG 150 and PEG 200, were membrane-permeant and did not cause liposome shrinkage, but they did partially activate ProP-(His)6. Biochemistry, 2001 Jun 19, 40(24), 7211 - 8 Interaction of fluorescence labeled single-stranded DNA with hexameric DNA-helicase RepA: a photon and fluorescence correlation spectroscopy study; Xu H et al.; Fluorescence correlation spectroscopy (FCS) was used to characterize the interaction of fluorescence labeled single-stranded DNA (ssDNA) with hexameric RepA DNA-helicase (hRepA) encoded by plasmid RSF1010 . The apparent dissociation constants, Kd(app), for the equilibrium binding of 12mer, 30mer, and 45mer ssDNA 5'-labeled with BFL to hRepA dimer in the presence of 0.5 mM ATPgammaS at pH 5.8 and 25 degrees C were determined to be 0.58 +/- 0.12, 0.52 +/- 0.07, and 1.66 +/- 0.32 microM, respectively . Binding curves are compatible with one binding site for ssDNA present on hRepA dimer, with no indication of cooperativity . At pH 7.6 in the presence of ATPgammaS and at pH 5.8 in the absence of ATPgammaS, complex formation between ssDNA and hRepA was too weak for measuring complete binding curves by FCS . Under these conditions, the dissociation constant, Kd(app), is in the range between 10 and 250 microM . The kinetics of complex formation at pH 5.8 are faster than the time resolution (approximately 10-20 s) of FCS experiments under pseudo-first-order conditions, with respect to BFL-ssDNA . Photon correlation spectroscopy (PCS) experiments yielded, within the experimental error range, the same values for the apparent hydrodynamic radii, R(h), of hRepA dimer and its complex with ssDNA as determined by FCS (R(h) = 6.6 +/- 1 nm) . hRepA starts to aggregate under acidic conditions (<pH 6.0) which are optimal for ssDNA binding . CD spectra taken at pH 5.8 in the presence of ATPgammaS showed a structural change induced by ssDNA binding to hRepA which is not visible at pH 7.6 and with ADP as nucleotide cofactor. Biochemistry, 2001 Jun 19, 40(24), 7174 - 9 The human interferon- and estrogen-regulated ISG20/HEM45 gene product degrades single-stranded RNA and DNA in vitro; Nguyen LH et al.; The human ISG20/HEM45 gene was identified independently on the basis of its increased level of expression in response to either interferon or estrogen hormone . Notably, the encoded protein is homologous with members of the 3' to 5' exonuclease superfamily that includes RNases T and D, and the proofreading domain of Escherichia coli DNA polymerase I . We provide here direct biochemical evidence that Isg20 acts as a 3' to 5' exonuclease in vitro . This protein displays a pH optimum of approximately 7.0, prefers Mn2+ as a metal cofactor, and degrades RNA at a rate that is approximately 35-fold higher than its rate for single-stranded DNA . Along with RNase L, Isg20 is the second known RNase regulated by interferon . Previous data showed that Isg20 is located in promyelocytic leukemia (PML) nuclear bodies, known sites of hormone-dependent RNA polymerase II transcription and oncogenic DNA viral transcription and replication . The combined data suggest a potential role for Isg20 in degrading viral RNAs as part of the interferon-regulated antiviral response and/or cellular mRNAs as a regulatory component of interferon and estrogen signaling. Biochemistry, 2001 Jun 19, 40(24), 6989 - 97 Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center; Thoden JB et al.; Dihydroorotase plays a key role in pyrimidine biosynthesis by catalyzing the reversible interconversion of carbamoyl aspartate to dihydroorotate . Here we describe the three-dimensional structure of dihydroorotase from Escherichia coli determined and refined to 1.7 A resolution . Each subunit of the homodimeric enzyme folds into a "TIM" barrel motif with eight strands of parallel beta-sheet flanked on the outer surface by alpha-helices . Unexpectedly, each subunit contains a binuclear zinc center with the metal ions separated by approximately 3.6 A . Lys 102, which is carboxylated, serves as a bridging ligand between the two cations . The more buried or alpha-metal ion in subunit I is surrounded by His 16, His 18, Lys 102, Asp 250, and a solvent molecule (most likely a hydroxide ion) in a trigonal bipyramidal arrangement . The beta-metal ion, which is closer to the solvent, is tetrahedrally ligated by Lys 102, His 139, His 177, and the bridging hydroxide . L-Dihydroorotate is observed bound to subunit I, with its carbonyl oxygen, O4, lying 2.9 A from the beta-metal ion . Important interactions for positioning dihydroorotate into the active site include a salt bridge with the guanidinium group of Arg 20 and various additional electrostatic interactions with both protein backbone and side chain atoms . Strikingly, in subunit II, carbamoyl L-aspartate is observed binding near the binuclear metal center with its carboxylate side chain ligating the two metals and thus displacing the bridging hydroxide ion . From the three-dimensional structures of the enzyme-bound substrate and product, it has been possible to propose a unique catalytic mechanism for dihydroorotase . In the direction of dihydroorotate hydrolysis, the bridging hydroxide attacks the re-face of dihydroorotate with general base assistance by Asp 250 . The carbonyl group is polarized for nucleophilic attack by the bridging hydroxide through a direct interaction with the beta-metal ion . During the cyclization of carbamoyl aspartate, Asp 250 initiates the reaction by abstracting a proton from N3 of the substrate . The side chain carboxylate of carbamoyl aspartate is polarized through a direct electrostatic interaction with the binuclear metal center . The ensuing tetrahedral intermediate collapses with C-O bond cleavage and expulsion of the hydroxide which then bridges the binuclear metal center. Biochem Biophys Res Commun, 2001 Apr 6, 282(3), 787 - 92 Purification and structural analysis of the hepatitis B virus preS1 expressed from Escherichia coli; Maeng CY et al.; The preS1 of hepatitis B virus (HBV) is located at the outermost part of the envelope protein and possesses several functionally important regions such as hepatocyte receptor-binding site and virus-neutralizing epitopes . As the first step to understand the structure-function relationship for the preS1 antigen, we have purified the preS1 and