Escherichia coli

Mol Microbiol, 2003 Jan, 47(2), 383 - 96
Proteome analysis of Escherichia coli K-12 by two-dimensional native-state chromatography and MALDI-MS; Champion MM et al.; To identify proteins expressed in Escherichia coli K-12 MG1655 during exponential growth in defined medium, we separated soluble proteins of E . coli over two dimensions of native-state high-performance liquid chromatography, and examined the components of the protein mixtures in each of 380 fractions by peptide mass fingerprinting . To date, we have identified the products of 310 genes covering a wide range of cellular functions . Validation of protein assignments was made by comparing the assignments of proteins to specific first-dimension fractions to proteins visualized by two-dimensional gel electrophoresis . Co-fractionation of proteins suggests the possible identities of components of multiprotein complexes . This approach yields high-throughput gel-independent identification of proteins . It can also be used to assign identities to spots visualized by two-dimensional gels, and should be useful to evaluate differences in expressed proteome content and protein complexes among strains or between different physiological states.

Mol Microbiol, 2003 Jan, 47(2), 345 - 55
A conserved sequence at the C-terminus of MinD is required for binding to the membrane and targeting MinC to the septum; Hu Z et al.; MinD is a key component of an oscillatory system that spatially regulates cell division in Escherichia coli . It is a peripheral membrane ATPase that recruits MinC and oscillates between the two halves of the cell in a MinE dependent manner . In vitro MinD binds to phospholipid vesicles in an ATP-dependent manner and is released through MinE-stimulated ATP hydrolysis . In this study we examined the function of the conserved C-terminus of MinD . Short truncations of three and ten amino acids dramatically decreased the ability of MinD to localize to the membrane and spatially regulate division . These truncations bound MinC but were deficient in targeting MinC to the septum . In vitro they dimerized, but were deficient in binding to phospholipid vesicles and undergoing MinE stimulation . We suggest a model in which the ATP-dependent dimerization of MinD affects the conformation of the C-terminal region, a potential amphipathic helix, triggering membrane binding.

J Med Chem, 2003 Jan 16, 46(2), 207 - 9
Potential tumor-selective nitroimidazolylmethyluracil prodrug derivatives: inhibitors of the angiogenic enzyme thymidine phosphorylase; Cole C et al.; Thymidine phosphorylase (TP) is an angiogenic growth factor and a target for anticancer drug design . Molecular modeling suggested that 2'-aminoimidazolylmethyluracils would be potent inhibitors of TP . The novel 5-halo-2-aminoimidazolylmethyluracils (4b/4c) were very potent inhibitors of E . coli TP (IC50 approximately 20 nM) . Contrastingly, the corresponding 2'-nitroimidazolylmethyluracil (as bioreductively activated) prodrugs (3b/3c) were 1000-fold less active (IC50 22-24 microM) . This approach may be used to selectively deliver TP inhibitors into hypoxic regions of solid tumors where TP is overexpressed.

An R Acad Nac Med (Madr), 2002, 119(2), 237 - 47; discussion 247-54
{Bone regeneration in oral surgery}; Sanz Casado JV; Bone regeneration is the technique of choice in order to repair small and medium sized bone defects in maxillary . It is used as an auxiliary medical treatment for dental implants . In this work, we show the different state of the art techniques used in bone lesions repair, and we also show experimental results obtained in our research group using bone morphogenetic protein BMP-2 . This protein has been expressed in an E . Coli strain previously modified with the protein nucleotide sequence . We also show a carrier developed in our laboratory that is able to bind the rhBMP-2 and to liberate it in its active form . The mechanical properties of the carrier can be chemically modified . We have tested Ti implants covered with films of this material activated with rhBMP-2 . The experimental in vivo results obtained with this kind of implants has been impressive.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jan, 35(1), 13 - 7
Expression, purification and in vitro N-myristoylation of human Src N-terminal region; Ma HH et al.; The DNA fragment encoding N-terminal region of human c-Src was amplified from Caco-2 cell total RNA by RT-PCR and cloned into vector pMFHT to obtain His-tag fusion expression plasmid pMF-SrcHT, which was based on T7 expression system . The fusion protein SrcHT was highly expressed in E.coli BL21(DE3) harboring the pMF-SrcHT and purified from bacterial lysate by Ni-IDA affinity chromatography . The assays using {(3)H}-labeled substrate demonstrate that the purified fusion protein SrcHT can be effectively N-myristoylated by recombinant human myristoyl-CoA: protein N-myristoyltransferase (NMT) in vitro . This work is a basis for further biochemical studies and development of new anti-cancer chemotherapeutic drugs based on specific inhibition of N-myristoylation of human Src.

Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 56 - 61 Epub 2002 Dec 23.
Addition of the keto functional group to the genetic code of Escherichia coli; Wang L et al.; Although the keto group is the most versatile of the functional groups in organic chemistry, it is absent in the genetically encoded amino acids . To overcome this natural limitation on protein biosynthesis, we have evolved an orthogonal tRNA-synthetase pair that makes possible the efficient incorporation of a keto amino acid, p-acetyl-l-phenylalanine, into proteins in E . coli with high translational fidelity in response to the amber nonsense codon . To demonstrate the utility of this keto amino acid, we have used it to modify a protein selectively with a small molecule fluorophore and biotin derivative . This additional genetically encoded amino acid should greatly expand our ability to manipulate protein structure and function both in vitro and in living cells.

Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 20 - 5 Epub 2002 Dec 23.
Genome-based peptide fingerprint scanning; Giddings MC et al.; We have implemented a method that identifies the genomic origins of sample proteins by scanning their peptide-mass fingerprint against the theoretical translation and proteolytic digest of an entire genome . Unlike previously reported techniques, this method requires no predefined ORF or protein annotations . Fixed-size windows along the genome sequence are scored by an equation accounting for the number of matching peptides, the number of missed enzymatic cleavages in each peptide, the number of in-frame stop codons within a window, the adjacency between peptides, and duplicate peptide matches . Statistical significance of matching regions is assessed by comparing their scores to scores from windows matching randomly generated mass data . Tests with samples from Saccharomyces cerevisiae mitochondria and Escherichia coli have demonstrated the ability to produce statistically significant identifications, agreeing with two commonly used programs, peptident and mascot, in 86% of samples analyzed . This genome fingerprint scanning method has the potential to aid in genome annotation, identify proteins for which annotation is incorrect or missing, and handle cases where sequencing errors have caused framing mistakes in the databases . It might also aid in the identification of proteins in which recoding events such as frameshifting or stop-codon read-through have occurred, elucidating alternative translation mechanisms . The prototype is implemented as a clientserver pair, allowing the distribution, among a set of cluster nodes, of a single or multiple genomes for concurrent analysis.

Blood, 2003 Apr 1, 101(7), 2529 - 33 Epub 2002 Nov 27.
Thromboembolic events in children with acute lymphoblastic leukemia (BFM protocols): prednisone versus dexamethasone administration; Nowak-Gottl U et al.; Alterations in hemostasis leading to symptomatic thromboembolism have been observed in patients with acute lymphoblastic leukemia (ALL) receiving Escherichia coli asparaginase (CASP) combined with steroids . Moreover, hereditary prothrombotic risk factors are associated with an increased risk for venous thromboembolism in pediatric ALL patients treated according to the BFM 90/95 protocols (including CASP combined with prednisone during induction therapy) . To assess whether the thromboembolic risk associated with established prothrombotic risk factors is modified by treatment modalities (prednisone or dexamethasone), the present analysis was performed . Three hundred thirty-six consecutively recruited leukemic children treated according to different BFM protocols (PRED group, n = 280, 60 mg/m(2) prednisone; DEXA group, n = 56, 10 mg/m(2) dexamethasone during induction therapy) were studied . Study end point was the onset of symptomatic vascular accidents during induction therapy . Cumulative thromboembolism-free survival was significantly reduced in children in the PRED group (thrombosis frequency, 10.4%) compared with children in the DEXA group (thrombosis frequency, 1.8%; P =.028) . Although no significant difference was found in the overall prevalence of prothrombotic risk factors, 46.5% of patients in the PRED group who experienced thromboembolic events were carriers of a prothrombotic risk factor, whereas no carrier in the DEXA group had a thromboembolism . At the time of maximum CASP activity, fibrinogen and activities of antithrombin, plasminogen, and protein S were significantly reduced in the PRED group . No significant correlation could be found between CASP activity and levels of coagulation factors . In conclusion, the use of dexamethasone instead of prednisone, administered with CASP, significantly reduced the onset of venous thromboembolism.

J Biol Chem, 2003 Mar 21, 278(12), 10157 - 61 Epub 2003 Jan 06.
The selectivity and inhibition of AlkB; Welford RW et al.; AlkB is one of four proteins involved in the adaptive response to DNA alkylation damage in Escherichia coli and is highly conserved from bacteria to humans . Recent analyses have verified the prediction that AlkB is a member of the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family of enzymes . AlkB mediates repair of methylated DNA by direct demethylation of 1-methyladenine and 3-methylcytosine lesions . Other members of the Fe(II) and 2OG-dependent oxygenase family, including those involved in the hypoxic response, are targets for therapeutic intervention . Assays measuring 2OG turnover were used to investigate the selectivity of AlkB . 1-Methyladenosine, 1-methyl-2'-deoxyadenosine, 3-methylcytidine, and 3-methyl-2'-deoxycytidine all stimulated 2OG turnover by AlkB but were not demethylated indicating an uncoupling of 2OG and prime substrate oxidation and that oligomeric DNA is required for hydroxylation and subsequent demethylation . In contrast the equivalent unmethylated nucleosides did not stimulate 2OG turnover indicating that the presence of a methyl group in the substrate is important in initiating oxidation of 2OG . Stimulation of 2OG turnover by 1-methyladenosine was highly dependent on the presence of a reducing agent, ascorbate or dithiothreitol . Following the observation that AlkB is inhibited by high concentrations of 2OG, analogues of 2OG, including 2-mercaptoglutarate, were found to specifically inhibit AlkB . The flavonoid quercetin inhibits both AlkB and the 2OG oxygenase factor-inhibiting hypoxia-inducible factor (FIH) in vitro . FIH inhibition by quercetin occurs in the presence of excess iron indicating a specific interaction, while the inhibition of AlkB by quercetin is, predominantly, due to nonspecific iron chelation.

Trends Biochem Sci, 2003 Jan, 28(1), 2 - 5
AlkB mystery solved: oxidative demethylation of N1-methyladenine and N3-methylcytosine adducts by a direct reversal mechanism; Begley TJ et al.; All organisms have multiple DNA repair pathways to protect against alkylation-induced mutation and cell death . For nearly two decades, we have known that the Escherichia coli alkB gene product protects against cell killing by S(N)2-alkylating agents, probably through DNA repair . Despite numerous attempts, a specific DNA repair activity could not be assigned to AlkB . Now, a breakthrough in biology and biochemistry, coupled with the discovery of an in silico protein structure, has uncovered a novel direct reversal DNA repair mechanism that is catalyzed by AlkB, namely the oxidative demethylation of N1-methyladenine or N3-methylcytosine DNA lesions . This reaction occurs on both single- and double-stranded DNA, and requires AlkB-bound non-heme Fe(2+), O(2) and alpha-ketogluterate to oxidize the offending methyl group . This is followed by the release of succinate, CO(2) and formaldehyde, and the restoration of undamaged A or C in DNA.

Mutat Res, 2003 Jan 28, 522(1-2), 33 - 44
Induction of SOS response, cellular efflux and oxidative stress response genes by chlorambucil in DNA repair-deficient Escherichia coli cells (ada, ogt and mutS); Salmelin C et al.; Chlorambucil (CLB) is a bifunctional alkylating drug widely used as an anticancer agent and as an immunosuppressant . It is known to be mutagenic, teratogenic and carcinogenic . The cellular actions of CLB have remained poorly investigated . It is very likely that DNA damage and its repair are the key elements determining the destiny of CLB-exposed cells . We investigated the role of two specific DNA repair pathways involved in CLB-induced mutagenicity and gene expression changes by using Escherichia coli strains lacking either (i) two DNA methyltransferase functions (O(6)-methylguanine-DNA methyltransferase I (ada) and II (ogt)), or (ii) mismatch repair (MutS (mutS)) . Mutagenicity was determined as the development of ciproxin and rifampicin resistance and the gene expression changes were assessed using expression profiling of all E . coli 4290 open reading frames (ORFs) by cDNA array . Chlorambucil-induced mutants in mutS cells, implying the importance of mismatch repair in preventing CLB-induced mutations . It also induced mutants in the ada, ogt strain, but to a lesser extent than in the wild-type strain . The simultaneous upregulation of several genes of the SOS response, cellular efflux and oxidative stress response, was demonstrated in both of the DNA repair-deficient strains but not in the wild-type cells . These and our previous results show that single-gene knock-out cells use specific gene regulation strategies to avoid mutations and cell death induced by agents such as chlorambucil .

Mutat Res, 2003 Jan 28, 522(1-2), 27 - 32
Mismatch repair in the antimutator Escherichia coli mud; Dzidic S et al.; Antimutators are genetic mutants that produce mutations at reduced rates compared to the wild type strain . They are interesting because they may provide insights into the mechanisms by which spontaneous mutations occur . We have investigated a reported antimutator strain of Escherichia coli termed mud for its possible mechanism . The mud strain exhibits a decrease in both spontaneous mutagenesis and mutability with alkylated agents and base analogs . These types of DNA lesions are known to be the substrates for the E . coli methyl-directed mismatch repair encoded by the mutHLSU system . We investigated whether the putative antimutator effect results from the increased expression or activity of the mutHLSU system . To directly measure the mismatch repair capacity of mud cells, we have transfected them with phage lambda heteroduplexes and scored the fraction of mixed (unrepaired) infective centers . This transfection system has been used routinely to assay mismatch repair capacity in E . coli and other organisms . No difference between mud and wild type cells is observed . From the results of the experiments we conclude that the reported antimutator effect of mud does not result from enhanced mismatch repair capacity . This conclusion is consistent with recently published evidence that the mud effect does not represent a real antimutator effect, but is an artifact due to impaired growth of mud cells under certain selective conditions .

Structure (Camb), 2003 Jan, 11(1), 31 - 42
Structure of Escherichia coli ribose-5-phosphate isomerase: a ubiquitous enzyme of the pentose phosphate pathway and the Calvin cycle; Zhang R et al.; Ribose-5-phosphate isomerase A (RpiA; EC 5.3.1.6) interconverts ribose-5-phosphate and ribulose-5-phosphate . This enzyme plays essential roles in carbohydrate anabolism and catabolism; it is ubiquitous and highly conserved . The structure of RpiA from Escherichia coli was solved by multiwavelength anomalous diffraction (MAD) phasing, and refined to 1.5 A resolution (R factor 22.4%, R(free) 23.7%) . RpiA exhibits an alpha/beta/(alpha/beta)/beta/alpha fold, some portions of which are similar to proteins of the alcohol dehydrogenase family . The two subunits of the dimer in the asymmetric unit have different conformations, representing the opening/closing of a cleft . Active site residues were identified in the cleft using sequence conservation, as well as the structure of a complex with the inhibitor arabinose-5-phosphate at 1.25 A resolution . A mechanism for acid-base catalysis is proposed.

Anim Biotechnol, 2002 Nov, 13(2), 179 - 93
Cloning, characterization, and expression studies in Escherichia coli of growth hormone cDNAs from Indian zebu cattle, reverine buffalo, and beetal goat; Mukhopadhyay UK et al.; The growth hormone cDNAs from three different economically important animal species of indian origin viz., indian zebu cattle (Bos indicus), indian reverine buffalo (Bubalus bubalis), and beetal goat (Capra hircus) were isolated by the RT-PCR technique . The amplified product was then cloned into phagemid pBluescriptIIKS- and the nucleotide sequence of the entire 573 base coding region for each product was determined . The genetic sequences as well as the translated protein sequence of these ruminant species were compared to that of closely related species like taurine cattle (Bos taurus) and sheep (Ovis aries) . A very high degree of nucleotide sequence homology, ranging between 97-98%, was observed . Subsequently, the buffalo and goat cDNAs were used for expression studies in Escherichia coli . Very low levels of expression resulted when the growth hormone cDNAs were directly placed under the strong E . coli (trc) or phage (T7) promoters with the approximate level being less than 0.1% and 1% of the intracellular E . coli proteins, respectively . The nearly 10-fold enhancement of the level of expression as observed was attributable to the nature of the untranslated leader sequence donated by the individual expression element . High level (about 20% of soluble E . coli protein) expression of buffalo/goat growth hormone was achieved as a fusion protein with glutathione-s-transferase (GST) in pGEX-KT . Further, although attempts at converting the GST-GH fusion protein system to a two-cistronic gene expression system were unsuccessful, the utilization of a short synthetic first cistron in the two-cistronic mode of expression resulted in high levels (approximately 30% of soluble protein cell fraction) of GH polypeptide with a native N-terminus in E . coli for all three cDNAs.

Microbiol Immunol, 2002, 46(11), 777 - 80
Monoclonal antibody to shiga toxin 1, which blocks receptor binding and neutralizes cytotoxicity; Nakao H et al.; A monoclonal antibody, 5-5B, which neutralizes Shiga toxin 1 (Stx1) cytotoxicity of Escherichia coli, was constructed . An epitope analysis indicated that Asn55 in Stx1 B subunit was an important residue . This result and our previous results using an anti-Stx2 monoclonal antibody indicate that the region around the cysteine residue of the disulfide bond might be important for the neutralization of Stx cytotoxicity, making it a potential vaccination candidate.

Eur J Immunol, 2002 Dec, 32(12), 3708 - 13
Endotoxin-free heat-shock protein 70 fails to induce APC activation; Bausinger H et al.; Previous work has suggested that the peptide-carrier, heat-shock protein (hsp)70, could directly activate APC . Here we show that this ability is related to endotoxin contamination of the human rhsp70 produced in Escherichia coli . Hence, the ability of 1-3 microg/ml of rhsp70 to induce the maturation of human monocyte-derived DC is abrogated in the presence of the LPS-antagonist polymyxin B or when the rhsp70 contains less than 60 IU/mg endotoxin . Such a level of contamination of the rhsp70 is, however, sufficient - in the presence of soluble rCD14, the LPS co-receptor - to induce cytokine secretion from monocytes and DC, despite the presence of polymyxin B . However, when endotoxin contamination is below 10 IU/mg, rhsp70 does not induce cytokine secretion - even in the presence of soluble rCD14 - or activate p38 mitogen-activated protein kinase signaling pathways, thus showing that an "endotoxin free" hsp70 does not activate APC.

