Escherichia coli

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 RecQ helicase, the immunohistochemistry of BLM was examined in human brains and visceral organs from fetuses to adults and an adult with BS, using anti-BLM antibodies . Purkinje cells exhibited positive BLM immunoreactivity from 21 gestational weeks (GW), which transiently increased at approximately 40 GW . Neurons of the pontine tegmentum were immunolabeled from the early fetal period . In visceral organs, positive BLM immunoreactivity was observed in the Hassal corpuscles in the thymus from 24 GW, in beta-cells in the Langerhans islets of the pancreas from 36 GW, and in sperm cells and sperms of the testes from 11 years of age . But in a patient with BS, it was negative in the pancreas and testis tissues examined . The characteristic effect of BLM on specific cells in different periods suggests that the BLM gene product is closely related to neuronal development as well as immune, insulin secretory and sperm functions, which appear in different periods, and disorders of which are major symptoms of BS.

Intensive Care Med, 2001 Feb, 27(2), 416 - 25
Hepatic oxygen exchange and energy metabolism in hyperdynamic porcine endotoxemia: effects of the combined thromboxane receptor antagonist and synthase inhibitor DTTX30; Trager K et al.; OBJECTIVE: We compared the effects of thromboxane receptor antagonist and synthase inhibitor DTTX30 on systemic and liver blood flow, oxygen (O2) exchange and energy metabolism during 24 h of hyperdynamic endotoxemia with untreated endotoxemia . DESIGN: Prospective, randomized, experimental study with repeated measures . SETTING: Investigational animal laboratory . SUBJECTS: Twenty-seven domestic pigs: 16 during endotoxemia with volume resuscitation alone; 11 with endotoxemia, volume resuscitation and treatment with DTTX30 . INTERVENTIONS: Continuous infusion of Escherichia coli lipopolysaccharide (LPS) for 24 h together with volume resuscitation . After 12 h of endotoxemia, DTTX30 was administered as a bolus of 0.12 mg kg-1 followed by 12 h continuous infusion of 0.29 mg kg-1 per h . MEASUREMENTS AND RESULTS: DTTX30 effectively counteracted the endotoxin-associated increase in TXB2 levels and increased 6-keto-PGF1 alpha with a significant shift of the thromboxane/prostacyclin ratio towards predominance of prostacyclin . DTTX30 prevented the significant progressive endotoxin-induced decrease of mean arterial pressure (MAP) below baseline while maintaining cardiac output (CO), and increased the fractional contribution of liver blood flow to CO without an effect on either hepatic O2 delivery or O2 uptake . The mean capillary hemoglobin O2 saturation (HbO2) on the liver surface and HbO2 frequency distributions remained unchanged as well . CONCLUSIONS: DTTX30 significantly attenuated the endotoxin-induced derangements of cellular energy metabolism as reflected by the diminished progressive decrease in hepatic lactate uptake rate and a blunted increase in hepatic venous lactate/pyruvate ratios . While endotoxin significantly increased the endogenous glucose production (EGP) rate, EGP returned towards baseline levels in the DTTX30-treated group . Thus, in our model DTTX30 resulted in hemodynamic stabilization concomitant with improved hepatic metabolic performance.

Exp Biol Med (Maywood), 2001 Jun, 226(6), 597 - 604
Anisodamine inhibits shiga toxin type 2-mediated tumor necrosis factor-alpha production in vitro and in vivo; Zhang HM et al.; Cytokines, in particular tumor necrosis factor (TNF), appear to be necessary to develop the pathological process of Shiga toxin-producing Escherichia coli (STEC) infection . In this study we examined the effect of anisodamine, a vasoactive drug, on TNF-alpha production in Shiga toxin type 2 (Stx2)-stimulated human monocytic cells in vitro and in Stx2-injected mice sera in vivo . Human monocytes and THP-1 cells were stimulated by Stx2 (1-100 ng/ml) with or without anisodamine addition (1-400 micrograms/ml) . For in vivo evaluations, C57BL/6 mice were given a single intraperitoneal injection of anisodamine (6-50 mg/kg) or saline after intraperitoneal injection of Stx2 (50 ng/kg) . The results showed that anisodamine suppressed Stx2-induced TNF-alpha production in a dose- and time-dependent manner . Anisodamine also suppressed Stx2-induced TNF-alpha mRNA expression . Further study showed that endogenous prostaglandin E2 may be involved in this inhibitory effect . In contrast to TNF-alpha mRNA, anisodamine at concentrations as high as 400 micrograms/ml did not decrease Stx2-induced IL-1 beta and IL-8 mRNA levels . In addition, anisodamine (> 50 micrograms/ml) increased Stx2-stimulated THP-1 cell viability . Levels of TNF-alpha in anisodamine-treated mice sera were significantly lower than those in the saline-treated group 1.5 and 24 hr after Stx2 injection . Anisodamine induced a lower percentage of death in Stx2-injected mice . Taken together, our results indicate that anisodamine has an important regulatory effect on Stx2-induced TNF-alpha production in vitro and in vivo . The present study suggested that this drug should be further investigated for its effects on Stx2-mediated diseases in humans.

Mol Biotechnol, 2001 Feb, 17(2), 97 - 108
The rescue by phage display of human Fabs to gp120 HIV-1 glycoprotein using EBV transformed lymphocytes; Kempf E et al.; Human hybridomas secreting monoclonal antibodies in a stable manner are difficult to develop . The main difficulties are the restricted techniques for B-cell immortalization, the low number of sensitized B cells in peripheral blood, and the impossibility, for ethical reasons, to immunize humans with most antigens . Phage display has proved to be a powerful method for the generation of recombinant antibody fragments . This technology relies on the construction of recombinant Fab or scFv libraries and their display on phage M13 . In order to rescue unstable B-cell clones secreting human antibodies we set up a method for the selection by phage display of human IgG fragments from Epstein-Barr virus (EBV)-transformed clones and applied it to the selection by phage display of Fabs directed against HIV-1 gp120, using a seropositive blood sample . The approach combines B-cell transformation by EBV of peripheral blood lymphocytes from a seropositive donor, preselection of specific IgG anti-gp120 producing clones, and the construction of a targeted human antibody library . In this library the percentage of heavy and light chain coding sequences expressed in Escherichia coli, amplified by a set of specific 5' primers for different antibody germ lines, was similar to that observed with the original untransformed B-cell sample . One round of panning was sufficient for the rescue of three Fabs specific for HIV-1 gp120 protein, which proves the efficiency of this technique.

Biochemistry, 2001 Jun 5, 40(22), 6653 - 9
Human endonuclease III acts preferentially on DNA damage opposite guanine residues in DNA; Eide L et al.; The human endonuclease III homologue (hNTH1) removes premutagenic cytosine damage from DNA . This includes 5-hydroxycytosine, which has increased potential for pairing with adenine, resulting in C --> T transition mutations . Here we report that hNTH1 acts on both 5-hydroxycytosine and abasic sites preferentially when these are situated opposite guanines in DNA . Discrimination against other opposite bases is strongly dependent on the presence of magnesium . To further elucidate this effect, we have introduced mutations in the helix-hairpin-helix domain of hNTH1 (K212S, P211R, +G212, and DeltaP211), and measured the kinetics of 5-hydroxycytosine removal of the mutants relative to wild type . The K212S and DeltaP211 (truncated hairpin) mutant proteins were both inactive, whereas the extended hairpin in the +G212 mutant diminished recognition and binding to 5-hydroxycytosine-containing DNA . The P211R mutant resembled native hNTH1, except for decreased specificity of binding . Despite the altered kinetic parameters, the active mutants retained the ability to discriminate against the pairing base, indicating that enzyme interactions with the opposite strand relies on other domains than the active site helix-hairpin-helix motif.

Crit Care Med, 2001 Jun, 29(6), 1181 - 8
Beneficial effect of glycoprotein IIb/IIIa inhibitor (AZ-1) on endothelium in Escherichia coli endotoxin-induced shock; Pu Q et al.; OBJECTIVE: To investigate the effects of AZ-1, a murine monoclonal antiglycoprotein-IIb/IIIa antibody, on endothelium and on hemostasis in a rabbit endotoxic shock model . DESIGN: Prospective laboratory study . SETTING: University laboratory . SUBJECTS: Thirty-five male New-Zealand rabbits . INTERVENTIONS: In vitro vascular reactivity, endothelium CD31-PECAM1 immunohistochemistry, plasma coagulation factors, and monocyte tissue factor determination were performed 1 day and/or 5 days after onset of endotoxic shock (0.5 mg/kg, intravenous bolus,Escherichia coli lipopolysaccharide) with or without treatment by AZ-1 (0.5 mg/kg intravenously) given 1 hr after lipopolysaccharide injection . MEASUREMENTS AND MAIN RESULTS: Metabolic acidosis and coagulation activation confirmed the presence of shock . AZ-1 treatment improved endothelial-dependent relaxation at 1 day (maximal effect = 87.2 +/- 4.0% vs . 60.9 +/- 5.2% in the nontreated group, p <.05) and at 5 days (maximal effect = 84.5 +/- 3.5% vs . 56.6 +/- 8.2% in the nontreated group, p <.05) . Endotoxin-induced endothelial injury was decreased significantly by AZ-1 at 1 day (6.4 +/- 1.9% vs . 10.3 +/- 0.8% in the nontreated group, p <.05) and at 5 days (6.3 +/- 2.0% vs . 20.2 +/- 1.2% in the nontreated group, p <.05) . Monocyte tissue factor expression was significantly reduced at 5 days . CONCLUSIONS: These data indicate that potent inhibition of platelet function via antiglycoprotein-IIb/IIIa receptor blockade can inhibit coagulation activation and protect against endothelial dysfunction and histologic injury in endotoxin-induced shock.

J Biol Chem, 2001 Aug 10, 276(32), 30414 - 22 Epub 2001 Jun 06.
Conformational changes in four regions of the Escherichia coli ArsA ATPase link ATP hydrolysis to ion translocation; Zhou T et al.; Structures of ArsA with ATP, AMP-PNP, or ADP.AlF(3) bound at the A2 nucleotide binding site were determined . Binding of different nucleotides modifies the coordination sphere of Mg(2+) . In particular, the changes elicited by ADP.AlF(3) provide insights into the mechanism of ATP hydrolysis . In-line attack by water onto the gamma-phosphate of ATP would be followed first by formation of a trigonal intermediate and then by breaking of the scissile bond between the beta- and gamma-phosphates . Motions of amino acid side chains at the A2 nucleotide binding site during ATP binding and hydrolysis propagate at a distance, producing conformational changes in four different regions of the protein corresponding to helices H4-H5, helices H9-H10, helices H13-H15, and to the S1-H2-S2 region . These elements are extensions of, respectively, the Switch I and Switch II regions, the A-loop (a small loop near the nucleotide adenine moiety), and the P-loop . Based on the observed conformational changes, it is proposed that ArsA functions as a reciprocating engine that hydrolyzes 2 mol of ATP per each cycle of ion translocation across the membrane.

J Bacteriol, 2001 Jul, 183(13), 4099 - 102
Characterization of Escherichia coli type 1 pilus mutants with altered binding specificities; Harris SL et al.; PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili . These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH . One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.

J Bacteriol, 2001 Jul, 183(13), 4012 - 23
Signal transduction cascade for regulation of RpoS: temperature regulation of DsrA; Repoila F et al.; Many environmental parameters modulate the amount of the RpoS sigma factor in Escherichia coli . Temperature control of RpoS depends on the untranslated RNA DsrA . DsrA activates RpoS translation by pairing with the leader of the mRNA . We find that temperature affects both the rate of transcription initiation of the dsrA gene and the stability of DsrA RNA . Both are increased at low temperature (25 degrees C) compared to 37 or 42 degrees C . The combination of these results is 25-fold-less DsrA at 37 degrees C and 30-fold less at 42 degrees C than at 25 degrees C . Using an adapted lacZ-based reporter system, we show that temperature control of transcription initiation of dsrA requires only the minimal promoter of 36 bp . Overall, transcription responses to temperature lead to a sixfold increase in DsrA synthesis at 25 degrees C over that at 42 degrees C . Furthermore, two activating regions and a site for LeuO negative regulation were identified in the dsrA promoter . The activating regions also activate transcription in vitro . DsrA decays with a half-life of 23 min at 25 degrees C and 4 min at 37 and 42 degrees C . These results demonstrate that the dsrA promoter and the stability of DsrA RNA are the thermometers for RpoS temperature sensing . Multiple inputs to DsrA accumulation allow sensitive modulation of changes in the synthesis of the downstream targets of DsrA such as RpoS.

J Bacteriol, 2001 Jul, 183(13), 3910 - 8
Activation from a distance: roles of Lrp and integration host factor in transcriptional activation of gltBDF; Paul L et al.; The leucine-responsive regulatory protein (Lrp) binds to three sites centered 252, 216, and 152 bp upstream of the transcription start site of the Escherichia coli glutamate synthase operon (gltBDF) and activates transcription . Activators of sigma(70)-dependent promoters usually bind closer to the -35 hexamer of the core promoter sequence . To study the mechanism by which Lrp-dependent activation occurs over this relatively large distance, the gltBDF upstream region was sequentially replaced with corresponding portions from the well-characterized sigma(70)-dependent promoter lacZYAp . The glt-lac promoter hybrids were placed upstream of lacZ, allowing transcriptional activity to be monitored via beta-galactosidase assays . Even replacing all gltBDF sequences downstream of and including the -35 hexamer did not eliminate Lrp-dependent activation of transcription . When a 91-bp region between the -35 hexamer and the proximal Lrp binding site (-48 to -128) was replaced with heterologous DNA of the same length, transcription was reduced nearly 40-fold . Based on the presence of a consensus binding sequence, this region seemed likely to be a binding site for integration host factor (IHF) . Experiments to study the effects of a himD mutant on expression of a gltB::lacZ transcriptional fusion, gel mobility shift analyses, and DNA footprinting assays were used to confirm the direct participation of IHF in gltBDF promoter regulation . Based on these results, we suggest that IHF plays a crucial architectural role, bringing the distant Lrp complex in close proximity to the promoter-bound RNA polymerase.

J Bacteriol, 2001 Jul, 183(13), 3848 - 54
Increased expression of Escherichia coli polynucleotide phosphorylase at low temperatures is linked to a decrease in the efficiency of autocontrol; Mathy N et al.; Polynucleotide phosphorylase (PNPase) synthesis is translationally autocontrolled via an RNase III-dependent mechanism, which results in a tight correlation between protein level and messenger stability . In cells grown at 18 degrees C, the amount of PNPase is twice that found in cells grown at 30 degrees C . To investigate whether this effect was transcriptional or posttranscriptional, the expression of a set of pnp-lacZ transcriptional and translational fusions was analyzed in cells grown at different temperatures . In the absence of PNPase, there was no increase in pnp-lacZ expression, indicating that the increase in pnp expression occurs at a posttranscriptional level . Other experiments clearly show that increased pnp expression at low temperature is only observed under conditions in which the autocontrol mechanism of PNPase is functional . At low temperature, the destabilizing effect of PNPase on its own mRNA is less efficient, leading to a decrease in repression and an increase in the expression level.

J Bacteriol, 2001 Jul, 183(13), 3842 - 7
Identification of a dedicated recycling pathway for anhydro-N-acetylmuramic acid and N-acetylglucosamine derived from Escherichia coli cell wall murein; Park JT; Turnover and recycling of the cell wall murein represent a major metabolic pathway of Escherichia coli . It is known that E . coli efficiently reuses, i.e., recycles, its murein tripeptide, L-alanyl-gamma-D-glutamyl-meso-diaminopimelate, to form new murein . However, the question of whether the cells also recycle the amino sugar moieties of cell wall murein has remained unanswered . It is demonstrated here that E . coli recycles the N-acetylglucosamine present in cell wall murein degradation products for de novo murein and lipopolysaccharide synthesis . Furthermore, E . coli also recycles the anhydro-N-acetylmuramic acid moiety by first converting it into N-acetylglucosamine . Based on the results obtained by studying mutants unable to recycle amino sugars, the pathway for recycling is revealed.

Bioinformatics, 2001 Jun, 17(6), 526 - 32; discussion 533-4
Database verification studies of SWISS-PROT and GenBank; Karp PD et al.; PROBLEM STATEMENT: We have studied the relationships among SWISS-PROT, TrEMBL, and GenBank with two goals . First is to determine whether users can reliably identify those proteins in SWISS-PROT whose functions were determined experimentally, as opposed to proteins whose functions were predicted computationally . If this information was present in reasonable quantities, it would allow researchers to decrease the propagation of incorrect function predictions during sequence annotation, and to assemble training sets for developing the next generation of sequence-analysis algorithms . Second is to assess the consistency between translated GenBank sequences and sequences in SWISS-PROT and TrEMBL . RESULTS: (1) Contrary to claims by the SWISS-PROT authors, we conclude that SWISS-PROT does not identify a significant number of experimentally characterized proteins . (2) SWISS-PROT is more incomplete than we expected in that version 38.0 from July 1999 lacks many proteins from the full genomes of important organisms that were sequenced years earlier . (3) Even if we combine SWISS-PROT and TrEMBL, some sequences from the full genomes are missing from the combined dataset . (4) In many cases, translated GenBank genes do not exactly match the corresponding SWISS-PROT sequences, for reasons that include missing or removed methionines, differing translation start positions, individual amino-acid differences, and inclusion of sequence data from multiple sequencing projects . For example, results show that for Escherichia coli, 80.6% of the proteins in the GenBank entry for the complete genome have identical sequence matches with SWISS-PROT/TrEMBL sequences, 13.4% have exact substring matches, and matches for 4.1% can be found using BLAST search; the remaining 2.0% of E.coli protein sequences (most of which are ORFs) have no clear matches to SWISS-PROT/TrEMBL . Although many of these differences can be explained by the complexity of the DB, and by the curation processes used to create it, the scale of the differences is notable.

Annu Rev Biochem, 2001, 70, 121 - 48
Radical mechanisms of enzymatic catalysis; Frey PA; Two classes of enzymatic mechanisms that proceed by free radical chemistry initiated by the 5'-deoxyadenosyl radical are discussed . In the first class, the mechanism of the interconversion of L-lysine and L-beta-lysine catalyzed by lysine 2,3-aminomutase (LAM) involves four radicals, three of which have been spectroscopically characterized . The reversible formation of the 5'-deoxyadenosyl radical takes place by the chemical cleavage of S-adenosylmethionine (SAM) reacting with the {4Fe-4S}+ center in LAM . In other reactions of SAM with iron-sulfur proteins, SAM is irreversibly consumed to generate the 5'-deoxyadenosyl radical, which activates an enzyme by abstracting a hydrogen atom from an enzymatic glycyl residue to form a glycyl radical . The glycyl radical enzymes include pyruvate formate-lyase, anaerobic ribonucleotide reductase from Escherichia coli, and benzylsuccinate synthase . Biotin synthase and lipoate synthase are SAM-dependent {4Fe-4S} proteins that catalyze the insertion of sulfur into unactivated C-H bonds, which are cleaved by the 5'-deoxyadenosyl radical from SAM . In the second class of enzymatic mechanisms using free radicals, adenosylcobalamin-dependent reactions, the 5'-deoxyadenosyl radical arises from homolytic cleavage of the cobalt-carbon bond, and it initiates radical reactions by abstracting hydrogen atoms from substrates . Three examples are described of suicide inactivation through the formation of exceptionally stable free radicals at enzymatic active sites.

Am J Pathol, 2001 Jun, 158(6), 1929 - 35
Adult-derived stem cells from the liver become myocytes in the heart in vivo; Malouf NN et al.; Recent evidence suggests that adult-derived stem cells, like their embryonic counterparts, are pluripotent . These simple, undifferentiated and uncommitted cells are able to respond to signals from their host tissue microenvironment and differentiate, producing progeny that display a phenotype characteristic of the mature cells of that tissue . We used a clonal stem cell line (termed WB-F344) that was derived from an adult male rat liver to investigate the possibility that uncommitted stem cells from a nonmyogenic tissue source would respond to the tissue microenvironment of the heart in vivo and differentiate into cardiac myocytes . Male WB-F344 cells that carry the Escherichia coli beta-galactosidase gene were identified in the left ventricular myocardium of adult female nude mice 6 weeks after transplantation . We confirmed the presence of a rat Y-chromosome-specific repetitive DNA sequence exclusively in the beta-galactosidase-positive myocytes by polymerase chain reaction and fluorescence in situ hybridization . Immunohistochemistry, using a cardiac troponin T-specific monoclonal antibody, and ultrastructural analysis confirmed a cardiac myocyte phenotype of the stem cell-derived myocytes . The beta-galactosidase-positive myocytes ranged from < 20 microm to 110 microm in length . The longer of these cells contained well-organized sarcomeres and myofibrils, and formed intercalated disks and gap junctions with endogenous (host-derived) myocytes, suggesting that WB-F344-derived myocytes participate in the function of the cardiac syncytium . These results demonstrate that adult liver-derived stem cells respond to the tissue microenvironment of the adult heart in vivo and differentiate into mature cardiac myocytes.

Vaccine, 2001 Jun 14, 19(27), 3759 - 68
Mucosal immunization of mice using CpG DNA and/or mutants of the heat-labile enterotoxin of Escherichia coli as adjuvants; McCluskie MJ et al.; Cholera toxin (CT) and the Escherichia coli heat-labile enterotoxin (LT) are potent mucosal adjuvants in animals associated, at least in part, with their ability to induce cAMP . While toxicity generally precludes their use in humans, a number of different subunit or genetically detoxified mutants of CT and LT have been developed . Another type of adjuvant that has been shown to be effective at mucosal surfaces comprises synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) . We have previously demonstrated a synergy between CpG ODN and native toxins after intranasal (IN) administration to mice, and herein have examined whether this synergy is linked to the cAMP activity . The adjuvanticity of CpG ODN was evaluated with IN and oral delivery of tetanus toxoid or the hepatitis B surface antigen, relative to and in combination with native LT holotoxin (LTh), three active site mutants (LTS61F, LTA69G, LTE112K), a protease site mutant (LTR192G), and the B subunit of LT (LTB) . At an equivalent dose, the adjuvants could generally be divided into two groups: one that included CpG ODN, LTh, LTR192G, and LTA69G which acted as strong adjuvants; and the second which comprised LTB, LTS61F, and LTE112K, which produced significantly weaker immune responses . When CpG ODN was co-administered with bacterial toxin-derivatives, in most cases, no synergy between CpG and the LT derivatives was found for strength of the humoral response . Nevertheless, for both routes and antigens, CpG ODN combined with any LT derivative induced a more Type 1-like response than LT derivative alone . These results suggest that while the synergy seen previously with native toxins may have been due in part to inherent cAMP activity, it may have also depended on the particular antigen used and the route of immunization.

Vaccine, 2001 Jun 14, 19(27), 3726 - 32
A bacterially expressed peptide prevents experimental infection of primates by the hepatitis E virus; Im SW et al.; A 23 kDa peptide of the major structural protein of the hepatitis E virus (HEV) expressed in E . coli was found to naturally interact with one another to form homodimers and the peptide was recognized strongly in its dimeric form by HEV reactive human sera . To determine if the peptide may confer protection against HEV infection, three monkeys were immunized with the purified peptide and three were given placebo . Both groups of animals were challenged with 10(5) genome equivalent dose of the homologous strain of HEV . All control animals excreted the virus for 10-12 days beginning 5 days after the infection . The viral genome was also present in the peripheral blood monocyte (PBMC) samples from two animals, but it was not detected in the plasma samples from any of the animals . The infection in two control animals was accompanied by HEV seroconversion . Immunization was found to abrogate HEV stool excretion in two animals and reduced the viral excretion to one day in the third . None of the immunized animals showed detectable HEV in plasma or PBMC samples nor did the animals showed evidence of HEV seroconversion . These results suggested that immunization with the bacterially expressed peptide may prevent experimental infection of primates with the homologous strain of HEV.

BMC Microbiol . 2001;1(1):7 . Epub 2001 May 24.
Site-specific mutations of FtsZ--effects on GTPase and in vitro assembly; Lu C et al.; BACKGROUND: FtsZ, the major cytoskeletal protein in bacterial cytokinesis, assembles in vitro into protofilaments, which can further associate into sheets, bundles or tubes . We have constructed 16 site-directed mutants of E . coli ftsZ, and tested them for GTP hydrolysis and assembly in vitro, and for their ability to complement the temperature sensitive ftsZ84 mutation in E . coli . RESULTS: The mutants were grouped into three classes . Benign mutants, which mapped mostly to the front and back surface of the protofilament, were able to complement ftsZ84 in vivo and showed normal assembly in vitro . GTP contact mutations had less than 10% of wild type GTPase activity . They could all assemble in vitro, and several of these mutants could complement ftsZ84 . A third, and newly discovered, class of mutations mapped to the sides of the protofilaments . These lateral mutants had mostly normal GTPase and assembly in vitro, but none of them complemented ftsZ84 . The non-complementing mutants showed greatly reduced expression from the pBS58 vector, suggesting possible dominant negative effects . CONCLUSIONS: Several mutants with greatly reduced GTPase could still complement ftsZ84, suggesting that the high level of GTPase observed in vitro is not essential for in vivo function . All of the lateral mutants failed to complement ftsZ84, which suggests that these surfaces of the protofilaments are important for function in cell division . These lateral surfaces may mediate association of FtsZ protofilaments into pairs or small sheets, although their structure is apparently different from the sheets assembled in DEAE dextran or calcium.

Biochem Biophys Res Commun, 2001 Jun 8, 284(2), 536 - 41
Structural and functional study of reconstituted peripheral benzodiazepine receptor; Lacapere JJ et al.; Recombinant mouse 18 kDa peripheral-type benzodiazepine receptor (PBR) protein was overexpressed in Escherichia coli and isolated using a His . Bind metal chelation resin . Recombinant PBR protein was purified with sodium dodecyl sulfate and reincorporated into liposomes using Bio-Beads SM2 as a detergent removing agent . Negative staining of the reconstituted PBR samples, examined by electron microscopy, showed the formation of proteoliposomes . Freeze-fracture of these proteoliposomes revealed the presence of transmembranous particles of an average size of 3.5 +/- 0.25 nm, consistent with the presence of a monomeric form of the recombinant PBR protein . The reconstituted protein exhibited the ability to bind both the PBR drug ligand isoquinoline carboxamide PK 11195 and cholesterol with nanomolar affinities . These data suggest that a PBR monomer is the minimal functional unit, binding drug ligands and cholesterol .

Biochem Biophys Res Commun, 2001 Jun 8, 284(2), 357 - 62
An eukaryotic-type serine/threonine protein kinase involved in the carbon source-dependent pigment biosynthesis in Amycolatopsis mediterranei U32; Yang L et al.; The structural gene, pkmA, was cloned and sequenced from a rifamycin SV-producing Amycolatopsis mediterranei U32 strain . The N-terminal portion of the deduced amino acid sequence of pkmA showed significant similarity to the family of serine/threonine protein kinases . It contains all the structural features which are highly conserved in protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein specific kinases . The protein possesses a region rich in Ala and Pro residues around the middle of pkmA open reading frame, which might be involved in the transmembrane function, as suggested by PhoA fusion protein analysis . The pkmA gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, and the protein was found to have the activity of autophosphorylation . A double crossover gene replacement was achieved by inserting an aparmycin resistance gene into pkmA in A . mediterranei chromosomal DNA . The phenotypic analysis of the mutant suggested that pkmA gene is involved in carbon source-dependent pigment formation in A . mediterranei U32 .