performed its structural characterization by circular dichroism (CD) spectroscopy . The preS1 was purified to near homogeneity from bacterially expressed glutathione S-transferase (GST)-preS1 fusion protein by two-step purification, affinity chromatography on glutathione-agarose column, and cation-exchange chromatography on Mono S column . The CD analysis showed that the purified preS1, which was largely unstructured in aqueous solution, acquired a significant (16%) alpha-helical structure when analyzed in 50% trifluoroethanol or 20 mM SDS . The results suggest that the preS1 assumes a mainly unstructured conformation and may form induced secondary structures upon binding to target proteins or under hydrophobic environment . Biochem Biophys Res Commun, 2001 Mar 30, 282(2), 562 - 9 A GrpE mutant containing the NH(2)-terminal "tail" region is able to displace bound polypeptide substrate from DnaK; Mehl AF et al.; A key feature to the dimeric structure for the GrpE heat shock protein is the pair of long helices at the NH(2)-terminal end followed by a presumable extended segment of about 30 amino acids from each monomer . We have constructed a GrpE deletion mutant protein that contains only the unique tail portion (GrpE1-89) and another that is missing this region (GrpE88-197) . Circular dichroism analysis shows that the GrpE1-89 mutant still contains one-third percent alpha-helical secondary structure . Using an assay that measures bound peptide to DnaK we show that the GrpE1-89 is able to lower the amount of bound peptide, whereas GrpE88-197 has no effect . Additionally, when the same peptide binding assay is carried out with the COOH-terminal domain of DnaK, the full-length GrpE and the two GrpE deletion mutants show little to no effect on peptide release . Furthermore, the GrpE88-197 mutant is able to enhance the off-rate of nucleotide from DnaK and the 1-89 mutant has no effect on the nucleotide release . Similar results of nucleotide release are observed with the NH(2)-terminal ATPase domain mutant of DnaK . The results presented show directly that there is interaction between the GrpE protein's "tail" region and the substrate COOH-terminal peptide binding domain of DnaK, although the effect is only fully manifest with an intact full-length DnaK molecule . Biochem Biophys Res Commun, 2001 Mar 30, 282(2), 436 - 41 In situ proteolytic digestion of inclusion body polypeptides occurs as a cascade process; Cubarsi R et al.; Misfolded proteins undergo a preferent degradation ruled by the housekeeping bacterial proteolytic system, but upon precipitation as inclusion bodies their stability dramatically increases . The susceptibility of aggregated polypeptides to proteolytic attack remains essentially unexplored in bacteria and also in eukaryotic cells . We have studied here the in vitro proteolysis of beta-galactosidase fusion proteins by trypsin treatment of purified inclusion bodies . A cascade digestion process similar to that occurring in vivo has been observed in the insoluble fraction of the digestion reaction . This suggests that major protease target sites are not either lost or newly generated by protein precipitation and that the digestion occurs in situ probably on solvent-exposed surfaces of inclusion bodies . In addition, the sequence of the proteolytic attack is influenced by protein determinants other than amino acid sequence, the early digestion steps having a dramatic influence on the further cleavage susceptibility of the intermediate degradation fragments . These observations indicate unexpected conformational changes of inclusion body proteins during their site-limited digestion, that could promote protein release from aggregates, thus partially accounting for the plasticity of in vivo protein precipitation and solubilization in bacteria . J Theor Biol, 2001 Mar 21, 209(2), 213 - 22 Promotion of evolution by intracellular coexistence of mutator and normal DNA polymerases; Aoki K et al.; The efficient evolution of a population requires both genetic diversity and stable reproduction of advantageous genotypes . The accuracy of DNA replication guarantees the stable reproduction, while errors during DNA replication produce the genetic diversity . Thus, one key to the promotion of evolution is inherent in DNA replication . In bacteria, replication forks progress bidirectionally from the single origin of replication on a genome . One replication fork contains two DNA polymerase molecules so that four DNA polymerases simultaneously carry out the replication of a genome . It is generally believed that the fidelity of the intracellular DNA polymerases is identical (parity strategy) . To test this, we examined the effects of the intracellular coexistence of a mutator polymerase with low fidelity and a normal polymerase with high fidelity on adaptive evolution (disparity strategy) . From the analysis using genetic algorithms based on the bacterial replication, it was found that the population using the disparity strategy could further expand its genetic diversity and preserve the advantageous genotypes more profoundly than the parity population . This strongly suggests that bacteria replicating with a disparity strategy may undergo rapid evolution, particularly during severe environmental changes . The implications of the conspicuous adaptability of Escherichia coli mutator strains are discussed in this context . Biochim Biophys Acta, 1974 Apr 27, 349(1), 23 - 31 The incorporation of wrong bases by DNA polymerase I following gamma-irradiation of DNA-like templates; Saffhill R; The synthesis of polydeoxyribose polymers by Escherichia coli DNA polymerase I has been investigated with control and gamma-irradiated DNA-like polymer templates containing only two bases . The results show that irradiation of a poly(dA) strand leads to the incorporation of dG, whereas irradiation of poly(dC) and poly(dG) strands both lead to the incorporation of dA . Irradiation of poly(dT) does not lead to the incorporation of any wrong base . The wrong bases are incorporated into the complementary strand of the newly synthesised DNA. Biochim Biophys Acta, 1974 Apr 27, 349(1), 131 - 4 Molecular structure of exonuclease I from Escherichia coli B; Mackay V et al.; Exonuclease I of Escherichia coli B (EC 3.1.4.25) was determined to be a monomeric protein of molecular weight approx . 