J Gene Med, 2003 Jan, 5(1), 30 - 7
Gene therapy for prostate cancer using the cytosine deaminase/uracil phosphoribosyltransferase suicide system; Miyagi T et al.; BACKGROUND: Cytosine deaminase (CD) activates prodrug 5-FC to 5-FU and is used for suicide gene therapy (the CD/5-FC system) . E . coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that directly converts 5-FU into 5-fluorouridine monophosphate and improves the antitumoral effect of 5-FU . This study demonstrates the effectiveness of transduction of the UPRT gene in addition to CD/5-FC cancer suicide gene therapy . METHODS: We investigated a combined suicide gene transduction therapy for human hormone independent prostate cancer cell line DU145 using two separate adenovirus vectors expressing the E . coli CD and E . coli UPRT genes and systemic 5-FC administration (the CD+UPRT/5-FC system) . RESULTS: Cells transfected with AdCA-UPRT showed approximately 57 times lower IC50 to 5-FU compared with those transfected with AdCA-LacZ . Furthermore, cells transfected with AdCA-CD and AdCA-UPRT proved to be more sensitive to 5-FC compared with those transfected with AdCA-CD . Intratumoral injection of AdCA-CD and AdCA-UPRT drastically suppressed the growth of tumors which had generated from DU145 cells inoculated into athymic (nude) mice compared with those injected with AdCA-LacZ or AdCA-LacZ and AdCA-CD . CONCLUSIONS: These results suggest that the CD+UPRT/5-FC system could be a powerful factor in human prostate cancer suicide gene therapy .

Zhonghua Zhong Liu Za Zhi, 2002 May, 24(3), 219 - 21
{Search for interacting proteins of esophageal cancer related gene-1 encoded protein through the yeast two-hybrid system}; Wang J et al.; OBJECTIVE: To understand the role that esophageal cancer related gene-1 (ECRG-1) plays and to search for ECRG-1-interacting proteins . METHODS: A DNA fragment encoding the carboxy-terminus of ECRG-1 (amino acids 40 - 418) was inserted into pGBKT7-DNA-BD vector and fused in-frame to the DNA-binding domain of GAL4 . Then, it was used as a bait to screen the human fetal liver cDNA library by yeast two-hybrid, with the cDNA fragment inserted into pACT2 vector and fused in-frame to the Gal4 activation domain . If ECRG-1 interacted with a protein encoded by a cDNA fragmant in the yeast, the transcription of reporter Gene could be activated . With the false positive clonies eliminated, the inserts in the positive plasmids were sequenced and compared to those in the GenBank . RESULTS: In approximately 3 x 10(6) independent tansformants screened, 23 clonies exhibited the expression of reporter gene . After eliminating the false positive clonies, two cDNA fragments were obtained . DNA sequencing revealed that one encoded Miz-1 (Myc-interacting Zn finger protein-1), and another encoded FLNA (actin-binding protein-280), Miz-1, being a Zn finger protein, could be bound to p15 promotor and activated the transcription . FLNA, being an actin-binding protein took part in the TGF-beta pathway via interaction with Smad . CONCLUSION: ECRG-1 is able to be specifically bound to Miz-1 and FLNA in the yeast . It may play a role in the regulation of cell cycle via interaction with Miz-1 and FLNA.

Biotechnol Appl Biochem, 2003 Jun, 37(Pt 3), 301 - 9
High-yield expression of properly folded insulinoma-associated protein intracellular domain (IA-2ic) in Escherichia coli; Sica MP et al.; The intracellular domain of insulinoma-associated protein (IA-2), IA-2ic, is a prominent antigen in autoimmune diabetes, and autoantibodies to it are early markers of the disease . The high-yield expression of properly folded IA-2ic is needed for basic research and crucial for low-cost immunoassays aimed at the detection of these autoantibodies in diagnostic and preventive medicine . In previous work, the expression of IA-2ic fused to glutathione S-transferase or to a biotinylatable peptide was reported; however, these methods had very poor yield . Here we show that, utilizing a codon-optimized gene, up to 80 mg of pure and properly folded autoantigen per litre of Escherichia coli culture may be obtained . Furthermore, the addition of a C-terminal His-tag greatly facilitates IA-2ic purification without compromising either its immunoreactivity or its expression yield . To take advantage of the recombinant antigen, an enzyme immunoassay format was developed which proved to be highly specific and sensitive.

Biochemistry, 2003 Jan 14, 42(1), 231 - 7
Phospholipid flop induced by transmembrane peptides in model membranes is modulated by lipid composition; Kol MA et al.; Since phospholipid synthesis is generally confined to one leaflet of a membrane, membrane growth requires phospholipid translocation (flip-flop) . It is generally assumed that this process is protein-mediated; however, the mechanism of flip-flop remains elusive . Previously, we have demonstrated flop of 2-{6-{(7-nitro-2,1,3-benzoxadiazol-4-yl)amino}caproyl} (C6NBD) phospholipids, induced by the presence of membrane-spanning peptides in vesicles composed of an Escherichia coli phospholipid extract, supporting the hypothesis that the presence of transmembrane stretches of proteins in the bilayer is sufficient to allow phospholipid flip-flop in the inner membrane of E . coli {Kol et al . (2001) Biochemistry 40, 10500} . Here, we investigated whether the specific phospholipid composition of E . coli is a prerequisite for transmembrane helix-induced flop of phospholipids . This was tested by determining the amount of C6NBD-phospholipid that was translocated from the inner leaflet to the outer leaflet of a model membrane in time, using a dithionite reduction assay . The transmembrane peptides GWWL(AL)8WWA (WALP23) and GKKL(AL)8KKA (KALP23) induced phospholipid flop in model membranes composed of various lipid mixtures . The rate of peptide-induced flop was found to decrease with increasing dioleoylphosphatidylethanolamine (DOPE) content of vesicles composed of DOPE and dioleoylphosphatidylcholine (DOPC), and the rate of KALP23-induced flop was shown to be stimulated by higher dioleoylphosphatidylglycerol (DOPG) content in model membranes composed of DOPG and DOPC . Furthermore, the incorporation of cholesterol had an inhibitory effect on peptide-induced flop . Finally, flop efficiency was strongly dependent on the phospholipid headgroup of the NBD-phospholipid analogue . Possible implications for transmembrane helix-induced flop in biomembranes in general are discussed.

Biochemistry, 2003 Jan 14, 42(1), 167 - 76
Amino acid residues of Escherichia coli acyl carrier protein involved in heterologous protein interactions; Worsham LM et al.; Acyl carrier protein (ACP) is a small, highly conserved protein with an essential role in a myriad of reactions throughout lipid metabolism in plants and bacteria where it interacts with a remarkable diversity of proteins . The nature of the proper recognition and precise alignment between the protein moieties of ACP and its many interactive proteins is not understood . Residues conserved among ACPs from numerous plants and bacteria were considered as possibly being crucial to ACP's function, including protein-protein interaction, and a method of identifying amino acid residue clusters of high hydrophobicity on ACP's surface was used to estimate residues possibly involved in specific ACP-protein interactions . On the basis of this information, single-site mutation analysis of multiple residues, one at a time, of ACP was used to probe the identities of potential contact residues of ACPSH or acyl-ACP involved in specific interactions with selected enzymes . The roles of particular ACP residues were more precisely defined by site-directed fluorescence analyses of various myristoyl-mutant-ACPs upon specific interaction with the Escherichia coli hemolysin-activating acyltransferase, HlyC . This was done by selectively labeling each mutated site, one at a time, with an environmentally sensitive fluoroprobe and observing its fluorescence behavior in the absence and presence of HlyC . Consequently, a picture of the portion of ACP involved in selected macromolecular interaction has emerged.

Biochemistry, 2003 Jan 14, 42(1), 72 - 9
A model of structure and catalysis for ketoreductase domains in modular polyketide synthases; Reid R et al.; A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes . This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction . Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate . The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis . In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position . The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue . In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides . The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains . Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin . Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.

J Org Chem, 2003 Jan 10, 68(1), 83 - 91
Biosynthesis of 4-methylproline in cyanobacteria: cloning of nosE and nosF genes and biochemical characterization of the encoded dehydrogenase and reductase activities; Luesch H et al.; The biosynthesis of the unusual amino acid 4-methylproline in the Nostoc genus of cyanobacteria was investigated on the genetic and enzymatic level . Two genes involved in the biosynthesis were cloned and the corresponding enzymes, a zinc-dependent long-chain dehydrogenase and a Delta(1)-pyrroline-5-carboxylic acid (P5C) reductase homologue, were overexpressed in Escherichia coli and biochemically characterized . Putative substrates were synthesized to test enzyme substrate specificities, and deuterium labeling studies were carried out to reveal the stereospecificities of the enzymatic reactions with respect to the substrates as well as to the coenzymes.

Exp Mol Med, 2002 Sep 30, 34(4), 265 - 72
Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation; Lee MJ et al.; In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis . However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline . We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium . Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle . With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz) . Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer . Luciferase expression increased as cation concentration of vehicle decreased and this result can be visualized by X-Gal staining . However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage . At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01) . These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1998 Mar, 12(1), 38 - 42
{Generation of anti-rhEPO ScFv by using phage display technology}; Zhong L et al.; In this paper, the recombinant phage antibody techniques was used to construct, clone, screen, and express anti-rhEPO single chain antibody ScFv . The variable region genes of antibody were amplified by PCR from a hybridoma cell line D3 which secreted monoclonal antibody to rhEPO . The ScFv gene fragments were successfully cloned into phagemid vector pCANTAB5E . The recombinant phages were panned by rhEPO which was coated on a microtiter plate . After three rounds of panning, 8 clones were determined specifically binding to rhEPO antigen . The positive recombinant phagemids were extracted and transformed into non-suppressed E . coli HB2151 . The soluble single chain antibody was expressed, and the specificity of the expressed ScFv was determined by ELISA . Western blot and Dot-blot . The result of SDS-PAGE indicated that the apparent molecular weight of the target peptide which is mainly in culture supernatant is 32 kD . The DNA sequence data showed that the ScFv gene included 783 bp, encoding 261 amino acids.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1998 Mar, 12(1), 33 - 7
{Construction and screening of type 1 human immunodeficiency virus specific phage antibodies combinatorial library}; He Y et al.; Human monoclonal antibodies to type 1 immunodeficiency virus (HIV-1) gp120 were generated from phage antibody combinatorial library . METHODS: The human immunoglobulin heavy chain Fd and light chain k genes were amplified by half-nested PCR from PBMC of patient infected with HIV . Phage antibody combinatorial library was constructed with the Fd and k chain genes using Pcomb3 as vector . The affinity selection and ELISA were adopted for generating specific phage antibodies . Partial DNA of a positive clone was sequenced and its soluble Fab was expressed in E coli . HIV-1 specific phage antibodies combinatorial library were constructed using the Fd and k genes and Pcomb3 vector . The library capacity was about 1.95 x 10(7) . The specific phage antibodies were highly enriched after three rounds of biopanning selection against HIV-1 gp120 and 32% positive clones were detected by ELISA screening . DNA fragment coding for CH1 and CL derived from a positive clone was sequenced and its product was successfully expressed as soluble Fab which was specific for HIV-1 gp120 . The HIV-1 specific phage antibody combinatorial library, and human monoclonal antibodies to HIV-1 gp120 have been used as tools for screening of neutralizing antibody to HIV-1, and the methods seem to be very crucial and applicable.

Nat Struct Biol, 2003 Feb, 10(2), 109 - 14
Structure of the proline dehydrogenase domain of the multifunctional PutA flavoprotein; Lee YH et al.; The PutA flavoprotein from Escherichia coli plays multiple roles in proline catabolism by functioning as a membrane-associated bi-functional enzyme and a transcriptional repressor of proline utilization genes . The human homolog of the PutA proline dehydrogenase (PRODH) domain is critical in p53-mediated apoptosis and schizophrenia . Here we report the crystal structure of a 669-residue truncated form of PutA that shows both PRODH and DNA-binding activities, representing the first structure of a PutA protein and a PRODH enzyme from any organism . The structure is a domain-swapped dimer with each subunit comprising three domains: a helical dimerization arm, a 120-residue domain containing a three-helix bundle similar to that in the helix-turn-helix superfamily of DNA-binding proteins and a beta/alpha-barrel PRODH domain with a bound lactate inhibitor . Analysis of the structure provides insight into the mechanism of proline oxidation to pyrroline-5-carboxylate, and functional studies of a mutant protein suggest that the DNA-binding domain is located within the N-terminal 261 residues of E . coli PutA.

J Vet Sci, 2002 Sep, 3(3), 207 - 12
Expression of recombinant porcine interleukin-2 and application of its antibody to immunoassays; Choi IS et al.; Interleukin-2 plays an important role in T lymphocyte proliferation and immune response regulations . In this study, porcine IL-2 cDNA was cloned from peripheral blood mononuclear cells, and recombinant porcine IL-2 (rpIL-2) was expressed in Escherichia coli . The size of rpIL-2 without signal peptides was about 15 kDa when determined by SDS-PAGE and Western blotting analysis . Anti-rpIL-2 antibody was produced from mice immunized with the purified rpIL-2, and its specificity was examined by Western blotting and ELISA . In the Western blotting assay, anti-rpIL-2 and anti-recombinant human IL-2 (rhIL-2) antibodies specifically recognized rpIL-2 and rhIL-2, respectively . However, anti-rpIL-2 antibody did not recognize rhIL-2, and anti-rhIL-2 antibody also did not react with rpIL-2 in the same assay . In ELISA, anti-rpIL-2 antibody strongly interacted with both rpIL-2 and rhIL-2, and anti-rhIL-2 antibody also efficiently recognized both proteins . Taken together, the specificity of anti-rpIL-2 antibody for rpIL-2 was demonstrated by Western blotting and ELISA . It was also shown that ELISA is more efficient than Western blotting in determining the species cross-reactivity of anti-rpIL-2 antibody.

Plant Cell Physiol, 2002 Dec, 43(12), 1542 - 57
Ordered cosmid library of the Mesorhizobium loti MAFF303099 genome for systematic gene disruption and complementation analysis; Hattori Y et al.; For effective exploitation of the genome sequence information of Lotus microsymbiont, Mesorhizobium loti MAFF303099, to discover gene functions, we have constructed an ordered and mutually overlapping cosmid library using an IncP broad host-range vector . The library consisted of 480 clones to cover approximately 99.6% of the genome with average insert size and overlap of 26.9 and 11.1 kbp, respectively . The genome of M . loti consists of a single chromosome and two plasmids . The chromosome (7,036,071 bp) was covered 99.68% by 445 clones with four gaps, although two clones were unstable in E . coli . The larger plasmid pMLa (351,911 bp) was completely covered by 23 clones, while the smaller pMLb (208,315 bp) was covered 98.85% by 12 clones with two gaps . We have also made ancillary plasmids to facilitate the construction of deletion mutants using derivatives of the library clones . As a pilot experiment to uncover regions which contain novel symbiotic genes, 13 deletion mutants were constructed to lack in total 180.5 kbp of the genome . All the mutants formed apparently normal nodules and supported symbiotic nitrogen fixation, however, one mutant that lacked a 5.3 kbp chromosomal region, 4,551,930-4,557,222, did not produce normal exopolysaccharides as judged by fluorescence on medium containing Calcofluor . The results supported the effectiveness of the approach to detect gene functions.

J Biol Chem, 2003 Mar 14, 278(11), 9203 - 11 Epub 2003 Jan 03.
Catalytic mechanism of thiol peroxidase from Escherichia coli . Sulfenic acid formation and overoxidation of essential CYS61; Baker LM et al.; Escherichia coli thiol peroxidase (Tpx, p20, scavengase) is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase to reduce alkyl hydroperoxides . Tpx contains three Cys residues, Cys(95), Cys(82), and Cys(61), and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin family . To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S, and C82S,C95S) . In rapid reaction kinetic experiments measuring steady-state turnover, C61S is inactive, C95S retains partial activity, and the C82S mutation only slightly affects reaction rates . Furthermore, a sulfenic acid intermediate at Cys(61) generated by cumene hydroperoxide (CHP) treatment was detected in UV-visible spectra of 4-nitrobenzo-2-oxa-1,3-diazole-labeled C82S,C95S, confirming the identity of Cys(61) as the peroxidatic center . In stopped-flow kinetic studies, Tpx and Trx1 form a Michaelis complex during turnover with a catalytic efficiency of 3.0 x 10(6) m(-1) s(-1), and the low K(m) (9.0 microm) of Tpx for CHP demonstrates substrate specificity toward alkyl hydroperoxides over H(2)O(2) (K(m) > 1.7 mm) . Rapid inactivation of Tpx due to Cys(61) overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid, but not H(2)O(2) . Unlike most other 2-Cys peroxiredoxins, which operate by an intersubunit disulfide mechanism, Tpx contains a redox-active intrasubunit disulfide bond yet is homodimeric in solution.

J Biol Chem, 2003 Mar 14, 278(11), 9647 - 54 Epub 2003 Jan 03.
Molecular dissection of the hydrophobic segments H3 and H4 of the yeast Ca2+ channel component Mid1; Tada T et al.; The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1-H4, and functions as a Ca(2+)-permeable, stretch-activated channel when expressed in mammalian cells . In some conditions Mid1 cooperates with Cch1, a yeast homolog of the alpha1 subunit of mammalian voltage-gated channels . To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro site-directed mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo . Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca(2+) accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself . Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1 . In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca(2+) accumulation with a slightly low complementing activity, G342A and Y355A . The importance of these amino acid residues will be discussed.

EMBO J, 2003 Jan 15, 22(2), 324 - 34
Hallmarks of homology recognition by RecA-like recombinases are exhibited by the unrelated Escherichia coli RecT protein; Noirot P et al.; Homologous recombination is a fundamental process for genome maintenance and evolution . Various proteins capable of performing homology recognition and pairing of DNA strands have been isolated from many organisms . The RecA family of proteins exhibits a number of biochemical properties that are considered hallmarks of homology recognition . Here, we investigated whether the unrelated Escherichia coli RecT protein, which mediates homologous pairing and strand exchange, also exhibits such properties . We found that, like RecA and known RecA homologs: (i) RecT promotes the co-aggregation of ssDNA with duplex DNA, which is known to facilitate homologous contacts; (ii) RecT binding to ssDNA mediates unstacking of the bases, a key step in homology recognition; (iii) RecT mediates the formation of a three-strand synaptic intermediate where pairing is facilitated by local helix destabilization, and the preferential switching of A:T base pairs mediates recognition of homology; and (iv) RecT-mediated pairing occurs from both 3'- and 5'-single-stranded ends . Taken together, our results show that RecT shares fundamental homology-recognition properties with the RecA homologs, and provide new insights on an underlying universal mechanism of homologous recognition.

EMBO J, 2003 Jan 15, 22(2), 315 - 23
Excess SeqA prolongs sequestration of oriC and delays nucleoid segregation and cell division; Bach T et al.; Following initiation of chromosomal replication in Escherichia coli, newly initiated origins (oriCs) are prevented from further initiations by a mechanism termed sequestration . During the sequestration period (which lasts about one-third of a cell cycle), the origins remain hemimethylated . The SeqA protein binds hemimethylated oriC in vitro . In vivo, the absence of SeqA causes overinitiation and strongly reduces the duration of hemimethylation . The pattern of immunostained SeqA complexes in vivo suggests that SeqA has a role in organizing hemimethylated DNA at the replication forks . We have examined the effects of overexpressing SeqA under different cellular conditions . Our data demonstrate that excess SeqA significantly increases the time oriC is hemimethylated following initiation of replication . In some cells, sequestration continued for more than one generation and resulted in inhibition of primary initiation . SeqA overproduction also interfered with the segregation of sister nucleoids and caused a delay in cell division . These results suggest that SeqA's function in regulation of replication initiation is linked to chromosome segregation and possibly cell division.