Biochem Biophys Res Commun, 2001 Jun 8, 284(2), 331 - 4
Colonocyte basolateral membranes contain Escherichia coli heat-stable enterotoxin receptors; Albano F et al.; Heat-stable enterotoxin (ST(a)) elaborated by E . coli is a major cause of diarrhea . The transmembrane protein guanylyl cyclase C (GC-C) is the acknowledged receptor for ST(a) and for the mammalian peptides guanylin and uroguanylin . Binding to GC-C results in generation of cGMP, activation of type II cGMP-dependent protein kinase, phosphorylation of CFTR and increased chloride and bicarbonate secretion . We had previously shown that ST(a) receptors (GC-C) are found on the brush border membranes of small intestinal enterocytes and of colonocytes . However, since it has subsequently been shown that the endogenous ligands for these receptors, guanylin and uroguanylin, circulate in blood, we proposed the existence of ST(a) binding sites on the basolateral membranes (BLM) of colonocytes . Specific binding of 125I-ST(a) to rat colonocyte BLM was seen . The kinetics of binding to the BLM were similar to binding to BBM . The nature of the BLM receptor is unknown . This suggests that circulating guanylin and uroguanylin, analogues of ST(a), may also function via the basolateral surface .

Fish Shellfish Immunol, 2001 Apr, 11(3), 245 - 56
Tumor necrosis factor alpha (TNFalpha)-like factor produced by macrophages in rainbow trout, Oncorhynchus mykiss; Qin QW et al.; The presence of a Tumor Necrosis Factor alpha (TNFalpha)-like molecule has been suggested in fish by biological assays and biological and antigenic cross-reactivities with human TNFalpha . In the present study, whether rainbow trout macrophages produce TNFalpha was examined . Murine recombinant TNFalpha (m-rTNFalpha) was used as the standard mammalian TNFalpha . The supernatants were harvested from trout macrophage culture stimulated with lipopolysaccharide (LPS) and then passed through a Polymyxin B column to remove LPS . Results show that trout macrophage culture supernatants exhibit TNF-like activities . The supernatants significantly enhanced neutrophil migration and macrophage respiratory burst activity as assessed by NBT reduction test . The supernatants were also highly cytotoxic to murine L929 cells, which are known to be sensitive to mammalian TNFalpha . The biological activities of TNF in the trout macrophage culture supernatant was determined as 2.6 U ml(-1) in the presence of actinomycin D . This indicates biological cross-reactivity of trout TNFalpha-like factor on mammalian cells . Moreover, these activities were inhibited by a rabbit anti-mTNFalpha antibody . These results suggest that rainbow trout macrophages produce a TNFalpha-like factor that is similar to the mammalian TNFalpha in functions.

Fish Shellfish Immunol, 2001 Apr, 11(3), 203 - 16
Antigenic comparison of a truncated form of VP2 of infectious pancreatic necrosis (IPN) virus expressed in four different cell types; Labus MB et al.; A truncated form of the structural protein VP2 (truncVP2) of infectious pancreatic necrosis (IPN) virus encompassing amino acids 147-307 was expressed in bacterial, yeast, piscine and mammalian cells . All four recombinant antigens were recognised by a VP2-specific monoclonal antibody by ELISA and immunoblot analysis . However, the minimum amount of r-truncVP2 needed for detection by these methods varies depending on the cell type used for expression . Furthermore, all four recombinant preparations, when used to immunise Atlantic salmon, were capable of inducing antibodies reactive with whole IPNV in ELISA.

Acta Virol, 2001 Feb, 45(1), 61 - 3
Preparation of recombinant coat protein of Prunus necrotic ringspot virus; Petrzik K et al.; The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli . CP-enriched fractions were prepared from whole cell lysate by differential centrifugation . The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP . This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

J Radiat Res (Tokyo), 2001 Mar, 42(1), 11 - 9
Role of the Escherichia coli and human DNA glycosylases that remove 5-formyluracil from DNA in the prevention of mutations; Zhang QM; Ionizing radiation induces a wide variety of modifications to purine and pyrimidine residues . The exocyclic methyl group of thymine does not escape oxidative damage . Any 5-hydroperoxymethyluracil produced is spontaneously decomposed to form 5-formyluracil (5-foU) and 5-hydroxymethyluracil . The yield of 5-foU by ionizing radiation is roughly the same as that of 8-oxoguanine . 5-foU is a potential mutagenic damage in vitro and in vivo . Mammalian cells have an activity that removes 5-foU from X-irradiated DNA . Furthermore, the Nth, Nei and MutM proteins of E . coli have DNA glycosylase/AP lyase activities that recognize and remove 5-foU in DNA . The mutation frequency of 5-foU-containing plasmid increases when replicated in E . coli nthneimutMalkA . Single mutations in the nth, nei or mutM gene do not affect the mutation frequency . Therefore, these gene products are likely backup enzymes used to repair 5-foU in DNA . Furthermore, the human hNTH1 enzyme, a homologue of E . coli Nth, is found to have similar DNA glycosylase activity to that of the Nth protein.

Parasitology, 2001 May, 122(Pt 5), 555 - 62
Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression; Mattsson JG et al.; The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies . So far, there is no in vitro propagation system for S . scabiei available, and mites used for various purposes must be isolated from infected hosts . Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity . It has also hampered the development of high performance serological assays . We have now constructed an S . scabiei cDNA expression library with mRNA purified from mites isolated from red foxes . Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein . Sequence similarity searches identified the protein as a paramyosin . Recombinant S . scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S . scabiei . We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule . The miniaturized protein was efficiently expressed in E . coli and was recognized by sera from immunized rabbits . These data demonstrate that the cDNA library can assist in the isolation of important S . scabiei antigens and that recombinant proteins can be useful for the study of scabies.

Evolution Int J Org Evolution, 2001 Apr, 55(4), 653 - 60
Perspective: reverse evolution; Teotonio H et al.; For some time, the reversibility of evolution was primarily discussed in terms of comparative patterns . Only recently has this problem been studied using experimental evolution over shorter evolutionary time frames . This has raised questions of definition, experimental procedure, and the hypotheses being tested . Experimental evolution has provided evidence for multiple population genetic mechanisms in reverse evolution, including pleiotropy and mutation accumulation . It has also pointed to genetic factors that might prevent reverse evolution, such as a lack of genetic variability, epistasis, and differential genotype-by-environment interactions . The main focus of this perspective is on laboratory studies and their relevance to the genetics of reverse evolution . We discuss reverse evolution experiments with Drosophila, bacterial, and viral populations . Field studies of the reverse evolution of melanism in the peppered moth are also reviewed.

Int J Cancer, 2001 Jul 1, 93(1), 123 - 30
Appropriate subcellular localisation of prodrug-activating enzymes has important consequences for suicide gene therapy; Spooner RA et al.; Escherichia coli B nitroreductase (NR) has been expressed stably in MDA-MB-361 human breast adenocarcinoma cells either as the wild-type protein (wtNR), which is distributed evenly between the cytoplasmic and nuclear compartments, or targeted to the mitochondrion (mtNR) . Whereas bacterial NR is active as a dimer, a proportion of wtNR is monomeric . In contrast, mtNR is mostly dimeric, suggesting that it adopts a more stable, native conformation . Despite this, when tested in gene-directed enzyme prodrug therapy cell cytotoxicity studies, cells expressing wtNR or mtNR had similar sensitivity to the prodrug CB1954 and mounted similar bystander killing effects . Furthermore, when short prodrug exposures were given, wtNR was more efficient at killing cells than mtNR . These data demonstrate that the site of enzyme expression and prodrug activation is an important variable that requires consideration in suicide gene therapy approaches .

Proc Natl Acad Sci U S A, 2001 Jun 5, 98(12), 6554 - 9
Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary; Low DW et al.; We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, {SeMet(7)}cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments . A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used . A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the alpha-amino group of one peptide segment to facilitate amide bond-forming ligation . The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis . Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product . The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule . Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered {SeMet(7)}cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by approximately 45 mV . The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.

Proc Natl Acad Sci U S A, 2001 Jun 19, 98(13), 7635 - 40 Epub 2001 Jun 05.
Transcription factor RF2a alters expression of the rice tungro bacilliform virus promoter in transgenic tobacco plants; Petruccelli S et al.; The promoter from rice tungro bacilliform badnavirus (RTBV) is expressed only in phloem tissues in transgenic rice plants . RF2a, a b-Zip protein from rice, is known to bind to the Box II cis element near the TATA box of the promoter . Here, we report that the full-length RTBV promoter and a truncated fragment E of the promoter, comprising nucleotides -164 to +45, result in phloem-specific expression of beta-glucuronidase (GUS) reporter genes in transgenic tobacco plants . When a fusion gene comprising the cauliflower mosaic virus 35S promoter and RF2a cDNA was coexpressed with the GUS reporter genes, GUS activity was increased by 2-20-fold . The increase in GUS activity was positively correlated with the amount of RF2a, and the expression pattern of the RTBV promoter was altered from phloem-specific to constitutive . Constitutive expression of RF2a did not induce morphological changes in the transgenic plants . In contrast, constitutive overexpression of the b-ZIP domain of RF2a had a strong effect on the development of transgenic plants . These studies suggest that expression of the b-Zip domain can interfere with the function of homologues of RF2a that regulate development of tobacco plants.

Microbiology, 2001 Jun, 147(Pt 6), 1651 - 6
A two-hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli; Di Lallo G et al.; The development of a convenient and promising alternative to the various two-hybrid methods that are used to study protein-protein interactions is described . In this system, a lambdoid chimeric operator is recognized by a hybrid repressor formed by two chimeric monomers whose C-terminal domains are composed of heterologous proteins (or protein domains) . Only if these proteins efficiently dimerize in vivo is a functional repressor formed able to bind the chimeric operator and shut off the synthesis of a downstream reporter gene . This new approach was tested with several interacting proteins ranging in size from less than 100 to more than 800 amino acids and, to date, no size or topology limit has been detected.

Microbiology, 2001 Jun, 147(Pt 6), 1581 - 9
Suppression of thermosensitive peptidyl-tRNA hydrolase mutation in Escherichia coli by gene duplication; Menez J et al.; Peptidyl-tRNA hydrolase (Pth) in Escherichia coli is required to recycle tRNA molecules that dissociate from the ribosome as peptidyl-tRNA during protein synthesis . At non-permissive temperatures, strains with a thermosensitive mutation affecting the enzyme accumulate peptidyl-tRNA, cease protein synthesis and die . The rate of reversion of this mutation to thermoresistance varies widely according to the genetic background of the cell and the temperature of selection; under certain conditions, reversion can occur at rates approaching 10(-3) per cell per generation . In such revertants, a chromosomal pth gene can be replaced by an inactivated gene, restoring thermosensitive growth in most cases . PCR amplification experiments and Southern blots show the presence of both normal and inactivated copies of the gene, demonstrating that gene duplication has occurred in the revertants . Estimation of intracellular peptidyl-tRNA hydrolase by Western blotting confirms this explanation of the mechanism of high-frequency reversion to thermoresistance.

Microbiology, 2001 Jun, 147(Pt 6), 1535 - 45
Cloning and functional analysis of a phosphopantetheinyl transferase superfamily gene associated with jadomycin biosynthesis in Streptomyces venezuelae ISP5230; Wang L et al.; Sequence analysis of a XhoI/SacI fragment of chromosomal DNA downstream of jadL in the Streptomyces venezuelae ISP5230 gene cluster for jadomycin biosynthesis detected a partial ORF similar in its deduced amino acid sequence to the hetI product involved in synthesizing a regulator of heterocyst spacing in ANABAENA: By probing a phage library of S . venezuelae DNA with the XhoI/SacI fragment, the authors identified and isolated a hybridizing clone . The nucleotide sequence of its DNA contained three complete ORFs (jadM, N and X) and one incomplete ORF (jadO) . The jadM ORF lay immediately downstream of, and partially overlapped, jadL . It contained 786 nucleotides encoding an amino acid sequence like those of enzymes in the phosphopantetheinyl transferase family . The jadN ORF contained 1794 nucleotides and encoded an amino acid sequence resembling acyl-CoA decarboxylases, thus suggesting a role in polyketide condensation reactions.The jadX ORF was not identified, but the partial jadO showed marked similarities in its deduced amino acid sequence to NDP-hexose-2,3-dehydratases, indicating a role in forming the sugar component of jadomycin B . Expression of jadM in Escherichia coli and examination of the product by SDS-PAGE established that the ORF encoded a 29.1 kDa protein, corresponding in size to the 262 amino acid polypeptide deduced from the jadM sequence . Evidence from a Northern hybridization indicated that jadM expression is correlated with jadomycin B synthesis . Cultures of S . venezuelae ISP5230 disrupted in jadM produced only 2-5% of the wild-type titre of jadomycin B, but grew well and produced chloramphenicol normally . The authors conclude that jadM encodes a holo-ACP synthase needed primarily for jadomycin B biosynthesis.

Microbiology, 2001 Jun, 147(Pt 6), 1483 - 98
Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli; Abdel-Hamid AM et al.; The metabolic importance of pyruvate oxidase (PoxB), which converts pyruvate directly to acetate and CO(2), was assessed using an isogenic set of genetically engineered strains of Escherichia coli . In a strain lacking the pyruvate dehydrogenase complex (PDHC), PoxB supported acetate-independent aerobic growth when the poxB gene was expressed constitutively or from the IPTG-inducible tac promoter . Using aerobic glucose-limited chemostat cultures of PDH-null strains, it was found that steady-states could be maintained at a low dilution rate (0.05 h(-1)) when PoxB is expressed from its natural promoter, but not at higher dilution rates (up to at least 0.25 h(-1)) unless expressed constitutively or from the tac promoter . The poor complementation of PDH-deficient strains by poxB plasmids was attributed to several factors including the stationary-phase-dependent regulation of the natural poxB promoter and deleterious effects of the multicopy plasmids . As a consequence of replacing the PDH complex by PoxB, the growth rate (mu(max)), growth yield (Y(max)) and the carbon conversion efficiency (flux to biomass) were lowered by 33%, 9-25% and 29-39% (respectively), indicating that more carbon has to be oxidized to CO(2) for energy generation . Extra energy is needed to convert PoxB-derived acetate to acetyl-CoA for further metabolism and enzyme analysis indicated that acetyl-CoA synthetase is induced for this purpose . In similar experiments with a PoxB-null strain it was shown that PoxB normally makes a significant contribution to the aerobic growth efficiency of E . coli . In glucose minimal medium, the respective growth rates (mu(max)), growth yields (Y(max)) and carbon conversion efficiencies were 16%, 14% and 24% lower than the parental values, and correspondingly more carbon was fluxed to CO(2) for energy generation . It was concluded that PoxB is used preferentially at low growth rates and that E . coli benefits from being able to convert pyruvate to acetyl-CoA by a seemingly wasteful route via acetate.

Mol Cell Biol, 2001 Jul, 21(13), 4149 - 61
Establishment of an oriP replicon is dependent upon an infrequent, epigenetic event; Leight ER et al.; Plasmids containing oriP, the latent origin of replication for Epstein-Barr virus, support efficient replication in selected cell clones when the viral protein EBNA-1 is provided, being lost at a rate of 2 to 4% per cell generation after removal of selection (A . L . Kirchmaier and B . Sugden, J . Virol . 69:1280-1283, 1995; B . Sugden and N . Warren, Mol . Biol . Med . 5:85-94, 1988) . We refer to these plasmids as established replicons in that they support efficient DNA synthesis and partitioning each cell cycle . Unexpectedly, we have found that upon introduction of oriP plasmids into a population of EBNA-1-positive cells, oriP plasmids replicate but are lost precipitously from cells during 2 weeks posttransfection (>25% rate of loss per cell generation) . Upon investigation of these disparate observations, we have found that only 1 to 10% of cells transfected with an oriP plasmid expressing EBNA-1 and hygromycin phosphotransferase give rise to drug-resistant clones in which the oriP replicon is established . A hereditable alteration in these drug-resistant cell clones, manifested at the genetic or epigenetic level, does not underlie the establishment of oriP, as newly introduced oriP plasmids replicate but are also lost rapidly from these cells . In addition, a genetic alteration in the oriP plasmid is not responsible for establishment, as oriP plasmids isolated from an established cell clone, propagated in Escherichia coli, and reintroduced into EBNA-1-positive cells are likewise established inefficiently . Our findings demonstrate that oriP replicons are not intrinsically stable in EBNA-1-positive cell lines . Rather, the establishment of an oriP replicon is conferred upon the replicon by a stochastic, epigenetic event that occurs infrequently and, therefore, is detected in only a minority of cells.

J Virol, 2001 Jul, 75(13), 6212 - 7
Expression of Moloney murine leukemia virus RNase H rescues the growth defect of an Escherichia coli mutant; Campbell AG; A 157-amino-acid fragment of Moloney murine leukemia virus reverse transcriptase encoding RNase H is shown to rescue the growth-defective phenotype of an Escherichia coli mutant . In vitro assays of the recombinant wild-type protein purified from the conditionally defective mutant confirm that it is catalytically active . Mutagenesis of one of the presumptive RNase H-catalytic residues results in production of a protein variant incapable of rescue and which lacks activity in vitro . Analyses of additional active site mutants demonstrate that their encoded variant proteins lack robust activity yet are able to rescue the bacterial mutant . These results suggest that genetic complementation may be useful for in vivo screening of mutant viral RNase H gene fragments and in evaluating their function under conditions that more closely mimic physiological conditions . The rescue system may also be useful in verifying the functional outcomes of mutations based on protein structural predictions and modeling.

J Virol, 2001 Jul, 75(13), 5778 - 95
The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles; Heljasvaara R et al.; The vaccinia virus (VV) A10L gene codes for a major core protein, P4a . This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly . To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system . Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields . This defect can be partially overcome by addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) . Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus . Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product . In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy . Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells . These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA . Some images suggest that these DNA structures enter into spherical immature viral particles . In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles . Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.

J Biol Chem, 2001 Aug 10, 276(32), 29864 - 70 Epub 2001 Jun 04.
Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer; Janiyani K et al.; Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis . The Escherichia coli biotin carboxylase is readily isolated from the other components of the acetyl-CoA carboxylase complex such that enzymatic activity is retained . The three-dimensional structure of biotin carboxylase, determined by x-ray crystallography, demonstrated that the enzyme is a homodimer consisting of two active sites in which each subunit contains a complete active site . To understand how each subunit contributes to the overall function of biotin carboxylase, we made hybrid molecules in which one subunit had a wild-type active site, and the other subunit contained an active site mutation known to significantly affect the activity of the enzyme . One of the two genes encoded a poly-histidine tag at its N terminus, whereas the other gene had an N-terminal FLAG epitope tag . The two genes were assembled into a mini-operon that was induced to give high level expression of both enzymes . "Hybrid" dimers composed of one subunit with a wild-type active site and a second subunit having a mutant active site were obtained by sequential chromatographic steps on columns of immobilized nickel chelate and anti-FLAG affinity matrices . In vitro kinetic studies of biotin carboxylase dimers in which both subunits were wild type revealed that the presence of the N-terminal tags did not alter the activity of the enzyme . However, kinetic assays of hybrid dimer biotin carboxylase molecules in which one subunit had an active site mutation (R292A, N290A, K238Q, or E288K) and the other subunit had a wild-type active site resulted in 39-, 28-, 94-, and 285-fold decreases in the activity of these enzymes, respectively . The dominant negative effects of these mutant subunits were also detected in vivo by monitoring the rate of fatty acid biosynthesis by {(14)C}acetate labeling of cellular lipids . Expression of the mutant biotin carboxylase genes from an inducible arabinose promoter resulted in a significantly reduced rate of fatty acid synthesis relative to the same strain that expressed the wild type gene . Thus, both the in vitro and in vivo data indicate that both subunits of biotin carboxylase are required for activity and that the two subunits must be in communication during enzyme function.

J Biol Chem, 2001 Aug 24, 276(34), 31651 - 6 Epub 2001 Jun 04.
Escherichia coli poly(A)-binding proteins that interact with components of degradosomes or impede RNA decay mediated by polynucleotide phosphorylase and RNase E; Feng Y et al.; The multifunctional ribonuclease RNase E and the 3'-exonuclease polynucleotide phosphorylase (PNPase) are major components of an Escherichia coli ribonucleolytic "machine" that has been termed the RNA degradosome . Previous work has shown that poly(A) additions to the 3' ends of RNA substrates affect RNA degradation by both of these enzymes . To better understand the mechanism(s) by which poly(A) tails can modulate ribonuclease action, we used selective binding in 1 m salt to identify E . coli proteins that interact at high affinity with poly(A) tracts . We report here that CspE, a member of a family of RNA-binding "cold shock" proteins, and S1, an essential component of the 30 S ribosomal subunit, are poly(A)-binding proteins that interact functionally and physically, respectively, with degradosome ribonucleases . We show that purified CspE impedes poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase by interfering with its digestion through the poly(A) tail and also inhibits both internal cleavage and poly(A) tail removal by RNase E . The ribosomal protein S1, which is known to interact with sequences at the 5' ends of mRNA molecules during the initiation of translation, can bind to both RNase E and PNPase, but in contrast to CspE, did not affect the ribonucleolytic actions of these enzymes . Our findings raise the prospect that E . coli proteins that bind to poly(A) tails may link the functions of degradosomes and ribosomes.

J Biol Chem, 2001 Aug 10, 276(32), 29826 - 32 Epub 2001 Jun 04.
Cyclophilin a binds to peroxiredoxins and activates its peroxidase activity; Lee SP et al.; Six distinct peroxiredoxin (Prx) proteins (Prx I-VI) from distinct genes have been identified in mammalian tissues . Prxs are members of a group of peroxidases that have conserved reactive cysteine residue(s) in the active site(s) . An immediate physiological electron donor for the peroxidase catalysis for five Prx proteins (Prx I-V) has been identified as thioredoxin (Trx), but that for Prx VI (1-Cys Prx) is still unclear . To identify an immediate electron donor and a binding protein for Prx VI, we performed a Prx VI protein overlay assay . A 20-kDa binding protein was identified by the Prx VI protein overlay assay with flow-through fractions from a High-Q column with rat lung crude extracts . Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MS-Fit, we identified the 20-kDa Prx VI-binding protein as a cyclophilin A (CyP-A) . The binding of recombinant human CyP-A (hCyP-A) to Prx VI was confirmed by using the hCyP-A protein overlay assay and Western immunoblot analysis with hCyP-A-specific antibodies . hCyP-A enhanced the antioxidant activity of Prx VI, as well as the other known mammalian Prx isotypes . hCyP-A supported antioxidant activity of Prx II and Prx VI both against thiol (dithiothreitol)-containing metal-catalyzed oxidation (MCO) systems and ascorbate-containing MCO systems . Prx II was reduced by hCyP-A without help from any other reductant, and the reduction was cyclosporin A-independent . These results strongly suggest that CyP-A not only binds to Prx proteins but also supports its peroxidase activity as an immediate electron donor . In addition, Cys(115) and Cys(161) of hCyP-A were found to be involved in the activation and the reduction of Prx.

Trends Microbiol, 2001 Jun, 9(6), 262 - 6
Is immune cell activation the missing link in the pathogenesis of post-diarrhoeal HUS?
Heyderman RS, Soriani M, Hirst TR.
Haemolytic uraemic syndrome (HUS), which is caused by Shiga toxin (Stx)-producing Escherichia coli, is the commonest cause of acute renal failure in childhood . It is widely believed that HUS develops following the release of Stx, an AB5 toxin that inhibits protein synthesis and has a direct toxic effect on the kidney endothelium . There remains, however, a mismatch between the current understanding of the pathogenesis of HUS and the evolution of the clinical signs of the disease . Our hypothesis is that Stx-mediated immune cell activation in the gut is the missing link in the pathogenesis of this condition, initiating the characteristic renal pathology of HUS either alone or in synergy with Stx . Validation of this hypothesis could lead to a targeted anti-inflammatory approach aimed at modulating immune cell function in HUS.

Vet Microbiol, 2001 Aug 8, 81(3), 235 - 42
Analysis of the functional domains of Arcanobacterium pyogenes pyolysin using monoclonal antibodies; Imaizumi K et al.; Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY) family of bacterial toxins . Four monoclonal antibodies (mAbs) to PLO were prepared for the analysis of functional domains of this toxin . Two (mAbs S and H) of these markedly inhibited the hemolytic activity of PLO, but the inhibiting activity of the other two antibodies (mAbs C and G) was weaker . Subsequently, nine truncated PLOs were derived from recombinant Escherichia coli by various deletions from the N-terminus . Strong hemolytic activity was recognized in truncates of PLO following the deletion of 30 or 55 amino acids, but not in the truncate with deletion of 74 residues . Truncated PLOs were used in immunoblotting experiments to locate the epitopes for the mAbs . The epitope for mAbs C and G lies within the undecapeptide region (amino acids 487-505) of the C-terminus of PLO, which seems to be the binding site to erythrocytes . In contrast, the epitopes for mAbs S and H, which showed strong neutralizing activity, were found to lie in the N-terminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively . From these results, it seems that the N-terminal region of PLO, in particular, the region of amino acids 55-74 is important for hemolytic activity.

J Control Release, 2001 May 14, 72(1-3), 101 - 13
Covalently conjugated VEGF--fibrin matrices for endothelialization; Zisch AH et al.; Vascular endothelial growth factor (VEGF) is a key factor in endothelial cell biology and blood vessel formation and a candidate therapeutic for the stimulation of angiogenesis-dependent tissue regeneration . The objective of this study was to confer the angiogenic activity of VEGF(121) upon the biomaterial fibrin, a natural substrate for endothelial cell growth and clinically accepted as 'fibrin glue' . To achieve this, we engineered fibrin-based hydrogels that were covalently modified with VEGF(121) . Our laboratory has recently developed novel methodology that allows the covalent incorporation of exogenous bioactive peptides by the transglutaminase activity of factor XIIIa into fibrin during coagulation . Here, this ability of factor XIIIa to crosslink additional proteins within fibrin was employed to covalently incorporate VEGF(121) . By recombinant DNA methodology, a mutant VEGF(121) variant, alpha(2)-PI(1--8)-VEGF(121), which contains an additional factor XIIIa substrate sequence NQEQVSPL at the aminoterminus, was expressed in E . coli . In soluble form, the mutant protein fully retained its mitogenic activity for endothelial cells . Using (125)I-labeled alpha(2)-PI(1--8)-VEGF(121), its covalent incorporation and the efficiency of incorporation into fibrin was demonstrated and characterized . The immobilized, fibrin-conjugated VEGF(121) protein remained an active and very efficient mitogen for human endothelial cells grown on two-dimensional VEGF(121)-modified fibrin surfaces, and the incorporation of increasing amounts of alpha(2)-PI(1--8)-VEGF(121) resulted in dose-dependent enhancement of endothelial cell growth . The VEGF-modified fibrin matrices can be formed as injectable gels in a single-step reaction under physiological conditions in vivo . When used as a ingrowth matrix, such VEGF incorporating materials could be useful in a variety of clinical situations that require an angiogenic response into an ischemic region or inplant.