72,000, as estimated by Sephadex gel filtration, sedimentation velocity centrifugation, and sodium dodecylsulfate polyacrylamide gel electrophoresis. Biochim Biophys Acta, 1974 Apr 27, 349(1), 125 - 30 Escherichia coli initiation factor IF3 binding to AUG and AUG-containing single strands and hairpin loops, and nonspecific binding to polymers; Wickstrom E; Nitrocellulose filter binding and equilibrium dialysis detected the binding of Escherichia coli initiation factor IF3 to AUG, An UGUm single strands and hairpin loops, poly(A,U,G), poly(U), and f2 RNA . No binding was detected for GUA, A8 U, or the hairpin loop A5 GC5 U5 . AUG-specific binding, per nucleotide, is strong; nonspecific binding, per nucleotide, is weak. Eur Cytokine Netw, 2001 Apr-Jun, 12(2), 260 - 7 Cytokine-mediated inflammatory hyperalgesia limited by interleukin-13; Lorenzetti BB et al.; The effect of interleukin-13 (IL-13) on hyperalgesic responses to intraplantar (i.pl.) injection of carrageenin, E . coli endotoxin (LPS), bradykinin, tumour necrosis factor a (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2)) was investigated in a model of mechanical hyperalgesia in rats . Also, the cellular source of the IL-13 was investigated . IL-13, administered 30 min before the stimulus, inhibited responses to carrageenin, LPS, bradykinin, and TNF-alpha, but not responses to IL-1 beta, IL-8 and PGE2 . IL-13, administered 2 hours before the injection of IL-1b, did not affect the response to IL-1b, whereas IL-13, administered 12 hours or 12 + 2 hours before the IL-1 beta, inhibited the hyperalgesia (- 35%, - 77%, respectively) . In murine peritoneal macrophages, IL-13 administered 2 hours before stimulation with LPS, inhibited the production of IL-1 beta (- 67%) and PGE(2) (- 56%) . IL-13 administered 12 hours before stimulation with LPS inhibited LPS-stimulated PGE(2) but not IL-1 beta . An anti-IL-13 serum potentiated responses to carrageenin, LPS, bradykinin and TNF-alpha (but not IL-1 beta and IL-8), as well as responses to bradykinin in rats depleted of mast cells with compound 40/80, but not in athymic rats . These data suggest that IL-13, released by lymphocytes, limits inflammatory hyperalgesia by the inhibition of the production TNF-alpha, IL-1 beta, IL-8 and PGs. Mol Immunol, 2000 Dec, 37(17), 1067 - 77 Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin; Cloutier SM et al.; Phage display is a powerful method of isolating of antibody fragments from highly diverse naive human antibody repertoires . However, the affinity of the selected antibodies is usually low and current methods of affinity maturation are complex and time-consuming . In this paper, we describe an easy way to increase the functional affinity (avidity) of single chain variable fragments (scFvs) by tetramerization on streptavidin, following their site-specific biotinylation by the enzyme BirA . Expression vectors have been constructed that enable addition of the 15 amino acid biotin acceptor domain (BAD) on selected scFvs . Different domains were cloned at the C-terminus of scFv in the following order: a semi-rigid hinge region (of 16 residues), the BAD, and a histidine tail . Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from human non-immune and murine immune phage display libraries . The scFvs were first synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then purified from the cytoplasmic extracts by Ni-NTA affinity chromatography . Purified biotinylated scFvs were tetramerized on the streptavidin molecule to create a streptabody (StAb) . The avidity of various forms of anti-CEA StAbs, tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the scFv monomer counterparts . Furthermore, the percentage of direct binding of 125I-labeled StAb or monomeric scFv on CEA-Sepharose beads and on CEA-expressing cells showed a dramatic increase for the tetramerized scFv (>80%), as compared with the monomeric scFv (<20%) . Interestingly, the percentage binding of 125I-labeled anti-CEA StAbs to CEA-expressing colon carcinoma cells was definitely higher (>80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%) . Another advantage of using scFvs in a StAb format was demonstrated by Western blot analysis, where tetramerized anti-CEA scFv could detect a small quantity of CEA at a concentration 100-fold lower than the monomeric scFv. J Mol Biol, 2001 Jun 22, 309(5), 1219 - 31 gyrB-225, a mutation of DNA gyrase that compensates for topoisomerase I deficiency: investigation of its low activity and quinolone hypersensitivity; Heddle JG et al.; The B subunit of DNA gyrase (GyrB) consists of a 43 kDa N-terminal domain, containing the site of ATP binding and hydrolysis, and a 47 kDa C-terminal domain that is thought to play a role in interactions with GyrA and DNA . In cells containing a deletion of topA (the gene encoding DNA topoisomerase I) a compensatory mutation is found in gyrB . This mutation (gyrB-225) results in a two amino acid insertion in the N-terminal domain of GyrB . We found that cells containing this mutation are more sensitive than wild-type cells to quinolone drugs with respect to bacteriostatic and lethal action . We have characterised the mutant GyrB protein in vitro and found it to have reduced DNA supercoiling, relaxation, ATPase, and cleavage activities . The mutant enzyme is up to threefold more sensitive to quinolones than wild-type . The mutation also increases the affinity of GyrB for GyrA and DNA, while the affinity of quinolone for the enzyme-DNA complex is unaffected . We propose that the loss in activity is due to misfolding of the GyrB-225 protein, providing an example in which misfolding of one protein, DNA gyrase, suppresses a deficiency of another, topoisomerase I . The increased quinolone sensitivity is proposed to be a consequence of an altered conformation of the protein that renders quinolones better able to disrupt, rather than generate, gyrase-drug-DNA complexes . J Mol Biol, 2001 Jun 22, 309(5), 1201 - 8 Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG {correction of non-CpA} sites; Gowher H et al.; We present the first in vitro study investigating the catalytic properties of a mammalian de novo DNA methyltransferase . Dnmt3a from mouse was cloned and expressed in Escherichia coli . It was shown to