Genes Dev, 2003 Jan 1, 17(1), 64 - 76
Checkpoint activation regulates mutagenic translesion synthesis; Kai M et al.; Cells have evolved checkpoint responses to arrest or delay the cell cycle, activate DNA repair networks, or induce apoptosis after genomic perturbation . Cells have also evolved the translesion synthesis processes to tolerate genomic lesions by either error-free or error-prone repair . Here, we show that after a replication perturbation, cells exhibit a mutator phenotype, which can be significantly affected by mutations in the checkpoint elements Cds1 and Rad17 or translesion synthesis polymerases DinB and Polzeta . Cells respond to genomic perturbation by up-regulation of DinB in a checkpoint activation-dependent manner . Moreover, association of DinB with chromatin is dependent on functional Rad17, and DinB physically interacts with the checkpoint-clamp components Hus1 and Rad1 . Thus, translesion synthesis is a part of the checkpoint response.

Appl Environ Microbiol, 2003 Jan, 69(1), 373 - 82
Enhanced heterologous expression of two Streptomyces griseolus cytochrome P450s and Streptomyces coelicolor ferredoxin reductase as potentially efficient hydroxylation catalysts; Hussain HA et al.; The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels . Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed . Analysis of the expression levels of the P450 proteins in several different E . coli expression hosts identified E . coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra . Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E . coli tRNA species . The level of its expression was improved in the modified forms of E . coli BL21(DE3), which contain extra copies of rare codon E . coli tRNA genes . The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins . Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160 . S . lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.

Appl Environ Microbiol, 2003 Jan, 69(1), 233 - 40
Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment; Sampaio MM et al.; We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-alpha-D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment . The strain carries a deletion in the manA gene, encoding phosphomannose isomerase . This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM) . The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer . Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose . A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-alpha-D-mannosyl-D-glycerate from GDP-mannose and endogenous glycerate . The rate-limiting step in 2-O-alpha-D-mannosyl-D-glycerate formation is the transfer of GDP-mannose to glycerate . 2-O-alpha-D-mannosyl-D-glycerate can be released from cells by treatment with cold-water shock . The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2002 Aug, 10(4), 327 - 31
{Apoptosis of Fas(+) Yac-1 cells induced with Fas ligand-transfected COS-7 cells}; Liu LB et al.; The possibility of immunotherapy for lymphoma by single FasL-Fas way was investigated . After pBillneo-mFasL was transformed into competent E . coli DH5alpha and amplified, the plasmid DNA was prepared and purified from the DH5alpha . To determine the primary structure and inserting direction of mFasL cDNA gene in pBillneo-mFasL, the plasmid DNA was cleaved by restriction enzyme, and the mFasL cDNA of pBillneo-mFasL was amplified by polymerase chain reaction (PCR), the DNA sequence of the PCR product was analysed by automatic DNA sequencing . After pBillneo-mFasL was transfected into COS-7 cells by liposome, the COS-7 cells were selected with G418 selective medium, and the expressing levels of mFasL cDNA on the COS-7 cell membrane was assayed by Western Blot . After the COS-7 cells higher expressing mFasL protein and mouse lymphoma cell line Yac-1 expressing Fas were cocultured for 5 hours, the suspending Yac-1 cells were collected and labeled by annexin V/PI kit . The apoptosis rate of the Yac-1 cells was tested by flow cytometry . The EcoRI cleaving products of pBillneo-mF asL included 920 bp and 7227 bp fragments . Its Hind III cleaving products included 1293 bp and 6807 bp fragments . These results showed: (1) the length of DNA sequence containing mFasL cDNA within pBillneo-mFasL is the same as theoretical length; (2) the inserting of mFasL cDNA in pBillneo-mFasL was in positive orientation . The expected 890 bp DNA fragments of mFasL cDNA (from ATG to +36 bp following TAA) emerged in PCR product with pBillneo-mFasL as a template . The sequencing result of the PCR product equaled the known mFasL cDNA sequence in the gene bank . The COS-7 cells transfected by pBillneo-mFasL and selected with G418 culture medium expressed more mFasL membrane protein assayed by Western Blot . After the COS-7 cells were cocultured with Fas(+) Yac-1 cells in different E:T ratios (1:1, 5:1 and 10:1) for 5 hours, the apoptosis rates of Yac-1 cells were (22 +/- 4.8)%, (32.18 +/- 7.8)%, and (51.8 +/- 5.4)%, respectively . These were obviously different from the control group (P < 0.01), in which the COS-7 cell was transfected by pBillneo (not carrying mFasL gene) . It was concluded that lymphoma cells highly expressing Fas can be effectively killed through single Fas-FasL way in vitro.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2002 Dec, 10(6), 540 - 3
{Molecular cloning of human vWF/A1 gene and its expression}; Zhu HP et al.; To study the mechanism of thrombogenesis and search new anti-thrombotic agent, the cDNA for human vWF A1 domain was high-level expressed in E . coli and recombinant protein of vWF A1 with biologic activity was obtained . The gene encoding A1 domain was amplified by PCR from plasmid containing full length cDNA of human vWF . After confirming by DNA sequencing analysis, the recombinant expression plasmid pQE31-vWF/A1 was constructed and introduced into E . coli M15 strain, then induced by IPTG; the expressed protein was purified with Ni-NTA agarose, identified by Western blotting . The results showed that the 854 bp DNA fragment was obtained by PCR from the plasmid containing full length cDNA for human vWF and its sequence was identical to the published sequence . High level expression of A1 protein was yielded after 5 hour-induction, which amounted to 30% of total bacteria protein in inclusion body . Western blot demonstrated it possessed good antigenicity and high specificity . It is concluded that cDNA for vWF/A1 had been cloned successfully, high level expression of A1 protein was achieved in E . oli . This study will provide a basis for the further clinical and basic research on the role of vWF in thrombosis and hemostasis.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2002 Dec, 10(6), 535 - 9
{Expression and purification of recombinant glycoprotein (GP) IIb/IIIa receptor antagonists}; Zha YP et al.; To investigate the effect of GST-KGDX (glutathione S-transferase-Lys-Gly-Asp-X) fusion protein, GP IIb/IIIa receptor antagonist, on platelet function in vitro . The KGDX (Lys-Gly-Asp-X) gene was assembled from 2 synthetic oligonucleotides, 36 bp in length, using BamH I and Xho I restriction enzyme sites at the end of the gene for cloning into the expression vector pGEX4T-1 . Expression of fusion protein was directed by the tac promoter . The Escherichia coli DH5a contained the plasmid pGEX-4T-1-KGDX was expressed by 37 degrees C heat induction . The fusion protein of KGDX with glutathione S-transferase (GST-KGDX) was purified in one step from the bacterial lysate by glutathione-agarose beads for affinity chromatography . GST-KGDX was found to be soluble and abundant, the yield of 35 mg/L of cultures was obtained . The GST-KGDX was expressed in E . coli to a level of 48.02% of total cellular protein . GST-KGDX inhibited ADP-induced human platelet aggregation stronger than GST (P < 0.05 or < 0.01) . In flow cytometry assay for fibrinogen binding, both GST and GST-KGDX inhibited platelet aggregation by binding with high affinity to GPIIb/IIIa . Mean fluorescence intensity of GST-KGDX fusion protein was significantly higher than that of GST . It is concluded that the GST-KGDX fusion protein can be produced by E . coli and used as an antiplatelet agent.

Biochem J, 2003 Apr 1, 371(Pt 1), 183 - 9
Human mismatch-repair protein MutL homologue 1 (MLH1) interacts with Escherichia coli MutL and MutS in vivo and in vitro: a simple genetic system to assay MLH1 function; Quaresima B et al.; A simple genetic system has been developed to test the effect of over-expression of wild-type or mutated human MutL homologue 1 (hMLH1) proteins on methyl-directed mismatch repair (MMR) in Escherichia coli . The system relies on detection of Lac(+) revertants using MMR-proficient or MMR-deficient E . coli strains carrying a lac +1 frameshift mutation expressing hMLH1 proteins . We report that expression of wild-type hMLH1 protein causes an approx . 19-fold increase in mutation rates . The mutator phenotype was due to the ability of hMLH1 protein to interact with bacterial MutL and MutS proteins, thereby interfering with the formation of complexes between MMR proteins and mismatched DNA . Conversely, expression of proteins encoded by alleles deriving from hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates, depending on the specific amino acid substitutions . These effects parallel the MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated proteins.

Kaohsiung J Med Sci, 2002 Nov, 18(11), 573 - 7
DKC1 gene mutation in a Taiwanese kindred with X-linked dyskeratosis congenita; Lin JH et al.; Dyskeratosis congenita (DKC) is a rare inherited disease characterized by the triad of abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia . Recent studies demonstrated mutations in the DKC1 gene encoding a protein named dyskerin, which is a component of human telomerase . In addition to the hypothesized function of pseudouridination in rRNA biosynthesis, ribosomal subunit assembly, and/or centromere/ microtubule binding, lower levels of telomerase activity in cells from patients with X-linked DKC have been observed . We report the mutation analysis of a Taiwanese family with X-linked DKC . The patient was a 19-year-old man who presented with progressive reticulate hyperpigmentation, nail dystrophy, alopecia, leukoplakia of the tongue, and pancytopenia . He died of enterocolitis and Escherichia coli sepsis at the age of 20 years . Only his mother's DNA was available for mutation analysis, which revealed a nucleotide transition of C to T (1058 C --> T), a hotspot mutation in DKC, resulting in an amino acid change from alanine to valine (A353V) in the DKC1 gene . Recent advances in the research of telomerase and its implications in the human aging process and cancer are discussed.

Mol Membr Biol, 2002 Oct-Dec, 19(4), 237 - 45
Non-permanent proteins in membranes: when proteins come as visitors (Review); Goni FM; The present review introduces the concept of 'non-permanent membrane proteins', to encompass the wide variety of proteins that are not found in a stable membrane-bound form under physiological conditions, yet they interact with the membrane at some stage of their specific course of action . Non-permanent membrane proteins can be codified by the cell's own genome, or else they may arise from a foreign genome . Non-permanent membrane proteins can be classified, according to the reversibility of the membrane interaction, into those with reversible and irreversible (very long-lived) membrane contacts . According to the nature (strength) of the interaction, non-permanent membrane proteins may be divided into those that interact weakly with the membrane, in an extrinsic-like form, and those that interact strongly with the membrane . The latter can in turn be classified into those that cause and those that do not cause covalent modification of the lipids, the latter behaving, after interacting with the membrane, in the way of the conventional intrinsic proteins . Multiple examples are provided for the different groups of non-permanent membrane proteins, and a more detailed description is given of three of them, representative of different groups, namely TrwD from plasmid R388, E . coli alpha-haemolysin and B . cereus sphingomyelinase.

Pol J Vet Sci, 2002, 5(4), 237 - 41
Modulation of murine macrophages and T lymphocytes by lysozyme dimer; Obminska-Mrukowicz B et al.; The effects of lysozyme dimer (2 and 20 microg/kg) administered i.p . once and four times to mice on the phagocytic and killing ability of peritoneal macrophages, interleukin-1 (IL-1) production by murine macrophages stimulated in vitro with lipopolisaccharide of E . coli and expression of thymocyte, splenocyte and mesenteric lymphonode cell CD3+, CD4+ and CD8+ markers were studied . It was found that lysozyme dimer administered once or four times at doses of 2 microg/kg and 20 microg/kg augments the phagocytic and killing activity of peritoneal macrophages . The strongest stimulating effect was noted after four injections of lysozyme dimer at a dose of 20 microg/kg . Moreover, lysozyme dimer is able to modulate the production of IL-1 by murine macrophages stimulated in vitro with LPS . Exposure to four doses of lysozyme dimer (20 microg/kg) enhances the synthesis and release of IL-1, but this drug administered once (2 microg/kg and 20 microg/kg) or four times (2 microg/kg) decreases IL-1 production by peritoneal macrophages . It was also found that administration of lysozyme dimer at a dose of 20 microg/kg, irrespective of the number of doses applied, increases the percentage of CD4+ thymocytes and splenocytes . Moreover, exposure to four doses of lysozyme dimer (2 and 20 microg/kg) increases the percentage of CD4+ and CD8+ mesenteric lymphonode cells.

Nature, 2003 Jan 2, 421(6918), 90 - 4
Structure of the Escherichia coli ribosomal termination complex with release factor 2; Klaholz BP et al.; Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site . Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site . Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis . Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear . Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM) . Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein . The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC) . By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site . Translational termination in eukaryotes is likely to be based on a similar mechanism.

Nature, 2003 Jan 2, 421(6918), 87 - 90
A cryo-electron microscopic study of ribosome-bound termination factor RF2; Rawat UB et al.; Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids . This process is terminated when a stop codon moves into the ribosomal decoding centre (DC) and is recognized by a class-1 release factor (RF) . RFs have a conserved GGQ amino-acid motif, which is crucial for peptide release and is believed to interact directly with the peptidyl-transferase centre (PTC) of the 50S ribosomal subunit . Another conserved motif of RFs (SPF in RF2) has been proposed to interact directly with stop codons in the DC of the 30S subunit . The distance between the DC and PTC is approximately 73 A . However, in the X-ray structure of RF2, SPF and GGQ are only 23 A apart, indicating that they cannot be at DC and PTC simultaneously . Here we show that RF2 is in an open conformation when bound to the ribosome, allowing GGQ to reach the PTC while still allowing SPF-stop-codon interaction . The results indicate new interpretations of accuracy in termination, and have implications for how the presence of a stop codon in the DC is signalled to PTC.

J Biol Chem, 2003 Mar 14, 278(11), 9176 - 84 Epub 2003 Jan 02.
Interactions between Na,K-ATPase alpha-subunit ATP-binding domains; Costa CJ et al.; The reaction mechanism of the Na,K-ATPase is thought to involve a number of ligand-induced conformational changes . The specific amino acid residues responsible for binding many of the important ligands have been identified; however, details of the specific conformational changes produced by ligand binding are largely undescribed . The experiments described in this paper begin to identify interactions between domains of the Na,K-ATPase alpha-subunit that depend on the presence of particular ligands . The major cytoplasmic loop (between TM4 and TM5), which we have previously shown contains the ATP-binding domain, was overexpressed in bacteria either with a His(6) tag or as a fusion protein with glutathione S-transferase . We have observed that these polypeptides associate in the presence of MgATP . Incubation with {gamma-(32)P}ATP under conditions that result in phosphorylation of the full-length Na,K-ATPase did not result in (32)P incorporation into either the His(6) tag or glutathione S-transferase fusion proteins . The MgATP-induced association was strongly inhibited by prior modification of the fusion proteins with fluorescein isothiocyanate or by simultaneous incubation with 10 microm eosin, indicating that the effect of MgATP is due to interactions within the nucleotide-binding domain . These data are consistent with Na,K-ATPase associating within cells via interactions in the nucleotide-binding domains . Although any functional significance of these associations for ion transport remains unresolved, they may play a role in cell function and in modulating interactions between the Na,K-ATPase and other proteins.

J Biol Chem, 2003 Mar 14, 278(11), 9107 - 15 Epub 2003 Jan 02.
Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein b23; Szebeni A et al.; Protein B23 is a multifunctional nucleolar protein whose molecular chaperone activity is proposed to play role in ribosome assembly . Previous studies (Szebeni, A., and Olson, M . O . J . (1999) Protein Sci . 8, 905-912) showed that protein B23 has several characteristics typical of molecular chaperones, including anti-aggregation activity, promoting the renaturation of denatured proteins, and preferential binding to denatured substrates . However, until now there has been no proposed mechanism for release of a bound substrate . Protein B23 can be phosphorylated by protein kinase CK2 (CK2) in a segment required for chaperone activity . The presence of bound substrate enhanced the rate of CK2 phosphorylation of protein B23 by 2-3-fold, and this enhancement was dependent on a nonpolar region in its N-terminal end . Formation of a complex between B23 and chaperone test substrates (rhodanese or citrate synthase) was inhibited by CK2 phosphorylation . Furthermore, CK2 phosphorylation of a previously formed B23-substrate complex promoted its dissociation . The dissociation of complexes between B23 and the human immunodeficiency virus-Rev protein required both CK2 phosphorylation and competition with a Rev nuclear localization signal peptide, suggesting that Rev binds B23 at two separate sites . These studies suggest that unlike many molecular chaperones, which directly hydrolyze ATP, substrate release by protein B23 is dependent on its phosphorylation by CK2.

J Bacteriol, 2003 Jan, 185(2), 660 - 3
Formation of an F' plasmid by recombination between imperfectly repeated chromosomal Rep sequences: a closer look at an old friend (F'(128) pro lac); Kofoid E et al.; Plasmid F'(128) was formed by an exchange between chromosomal Rep sequences that placed lac near dinB between many pairs of Rep sequences . Plasmid F'(128) is critical for selection-enhanced lac reversion (adaptive mutation), which requires prior lac amplification . The structure of F'(128) supports the idea that amplification is initiated by Rep-Rep recombination and that general mutagenesis requires coamplification of dinB (error-prone polymerase) with lac.

J Bacteriol, 2003 Jan, 185(2), 630 - 44
Escherichia coli cells with increased levels of DnaA and deficient in recombinational repair have decreased viability; Grigorian AV et al.; The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, beta clamp of DNA polymerase III holoenzyme, and RecF . When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs . Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression . To test this, we examined the effects of increasing the intracellular concentrations of DnaA, beta clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability . The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression . A shorter C period was not observed with increased expression of the beta clamp; in fact, many chromosomes did not complete replication in runout experiments . Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability . When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 micro m) with extremely low viability and, in runout experiments, most chromosomes had not completed replication . The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways . The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional approximately 100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.

J Bacteriol, 2003 Jan, 185(2), 620 - 9
Transcriptional analysis of the sfa determinant revealing mmRNA processing events in the biogenesis of S fimbriae in pathogenic Escherichia coli; Balsalobre C et al.; Among the virulence factors present in pathogenic extraintestinal Escherichia coli strains, expression of fimbrial adhesins is necessary for attachment to the host tissues and subsequent colonization . Occurrence of the sfa determinant coding for the S fimbriae is widespread among the uropathogens and meningitis isolates . The sfa operon consists of nine genes . In the biogenesis of S fimbriae, the proteins encoded by the sfa genes are presumably required in a specific stoichiometry . In the present work we studied how differential expression of the sfa operon genes occurs . Our findings indicate that a number of endoribonucleolytic cleavages occur in the mRNA from the sfa operon, and we detected the presence of different distinct transcriptional products, including sfaBA, sfaA, sfaADE, and sfaGSH . The sfaGSH transcript represents the three distal genes of the sfa operon, which code for the minor subunits of the S fimbriae . Analysis of the proteins in S fimbriae suggested that expression of the sfaGSH transcript provides equimolar amounts of the minor subunits . Furthermore, we showed that in the generation of the major sfaA transcript, the processing included RNase E endoribonuceolytic cleavage of the precursor sfaBA transcript . We suggest that posttranscriptional mRNA processing events result in differential gene expression important to achieve the stoichiometry necessary for fimbrial adhesin biogenesis.