Vet Immunol Immunopathol, 2001 May 30, 79(3-4), 151 - 65
Regulation of bovine E-selectin expression by recombinant tumor necrosis factor alpha and lipopolysaccharide; Van Kampen C et al.; Induction of adhesion molecules by cytokines and LPS is an important mechanism of regulating leukocyte migration into tissue . Expression and regulation of E-selectin may be differentially influenced by the stimuli involved with effects on mRNA or surface protein kinetics . Surface protein and mRNA expression kinetics of bovine E-selectin were measured and compared in primary cultures of bovine aortic endothelial cells (BAEC) stimulated for various periods of time with recombinant bovine tumor necrosis factor alpha (rbTNF-alpha) or Escherichia coli lipopolysaccharide (LPS) . E-selectin mRNA expression was measured via quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) using a construct that contained multiple synthetic oligonucleotides for several bovine adhesion molecules and cytokines . Surface expression of E-selectin was measured by flow cytometry . Unstimulated BAECs expressed minimum or no E-selectin on the surface . A low number of endothelial cells expressed surface E-selectin as early as 1h post-stimulation and surface expression was sustained after both stimuli for 24-72h . Mean fluorescence intensity (MFI) indicated peak surface concentration of E-selectin at 6 h post-stimulation after LPS followed by a gradual decrease to 72h without returning to baseline values . Mean fluorescence intensity following stimulation with TNF-alpha increased slightly between 0 and 72h . The pattern of mRNA expression differed between stimuli . LPS-stimulated BAECs expressed peak amounts of E-selectin mRNA at 6 h, followed by a decline to baseline by 24 h . Conversely, BAECs stimulated with rbTNF-alpha expressed significantly (p pound 0.05) higher amounts of mRNA at 1h than compared to unstimulated controls (0 h), but this decreased to below baseline levels by 6h; followed by a gradual increase and eventually a sharp increase between 18 and 72 h . To account for the lack of correlation between mRNA and protein expression, it was hypothesized that shedding of surface E-selectin accounted at least in part, for the large increase in mRNA expression seen at 18-72h . Culture supernatants from rbTNF-alpha-treated BAECs were harvested, and tested for the presence of shed E-selectin using ELISA . Unstimulated culture supernatants contained little or no E-selectin . Between 6 and 48 h, the concentration of E-selectin in culture supernatants from rbTNF-alpha-stimulated BAECs increased approximately two-fold, suggesting that the sharp increase in E-selectin mRNA expression around 18 h may be related to significant loss of surface E-selectin during this period.

FEBS Lett, 2001 Jun 1, 498(1), 87 - 92
A discrete amino terminal domain of Kv1.5 and Kv1.4 potassium channels interacts with the spectrin repeats of alpha-actinin-2; Cukovic D et al.; The interaction between the amino terminus of Kv1-type potassium channels and alpha-actinin-2 has been investigated . Using a combination of yeast two-hybrid analysis and in vitro binding assays, alpha-actinin-2 was found to bind to the N-termini of both Kv1.4 and Kv1.5 but not to the equivalent segments of Kv1.1, Kv1.2 or Kv1.3 . Deletion analysis in the in vitro binding assays delineated the actinin binding region of Kv1.5 to between amino acids 73 and 148 of the channel . The Kv1.5 binding sites in alpha-actinin-2 were found to lie within actinin's internal spectrin repeats . Unlike the reported interaction between actinin and the NMDA receptor, calmodulin was found to have no effect on actinin binding to Kv1.5.

FEBS Lett, 2001 Jun 1, 498(1), 42 - 5
Dipeptide synthesis by an isolated adenylate-forming domain of non-ribosomal peptide synthetases (NRPS); Dieckmann R et al.; A deletion mutant of tyrocidine synthetase 1 (DeltaDeltaTY1), comprising the adenylation domain of TY1 as an independent functional adenylate-forming unit, was used to investigate the ability of the adenylation domain in non-ribosomal peptide synthetases to catalyse peptide bond formation from the aminoacyl adenylate intermediate . The results demonstrate that only one substrate amino acid needs to be activated as an aminoacyl adenylate . In view of the potential exploitation of peptide synthetases for enzymatic synthesis of dipeptides of choice, it is important to note that this does not necessarily require a dimodular construct or an intermediate acyl transfer step.

FEBS Lett, 2001 Jun 1, 498(1), 16 - 21
Do mammalian cells synthesize lipoic acid? Identification of a mouse cDNA encoding a lipoic acid synthase located in mitochondria; Morikawa T et al.; Lipoic acid is a coenzyme essential to the activity of enzymes such as pyruvate dehydrogenase, which play important roles in central metabolism . However, neither the enzymes responsible for biosynthesis nor the biosynthetic event of lipoic acid has been reported in mammalian cells . In this study, a mouse mLIP1 cDNA for lipoic acid synthase has been identified . We have shown that the cDNA encodes a lipoic acid synthase by its ability to complement a mutant of Escherichia coli defective in lipoic acid synthase and that mLIP1 is targeted into the mitochondria . These findings suggest that mammalian cells are able to synthesize lipoic acid in mitochondria.

Mol Cell, 2001 May, 7(5), 1037 - 45
Localization of the ribosomal protection protein Tet(O) on the ribosome and the mechanism of tetracycline resistance; Spahn CM et al.; Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance . It is a G protein that shows significant sequence similarity to elongation factor EF-G . Here we present a cryo-electron microscopic reconstruction, at 16 A resolution, of its complex with the E . coli 70S ribosome . Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site . Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G . However, there are important differences . One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site . Our findings give insights into the mechanism of tetracycline resistance.

Eur J Biochem, 2001 Jun, 268(11), 3354 - 9
Benzalacetone synthase . A novel polyketide synthase that plays a crucial role in the biosynthesis of phenylbutanones in Rheum palmatum; Abe I et al.; Benzalacetone synthase (BSA) is a novel plant-specific polyketide synthase that catalyzes a one step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 skeleton of phenylbutanoids in higher plants . A cDNA encoding BAS was for the first time cloned and sequenced from rhubarb (Rheum palmatum), a medicinal plant rich in phenylbutanoids including pharmaceutically important phenylbutanone glucoside, lindleyin . The cDNA encoded a 42-kDa protein that shares 60-75% amino-acid sequence identity with other members of the CHS-superfamily enzymes . Interestingly, R . palmatum BAS lacks the active-site Phe215 residue (numbering in CHS) which has been proposed to help orient substrates and intermediates during the sequential condensation of 4-coumaroyl-CoA with malonyl-CoA in CHS . On the other hand, the catalytic cysteine-histidine dyad (Cys164-His303) in CHS is well conserved in BAS . A recombinant enzyme expressed in Escherichia coli efficiently afforded benzalacetone as a single product from 4-coumaroyl-CoA and malonyl-CoA . Further, in contrast with CHS that showed broad substrate specificity toward aliphatic CoA esters, BAS did not accept hexanoyl-CoA, isobutyryl-CoA, isovaleryl-CoA, and acetyl-CoA as a substrate . Finally, besides the phenylbutanones in rhubarb, BAS has been proposed to play a crucial role for the construction of the C6-C4 moiety of a variety of natural products such as medicinally important gingerols in ginger plant.

Eur J Biochem, 2001 Jun, 268(11), 3296 - 303
The pro-sequence facilitates folding of human nerve growth factor from Escherichia coli inclusion bodies; Rattenholl A et al.; Nerve growth factor (beta-NGF), a neurotrophin required for the development and survival of specific neuronal populations, is translated as a prepro-protein in vivo . While the presequence mediates translocation into the endoplasmic reticulum, the function of the pro-peptide is so far unknown . As the pro-sequences of several proteins are known to promote folding of the mature part, the renaturation behaviour of recombinant human beta-NGF pro-protein was compared to that of the mature form . Expression of rh-pro-NGF in Escherichia coli led to the formation of inclusion bodies (IBs) . The presence of the covalently attached pro-sequence significantly increased the yield and rate of refolding with concomitant disulfide bond formation when compared to the in vitro refolding of mature NGF (rh-NGF) . Physicochemical characterization revealed that rh-pro-NGF is a dimer . The pro-peptide could be removed by limited proteolysis with trypsin yielding biologically active, mature rh-NGF . Furthermore, rh-pro-NGF exhibited biological activity in the same concentration range as rh-NGF.

Eur J Biochem, 2001 Jun, 268(11), 3223 - 32
Molecular cloning, characterization and regulation by cadmium of a superoxide dismutase from the ectomycorrhizal fungus Paxillus involutus; Jacob C et al.; The gene encoding a superoxide dismutase (PiSOD) was cloned by suppressive subtractive hybridization from cDNA library of the ectomycorrhizal fungus, Paxillus involutus, grown under cadmium-stress conditions . The encoded protein was presumed to be localized in the peroxisomes because it contained a C-terminal peroxisomal localization peptide (SKL) and lacked an N-terminal mitochondrial transit peptide . Complementation of an Escherichia coli SOD null strain that is unable to grow in the presence of paraquat or cadmium indicated that cloned Pisod encoded a functional superoxide dismutase . Sensitivity of PiSOD activity to H2O2 but not KCN, and sequence homologies to other SODs strongly suggest that it is a manganese-containing superoxide dismutase . Monitoring PiSOD transcript, immunoreactive polypeptide and superoxide dismutase activity following cadmium stress suggests that the principal level of control is post-translational . This is, to our knowledge, the first insight in the characterization of molecular events that take place in an ectomycorrhizal fungus during exposure to heavy metals.

Eur J Biochem, 2001 Jun, 268(11), 3205 - 13
Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase . An essential residue in catalysis; Cho SW et al.; It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH) . There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function . We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5'-phosphate (PLP) . For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors . The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues . Despite an approximately 400-fold decrease in the respective apparent Vmax of the Lys130 mutant enzymes, apparent Km values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate . Unlike the wild-type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding . The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH.

Eur J Biochem, 2001 Jun, 268(11), 3190 - 7
Biosynthesis of terpenoids . 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) from Plasmodium falciparum; Rohdich F et al.; The putative catalytic domain of an open reading frame from Plasmodium falciparum with similarity to the ispF gene of Escherichia coli specifying 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase was expressed in a recombinant E . coli strain . The recombinant protein was purified to homogeneity and was found to catalyze the formation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate at a rate of 4.3 micromol x mg(-1) x min(-1) . At lower rates, the recombinant protein catalyzes the formation of 2-phospho-2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate and the formation of 2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol . Divalent metal ions such as magnesium or manganese are required for catalytic activity . The enzyme has a pH optimum at pH 7.0 . Recombinant expression of the full-length open reading frame afforded insoluble protein that could not be folded in vitro . The enzyme is a potential target for antimalarial drugs directed at the nonmevalonate pathway of isoprenoid biosynthesis.

Eur J Biochem, 2001 Jun, 268(11), 3154 - 62
HMG-1 enhances HMG-I/Y binding to an A/T-rich enhancer element from the pea plastocyanin gene; Webster CI et al.; High-mobility-group proteins HMG-1 and HMG-I/Y bind at overlapping sites within the A/T-rich enhancer element of the pea plastocyanin gene . Competition binding experiments revealed that HMG-1 enhanced the binding of HMG-I/Y to a 31-bp region (P31) of the enhancer . Circularization assays showed that HMG-1, but not HMG-I/Y, was able to bend a linear 100-bp DNA containing P31 so that the ends could be ligated . HMG-1, but not HMG-I/Y, showed preferential binding to the circular 100-bp DNA compared with the equivalent linear DNA, indicating that alteration of the conformation of the DNA by HMG-1 was not responsible for enhanced binding of HMG-I/Y . Direct interaction of HMG-I/Y and HMG-1 in the absence of DNA was demonstrated by binding of 35S-labeled proteins to immobilized histidine-tagged proteins, and this was due to an interaction of the N-terminal HMG-box-containing region of HMG-1 and the C-terminal AT-hook region of HMG-I/Y . Kinetic analysis using the IAsys biosensor revealed that HMG-1 had an affinity for immobilized HMG-I/Y (Kd = 28 nM) similar to that for immobilized P31 DNA . HMG-1-enhanced binding of HMG-I/Y to the enhancer element appears to be mediated by the formation of an HMG-1-HMG-I/Y complex, which binds to DNA with the rapid loss of HMG-1.

Biochem J, 2001 Jun 15, 356(Pt 3), 891 - 7
Identification of a new subfamily of sulphotransferases: cloning and characterization of canine SULT1D1; Tsoi C et al.; Sulphation is an important conjugation pathway in drug metabolism that has been studied in several species including humans . However, few studies have been performed using the dog as a subject . In this report we describe the cloning and characterization of a canine cytosolic sulphotransferase (SULT) . The overall primary structure of this enzyme is very similar to that of a rat phenol-sulphating enzyme found in the EMBL Database and to a mouse SULT termed amine-N-sulphotransferase (81% identity) . The expressed canine SULT conjugates small phenols and aromatic amines such as dopamine, minoxidil, p-nitrophenol and 5-hydroxytryptamine, but not dehydroepiandrosterone or beta-oestradiol . These results are in agreement with the results reported for the mouse SULT . In contrast with the mouse enzyme, the canine SULT does not conjugate eicosanoid compounds, i.e . prostaglandins, thromboxane B(2) or leukotriene E(4) . The canine SULT is expressed at high levels in the colon of both genders; it is also expressed in the small intestine, kidney and liver . Furthermore, because the canine, mouse and rat SULT forms exhibit significant sequence identity (more than 80%), they seem to represent a distinct group in the SULT family tree . This suggestion is strengthened by the low identity with other SULTs . The subfamily that is most similar to this new group is SULT1A, with approx . 60% similarity . However, the mouse and canine enzymes are not characterized by the efficient sulphation of p-nitrophenol, dopamine, beta-oestradiol or oestrone . Thus these results seem to exclude them from the SULT1A subfamily . We therefore propose a new subfamily in the phenol SULT family, designated SULT1D, and consequently the canine enzyme is termed SULT1D1.

Biochem J, 2001 Jun 15, 356(Pt 3), 867 - 73
Conjugation of an antibody Fv fragment to a virus coat protein: cell-specific targeting of recombinant polyoma-virus-like particles; Stubenrauch K et al.; The development of cell-type-specific delivery systems is highly desirable for gene-therapeutic applications . Current virus-based vector systems show broad cell specificity, which results in the need to restrict the natural tropism of these viral systems . Here we demonstrate that tumour-cell-specific virus-like particles can be functionally assembled in vitro from recombinant viral coat protein expressed in Escherichia coli . The insertion of a negatively charged peptide in the HI loop of polyoma VP1 interferes with the binding of VP1 to the natural recognition site on mammalian cells and also serves as an adapter for the coupling of antibody fragments that contain complementary charged fusion peptides . A recombinant antibody fragment of the tumour-specific anti-(Lewis Y) antibody B3 could be coupled to the mutant VP1 by engineered polyionic peptides and an additional disulphide bond . With this system an entirely recombinant cell-specific delivery system assembled in vitro could be generated that transfers genes preferentially to cells presenting the tumour-specific antigen on the cell surface.

Biotechnol Appl Biochem, 2001 Jun, 33(Pt 3), 209 - 14
Running-buffer composition influences DNA-protein and protein-protein complexes detected by electrophoretic mobility-shift assay (EMSA); Roder K et al.; The gel-shift assay is a rapid, extremely sensitive, technically simple and widely used method for investigating nucleic acid-protein interaction based on the observation that binding of protein to DNA or RNA fragments usually leads to a reduction in the electrophoretic mobility of the fragment in non-denaturing gels . Here we report on the critical role of the running buffer and show that its importance ranks equally with other factors affecting complex formation and stability such as binding buffer, temperature, non-specific competitor or gel concentration and/or composition . We demonstrate differences in the binding patterns obtained with oligonucleotides containing binding sites for the ubiquitously expressed transcription factors Sp1 (stimulatory protein 1), NF-Y (nuclear factor Y) and USF (upstream stimulatory protein), which are dependent on the ionic strength of the running buffer used . Furthermore, we show the influence of glycine concentration on Sp1 binding using recombinant glutathione S-transferase-Sp1 fusion protein expressed in Escherichia coli.

Biotechnol Appl Biochem, 2001 Jun, 33(Pt 3), 173 - 82
Purification and conformational properties of a human interferon alpha2b produced in Escherichia coli; Beldarrain A et al.; Recombinant human interferon alpha2b was expressed intracellularly in Escherichia coli as insoluble aggregates using a new expression vector, and was purified to homogeneity using essentially two-step chromatographic procedures, i.e . immobilized metal-ion-affinity chromatography and reversed-phase HPLC . The established purification process is highly reproducible and leads to a total recovery of approx . 12% with a specific biological activity of higher than 1x10(8) i.u./mg of protein, which is comparable with the international requirement for interferon alpha2b . For purified protein we report conformational stability as a function of pH and temperature using differential scanning calorimetry and CD . Thermal unfolding as a function of pH showed only one endotherm at a temperature higher than 45 degrees C, and was reversible at pH 2-3.75 and irreversible at pH 4-10 . At pH 7.0, the most stable condition, the conformational stability depends on protein concentration and ionic strength . The highly helical secondary structure is very conserved over the whole pH range studied, including at high temperatures.

Biochemistry, 2001 Jun 12, 40(23), 6836 - 44
Identification of the regulatory subunit of Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit; Lee YT et al.; Acetohydroxyacid synthase (EC 4.1.3.18; AHAS) catalyzes the initial step in the formation of the branched-chain amino acids . The enzyme from most bacteria is composed of a catalytic subunit, and a smaller regulatory subunit that is required for full activity and for sensitivity to feedback regulation by valine . A similar arrangement was demonstrated recently for yeast AHAS, and a putative regulatory subunit of tobacco AHAS has also been reported . In this latter case, the enzyme reconstituted from its purified subunits remained insensitive to feedback inhibition, unlike the enzyme extracted from native plant sources . Here we have cloned, expressed in Escherichia coli, and purified the AHAS regulatory subunit of Arabidopsis thaliana . Combining the protein with the purified A . thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine, leucine, and isoleucine . Moreover, there is a strong synergy between the effects of leucine and valine, which closely mimics the properties of the native enzyme . The regulatory subunit contains a sequence repeat of approximately 180 residues, and we suggest that one repeat binds leucine while the second binds valine or isoleucine . This proposal is supported by reconstitution studies of the individual repeats, which were also cloned, expressed, and purified . The structure and properties of the regulatory subunit are reminiscent of the regulatory domain of threonine deaminase (EC 4.2.1.16), and it is suggested that the two proteins are evolutionarily related.

Biochemistry, 2001 Jun 12, 40(23), 6805 - 18
Mechanistic implications of methylglyoxal synthase complexed with phosphoglycolohydroxamic acid as observed by X-ray crystallography and NMR spectroscopy; Marks GT et al.; Methylglyoxal synthase (MGS) and triosephosphate isomerase (TIM) share neither sequence nor structural similarities, yet the reactions catalyzed by both enzymes are similar, in that both initially convert dihydroxyacetone phosphate to a cis-enediolic intermediate . This enediolic intermediate is formed from the abstraction of the pro-S C3 proton of DHAP by Asp-71 of MGS or the pro-R C3 proton of DHAP by Glu-165 of TIM . MGS then catalyzes the elimination of phosphate from this enediolic intermediate to form the enol of methylglyoxal, while TIM catalyzes proton donation to C2 to form D-glyceraldehyde phosphate . A competitive inhibitor of TIM, phosphoglycolohydroxamic acid (PGH) is found to be a tight binding competitive inhibitor of MGS with a K(i) of 39 nM . PGH's high affinity for MGS may be due in part to a short, strong hydrogen bond (SSHB) from the NOH of PGH to the carboxylate of Asp-71 . Evidence for this SSHB is found in X-ray, 1H NMR, and fractionation factor data . The X-ray structure of the MGS homohexamer complexed with PGH at 2.0 A resolution shows this distance to be 2.30-2.37 +/- 0.24 A . 1H NMR shows a PGH-dependent 18.1 ppm signal that is consistent with a hydrogen bond length of 2.49 +/- 0.02 A . The D/H fractionation factor (phi = 0.43 +/- 0.02) is consistent with a hydrogen bond length of 2.53 +/- 0.01 A . Further, 15N NMR suggests a significant partial positive charge on the nitrogen atom of bound PGH, which could strengthen hydrogen bond donation to Asp-71 . Both His-98 and His-19 are uncharged in the MGS-PGH complex on the basis of the chemical shifts of their Cdelta and C(epsilon) protons . The crystal structure reveals that Asp-71, on the re face of PGH, and His-19, on the si face of PGH, both approach the NO group of the analogue, while His-98, in the plane of PGH, approaches the carbonyl oxygen of the analogue . The phosphate group of PGH accepts nine hydrogen bonds from seven residues and is tilted out of the imidate plane of PGH toward the re face . Asp-71 and phosphate are thus positioned to function as the base and leaving group, respectively, in a concerted suprafacial 1,4-elimination of phosphate from the enediolic intermediate in the second step of the MGS reaction . Combined, these data suggest that Asp-71 is the one base that initially abstracts the C3 pro-S proton from DHAP and subsequently the 3-OH proton from the enediolic intermediate . This mechanism is compared to an alternative TIM-like mechanism for MGS, and the relative merits of both mechanisms are discussed.

Proc Natl Acad Sci U S A, 2001 Jun 5, 98(12), 6765 - 70 Epub 2001 May 29.
In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair; Burdett V et al.; Biochemical studies with model DNA heteroduplexes have implicated RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X in Escherichia coli methyl-directed mismatch correction . However, strains deficient in the four exonucleases display only a modest increase in mutation rate, raising questions concerning involvement of these activities in mismatch repair in vivo . The quadruple mutant deficient in the four exonucleases, as well as the triple mutant deficient in RecJ exonuclease, exonuclease VII, and exonuclease I, grow poorly in the presence of the base analogue 2-aminopurine, and exposure to the base analogue results in filament formation, indicative of induction of SOS DNA damage response . The growth defect and filamentation phenotypes associated with 2-aminopurine exposure are effectively suppressed by null mutations in mutH, mutL, mutS, or uvrD/mutU, which encode activities that act upstream of the four exonucleases in the mechanism for the methyl-directed reaction that has been proposed based on in vitro studies . The quadruple exonuclease mutant is also cold-sensitive, having a severe growth defect at 30 degrees C . This phenotype is suppressed by a uvrD/mutU defect, and partially suppressed by mutH, mutL, or mutS mutations . These observations confirm involvement of the four exonucleases in methyl-directed mismatch repair in vivo and suggest that the low mutability of exonuclease-deficient strains is a consequence of under recovery of mutants due to a reduction in viability and/or chromosome loss associated with activation of the mismatch repair system in the absence of RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X.

Proc Natl Acad Sci U S A, 2001 Jun 5, 98(12), 6565 - 70 Epub 2001 May 29.
Localization of GAR transformylase in Escherichia coli and mammalian cells; Gooljarsingh LT et al.; Enzymes of the de novo purine biosynthetic pathway may form a multienzyme complex to facilitate substrate flux through the ten serial steps constituting the pathway . One likely strategy for complex formation is the use of a structural scaffold such as the cytoskeletal network or subcellular membrane of the cell to mediate protein-protein interactions . To ascertain whether this strategy pertains to the de novo purine enzymes, the localization pattern of the third purine enzyme, glycinamide ribonucleotide transformylase (GAR Tfase) was monitored in live Escherichia coli and mammalian cells . Genes encoding human as well as E . coli GAR Tfase fused with green fluorescent protein (GFP) were introduced into their respective cells with regulated expression of proteins and localization patterns monitored by using confocal fluorescence microscopy . In both instances images showed proteins to be diffused throughout the cytoplasm . Thus, GAR Tfase is not localized to an existing cellular architecture, so this device is probably not used to concentrate the members of the pathway . However, discrete clusters of the pathway may still exist throughout the cytoplasm.

Proc Natl Acad Sci U S A, 2001 Jun 5, 98(12), 6617 - 22 Epub 2001 May 29.
An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors; Zagnitko O et al.; cDNA fragments encoding the carboxyltransferase domain of the multidomain plastid acetyl-CoA carboxylase (ACCase) from herbicide-resistant maize and from herbicide-sensitive and herbicide-resistant Lolium rigidum were cloned and sequenced . A Leu residue was found in ACCases from herbicide-resistant plants at a position occupied by Ile in all ACCases from sensitive grasses studied so far . Leu is present at the equivalent position in herbicide-resistant ACCases from other eukaryotes . Chimeric ACCases containing a 1000-aa fragment of two ACCase isozymes found in a herbicide-resistant maize were expressed in a yeast ACC1 null mutant to test herbicide sensitivity of the enzyme in vivo and in vitro . One of the enzymes was resistant/tolerant, and one was sensitive to haloxyfop and sethoxydim, rendering the gene-replacement yeast strains resistant and sensitive to these compounds, respectively . The sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site . Additionally, a single Ile to Leu replacement at an equivalent position changes the wheat plastid ACCase from sensitive to resistant . The effect of the opposite substitution, Leu to Ile, makes Toxoplasma gondii apicoplast ACCase resistant to haloxyfop and clodinafop . In this case, inhibition of the carboxyltransferase activity of ACCase (second half-reaction) of a large fragment of the Toxoplasma enzyme expressed in Escherichia coli was tested . The critical amino acid residue is located close to a highly conserved motif of the carboxyltransferase domain, which is probably a part of the enzyme active site, providing the basis for the activity of fop and dim herbicides.

J Biol Chem, 2001 Jun 8, 276(23), 20167 - 74 Epub 2001 Feb 28.
Investigation of Escherichia coli dimethyl sulfoxide reductase assembly and processing in strains defective for the sec-independent protein translocation system membrane targeting and translocation; Sambasivarao D et al.; Dimethyl sulfoxide reductase is a heterotrimeric enzyme (DmsABC) localized to the cytoplasmic surface of the inner membrane . Targeting of the DmsA and DmsB catalytic subunits to the membrane requires the membrane targeting and translocation (Mtt) system . The DmsAB dimer is a member of a family of extrinsic, cytoplasmic facing membrane subunits that require Mtt in order to assemble on the membrane . We show that the MttA(2), MttB, and presumably MttA(1) but not the MttC proteins are required for targeting DmsAB to the membrane . Unlike other Mtt substrates such as trimethylamine N-oxide reductase, the soluble cytoplasmic DmsAB dimer that accumulates in the mtt deletions is very labile . Deletion of the mttA(2) or mttB genes also prevents anaerobic growth on fumarate even though fumarate reductase does not require Mtt for assembly . This was due to the lethality of membrane insertion of DmsC in the absence of the DmsAB subunits . In the absence of DmsC, DmsAB accumulates in the cytoplasm . A 45-amino acid leader on DmsA is removed during assembly . Processing does not require DmsC but does require Mtt . Translocation of DmsAB to the periplasm is not required for processing . The leader may be cleaved by a novel leader peptidase, or the long DmsA leader may traverse the membrane through the Mtt system resulting in cleavage by the periplasmic leader peptidase I followed by release of DmsA into the cytoplasm.