be catalytically active in E . coli cells in vivo . The methylation activity of the purified protein was highest at pH 7.0 and 30 mM KCl . Our data show that recombinant Dnmt3a protein is indeed a de novo methyltransferase, as it catalyzes the transfer of methyl groups to unmethylated substrates with similar efficiency as to hemimethylated substrates . With oligonucleotide substrates, the catalytic activity of Dnmt3a is similar to that of Dnmt1: the K(m) values for the unmethylated and hemimethylated oligonucleotide substrates are 2.5 microM, and the k(cat) values are 0.05 h(-1) and 0.07 h(-1), respectively . The enzyme catalyzes the methylation of DNA in a distributive manner, suggesting that Dnmt3a and Dnmt1 may cooperate during de novo methylation of DNA . Further, we investigated the methylation activity of Dnmt3a at non-canonical sites . Even though the enzyme shows maximum activity at CpG sites, with oligonucleotide substrates, a high methylation activity was also found at CpA sites, which are modified only twofold slower than CpG sites . Therefore, the specificity of Dnmt3a is completely different from that of the maintenance methyltransferase Dnmt1, which shows a 40 to 50-fold preference for hemimethylated over unmethylated CpG sites and has almost no methylation activity at non-CpG sites . J Mol Biol, 2001 Jun 22, 309(5), 1017 - 27 Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii; Dzierszinski F et al.; The precise molecular mechanisms underlying the switch between the two developmental stages of Toxoplasma gondii, and the metabolic adaptations occurring during this stage conversion are poorly understood . Because inhibitors of mitochondrial respiration are known to trigger differentiation from tachyzoite into bradyzoite stages, we believe that some of the switch components may be sought in the regulation of central carbohydrate metabolism . We have previously described a cDNA encoding a bradyzoite-specific enolase, ENO1 . We now report the isolation and characterization of another enolase-encoding cDNA (ENO2) that is expressed preferentially in the tachyzoite stage . The deduced amino acid sequences of ENO1 and ENO2 share 73.65 % identity . They both display significant homologies to plant enolases with the presence of two plant-like peptide insertions, a pentapeptide EWGW(Y)C(S) and a dipeptide EK (or DK) . We demonstrate that deletions of the ENO1 pentapeptide motif on its own or together with the dipeptide reduce drastically the affinity for the 2PGA substrate, suggesting that the evolutionary acquisition of these peptides in enolases of land plants and apicomplexan parasites contribute a specific function to their enzymatic activities . T . gondii ENO1 and ENO2 were also expressed as active recombinant enzymes in Escherichia coli . While ENO1 and ENO2 display similar K(m) values, the pure tachyzoite-specific enzyme (ENO2) has a threefold specific activity at V(max) compared with that of the bradyzoite-specific enolase (ENO1) . Moreover, immunoblot analyses performed using polyclonal antibodies raised against the recombinant enzymes revealed that the native enolase in tachyzoite and bradyzoite are also antigenically distinct . Taken together, our results indicate that the differences witnessed between the two activities may be instrumental in maintaining glycolysis in pace with the distinct stage-specific requirements of carbohydrate metabolism . J Mol Biol, 2001 Jun 15, 309(4), 961 - 74 Ligand-induced structural changes to maltodextrin-binding protein as studied by solution NMR spectroscopy; Evenas J et al.; Solution NMR studies on the physiologically relevant ligand-free and maltotriose-bound states of maltodextrin-binding protein (MBP) are presented . Together with existing data on MBP in complex with beta-cyclodextrin (non-physiological, inactive ligand), these new results provide valuable information on changes in local structure, dynamics and global fold that occur upon ligand binding to this two-domain protein . By measuring a large number of different one-bond residual dipolar couplings, the domain conformations, critical for biological function, were investigated for all three states of MBP . Structural models of the solution conformation of MBP in a number of different forms were generated from the experimental dipolar coupling data and X-ray crystal structures using a quasi-rigid-body domain orientation algorithm implemented in the structure calculation program CNS . Excellent agreement between relative domain orientations in ligand-free and maltotriose-bound solution conformations and the corresponding crystal structures is observed . These results are in contrast to those obtained for the MBP/beta-cyclodextrin complex where the solution state is found to be approximately 10 degrees more closed than the crystalline state . The present study highlights the utility of residual dipolar couplings for orienting protein domains or macromolecules with respect to each other . J Mol Biol, 2001 Jun 15, 309(4), 949 - 60 The solution structure of the N-terminal domain of riboflavin synthase; Truffault V et al.; The structure of the amino-terminal domain of Escherichia coli riboflavin synthase (RiSy) has been determined by NMR spectroscopy with riboflavin as a bound ligand . RiSy is functional as a 75 kDa homotrimer, each subunit of which consists of two domains which share very similar sequences and structures . The N-terminal domain (RiSy-N; 97 residues) forms a 20 kDa homodimer in solution which binds riboflavin with high affinity . The structure features a six-stranded antiparallel beta-barrel with a Greek-key fold, both ends of which are closed by an alpha-helix . One riboflavin molecule is bound per monomer in a site at one end of the barrel which is comprised of elements of both monomers . The structure and ligand binding are similar to that of the FAD binding domains of ferrodoxin reductase family proteins . The structure provides insights into the structure of the whole enzyme, the organisation of the functional trimer and the mechanism of riboflavin synthesis . C48 from the N-terminal domain is identified as the free cysteine implicated in a nucleophilic role in the synthesis mechanism, while H102 from the C-terminal domains is also likely to play a key role . Both are invariant in all known riboflavin synthase sequences . APMIS, 2001 Feb, 109(2), 89 - 95 Genotoxic potential of xenobiotic growth promoters and their metabolites; Metzler M et al.; This paper reviews data reported in the literature as well as recent and unpublished studies from our laboratory on the metabolism and genotoxicity of the xenobiotic growth promoters 17beta-trenbolone, melengestrol acetate and zeranol . In our metabolic study, the oxidative in vitro metabolites generated by hepatic microsomes from rats, bovine and humans were analyzed by HPLC and GC/MS . 