J Bacteriol, 2003 Jan, 185(2), 581 - 91
Functional and mutational analysis of conjugative transfer region 2 (Tra2) from the IncHI1 plasmid R27; Lawley TD et al.; The transfer 2 region (Tra2) of the conjugative plasmid drR27 (derepressed R27) was analyzed by PSI-BLAST, insertional mutagenesis, genetic complementation, and an H-pilus assay . Tra2 contains 11 mating-pair formation (Mpf) genes that are essential for conjugative transfer, 9 of which are essential for H-pilus production (trhA, -L, -E, -K, -B, -V, -C, -P, and -W) . TrhK has similarity to secretin proteins, suggesting a mechanism by which DNA could traverse the outer membrane of donors . The remaining two Mpf genes, trhU and trhN, play an auxiliary role in H-pilus synthesis and are proposed to be involved in DNA transfer and mating-pair stabilization, respectively . Conjugative transfer abilities were restored for each mutant when complemented with the corresponding transfer gene . In addition to the essential Mpf genes, three genes, trhO, trhZ, and htdA, modulate R27 transfer frequency . Disruption of trhO and trhZ severely reduced the transfer frequencies of drR27, whereas disruption of htdA greatly increased the transfer frequency of wild-type R27 to drR27 levels . A comparison of the essential transfer genes encoded by the Tra2 and Tra1 (T . D . Lawley, M . W . Gilmour, J . E . Gunton, L . J . Standeven, and D . E . Taylor, J . Bacteriol . 184:2173-2183, 2002) of R27 to other transfer systems illustrates that the R27 conjugative transfer system is a chimera composed of IncF-like and IncP-like transfer systems . Furthermore, the Mpf/type IV secretion systems encoded by IncH and IncF transfer systems are distinct from that of the IncP transfer system . The phenotypic and ecological significance of these observations is discussed.

J Bacteriol, 2003 Jan, 185(2), 544 - 52
Different evolutionary constraints on chemotaxis proteins CheW and CheY revealed by heterologous expression studies and protein sequence analysis; Alexandre G et al.; CheW and CheY are single-domain proteins from a signal transduction pathway that transmits information from transmembrane receptors to flagellar motors in bacterial chemotaxis . In various bacterial and archaeal species, the cheW and cheY genes are usually encoded within homologous chemotaxis operons . We examined evolutionary changes in these two proteins from distantly related proteobacterial species, Escherichia coli and Azospirillum brasilense . We analyzed the functions of divergent CheW and CheY proteins from A . brasilense by heterologous expression in E . coli wild-type and mutant strains . Both proteins were able to specifically inhibit chemotaxis of a wild-type E . coli strain; however, only CheW from A . brasilense was able to restore signal transduction in a corresponding mutant of E . coli . Detailed protein sequence analysis of CheW and CheY homologs from the two species revealed substantial differences in the types of amino acid substitutions in the two proteins . Multiple, but conservative, substitutions were found in CheW homologs . No severe mismatches were found between the CheW homologs in positions that are known to be structurally or functionally important . Substitutions in CheY homologs were found to be less conservative and occurred in positions that are critical for interactions with other components of the signal transduction pathway . Our findings suggest that proteins from the same cellular pathway encoded by genes from the same operon have different evolutionary constraints on their structures that reflect differences in their functions.

J Bacteriol, 2003 Jan, 185(2), 534 - 43
FlhD/FlhC is a regulator of anaerobic respiration and the Entner-Doudoroff pathway through induction of the methyl-accepting chemotaxis protein Aer; Pruss BM et al.; The regulation by two transcriptional activators of flagellar expression (FlhD and FlhC) and the chemotaxis methyl-accepting protein Aer was studied with glass slide DNA microarrays . An flhD::Kan insertion and an aer deletion were independently introduced into two Escherichia coli K-12 strains, and the effects upon gene regulation were investigated . Altogether, the flhD::Kan insertion altered the expression of 29 operons of known function . Among them was Aer, which in turn regulated a subset of these operons, namely, the ones involved in anaerobic respiration and the Entner-Doudoroff pathway . In addition, FlhD/FlhC repressed enzymes involved in aerobic respiration and regulated many other metabolic enzymes and transporters in an Aer-independent manner . Expression of 12 genes of uncharacterized function was also affected . FlhD increased gltBD, gcvTHP, and ompT expression . The regulation of half of these genes was subsequently confirmed with reporter gene fusions, enzyme assays, and real-time PCR . Growth phenotypes of flhD and flhC mutants were determined with Phenotype MicroArrays and correlated with gene expression.

J Bacteriol, 2003 Jan, 185(2), 496 - 503
Characterization of a novel intracellular endopeptidase of the alpha/beta hydrolase family from Streptomyces coelicolor A3(2); Nagy I et al.; In a proteasome-lacking mutant of Streptomyces coelicolor A3(2), an intracellular enzyme with chymotrypsin-like activity, absent from the wild type, was detected . Complementation that restored proteasome function did not suppress expression of the endopeptidase . Since the enzyme was not found in two other S . coelicolor proteasome mutants, its expression probably resulted from a secondary mutation arisen in the proteasome mutant . Purification of the endopeptidase revealed its identity to SCO7095, a putative hydrolase encoded by the S . coelicolor A3(2) genome with no known homologue . Based on the prediction of a Ser-Asp-His catalytic triad and an alpha/beta hydrolase fold, SCO7095 was assigned to peptidase clan SC . N-terminally His-tagged SCO7095 was efficiently expressed in Escherichia coli cells and purified for further characterization . Although SCO7095 is distantly related to several proline iminopeptidases, including Thermoplasma acidophilum tricorn-interacting F1, no aminopeptidase activity was detected . On synthetic substrates, the monomeric enzyme exhibited not only chymotrypsin-like activity but also thrombin-like activity.

J Bacteriol, 2003 Jan, 185(2), 444 - 52
Expression of spoT in Borrelia burgdorferi during serum starvation; Concepcion MB et al.; Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the tick Ixodes scapularis . A 2.9-kb fragment containing a putative spoT gene was isolated from B . burgdorferi genomic DNA by PCR amplification and cloned into a pBAD24 vector . The cloned gene complemented Escherichia coli mutant strain CF1693, which contains deletions of both the relA and spoT genes . The spoT gene in E . coli encodes a bifunctional enzyme capable of synthesizing and degrading (p)ppGpp, which mediates the stringent response during carbon source starvation . B . burgdorferi has been reported to have a stress response to serum starvation . Thin-layer chromatography was used to detect (p)ppGpp extracted from H(3)(32)PO(4)-labeled B . burgdorferi cells starved for serum in RPMI . B . burgdorferi spoT gene expression was characterized during fatty acid starvation . Northern analysis of spoT revealed detectable message at 2.5 min of starvation in RPMI . Expression of spoT during serum starvation increased approximately 6-fold during the 30 min that starvation conditions were maintained . Further, expression of spoT decreased when serum was added to serum-starved cells . Reverse transcriptase PCR (RT-PCR) was used to detect spoT mRNA from approximately 10(6) cells starved for serum in RPMI for 2.5 to 30 min or incubated in tick saliva for 15 min . Northern blot analysis suggests that spoT transcript was approximately 900 nucleotides in length . RT-PCR amplification of the transcript using several sets of primers confirmed this finding . Additionally, a truncated clone containing only the first 950 bp of the 2,001-bp spoT open reading frame was able to complement E . coli CF1693 . The data suggest that B . burgdorferi exhibits a stringent response to serum starvation and during incubation in tick saliva.

J Bacteriol, 2003 Jan, 185(2), 405 - 12
Biochemical characterization of a mutationally altered protein translocase: proton motive force stimulation of the initiation phase of translocation; Mori H et al.; Protein translocation across the Escherichia coli plasma membrane is facilitated by concerted actions of the SecYEG integral membrane complex and the SecA ATPase . A secY mutation (secY39) affects Arg357, an evolutionarily conserved and functionally important residue, and impairs the translocation function in vivo and in vitro . In this study, we used the "superactive" mutant forms of SecA, which suppress the SecY39 deficiency, to characterize the mutationally altered SecY39EG translocase . It was found that SecY39-mediated preprotein translocation exhibited absolute dependence on the proton motive force . The proton motive force-dependent step proved to lie before signal peptide cleavage . We suggest that the proton motive force assists in the initiation phase of protein translocation.

Immunopharmacol Immunotoxicol, 2002 Nov, 24(4), 567 - 82
Helicobacter pylori organisms induce expression of activation and apoptotic surface markers on human lymphocytes and AGS cells: a cytofluorimetric evaluation; Lembo A et al.; Human peripheral blood mononuclear cells (PBMCs) were treated with Helicobacter pylori (Hp) organisms alone or with Hp-stimulated AGS cells (a gastric adenocarcinoma cell line) . Hp organisms were able per se to increase the percentage of CD8 +/- CD95 +/- cells, while number of CD25+ cells and HLA-DR molecule expression increased following pretreatment with Hp-stimulated AGS cells . A comparison was made with a test system in which PBMCs were stimulated with Escherichia coli (Ec) organisms and colo-cells (a colon carcinoma cell line) . In this case, CD95+ cells and CD25+ cells increased when the combination Ec organisms/colo-cells was present in the culture . On the other hand, Hp bacteria in combination with colo-cells were not able to induce activation and/or apoptotic surface markers on PBMCs, while Ec-stimulated AGS cells increased the expression of CD95 on PBMC . Finally, the direct interaction of AGS cells with Hp was able to induce higher expression of CD95 on gastric epithelial cells than Hp-stimulated PBMCs . Taken together, these data support the interplay between bacteria and epithelial cells in the course of Hp-mediated gastropathy.

Anal Chem, 2002 Dec 15, 74(24), 6408 - 12
Direct isolation of purines and pyrimidines from nucleic acids using sublimation; Glavin DP et al.; A sublimation technique was developed to isolate purines and pyrimidines directly from lambda-deoxyribonucleic acid (lambda-DNA) and Escherichia coli cells . The sublimation of adenine, cytosine, guanine, and thymine from lambda-DNA was tested under reduced pressure (approximately 0.5 Torr) at temperatures of >150 degrees C . With the exception of guanine, approximately 60-75% of each base was sublimed directly from the lambda-DNA and recovered on a coldfinger of the sublimation apparatus after heating to 450 degrees C . Several nucleobases including adenine, cytosine, thymine, and uracil were also recovered from E . coli bacteria after heating the cells to the same temperature, although some thermal decomposition of the bases also occurred . These results demonstrate the feasibility of using sublimation to isolate purines and pyrimidines from native E . coli DNA and RNA without any chemical treatment of the cells.

Anal Chem, 2002 Dec 15, 74(24), 6349 - 54
Electrochemical and Raman studies of the biointeraction between Escherichia coli and mannose in polydiacetylene derivative supported on the self-assembled monolayers of octadecanethiol on a gold electrode; Li Y et al.; Here, we describe a new method to study the biointeraction between Escherichia coli and mannose by using supramolecular assemblies composed of polydiacetylene supported on the self-assembled monolayer of octadecanethiol on a gold electrode . These prepared bilayer materials simply are an excellent protosystem to study a range of important sensor-related issues . The experimental results from UV-vis spectroscopy, resonance Raman spectroscopy, and electrochemistry confirm that the specific interactions between E . coli and mannose can cause conformational changes of the polydiacetylene backbone rather than simple nonspecific adsorption . Moreover, the direct electrochemical detection by polydiacetylene supramolecular assemblies not only opens a new path for the use of these membranes in the area of biosensor development but also offers new possibilities for diagnostic applications and screening for binding ligands.

Protein Expr Purif, 2003 Jan, 27(1), 186 - 93
Expression, purification, crystallization, and preliminary X-ray analysis of recombinant human saposin B; Ahn VE et al.; Saposin B (also known as cerebroside sulfate activator or CSAct) is a small non-enzymatic glycoprotein required for the breakdown of cerebroside sulfates (sulfatides) in lysosomes . Saposin B contains three intramolecular disulfide bridges, exists as a dimer and is remarkably heat, protease, and pH stable . We have expressed the protein in a thioredoxin reductase deficient strain of Escherichia coli and purified the protein by heat treatment, followed by ion-exchange, gel filtration, and hydrophobic interaction chromatographies . The protein is properly folded as judged by the observed disulfide bond topology, the hydrogen-deuterium exchange rate, and the level of stimulation of sulfatide hydrolysis by arylsulfatase A . Crystals of human saposin B were grown by vapor diffusion and diffract to a resolution of 2.2A . Despite obtaining only merohedrally twinned P3(1) native crystals, an untwined seleomethionine-substituted crystal belonging to space group P3(1)21 was also grown . The three-dimensional structure of saposin B protein will provide insights into how this 79 amino acid protein is able to solubilize relatively large membrane-bound lipid ligands.

Protein Expr Purif, 2003 Jan, 27(1), 165 - 70
Cloning, expression, and purification of His-tagged rat mevalonate kinase; Chu X et al.; Mevalonate kinase catalyzes the phosphorylation of mevalonic acid to form mevalonate 5-phosphate, which plays a key role in regulating cholesterol biosynthesis in animal cells . Deficiency of mevalonate kinase activity in the human body has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS) . We cloned the gene of rat mevalonate kinase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5(') of the gene . The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal affinity column in 90% yield to apparent homogeneity . The purified rat mevalonate kinase had a dimeric structure composed of identical subunits . Based on SDS-PAGE, the subunit was 42 kDa . The specific activity of the purified His-tagged rat mevalonate kinase was 32.7 micromol/min/mg and the optimal pH was found to be 7.0-8.0 in phosphate buffer . The Michaelis constant K(M) was 35 microM for (RS)-mevalonate and 953 microM for ATP, respectively . The V(max) was determined to be 38.7 micromol/min/mg . The overexpression of rat mevalonate kinase in E . coli and one-step purification of the highly active rat mevalonate kinase will facilitate further our investigation of this enzyme through site-directed mutagenesis and enzyme-catalyzed reactions with substrate analogs.

Protein Expr Purif, 2003 Jan, 27(1), 158 - 64
Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase; Silva RG et al.; Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides . A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect . Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas . Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation . In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (P(i)) and continuous assay of reactions that generate P(i) such as those catalyzed by ATPases and GTPases . Human PNP may therefore be an important biotechnological tool for P(i) detection . However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P(i) detection method . Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme . Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein . Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control . Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1L cell culture, with a specific activity value of 80 Umg(-1).

Protein Expr Purif, 2003 Jan, 27(1), 150 - 7
High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli; Hiszczynska-Sawicka E et al.; This report describes a simple, highly efficient and reproducible method for obtaining large quantities of highly pure recombinant Toxoplasma gondii antigens, which can be used for diagnostic application . The obtained T . gondii SAG1, GRA1, and GRA7 antigens (as fusion proteins), expressed in Escherichia coli, contained polyhistidine tags at the N- and C-ends that allowed single-step isolation by metal-affinity chromatography on Ni(2+)-IDA-Sepharose columns . The immunoreactivity of the recombinant antigens was tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of T . gondii infection.

Protein Expr Purif, 2003 Jan, 27(1), 143 - 9
Expression, refolding, and activation of the catalytic domain of human blood coagulation factor XII; Shan J et al.; Human blood coagulation factor XII (FXII; 80 kDa) contains a C-terminal serine protease zymogen domain, which becomes activated upon contacting a negative surface . Activated FXII (alphaFXIIa) brings about reciprocal activation of FXII and kallikrein that by further hydrolysis produces the free catalytic domain (betaFXIIa; 28 kDa) . Increased levels of alphaFXIIa are associated with coronary heart disease, sepsis, and diabetes . Biophysical investigation of the structural basis of activation, substrate specificity, and regulation of FXII requires an efficient bacterial system for producing the wild-type and mutant recombinant proteins . Here, the cDNA of the zymogen domain of FXII (betaFXII) was cloned into the pET-28a(+) vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) and overexpressed . The multi-disulfide, recombinant protein, His(6)-betaFXII (rbetaFXII), expressed as an inclusion body, was purified by means of a Ni(2+)-charged resin . The matrix-bound rbetaFXII was subjected to refolding with the glutathione redox system and activated by the in vivo activator, kallikrein . The active form, rbetaFXIIa, obtained in milligram quantities, exhibited similar structural and comparable functional properties relative to human betaFXIIa, as indicated by circular dichroism spectroscopy and kinetics of substrate hydrolysis . Thermodynamics of enzyme:inhibitor complex formation, including the expected 1:1 stoichiometry, was determined for rbetaFXIIa by isothermal calorimetric titration with a specific recombinant protein inhibitor, Cucurbita maxima trypsin inhibitor-V (rCMTI-V; 7kDa).

Protein Expr Purif, 2003 Jan, 27(1), 134 - 42
Effects of codon usage versus putative 5'-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm; Griswold KE et al.; Matching the codon usage of recombinant genes to that of the expression host is a common strategy for increasing the expression of heterologous proteins in bacteria . However, while developing a cytoplasmic expression system for Fusarium solani cutinase in Escherichia coli, we found that altering codons to those preferred by E . coli led to significantly lower expression compared to the wild-type fungal gene, despite the presence of several rare E . coli codons in the fungal sequence . On the other hand, expression in the E . coli periplasm using a bacterial PhoA leader sequence resulted in high levels of expression for both the E . coli optimized and wild-type constructs . Sequence swapping experiments as well as calculations of predicted mRNA secondary structure provided support for the hypothesis that differential cytoplasmic expression of the E . coli optimized versus wild-type cutinase genes is due to differences in 5(') mRNA secondary structures . In particular, our results indicate that increased stability of 5(') mRNA secondary structures in the E . coli optimized transcript prevents efficient translation initiation in the absence of the phoA leader sequence . These results underscore the idea that potential 5(') mRNA secondary structures should be considered along with codon usage when designing a synthetic gene for high level expression in E . coli.