J Biol Chem, 2001 Aug 3, 276(31), 29582 - 7 Epub 2001 Jun 01.
Role of the linker region of the anion-stimulated ATPase ArsA . Effect of deletion and point mutations in the linker region; Jia H et al.; The anion-stimulated ATPase ArsA in Escherichia coli consists of two homologous halves, A1 and A2, which are connected by a 40-amino acid long stretch of residues designated as the linker region . The linker region of ArsA lies in close proximity of the nucleotide-binding domain(s) of ArsA and is involved in significant conformational changes on binding of the substrates . Hence, it has been suggested earlier that the linker may play an important role in the function of ArsA . The aim of the present study was to determine the role of the linker by deletion and by site-directed mutagenesis of specific residues . Effect of deletion of the linker was determined by using the in vivo complementation approach where two halves of ArsA were co-expressed either with or without the linker region . Two co-expressed halves of ArsA conferred arsenite resistance only if the linker region was present on one of the halves . Of the six different point mutations created in the linker region, three (G284S, R290S, and D303G) were seen to drastically affect the catalytic function of ArsA . In addition, these three mutant alleles conferred arsenite sensitivity in cells carrying the wild type arsB gene . Trypsin proteolysis studies carried out with the purified proteins showed that the A1 nucleotide-binding domain in D303G protein has a conformation different from the wild type ArsA, suggesting that the linker region interacts with the nucleotide-binding domain(s) of ArsA . Based on the studies presented here, we propose that, in addition to providing flexibility, the nature of the residues themselves in the linker region is important for the conformation of the nucleotide-binding domains and for the catalytic function of ArsA.

J Biol Chem, 2001 Aug 17, 276(33), 31151 - 5 Epub 2001 Jun 01.
Alteration of the specificity of malate dehydrogenase by chemical modulation of an active site arginine; Wright SK et al.; Malate dehydrogenase from Escherichia coli is highly specific for the oxidation of malate to oxaloacetate . The technique of site-specific modulation has been used to alter the substrate binding site of this enzyme . Introduction of a cysteine in place of the active site binding residue arginine 153 results in a mutant enzyme with diminished catalytic activity, but with K(m) values for malate and oxaloacetate that are surprisingly unaffected . Reaction of this introduced cysteine with a series of amino acid analog reagents leads to the incorporation of a range of functional groups at the active site of malate dehydrogenase . The introduction of a positively charged group such as an amine or an amidine at this position results in improved affinity for several inhibitors over that observed with the native enzyme . However, the recovery of catalytic activity is less dramatic, with less than one third of the native activity achieved with the optimal reagents . These modified enzymes do have altered substrate specificity, with alpha-ketoglutarate and hydroxypyruvate no longer functioning as alternative substrates.

J Biochem (Tokyo), 2001 Jun, 129(6), 979 - 86
Cryoprotective effect of the serine-rich repetitive sequence in silk protein sericin; Tsujimoto K et al.; The silk proteins, fibroin and sericin, are produced in the silk gland of Bombyx mori, and hydrophilic sericin envelops fibroin with successive sticky layers in the formation of a cocoon . To study the biological functions of sericin, we focused on the serine-rich sericin peptide consisting of 38 amino acids, which is a highly conserved and internally repetitive sequence of a sericin protein . The corresponding gene was chemically synthesized, and the PCR-amplified gene was ligated to oligomerize sericin peptide and fused at the amino terminus to a His-tagged and proteolytic cleavage sequence in an inducible expression vector . When the dimers of sericin peptides were overexpressed in Escherichia coli, the transformants showed a prominent increase in cell viability after freezing in medium . Further, the purified dimeric sericin peptide from E . coli was found to be effective in protecting lactate dehydrogenase from denaturation caused by freeze-thaw . Both of these protective effects against freezing stress in cells and proteins were also observed with sericin hydrolysate . These results indicate that this unique sericin peptide, like sericin, has a high cryoprotective activity and will be valuable as a new biomaterial for industrial use.

J Biochem (Tokyo), 2001 Jun, 129(6), 943 - 8
Demonstration of the importance and usefulness of manipulating non-active-site residues in protein design; Shimotohno A et al.; Do non-active-site residues participate in protein function in a more direct way than just by holding the static framework of the protein molecule? If so, how important are they? As a model to answer these questions, ATB17, which is a mutant of aspartate aminotransferase created by directed evolution, is an ideal system because it shows a 10(6)-fold increase in the catalytic efficiency for valine but most of its 17 mutated residues are non-active-site residues . To analyze the roles of the mutations in the altered function, we divided the mutations into four groups, namely, three clusters and the remainder, based on their locations in the three-dimensional structure . Mutants with various combinations of the clusters were constructed and analyzed, and the data were interpreted in the context of the structure-function relationship of this enzyme . Each cluster shows characteristic effects: for example, one cluster appears to enhance the catalytic efficiency by fixing the conformation of the enzyme to that of the substrate-bound form . The effects of the clusters are largely additive and independent of each other . The present results illustrate how a protein function is dramatically modified by the accumulation of many seemingly inert mutations of non-active-site residues.

J Biochem (Tokyo), 2001 Jun, 129(6), 851 - 60
Codon and base biases after the initiation codon of the open reading frames in the Escherichia coli genome and their influence on the translation efficiency; Sato T et al.; Nucleotide sequences around the boundaries of all open reading frames in the Escherichia coli whole genome were analyzed . Characteristic base biases were observed after the initiation codon and before the termination codon . We examined the effect of the base sequence after the initiation codon on the translation efficiency, by introducing mutations after the initiation codon of the E . coli dihydrofolate reductase (DHFR) gene, considering codon and base biases, and using in vitro and in vivo translation systems . In both assay systems, the two most frequent second codons, AAA and AAU, enhanced the translation efficiency compared with the wild type, whereas the effects of lower frequency codons were not significant . Experiments using 16S rRNA variants with mutations in the putative complementary sequence to the region downstream of the initiation codon showed that the translation efficiency of none of the DHFR mutants was affected . These results demonstrate that the statistically most frequent sequences for the second codon enhance translation efficiency, and this effect seems to be independent of base pairing between mRNA and 16S rRNA.

Protein Expr Purif, 2001 Jun, 22(1), 148 - 58
Isolation, expression, and characterization of fully functional nontoxic BiP/GRP78 mutants; King LS et al.; Mammalian BiP/GRP78 and Escherichia coli DnaK belong to the highly conserved hsp70 family and function as molecular chaperones in the endoplasmic reticulum or the cytosol, respectively . Induction of murine BiP/GRP78 expression in E . coli leads to growth arrest and cell death, independent of the bacterial strain and vector used . Analysis of various BiP constructs and mutants shows that the dominant-lethal phenotype is induced specifically by the expression of the 13.7-kDa C-terminal domain and abolished by a single substitution in that region . Deletion of that region also results in nontoxic gene products that can be overexpressed and purified to homogeneity . The nontoxic mutants are highly expressed in E . coli, representing up to 20% of the soluble fraction . They are catalytically active, depolymerize upon binding ATP or synthetic peptide, and interact with the J-domain of the DnaJ-like accessory protein, MTJ1, with near wild-type affinity . Our data indicate that the cytotoxic effect encountered during overexpression of recombinant proteins can be caused by a single domain and can be alleviated by a specific mutation or deletion in that region without altering the catalytic properties of the enzyme .

Protein Expr Purif, 2001 Jun, 22(1), 128 - 34
Systematic optimization of expression and refolding of the Plasmodium falciparum cysteine protease falcipain-2; Sijwali PS et al.; The Plasmodium falciparum cysteine protease falcipain-2 is a potential new target for antimalarial chemotherapy . In order to obtain large quantities of active falcipain-2 for biochemical and structural analysis, a systematic assessment of optimal parameters for the expression and refolding of the protease was carried out . High-yield expression was achieved using M15(pREP4) Escherichia coli transformed with the pQE-30 plasmid containing a truncated profalcipain-2 construct . Recombinant falcipain-2 was expressed as inclusion bodies, solubilized, and purified by nickel affinity chromatography . A systematic approach was then used to optimize refolding parameters . This approach utilized 100-fold dilutions of reduced and denatured falcipain-2 into 203 different buffers in a microtiter plate format . Refolding efficiency varied markedly . Optimal refolding was obtained in an alkaline buffer containing glycerol or sucrose and equal concentrations of reduced and oxidized glutathione . After optimization of the expression and refolding protocols and additional purification with anion-exchange chromatography, 12 mg of falcipain-2 was obtained from 5 liters of E . coli, and crystals of the protease were grown . The systematic approach described here allowed the rapid evaluation of a large number of expression and refolding conditions and provided milligram quantities of recombinant falcipain-2 .

Protein Expr Purif, 2001 Jun, 22(1), 108 - 12
Accurate disulfide formation in Escherichia coli: overexpression and characterization of the first domain (HF6478) of the multiple Kazal-type inhibitor LEKTI; Lauber T et al.; The human hemofiltrate peptide HF6478, a putative serine proteinase inhibitor, which is part of the precursor protein LEKTI, was cloned, overexpressed, and purified . HF6478 contains two disulfide bridges with 1-4, 2-3 connectivity, sharing partial homology to Kazal-type domains and other serine proteinase inhibitors . It was expressed as thioredoxin (Trx) fusion protein, and disulfide formation occurred in the oxidative cytoplasm of Escherichia coli Origami (DE3) strain which carries a trxB(-)/gor522(-) double mutation . The soluble fusion protein was purified using metal-chelating affinity chromatography . Cleavage of the Trx fusion protein with factor Xa and subsequent purification yielded the final product in amounts sufficient for structural studies . Characterization of recombinant HF6478 was done by amino acid sequencing, mass spectrometry, capillary zone electrophoresis, and CD spectroscopy . Taking the blood filtrate peptide HF6478 as example, we present a strategy which should facilitate the expression of different extracellular proteins in the E . coli cytoplasm .

Protein Expr Purif, 2001 Jun, 22(1), 84 - 91
Expression of mammalian geranylgeranyltransferase type-II in Escherichia coli and its application for in vitro prenylation of Rab proteins; Kalinin A et al.; Mammalian geranylgeranyltransferase type II (GGTase-II) is a 100-kDa heterodimer that catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab GTPases . This modification is essential for the biological activity of Rab proteins . Geranylgeranylation can be performed in vitro using recombinant GGTase-II but so far large-scale production of the enzyme was challenging . We report here the design of a two plasmid expression system that will produce GGTase-II at levels as high as 15 mg/L in Escherichia coli . The protein was produced as a heterodimer with the alpha subunit bearing a cleavable tandem 6His-glutathione S-transferase (GST) tag that was used for two-step purification of the enzyme . Purified enzyme was functionally active as determined by in vitro prenylation and phosphoisoprenoid binding assay . Furthermore, the GST-tagged GGTase-II was used for preparative in vitro prenylation of the Rab7:REP-1 complex . Using this procedure, 10 mg of doubly prenylated Rab7:REP-1 complex were obtained .

Protein Expr Purif, 2001 Jun, 22(1), 75 - 81
Expression and purification of Escherichia coli beta-glucuronidase; Aich S et al.; A strong and constitutive expression vector of Escherichia coli beta-glucuronidase with the isocitrate dehydrogenase promoter has been developed for producing a large amount of recombinant protein . More than 95% pure enzyme was obtained by a four step purification procedure-ammonium sulfate precipitation, DEAE ion-exchange chromatography, Superose 12 gel filtration, and hydroxyapatite steric ion-exchange chromatography . The overexpressed gene can produce 23 mg of pure enzyme from one liter of bacterial culture .

Protein Expr Purif, 2001 Jun, 22(1), 60 - 9
Recombinant expression of biologically active rat leptin in Escherichia coli; Park JH et al.; Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight . While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available . In the present study, rat leptin was recombinantly expressed in Escherichia coli and purified in a bioactive form to provide a further tool for the analysis of leptin functions in rats . Leptin cDNA was cloned by RT-PCR from total RNA of SD rat adipocytes, and overexpression was achieved by subcloning the leptin cDNA into the pET-29a vector, which enabled the recombinant expression of rat leptin as an S-peptide-tagged fusion protein . Since the fusion proteins were expressed in inclusion bodies, after purification of the insoluble fraction, leptin proteins were refolded by sequential dialysis into physiological buffers . The biological activity of this recombinant protein was confirmed in proliferation assays using leptin-sensitive rat insulinoma cells as well as a newly developed leptin-sensitive luciferase assay system . The specific binding of the S-tagged leptin to leptin-receptor-expressing cells was further shown by flow cytometry using fluorescence-conjugated S-proteins .

Protein Expr Purif, 2001 Jun, 22(1), 38 - 44
One-step purification of a fully active hexahistidine-tagged human hexokinase type I overexpressed in Escherichia coli; Palma F et al.; The conversion of glucose into glucose 6-phosphate (Glc 6-P)1 traps glucose in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism . In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation . These enzymes are constituted by a single polypeptide chain with a molecular weight of approximately 100 kDa . Among these isoenzymes, hexokinase type I is the most widely expressed in mammalian tissues and shows reversion of Glc 6-P inhibition by physiological levels of inorganic phosphate . In this work the hexokinase I from human brain was overexpressed in Escherichia coli, as a hexahistidine-tagged protein with the tag extending the C-terminal end . An average of 900 U per liter of culture was obtained . The expressed protein was one-step purified by metal chelate affinity chromatography performed in NTA-agarose column charged with Ni(2+) ions . In order to stabilize the enzymatic activity 0.5 M ammonium sulfate was added to elution buffer . The specific activity of purified hexokinase I was 67.8 U/mg . The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thus it can be used for biophysical and biochemical investigation .

Protein Expr Purif, 2001 Jun, 22(1), 31 - 7
Two-cistron system overexpression of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunit B and B-derivatives from spinach in Escherichia coli; Tang GL et al.; A gene coding for the subunit B (GapB) of chloroplast glyceraldehyde-3-phosphate dehydrogenase from spinach and its two derivatives (GapBc) lacking the GapB-specific C-terminal extension have been cloned by RT-PCR . These three genes have been overexpressed with full activity in Escherichia coli when a two-cistron expression system controlled by an inducible promoter P(trc) is used . With a suitable base composition of the first cistron, the expression level of GapB and the derivatives GapBc are expressed up to 15-20% of the total cell protein and around 20 mg of recombinant GapBcs with full activity are purified from 1 liter of cultured bacteria . The specific activity of the two derivatives GapBc (40-60 u/mg) is similar to that of GapA (50-70 u/mg) and lower than that of reported GapBc derivative (E . Baalmann, R . Scheibe, R . Cerff, and W . Martin, 1996, Plant Mol . Biol . 32, 505-513) .

Protein Expr Purif, 2001 Jun, 22(1), 25 - 30
Expression of Giardia duodenalis beta-tubulin as a soluble protein in Escherichia coli; MacDonald LM et al.; The beta-tubulin gene of the parasitic protozoan Giardia duodenalis has been expressed for the first time using a novel and direct method . The protein was expressed in both soluble and insoluble forms in an Escherichia coli-based expression system . The level of expression was found to be affected by several variables including the incubation temperature, length of time for which expression was carried out, and the E . coli culture volume . The protein expression system contributed no additional amino acids to the final fusion protein and the polyhistidine fusion sequence was easily removed from the beta-tubulin protein using a specific enterokinase enzyme . The expression system also provided a means of preparing a soluble protein and purifying it by a relatively straightforward affinity chromatography method to give a very high level of protein purity . This makes the protein suitable for a number of applications for characterization including beta-tubulin antibody assays, alpha-/beta-tubulin-binding regions, and beta-tubulin folding intermediates .

Protein Expr Purif, 2001 Jun, 22(1), 11 - 24
Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80/CD86-CD28 costimulatory pathway; Dincq S et al.; The development of recombinant techniques for rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering . By fusion to heterologous protein domains, chain shuffling, or inclusion of self-assembly motifs, novel molecules such as bispecific Abs can be generated that possess the subset of functional properties designed to fit the intended application . We describe the engineering of Ab fragments produced in bacteria for blocking the CD28-CD80/CD86 costimulatory interaction in order to induce tolerance against transplanted organs . We designed single-chain Fv antibodies, monospecific and bispecific diabodies, and a bispecific tetravalent antibody (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules . These recombinant Ab molecules were expressed in Escherichia coli, followed by purification and evaluation for specific interaction with their respective antigen in an enzyme-linked immunosorbent assay (ELISA) . A specific sandwich ELISA confirmed the bispecificity of the bispecific diabodies and the BiTAb .

Life Sci, 2001 Mar 16, 68(17), 1939 - 49
Acute alcohol intoxication and endotoxemia desensitize HIV-1 gp120-induced CC-chemokine production by Kupffer cells; Bautista AP; Chemokines are involved in the inhibition of HIV-1 infection and in the pathogenesis of tissue injury in a number of conditions, including endotoxemia and alcoholic liver disease . CC chemotactic peptides (MIP-1alpha, MCP-1 and RANTES) are produced by a wide variety of cell types in response to immunological stimuli, bacterial endotoxin and gp120 from HIV-1 and HIV-2 . This work tests the hypothesis that prior exposure to endotoxin and/or ethanol in vivo inhibits the production of CC-chemokines following a secondary challenge with HIV-1 gp120 in vitro . Male Sprague-Dawley rats received in intravenous infusion of ethanol to maintain blood ethanol level at 170 mg/dl for 3 hr . Escherichia coli LPS (1 mg/Kg) was given intravenously 5 min after the ethanol bolus was injected . Control groups received similar volumes of saline . Three hr after LPS treatment, Kupffer cells were obtained and treated with HIV-1 gp120 (5 microg/10(6) cells/24 hr) . At the end of the incubation period, cells were obtained for RT-PCR analysis of CC-chemokine mRNA expression . Chemokine release in culture supernatants was measured by ELISA . Results show that in vivo ethanol was associated with downregulation of MIP-1alpha and MCP-1 mRNA expression and protein release in primary cultures of Kupffer cells . However, ethanol alone primed isolated Kupffer cells for enhanced RANTES mRNA and protein release in the presence or absence of HIV-1 gp120 . These results demonstrate that acute ethanol intoxication and endotoxemia may selectively act as a desensitizing agent in response to a secondary challenge with bacterial or viral products.

Life Sci, 2001 Feb 9, 68(12), 1395 - 403
Opposing pharmacological actions of cepharanthin on lipopolysaccharide-induced histidine decarboxylase activity in mice spleens; Sogawa N et al.; A biscoclaurin alkaloid preparation, cepharanthin (Ceph), is reported to have opposing pharmacological effects, enhancement or depression, on several cells and tissues, although detailed mechanisms remain unclear . Previously, we reported that Ceph enhanced lipopolysaccharide (LPS)-induced histidine decarboxylase (HDC) activity in mice spleens by consecutive pre-administration . In this study, we examined the pharmacological effects on HDC activity of a single Ceph pre-administration to test the influence of the administration method . Consequently, HDC activities were decreased by a single administration 15 minutes before LPS challenge in ddY and ICR mice spleens . Moreover, to further examine this suppressing effect, we employed genetically mast cell-deficient WBB6F1 W/Wv (W/Wv) mice to avoid the influence of mast cells . In W/Wv mice, HDC activity was enhanced, but not in the congenic WBB6F1 +/+ mice . These findings suggest that mast cells influence the depressant effect on HDC activity by a Ceph single administration in mast cell sufficient mice.

Med Microbiol Immunol (Berl), 2001 Apr, 189(3), 133 - 6
The detergent octylglucoside neutralizes lipopolysaccharide in a specific manner; Henrich B et al.; The stimulatory effect of lipopolysaccharide (LPS) on human macrophages was found to be neutralized by the detergent octylglucoside (OG) . Both macrophage stimulation and reactivity in a limulus amebocyte lysate test were suppressed by suspension of LPS in OG at concentrations between 0.25 and 2.5 mM, whereas other stimulatory lipopeptides and lipid containing stimulants were unaffected by OG . LPS at concentrations causing maximal stimulation of macrophages could be completely neutralized by non-toxic concentrations of OG . In addition, it was found that the neutralization in complex mixtures of macromolecules, such as bacterial cell lysate, was specific for LPS and that the stimulatory activity of the other substances in the mixture was not affected by the OG.

Biosci Biotechnol Biochem, 2001 Apr, 65(4), 969 - 72
Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor Sca from sunflower seeds; Kouzuma Y et al.; Sunflower cystatin a (Sca) is distinguished from other phytocystatins by its lack of the N-terminal about 20 amino acids, resulting in the absence of the evolutionarily conserved Gly residue . The cDNA encoding Sca was amplified by PCR methods . The cDNA consists of 520 nucleotides and includes an open reading frame encoding a polypeptide of 98 amino acids . Comparison of the deduced amino acid sequence with the Sca protein sequence indicated that the deduced sequence has an extra 15 amino acids and one amino acid at the N- and C-termini, respectively . This result suggests that Sca is synthesized as a preprotein (preSca) and proteolytic cleavages at peptide bonds may give rise to the mature Sca . To address this assumption and also to investigate the significance of the N-terminal extension sequence to Sca for inhibitory activity, a recombinant pre-Sca (rpre-Sca), in which the N-terminal extension was fused to the matured Sca, and a recombinant matured Sca (rSca) were overproduced in Escherichia coli cells . Incubation of the rpre-Sca with a seed extract resulted in a mobility by SDS-PAGE that was the same as rSca, demonstrating a proteolytic cleavage by endogenous proteinases . The rSca and rpre-Sca proteins were further characterized with respect to inhibitory activity and sensorgrams of the interaction with papain . The result showed that rpre-Sca had stronger inhibitory activity than rSca, and that the increased activity toward papain was due to a lower dissociation rate constant . This finding indicates that the N-terminal region of rpre-Sca increases the inhibitory activity by stabilizing the rpre-Sca and papain complex.

Biosci Biotechnol Biochem, 2001 Apr, 65(4), 865 - 74
Increase in the stability of serine acetyltransferase from Escherichia coli against cold inactivation and proteolysis by forming a bienzyme complex; Mino K et al.; Cysteine synthetase from Escherichia coli is a bienzyme complex composed of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS) . The effects of the complex formation on the stability of SAT against cold inactivation and proteolysis were investigated . SAT was reversibly inactivated on cooling to 0 degrees C . Ultracentrifugal analysis showed that SAT (a hexamer) was dissociated mostly into two trimers on cooling to 0 degrees C in the absence of OASS, while in the presence of OASS one trimer of the SAT subunits formed a complex with one dimer of OASS subunits . In the presence of OASS, not only the cold inactivation rate was reduced but also the reactivation rate was increased . Furthermore, SAT became stable against proteolytic attack by alpha-chymotrypsin and V8 protease by forming the complex with OASS . On the other hand, SAT was degraded by trypsin in the same manner both in the presence and in the absence of OASS . The different tendency in the stability against proteolysis with the different proteases was discussed with respect to the substrate specificity of the proteases and amino acid sequence of the C-terminal region of SAT that interacts with OASS.

J Biol Chem, 2001 Aug 3, 276(31), 29275 - 81 Epub 2001 May 31.
RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor Activity; Kimple RJ et al.; The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling . RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site . Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14 . Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms . In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1) . Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-) . Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity . The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.

J Biol Chem, 2001 Aug 3, 276(31), 29188 - 94 Epub 2001 May 31.
Reducing the environmental sensitivity of yellow fluorescent protein . Mechanism and applications; Griesbeck O et al.; Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions . However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 degrees C . Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pK(a) (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 degrees C and in organelles . The halide resistance is explained by a 2.2-A x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore . Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators . These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free {Ca(2+)} in the Golgi . Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiological signals.

EMBO J, 2001 Jun 1, 20(11), 2977 - 86
Specific interaction between the ribosome recycling factor and the elongation factor G from Mycobacterium tuberculosis mediates peptidyl-tRNA release and ribosome recycling in Escherichia coli; Rao AR et al.; Once the translating ribosomes reach a termination codon, the nascent polypeptide chain is released in a factor-dependent manner . However, the P-site-bound deacylated tRNA and the ribosomes themselves remain bound to the mRNA (post-termination complex) . The ribosome recycling factor (RRF) plays a vital role in dissociating this complex . Here we show that the Mycobacterium tuberculosis RRF (MtuRRF) fails to rescue Escherichia coli LJ14, a strain temperature-sensitive for RRF (frr(ts)) . More interestingly, co-expression of M.tuberculosis elongation factor G (MtuEFG) with MtuRRF rescues the frr(ts) strain of E.coli . The simultaneous expression of MtuEFG is also needed to cause an enhanced release of peptidyl-tRNAs in E.coli by MtuRRF . These observations provide the first genetic evidence for a functional interaction between RRF and EFG . Both the in vivo and in vitro analyses suggest that RRF does not distinguish between the translating and terminating ribosomes for their dissociation from mRNA . In addition, complementation of E.coli PEM100 (fusA(ts)) with MtuEFG suggests that the mechanism of RRF function is independent of the translocation activity of EFG.

EMBO J, 2001 Jun 1, 20(11), 2966 - 76
Transfer RNA(Ala) recognizes transfer-messenger RNA with specificity; a functional complex prior to entering the ribosome?
Gillet R, Felden B.
tmRNA (SsrA or 10Sa RNA) functions as both a transfer RNA and a messenger RNA, rescues stalled ribosomes and clears the cell of incomplete polypeptides . We report that native Escherichia coli tmRNA interacts specifically with native or synthetic E.coli tRNA alanine (tRNA(Ala)) in vitro, alanine being the first codon of the tmRNA internal open reading frame . Aminoacylatable RNA microhelices also bind tmRNA . Complex formation was monitored by gel retardation assays combined with structural probes . Nucleotides from the acceptor stem of tRNA(Ala) are essential for complex formation with tmRNA . tRNA(Ala) isoacceptors recognize tmRNA with different affinities, with an important contribution from tRNA(Ala) post-transcriptional modifications . The most abundant tRNA(Ala) isoacceptor in vivo binds tmRNA with the highest affinity . A complex between tRNA(Ala) and tmRNA might involve up to 140 tmRNA molecules out of 500 present per E.coli cell . Our data suggest that tmRNA interacts with the tRNA that decodes the resume codon prior to entering the ribosome . Biological implications of promoting specific complexes between tmRNA and aminoacylatable RNAs are discussed, with emphasis on primitive versions of the translation apparatus.