17beta-Trenbolone gave rise to at least 13 monohydroxylated products, whereas 12 mono- and dihydroxylated metabolites were obtained with melengestrol acetate and at least 5 with zeranol . The genotoxic potential of the parent compounds was studied using the following endpoints: induction of HPRT mutations in cultured V79 cells and of lacI mutations in E . coli; induction of micronuclei in V79 cells; and formation of DNA adducts in cultured primary rat hepatocytes . Negative results were obtained in most of these assay systems . Only the micronucleus induction was marginally positive with 17beta-trenbolone and zeranol at near-cytotoxic concentrations . Commercial melengestrol acetate was found to contain an impurity causing apoptosis in V79 cells . The genotoxic potential of the numerous oxidative metabolites of the xenobiotic growth promoters remains to be studied. Intensive Care Med, 2001 Apr, 27(4), 757 - 66 Increased ileal-mucosal-arterial PCO2 gap is associated with impaired villus microcirculation in endotoxic pigs; Tugtekin IF et al.; OBJECTIVE: To investigate whether an increased ileal-mucosal-arterial PCO2 gap (delta PCO2) during hyperdynamic porcine endotoxemia is associated with impaired villus microcirculation . DESIGN: Prospective, randomized, controlled, experimental study . SETTING: Animal research laboratory . ANIMALS: Twenty-two domestic pigs . INTERVENTIONS: After baseline measurements, anesthetized and ventilated pigs received continuous i.v . endotoxin (ETX, n = 12) for 24 h or placebo (SHAM, n = 10) . MEASUREMENTS AND RESULTS: Before, as well as 12 and 24 h after, the start of endotoxin or saline portal venous blood flow (QPV, ultrasound flow probe) and lactate/pyruvate ratios (L/P), the ileal-mucosal-arterial delta PCO2 (fiberoptic sensor) and bowel-wall capillary hemoglobin O2 saturation (%Hb-O2-cap, remission spectrophotometry) were assessed together with intravital video records of the ileal-mucosal microcirculation (number of perfused/heterogeneously perfused/unperfused villi) using orthogonal polarization spectral imaging (CYTOSCAN A/R) via an ileostomy . At 12 and 24 h endotoxin infusion, about half of the evaluated villi were heterogeneously or unperfused which was paralleled by a progressive significant increase of the ileal-mucosal-arterial delta PCO2 and portal venous L/P ratios, whereas QPV as well as both the mean %Hb-O2-cap and the %Hb-O2-cap frequency distributions remained unchanged . By contrast, in the SHAM-group, mucosal microcirculation was well-preserved, and none of the other parameters were influenced . CONCLUSIONS: We conclude that an increased ileal-mucosal-arterial delta PCO2 during porcine endotoxemia is related to impaired villus microcirculation . A putative contribution of disturbed cellular oxygen utilization resulting from "cytopathic hypoxia" may also assume importance. J Infect Dis, 2001 Jul 1, 184(1), 43 - 51 Epub 2001 Jun 08. T cell receptor dynamism of mucosal and systemic CD4+ T cells in the course of an immune response to Escherichia coli heat-labile enterotoxin; Kim JK et al.; The changes in T cell receptor (TCR) Vbeta expression, use, and clonality in mice orally challenged with Escherichia coli heat-labile enterotoxin (LT) were assessed . Use of the TCR Vbeta family and clonality were significantly changed at the single-cell level . In Peyer's patches of treated mice, use of TCR Vbeta6, Vbeta8, and Vbeta14 increased in CD4(+)CD44(+) T cells, compared with use in nontreated mice . On the other hand, use of TCR Vbeta1 and Vbeta8 was enhanced in splenic CD4(+)CD44(+) T cells . Intraepithelial lymphocytes isolated from LT-challenged mice showed expanded clonality (e.g., Vbeta1, Vbeta2, Vbeta9, and Vbeta18) and altered TCR Vbeta use (e.g., Vbeta15, Vbeta16, and Vbeta17) . These findings reveal that oral administration of LT has distinct effects on mucosal versus systemic alphabeta T cells for induction of CD4(+) T cells with selected Vbeta use . This most likely reflects the function of LT as a mucosal modulator. J Allergy Clin Immunol, 2001 Jun, 107(6), 977 - 84 The molecular basis of antigenic cross-reactivity between the group 2 mite allergens; Smith AM et al.; BACKGROUND: Mite group 2 allergens Der p 2, Der f 2, and Eur m 2 are 14-kDa proteins of unknown function that share 83% to 85% amino acid sequence identity . Isoforms of the allergens within each genus have been identified which differ by 3 or 4 amino acids, but little is known of the influence of group 2 polymorphisms on human IgE antibody binding . OBJECTIVE: The purpose of this study was to investigate the importance of interspecies and isoform substitutions on murine mAb and IgE antibody binding and on the molecular structure of the group 2 allergens . METHODS: Site-directed mutagenesis was used to incorporate the isoform amino acid substitutions onto the Der p 2.0101 sequence . Recombinant allergens were expressed and purified from Escherichia coli and used to evaluate antibody binding by enzyme-linked immunosorbent assay (ELISA) . Molecular modeling of the tertiary structure was used to analyze structural differences between the various group 2 allergens . RESULTS: The substitution of asparagine for aspartic acid at position 114 restored mAb binding of rDer p 2.0101; the other Der p 2 isoforms and the 3 rDer f 2 isoforms also reacted in the 2-site ELISA . The correlation of IgE binding to the Der p 2 isoforms was excellent and tended to be higher in the isoforms with the asparagine 114 substitution (r (2) = 0.87 vs r (2) = 0.95) . rEur m 2.0101 bound to all mAb except 7A1; when compared with rDer p 2 for IgE binding, rEur m 2.0101 gave a correlation coefficient of r (2) = 0.68 . Molecular modeling revealed that Eur m 2 and the storage mite homologs Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2 . Eur m 2 has a conserved surface, whereas Lep d 2 and Tyr p 2 present most of the amino acid substitutions on this surface . Lep d 2 and Tyr p 2 did not react with mAb or with sera from patients with IgE to Dermatophagoides species . CONCLUSION: The isoform substitutions of rDer p 2 can be distinguished by mAb . The allergenic cross-reactivity between Der p 2, Der f 2, and Eur m 2 is a direct result of the conserved antigenic surface, whereas the lack of cross-reactivity with Lep d 2 and Tyr p 2 is a result of the multiple substitutions across this surface. Science, 2001 Jun 29, 292(5526), 2488 - 92 Epub 2001 Jun 07. Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis; Outten CE et al.; Intracellular zinc is thought to be available in a cytosolic pool of free or loosely bound Zn(II) ions in the micromolar to picomolar range . To test this, we determined the mechanism of zinc sensors that control metal uptake or export in Escherichia coli and calibrated their response against the thermodynamically defined free zinc concentration . Whereas the cellular zinc quota is millimolar, free Zn(II) concentrations that trigger transcription of zinc uptake or efflux machinery are femtomolar, or six orders of magnitude less than one atom per cell . This is not consistent with a cytosolic pool of free Zn(II) and suggests an extraordinary intracellular zinc-binding capacity . Thus, cells exert tight control over cytosolic metal concentrations, even for relatively low-toxicity metals such as zinc. J Biol Chem, 2001 Aug 10, 276(32), 30374 - 80 Epub 2001 Jun 07. Identification and characterization of a new mammalian glutaredoxin (thioltransferase), Grx2; Gladyshev VN et al.; A thiol/disulfide oxidoreductase component of the GSH system, glutaredoxin (Grx), is involved in the reduction of GSH-based mixed disulfides and participates in a variety of cellular redox pathways . A single cytosolic Grx (Grx1) was previously described in mammals . We now report identification and characterization of a second mammalian Grx, designated Grx2 . Grx2 exhibited 36% identity with Grx1 and had a disulfide active center containing the Cys-Ser-Tyr-Cys motif . Grx2 was encoded in the genomes of mammals and birds and expressed in a variety of cell types . The gene for human Grx2 consisted of four exons and three introns, spanned 10 kilobase pairs, and localized to chromosome 1q31.2-31.3 . The coding sequence was present in all exons, with the first exon encoding a mitochondrial signal peptide . The mitochondrial leader sequence was also present in mouse and rat Grx2 sequences and was shown to direct either Grx2 or green fluorescent protein to mitochondria . Alternative splicing forms of mammalian Grx2 mRNAs were identified that differed in sequences upstream of exon 2 . To functionally characterize the new protein, human and mouse Grx2 proteins were expressed in Escherichia coli, and the purified proteins were shown to reduce mixed disulfides formed between GSH and S-sulfocysteine, hydroxyethyldisulfide, or cystine . Grx1 and Grx2 were sensitive to inactivation by iodoacetamide and H(2)O(2) and exhibited similar pH dependence of catalytic activity . However, H(2)O(2)-inactivated Grx2 could only be reactivated with 5 mm GSH, whereas Grx1 could also be reactivated with dithiothreitol or thioredoxin/thioredoxin reductase . The Grx2 structural model suggested a common reaction mechanism for this class of proteins . The data provide the first example of a mitochondrial Grx and also indicate the occurrence of a second functional Grx in mammals. Enzyme Microb Technol, 2001 Jun 7, 28(9-10), 785 - 791 L(-)-carnitine production using a recombinant Escherichia coli strain; Castellar MR et al.; The L(-)-carnitine production by biotransformation using the recombinant strain Escherichia coli pT7-5KE32 has been studied and optimized with crotonobetaine and D(+)-carnitine as substrates . A resting rather than a growing cells system for L(-)-carnitine production was chosen, crotonobetaine being the best substrate . High biocatalytic activity was obtained after growing the cells under anaerobic conditions at 37 degrees C and with crotonobetaine or L(-)-carnitine as inducer . The growth incubation temperature (37 degrees C) was high enough as to activate the heat-inducible lambdap(L) promoter inserted in the plasmid pGP1-2 . The best biotransformation conditions were with resting cells, under aerobiosis, with 4 g l(-1) and 100 mM biomass and substrate concentrations respectively . Under these conditions the biotransformation time (1 h) was shorter and the L(-)-carnitine yield (70%) higher than previously reported . Consequently productivity value (11.3 g l(-1)h(-1)) was highly improved when comparing with other published works . The resting cells could be reused until eight times maintaining product yield levels well over 50% that meant to increase ten times the L(-)-carnitine obtained per gram of biomass. Chem Biol Interact, 2001 Jun 1, 135-136, 325 - 41 Mutational spectrum of 1,3-butadiene and metabolites 1,2-epoxybutene and 1,2,3,4-diepoxybutane to assess mutagenic mechanisms; Recio L et al.; 1,3-Butadiene (BD) is a multisite carcinogen and is mutagenic in multiple tissues of B6C3F1 mice . BD is bioactivated to at least three directly mutagenic metabolites: 1,2-epoxybutene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB) . However, the contribution of these individual metabolites to the carcinogenicity and in vivo mutatidnal spectrum of BD is uncertain . To assess the role of two BD metabolites EB and DEB in the in vivo mutagenicity of the parent compound BD, we examined the in vitro mutational spectra of EB and DEB in human and rodent cells . We also examined the in vivo mutagenicity and mutational spectrum of inhaled EB in the lung . In the bone marrow and spleen of B6C3F1 laci transgenic mice, BD-induced an increased frequency of the identical class of point mutations at A:T base pairs: AT-->GC transitions and AT-->TA transversions . BD exposure also induced an increased frequency of GC-->AT transitions in the spleen that was not observed in bone marrow, demonstrating tissue-specific differences in mutation spectrum . Exposure of Rat2 laci transgenic cells and human TK6 lymphoblasts to EB-induced an increased