Protein Expr Purif, 2003 Jan, 27(1), 104 - 8
Expression, purification, and characterization of human tyrosyl-tRNA synthetase; Jia J et al.; Human tyrosyl-tRNA synthetase is a homodimeric enzyme and each subunit is near 58 KD . It catalyzes the aminoacylation of tRNA(Tyr) by L-tyrosine . The His(6)-tagged human TyrS gene was obtained by RT-PCR from total RNA of human lung giant-cell cancer strain 95 D . It was confirmed by sequencing and cloned into the expression vector pET-24 a (+) to yield pET-24 a (+)-HTyrRS, which was transfected into Escherichia coli BL21-CodonPlus-RIL . The induced-expression level of His(6)-tagged human TyrRS was about 24% of total cell proteins under IPTG inducing . The recombinant protein was conveniently purified in a single step by metal (Ni(2+)) chelate affinity chromatography . About 22.3mg purified enzyme could be obtained from 1L cell culture . The k(cat) value of His(6)-tagged human TyrRS in the second step of tRNA(Tyr) aminoacylation was 1.49 s(-1) . The K(m) values of tyrosine and tRNA(Tyr) were 0.3 and 0.9 microM . Six His residues at the C terminus of human TyrRS have little effect on the activities of the enzyme compared with other eukaryotic TyrRSs.

Protein Expr Purif, 2003 Jan, 27(1), 75 - 84
Structural investigation of mutant Mycobacterium smegmatis arylamine N-acetyltransferase: a model for a naturally occurring functional polymorphism in Mycobacterium tuberculosis arylamine N-acetyltransferase; Kawamura A et al.; Arylamine N-acetyltransferase (NAT) acetylates the front-line anti-tuberculosis drug isoniazid (INH) and has been identified in Mycobacterium tuberculosis . A naturally occurring single nucleotide polymorphism (SNP) was recently found in the NAT gene in clinical isolates of M . tuberculosis . The nucleotide change from G-->A (619) produces an amino acid change Gly(207) Arg, which appears to reduce the activity of the NAT from M . tuberculosis (TBNAT) . It has not been possible to generate sufficient soluble recombinant TBNAT for 3D structural studies . Therefore, Mycobacterium smegmatis NAT (SMNAT), which has 60% identity to TBNAT and has Gly at 207, was used as a model to investigate the possible structural effects of the G-->A 619 SNP . The mutant form of SMnat (SM207Rnat) was constructed by in vitro site-directed mutagenesis and was heterologously expressed with an N-terminal His tag in Escherichia coli, for comparison with the SMNAT . Both recombinant SMNATs were purified using Ni affinity chromatography and treated with thrombin to cleave the tag . Both proteins were produced with average yields of over 10 mg/L and were active . Substrate specificity and thermal stability of SM207RNAT were assessed and compared with the wild type SMNAT using kinetic assays and circular dichroism spectroscopy . SM207RNAT was crystallised and a data set of 2.00 A resolution was obtained . The SM207RNAT had different substrate specificities to the wild type protein and the 3D structures revealed that the Gly(207) Arg mutation caused slight changes in the orientation of His(203) in SMNAT.

Protein Expr Purif, 2003 Jan, 27(1), 63 - 74
Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening; Cheng D et al.; Gelatinase A represents an attractive therapeutic target for cancer invasion and metastasis . In order to screen for gelatinase A inhibitors, we have cloned, overexpressed in a bacterial system, and purified the catalytic domain of human gelatinase A with (GaCDfn) or without (GaCD) fibronectin-like insert . GaCDfn and GaCD were purified to homogeneity and refolded in vitro . GaCDfn was refolded to a stable and active form in the presence of calcium and zinc ions . GaCD was refolded through direct dialysis against Tris-HCl (pH 7.5) buffer without calcium and zinc ions . GaCD is unstable in the presence of calcium and zinc ions . The enzymatic activities of GaCDfn and GaCD require calcium and zinc ions, but high concentration of zinc and calcium ions inhibited the activities . The GaCDfn and GaCD cleaved several synthetic substrates including a chromogenic thiopeptolide (TPL) and fluorogenic peptides with optimal activity around pH 7.5 . Moreover, GaCDfn and GaCD cleave gelatin and collagen VII and display similar cleavage patterns on the gel, but the digestion rate of these protein substrates by GaCD is apparently slower than GaCDfn . EDTA, 1,10-phenanthroline, and reference inhibitors potently blocked GaCDfn and GaCD enzymatic activities . A set of 3596 compounds from our center collection were screened by using GaCDfn and GaCD to cleave TPL . Further analysis by using MMP inhibitors indicated there is a correlation between IC(50) values on GaCDfn and GaCD . A few compounds with selectivity toward gelatinase A catalytic domain were identified for structure modification.

Protein Expr Purif, 2003 Jan, 27(1), 12 - 8
Cloning, expression, purification, and crystallisation of HIV-2 reverse transcriptase; Bird LE et al.; A purification procedure is described for the isolation of recombinant HIV-2 reverse transcriptase expressed in Escherichia coli . The p68 subunit is expressed, in the absence of induction, and use of a heparin-Sepharose column produces substantially pure protein . Concentration of the homodimeric p68 reverse transcriptase pool, followed by incubation at room temperature for several days, results in full conversion by E . coli proteases to the heterodimer (p68/p55) . This extended incubation simplifies the purification process and improves the yield of heterodimeric reverse transcriptase, which shows a truncation of the smaller subunit to 427 residues . The protein is then purified further by hydroxyapatite and gel-filtration chromatography to homogeneity . The HIV-2 RT is active and has been used to produce crystals that diffract to beyond 3.0 A.

Protein Expr Purif, 2003 Jan, 27(1), 1 - 11
Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system; Bagnall W et al.; The anabolic effects and bioavailability of insulin-like growth factors I and II (IGF-I, IGF-II) are regulated in part by a family of IGF-binding proteins (IGFBPs) . There are six known members of the IGFBP family, which share distinct structural characteristics and functional activities . To study the binding properties of these proteins, we have expressed recombinant IGFBP-3 and IGFBP-4 using the LCR/Mel expression system . Using this system, we found that recombinant IGFBP-3 was secreted by Mel cells and had a glycosylation pattern similar to that of native IGFBP-3 . Recombinant IGFBP-4 secreted from Mel cells had a molecular size identical to that of non-glycosylated native IGFBP-4 . The binding kinetics of recombinant IGFBPs was measured using a solid-phase ligand-binding assay, an in vitro solution-binding assay, and a cellular proliferation assay . IGF-I bound with high affinity to recombinant IGFBP-3 and IGFBP-4 with K(D)s of <0.25 nmol . As reported for native IGFBPs, IGF-II bound with affinity higher than IGF-I to recombinant IGFBP-3 and IGFBP-4 (K(D) of <0.05 nmol) . Recombinant IGFBP-3 and IGFBP-4 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the IGF-I receptor . We have compared the binding kinetics of Mel cell-expressed IGFBPs with that of recombinant protein expressed in Escherichia coli and found them to be equivalent . Here, we show that the LCR/Mel expression system represents an effective route for expression of biologically active IGFBPs.

Zhonghua Yi Xue Za Zhi, 2002 Nov 10, 82(21), 1493 - 7
{Construction and characterization of bispecific single-chain antibody fragments SZ-2/SZ-21 against platelet glycoprotein Ib alpha and beta3}; Dai K et al.; OBJECTIVE: To generate bispecific single-chain Fv (bis-scFv) antibody fragment SZ-2/SZ-21 against platelet glycoprotein (GP) Ib alpha and GP IIIa . METHODS: The SZ-2/SZ-21 bispecific single-chain antibody expression vector pET22-21-L-2scFv was constructed by gene recombination technique with a interdomain linker fragment, SZ-21 single-chain antibody (anti- GP IIIa) gene and SZ-2 single-chain antibody (anti- GP Ib alpha) gene, identified by sequencing, and introduced into Escherichia coli strain BL21 (DE3) Plys . The bispecific protein accumulated in bacteria as insoluble inclusion bodies was harvested, denatured, refolded, and affinity-purified in vitro . SZ-2/SZ-21 bispecific single-chain antibody was added into platelet-rich plasma (PRP) and the binding of SZ-2/SZ-21 bispecific single-chain antibody with platelets was examined by histochemistry and cytometry, ELISA, or Western blotting . Restocetin, ADP, or thrombin was added into PRP with SZ-2/SZ-21 bispecific single-chain antibody to observe the platelet aggregation . RESULTS: The expression vector pET22-21-L-2scFv was correctly constructed . Its recombinant protein was expressed mostly in the form of inclusion bodies, and the yield accounted for 14% of the total cell protein . The bispecific protein could bind to platelet . The platelet aggregation induced by restocetion, ADP, or thrombin in PRP with SZ-2/SZ-21 bispecific single-chain antibody was significantly reduced . CONCLUSION: An SZ-2/SZ-21 bispecific single-chain antibody against two target antigens in platelet was developed and characterized with the ability to inhibit platelet aggregation induced by restocetin, ADP, and thrombin.

Plant Cell, 2003 Jan, 15(1), 133 - 49
Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans; Hussain H et al.; Isoamylases are debranching enzymes that hydrolyze alpha-1,6 linkages in alpha-1,4/alpha-1,6-linked glucan polymers . In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated . cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato . The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis . Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms . This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity . Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex . Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis . The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.

Mol Cell Biol, 2003 Jan, 23(2), 687 - 98
Eukaryotic initiation factors 4G and 4A mediate conformational changes downstream of the initiation codon of the encephalomyocarditis virus internal ribosomal entry site; Kolupaeva VG et al.; Initiation of translation of encephalomyocarditis virus mRNA is mediated by an internal ribosome entry site (IRES) comprising structural domains H, I, J-K, and L immediately upstream of the initiation codon AUG at nucleotide 834 (AUG834) . Assembly of 48S ribosomal complexes on the IRES requires eukaryotic initiation factor 2 (eIF2), eIF3, eIF4A, and the central domain of eIF4G to which eIF4A binds . Footprinting experiments confirmed that eIF4G binds a three-way helical junction in the J-K domain and showed that it interacts extensively with RNA duplexes in the J-K and L domains . Deletion of apical hairpins in the J and K domains synergistically impaired the binding of eIF4G and IRES function . Directed hydroxyl radical probing, done by using Fe(II) tethered to surface residues in eIF4G's central domain, indicated that it is oriented with its N terminus towards the base of domain J and its C terminus towards the apex . eIF4G recruits eIF4A to a defined location on the IRES, and the eIF4G/eIF4A complex caused localized ATP-independent conformational changes in the eIF4G-binding region of the IRES . This complex also induced more extensive conformational rearrangements at the 3' border of the ribosome binding site that required ATP and active eIF4A . We propose that these conformational changes prepare the region flanking AUG834 for productive binding of the ribosome.

J Biol Chem, 2003 Mar 14, 278(11), 9092 - 9 Epub 2002 Dec 30.
Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide; Bulieris PV et al.; We have studied the folding pathway of a beta-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example . The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria . We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway . In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA . In addition, lipopolysaccharide (LPS) was required . OmpA remained unfolded when bound to Skp and LPS in solution . From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy . The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent . Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS . Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA . Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.

J Biol Chem, 2003 Mar 7, 278(10), 8653 - 60 Epub 2002 Dec 31.
Topoisomerase III can serve as the cellular decatenase in Escherichia coli; Nurse P et al.; topB, encoding topoisomerase III, was identified as a high copy suppressor of the temperature-sensitive parC1215 allele, encoding one of the subunits of topoisomerase IV . Overexpression of topoisomerase III at the nonpermissive temperature was shown subsequently to restore timely chromosome decatenation and suppress lethality in strains carrying either temperature-sensitive parE or parC alleles . By developing an assay in vitro for precatenane unlinking, we demonstrated directly that both topoisomerase III and topoisomerase IV were efficient at this task, whereas DNA gyrase was very inefficient at precatenane removal . These observations suggest that precatenane unlinking is sufficient to sustain decatenation of replicating daughter chromosomes in the cell.

DNA Repair (Amst), 2002 Jan 22, 1(1), 77 - 93
A model for the structure of the Escherichia coli SOS-regulated UmuD2 protein; Sutton MD et al.; The ubiquitous Y-family of DNA polymerases, exemplified by the Escherichia coli UmuC protein (the catalytic subunit of DNA Pol V), possess the remarkable ability to replicate imperfect DNA templates that cannot be replicated by other types of DNA polymerases . Since this ability comes at the cost of a reduced fidelity, it is important that organisms manage these unique polymerases to coordinate their actions with those of the replication machinery . In E . coli, it is becoming evident that a sophisticated series of protein-protein interactions involving the two forms of the umuD gene product, UmuD and UmuD' and components of the replicative DNA polymerase serve to manage the actions of the umuC-encoded DNA polymerase . The purpose of this study was to better understand how structural differences between UmuD2 and UmuD2' help to determine which biological role the umuDC gene products will play; the UmuD2C complex functions as a DNA damage checkpoint effector, while the UmuD2'C complex participates in translesion DNA synthesis, which serves as the mechanistic basis for most chemical and UV light mutagenesis . Based on the results of a combination of disulfide cross-linking experiments, measurements of solvent accessibility and electron paramagnetic spin resonance (EPR) studies, we have developed a refined model for the structure of the UmuD2 homodimer . In the model that we are proposing, the N-terminal arms of UmuD (residues 1-39) form an extended interface in the UmuD2 homodimer by folding down over the globular domains of their intradimer partners . As a result, significant portions of the surface of each globular domain are buried in the UmuD2 homodimer . Based on the structure of the UmuD2' homodimer, both in the crystal and in solution, these same surfaces are exposed . Implications of these structural differences between the UmuD2 and the UmuD2' homodimers with respect to their roles in managing the actions of the umuC-encoded DNA polymerase are discussed.

DNA Repair (Amst), 2002 Aug 6, 1(8), 699 - 701
A DNA damage checkpoint in Escherichia coli; Cairns J; An early attempt to find out if the DNA double helix is actively unwound before being replicated was not conclusive, but it did disclose the existence of a unique moment in the life cycle of Escherichia coli when the cell registers whether or not its DNA is intact . If not, the cell embarks on rapid breakdown of its DNA, like "apoptosis" in eukaryotic cells.

DNA Repair (Amst), 2002 Sep 4, 1(9), 731 - 41
Incision of a 1,3-intrastrand d(GpTpG)-cisplatin adduct by nucleotide excision repair proteins from yeast; Kong SE et al.; The protein machinery responsible for nucleotide excision repair (NER) is highly conserved from yeast to man . NER can be reconstituted with purified proteins, and the incision sites around a defined DNA lesion have been defined to the nucleotide level in a mammalian NER system . Here, we reconstitute NER in yeast whole cell extracts, as well as with partially purified yeast NER components . We show that NER activity can be isolated partly as a large protein complex, and map the sites of nucleotide incision around a cisplatin-induced DNA lesion . Our data indicate that yeast NER proteins excise an oligonucleotide of 23-26 bases containing the DNA lesion (rather than 26-30 bases as in humans), and that the 3' incision occurs around position 17 (rather than at position 9 as in humans).

DNA Repair (Amst), 2003 Jan 2, 2(1), 61 - 71
Lethality of visible light for Escherichia coli hemH1 mutants influence of defects in DNA repair; Sikora A et al.; The hemH gene encodes ferrochelatase, the final enzyme of the heme biosynthetic pathway . Defects of this enzyme lead to accumulation of protoporphyrin IX and an increase in reactive oxygen species, causing susceptibility to blue and white light in bacteria and protoporphyria in humans . Here we show that the photosensitivity of hemH1 strains is much increased when the bacteria are devoid of ability to repair abasic sites . The sensitivity is increased 10- or 50-fold, in mutants bearing single xth or triple xth-nth-nfo mutations, respectively, but is not changed in mutants bearing nth, fpg, mutY, and mutT that are positive or negative for uvrA . This may indicate that in hemH1 mutants abasic sites are accumulated to a greater degree than oxidised bases, and/or that protoporphyrin, in the presence of abasic sites, increases the photosensitivity of hemH1 cells . It was shown in this work that the level of abasic sites (and/or strand breaks) in DNA of hemH1 strains increases greatly . Abasic sites and oxidative bases are potential mutagenic lesions . Nevertheless, the sensitivity of hemH1 bacteria to the lethal effect of visible light is not accompanied by increase in mutations . One of the possible explanations is that the genotoxic effect due to damage of hemH, shortage of heme and/or accumulating of protoporphyrin IX makes mutagenesis impossible.

DNA Repair (Amst), 2002 Feb 28, 1(2), 111 - 23
In pursuit of a molecular mechanism for adaptive gene amplification; Hastings PJ et al.; "Adaptive" or "stationary-phase" mutation is a collection of apparent stress responses in which cells exposed to a growth-limiting environment generate genetic changes, some of which can allow resumption of rapid growth . In the well-characterized Lac system of Escherichia coli, reversions of a lac frameshift allele give rise to adaptive point mutations . Also in this system, adaptive gene amplification has been documented as a separate and parallel response that allows growth on lactose medium without acquisition of a compensatory frameshift mutation . In amplification, the DNA region containing the weakly functional lac allele becomes amplified to multiple copies, which produce sufficient enzyme activity to allow growth on the otherwise growth-limiting lactose medium . The amplifications are "adaptive" in that they occur after cells encounter the growth-limiting environment . Adaptive amplification is a reversible genetic change that allows adaptation and growth . It may be similar to chromosomal instability observed in the origins and progression of many cancers . We explore possible molecular mechanisms of adaptive amplification in the bacterial system and note parallels to chromosomal instability in other systems.

DNA Repair (Amst), 2002 Mar 28, 1(3), 251 - 7
Opposite base specificity in excision of pyrimidine ring-opened 1,N6-ethenoadenine by thymine glycol-DNA-glycosylases; Bajek M et al.; A highly mutagenic DNA lesion, 1,N6-ethenoadenine ( epsilon A) is chemically unstable and either depurinates or converts to a pyrimidine ring-opened product upon water molecule addition to the C(2)z.sbnd;N(3) bond in epsilon dA (compound B) . Compound B subsequently undergoes deformylation to yield compound C, which depurinates in the final step of the epsilon A rearrangement pathway . We have previously shown that epsilon A rearrangement products are not repaired by human N-methylpurine-DNA-glycosylase, which excises parental epsilon A . Compound B was shown to be eliminated from a B:T pair by Escherichia coli formamidopyrimidine-DNA-glycosylase (Fpg protein) and endonuclease III (Nth protein) . Fpg protein excised B also from a B:C pair, and much less efficiently from B:A and B:G pairs {J . Biol . Chem . 276 (2001) 21821} . Here we show that efficiency of B excision by the Nth protein also depends on the opposite base in the pair . Most efficient repair is observed when this derivative is paired with dG (Km=18nM, kcat=12) and is less favourable when paired with dC (Km=40nM, kcat=13) and dT (Km=32nM, kcat=11) . In physiological conditions, compound B is probably not excised by the Nth-glycosylase from a B:A pair, or from a single-stranded DNA, since kinetic constants in these conditions are an order or two orders of magnitude higher than when B is paired with T, C or G . A similar specificity for B excision was found for Saccharomyces cerevisiae Ntg2-glycosylase . Thus, when paired with A, an epsilon A derivative might be more persistent than when paired with other bases and give rise to AT-->TA transversions.