Hum Gene Ther, 2001 May 20, 12(8), 871 - 81
In vivo gene transfer in mouse skeletal muscle mediated by baculovirus vectors; Pieroni L et al.; Baculovirus vectors are efficient tools for gene transfer into mammalian cells in vitro . However, in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system . To characterize further the gene transfer efficacy of baculovirus we examined the vector transduction efficiency in skeletal muscle . Vectors expressing vesicular stomatitis virus glycoprotein (VSV-G) in the viral envelope were generated by inserting the VSV-G coding sequence downstream of the polyhedrin promoter . Two viruses were constructed to carry either the Escherichia coli beta-galactosidase (beta-Gal) gene or the mouse erythropoietin (EPO) cDNA cloned downstream of the cytomegalovirus immediate-early promoter and enhancer . The greater gene transduction efficiency of the Bac-G-betaGal vector was confirmed by comparing the beta-Gal expression level in a variety of human and mouse cell lines with that obtained on infection with Bac-betaGal, a vector that lacks VSV-G . Similarly, a 5- to 10-fold increase in beta-Gal expression between Bac-G-betaGal and Bac-betaGal was observed when mouse myoblasts and myotubes were infected . The same increase in beta-Gal expression was detected on injection of the Bac-G-betaGal vector in the quadriceps of BALB/c and C57BL/6 mice . In contrast, a 2-fold difference in transduction was observed between these two vectors in DBA/2J mouse strain . Last, expression of EPO cDNA was detected for at least 178 days in DBA/2J mice on Bac-G-EPO injection into the quadriceps whereas EPO expression declined to normal values by 35 days postinfection in BALB/c and C57BL/6 mice . Thus, these results indicate that baculovirus may be considered a useful vector for gene transfer in mouse skeletal muscle and that persistence of expression may depend on the mouse strain used.

Biotechnol Prog, 2001 May-Jun, 17(3), 573 - 6
Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes; Morita M et al.; Self-disruptive Escherichia coli that produces foreign target protein was developed . E . coli was co-transformed with two vector plasmids, a target gene expression vector and a lysis gene expression vector . The lytic protein was produced after the expression of the target gene, resulting in simplification of the cell disruption process . In this study, the expression of cloned T4 phage gene e or t was used for the disruption of E . coli that produced beta-glucuronidase (GUS) as a model target protein . The expression of gene e did not lead to prompt cell disruption but weakened the cell wall . Resuspension with deionized water facilitated cell lysis, and GUS activity was observed in the resuspended liquid . Expression of gene e at mid logarithmic growth phase was the optimal induction period for GUS production and release . On the other hand, the expression of gene t induced immediate cell lysis, and intracellular GUS was released to the culture medium . Maximum GUS production was obtained when gene t was induced at late logarithmic growth phase.

Biotechnol Prog, 2001 May-Jun, 17(3), 537 - 42
Structural and functional stabilization of L-asparaginase via multisubunit immobilization onto highly activated supports; Balcao VM et al.; A new protocol for the stabilization of the quaternary structure of multimeric enzymes has been attempted using as model enzyme (tetrameric) L-asparaginase from Escherichia coli . Such strategy is based upon multisubunit covalent immobilization of the enzyme onto activated supports (agarose-glutaraldehyde) . Supports activated with different densities of reactive groups were used; the higher the density of groups, the higher the stabilization attained . However, because of the complexity of that enzyme, even the use of the highest densities of reactive groups was not enough to encompass all four subunits in the immobilization process . Therefore, a further chemical intersubunit cross-linking with aldehyde-dextran was pursued; these derivatives displayed a fully stabilized multimeric structure . In fact, boiling the modified enzyme derivative in the presence of sodium dodecyl sulfate and beta-mercaptoethanol did not lead to release of any enzyme subunit into the medium . Such a derivative, prepared under optimal conditions, retained ca . 40% of the intrinsic activity of the free enzyme and was also functionally stabilized, with thermostabilization enhancements of ca . 3 orders of magnitude when compared with its soluble counterpart . This type of derivative may be appropriate for extracorporeal devices in the clinical treatment of acute leukemia and might thus bring about inherent advantages in that all subunits are covalently bound to the support, with a longer half-life and a virtually nil risk of subunit release into the circulating blood stream.

Biotechnol Prog, 2001 May-Jun, 17(3), 490 - 4
L-asparaginase release from Escherichia coli cells with K2HPO4 and Triton X100; Zhao F et al.; A method to release L-asparaginase (EC 3.5.1.1) from ATCC Escherichia coli 11303 cells by chemical permeabilization was studied . It was found that a combination of K2HPO4 and Triton X100 was effective . The influences of K2HPO4 concentration, Triton concentration, E . coli cell concentration and pH on the release of enzyme and proteins were investigated in detail . Experimental results showed that 12.5% (w/v) K2HPO4, 2% (w/v) Triton X100 and 3 x 10(8) cells/mL made the amount of enzyme released over 70% . L-Asparaginase in K2HPO4 and Triton solution could remain stable at least for 24 h . The release effect of K2HPO4 and Triton X100 used simultaneously was better than that of K2HPO4 and Triton X100 used separately in succession . Electron microscopy indicated that the chemical treatment altered the surface structure of E . coli cells but did not break them . As the method does not produce a large amount of cell fragments and the amount of enzyme released is relatively high, it can be thought to be an valuable and economic method to release intracellular enzyme.

Biotechnol Prog, 2001 May-Jun, 17(3), 407 - 11
Whole-cell immobilization using cell surface-exposed cellulose-binding domain; Wang AA et al.; Specific adhesion of Eshcherichia coli with surface-exposed cellulose-binding domain (CBD) to cellulosic materials was investigated . Whole-cell immobilization was very specific, forming essentially a monolayer of cells onto the different supports . Cells with surface-exposed CBD bound specifically and tightly to cellulose supports at a wide range of pH . In contrast to CBD, which shows the highest binding to cellulose at 4 degrees C, highest cell loading was observed at 37 degrees C . The extent of immobilization was dependent on the amount of surface-exposed CBD . Cells binding increased with increasing amount of CBD until binding was saturated . Even induction of very low level of CBD (0.05 mM IPTG) was sufficient to provide specific and tight binding to cellulose support . Because optimal binding can be obtained under physiological conditions such as pH 7 and 37 degrees C, the results demonstrate the general utility of surface-exposed CBD as an efficient means of whole-cell immobilization.

Mol Genet Metab, 2001 Jun, 73(2), 126 - 37
Identification of Caenorhabditis elegans isovaleryl-CoA dehydrogenase and structural comparison with other acyl-CoA dehydrogenases; Mohsen AW et al.; Isovaleryl-CoA dehydrogenase (IVD) is a flavoenzyme, which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway and transfers electrons to the electron-transferring flavoprotein (ETF) . IVDs from human and rat have been identified and characterized previously . In this study, the gene coding for Caenorhabditis elegans IVD has been identified from a published cDNA sequence and molecular modeling has been performed using the human IVD atomic coordinates . The coding sequence for the mature form of the enzyme was expressed in Escherichia coli, and the recombinant nematode IVD enzyme was purified to essential homogeneity . Its spectrum is typical of recombinant FAD-containing acyl-CoA dehydrogenases and shows a minor broad absorption band at 650-700 nm characteristic of an IVD:CoA persulfide charge-transfer complex . Following treatment of the enzyme with sodium dithionite to remove the bound CoA persulfide, the K(m) values for isovaleryl-, butyryl-, valeryl-, and hexanoyl-CoA were estimated to be 2.5, 36.2, 10.5, and 33.8 microM, respectively, using the ETF fluorescence reduction assay . The catalytic efficiency (k(cat)/K(m)) for these substrates was 56.9, 1.3, 13.7, and 3.2 microM(-1) . min(-1) per mole of FAD, respectively . The apparent binding constant (K(D app)) of the recombinant IVD determined spectrally for isovaleryl-CoA was 0.34 microM . These kinetic parameters confirm that isovaleryl-CoA is the preferred substrate for the purified enzyme . The variability in the protein structure among known and putative IVDs from various species is discussed in the context of possible mechanisms for modulating enzyme activity .

Microbiol Immunol, 2001, 45(4), 319 - 22
Identification of shiga toxin-producing Escherichia coli possessing insertionally inactivated Shiga toxin gene; Okitsu T et al.; We have investigated the Shiga toxin genes of Shiga toxin-producing Escherichia coli (STEC) strains, using polymerase chain reaction (PCR) amplifying the full lengths of these genes . As a result, we found the Shiga toxin 2 gene which was insertionally inactivated by an insertion sequence (IS) . This IS element was identical to IS1203v which has been also found in inactivated Shiga toxin 2 genes, and was inserted at the same site as in the previous paper . On the other hand, both Shiga toxin 2 genes were different (98.3% identity) . These suggested that IS1203v independently inserted into each Shiga toxin 2 genes, and STEC strains possessing the insertionally inactivated Shiga toxin genes are most likely to have a wide distribution . Amplification of the full length of the Shiga toxin gene is one of the effective methods to detect the gene no matter where the IS element is included, i.e., the insertion can be reflected in the size of amplicon.

Antonie Van Leeuwenhoek, 2000 Dec, 78(3-4), 277 - 85
Transcriptional and functional analysis of the gene for factor C, an extracellular signal protein involved in cytodifferentiation of Streptomyces griseus; Biro S et al.; Factor C is an extracellular signal protein involved in cellular differentiation in Streptomyces griseus . Nuclease S1 mapping experiments revealed that transcription of the gene takes place from a single promoter in a developmental-stage specific manner . The latter was also confirmed by in vivo promoter probing . The sequence of its promoter suggests that the gene is not transcribed by the major sigma factor . The cloned gene expressed from its own promoter in low- and high-copy-number vectors restored normal sporulation to a bald mutant of Streptomyces griseus . Computer analysis of the amino acid sequence revealed the presence of a transmembrane localization segment with the N-terminus positioned inside the cell . These data fit well into our working model that points at an important role for factor C in the morphogenesis of Streptomyces griseus.

Mikrobiologiia, 2001 Mar-Apr, 70(2), 248 - 52
{Effect of mechanical stress on lon mutant strain of Escherichia coli K-12}; Airapetian SN et al.; It was found that, depending on their frequency, mechanical vibrations (MVs) can either stimulate (4 Hz) or inhibit (50 Hz) the growth and the division of the lon mutant of Escherichia coli K-12 . Similar effects were observed when the MV-treated nutrient medium was inoculated with untreated mutant cells . MVs enhanced the motility of mutant cells and the fragmentation of filament cells always present in the populations of lon mutants.

Mikrobiologiia, 2001 Mar-Apr, 70(2), 168 - 73
{The role of putrescine in of oxidative stress defense genes expression regulation in Escherichia coli}; Tkachenko AG et al.; The role of putrescine in the adaptive response of Escherichia coli grown aerobically in synthetic M9 medium with glucose to the H2O2-induced oxidative stress was studied . Under oxidative stress, the expression of the single-copy reporter gene fusions oxyR::lacZ and katG::lacZ was found to undergo biphasic changes, which were most pronounced in glucose-starved E . coli cells . The concentration-dependent activating effect of putrescine on the expression of the oxyR regulon genes was maximum when the oxyR gene was inhibited by high concentrations of hydrogen peroxide.

Cancer Biother Radiopharm, 2001 Apr, 16(2), 109 - 23
Preclinical evaluation of a humanized NR-LU-10 antibody-streptavidin fusion protein for pretargeted cancer therapy; Goshorn S et al.; A humanized single chain Fv antibody fragment specific to the EGP40 antigen was genetically engineered as a streptavidin fusion (scFvSA) for use in pretargeted radioimmunotherapy . The scFvSA construct was expressed as a soluble, tetrameric species in the Escherichia coli periplasm at 110-140 mg/liter . The fusion protein was purified from crude lysates by iminobiotin affinity chromatography with an overall yield of 50-60% . Characterization of the purified protein by SDS-PAGE, light scattering, and size exclusion chromatography demonstrated that the fusion protein was tetrameric with a molecular weight of approximately 172,000 . Competitive immunoreactivity assays showed a two-fold greater binding to the antigen than the comparable whole antibody . The purified protein had a biotin disassociation rate identical to recombinant streptavidin and bound an average of three of four possible biotins per molecule . The radiolabeled fusion protein showed a faster blood clearance rate in normal mice than the corresponding whole antibody-streptavidin chemical conjugate . Tumor-specific targeting of a subsequently administered radionuclidechelate/biotin molecule was demonstrated in nude mice bearing SW1222 human colon carcinoma xenografts . A single dose of 800 microCi of 90Y-DOTA-biotin produced cures in mice with established subcutaneous human small cell lung or colon cancer xenografts.

Nature, 2001 May 31, 411(6837), 595 - 9
ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology; Perraud AL et al.; Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems . Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases . Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR . The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2 . These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.

Nat Biotechnol, 2001 Jun, 19(6), 548 - 52
A plant-based multicomponent vaccine protects mice from enteric diseases; Yu J et al.; Cholera toxin (CT) B and A2 subunit complementary DNAs (cDNAs) were fused to a rotavirus enterotoxin and enterotoxigenic Escherichia coli fimbrial antigen genes and transferred into potato . Immunoblot and enzyme-linked immunosorbent assay (ELISA) results indicated that the fusion antigens were synthesized in transformed tuber tissues and assembled into cholera holotoxin-like structures that retained enterocyte-binding affinity . Orally immunized mice generated detectable levels of serum and intestinal antibodies against the pathogen antigens . Elevated levels of interleukin 2 (IL2) and interferon gamma (INFgamma) detected in immunogen-challenged spleen cells from the immunized mice indicated the presence of a strong Th1 immune response to the three plant-synthesized antigens . This result was supported by flow cytometry analysis of immunized mouse spleen cells that showed a significant increase in CD4+ lymphocyte numbers . Diarrhea symptoms were reduced in severity and duration in passively immunized mouse neonates following rotavirus challenge . The results suggest that food plants can function as vaccines for simultaneous protection against infectious virus and bacterial diseases.

J Nutr, 2001 Jun, 131(6), 1668 - 74
Dietary conjugated linoleic acid reduces adiposity in lean but not obese Zucker rats; Sisk MB et al.; Recent studies have demonstrated a reduction in body fat in growing animals fed conjugated linoleic acid (CLA) . Two experiments were conducted to extend these observations to obese rats so that the mechanism of the actions of CLA might be more easily elucidated . In experiment 1, male lean and obese Zucker rats were fed diets containing either 0 or 0.5% CLA for 5 wk . There was no effect of diet on growth rate or food intake . Dietary CLA reduced retroperitoneal and inguinal fat pad weights in the lean rats but increased fat pad weights in the obese genotype (diet x genotype interaction; P < 0.05) . Determination of fat pad cellularity indicated that these changes in fat pad weight were due to a reduction or increase in average fat cell size for the lean and obese Zucker rats, respectively . In experiment 2, we sought to reproduce these effects on fat pad size, as well as to determine the effect of dietary CLA on the catabolic response to bacterial endotoxin injection in obese Zucker rats . Growing female lean and obese Zucker rats were fed diets containing 0 or 0.5% CLA for 8 wk . On d 28, each rat was injected intraperitoneally with lipopolysaccharide from Escherichia coli serotype 055:B5 (1 mg/kg body weight) and body weight was determined over the next 96 h . There was a diet x genotype interaction (P < 0.05) for the body weight response to lipopolysaccharide 24 h postinjection . Lean rats fed CLA lost less weight than did lean controls, but obese rats fed CLA lost more weight than did obese controls . As in the first experiment, there was a diet x genotype (P < 0.05) for the effect of treatment on retroperitoneal fat pad weights determined at the end of the experiment . Lean rats fed CLA had smaller RP fat pads than did lean controls, but obese rats fed CLA once again had heavier RP fat pads than did obese controls . These results indicate that CLA reduces body fat and catabolic response to endotoxin injection in lean Zucker rats but not in the obese genotype . The observed interaction between diet and genotype warrants additional investigation into the specific mechanism(s) of the biological activities of CLA.

J Biol Chem, 2001 Aug 3, 276(31), 29116 - 25 Epub 2001 May 30.
Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein; Koutsodontis G et al.; In the present study we present evidence for the critical role of Sp1 in the mechanism of transactivation of the human cell cycle inhibitor p21(WAF1/Cip1) (p21) gene promoter by the tumor suppressor p53 protein . We found that the distal p53-binding site of the p21 promoter acts as an enhancer on the homologous or heterologous promoters in hepatoma HepG2 cells . In transfection experiments, p53 transactivated the p21 promoter in HaCaT cells that express Sp1 but have a mutated p53 form . In contrast, p53 could not transactivate the p21 promoter in the Drosophila embryo-derived Schneider's SL2 cells that lack endogenous Sp1 or related factors . Cotransfection of SL2 cells with p53 and Sp1 resulted in a synergistic transactivation of the p21 promoter . Synergistic transactivation was greatly decreased in SL2 cells and HaCaT cells by mutations in either the p53-binding site or in the -82/-77 Sp1-binding site indicating functional cooperation between Sp1 and p53 in the transactivation of the p21 promoter . Synergistic transactivation was also decreased by mutations in the transactivation domain of p53 . Physical interactions between Sp1 and p53 proteins were established by glutathione S-transferase pull-down and coimmunoprecipitation assays . By using deletion mutants we found that the DNA binding domain of Sp1 is required for its physical interaction with p53 . In conclusion, Sp1 must play a critical role in regulating important biological processes controlled by p53 via p21 gene activation such as DNA repair, cell growth, differentiation, and apoptosis.

J Biol Chem, 2001 Aug 31, 276(35), 32591 - 6 Epub 2001 May 30.
Physical interaction of CcmC with heme and the heme chaperone CcmE during cytochrome c maturation; Ren Q et al.; Biogenesis of c-type cytochromes requires the covalent attachment of heme to the apoprotein . In Escherichia coli, this process involves eight membrane proteins encoded by the ccmABCDEFGH operon . CcmE binds heme covalently and transfers it to apocytochromes c in the presence of other Ccm proteins . CcmC is necessary and sufficient to incorporate heme into CcmE . Here, we report that the CcmC protein directly interacts with heme . We further show that CcmC co-immunoprecipitates with CcmE . CcmC contains two conserved histidines and a signature sequence, the so-called tryptophan-rich motif, which is the only element common to cytochrome c maturation proteins of bacteria, archae, plant mitochondria, and chloroplasts . We report that mutational changes of these motifs affecting the function of CcmC in cytochrome c maturation do not influence heme binding of CcmC . However, the mutants are defective in the CcmC-CcmE interaction, suggesting that these motifs are involved in the formation of a CcmC-CcmE complex . We propose that CcmC, CcmE, and heme interact directly with each other, establishing a periplasmic heme delivery pathway for cytochrome c maturation.

J Biol Chem, 2001 Aug 3, 276(31), 29353 - 60 Epub 2001 May 30.
Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II; Singla SI et al.; Autophosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) at Thr(286) results in calmodulin (CaM) trapping, a >10,000-fold decrease in the dissociation rate of CaM from the enzyme . Here we present the first site-directed mutagenesis study on the dissociation of the high affinity complex between CaM and full-length CaM kinase II . We measured dissociation kinetics of CaM and CaM kinase II proteins by using a fluorescently modified CaM that is sensitive to binding to target proteins . In low {Ca(2+)}, the phosphorylated mutant kinase F293A and the CaM mutant E120A/M124A exhibited deficient trapping compared with wild-type . In high {Ca(2+)}, the CaM mutations E120A, M124A, and E120A/M124A and the CaM kinase II mutations F293A, F293E, N294A, N294P, and R297E increased dissociation rate constants by factors ranging from 2.3 to 116 . We have also identified residues in CaM and CaM kinase II that interact in the trapped state by mutant cycle-based analysis, which suggests that interactions between Phe(293) in the kinase and Glu(120) and Met(124) in CaM specifically stabilize the trapped CaM-CaM kinase II complex . Our studies further show that Phe(293) and Asn(294) in CaM kinase II play dual roles, because they likely destabilize the low affinity state of CaM complexed to unphosphorylated kinase but stabilize the trapped state of CaM bound to phosphorylated kinase.

J Immunol Methods, 2001 Jul 1, 253(1-2), 1 - 11
Specific recognition of cytosolic thymidine kinase in the human lung tumor by monoclonal antibodies raised against recombinant human thymidine kinase; Kuroiwa N et al.; Anti-TK monoclonal antibodies (mAbs) were raised against recombinant human cytosolic thymidine kinase (rhTK) and characterized by Western immunoblotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining of tumor cells . Twenty-three clones of TK mAbs were characterized to recognize specifically not only rhTK produced by Escherichia coli but also TK subunit of 25 kDa in human lung cancer . The anti-TK mAbs reacted specifically with cytosolic TK but not with mitochondrial TK . Only one clone of the mAbs inhibited the catalytic activity of TK . By solid phase sandwich enzyme immunoassay using these mAbs, we could quantitate the cytosolic TK content in tissues . Immunohistochemical staining analysis using one of the TK mAbs showed that human lung adenocarcinoma and squamous cell carcinoma exhibited much higher staining intensity than stromal cells . These mAbs are useful for biochemical studies on the regulation of human TK in proliferating cells such as tumor cells and for diagnosis of highly proliferating tumors.

Virology, 2001 Jun 5, 284(2), 297 - 307
The use of recombinant baculoviruses for sustained expression of human cytomegalovirus immediate early proteins in fibroblasts; Dwarakanath RS et al.; The isolation of viruses with mutations in essential genes requires that they be propagated in cells expressing the wild-type proteins . This has been a particularly challenging problem for studying mutations in the human cytomegalovirus (HCMV) immediate early (IE) gene, IE2 86 . In the past, we tried a number of approaches to derive human fibroblasts expressing wild-type IE2 86, but were unable to maintain expression of a fully functional protein . To overcome this obstacle, we developed a strategy whereby recombinant baculoviruses were used as vectors for the expression of HCMV IE proteins in primary human fibroblasts (FFs) . The IE2 86 and IE1 72 cDNAs, as well as the genomic fragment of the UL122-123 region under the control of a chicken actin promoter, were introduced into the baculovirus genome by site-specific transposition in Escherichia coli . Recombinant "bacmid" DNAs were then transfected into Sf9 cells to generate recombinant baculoviruses . FFs infected at high m.o.i . with these baculoviruses expressed high levels of the HCMV protein for at least 1 week, as determined by immunofluorescence assays and Western blots . Moreover, the IE2 86 protein was found to be fully functional with respect to its ability to activate the HCMV UL112-113 early promoter . Recombinant baculoviruses expressing IE1 72 were also able to efficiently complement HCMV ie1 mutants . These data demonstrate the potential of using recombinant baculoviruses as vectors for the expression of toxic viral genes in human cells and for subsequent isolation of mutant HCMV lacking these essential genes .

Plant Cell Physiol, 2001 May, 42(5), 475 - 81
Cloning and characterization of a hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase induced in response to UV-C and wounding from Capsicum annuum; Back K et al.; Hydroxycinnamoyl-CoA : tyramine N-(hydroxycinnamoyl) transferase (THT) is a pivotal enzyme in the synthesis of N-(hydroxycinnamoyl)-amines, which are associated with cell wall fortification in plants . The cDNA encoding THT was cloned from the leaves of UV-C treated Capsicum annuum (hot pepper) using a differential screening strategy . The predicted protein encoded by the THT cDNA is 250 amino acids in length and has a relative molecular mass of 28,221 . The protein sequence derived from the cDNA shares 76% and 67% identity with the potato and tobacco THT protein sequences, respectively . The recombinant pepper THT enzyme was purified using a bacterial overexpression system . The purified enzyme has a broad substrate specificity including acyl donors such as cinnamoyl-, sinapoyl-, feruloyl-, caffeoyl-, and 4-coumaroyl-CoA and acceptors such as tyramine and octopamine . In UV-C treated plants, the THT mRNA was strongly induced in leaves, and the elevated level of expression was stable for up to 36 h . THT mRNA also increased in leaves that were detached from the plant but not treated with UV-C . THT expression was measured in different plant tissues, and was constitutive at a similar level in leaf, root, stem, flower and fruit . Induction of THT mRNA was correlated with an increase in THT protein.

J Biol Chem, 2001 Aug 3, 276(31), 28700 - 9 Epub 2001 May 29.
Interaction of two structurally distinct sequence types with the clathrin terminal domain beta-propeller; Drake MT et al.; The amino-terminal domain of the clathrin heavy chain, which folds into a seven-bladed beta-propeller, binds directly to several endocytic proteins via short sequences based on the consensus residues LLDLD . In addition to a single LLDLD-based, type I clathrin-binding sequence, both amphiphysin and epsin contain a second, distinct sequence that is also capable of binding to clathrin directly . Here, we analyzed these sequences, which we term type II sequences, and show that the (257)LMDLA sequence in rat epsin 1 appears to be a weak clathrin-binding variant of the sequence (417)PWDLW originally found in human amphiphysin II . The structural features of the type II sequence required for association with clathrin are distinct from the LLDLD-based sequence . In the central segment of amphiphysin, the type I and type II sequences cooperate to effect optimal clathrin binding and the formation of sedimentable assemblies . Together, the data provide evidence for two interaction surfaces upon certain endocytic accessory proteins that could cooperate with other coat components to enhance clathrin bud formation at the cell surface.

J Biol Chem, 2001 Aug 10, 276(32), 30442 - 51 Epub 2001 May 29.
Apg2 is a novel protein required for the cytoplasm to vacuole targeting, autophagy, and pexophagy pathways; Wang CW et al.; To survive starvation conditions, eukaryotes have developed an evolutionarily conserved process, termed autophagy, by which the vacuole/lysosome mediates the turnover and recycling of non-essential intracellular material for re-use in critical biosynthetic reactions . Morphological and biochemical studies in Saccharomyces cerevisiae have elucidated the basic steps and mechanisms of the autophagy pathway . Although it is a degradative process, autophagy shows substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that delivers resident hydrolases to the vacuole . Recent molecular genetics analyses of mutants defective in autophagy and the Cvt pathway, apg, aut, and cvt, have begun to identify the protein machinery and provide a molecular resolution of the sequestration and import mechanism that are characteristic of these pathways . In this study, we have identified a novel protein, termed Apg2, required for both the Cvt and autophagy pathways as well as the specific degradation of peroxisomes . Apg2 is required for the formation and/or completion of cytosolic sequestering vesicles that are needed for vacuolar import through both the Cvt pathway and autophagy . Biochemical studies revealed that Apg2 is a peripheral membrane protein . Apg2 localizes to the previously identified perivacuolar compartment that contains Apg9, the only characterized integral membrane protein that is required for autophagosome/Cvt vesicle formation.

Exp Anim, 2001 Apr, 50(2), 139 - 45
In vitro migratory responses of swine neutrophils to actinobacillus pleuropneumoniae; Wang FI et al.; Swine neutrophils were quantitatively examined for the direct and indirect migratory responses to Actinobacillus pleuropneumoniae (APP) in vitro and the effects of pseudorabies virus (PrV), frequently co-infecting with APP, were also observed . About 30% of swine neutrophils responded to viable APP, while 3.2% of the neutrophils responded to 0.1% casein which served as the control . The migration of APP was not affected by preincubation of neutrophils with PrV, which inhibited the random migration . When the random migration was normalized to 1, the chemotactic indices for APP, opsonized-APP and casein were 64, 70 and 8.5, respectively . Heat-killed APP or E . coli lipopolysaccharide stimulated the production of interleukin-8 activity by adherent peripheral blood mononuclear cells (PBMC) . Preincubation of PBMC with PrV inhibited the production of neutrophil attractant activity when stimulated with heat-killed APP . The results suggested that the direct chemotaxis of neutrophils to viable APP might contribute to early infiltration in Actinobacillus pleuropneumonia, and that PrV might inhibit indirect recruitment of neutrophils to infected lungs by compromising the functions of PBMC.