frequency of AT-->TA transversions . DEB exposure induced an increased frequency of AT-->TA transversions and partial deletions at hprt in human cells . In Rat laci transgenic cells, DEB was not mutagenic at laci but induced an increased frequency of micronuclei . In contrast to inhaled BD, inhaled DEB and EB were not mutagenic in the bone marrow or spleen . However, EB was mutagenic in the lungs . In the lung of mice, EB-induced specific increases in GC-->AT transitions, AT-->TA transversions, and deletion events . AT-->TA transversions are the most consistent mutation observed across biological systems following in vivo exposure to BD or in vitro exposures to EB and DEB . Although, BD exposure in mice induces chromosomal alterations and single base substitutions, the specific BD metabolite that induces the genetic events leading to tumors is uncertain . At present, it appears that only DEB can effectively induce this range of mutagenic events at levels of this metabolite that occur in the blood of mice exposed to BD . Detailed investigations to identify relevant biomarkers of BD exposure and response, particularly DNA adducts or lesions, that can be biologically linked to the range of genotoxic events known to occur in mice exposed to BD are needed. J Mol Biol, 2001 Jun 8, 309(3), 817 - 32 The allosteric activator Mg-ATP modifies the quaternary structure of the R-state of Escherichia coli aspartate transcarbamylase without altering the T<-->R equilibrium; Fetler L et al.; The allosteric enzyme aspartate transcarbamylase from Escherichia coli (ATCase) displays regulatory properties that involve various conformational changes, including a large quaternary structure rearrangement . This entails a major change in its solution X-ray scattering curve upon binding substrate analogues . We show here that, in the presence of the nucleotide effector ATP, known to stimulate the enzyme activity, the scattering profiles show a marked dependence on the metal bound to ATP . Whereas ATP has no major effect on the scattering pattern of ATCase, a saturating concentration of Mg-ATP notably modifies the scattering profile of the enzyme, either in the absence or in the presence of the bisubstrate analogue N-(phosphonacetyl)-l-aspartate (PALA) . The transition with PALA in the presence of this metal-nucleotide complex remains concerted . Furthermore, Mg-ATP, as already observed with ATP, has no detectable direct effect on the T to R transition . The experimental scattering curves in the presence of Mg-ATP were fitted by a modeling approach using rigid body movements of the regulatory subunits and the catalytic trimers in the crystal structures . While the differences observed in the T-state in the presence of Mg-ATP are essentially attributed to the binding per se of the nucleotide, the solution structure of the R-state complexed to Mg-ATP is even more extended along the 3-fold axis than the previously described R solution structure, which is already more stretched out along the same axis than the crystal R structure . Based on the crystal structure of the enzyme in the R-state complexed with free ATP, a proposal is made to account for the effect of magnesium . J Mol Biol, 2001 Jun 8, 309(3), 561 - 72 Domain 1.1 of the sigma(70) subunit of Escherichia coli RNA polymerase modulates the formation of stable polymerase/promoter complexes; Vuthoori S et al.; The sigma 70 (sigma(70)) subunit of Escherichia coli RNA polymerase specifies transcription from promoters that are responsible for basal gene expression during vegetative growth . When sigma(70) is present within polymerase holoenzyme, two of its domains, 2.4 and 4.2, interact with sequences within the -10 and -35 regions, respectively, of promoter DNA . However, in free sigma(70), DNA binding is prevented by domain 1.1, the N-terminal domain of the protein . Previous work has demonstrated that the presence of domain 1.1 is required for efficient transcription initiation at the lambda promoter P(R) . To investigate whether this is a general property of domain 1.1, we have used five promoters to compare polymerases with and without domain 1.1 in in vitro transcription assays, and in assays assessing the formation and decay of stable, pretranscription complexes . We find that the absence of domain 1.1 does not render the polymerase defective at all of these promoters . Depending on the promoter, the absence of domain 1.1 can promote or inhibit transcription initiation by affecting the formation of stable pretranscription complexes . However, domain 1.1 does not affect the stability of these complexes once they are formed . For polymerases containing domain 1.1, the efficiency of stable complex formation correlates with how well the -10 and -35 regions of a promoter match the ideal sigma(70) recognition sequences . However, when domain 1.1 is absent, having this match becomes less important in determining how efficiently stable complexes are made . We suggest that domain 1.1 influences initiation by constraining polymerase to assess a promoter primarily by the fitness of its -10 and -35 regions to the canonical sequences . Biochem Biophys Res Commun, 2001 Jun 15, 284(3), 845 - 9 Plant sterol 14 alpha-demethylase affinity for azole fungicides; Lamb DC et al.; Azole fungicides were thought to have much greater affinity for the fungal cytochrome P450 enzyme, sterol 14 alpha-demthylase (CYP51) than the plant orthologue . Using purified CYP51 from the plant Sorghum bicolor L Moenech, a direct comparison of the sensitivity to the fungicides triadimenol and tebuconazole has been carried out . S . bicolor CYP51 was purified to homogenity as determined by SDS--PAGE and specific heme content . Addition of the azole fungicides triadimenol and tebuconazole induced type II spectral changes, with saturation occurring at equimolar azole/P450 concentrations . Inhibition of reconstituted activities revealed only a threefold insensitivity of the plant CYP51 compared to a fungal CYP51, from the phytopathogen Ustilago maydis, as judged by IC(50) values . The implications for fungicide mode of action and application are discussed . Biochem Biophys Res Commun, 2001 Jun 15, 284(3), 785 - 91 N- and C-terminal halves of human annexin VI differ in ability to form low pH-induced ion channels; Golczak M et