DNA Repair (Amst), 2002 Apr 29, 1(4), 261 - 73
Repair of 8-oxoG is slower in endogenous nuclear genes than in mitochondrial DNA and is without strand bias; Thorslund T et al.; DNA is vulnerable to the attack of certain oxygen radicals and one of the major DNA lesions formed is 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic lesion that can mispair with adenine . The repair of 8-oxoG was studied by measuring the gene specific removal of 8-oxoG after treatment of Chinese hamster ovary (CHO) fibroblasts with the photosensitizer Ro19-8022 . This compound introduces 8-oxoG lesions, which can then be detected with the Escherichia coli formamidopyrimidine DNA glycosylase (FPG) . In this report we present gene specific repair analysis of endogenous genes situated in different important cellular regions and also the first analysis of strand specific DNA repair of 8-oxoG in an endogenous gene . We were not able to detect any preferential repair of transcribed genes compared to non-transcribed regions and we did not detect any strand-bias in the repair of the housekeeping gene, dihydrofolate reductase (DHFR) . In vivo, mitochondrial DNA is highly exposed to reactive oxygen species (ROS), and we find that the repair of 8-oxoG is more efficient in the mitochondrial DNA than in the nuclear DNA.

DNA Repair (Amst), 2002 May 30, 1(5), 391 - 5
Fold-recognition analysis predicts that the Tag protein family shares a common domain with the helix-hairpin-helix DNA glycosylases; Bujnicki JM et al.; The Escherichia coli protein Tag is traditionally regarded as an archetype of one of four classes of N-alkylpurine DNA glycosylases . However, its structure and phylogenetic relationship to other glycosylases remains a mystery . Fold-recognition and sequence profile analyses suggest that Tag shares the catalytic domain with helix-hairpin-helix (HhH) glycosylases such as MutY, AlkA and EndoIII, but its N- and C-termini together form a unique His2Cys2 cluster . The findings presented in this paper provide insight into sequence-structure-function relationships in the Tag family and should aid in a more precise definition of the common core of the HhH superfamily of glycosylases involved in DNA repair.

DNA Repair (Amst), 2002 May 30, 1(5), 343 - 58
Structure-based interpretation of missense mutations in Y-family DNA polymerases and their implications for polymerase function and lesion bypass; Boudsocq F et al.; Our understanding of the molecular mechanisms of error-prone lesion bypass has changed dramatically in the past few years . The concept that the key participants in the mutagenic process were accessory proteins that somehow modified the ability of the cell's main replicase to facilitate bypass of normally blocking lesions has been replaced with one in which the replicase is displaced by a polymerase specialized in lesion bypass . The participants in this process remain the same, only their function has been reassigned . What was once known as the UmuC/DinB/Rev1/Rad30 superfamily of mutagenesis proteins, is now known as the Y-family of DNA polymerases . Quite remarkably, within the space of 3 years, the field has advanced from the initial discovery of intrinsic polymerase function, to the determination of the tertiary structures of several Y-family DNA polymerases.A key to determining the biochemical properties of each DNA polymerase is through structure-function studies that result in the site-specific substitution of particular amino acids at critical sites within each DNA polymerase . However, we should not forget the power of genetic selection that allows us to identify residues within each polymerase that are generated by "random mutagenesis" and which are important for both a gain or loss of function in vivo . In this review, we discuss the structural ramifications of several missense mutations previously identified in various Y-family DNA polymerase and speculate on how each amino acid substitution might modify the enzymatic activity of the respective polymerase or possibly perturb protein-protein interactions necessary for efficient translesion replication in vivo.

DNA Repair (Amst), 2002 Jun 21, 1(6), 437 - 47
Excision of 8-methylguanine site-specifically incorporated into oligonucleotide substrates by the AlkA protein of Escherichia coli; Gasparutto D et al.; 8-Methyl-2'-deoxyguanosine (8-medGuo) has been shown to be a major stable alkylation product of 2'-deoxyguanosine induced by methyl radical attack on DNA . Moreover, by using primer extension assays, the latter DNA modification has recently been reported to be a miscoding lesion by generating G to C and G to T transversions and deletions in vitro . However, no data have been reported up to now, concerning the processing of this C8-alkylated nucleoside by the DNA repair machinery . Therefore, we have investigated the capability of excision of 8-methylguanine (8-meGua) site specifically incorporated into oligonucleotide substrates by several bacterial, yeast and mammalian DNA N-glycosylases . The results show that the 3-methyladenine (3-meAde) DNA glycosylase II (AlkA protein) from Escherichia coli is the only DNA N-glycosylase tested able to remove 8-meGua from double-stranded DNA fragments . Moreover, the activity of AlkA for 8-meGua varied markedly depending on the opposite base in DNA, being the highest with Adenine and Thymine and the lowest with Cytosine and Guanine . The removal of 8-meGua by AlkA protein was compared to that of 7-methylguanine (7-meGua) and hypoxanthine (Hx) . The rank of damage as a substrate for AlkA being 7-meGua>8-meGua>Hx . In contrast, the human 3-meAde DNA N-glycosylase (Mpg) is not able to release 8-meGua paired with any of the four DNA bases . We also show that, DNA N-glycosylases involved in the removal of oxidative damage, such as Fpg or Nth proteins from E . coli, Ntg1, Ntg2 or Ogg1 proteins of Saccharomyces cerevisiae, or human Ogg1 do not release 8-meGua placed opposite any of the four DNA bases . Furthermore, HeLa and Chinese hamster ovary (CHO) cell free protein extracts do not show any cleavage activity at 8-meGua paired with adenine or cytosine, which suggests the absence of base excision repair (BER) of this lesion in mammalian cells.

DNA Repair (Amst), 2002 Jul 17, 1(7), 571 - 6
The oxidized pyrimidine ribonucleotide, 5-hydroxy-CTP, is hydrolyzed efficiently by the Escherichia coli recombinant Orf135 protein; Fujikawa K et al.; The Escherichia coli orf135 gene encodes a 15.4kDa protein with homology to the MutT family of nucleotide hydrolases . The orf135 gene was cloned within a glutathione S-transferase (GST) fusion protein expression vector, which was used to overproduce the GST-Orf135 fusion protein in E . coli . The fusion protein thus obtained was purified by affinity column chromatography and gel filtration chromatography from the crude extract . The recombinant Orf135 protein was obtained by removing the GST tag from the purified fusion protein . Various oxidized nucleotides were tested as substrates for the recombinant Orf135 protein . As a result, we found a novel 5-hydroxy-CTPase activity of Orf135, but the hydrolyzing activities for the other nucleotides, including 5-hydroxy-dCTP, were very low . The activation constant (K(a)) of Mg(2+) for the 5-hydroxy-CTPase activity was 1.2 mM, and the pH optimum was 8.5 . The catalytic efficiency (k(cat)/K(m)) for this activity was 630 s(-1) mM(-1) at 30 degrees C, which was 30-fold higher than that for the CTPase activity . This result indicates that 5-hydroxy-CTP is the best substrate of Orf135 among the nucleotides tested .

World J Gastroenterol, 2003 Jan, 9(1), 89 - 93
Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma; Liao C et al.; AIM: To find the point mutations meaningful for inactivation of liver-related putative tumor suppressor gene (LPTS) gene, a human novel liver-related putative tumor suppressor gene and telomerase inhibitor in hepatocellular carcinoma . METHODS: The entire coding sequence of LPTS gene was examined for mutations by single strand conformation polymorphism (SSCP) assay and PCR products direct sequencing in 56 liver cancer cell lines, 7 ovarian cancer and 7 head neck tumor cell lines and 70 pairs of HCC tissues samples . The cDNA fragment coding for the most frequent mutant protein was subcloned into GST fusion expression vector . The product was expressed in E.coli and purified by glutathione-agarose column . Telomeric repeat amplification protocol (TRAP) assays were performed to study the effect of point mutation to telomerase inhibitory activity . RESULTS: SSCP gels showed the abnormal shifting bands and DNA sequencing found that there were 5 different mutations and/or polymorphisms in 12 tumor cell lines located at exon2, exon5 and exon7 . The main alterations were A(778)A/G and A(880)T in exon7 . The change in site of 778 could not be found in HCC tissue samples, while the mutation in position 880 was seen in 7 (10 %) cases . The mutation in the site of 880 had no effect on telomerase inhibitory activity . CONCLUSION: Alterations identified in this study are polymorphisms of LPTS gene . LPTS mutations occur in HCC but are infrequent and of little effect on the telomerase inhibitory function of the protein . Epigenetics, such as methylation, acetylation, may play the key role in inactivation of LPTS.

J Infect Dis, 2003 Jan 1, 187(1), 154 - 8 Epub 2002 Dec 13.
Induction of apoptosis of human brain microvascular endothelial cells by shiga toxin 1; Ergonul Z et al.; Brain injury is the most frequent cause of mortality among patients with the hemolytic-uremic syndrome . Human brain endothelial cells (HBECs) are resistant to Escherichia coli-derived Shiga toxin (Stx); however, inflammatory cytokines markedly increase HBEC sensitivity to Stx cytotoxicity . HBECs were exposed to tumor necrosis factor (TNF)-alpha, with and without Stx-1, and cell survival, (125)I-Stx1 binding, globotriaosylceramide content, cell necrosis, and cell apoptosis levels were determined . TNF greatly increased Stx-1 cytotoxicity, primarily through induction of apoptosis, in HBEC.

Res Vet Sci, 2003 Feb, 74(1), 31 - 6
Cytokines in mammary lymph and milk during endotoxin-induced bovine mastitis; Persson Waller K et al.; Cytokine kinetics were examined in milk and in afferent and efferent lymph of the supramammary lymph node after intramammary infusion of endotoxin from Escherichia coli . Cows were sampled 0, 2 and 4h after infusion (p.i.) . Neutrophils appeared in afferent lymph 2h p.i., and in efferent lymph and milk 4h p.i . The milk contained high concentrations of interleukin-8 (IL-8) at 2 and 4h p.i . IL-8 was also found in lymph, but at lower concentrations . The tumor necrosis factor-alpha (TNF-alpha) concentration tended to increase in afferent lymph at 2h p.i., and increased in milk at 4h p.i . The level of IL-1beta increased at 4h p.i . in milk, but was not detected in lymph . Interferon-gamma was not detected in any sample, at any time . The results indicate a primary role for IL-8 in the recruitment of neutrophils into the gland, and suggest that IL-1beta and TNF-alpha are not necessary for IL-8 production and release in response to endotoxin.

Biochem Biophys Res Commun, 2003 Jan 17, 300(3), 699 - 705
Identification of functional domains of the Escherichia coli SeqA protein; Fujikawa N et al.; The Escherichia coli SeqA protein, a negative regulator of chromosomal DNA replication, prevents the overinitiation of replication within one cell cycle by binding to hemimethylated G-mA-T-C sequences in the replication origin, oriC . In addition to the hemimethylated DNA-binding activity, the SeqA protein has a self-association activity, which is also considered to be essential for its regulatory function in replication initiation . To study the functional domains responsible for the DNA-binding and self-association activities, we performed a deletion analysis of the SeqA protein and found that the N-terminal (amino acid residues 1-59) and the C-terminal (amino acid residues 71-181) regions form structurally distinct domains . The N-terminal domain, which is not involved in DNA binding, has the self-association activity . In contrast, the C-terminal domain, which lacks the self-association activity, specifically binds to the hemimethylated G-mA-T-C sequence . Therefore, two essential SeqA activities, self-association and DNA-binding, are independently performed by the structurally distinct N-terminal and C-terminal domains, respectively.

Biochem Biophys Res Commun, 2003 Jan 17, 300(3), 645 - 8
Purification of antifreeze proteins by adsorption to ice; Kuiper MJ et al.; Antifreeze proteins (AFPs) can protect organisms from freezing injury by adsorbing to ice and inhibiting its growth . We describe here a method where ice, grown on a cold finger, is used to selectively adsorb and purify these ice-binding proteins from a crude mixture . Type III recombinant AFP was enriched approximately 50-fold after one round of partitioning into ice and purified to homogeneity by a second round . This method can also be used to purify non-ice-binding proteins by linkage to AFP domains as demonstrated by the recovery of a 50 kDa maltose-binding protein-AFP fusion from a crude lysate of Escherichia coli.

J Mol Biol, 2003 Jan 24, 325(4), 809 - 23
Repositioning about the dimer interface of the transcription regulator CooA: a major signal transduction pathway between the effector and DNA-binding domains; Kerby RL et al.; Activation of the homodimeric transcriptional regulator CooA depends on the coupling of CO binding at an effector domain heme with the allosteric repositioning of the DNA-binding domain F-helix that promotes specific DNA interaction . By analogy to the homologous cAMP receptor protein (CRP), it has been proposed that effector binding elicits subunit reorientation about their coiled-coil C-helix interface, and that this effector domain reorientation stabilizes the active position of the DNA-binding domains . Here, we describe experiments in which effector-independent "CooA*" variants were selected following randomization of a six-residue portion of the C-helix dimerization domain . Subsequent activity analyses, both in vivo and in vitro, were consistent with a model wherein improved C-helix "leucine zipper" interactions modestly shifted the regulator population equilibrium towards the active conformation, although full activation remained CO-dependent . However, in addition to the improved leucine zipper, maximal CooA* activity required additional C-helix changes which in a WT background decreased normal CO-dependent DNA-binding 100-fold . This seemingly paradoxical combination suggested that maximal CooA* activity depended both on the improved coiled-coil interactions and the decoupling of the signal pathway within the effector domain . Both types of C-helix changes indicate that its repositioning is crucial for the allosteric shift in the inactive/active equilibrium of the DNA-binding domain.

J Mol Biol, 2003 Jan 24, 325(4), 697 - 709
Importance of the +73/294 interaction in Escherichia coli RNase P RNA substrate complexes for cleavage and metal ion coordination; Brannvall M et al.; We have studied an interaction, the "73/294-interaction", between residues 294 in M1 RNA (the catalytic subunit of Escherichia coli RNase P) and +73 in the tRNA precursor substrate . The 73/294-interaction is part of the "RCCA-RNase P RNA interaction", which anchors the 3' R(+73)CCA-motif of the substrate to M1 RNA (interacting residues underlined) . Considering that in a large fraction of tRNA precursors residue +73 is base-paired to nucleotide -1 immediately 5' of the cleavage site, formation of the 73/294-interaction results in exposure of the cleavage site . We show that the nature/orientation of the 73/294-interaction is important for cleavage site recognition and cleavage efficiency . Our data further suggest that this interaction is part of a metal ion-binding site and that specific chemical groups are likely to act as ligands in binding of Mg(2+) or other divalent cations important for function . We argue that this Mg(2+) is involved in metal ion cooperativity in M1 RNA-mediated cleavage . Moreover, we suggest that the 73/294-interaction operates in concert with displacement of residue -1 in the substrate to ensure efficient and correct cleavage . The possibility that the residue at -1 binds to a specific binding surface/pocket in M1 RNA is discussed . Our data finally rationalize why the preferred residue at position 294 in M1 RNA is U.

J Mol Biol, 2003 Jan 24, 325(4), 629 - 35
Determinants that target the integrase of phage HK022 into the mammalian nucleus; Kolot M et al.; The integrase (Int) protein of coliphage HK022 catalyzes the site-specific integration and excision of the phage into and from its Escherichia coli host chromosome . Int expressed from a plasmid in COS1 monkey cells is localized in the nucleus, as is a fusion protein between Int and the green fluorescent protein (GFP) . Mutation analysis of the GFP-Int fusion has revealed in Int two regions of positively charged amino acid residues that cooperate in the nuclear localization . One region harbors residues Arg90 and Arg93 . The other, which spans residues 307-340 belongs to the catalytic domain of Int, is rich in basic residues and is strongly conserved within the Int protein family . Being localized in the nucleus renders Int of HK022 as a potential recombinase for site-specific gene manipulations in mammals.

Cell, 2002 Dec 27, 111(7), 1027 - 39
Directed evolution of substrate-optimized GroEL/S chaperonins; Wang JD et al.; GroEL/S chaperonin ring complexes fold many unrelated proteins . To understand the basis and extent of the chaperonin substrate spectrum, we used rounds of selection and DNA shuffling to obtain GroEL/S variants that dramatically enhanced folding of a single substrate-green fluorescent protein (GFP) . Changes in the substrate-optimized chaperonins increase the polarity of the folding cavity and alter the ATPase cycle . These findings reveal a surprising plasticity of GroEL/S, which can be exploited to aid folding of recombinant proteins . Our studies also reveal a conflict between specialization and generalization of chaperonins as increased GFP folding comes at the expense of the ability of GroEL/S to fold its natural substrates . This conflict and the nature of the ring structure may help explain the evolution of cellular chaperone systems.

Biosci Biotechnol Biochem, 2002 Nov, 66(11), 2382 - 7
Equilibrium dialysis measurements of the Ca2+-binding properties of recombinant radish vacuolar Ca2+-binding protein expressed in Escherichia coli; Yuasa K et al.; Vacuoles of radish (Raphanus sativus) contained a Ca2+-binding protein (RVCaB) of 43 kDa . We investigated the Ca2+-binding properties of the protein . RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography . Ca2+-binding properties of the recombinant protein were examined by equilibrium dialysis with 45Ca2+ and small dialysis buttons . The protein was estimated to bind 19Ca2+ ions per molecule with a Kd for Ca2+ of 3.4 mM . Ca2+ was bound to the protein even in the presence of high concentrations of Mg2+ or K+ . The results suggested that the protein bound Ca2+ with high ion selectivity, high capacity, and low affinity.

Biosci Biotechnol Biochem, 2002 Nov, 66(11), 2367 - 75
Stable form of ascorbate peroxidase from the red alga Galdieria partita similar to both chloroplastic and cytosolic isoforms of higher plants; Kitajima S et al.; Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions . Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B) . The electrophoretic mobility, Km values, kcat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured . Recombinant APX-B remained active for at least 180 min after depletion of ascorbate . The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs . In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform . The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.

Mol Reprod Dev, 2003 Feb, 64(2), 136 - 43
Expression in Escherichia coli and immunological characterization of three zona pellucida proteins (ZP1, ZP2, and ZP3) from a marsupial, the brushtail possum (Trichosurus vulpecula); Mate KE et al.; The brushtail possum (Trichosurus vulpecula) zona pellucida (ZP) is composed of three major glycoproteins, designated ZP1, ZP2, and ZP3 based on their size and homology with eutherian ZP proteins . These proteins are candidate antigens for the development of an immunocontraceptive vaccine to control the fertility of the brushtail possum in New Zealand, where it is an introduced pest . In order to further their immunological and functional characterization, recombinant possum ZP proteins were produced in Escherichia coli (E . coli) strain JM109, M15, SG13009, or BL21 codon plus . Each of the proteins produced possessed a N-terminal six histidine tag (His)(6) to facilitate purification and consisted of amino acid (aa) residues 18-471 of possum ZP1, aa residues 40-311 of ZP2 (ZP2-N), aa residues 305-634 of ZP2 (ZP2-C), and aa residues 23-342 of ZP3 . Immunoblot using anti-RGS(His)(4) antibodies and polyclonal rabbit anti-porcine ZP antibodies detected major bands at 54 kDa for ZP1, 32 kDa for ZP2-N, 39 kDa for ZP2-C, and 40 kDa for ZP3 . Immunization of male and female rabbits with ZP2-N, ZP2-C, and ZP3 purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with recombinant ZP proteins on Western blot and with native ZP proteins in possum ovarian sections using immunofluorescence . Antibodies generated against ZP1 in the same way were reactive with recombinant ZP proteins on Western blot only . The recombinant possum ZP proteins and specific antibodies produced in this study give an indication of the antigenic relationship of the possum ZP proteins and are vital tools for future studies of sperm-ZP binding in marsupials and for the evaluation of ZP-based contraceptive vaccines in possums and other marsupials .

Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 32 - 7 Epub 2002 Dec 27.
Convergence and divergence in the mechanism of SNARE binding by Sec1/Munc18-like proteins; Dulubova I et al.; Sec1Munc18-like (SM) proteins functionally interact with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) in membrane fusion, but the mechanisms of these interactions differ . In vertebrates, SM proteins that mediate exocytosis (Munc18-1, 18-2, and 18c) bind to the closed conformation of syntaxins 1-4, which requires the N-terminal H(abc) domains and SNARE motifs of these syntaxins . In contrast, SM proteins that mediate Golgi and endoplasmic reticulum fusion (Sly1 and Vps45) bind only to short N-terminal sequences of syntaxins 5, 16, or 18, independently of their H(abc) domains and SNARE motifs . We now show that Munc18-1, Sly1, and Vps45 interact with cognate syntaxins via similar, autonomously folded N-terminal domains, but the syntaxin 5-binding surface of the Sly1 N-terminal domain is opposite to the syntaxin 1-binding surface of the Munc18-1 N-terminal domain . In transfected cells, the N-terminal domain of Sly1 specifically disrupts the structure of the Golgi complex, supporting the notion that the interaction of Sly1 with syntaxin 5 is essential for fusion . These data, together with previous results, suggest that a relatively small N-terminal domain of SM proteins is dedicated to mechanistically distinct interactions with SNAREs, leaving the remaining large parts of SM proteins free to execute their as yet unknown function as effector domains.

EMBO J, 2003 Jan 2, 22(1), 140 - 50
Stable co-existence of separate replicons in Escherichia coli is dependent on once-per-cell-cycle initiation; Skarstad K et al.; DNA replication in most organisms is regulated such that all chromosomes are replicated once, and only once, per cell cycle . In rapidly growing Escherichia coli, replication of eight identical chromosomes is initiated essentially simultanously, each from the same origin, oriC . Plasmid-borne oriC sequences (minichromosomes) are also initiated in synchrony with the eight chromosomal origins . We demonstrate that specific inactivation of newly formed, hemimethylated origins (sequestration) was required for the stable co-existence of oriC-dependent replicons . Cells in which initiations were not confined to a short interval in the cell cycle (carrying mutations in sequestration or initiation genes or expressing excess initiator protein) could not support stable co-existence of several oriC-dependent replicons . The results show that such stable co-existence of oriC-dependent replicons is dependent on both a period of sequestration that is longer than the initiation interval and a reduction of the initiation potential during the sequestration period . These regulatory requirements are the same as those required to confine initiation of each replicon to once, and only once, per cell cycle.

EMBO J, 2003 Jan 2, 22(1), 36 - 46
Domain organization of the MscS mechanosensitive channel of Escherichia coli; Miller S et al.; The major structural features of the Escherichia coli MscS mechanosensitive channel protein have been explored using alkaline phosphatase (PhoA) fusions, precise deletions and site-directed mutations . PhoA protein fusion data, combined with the positive-inside rule, strongly support a model in which MscS crosses the membrane three times, adopting an N(out)-C(in) configuration . Deletion data suggest that the C-terminal domain of the protein is essential for the stability of the MscS channel, whereas the protein will tolerate small deletions at the N-terminus . Four mutants that exhibit either gain-of-function (GOF) or loss-of-function have been identified: a double mutation I48D/S49P inactivates MscS, whereas the MscS mutants T93R, A102P and L109S cause a strong GOF phenotype . The similarity of MscS to the last two domains of MscK (formerly KefA) is reinforced by the demonstration that expression of a truncated MscK protein can substitute for MscL and MscS in downshock survival assays . The data derived from studies of the organization, conservation and the influence of mutations provide significant insights into the structure of the MscS channel.

Int J Food Microbiol, 2003 Jan 26, 82(1), 33 - 43
Modelling the effects of temperature, water activity, pH and lactic acid concentration on the growth rate of Escherichia coli; Ross T et al.; An extended square root-type model describing Escherichia coli growth rate was developed as a function of temperature (7.63-47.43 degrees C), water activity (0.951-0.999, adjusted with NaCl), pH (4.02-8.28) and lactic acid concentration (0-500 mM).The new model, based on 236 growth rate data, combines and extends previously published square root-type models and incorporates terms for upper and lower limiting temperatures, upper and lower limiting pH, minimum inhibitory concentrations of dissociated and undissociated lactic acid and lower limiting water activity . A term to describe upper limiting water activity was developed but could not be fitted to the E . coli data set because of the difficulty of generating data in the super-optimal water activity range (i.e . >0.998) . All data used to generate the model are presented.The model provides an excellent description of the experimental data.

Toxicology, 2002 Dec 27, 181-182, 219 - 22
Oxy radicals, lipid peroxidation and DNA damage; Marnett LJ; Oxygen radicals react with polyunsaturated fatty acid residues in phospholipids resulting in the production of a plethora of products, many of them reactive toward protein and DNA . One of the most abundant carbonyl products of lipid peroxidation is malondialdehyde (MDA), which also is generated as a side-product of prostaglandin biosynthesis . It reacts with DNA to form adducts to deoxyguanosine, deoxyadenosine, and deoxycytidine . The deoxyguanosine adduct (M(1)G) has been detected in liver, white blood cells, colon, pancreas, and breast from healthy human beings at levels ranging from 1 to 120 per 10(8) nucleotides . Random and site-specific mutagenesis experiments indicate that MDA-DNA adducts are mutagenic in bacteria and in mammalian cells . M(1)G is highly mutagenic when incorporated into viral genomes then replicated in E . coli . It is repaired by the nucleotide excision repair pathway . Lipid peroxidation appears to be a major source of endogenous DNA damage in humans that may contribute significantly to cancer and other genetic diseases linked to lifestyle and dietary factors.

FEBS Lett, 2003 Jan 2, 533(1-3), 119 - 23
Interdomain interaction through helices A and B of DnaK peptide binding domain; Moro F et al.; In order to better define the structural elements involved in allosteric signalling, wild-type DnaK and three deletion mutants of the peptide binding domain have been characterized by biophysical (steady-state and time-resolved fluorescence) and biochemical methods . In the presence of ATP the chemical environment of the single tryptophan residue of DnaK, located in the ATPase domain, becomes less polar, as seen by a blue shift of the emission maximum and a shortening of the fluorescence lifetime, and its accessibility to polar quenchers is drastically reduced . These nucleotide-dependent modifications are also observed for the deletion mutant DnaK1-537, but not for DnaK1-507 or DnaK1-385, and thus rely on the presence of residues 507-537 (helices A and the N-terminal half of B) of the peptide binding domain . These data indicate that alphaA and half alphaB contribute to the allosteric communication of DnaK . In the presence of ATP, they promote a conformational change that displaces a residue(s) of the peptide binding domain towards a region of the ATPase domain where the tryptophan residue (W102) is located . A putative role for these helical segments as regulators of the position of the lid is discussed.

FEBS Lett, 2003 Jan 2, 533(1-3), 115 - 8
Engineering of Escherichia coli beta-galactosidase for solvent display of a functional scFv antibody fragment; Alcala P et al.; Protein engineering allows the generation of hybrid polypeptides with functional domains from different origins and therefore exhibiting new biological properties . We have explored several permissive sites in Escherichia coli beta-galactosidase to generate functional hybrid enzymes displaying a mouse scFv antibody fragment . When this segment was placed at the amino-terminus of the enzyme, the whole fusion protein was stable, maintained its specific activity and interacted specifically with the target antigen, a main antigenic determinant of foot-and-mouth disease virus . In addition, the antigen-targeted enzyme was enzymatically active when bound to the antigen and therefore useful as a reagent in single-step immunoassays . These results prove the flexibility of E . coli beta-galactosidase as a carrier for large-sized functional domains with binding properties and prompt the further exploration of the biotechnological applicability of the scFv enzyme targeting principle for diagnosis or other biomedical applications involving antigen tagging.

FEBS Lett, 2003 Jan 2, 533(1-3), 95 - 8
Kinetic properties and structural characteristics of an unusual two-domain arginine kinase of the clam Corbicula japonica; Suzuki T et al.; Arginine kinase (AK) from the clam Corbicula japonica is a unique enzyme in that it has an unusual two-domain structure with molecular mass of 80 kDa . It lacks two functionally important amino acid residues, Asp-62 and Arg-193, which are conserved in other 40 kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure . K m arg and Vmax values for the recombinant two-domain AK were determined . These values were close to those of usual 40 kDa AKs, although Corbicula AK lacks the functionally important Asp-62 and Arg-193 . Domain 2 of Corbicula AK was separated from the two-domain enzyme and was expressed in Escherichia coli . Domain 2 still exhibited activity . However, kinetic parameters for domain 2 appeared to be slightly, but significantly, different from those of two-domain AK . Thus, it is likely that the formation of the contiguous dimer alters the kinetic properties of its constituent domains significantly . Comparison of K d arg and K m arg for two-domain AK and its domain 2 showed that the affinity of the enzyme for arginine is greater in the presence of substrate ATP than in its absence . Presumably this difference is correlated with the large structural differences in the enzyme in the presence or absence of substrate, namely open and closed structures . We expressed three mutants of Corbicula AK domain 2 (His-60 to Gly or Arg, Asp-197 to Gly), and determined their K m arg and Vmax values . The affinity for the substrate arginine in mutant enzymes was reduced considerably, accompanied by a decrease in Vmax . These results suggest that His-60 and Asp-197 affect the substrate binding system, and are consistent with the hypothesis that a hydrogen bond is formed between His-60 and Asp-197 in Corbicula AK as a substitute for the Asp-62 and Arg-193 bond in normal AKs.

Arch Biochem Biophys, 2003 Jan 15, 409(2), 385 - 94
Probing sesquiterpene hydroxylase activities in a coupled assay with terpene synthases; Greenhagen BT et al.; 5-epi-Aristolochene dihydroxylase (EAH) catalyzes unique stereo- and regiospecific hydroxylations of a bicyclic sesquiterpene hydrocarbon to generate capsidiol . To define functional and mechanistic features of the EAH enzyme, the utility of a coupled assay using readily available sesquiterpene synthases and microsomes from yeast overexpressing the EAH enzyme was determined . Capsidiol and deoxycapsidiol biosyntheses were readily measured in coupled assays consisting of 5-epi-aristolochene synthase and EAH as determined by the incorporation of radiolabeled farnesyl diphosphate into thin-layer chromatography-isolated products and verified by gas chromatography-mass spectrometry analysis . The assays were dependent on the amounts of synthase and hydroxylase protein added, the incubation times, and the presence of nicotinamide adenine dinucleotide phosphate . The utility of this coupled assay was extended by examining the relative efficiency of the EAH enzyme to catalyze hydroxylations of different sesquiterpene skeletons generated by other terpene synthases.

Arch Biochem Biophys, 2003 Jan 15, 409(2), 305 - 14
Cytosine-block telomeric type DNA-binding activity of hnRNP proteins from human cell lines; Bandiera A et al.; Following the observation of the presence in mammalian nuclear extracts of a DNA binding activity quite specific for the single-stranded C-rich telomeric motif, we have isolated from the K562 human cell line by affinity chromatography and identified by mass spectrometry a number of proteins able to bind to this sequence . All of them belong to different heterogeneous nuclear ribonucleoprotein subgroups (hnRNP) . Whereas many of them, namely hnRNP K, two isoforms of hnRNP I, and the factor JKTBP, appear to bind to this sequence with limited specificity after isolation, an isoform of hnRNP D (alias AUF1) and particularly hnRNP E1 (alias PCBP-1) show a remarkable specificity for the (CCCTAA)n repeated motif . Both have been obtained also as recombinant proteins expressed in Escherichia coli and have been shown to retain their binding specificity toward the C-block repeated sequence . In the light of the current knowledge about these proteins, their possible involvement in telomere functioning is discussed.

Arch Biochem Biophys, 2003 Jan 15, 409(2), 251 - 61
Comparison of ligand polarization and enzyme activation in medium- and short-chain acyl-coenzyme A dehydrogenase-novel analog complexes; Lamm TR et al.; Spectroelectrochemical and off-resonance Raman indicate that substrate/product binding to medium-chain acyl-coenzyme A (CoA) dehydrogenase (pMCAD) results in ligand polarization and positive flavin potential shifts, which activate the enzyme for electron transfer . Bacterial short-chain acyl-CoA dehydrogenase (bSCAD) typically exhibits smaller potential shifts upon substrate/product binding that have not been linked to ligand polarization . To further investigate the roles of ligand binding and polarization in activation, several novel aromatic carboxyloyl-CoAs were designed . These analogs allowed for the first direct comparison of pMCAD and bSCAD mechanisms . The results indicate that pMCAD activation can occur without perceptible analog polarization . bSCAD data provide the first spectral evidence of ligand polarization . The potential alterations exhibited by ligand-bound bSCAD are smaller than those of pMCAD, while their directionality and magnitude suggest differing enzyme-analog interactions . Such data provide the first indication of variations in the activation mechanism of these enzymes, which were thought to be comparable in both structure and function.

Mutat Res, 2003 Jan 10, 534(1-2), 173 - 86
Spontaneous tandem-base mutations (TBM) show dramatic tissue, age, pattern and spectrum specificity; Hill KA et al.; To supplement a previous analysis of spontaneous tandem-base mutations (TBM) in the lacI gene of Big Blue((R)) mice, 2658 additional mutants were sequenced from 13 tissues and 44 spontaneous TBM were identified (tripling the sample size) . Previous findings were confirmed and generalized and several new observations were made . TBM differ from single and other double mutations in that TBM frequency varies dramatically with tissue type . In certain tissues, most notably male germ cells, no TBM are observed despite screening as many as 26 million plaque forming units . TBM are most frequent in kidney and liver (3.45 and 2x10(-6), respectively), accounting for 7.6 and 4.8% of all mutational events in kidney and liver, respectively . There is a trend for elevated TBM frequency in thymic lymphomas in p53-deficient mice . TBM are more frequent in old age in both liver and kidney . TBM differ from single mutations and other double mutations because they display a marked difference in pattern and dramatic tissue specificity for target sequence . Five of the 78 possible TBM outcomes comprise 79% of those observed, and mutations at GG/CC predominate . TBM in mice were compared with TBM found in human mutation databases . TBM are also rare in the human germline (one in 5133 germline mutations reported in five human mutation databases) . In general, the types of somatic TBM are similar in mice and humans except for an excess of TG/CA to CA/TG TBM in humans (TBM related to ultraviolet light-induced skin cancer were excluded) . TBM may be the result of unknown mechanisms that may have some similarities in mice and humans.

Curr Opin Struct Biol, 2002 Dec, 12(6), 709 - 20
Structural biology of enzymes involved in NAD and molybdenum cofactor biosynthesis; Rizzi M et al.; The structural analysis of all enzymes in a metabolic pathway is a prerequisite to answering fascinating questions, such as those relating to the evolutionary relationships between enzymes within the same and related pathways . Furthermore, the observed impressive diversity of catalytic functions displayed by these enzymes can lead to the synthesis of highly complex or unstable molecules, frequently involving unusual chemical reactions . Moreover, a detailed description of the active site of each enzyme in a pathway is of immense importance for the rational design of new drugs . The recent progress made in the structural biology of enzymes involved in NAD and molybdenum cofactor biosynthesis presents a significant step toward these goals.

Biomaterials, 2003 Mar, 24(6), 1059 - 66
MG63 osteoblastic cell adhesion to the hydrophobic surface precoated with recombinant osteopontin fragments; Lee YJ et al.; The hydrophobicity of biomaterials has been recognized as a limitation to the adequate function of anchorage-dependent cells when hydrophobic biomaterials are used for tissue engineering . This is due to flawed solid-state signals from cell adhesion . In this study, a recombinant osteopontin (rOPN17-169) fragment containing the cell adhesion motifs was expressed in E . coli and was precoated on the hydrophobic surface prior to osteoblastic MG63 cell culture . Precoating the hydrophobic surface with rOPN17-169 improved osteoblastic cell adhesion, which was blocked by soluble RGDS . The adhesion of MG63 cells to rOPN17-169 pre-coated surface-activated mitogen-activated protein kinases (MAPK) such as extracellular signal-receptor kinase 1/2, p38, and c-Jun N-terminal kinase (JNK) . In addition, p38 MAPK was activated in response to a soluble factor of transforming growth factor-beta in the cells adhered to the hydrophobic surface via rOPN17-169 . This suggests that rOPN17-169 precoated on the hydrophobic surface can allow osteoblastic cells to generate adhesion signals sufficient for cell adhesion, MAPK activation, and the cytokine activation of osteoblastic cells.

Biochimie, 2002 Oct, 84(10), 1021 - 9
Identification of the cofilin-binding sites in the large cytoplasmic domain of Na,K-ATPase; Kim M et al.; Na,K-ATPase, an alpha, beta heterodimer, is found in the plasma membrane of all animal cells . The alpha chain is believed to have 10 transmembrane regions and a large cytoplasmic domain between the 4th and 5th transmembrane regions (H4-H5) . In our previous report, the large (3rd) cytoplasmic domains of the alpha1 and alpha2 isoform were found to interact with cofilin, an actin-modulating protein, by the yeast two-hybrid system . Here we show that cofilin interacts only with the 3rd cytoplasmic domain of the alpha2 subunit but not with the 2nd, 4th, and 5th cytoplasmic domains or the cytoplasmic region of the beta subunit of Na,K-ATPase . We also demonstrate that cofilin interacts with the large cytoplasmic domains of the alpha1, alpha2 and alpha3 isoforms of Na,K-ATPase, but not with those of glucose transporter 1, glucose transporter 4, cystic fibrosis transmembrane conductance regulator and plasma membrane Ca-ATPase . We introduced 10 mutations into the 3rd cytoplasmic domain of Na,K-ATPase to identify the binding sites with cofilin . Eight of these mutants were single amino acid substitutions (R417Q, K470Q, K654G, D672A, K691A, R700G, R700A and D710G) and two were double mutant (K654GR700G and K719AK720A) . Analysis of the activity of the reporter gene of these mutants shows that residues D672 and R700 of the 3rd cytoplasmic domain of Na,K-ATPase are involved in the interaction with cofilin.