Kidney Int, 2001 Jun, 59(6), 2243 - 9
Endotoxemic renal failure in mice: Role of tumor necrosis factor independent of inducible nitric oxide synthase; Knotek M et al.; BACKGROUND: Renal failure is a frequent complication of sepsis with a high mortality . Tumor necrosis factor (TNF) has been suggested to be a factor in the acute renal failure in sepsis or endotoxemia . Recent studies also suggest involvement of nitric oxide (NO), generated by inducible NO synthase (iNOS), in the pathogenesis of endotoxin-induced renal failure . The present study tested the hypothesis that the role of TNF in endotoxic renal failure is mediated by iNOS-derived NO . METHODS: Renal function was evaluated in endotoxemic {Escherichia coli lipopolysaccharide (LPS), 5 mg/kg IP} wild-type and iNOS knockout mice . The effect of TNF neutralization on renal function during endotoxemia in mice was assessed by a TNF-soluble receptor (TNFsRp55) . RESULTS: An injection of LPS to wild-type mice resulted in a 70% decrease in glomerular filtration rate (GFR) and in a 40% reduction in renal plasma flow (RPF) 16 hours after the injection . The results occurred independent of hypotension, morphological changes, apoptosis, and leukocyte accumulation . In mice pretreated with TNFsRp55, only a 30% decrease in GFR without a significant change in RPF in response to LPS, as compared with vehicle-treated mice, was observed . Also, the serum NO concentration was significantly lower in endotoxemic wild-type mice pretreated with TNFsRp55, as compared with untreated endotoxemic wild-type mice (260 +/- 52 vs . 673 +/- 112 micromol/L, P < 0.01) . In LPS-injected iNOS knockout mice and wild-type mice treated with a selective iNOS inhibitor, 1400W, the development of renal failure was similar to that in wild-type mice . As in wild-type mice, TNFsRp55 significantly attenuated the decrease in GFR (a 33% decline, as compared with 75% without TNFsRp55) without a significant change in RPF in iNOS knockout mice given LPS . CONCLUSIONS: These results demonstrate a role of TNF in the early renal dysfunction (16 h) in a septic mouse model independent of iNOS, hypotension, apoptosis, leukocyte accumulation, and morphological alterations, thus suggesting renal hypoperfusion secondary to an imbalance between, as yet to be defined, renal vasoconstrictors and vasodilators.

Int J Androl, 2001 Jun, 24(3), 165 - 74
Rabbit sex hormone-binding globulin: expression in the liver and testis during postnatal development and structural characterization by truncated proteins; Wong AS et al.; Although sex hormone binding globulin (SHBG) is found in the blood plasma of adult humans and rabbits and the gene is expressed in their livers, it is not detected in the plasma of adult rodents nor is it expressed in adult rodent livers . Thus the rabbit represents a good model to study the metabolism and function of SHBG in the blood . We have used a cloned rabbit SHBG cDNA to detect mRNA expression in rabbits during the postnatal period, and to construct truncated SHBG proteins for structure/function analysis . The SHBG mRNA appeared in the testis as early as 3 days after birth . The level increased gradually in abundance throughout postnatal development, and attained a maximum at 12 weeks of age when the gonads were fully matured . In contrast, SHBG mRNA in the livers of male and female animals increased to a maximum by 4 weeks of age, and were maintained at this level until 12 weeks before subsiding to the initial levels . The increase and decrease in SHBG mRNA levels in the liver were accompanied by similar changes in serum SHBG . This suggests that SHBG in the blood circulation comes from the liver and this might also provide a source of SHBG for the male reproductive tract before formation of the blood-testis barrier . To elucidate the minimal sequence of rabbit SHBG responsible for steroid-binding, a panel of 13 truncated SHBG proteins was constructed, expressed in Escherichia coli, and biochemically purified for study . It was shown that the complete protein sequence of rabbit SHBG was important for maintaining a stable steroid-protein complex . Unlike human SHBG for which a truncated protein of the first 206 residues of the 373 amino acid protein can still bind steroid, removal of 43 or more residues from the C-terminus of rabbit SHBG completely abolished steroid-binding.

Genes Cells, 2001 May, 6(5), 389 - 401
Conformational switching of Escherichia coli RNA polymerase-promoter binary complex is facilitated by elongation factor GreA and GreB; Sen R et al.; BACKGROUND: The initiation arrest at a modified lambdaPR promoter is caused by irreversible divergence of the reaction pathway into productive and arrested branches . Escherichia coli GreA and GreB induce cleavage of the nascent transcript and relieve arrest in elongation . They also reduce abortive synthesis at several promoters and relieve initiation arrest . Their mechanism of action during initiation, and its relationship to the branched initiation pathway are unknown . RESULTS: The Gre factors mitigated initiation arrest only when they were added to the binary complex of the holoenzyme bound to the lambdaPR promoter, prior to RNA synthesis . They exerted little effect when they were added to ternary initiation complexes . They accelerated the exchange of the binary complex with its free components by 6-9-fold . When they are present, a high concentration of the initiating nucleotide increased yield of the full-length transcript, whereas a low concentration did not . CONCLUSIONS: All the results presented above can be explained by a model where the productive and arrested pathways diverge at the binary complex stage . The Gre factors relieve the initiation arrest by introducing reversibility between subspecies of the binary complex that are precursors of the two pathways . RNA cleavage is unlikely to cause relief of initiation arrest.

Biochemistry, 2001 Jun 5, 40(22), 6699 - 705
Acid-base catalysis by UDP-galactose 4-epimerase: correlations of kinetically measured acid dissociation constants with thermodynamic values for tyrosine 149; Berger E et al.; The steady-state kinetic parameters for epimerization of UDP-galactose by UDP-galactose 4-epimerase from Escherichia coli (GalE), Y149F-GalE, and S124A-GalE have been measured as a function of pH . The deuterium kinetic isotope effects for epimerization of UDP-galactose-C-d(7) by these enzymes have also been measured . The results show that the activity of wild-type GalE is pH-independent in the pH range of 5.5-9.3, and there is no significant deuterium kinetic isotope effect in the reaction of UDP-galactose-C-d(7) . It is concluded that the rate-limiting step for epimerization by wild-type GalE is not hydride transfer and must be either a diffusional process or a conformational change . Epimerization of UDP-galactose-C-d(7) by Y149F-GalE proceeds with a pH-dependent deuterium kinetic isotope effect on k(cat) of 2.2 +/- 0.4 at pH 6.2 and 1.1 +/- 0.5 at pH 8.3 . Moreover, the plot of log k(cat)/K(m) breaks downward on the acid side with a fitted value of 7.1 for the pK(a) . It is concluded that the break in the pH-rate profile arises from a change in the rate-limiting step from hydride transfer at low pH to a conformational change at high pH . Epimerization of UDP-galactose-C-d(7) by S124A-GalE proceeds with a pH-independent deuterium kinetic isotope effect on k(cat) of 2.0 +/- 0.2 between pH 6 and 9 . Both plots of log k(cat) and log k(cat)/K(m) display pH dependence . The plot of log k(cat) versus pH breaks downward with a pK(a) of 6.35 +/- 0.10 . The plot of log k(cat)/K(m) versus pH is bell-shaped, with fitted pK(a) values of 6.76 +/- 0.09 and 9.32 +/- 0.21 . It is concluded that hydride transfer is rate-limiting, and the pK(a) of 6.7 for free S124A-GalE is assigned to Tyr 149, which displays the same value of pK(a) when measured spectrophotometrically in this variant . Acid-base catalysis by Y149F-GalE is attributed to Ser 124, which is postulated to rescue catalysis of proton transfer in the absence of Tyr 149 . The kinetic pK(a) of 7.1 for free Y149F-GalE is lower than that expected for Ser 124, as proven by the pH-dependent kinetic isotope effect . Epimerization by the doubly mutated Y149F/S124A-GalE proceeds at a k(cat) that is lower by a factor of 10(7) than that of wild-type GalE . This low rate is attributed to the synergistic actions of Tyr 149 and Ser 124 in wild-type GalE and to the absence of any internal catalysis of hydride transfer in the doubly mutated enzyme.

Biochemistry, 2001 Jun 5, 40(22), 6660 - 9
Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I; Zhuang P et al.; The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated . The bulky lesion was derived from the binding of a benzo{a}pyrene diol epoxide isomer {(+)-anti-BPDE} to N(2)-guanine (G*) . Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end . All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T) . In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G) . Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies) . A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T . Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies) . The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated . Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T . These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base . The latter depend on the base Y and on the base preferentially inserted opposite the adduct . Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis.

Biochemistry, 2001 Jun 5, 40(22), 6628 - 35
High-yield expression and functional analysis of Escherichia coli glycerol-3-phosphate transporter; Auer M et al.; The glycerol-3-phosphate (G3P) transporter, GlpT, from Escherichia coli mediates G3P and inorganic phosphate exchange across the bacterial inner membrane . It possesses 12 transmembrane alpha-helices and is a member of the Major Facilitator Superfamily . Here we report overexpression, purification, and characterization of GlpT . Extensive optimization applied to the DNA construct and cell culture has led to a protocol yielding approximately 1.8 mg of the transporter protein per liter of E . coli culture . After purification, this protein binds substrates in detergent solution, as measured by tryptophan fluorescence quenching, and its dissociation constants for G3P, glycerol-2-phosphate, and inorganic phosphate at neutral pH are 3.64, 0.34, and 9.18 microM, respectively . It also shows transport activity upon reconstitution into proteoliposomes . The phosphate efflux rate of the transporter in the presence of G3P is measured to be 29 micromol min(-1) mg(-1) at pH 7.0 and 37 degrees C, corresponding to 24 mol of phosphate s(-1) (mol of protein)(-1) . In addition, the glycerol-3-phosphate transporter is monomeric and stable over a wide pH range and in the presence of a variety of detergents . This preparation of GlpT provides ideal material for biochemical, biophysical, and structural studies of the glycerol-3-phosphate transporter.

Biochemistry, 2001 Jun 5, 40(22), 6598 - 610
Probing catalysis by Escherichia coli dTDP-glucose-4,6-dehydratase: identification and preliminary characterization of functional amino acid residues at the active site; Hegeman AD et al.; A model of the Escherichia coli dTDP-glucose-4,6-dehydratase (4,6-dehydratase) active site has been generated by combining amino acid sequence alignment information with the 3-dimensional structure of UDP-galactose-4-epimerase . The active site configuration is consistent with the partially refined 3-dimensional structure of 4,6-dehydratase, which lacks substrate-nucleotide but contains NAD(+) (PDB file ) . From the model, two groups of active site residues were identified . The first group consists of Asp135(DEH), Glu136(DEH), Glu198(DEH), Lys199(DEH), and Tyr301(DEH) . These residues are near the substrate-pyranose binding pocket in the model, they are completely conserved in 4,6-dehydratase, and they differ from the corresponding equally well-conserved residues in 4-epimerase . The second group of residues is Cys187(DEH), Asn190(DEH), and His232(DEH), which form a motif on the re face of the cofactor nicotinamide binding pocket that resembles the catalytic triad of cysteine-proteases . The importance of both groups of residues was tested by mutagenesis and steady-state kinetic analysis . In all but one case, a decrease in catalytic efficiency of approximately 2 orders of magnitude below wild-type activity was observed . Mutagenesis of each of these residues, with the exception of Cys187(DEH), which showed near-wild-type activity, clearly has important negative consequences for catalysis . The allocation of specific functions to these residues and the absolute magnitude of these effects are obscured by the complex chemistry in this multistep mechanism . Tools will be needed to characterize each chemical step individually in order to assign loss of catalytic efficiency to specific residue functions . To this end, the effects of each of these variants on the initial dehydrogenation step were evaluated using a the substrate analogue dTDP-xylose . Additional steady-state techniques were employed in an attempt to further limit the assignment of rate limitation . The results are discussed within the context of the 4,6-dehydratase active site model and chemical mechanism.

Int Immunopharmacol, 2001 May, 1(5), 911 - 23
Comparison of the effect of lidocaine-epinephrine and prilocaine-felypressine to alter macrophage functions; Azuma Y et al.; In vitro treatment of macrophages with lidocaine-epinephrine or prilocaine-felypressine resulted in inhibition of their adhesion, chemotaxis and phagocytosis . However, prilocaine-felypressine was a much more potent inhibitor of adhesion and phagocytosis than lidocaine-epinephrine . On the other hand, lidocaine-epinephrine induced transient potentiation of superoxide anion production by macrophages, while prilocaine-felypressine consistently inhibited this . Moreover, lidocaine-epinephrine and prilocaine-felypressine both inhibited the production of hydrogen peroxide . In contrast, epinephrine strongly potentiated superoxide anion production, while markedly inhibiting hydrogen peroxide production . This potentiation by epinephrine was not prevented by adrenergic antagonists . In addition, superoxide dismutase potentiated the production of hydrogen peroxide, which was in part prevented by epinephrine . These results suggest that lidocaine-epinephrine and prilocaine-felypressine inhibit adhesion, chemotaxis, phagocytosis, and the production of hydrogen peroxide by macrophages . In addition, lidocaine-epinephrine evidently differs from prilocaine-felypressine regarding the molecular mechanisms underlying the modulation of superoxide anion production by macrophages.

Int J Biochem Cell Biol, 2001 Jun, 33(6), 591 - 602
Oxy5, a novel protein from Arabidopsis thaliana, protects mammalian cells from oxidative stress; Kush A et al.; The use of molecular oxygen in various cellular processes results in the generation of toxic intracellular by-products termed reactive oxygen species (ROS) . In Escherichia coli the oxyR gene product is a transcriptional regulator of the oxyR regulon that is induced in response to hydrogen peroxide-induced oxidative stress (OS) . We have previously shown that an annexin-like protein from Arabidopsis thaliana, termed Oxy5, can replace the obligatory role of OxyR in E . coli . Here, we have investigated as to whether oxy5 can function across evolutionary boundaries to protect mammalian cells from OS . Overexpression of the oxyR gene in mammalian tumor cell lines protects them from hydrogen peroxide-induced cell death, and these cells are also highly resistant to the superoxide ion producing compound paraquat . Oxy5 appears to be involved in the detection of calcium flux, as it binds to Ca2+ ions during hydrogen peroxide stress . Moreover, overexpression of Oxy5 leads to lowered protein kinase C activity . Thus, Oxy5 probably functions to sense and initiate protective responses to OS . In addition, Oxy5-overexpressing cells exhibit a reduction in endogenous superoxide ion levels, which concomitantly results in a dramatic decrease in their tumorigenic potential . Taken together, the results demonstrate an antioxidant role for plant oxy5 gene in mammalian cells, which can be potentially utilized in gene therapy programs aimed at reducing the deleterious effects of ROS.

Int J Biochem Cell Biol, 2001 Jun, 33(6), 577 - 89
Phosphorylation of the Fas associated factor FAF1 by protein kinase CK2 and identification of serines 289 and 291 as the in vitro phosphorylation sites; Jensen HH et al.; We previously identified the human Fas associated factor (FAF1) as one of the interacting partners of protein kinase CK2 beta subunit . Since FAF1 is a phosphoprotein we investigated whether it is a substrate for CK2 . Here, we report the full length human FAF1 cDNA sequence, expression of FAF1 in Escherichia coli and purification and characterization of FAF1 as a substrate for CK2 . FAF1 as well as an N-terminal 40 kDa degradation product serve as substrates for both the recombinant CK2 holoenzyme (km 100 microM) and the isolated catalytic alpha subunit (km 200 microM) . Despite the high k(m) values, we obtained evidence that CK2 is the major cellular kinase responsible for FAF1 phosphorylation, using tissue extracts as kinase sources . By MALDI-MS we identified the two serine residues at positions 289 and 291 as the major in vitro CK2 phosphorylation sites . These data may help us elucidate the functions of FAF1 and the involvement of CK2 mediated phosphorylation in processes such as apoptotic signaling, ubiquitination, nuclear translocation and embryonic development.

Mol Biochem Parasitol, 2001 May, 114(2), 217 - 26
Merozoite surface protein 8 of Plasmodium falciparum contains two epidermal growth factor-like domains; Black CG et al.; By motif searching of the unfinished sequences in the Malaria Genome Sequencing Project databases we have identified a novel EGF-like domain-containing protein of Plasmodium falciparum . The sequence lies within a single open reading frame of 1791 bp and is predicted to encode a polypeptide of 597 amino acids . There are hydrophobic regions at the extreme N- and C-termini, which could represent secretory signal peptide and GPI attachment sites, respectively . Similar to MSP1, there are two EGF-like domains located near the C-terminus . RT-PCR analysis of the novel gene shows that it is transcribed in asexual stages of the malaria parasite . We have expressed portions of the protein as recombinant GST fusions in Escherichia coli and raised antisera in rabbits . Antibodies to the EGF-like domains of the novel protein are highly specific and do not cross-react with the EGF-like domains of MSP1, MSP4 or MSP5 expressed as GST fusion proteins . Antiserum raised to the most C-terminal region of the protein reacts with four bands of 98, 50, 25 and 19 kDa in P . falciparum parasite lysates whereas antisera to the N-terminal fusion proteins recognise the 98 and 50 kDa bands, suggesting that the novel protein may undergo processing in a similar way to MSP1 . Immunoblot analysis of stage-specific parasite samples reveals that the protein is present throughout the parasite asexual life cycle and in isolated merozoites, with the smaller fragments present in ring stage parasites . The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites, schizonts and free merozoites by indirect immunofluorescence . Antisera to the C-terminus stain the surface of rings, whereas antisera to the N-terminus do not, suggesting that a fragment of the protein is carried into the developing ring stage parasite . Based on the accepted nomenclature in the field we designate this protein MSP8 . We have shown that the MSP8 fusion proteins are in a conformation that can be recognised by human immune sera and that there is very limited diversity in the MSP8 gene sequences from various P . falciparum laboratory isolates . MSP8 shows significant similarity to the recently reported sequence of the protective P . yoelii merozoite surface protein pypAg-2 {Burns JM, Belk CC, Dunn PD . Infect Immun 2000;68:6189-95.} suggesting that the two proteins are homologues . Taken together, these findings suggest that MSP8/pypAg-2 may play an important role in the process of red cell invasion and is a potential malaria vaccine candidate.

J Steroid Biochem Mol Biol, 2001 May, 77(2-3), 159 - 65
Human dehydroepiandrosterone sulfotransferase: purification and characterization of a recombinant protein; Chang HJ et al.; Dehydroepiandrosterone sulfate is the most abundant sulfated steroid transformed in human tissues and serves as a precursor for steroid hormones . Recombinant human dehydroepiandrosterone sulfotransferase (DHEA-ST) expressed in glutathione sulfotransferase fusion form in E . coli was purified using glutathione sepharose 4B affinity adsorption chromatography, a Factor Xa cleavage step, and Q-sepharose fast flow column chromatography . The homogeneous preparation had an activity toward dehydroepiandrosterone (DHEA) of 150+/-40 nmol/min per mg of protein under the assay conditions at an overall yield of 38.4% . The recombinant human DHEA-ST was shown to have a subunit mass of 34 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, while having a molecular mass of 67.2 kDa by Superose-12 gel filtration . Our results indicate that the active recombinant enzyme expressed in E . coli is a homodimer.Biochemical properties for purified DHEA-ST were studied using DHEA as a substrate . The optimum pH ranged from pH 7 to 8, and the optimum temperature 40-45 degrees C . Ninety percent of basal DHEA-ST activity remained even after the enzyme was treated at 45 degrees C for 15 min . The 50% inactivation concentration of NaCl for DHEA-ST activity was determined to be around 500 mM . The K(m) value for DHEA was 1.9+/-0.3 microM and V(max)=190+/-18 nmol/min per mg of protein at 37 degrees C, pH 7.5.

Trends Genet, 2001 Jun, 17(6), 318 - 21
Double-strand break repair: are Rad51/RecA--DNA joints barriers to DNA replication?
Aguilera A.
The central step of homologous recombination is the DNA strand exchange reaction catalyzed by bacterial RecA or eukaryotic Rad51 . Besides Rad51-mediated synthesis-dependent strand annealing (SDSA), DNA ends can promote replication in Escherichia coli (recombination-dependent replication, RDR) and yeast (break-induced replication, BIR) . However, what causes a DNA end to be repaired via SDSA or via BIR/RDR? I propose that Rad51/RecA--DNA plectonemic joints act as barriers to DNA replication and that BIR/RDR is only possible when the DNA polymerase that synthesizes DNA from the invading 3' end does not encounter RecA/Rad51--DNA joints in its path.

J Biotechnol, 2001 Jun 1, 88(1), 67 - 75
Removal of tightly bound endotoxin from biological products; Wilson MJ et al.; The method for endotoxin removal described in this paper is useful for separation of tightly bound endotoxin from biological products, particularly those produced in Escherichia coli in the form of inclusion bodies for which a denaturation step is required to solubilise the product . We employed guanidine hydrochloride and ammonium sulphate in combination with hydrophobic interaction chromatography (HIC) . These conditions enable binding of the endotoxin to the matrix, giving unbound product in the column flow-through . This makes the method generally applicable to biological products . An endotoxin reduction of about 3.7 logs was achieved; from as much as 1,100,000 EU mg(-1) in the solubilised material to about 200 EU mg(-1) in the product purified by this method . The method was developed for a cervical dysplasia vaccine, a fusion protein comprising L2, E7 and E6 from Human Papilloma Virus type 16, because both conventional and commercially available methods of endotoxin removal were ineffective in removing the tightly bound endotoxin from this product.

Mol Biochem Parasitol, 2001 Jun, 115(1), 41 - 53
Plasmodium vivax merozoite surface proteins-3beta and-3gamma share structural similarities with P . vivax merozoite surface protein-3alpha and define a new gene family; Galinski MR et al.; The genes encoding two merozoite surface proteins of Plasmodium vivax that are related to PvMSP3 {1} are reported . One of these genes was identified within P . vivax lambdagt11 clone 5.4, which was selected by immunoscreening with a Saimiri monkey antiserum . The insert DNA of this clone was used as a probe to isolate the complete gene from a P . vivax lambdaDASH genomic (g) DNA library . Antibodies to recombinant 5.4 and subsequent fusion proteins produce a pattern of circumferential surface fluorescence by indirect immunofluorescence assays (IFA) on segmented schizonts and free intact merozoites, and recognize a 125 kDa protein via western immunoblots . The gene, however, encodes a protein with a calculated size of 75677 Da, and 3' and 5' RACE analyses were employed to confirm the size of the gene and its coding region . The second related P . vivax gene was isolated by hybridization of a fragment of an orthologous P . knowlesi gene . The encoded proteins of all three related P . vivax genes have putative signal peptides, large central domains that contain >20% alanine residues bound by charged regions, are predicted to form alpha-helices with heptad repeat coiled-coil structures, and do not have a hydrophobic region that could anchor them to the surface of the merozoite . Although the overall identity in amino acid alignment among the three encoded proteins is low (<40%), the shared predicted structural features and motifs indicate that they are members of an intra-species family, which we are designating as the PvMSP-3 family with the reported members being Pvmsp-3alpha, Pvmsp-3beta, and Pvmsp-3gamma . We further demonstrate that this family also includes related proteins from P . knowlesi and P . falciparum.

J Virol Methods, 2001 Jun, 95(1-2), 1 - 10
Phosphorylation of purified recombinant hepatitis B virus-X protein by mitogen-activated protein kinase and protein kinase C in vitro; Lee YI et al.; The recombinant human hepatitis B virus-X protein (rhHBx) has been expressed as inclusion bodies in Escherichia coli and purified . By sequential dialysis of urea, rhHBx was folded into the native structure, which was demonstrated by both the efficacy of its transcriptional activation of the adenovirus major late promoter, fluorescence and circular dichroism (CD) analysis . The increase in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of HBx to refold in lower concentrations of urea to produce the active protein . After purification and renaturation, the rhHBx protein was found to be phosphorylated by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) . In vivo phosphorylation of HBx was also demonstrated . Although PKC and MAPK enhance the HBx phosphorylation in vitro, neither protein kinase A nor caseine kinase II (CKII) phosphorylate HBx protein, though there are possible substrate residues of both kinases in HBx protein . Phosphoamino acid analysis of the total acid hydrolyzed HBx showed that serine residues can be phosphorylated by PKC or MAPK.

FEBS Lett, 2001 May 25, 497(2-3), 131 - 6
The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity; Shehi E et al.; Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus . Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded beta-sheet across the minor groove . We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli . This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions . In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA . If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases . Apparently, the more readily attacked bonds were those intrinsically more unstable.

FEBS Lett, 2001 May 25, 497(2-3), 90 - 4
Cleavage of mitochondria-like transfer RNAs expressed in Escherichia coli; Bourdeau V et al.; Mitochondrial (mt) transfer RNAs (tRNAs) often harbor unusual structural features causing their secondary structure to differ from the conventional cloverleaf . tRNAs designed with such irregularities, termed mt-like tRNAs, are active in Escherichia coli as suppressors of reporter genes, although they display low steady-state levels . Characterization of fragments produced during mt-like tRNA processing in vitro and in vivo suggests that these RNAs are not fully processed at their 5' ends and are cleaved internally . These abnormal processing events may account for the low levels of mature mt-like RNAs in vivo and are most likely related to defective processing by RNase P.

Structure (Camb), 2001 May 9, 9(5), 367 - 75
The 2.2 A crystal structure of Hsp33: a heat shock protein with redox-regulated chaperone activity; Vijayalakshmi J et al.; BACKGROUND: One strategy that cells employ to respond to environmental stresses (temperature, oxidation, and pathogens) is to increase the expression of heat shock proteins necessary to maintain viability . Several heat shock proteins function as molecular chaperones by binding unfolded polypeptides and preventing their irreversible aggregation . Hsp33, a highly conserved bacterial heat shock protein, is a redox-regulated molecular chaperone that appears to protect cells against the lethal effects of oxidative stress . RESULTS: The 2.2 A crystal structure of a truncated E . coli Hsp33 (residues 1-255) reveals a domain-swapped dimer . The core domain of each monomer (1-178) folds with a central helix that is sandwiched between two beta sheets . The carboxyl-terminal region (179-235), which lacks the intact Zn binding domain of Hsp33, folds into three helices that pack on the other subunit . The interface between the two core domains is comprised of conserved residues, including a rare Glu-Glu hydrogen bond across the dyad axis . Two potential polypeptide binding sites that span the dimer are observed: a long groove containing pockets of conserved and hydrophobic residues, and an intersubunit 10-stranded beta sheet "saddle" with a largely uncharged or hydrophobic surface . CONCLUSIONS: Hsp33 is a dimer in the crystal structure . Solution studies confirmed that this dimer reflects the structural changes that occur upon activation of Hsp33 as a molecular chaperone . Patterns of conserved residues and surface charges suggest that two grooves might be potential binding sites for protein folding intermediates.