al.; Human recombinant annexin VI (AnxVI) or its N- (AnxVIA) and C-terminal (AnxVIB) fragments were expressed in E . coli . Their ability to form voltage-dependent ion channels in membranes, induced by low pH, was measured to verify the hypothesis that, upon acidification, the hydrophobicity of AnxVI at a specific domain significantly increases allowing the AnxVI interaction with lipids in a Ca(2+)-independent manner . By theoretically analyzing changes in protein hydrophobicity, we found that hydrophobicity of AnxVIA significantly differed from that of AnxVIB at low pH . These predictions were confirmed experimentally by using planar lipid bilayers and liposome pull-down assay . We found striking difference between AnxVIA and AnxVIB in the ion channel activity, as well as in the membrane binding, suggesting that the halves of AnxVI maybe functionally different . Moreover, we calculated and predicted that the ion channel activity at low pH should appear in other human annexins, as AnxII, AnxV (as known), AnxVIII, and AnxXIII . The possibility that AnxVI acts as cytosolic component of a transmembrane pH-sensing mechanism is proposed . Biochem Biophys Res Commun, 2001 Jun 15, 284(3), 556 - 62 Processing of DNase domain during translocation of colicin E7 across the membrane of Escherichia coli; Liao CC et al.; Translocation of colicin across the membrane of sensitive cells has been studied extensively . However, processing of the toxicity domain of colicin during translocation has been the subject of much controversy . To investigate the final translocation product of colicin across the membrane of Escherichia coli, an endogenously expressed His-tagged Im7 protein was constructed to detect any translocation product containing the DNase domain traversed the inner membrane into cytoplasm of the E . coli cells . As a result, a final processed DNase domain of ColE7 was identified in the intracellular space of the cells treated with Col-Im complex . In the presence of periplasmic extracts, in vitro processing of DNase domain of ColE7 was also observed . These results suggest that the processing of ColE7 has occurred for translocation of the DNase-type colicin across the membrane and the process is probably taking place in the periplasmic space of the membrane . Arch Biochem Biophys, 2001 Jun 15, 390(2), 279 - 86 Demonstration that menthofuran synthase of mint (Mentha) is a cytochrome P450 monooxygenase: cloning, functional expression, and characterization of the responsible gene; Bertea CM et al.; (+)-Menthofuran is an undesirable monoterpenoid component of peppermint (Mentha x piperita) essential oil that is derived from the alpha,beta-unsaturated ketone (+)-pulegone . Microsomal preparations, from the oil gland secretory cells of a high (+)-menthofuran-producing chemotype of Mentha pulegium, transform (+)-pulegone to (+)-menthofuran in the presence of NADPH and molecular oxygen, implying that menthofuran is synthesized by a mechanism analogous to that of mammalian liver cytochrome P450s involving the hydroxylation of the syn-methyl group of (+)-pulegone, spontaneous intramolecular cyclization to the hemiketal, and dehydration to the furan . An abundant cytochrome P450 clone from a peppermint oil gland cell cDNA library was functionally expressed in Saccharomyces cerevisiae and Escherichia coli and shown to encode the (+)-menthofuran synthase (i.e., (+)-pulegone-9-hydroxylase) . The full-length cDNA contains 1479 nucleotides, and encodes a protein of 493 amino acid residues of molecular weight 55,360, which bears all of the anticipated primary structural elements of a cytochrome P450 and most closely resembles (35% identity) a cytochrome P450 monoterpene hydroxylase, (+)-limonene-3-hydroxylase, from the same source . The availability of this gene permits transgenic manipulation of peppermint to improve the quality of the derived essential oil . Arch Biochem Biophys, 2001 Jun 15, 390(2), 215 - 21 Reconstitution of the enzymatic activities of cytochrome P450s using recombinant flavocytochromes containing rat cytochrome b(5) fused to NADPH--cytochrome P450 reductase with various membrane-binding segments; Gilep AA et al.; The role of the hydrophobic membrane-binding segments of NADPH-cytochrome P450 reductase (CPR) and cytochrome b(5) remain undefined . We have expressed four different recombinant flavocytochromes containing b(5) linked to CPR with different hydrophobic segments as linkers . These fusion proteins have been expressed in Escherichia coli and purified and some of their physical properties and electron transfer activities described in the accompanying paper . Of interest is the presence of internal "membrane-binding" hydrophobic segments in these flavocytochromes . This paper describes the ability of these flavocytochromes to reconstitute in vitro two P450 activities that have been reported to be stimulated by the addition of b(5) (the 17,20-lyase activity of CYP17A and the 6 beta hydroxylation of testosterone catalyzed by CYP3A4) and two P450 reactions that do not respond to the presence of b(5) (the 17 alpha-hydroxylation of progesterone catalyzed by CYP17A and the omega hydroxylation of lauric acid catalyzed by CYP4A1) . The present study shows that a hydrophobic "membrane-binding" segment must be present in the artificial flavocytochromes in order to successfully reconstitute in vitro hydroxylation activities with P450s . Differences in the effectiveness of the different flavocytochromes to reconstitute enzymatic activities depends on the P450 tested and the nature of the hydrophobic linker segment present in the purified recombinant flavocytochromes . The hypothesis is proposed that differences in the surface topology of a P450 may dictate differences in their docking with the CPR or b(5) component of a fusion protein, resulting in differences in the rates of electron transfer to the P450 . Neuropathology, 2001 Jun, 21(2), 123 - 8 Immunohistochemical expression and pathogenesis of BLM in the human brain and visceral organs; Hachiya Y et al.; Bloom syndrome (BS) involves the clinical features of telangiectatic erythema, immunodeficiency, and an increased risk for cancer . In order to clarify the pathogenetic significance of the responsible gene, BLM, which encodes a protein possessing homology to Escherichia coli