Mutat Res, 2001 Jun 2, 477(1-2), 119 - 24
LacI transgenic animal study: relationships among DNA-adduct levels, mutant frequencies and cancer incidences; Nagao M et al.; In the processes of carcinogenesis caused by genotoxic carcinogens, DNA-adduct formation and resultant genetic changes are crucially important . In this report, the relationship between DNA-adduct levels and mutant frequencies (MFs), DNA-adduct levels and cancer incidences, and MFs and cancer incidences induced by heterocyclic amines (HCAs), to which humans are exposed on daily basis were investigated . There was no direct correlation between adduct levels and MFs detected after feeding Big Blue mice with 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ), in a comparison among various organs . Further, there was no direct correlation between DNA-adduct levels and cancer incidences, in a comparison among various organs of F344 rats . Since DNA-adducts are fixed as mutations after cell proliferation, and mutations in cancer-related genes result in cancer development, it is expected that MFs directly correlate with cancer incidences . However, there was no direct correlation between MFs and cancer incidences . Possible mechanisms involved in the discordance between DNA damage markers and cancer incidences are discussed.

Mutat Res, 2001 Jun 2, 477(1-2), 41 - 9
Importance of DNA repair in carcinogenesis: evidence from transgenic and gene targeting studies; Ishikawa T et al.; We have generated transgenic mice by introducing copies of the E . coli O6-methylguanine-DNA methyltransferase gene, ada . Liver extracts from homozygotes demonstrate about three times the control enzyme activity and increase up to about eight-fold can be induced by treatment with zinc, since the metal-responsive metallothionein promoter is attached to the ada gene . Furthermore, studies of liver carcinogenesis in our transgenic mice demonstrated significantly reduced rates of development of hepatocellular tumors after treatment with dimethylnitrosamine or diethylnitrosamine . It is well known that xeroderma pigmentosum (XP) patients are deficient in DNA repair . The availability of XPA (XP group A complementing) knockout mice has enabled us to investigate the functional role of the XPA nucleotide excision repair gene in carcinogenesis in vivo, first using the mouse skin as a model system . XPA-/- mice demonstrated skin ulcers 5-7 days after 7,12-dimethylbenz{a}anthracene (DMBA) treatment and papilloma development within 4 weeks prior to promotion, skin tumor incidence being also much higher than in heterozygous and wild-type mice . Experiments targeting the lung, liver and tongue have also been conducted to answer the question of whether the internal organs of these mice are also susceptible to chemical carcinogens . For lung carcinogenesis, mice were instilled intratracheally with a small dose of benzo{a}pyrene . The pulmonary tumor incidence in XPA-/- mice was significantly higher than in XPA+/- and XPA+/+ mice . XPA-/- mice were also found to be have enhanced sensitivity to aflatoxin B1 regarding liver tumor induction . In addition, administration of 4-nitroquinoline-1-oxide in drinking water for 50 weeks resulted in tongue tumors only in XPA-/- mice . These studies, thus, provided convincing evidence that XPA mice are also sensitive to carcinogenesis in organs other than the skin.

FEBS Lett, 2001 May 18, 497(1), 55 - 8
Mutations in the interdomain linker region of DnaK abolish the chaperone action of the DnaK/DnaJ/GrpE system; Han W et al.; Hsp70s assist the folding of proteins in an ATP-dependent manner . DnaK, the Hsp70 of Escherichia coli, acts in concert with its co-chaperones DnaJ and GrpE . Amino acid substitutions (D388R and L391S/L392G) in the linker region between the ATPase and substrate-binding domain did not affect the functional domain coupling and oligomerization of DnaK . The intrinsic ATPase activity was enhanced up to 10-fold . However, the ATPase activity of DnaK L391S/L392G, if stimulated by DnaJ plus protein substrate, was five times lower than that of wild-type DnaK and DnaK D388R . This defect correlated with the complete loss of chaperone action in luciferase refolding . Apparently, the conserved leucine residues in the linker mediate the synergistic effects of DnaJ and protein substrate on ATPase activity, a function which might be essential for chaperone action.

FEBS Lett, 2001 May 18, 497(1), 50 - 4
Design and production of genetically modified soybean protein with anti-hypertensive activity by incorporating potent analogue of ovokinin(2-7); Matoba N et al.; The potent anti-hypertensive peptide, RPLKPW, has been designed based on the structure of ovokinin(2-7) . The sequence encoding this peptide was introduced into three homologous sites in the gene for soybean beta-conglycinin alpha' subunit . The native alpha' subunit as well as the modified, RPLKPW-containing alpha' subunit were expressed in Escherichia coli, recovered from the soluble fraction and then purified by ion-exchange chromatography . The RPLKPW peptide was released from recombinant RPLKPW-containing alpha' subunit after in vitro digestion by trypsin and chymotrypsin . Moreover, the undigested RPLKPW-containing alpha' subunit given orally at a dose of 10 mg/kg exerted an anti-hypertensive effect in spontaneously hypertensive rats, unlike the native alpha' subunit . These results provide evidence for the first time that a physiologically active peptide introduced into a food protein by site-directed mutagenesis could practically function in vivo even at a low dose.

Nucleic Acids Res, 2001 Jun 1, 29(11), E54 - 4
Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure; Wharam SD et al.; The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets . The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure . One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence . This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA) . The sequence of the RNA signal is always the same, regardless of the original target sequence . The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA . The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli . It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required . The assay is simple to perform and easily adaptable to different targets.

Nucleic Acids Res, 2001 Jun 1, 29(11), 2234 - 43
Human DNA mismatch repair in vitro operates independently of methylation status at CpG sites; Drummond JT et al.; Whereas in Escherichia coli DNA mismatch repair is directed to the newly synthesized strand due to its transient lack of adenine methylation, the molecular determinants of strand discrimination in eukaryotes are presently unknown . In mammalian cells, cytosine methylation within CpG sites may represent an analogous and mechanistically plausible means of targeting mismatch correction . Using HeLa nuclear extracts, we conducted a systematic analysis in vitro to determine whether cytosine methylation participates in human DNA mismatch repair . We prepared a set of A.C heteroduplex molecules that were either unmethylated, hemimethylated or fully methylated at CpG sequences and found that the methylation status persisted under the assay conditions . However, no effect on either the time course or the magnitude of mismatch repair events was evident; only strand discontinuities contributed to strand bias . By western analysis we demonstrated that the HeLa extract contained MED1 protein, which interacts with MLH1 and binds to CpG-methylated DNA; supplementation with purified MED1 protein was without effect . In summary, human DNA mismatch repair operates independently of CpG methylation status, and we found no evidence supporting a role for CpG hemimethylation as a strand discrimination signal.

Nucleic Acids Res, 2001 Jun 1, 29(11), 2205 - 16
Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins; Lewis JA et al.; Similarity between the DNA substrates and products of integrase-mediated site-specific recombination reactions results in a single recombinase enzyme being able to catalyze both the integration and excision reactions . The control of directionality in these reactions is achieved through a class of small accessory factors that favor one reaction while interfering with the other . These proteins, which we will refer to collectively as recombination directionality factors (RDFs), play architectural roles in reactions catalyzed by their cognate recombinases and have been identified in conjunction with both tyrosine and serine integrases . Previously identified RDFs are typically small, basic and have diverse amino acid sequences . A subset of RDFs, the cox genes, also function as transcriptional regulators . We present here a compilation of all the known RDF proteins as well as those identified through database mining that we predict to be involved in conferring recombination directionality . Analysis of this group of proteins shows that they can be grouped into distinct sub-groups based on their sequence similarities and that they are likely to have arisen from several independent evolutionary lineages . This compilation will prove useful in recognizing new proteins that confer directionality upon site-specific recombination reactions encoded by plasmids, transposons, phages and prophages.

J Biol Chem, 2001 Jun 1, 276(22), 19046 - 51 Epub 2001 Mar 16.
LeuO expression in response to starvation for branched-chain amino acids; Majumder A et al.; The recently identified role of LeuO in the regulation of transcription has prompted us to search for the specific function(s) of LeuO in bacterial physiology . The cryptic nature of expression of leuO has previously limited such analysis . A conditional leuO expression was found when bacteria enter stationary phase and was shown to be guanosine 3',5'-bispyrophosphate-dependent . Multiple physiological events, including the stringent response, are induced upon the increase of the bacterial stress signal, guanosine 3',5'-bispyrophosphate . In this study, we tested whether LeuO was directly involved in the bacterial stringent response . LeuO was shown to be indispensable for growth resumption following a 2-h growth arrest caused by starvation for branched-chain amino acids in an E . coli K-12 relA1 strain . This result supports a functional role for LeuO in the bacterial stringent response.

J Biol Chem, 2001 Aug 10, 276(32), 30064 - 71 Epub 2001 May 25.
Sigma38 (rpoS) RNA polymerase promoter engagement via -10 region nucleotides; Lee SJ et al.; Band shift assays using DNA probes that mimic closed and open complexes were used to explore the determinants of promoter recognition by sigma38 (rpoS) RNA polymerase . Duplex recognition was found to be much weaker than that observed in sigma70 promoter usage . However, binding to fork junction probes, which attempt to mimic melted DNA, was very strong . This binding occurs via the non-template strand with the identity of the two conserved junction nucleotides (-12T and -11A) being of paramount importance . A modified promoter consensus sequence identified these two nucleotides as among only four (underlined) that are highly conserved, and all four were in the -10 region (CTAcacT from -13 to -7) . The remaining two nucleotides were shown to have different roles, -13C in preventing recognition by the heterologous sigma70 polymerase and -7T in directing enzyme isomerization . These -10 region nucleotides appear to have their primary function prior to full melting because probes that had a melted start site were relatively insensitive to substitution at these positions . These results suggest the sigma38 mechanism differs from the sigma70 mechanism, and this difference likely contributes to selective use of sigma38 under conditions that exist during stationery phase.

J Biol Chem, 2001 Aug 3, 276(31), 28824 - 8 Epub 2001 May 25.
Identification of the catalytic residues of bifunctional glycogen debranching enzyme; Nakayama A et al.; Eukaryotic glycogen debranching enzyme (GDE) possesses two different catalytic activities (oligo-1,4-->1,4-glucantransferase/amylo-1,6-glucosidase) on a single polypeptide chain . To elucidate the structure-function relationship of GDE, the catalytic residues of yeast GDE were determined by site-directed mutagenesis . Asp-535, Glu-564, and Asp-670 on the N-terminal half and Asp-1086 and Asp-1147 on the C-terminal half were chosen by the multiple sequence alignment or the comparison of hydrophobic cluster architectures among related enzymes . The five mutant enzymes, D535N, E564Q, D670N, D1086N, and D1147N were constructed . The mutant enzymes showed the same purification profiles as that of wild-type enzyme on beta-CD-Sepharose-6B affinity chromatography . All the mutant enzymes possessed either transferase activity or glucosidase activity . Three mutants, D535N, E564Q, and D670N, lost transferase activity but retained glucosidase activity . In contrast, D1086N and D1147N lost glucosidase activity but retained transferase activity . Furthermore, the kinetic parameters of each mutant enzyme exhibiting either the glucosidase activity or transferase activity did not vary markedly from the activities exhibited by the wild-type enzyme . These results strongly indicate that the two activities of GDE, transferase and glucosidase, are independent and located at different sites on the polypeptide chain.

EMBO Rep, 2001 May, 2(5), 403 - 8
Anionic lipids stimulate Sec-independent insertion of a membrane protein lacking charged amino acid side chains; Ridder AN et al.; We have investigated the influence of the different lipid classes of Escherichia coli on Sec-independent membrane protein insertion, using an assay in which a mutant of the single-spanning Pf3 coat protein is biosynthetically inserted into liposomes . It was found that phosphatidylethanolamine and other non-bilayer lipids do not have a significant effect on insertion . Surprisingly, the anionic lipids phosphatidylglycerol and cardiolipin stimulate N-terminal translocation of the protein, even though it has no charged amino acid side chains . This novel effect is general for anionic lipids and depends on the amount of charge on the lipid headgroup . Since the N-terminus of the protein is at least partially positively charged due to a helix dipole moment, apparently negatively charged lipids can stimulate translocation of slightly positively charged protein segments in a direction opposite to the positive-inside rule . A mechanism is proposed to explain these results.

EMBO Rep, 2001 May, 2(5), 399 - 402
Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage; Agafonov DE et al.; We have recently isolated and characterized a novel protein associated with Escherichia coli ribosomes and named protein Y (pY) . Here we show that the ribosomes from bacterial cells growing at a normal physiological temperature contain no pY, whereas a temperature downshift results in the appearance of the protein in ribosomes . The protein also appears in the ribosomes of those cells that reached the stationary phase of growth at a physiological temperature . Our experiments with cell-free translation systems demonstrate that the protein inhibits translation at the elongation stage by blocking the binding of aminoacyl-tRNA to the ribosomal A site . The function of the protein in adaptation of cells to environmental stress is discussed.

Acta Crystallogr D Biol Crystallogr, 2001 Jun, 57(Pt 6), 909 - 11 Epub 2001 May 25.
Cloning, expression, purification and preliminary X-ray crystallographic studies of Escherichia coli Hsp100 ClpB nucleotide-binding domain 1 (NBD1); Li J et al.; Escherichia coli Hsp100 ClpB plays critical roles in multi-chaperone systems in cell physiology . After being activated by protein or peptide binding, ClpB disaggregates denatured polypeptides by employing ATP hydrolysis and allows other molecular chaperones such as Hsp70 DnaK and Hsp40 DnaJ to refold the non-native polypeptides . ClpB contains two nucleotide-binding domains with Walker A and B motifs within their primary sequences . Therefore, ClpB can be classified as a member of the large ATPase family known as ATPases associated with various cellular activities (AAAs) . The mechanisms by which the ClpB acts as a molecular chaperone to disaggregate denatured polypeptides are unknown . To investigate how the nucleotide-binding domain participates in ClpB chaperone activity, we have cloned and crystallized ClpB nucleotide-binding domain 1 (NBD1) . The ClpB NBD1 crystals diffract to 1.80 A using a synchrotron X-ray source and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 38.41, b = 65.48, c = 79.13 A . Structure determination by the MAD method is under way.

Acta Crystallogr D Biol Crystallogr, 2001 Jun, 57(Pt 6), 906 - 8 Epub 2001 May 25.
Crystallization and preliminary crystallographic characterization of catechol-O-methyltransferase in complex with its cosubstrate and an inhibitor; Rodrigues ML et al.; Catechol-O-methyltransferase (COMT) is involved in the metabolism of catecholamines, catechol steroids and xenobiotic catechols . A precise knowledge of the enzyme-inhibitor structural interactions could help in the design of better inhibitors . Soluble rat COMT was expressed in Escherichia coli and the recombinant protein was crystallized with a new tight-binding inhibitor, BIA 3-335 {1-(3,4-dihydroxy-5-nitrophenyl)-3-(n-3'-trifluoromethylphenyl)piperazine-1-propanone dihydrochloride} . The crystals were obtained by the sitting-drop vapour-diffusion method using PEG 6K as a precipitant . These crystals diffracted to better than 1.9 A and belong to the trigonal space group P3(2)21 . The unit-cell parameters for the crystal measured at room temperature were a = b = 51.5, c = 168.3 A; each shrank by about 1 A on freezing.

Acta Crystallogr D Biol Crystallogr, 2001 Jun, 57(Pt 6), 896 - 7 Epub 2001 May 25.
The MinD protein from the hyperthermophilic archaeon Pyrococcus horikoshii: crystallization and preliminary X-ray analysis; Sakai N et al.; MinD is one of the proteins regulating cell division . MinD from Escherichia coli has been designated as a type of motor protein which has an ATPase activity . This paper deals with the first crystallization and preliminary crystallographic analysis of recombinant MinD from Pyrococcus horikoshii (molecular weight 26.3 kDa) expressed in E . coli . Crystals of MinD were obtained by the hanging-drop vapour-diffusion method . MinD crystals belong to space group P2(1)3, with unit-cell parameters a = b = c = 98.5 A, and diffract to 3.0 A resolution . The asymmetric units each contain one molecule of MinD, giving a crystal volume per protein mass (V(M)) of 3.0 A(3) Da(-1) and a solvent content of 59.0%.

Acta Crystallogr D Biol Crystallogr, 2001 Jun, 57(Pt 6), 893 - 5 Epub 2001 May 25.
Purification, crystallization and preliminary X-ray diffraction analysis of the yeast Sec12Deltap protein, a guanine nucleotide-exchange factor involved in vesicle transport; Dumon-Seignovert L et al.; Sec12 is a guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Sar1 . Its GEF activity on Sar1 makes it a key element in vesicle budding from the endoplasmic reticulum to the Golgi apparatus in yeast . Sec12 is an integral membrane glycoprotein of 70 kDa . A 38.5 kDa N-cytoplasmic domain (Sec12Deltap) has been expressed in Saccharomyces cerevisiae and in Escherichia coli, purified to homogeneity and crystallized . Two crystal forms were obtained . Crystal form I belongs to space group P6(2)/P6(4), with unit-cell parameters a = b = 191.7, c = 53.3 A, gamma = 120 degrees, and diffracts to 2.6 A resolution . Crystal form II belongs to space group P1, with unit-cell parameters a = 52.6, b = 53.0, c = 116.8 A, alpha = 98.0, beta = 97.4, gamma = 93.4 degrees, and diffracts to 2.0 A resolution.

Acta Crystallogr D Biol Crystallogr, 2001 Jun, 57(Pt 6), 800 - 5 Epub 2001 May 25.
Solving a 300 kDa multimeric protein by low-resolution MAD phasing and averaging/phase extension; Gomis-Ruth FX et al.; The structure of the conjugative coupling protein TrwBDeltaN70 from Escherichia coli plasmid R388 was solved using two crystal forms . This large multimeric membrane protein of 437 residues per monomer is involved in cell-to-cell single-strand DNA transfer . Diffraction data to 2.4 A were available from trigonal crystals obtained from ammonium sulfate and to 2.5 A from monoclinic crystals grown from tartrate . A single tantalum bromide (Ta(6)Br(12)(2+)) derivative of the trigonal form, which presented a protein hexamer with C6 local symmetry in the asymmetric unit, was used in a three-wavelength MAD experiment to achieve 4.5 A resolution for initial phases . Sixfold averaging and phase extension increased the effective phasing resolution and eventually produced a straightforwardly traceable electron-density map . The monoclinic structure was solved by molecular replacement, i.e . a hexamer of the trigonal form was used as a search model . Two such hexamers are present in the asymmetric unit.

Acta Crystallogr D Biol Crystallogr, 2001 Jun, 57(Pt 6), 767 - 74 Epub 2001 May 25.
Atomic resolution structure of Escherichia coli dUTPase determined ab initio; Gonzalez A et al.; Cryocooled crystals of a mercury complex of Escherichia coli dUTPase diffract to atomic resolution . Data to 1.05 A resolution were collected from a derivative crystal and the structure model was derived from a Fourier map with phases calculated from the coordinates of the Hg atom (one site per subunit of the trimeric enzyme) using the program ARP/wARP . After refinement with anisotropic temperature factors a highly accurate model of the bacterial dUTPase was obtained . Data to 1.45 A from a native crystal were also collected and the 100 K structures were compared . Inspection of the refined models reveals that a large part of the dUTPase remains rather mobile upon freezing, with 14% of the main chain being totally disordered and with numerous side chains containing disordered atoms in multiple discrete conformations . A large number of those residues surround the active-site cavity . Two glycerol molecules (the cryosolvent) occupy the deoxyribose-binding site . Comparison between the native enzyme and the mercury complex shows that the active site is not adversely affected by the binding of mercury . An unexpected effect seems to be a stabilization of the crystal lattice by means of long-range interactions, making derivatization a potentially useful tool for further studies of inhibitor-substrate-analogue complexes of this protein at very high resolution.

Circ Res, 2001 May 25, 88(10), 1066 - 71
ADAR1 is involved in the development of microvascular lung injury; Rabinovici R et al.; Deamination of adenosine on pre-mRNA to inosine is a recently discovered process of posttranscription modification of pre-mRNA, termed A-to-I RNA editing, which results in the production of proteins not inherent in the genome . The present study aimed to identify a role for A-to-I RNA editing in the development of microvascular lung injury . To that end, the pulmonary expression and activity of the RNA editase ADAR1 were evaluated in a mouse model of endotoxin (15 mg/kg IP)-induced microvascular lung injury (n=5) as well as in cultured alveolar macrophages stimulated with endotoxin, live bacteria, or interferon . ADAR1 expression and activity were identified in sham lungs that were upregulated in lungs from endotoxin-treated mice (at 2 hours) . Expression was localized to polymorphonuclear and monocytic cells . These events preceded the development of pulmonary edema and leukocyte accumulation in lung tissue and followed the local production of interferon-gamma, a known inducer of ADAR1 in other cell systems . ADAR1 was found to be upregulated in alveolar macrophages (MH-S cells) stimulated with endotoxin (1 to 100 microg/mL), live Escherichia coli (5x10(7) colony-forming units), or interferon-gamma (1000 U/mL) . Taken together, these data suggest that ADAR1 may play a role in the pathogenesis of microvascular lung injury possibly through induction by interferon.

Appl Environ Microbiol, 2001 Jun, 67(6), 2445 - 52
Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence; Kannan Y et al.; The gene encoding subtilisin-like protease T . kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 . T . kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783 . It consists of a putative presequence, prosequence, and catalytic domain . Like bacterial subtilisins, T . kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule . However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T . kodakaraensis subtilisin was refolded in a form with a putative prosequence . This refolded protein of recombinant T . kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly(-82) to Gly(316)), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties . The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T . kodakaraensis subtilisin exists in a monomeric form . T . kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca(2+) ion with an optimal pH and temperature of pH 9.5 and 80 degrees C . Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues . However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80 degrees C, 20 min at 90 degrees C, and 7 min at 100 degrees C.

Biochem Biophys Res Commun, 2001 Jun 1, 284(1), 179 - 84
An in vivo approach to identifying sequence context of 8-oxoguanine mutagenesis; Watanabe T et al.; Base substitution mutations are not distributed randomly in that most are located at a few specific hotspots sites . We have been studying 7,8-dihydro-8-oxoguanine mutagenesis in Escherichia coli in the supF gene carried in a plasmid . Among hotspots, guanine within the 5'-AGA-3' located in the anticodon site was susceptible to the induction of G:C-->T:A transversion . In this study, we constructed variants of the supF gene in which the hotspot 5'-AGA-3' was modified to 5'-AGT-3', 5'-AGG-3' and 5'-AGC-3' to determine the influence of 3' neighboring base on G:C-->T:A mutational activity . Using these variant supF genes propagated in a 7,8-dihydro-8-oxoguanine repair-deficient host, we found that guanine within 5'-AGA-3' and 5'-AGG-3' produce G:C-->T:A, but guanine within 5'-AGT-3' and 5'-AGC-3' reduce the formation of G:C-->T:A . These changes were thus due to the effect of sequence context on the efficiency of mutation formation at the sites of 7,8-dihydro-8-oxoguanine . We also observed a longer range base-pair effect on hotspot formation .

Biochem Biophys Res Commun, 2001 Jun 1, 284(1), 57 - 64
Transcription of the ibpB heat-shock gene is under control of sigma(32)- and sigma(54)-promoters, a third regulon of heat-shock response; Kuczynska-Wisnik D et al.; The expression of the ibpAibpB heat-shock operon of Escherichia coli was found previously not to conform to the known pattern of expression of the sigma(32)-regulated operons because the rpoH gene mutation inactivating the sigma(32) protein did not abolish the ibp induction . We show here that this effect can depend partly on the sigma(54)-promoter that is inducible by heat shock, located upstream of the ibpB, the distal gene of the operon . It may also depend on a metabolic signal, postulated by others, and possibly required for the expression of the ibpAB genes . Thus, the ibpB gene can be translated from the transcript covering the whole operon starting from the sigma(32)-promoter and from the ibpB gene transcript starting from the sigma(54)-promoter . These results indicate that the ibpB gene is a second member of the sigma(54)-heat-shock regulon in E . coli besides pspA-E operon . Thus, heat-shock response involves three regulons controlled by sigma(32), sigma(24), and sigma(54) RNA polymerase subunits .

Folia Histochem Cytobiol, 2001, 39(2), 177 - 8
Immunoreactivity of iNOS in porcine uterus after infusions of Escherichia coli endotoxin; Jana B et al.; The aim of this study was to investigate the distribution of inducible isoform of nitric oxide synthase (iNOS) in the porcine uterus after infusions of Escherichia coli endotoxin (lipopolysaccharide, LPS) . In the group I (treated; n=6), 1 mg of LPS was infused into both the left and right uterine horn starting from the 4th to the 10th day of the estrous cycle, twice a day . In the group II (control; n=6), saline was infused into the uterus . The uterine horns were collected on the 14th day of the estrous cycle . Cryostat sections from the paraformaldehyde fixed tissues were stained immunohistochemically to estimate the distribution of iNOS . The luminal and glandular epithelium was stained more intensely for iNOS in the LPS-treated gilts than in the control animals . After LPS infusions, iNOS staining in vascular endothelial cells was also more intense than that observed in the controls . The present study has revealed that infusions of LPS into the porcine uterus result in an increase in the intensity of iNOS staining in some structures of this organ and supports our earlier data that NO can mediate an inflammatory effect of LPS in the uterus.

J Inorg Biochem, 2001 Apr, 84(3-4), 287 - 92
Structural characterization and cytostatic activity of chlorobischolylglycinatogold(III); Carrasco J et al.; Based on the ability of bile acids for vectorializing the cytostatic activity of other agents, we have designed and synthesized a new bile acid cholylglycinato Au(III) complex, named Bamet-A1 . It has been characterized by means of EA (elemental analysis), FT-IR, NMR, FAB-MS (fast atom bombardment-mass spectrometry) and Vis-UV techniques . This characterization allowed us to propose a structure of the type {Au CG(O) CG(N,O) Cl} for the neutral complex, which has the composition C522H84N2O12AuCl and is very soluble in water, methanol, ethanol and DMSO (dimethylsulfoxide) . The study in aqueous solution suggested a redox process for its transformation, which is accompanied by the appearance of colloidal gold phase . The behavior in 4 mM NaCl water (in order to mimic the cytoplasmatic fluid) was similar to that observed in water, while in a 150 mM NaCl (similar to extracellular fluid and serum), the apparition of a dark blue precipitate was observed . This complex displays fluorescence, which does not change when incubated with DNA obtained from E . coli . Bamet-A1 was found to inhibit the growth of a variety of cell lines . The cytostatic effect was mild against human hepatoma HepG2, mouse hepatoma Hepa 1-6, rat hepatoma McA RH-7777 and human colon adenocarcinoma LS-174T, and stronger against mouse sarcoma S180-II and mouse leukemia L-1210 cells . The appearance of colloidal Au during the process of hydrolysis under physiological conditions may explains the low cytostatic activity.

Langenbecks Arch Surg, 2001 Mar, 386(2), 146 - 9
Treatment of enterogenic endotoxinemia with lactoferrin in rats; Nebermann L et al.; Enterogenic endotoxinemia was induced in 28 Wistar rats by means of intraperitoneal injection of 80 mg/kg carrageenan and intraduodenal administration of 5 x 10(10)/kg Escherichia coli bacteria and 10 mg/kg nebacetin . The control group A received 600 mg/kg albumin in addition via the duodenal probe, and the groups B, C and D received 20, 40 and 80 mg/kg lactoferrin, respectively . The therapeutic effect was investigated by determining the endotoxin activity in the plasma every hour . In addition, the bacterial contamination of peritoneal lavages and mesenteric lymph nodes was checked by incubation for 48 h at 37 degrees C . The period of observation was 5 h . There was a dose-dependent improvement of the endotoxin activity in plasma and the bacterial contamination of the peritoneum cavity and mesenteric lymph nodes after lactoferrin administration . The maximum plasma endotoxin activity could be reduced by 89% with 80 mg/kg lactoferrin.

Nature, 2001 May 24, 411(6836), 498 - 501
Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide; Polacek N et al.; Peptide bond formation is the principal reaction of protein synthesis . It takes place in the peptidyl transferase centre of the large (50S) ribosomal subunit . In the course of the reaction, the polypeptide is transferred from peptidyl transfer RNA to the alpha-amino group of amino acyl-tRNA . The crystallographic structure of the 50S subunit showed no proteins within 18 A from the active site, revealing peptidyl transferase as an RNA enzyme . Reported unique structural and biochemical features of the universally conserved adenine residue A2451 in 23S ribosomal RNA (Escherichia coli numbering) led to the proposal of a mechanism of rRNA catalysis that implicates this nucleotide as the principal catalytic residue . In vitro genetics allowed us to test the importance of A2451 for the overall rate of peptide bond formation . Here we report that large ribosomal subunits with mutated A2451 showed significant peptidyl transferase activity in several independent assays . Mutations at another nucleotide, G2447, which is essential to render catalytic properties to A2451 (refs 2, 3), also did not dramatically change the transpeptidation activity . As alterations of the putative catalytic residues do not severely affect the rate of peptidyl transfer the ribosome apparently promotes transpeptidation not through chemical catalysis, but by properly positioning the substrates of protein synthesis.

Nat Struct Biol, 2001 Jun, 8(6), 510 - 4
Aminoglycoside binding displaces a divalent metal ion in a tRNA-neomycin B complex; Mikkelsen NE et al.; Aminoglycosides bind to RNA and interfere with its function, and it has been suggested that aminoglycoside binding to RNA displaces essential divalent metal ions . Here we demonstrate that addition of various aminoglycosides inhibited Pb2+-induced cleavage of yeast tRNA(Phe) . Cocrystallization of yeast tRNA(Phe) and an aminoglycoside, neomycin B, resulted in crystals that diffracted to 2.6 A and the structure of the complex was solved by molecular replacement . The structure shows that the neomycin B binding site overlaps with known divalent metal ion binding sites in yeast tRNA(Phe), providing direct evidence for the hypothesis that aminoglycosides displace metal ions . Additionally, the neomycin B binding site overlaps with major determinants for Escherichia coli phenylalanyl-tRNA-synthetase . Here we present data demonstrating that addition of neomycin B inhibited aminoacylation of E . coli tRNA(Phe) in the mid microM range . Given that aminoglycoside and metal ion binding sites overlap, we discuss that aminoglycosides can be considered as 'metal mimics'.

Crit Care Med, 2001 Apr, 29(4), 839 - 46
Sustained endotoxemia leads to marked down-regulation of early steps in the insulin-signaling cascade; McCowen KC et al.; OBJECTIVES: To determine the effects of sustained, 3-day endotoxin infusion on early steps of the insulin-signaling pathway in rat liver and skeletal muscle in vivo; to examine insulin signaling in well-established acute endotoxin models of insulin resistance . DESIGN: Prospective, controlled animal study . SETTING: University research laboratory . SUBJECTS: Male Sprague-Dawley rats: 24 in the 3-day endotoxin study, 22 in each acute endotoxin study . INTERVENTIONS: In prolonged endotoxemia studies, endotoxin (1 mg.kg-1.24 hrs-1) was administered via jugular venous catheter for 74 hrs . Insulin was then injected, and liver and skeletal muscle were removed after 5 mins . In acute endotoxemia studies, an endotoxin bolus (1 mg/kg) was administered, and insulin-signaling responses were studied after 4 hrs . MEASUREMENTS AND MAIN RESULTS: In liver of rats with sustained endotoxemia, there were significant decreases in insulin-stimulated tyrosine phosphorylation of insulin receptors (74%), insulin receptor substrate (IRS)-1 (74%), and IRS2 (53%); binding of the p85 subunit of phosphatidylinositide 3-kinase to IRS1 (80%); and IRS1-precipitable phosphatidylinositide 3-kinase activity (>90%) . These findings were associated with significant reductions in abundance of insulin receptors (37%), IRS1 (60%), and IRS2 (23%) . Signaling in skeletal muscle was similarly affected, with reduced IRS1 phosphorylation (49%), IRS1 abundance (50%), and binding of p85 to IRS1 (57%) . Insulin signaling 4 hrs after endotoxin administration was not different from controls . CONCLUSIONS: Prolonged endotoxemia is associated with marked deficits in early steps of the insulin-signaling pathway, which are at least partly explained by reduced abundance of the insulin receptor and IRS proteins . Signaling defects were not evident 4 hrs after endotoxin administration under conditions of adequate nutrition, indicating that insulin resistance develops gradually, may require concomitant malnutrition, and is not reversed by the development of endotoxin tolerance.

Crit Care Med, 2001 Apr, 29(4), 776 - 81
Modulation of soluble phases of endothelial/leukocyte adhesion molecule 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 with interleukin-1beta after experimental endotoxic challenge; Tellez Gil L et al.; OBJECTIVE: To evaluate the effect of treatment with interleukin 1beta (IL-1beta) on the concentrations of soluble adhesion molecules after an endotoxic challenge . DESIGN: Randomized, controlled study . SETTING: Experimental Unit, Virgen de las Nieves University Hospital . SUBJECTS: Seventy-two female CBA/H mice of 20 to 21 g, supplied by the animal center of the Experimental Unit . INTERVENTION: The mice were randomized into three groups of 24 . Group 1 (sham) received two intraperitoneal (ip) doses of 0.1 mL of phosphate-buffered saline; group 2 (lipopolysaccharide) was injected with 125 mg/kg lipopolysaccharide (Escherichia coli) (i.p.) 24 hrs after 0.1 mL of phosphate-buffered saline; group 3 was pretreated with 80 ng (i.p.) of IL-1beta per mouse 24 hrs before the endotoxic challenge . MEASUREMENTS AND MAIN RESULTS: At 1, 2, 4, and 24 hrs after the endotoxic challenge, the concentrations of soluble endothelial/leukocyte adhesion molecule 1 (ELAM-1), intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) were measured in the three groups . There was a significant increase (p <.01) in these concentrations at these times in comparison with the sham group . The use of IL-1beta produced a significant decrease (p <.05) in the three molecules among the treated group versus the group submitted only to the challenge; concentrations of ELAM-1 significantly decreased to below those of the sham group, and those of VCAM-1 reduced to levels that did not significantly differ from those of the sham group . CONCLUSION: Endotoxin administration significantly increases the concentrations of soluble ELAM-1, ICAM-1, and VCAM-1 in mice . Treatment with IL-1beta significantly decreases these concentrations, probably attenuating cell injury and organ dysfunction.

Crit Care Med, 2001 Apr, 29(4), 703 - 8
Inosine improves gut permeability and vascular reactivity in endotoxic shock; Garcia Soriano F et al.; OBJECTIVE: To investigate the effects of inosine administration on vascular reactivity, gut permeability, neutrophil accumulation and lipid peroxidation in tissues in murine endotoxin shock . DESIGN: Randomized, prospective laboratory study . SETTING: Research laboratory . SUBJECTS: BALB/c mice 6-8 wks age . INTERVENTIONS: BALB/c mice were randomly assigned to one of five groups: a) vehicle controls, which received saline intraperitoneally; b) inosine controls, which received inosine alone (100 mg/kg, ip); c) lipopolysaccharide (LPS)-treated animals, which received LPS (40 and 100 mg/kg, ip, depending on the experimental protocol); d) inosine pretreatment group, which received inosine (100 mg/kg, ip) 30 mins before LPS; and finally, e) inosine posttreatment group, which received inosine (100 mg/kg, ip) 60 mins after LPS . MEASUREMENTS AND MAIN RESULTS: The passage of fluorescein isothiocyanate-conjugated dextran (4 kDa, FD4) was analyzed in everted gut ileal sacs incubated ex vivo as an index of gut permeability . LPS induced a significant intestinal hyperpermeability, and inosine exerted protective effects both in pre- and posttreatment regimens . Myeloperoxidase and malondialdehyde were also measured to study neutrophil accumulation and lipid peroxidation in selected tissues . Inosine, both in pre- and posttreatment regimens ameliorated the increases in myeloperoxidase and malondialdehyde in the lung and gut . LPS-treated animals showed decreased contractile and relaxant responses, and inosine pretreatment (but not posttreatment) partially improved these responses . CONCLUSIONS: Taken together, inosine has organ protective effects during shock . A significant portion of its protective action is maintained even in the posttreatment scenario.

Crit Care Med, 2001 Mar, 29(3), 628 - 34
Effects of a dual inhibitor of tumor necrosis factor-alpha and interleukin-1 on lipopolysaccharide-induced lung injury in rats: involvement of the p38 mitogen-activated protein kinase pathway; Yoshinari D et al.; OBJECTIVE: Sepsis is a major cause of adult respiratory distress syndrome . In this study, we evaluated the effect of FR167653, which is a potent suppressant of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 production, on lipopolysaccharide (LPS)-induced lung injury and lethality in rats, and we examined the involvement of p38 mitogen-activated protein (MAP) kinase in the action of FR167653 . DESIGN: Prospective, randomized study . SETTING: Animal research facility in a university . SUBJECTS: Male Sprague-Dawley rats weighing 200-270 g . INTERVENTIONS: All the animals were assigned to one of the following four groups: control group, FR-only group, LPS-only group, and LPS/FR group . Animals in the LPS-only and LPS/FR groups received 6 mg/kg of LPS intravenously . The animals in the FR-only and LPS/FR groups also received an infusion of FR167653 at 0.2 mg x kg(-1) x hr(-1), commencing 30 mins before the LPS (or vehicle) injection and continuing for 5.5 hrs . MEASUREMENTS AND MAIN RESULTS: LPS significantly induced the accumulation of pulmonary neutrophils and lung edema, both of which were significantly attenuated by treatment with FR167653 . FR167653 also significantly decreased the LPS-induced lethality . Histologically, tissue damage was milder in the LPS/FR group than in the LPS-only group . Serum concentrations of TNF-alpha and IL-1beta and plasma concentrations of thromboxane B2 were all suppressed in the LPS/FR group compared with the LPS-only group . Western blot analysis revealed that FR167653 inhibited the phosphorylation of p38 MAP kinase in lung tissues . CONCLUSIONS: FR167653 administration decreased serum TNF-alpha and IL-1beta concentrations, which was associated with decreased lung injury and lethality . The mechanism responsible for the decreased TNF-alpha and IL-1 may be related to the inhibitory effect of FR167653 on p38 MAP kinase activation.

Crit Care Med, 2001 Mar, 29(3), 581 - 8
Effects of alpha - and beta -adrenergic stimulation on hepatosplanchnic perfusion and oxygen extraction in endotoxic shock; Zhang H et al.; OBJECTIVE: To examine the effects of adrenergic stimulation on hepatosplanchnic perfusion, oxygen extraction, and tumor necrosis factor-alpha production during endotoxic shock . DESIGN: In vivo, prospective, randomized, controlled, repeated-measures, experimental study . SETTING: Experimental physiology laboratory in a university teaching hospital . SUBJECTS: Twenty-one anesthetized and mechanically ventilated dogs . INTERVENTIONS: An intrapericardial catheter was positioned . Catheters for blood sampling were inserted into the right femoral artery, hepatic vein, portal vein, and pulmonary artery . Ultrasonic flow probes were placed around the portal vein, the hepatic artery, the mesenteric artery, the left renal artery, and the left femoral artery . Animals received 2 mg/kg of Escherichia coli endotoxin, followed by fluid resuscitation . Seven dogs received intravenous isoproterenol (0.1 microg/kg x min(-1)), seven received phenylephrine (1 microg/kg x min(-1)), and seven served as controls . Thirty minutes later, cardiac tamponade was introduced to study organ perfusion and tissue oxygen extraction capabilities . MAIN RESULTS: The isoproterenol group had a higher cardiac index and stroke index and lower systemic vascular resistance than the other groups . The phenylephrine group had a higher arterial pressure but a lower cardiac index than the isoproterenol group . The isoproterenol group had a higher hepatic artery blood flow than the other groups and a higher portal and mesenteric flow than the control group . Liver and gut mucosal blood flow was greater in the isoproterenol than in the phenylephrine group . The isoproterenol group had a lower global critical oxygen delivery than the other groups (8.8 +/- 1.3 vs . 13.1 +/- 2.0 (control) and 11.8 +/- 3.3 mL/kg x min(-1) (phenylephrine); both p < .05) and a higher liver critical oxygen extraction ratio than the control group . Isoproterenol tended to attenuate, but phenylephrine significantly increased, blood tumor necrosis factor levels . CONCLUSIONS: During endotoxic shock, beta-stimulation can improve hepatosplanchnic perfusion and enhance tissue oxygen extraction capabilities, whereas alpha-stimulation does not . In addition, alpha-adrenergic stimulation can increase tumor necrosis factor levels.

J Biol Chem, 2001 Jul 27, 276(30), 28509 - 15 Epub 2001 May 23.
Identification of endogenous SsrA-tagged proteins reveals tagging at positions corresponding to stop codons; Roche ED et al.; The SsrA.SmpB quality control system adds a C-terminal degradation peptide (AANDENYALAA) to nascent chains on stalled ribosomes, thereby freeing the ribosome and ensuring proteolysis of the tagged protein . An SsrA mutant with the tag sequence AANDEHHHHHH was used to slow degradation and facilitate Ni2+-nitrilotriacetic acid affinity purification . Display of affinity-purified Escherichia coli proteins on two-dimensional gels revealed small quantities of a diverse set of SsrA-H6-tagged proteins, and mass spectroscopy identified LacI repressor, lambda cI repressor, YbeL, GalE, RbsK, and a SlyD-kan(R) fusion protein as members of this set . For lambda repressor and YbeL, the SsrA-H6 tag was added after the natural C terminus of the protein, suggesting that tagging occurred while the ribosome idled at the termination codon of these genes . Potential causes of tagging for the other proteins include interference from translation of downstream reading frames, rare codons, and gene disruption . These and previous results support a broad role for the SsrA.SmpB system in freeing stalled ribosomes and in directing degradation of the products of these frustrated protein synthesis reactions.

J Biol Chem, 2001 Jul 27, 276(30), 27806 - 15 Epub 2001 May 23.
Identification of phosphorylation sites for Bruton's tyrosine kinase within the transcriptional regulator BAP/TFII-I; Egloff AM et al.; Bruton's tyrosine kinase (Btk), a member of the Tec family of cytosolic kinases, is essential for B cell development and function . BAP/TFII-I, a protein implicated in transcriptional regulation, is associated with Btk in B cells and is transiently phosphorylated on tyrosine following B cell receptor engagement . BAP/TFII-I is a substrate for Btk in vitro and is hyperphosphorylated on tyrosine upon coexpression with Btk in mammalian cells . In an effort to understand the physiologic consequences of BAP/TFII-I tyrosine phosphorylation following B cell receptor stimulation, site-directed mutagenesis and phosphopeptide mapping were used to locate the predominant sites of BAP/TFII-I phosphorylation by Btk in vitro . These residues, Tyr248, Tyr357, and Tyr462, were also found to be the major sites for Btk-dependent phosphorylation of BAP/TFII-I in vivo . Residues Tyr357 and Tyr462 are contained within the loop regions of adjacent helix-loop-helix-like repeats within BAP/TFII-I . Mutation of either Tyr248, Tyr357, or Tyr462 to phenylalanine reduced transcription from a c-fos promoter relative to wild-type BAP/TFII-I in transfected COS-7 cells, consistent with the interpretation that phosphorylation at these sites contributes to transcriptional activation . Phosphorylation of BAP/TFII-I by Btk may link engagement of receptors such as surface immunoglobulin to modulation of gene expression.

J Biol Chem, 2001 Jul 27, 276(30), 27967 - 74 Epub 2001 May 23.
Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II; Kremer L et al.; Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis . MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis . To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA:AcpM transacylase) from M . tuberculosis . According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry . The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E . coli fabD mutant . Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro . Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD . In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated . All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation . The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M . tuberculosis . Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.

Anal Biochem, 2001 Jun 1, 293(1), 38 - 42
Heparin-enhanced zymographic detection of matrilysin and collagenases; Yu WH et al.; Unlike the gelatinases (MMP-2 and -9), matrilysin (MMP-7) and collagenases (MMP-1 and -13) are difficult to detect at low levels in conventional casein or gelatin zymography . In this report, heparin was used to enhance the zymographic assays for MMP-7, -1, and -13 . With the addition of heparin to the enzyme sample, MMP-7 can be detected at a level of 30 pg in transferrin zymography and MMP-1 and -13 can be detected at a level of 0.2 ng in gelatin zymography . Carboxymethylated transferrin is used instead of casein as a substrate for assaying rat MMP-7 . This substrate does not require a prerun step or substrate cross-linking to give uniform staining and clear band formation . It is necessary for heparin to run to the same region of the gel as the enzyme to produce its enhancing effect . For MMP-7 movement of heparin and enzyme is almost equal; for the collagenases it is necessary to add heparin to each well after the electrophoretic run is underway . Possible mechanisms of activity enhancement are discussed .

Anal Biochem, 2001 Jun 1, 293(1), 31 - 7
Development of a novel helicase assay using electrochemiluminescence; Zhang L et al.; DNA helicases are ubiquitous enzymes involved in DNA replication, recombination, and repair . These enzymes use the energy of ATP to unwind duplex DNA . A common approach to measure DNA helicase activity utilizes electrophoresis to visualize product formation in a gel . The present study develops a rapid helicase assay based on electrochemiluminescence . The assay is adaptable to rapid high-throughput screening for chemical inhibitors . The assay is applied to Escherichia coli DnaB . Three other technologies have also been applied to DnaB and are compared to results of the electrochemiluminescence-based assay .

J Trauma, 2001 May, 50(5), 882 - 6
Hypoxic pulmonary vasoconstriction increases during endotoxemia in the perfused rat lung; Castaneda J et al.; BACKGROUND: Several investigations have studied hypoxic pulmonary vasoconstriction (HPV) during endotoxemia, as in this situation there is an increase in the activation of the inducible nitric oxide synthases, producing a greater liberation of nitric oxide (NO) in the pulmonary vessels . However, these studies yielded conflicting or at times contradictory results, since reference has been made to both enhancement and inhibition of HPV . Our objective was to determine the effect of hypoxia on the isolated blood-perfused lung of endotoxemic rats, and to give at least a partial explanation of its production mechanism . METHODS: Pulmonary arterial pressure (PAP) was measured in a blood-perfused lung preparation from Wistar rats in normoxia (O2, 20%; CO2, 5%; N, 75%) and hypoxia (O2, 2%; CO2, 5%; N, 93%) . There were three experimental protocols . We studied the effect of hypoxia in a control group (CG) and an endotoxemic group (EG) . Second, we studied the effect of hypoxia in endotoxemic rats pretreated with indomethacin (E+IG) . Third, we assessed the effect of two inhibitors of NO synthesis: N-methyl-l-arginine (NMLA) and methylene blue (MB) on two subgroups of groups CG (CGnmla and CGmb) and EG (EGnmla and EGmb) . With the exception of the CG, all specimens were pretreated with a 20-mg/kg intraperitoneal injection of Escherichia coli lipopolysaccharide . RESULTS: DeltaPAP elicited by hypoxia in the EG group (15.90 +/- 4.75 mm Hg) was 2.30 times higher than in the CG (6.89 +/- 1.96 mm Hg) . In the E+IG group, hypoxia produced a DeltaPAP of 15.20 +/- 3.56 mm Hg, similar to that in the EG . The addition of MB in the EGmb subgroup increased base PAP during normoxia from 19.1 +/- 1.23 mm Hg to 32.2 +/- 6.1 mm Hg (p < 0.05) . CONCLUSION: In an isolated-perfused rat model, E . coli lipopolysaccharide (20 mg/kg) significantly increased HPV . This response is maintained over time . Inhibition of NO release by hypoxia may be responsible for the enhanced HPV after endotoxin.

J Biol Chem, 2001 Jul 27, 276(30), 28291 - 9 Epub 2001 May 22.
Interaction of Escherichia coli MutS and MutL at a DNA mismatch; Schofield MJ et al.; MutS and MutL are both required to activate downstream events in DNA mismatch repair . We examined the rate of dissociation of MutS from a mismatch using linear heteroduplex DNAs or heteroduplexes blocked at one or both ends by four-way DNA junctions in the presence and absence of MutL . In the presence of ATP, dissociation of MutS from linear heteroduplexes or heteroduplexes blocked at only one end occurs within 15 s . When both duplex ends are blocked, MutS remains associated with the DNA in complexes with half-lives of 30 min . DNase I footprinting of MutS complexes is consistent with migration of MutS throughout the DNA duplex region . When MutL is present, it associates with MutS and prevents ATP-dependent migration away from the mismatch in a manner that is dependent on the length of the heteroduplex . The rate and extent of mismatch-provoked cleavage at hemimethylated GATC sites by MutH in the presence of MutS, MutL, and ATP are the same whether the mismatch and GATC sites are in cis or in trans . These results suggest that a MutS-MutL complex in the vicinity of a mismatch is involved in activating MutH.

J Bacteriol, 2001 Jun, 183(12), 3804 - 10
Oxaloacetate synthesis in the methanarchaeon Methanosarcina barkeri: pyruvate carboxylase genes and a putative Escherichia coli-type bifunctional biotin protein ligase gene (bpl/birA) exhibit a unique organization; Mukhopadhyay B et al.; Evidence is presented that, in Methanosarcina barkeri oxaloacetate synthesis, an essential and major CO(2) fixation reaction is catalyzed by an apparent alpha(4)beta(4)-type acetyl coenzyme A-independent pyruvate carboxylase (PYC), composed of 64.2-kDa biotinylated and 52.9-kDa ATP-binding subunits . The purified enzyme was most active at 70 degrees C, insensitive to aspartate and glutamate, mildly inhibited by alpha-ketoglutarate, and severely inhibited by ATP, ADP, and excess Mg(2+) . It showed negative cooperativity towards bicarbonate at 70 degrees C but not at 37 degrees C . The organism expressed holo-PYC without an external supply of biotin and, thus, synthesized biotin . pycA, pycB, and a putative bpl gene formed a novel operon-like arrangement . Unlike other archaeal homologs, the putative biotin protein ligases (BPLs) of M . barkeri and the closely related euryarchaeon Archaeoglobus fulgidus appeared to be of the Escherichia coli-type (bifunctional, with two activities: BirA or a repressor of the biotin operon and BPL) . We found the element Tyr(Phe)ProX(5)Phe(Tyr) to be fully conserved in biotin-dependent enzymes; it might function as the hinge for their "swinging arms."

J Bacteriol, 2001 Jun, 183(12), 3800 - 3
Cs(+) induces the kdp operon of Escherichia coli by lowering the intracellular K(+) concentration; Jung K et al.; Cs(+) was found to induce expression of the kdpFABC operon, encoding a high-affinity K(+) uptake system of Escherichia coli . Quantitative expression analyses at the transcriptional and translational levels reveal that CsCl causes much higher induction of kdpFABC than does NaCl . A decrease of the intracellular K(+) concentration is found in cells exposed to CsCl . The results indicate that kdpFABC expression is induced when the intracellular K(+) concentration is lowered . Moreover, the results imply that the signal transduction cascade mediated by KdpD and KdpE is able to integrate multiple signals.

J Bacteriol, 2001 Jun, 183(12), 3631 - 5
Very-short-patch repair in Escherichia coli requires the dam adenine methylase; Bell DC et al.; Strains of Escherichia coli which lack the dam-encoded adenine methylase are mutators due to a reduction in the efficiency of postreplication mismatch repair . In this study, we show that Dam(-) strains are also defective in very-short-patch repair, the system which corrects T/G mismatches arising from the deamination of 5-methylcytosine . This defect is associated with decreased levels of Vsr, the endonuclease which initiates short-patch repair . We also show that production of the dcm-encoded cytosine methylase is unaffected in Dam(-) strains . Since the dcm and vsr genes are cotranscribed, the regulation of Vsr by Dam is probably posttranscriptional.

Biophys J, 2001 Jun, 80(6), 2954 - 67
Visualization and tracking of single protein molecules in the cell nucleus; Kues T et al.; A recently developed laser fluorescence videomicroscopy method was used to determine for the first time the intranuclear trajectories of single protein molecules . Using the recombinant Escherichia coli beta-galactosidase protein P4K, labeled with an average of 4.6 ALEXA 488 chromophores per tetramer, single P4K molecules could be localized and tracked in the nuclei of permeabilized 3T3 cells at a spatial accuracy of approximately 30 nm and a time resolution of 18 ms . Our previous photobleaching measurements indicated that P4K had two fractions inside the nucleus, a larger mobile and a smaller immobile fraction . The present study supported this observation but revealed a much larger variety of mobility classes . Thus, a fraction of P4K molecules appeared to be truly immobile while another fraction was mobile but confined to very small areas . In addition, a large fraction of the P4K molecules appeared to be mobile and to move over extended distances by diffusion . However, a quantitative analysis showed that at least two subpopulations were present differing widely in diffusion coefficients . Importantly, both the diffusion coefficients and the fractions of these subpopulations were time-dependent . Our results suggest that proteins can move inside the nucleus over extended distances by diffusion . However, intranuclear protein diffusion is severely restricted, most likely by multiple association-dissociation events and/or impermeable obstacles.

Biophys J, 2001 Jun, 80(6), 2649 - 57
Rapid stiffening of integrin receptor-actin linkages in endothelial cells stimulated with thrombin: a magnetic bead microrheology study; Bausch AR et al.; By using magnetic bead microrheology we study the effect of inflammatory agents and toxins on the viscoelastic moduli of endothelial cell plasma membranes in real time . Viscoelastic response curves were acquired by applying short force pulses of ~500 pN to fibronectin-coated magnetic beads attached to the surface membrane of endothelial cells . Upon addition of thrombin, a rapid stiffening of the membrane was observed within 5 s, followed by recovery of the initial deformability within 2 min . By using specific inhibitors, two known pathways by which thrombin induces actin reorganization in endothelial cells, namely activation of Ca2+-calmodulin-dependent myosin light chain kinase and stimulation of Rho/Rho-kinase, were excluded as possible causes of the stiffening effect . Interestingly, the cytotoxic necrotizing factor of Escherichia coli, a toxin which, in addition to Rho, activates the GTPases Rac and CDC42Hs, also induced a dramatic stiffening effect, suggesting that the stiffening may be mediated through a Rac- or Cdc42Hs-dependent pathway . This work demonstrates that magnetic bead microrheometry is not only a powerful tool to determine the absolute viscoelastic moduli of the composite cell plasma membrane, but also a valuable tool to study in real time the effect of drugs or toxins on the viscoelastic parameters of the plasma membrane.

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