Biochem Biophys Res Commun, 2003 Jan 10, 300(2), 555 - 62
Interaction of a GATA factor with cis-acting elements involved in light regulation of nuclear genes encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase in Arabidopsis; Jeong MJ et al.; We have previously identified a cis-acting element, named the XXIII box, that is essential for light-regulated expression of the nuclear gene GAPB, which encodes the B subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana . Examination of the sequences indicated that there are two GATA motifs within the XXIII box . Based on the degree of the amino-acid sequence identity in the DNA binding domains, we divided the 25 GATA factors encoded in the Arabidopsis genome into three classes . We chose GATA-1 and GATA-20 from Class I and Class II, which include the majority of GATA factors, for overexpression in an Escherichia coli expression system . Gel mobility shift assays showed that GATA-1, but not GATA-20, binds specifically to the two GATA motifs within the XXIII fragment . In addition, we showed that GATA-1 could also bind specifically to a cis-acting element in the promoter of the GAPA gene, which is coordinately regulated by light with the GAPB gene . Based on these results, we propose that light controls the expression of GAPA and GAPB genes in part by regulating the binding of the same transcription factor at their GATA motifs.

Biochem Biophys Res Commun, 2003 Jan 10, 300(2), 531 - 40
Human serum amyloid A3 peptide enhances intestinal MUC3 expression and inhibits EPEC adherence; Larson MA et al.; We previously determined that the N-terminal region of bovine mammary-associated serum amyloid A3 (M-SAA3) increased intestinal mucin MUC3 levels in HT29 human intestinal cells by approximately 2.5-fold, relative to untreated cells . This study shows that the human M-SAA3 N-terminal peptide further enhances MUC3 transcript levels by approximately 4.3-fold in these cells (p<0.02), implicating a species-specific interaction . Furthermore, immunofluorescence and immunoblot analysis using a MUC3-specific monoclonal antibody confirms that the human M-SAA3 peptide stimulates MUC3 protein expression and secretion by the HT29 cells . More importantly, pretreatment of the cells with the peptide causes a subsequent 73% decrease in the adherence of enteropathogenic Escherichia coli (EPEC) to these cells, relative to untreated cells (p<0.01) . The intestinal mucin MUC3 has been shown to provide a protective barrier in the gut and inhibit adherence of pathogens to the gut wall . Therefore, a means to increase MUC3 protein expression by a colostrum-associated peptide or protein may be a highly effective prophylactic treatment for the prevention of gastrointestinal diseases such as necrotizing enterocolitis and infectious diarrhea.

Biochem Biophys Res Commun, 2003 Jan 10, 300(2), 378 - 82
A methionine sulfoxide reductase in Escherichia coli that reduces the R enantiomer of methionine sulfoxide; Etienne F et al.; It is known that Escherichia coli methionine mutants can grow on both enantiomers of methionine sulfoxide (met(o)), i.e., met-R-(o) or met-S-(o), indicating the presence of enzymes in E . coli that can reduce each of these enantiomers to methionine (met) . Previous studies have identified two members of the methionine sulfoxide reductase (Msr) family of enzymes, MsrA and fSMsr, that could reduce free met-S-(o), but the reduction of free met-R-(o) to met has not been elucidated . One possible candidate is MsrB which is known to reduce met-R-(o) in proteins to met . However, free met-R-(o) is a very poor substrate for MsrB and the level of MsrB activity in E . coli extracts is very low . A new member of the Msr family (fRMsr) has been identified in E . coli extracts that reduces free met-R-(o) to met . Partial purification of FRMsr has been obtained using extracts from an MsrA/MsrB double mutant of E . coli.

Mol Cell, 2002 Dec, 10(6), 1345 - 53
Molecular basis for the recognition of a nonclassical nuclear localization signal by importin beta; Cingolani G et al.; Nuclear import of proteins containing a classical nuclear localization signal (NLS) involves NLS recognition by importin alpha, which associates with importin beta via the IBB domain . Other proteins, including parathyroid hormone-related protein (PTHrP), are imported into the nucleus by direct interaction with importin beta . We solved the crystal structure of a fragment of importin beta-1 (1-485) bound to the nonclassical NLS of PTHrP . The structure reveals a second extended cargo binding site on importin beta distinct from the IBB domain binding site . Using a permeabilized cell import assay we demonstrate that importin beta (1-485) can import PTHrP-coupled cargo in a Ran-dependent manner . We propose that this region contains a prototypical nuclear import receptor domain, which could have evolved into the modern importin beta superfamily.

Environ Toxicol Chem, 2003 Jan, 22(1), 1 - 6
Analysis of arsenic bioavailability in contaminated soils; Turpeinen R et al.; The bioavailable arsenic (As) content of contaminated soils was determined by joint analyses of acid-soluble, total water-soluble, and biovailable As by using a luminescent bacterial sensor, Escherichia coli MC1061(pTOO31) . According to the results of this study, a significant positive correlation was found between the concentration of total water-soluble As and the bioavailability of As . However, the bioavailability of As in soil varied between sampling sites and was not equal when compared to the concentration of total water-soluble As; bioavailable As was 3 to 77% of total water-soluble As in soil . Our experiments also showed that aging and sequestration of As occurs in contaminated soils and As compounds thus become progressively less bioavailable with time . As a consequence, the bioavailability and toxicity of As should be considered when evaluating the ecological risks of contaminated soils.

Folia Microbiol (Praha), 2002, 47(5), 499 - 505
Response regulator ChiR regulates expression of chitinase gene, chiC, in Streptomyces coelicolor; Homerova D et al.; Transcription from the chiC promoter, directing expression of the chitinase gene, chiC, in Streptomyces coelicolor, was analyzed using xylE reporter gene and high-resolution S1-nuclease mapping . The transcription from the chiC promoter was induced by chitin, and this induction was dramatically reduced in the S . coelicolor chiR-disrupted strain . This indicated a dependence of chiC expression upon the chiR gene encoding a response regulator protein . To investigate this relationship, the S . coelicolor ChiR was overproduced using Escherichia coli T7 RNA polymerase expression system . However, gel mobility shift-assay with such a purified ChiR showed no binding in the chiC promoter region, which indicates a lack of specific phosphorylation of E . coli overproduced ChiR that is necessary for DNA-binding activity of response regulators.

Jpn J Hum Genet, 1997 Sep, 42(3), 389 - 99
Roles of DNA repair methyltransferase in mutagenesis and carcinogenesis; Sekiguchi M et al.; Alkylation of DNA at the O6-position of guanine is one of the most critical events leading to induction of mutation as well as cancer . An enzyme, O6-methylguanine-DNA methyltransferase, is present in various organisms, from bacteria to human cells, and appears to be responsible for preventing the occurrence of such mutations . The enzyme transfers methyl groups from O6-methylguanine and other methylated moieties of the DNA to its own molecule, thereby repairing DNA lesions in a single-step reaction . To elucidate the role of methyltransferase in preventing cancer, animal models with altered levels of enzyme activity were generated . Transgenic mice carrying extra copies of the foreign methyltransferase gene showed a decreased susceptibility to alkylating carcinogens, with regard to tumor formation . By means of gene targeting, mouse lines defective in both alleles of the methyltransferase gene were established . Administration of methylnitrosourea to these gene-targeted mice led to early death while normal mice treated in the same manner showed no untoward effects . Numerous tumors were formed in the gene-defective mice exposed to a low dose of methylnitrosourea, while none or only few tumors were induced in the methyltransferase-proficient mice . It seems apparent that the DNA repair methyltransferase plays an important role in lowering a risk of occurrence of cancer in organisms.

J Virol, 2003 Jan, 77(2), 1415 - 26
The 3C-like proteinase of an invertebrate nidovirus links coronavirus and potyvirus homologs; Ziebuhr J et al.; Gill-associated virus (GAV), a positive-stranded RNA virus of prawns, is the prototype of newly recognized taxa (genus Okavirus, family Roniviridae) within the order NIDOVIRALES: In this study, a putative GAV cysteine proteinase (3C-like proteinase {3CL(pro)}), which is predicted to be the key enzyme involved in processing of the GAV replicase polyprotein precursors, pp1a and pp1ab, was characterized . Comparative sequence analysis indicated that, like its coronavirus homologs, 3CL(pro) has a three-domain organization and is flanked by hydrophobic domains . The putative 3CL(pro) domain including flanking regions (pp1a residues 2793 to 3143) was fused to the Escherichia coli maltose-binding protein (MBP) and, when expressed in E . coli, was found to possess N-terminal autoprocessing activity that was not dependent on the presence of the 3CL(pro) C-terminal domain . N-terminal sequence analysis of the processed protein revealed that cleavage occurred at the location (2827)LVTHE downward arrow VRTGN(2836) . The trans-processing activity of the purified recombinant 3CL(pro) (pp1a residues 2832 to 3126) was used to identify another cleavage site, (6441)KVNHE downward arrow LYHVA(6450), in the C-terminal pp1ab region . Taken together, the data tentatively identify VxHE downward arrow (L,V) as the substrate consensus sequence for the GAV 3CL(pro) . The study revealed that the GAV and potyvirus 3CL(pro)s possess similar substrate specificities which correlate with structural similarities in their respective substrate-binding sites, identified in sequence comparisons . Analysis of the proteolytic activities of MBP-3CL(pro) fusion proteins carrying replacements of putative active-site residues provided evidence that, in contrast to most other 3C/3CL(pro)s but in common with coronavirus 3CL(pro)s, the GAV 3CL(pro) employs a Cys(2968)-His(2879) catalytic dyad . The properties of the GAV 3CL(pro) define a novel RNA virus proteinase variant that bridges the gap between the distantly related chymotrypsin-like cysteine proteinases of coronaviruses and potyviruses.

Int Immunol, 2003 Jan, 15(1), 71 - 7
Potential preventive effects of follistatin-related protein/TSC-36 on joint destruction and antagonistic modulation of its autoantibodies in rheumatoid arthritis; Tanaka M et al.; We previously reported that follistatin-related protein (FRP)/TSC-36 was one of the target antigens of autoantibodies in rheumatoid arthritis (RA) and that the appearance of serum autoantibodies to FRP correlated to disease activity in RA . However, the significance of FRP in autoimmunity remained to be explained due to the unknown function of FRP . Here, we disclose in part the function of FRP . Transforming growth factor (TGF)-beta augmented FRP gene expression in synovial cells . FRP reduced synovial production of matrix metalloproteinase (MMP)-1, MMP-3 and prostaglandin E(2), potent agonists of joint destruction in RA . In contrast, autoantibodies to FRP from patients with RA increased their production by blocking FRP activity, probably in the autocrine system . Moreover, FRP down-regulated synovial expression of FOS (c-fos), which seemed responsible for the reduction in MMP-1 and MMP-3 caused by FRP . Therefore, FRP and its autoantibody can be regarded as defensive and offensive factors respectively in rheumatoid arthropathy . The major epitope of autoantibodies to FRP was mapped to the sequence LKFVEQNE (residues 169-176) and homologous sequences were found in proteins from Escherichia coli, Epstein-Barr virus, etc . FRP and its autoantibody may provide some clues to elucidate the process of disease development and a new approach to the design of therapeutics in RA.

J Biol Chem, 2003 Mar 7, 278(10), 8065 - 74 Epub 2002 Dec 26.
The FAR protein family of the nematode Caenorhabditis elegans . Differential lipid binding properties, structural characteristics, and developmental regulation; Garofalo A et al.; Parasitic nematodes of humans and plants secrete a structurally novel type of fatty acid- and retinol-binding protein, FAR, into the tissues they occupy . These proteins may interfere with intercellular lipid signaling to manipulate the defense reactions of the host or acquire essential lipids for the parasites . The genome of the nematode Caenorhabditis elegans encodes eight FAR-like proteins (Ce-FAR-1 to -8) . These fall into three discrete groups as indicated by phylogenetic sequence comparisons and intron positions, the proteins from parasitic nematodes falling into group A . Recombinant Ce-FAR-1 to -7 were produced in Escherichia coli and tested for lipid binding in fluorescence-based assays . Ce-FAR-1 to -6 bound DAUDA (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid), cis-parinaric acid, and retinol with dissociation constants in the micromolar range, whereas Ce-FAR-7 bound the latter two lipids relatively poorly . Each protein produced a characteristic shift in peak fluorescence emission of DAUDA, and one (Ce-FAR-5) produced a shift greater than has been observed previously for any lipid-binding protein . Selected Ce-FAR proteins were analyzed by circular dichroism (CD) and differential scanning calorimetry, were found to be helix-rich, and exhibited high thermal stability (transition midpoint, 82.7 degrees C) . CD and secondary structure predictions, however, both indicated that Ce-FAR-7 possesses substantially less helix than the other FAR proteins . The genes encoding the Ce-FAR proteins were found to be transcribed differentially through the life cycle of C . elegans, such that Ce-far-4 was transcribed at highest levels in the fourth larval stage, and Ce-far-3 and -7 predominated in males.

Microbiol Res, 2002, 157(4), 257 - 65
Control of Escherichia coli growth rate through cell density; Carbonell X et al.; The transition from the exponential to the stationary phase of Escherichia coli cultures has been investigated regarding nutrient availability . This analysis strongly suggests that the declining of the cell division rate is not caused by mere nutrient limitation but also by an immediate sensing of cell concentration . In addition, both the growth rate and the final biomass achieved by a batch culture can be manipulated by altering its density during the early exponential phase . This result, which has been confirmed by using different experimental approaches, supports the hypothesis that the E . coli quorum sensing is not only determined by the release of soluble cell-to-cell communicators . Cell-associated sensing elements might also be involved in modulating the bacterial growth even in the presence of non-limiting (although declining) nutrient concentrations, thus promoting their economical utilisation in dense populations.

Sheng Li Ke Xue Jin Zhan, 1998 Apr, 29(2), 103 - 8
{Refolding of recombinant proteins in vitro}; Long JY et al.; Refolding of recombinant proteins is an important event in the downstream process of genetic engineering . On the basis of the mechanism of protein folding in vitro, general strategy of recombinant protein refolding is postulated, and major developments in recent years on this field are reviewed, including: molecular chaperone mediated refolding, detergent-assisted refolding, protein refolding in reverse micelle, addition of folding enhancer and methods for the formation of disulfide bond during protein refolding.

J Gen Appl Microbiol, 1999 Apr, 45(2), 69 - 75
Oxidation of chlorinated olefins by Escherichia coli transformed with dimethyl sulfide monooxygenase genes or cumene dioxygenase genes; Takami W et al.; In the present work, it was shown that the dimethyl sulfide (DMS) monooxygenase and the cumene dioxygenase catalyzed oxidation of various chlorinated ethenes, propenes, and butenes . The specific activities of these oxygenases were determined for C(2) to C(4) chlorinated olefins, and the oxidation rates ranged from 0.19 to 4.18 nmol.min(-1).mg(-1) of dry cells by the DMS monooxygenase and from 0.19 to 1.29 nmol.min(-1).mg(-1) of dry cells by the cumene dioxygenase . The oxidation products were identified by gas chromatography-mass spectrometry . Most chlorinated olefins were monooxygenated by the DMS monooxygenase to yield chlorinated epoxides . In the case of the cumene dioxygenase, the substrates lacking any chlorine atom on double-bond carbon atoms were dioxygenated, and those with chlorine atoms attaching to double-bond carbon atoms were monooxygenated to yield allyl alcohols.

J Gen Appl Microbiol, 1999 Apr, 45(2), 57 - 61
Molecular cloning, expression, and characterization of an endo-beta-1,4-glucanase cDNA from Epidinium caudatum1; Takenak A et al.; An endo-beta-1,4-glucanase gene (epi3) from the rumen ciliated protozoan Epidinium caudatum was cloned from a cDNA library constructed by using the lambda ZAP II vector . The enzymatic activity of the gene product was detected by the Congo red assay, using carboxymethyl cellulose (CMC) as substrate . The nucleotide sequence of epi3 revealed 1,253 nucleotides with an open reading frame for a protein (Epi3) of 356 amino acids (Mr -41,014) . Epi3 shows high homology with family 5 endoglucanase genes and with genes from protozoa isolated from sources other than the rumen . The specific activity of Epi3 produced in Escherichia coli was 5.544, 2.754, and 0.295 mmol of glucose min(-1) mg(-1) protein when the substrates used were CMC, beta-glucan, and xylan, respectively . A beta-1,4-linked trisaccharide of glucose was the preferred substrate of Epi3, as determined by analysis with the p-nitrophenyl form of the substrate . To our knowledge, this is the first report of the isolation of an endoglucanase gene from a rumen protozoan.

J Gen Appl Microbiol, 1997 Aug, 43(4), 199 - 208
Ability of high hydrostatic pressure treated plasmids and cells of Escherichia coli to genetically transform; Sharma A et al.; The exposure of plasmid pUC18 and pBR322 DNA to high hydrostatic pressure increased the ability of plasmids to transform competent Escherichia coli cells . For pUC18 plasmid, a pressure of 400 MPa, and for pBR322, a pressure of 200 MPa was found to provide the highest transformation efficiency . The DNA duplexes of the two plasmids were found to be the most stable for melting conditions at these pressures . At pressures higher than these, both the stability of the duplex DNA and the transformation efficiency were affected . The stabilizing effect of high hydrostatic pressure on the hydrogen bond may be responsible for the observed increase in transformation efficiency of the pressure-exposed plasmid DNA . The possibility of pressure-induced changes in the structure and conformation of DNA was studied using various techniques . In agarose gel electrophoresis, pressure-treated plasmids (pUC18 at 400 MPa and pBR322 at 200 MPa) consistently showed visibly distinct higher mobility compared to untreated plasmids . Pressure-treated pUC18 as well as pBR322 DNA showed significant reduction in ethidium bromide binding as is evident from the reduced intensity of fluorescence of the dye bound pressure-treated DNA . Spectroscopic studies using circular dichroism and Fourier transform infrared (FTIR) spectroscopy also showed significant differences in the absorption profiles of pressure-treated plasmids as compared to an untreated control . These studies revealed that the pressure-induced changes in the conformation of these DNAs may be responsible for the observed increase in the transformation ability of the plasmids . On the other hand, the exposure of competent cells of E . coli to a high hydrostatic pressure of 50 MPa not only reduced their colony-forming ability but also drastically reduced their ability to take up plasmid DNA.

Biochemistry, 2002 Dec 31, 41(52), 15861 - 6
Mutational analysis of synaptobrevin transmembrane domain oligomerization; Bowen ME et al.; Synaptobrevin 2 is thought to facilitate fusion of synaptic vesicles with the presynaptic membrane through formation of a soluble NSF attachment protein receptor complex (SNARE) with syntaxin 1a and a synaptosomal associated protein of 25 kDa (SNAP-25) . Previous reports have described a homodimer of synaptobrevin that is dependent on the transmembrane domain . However, these reports disagree about the magnitude of dimerization, which makes it difficult to assess the biological relevance of this interaction . We used SDS-PAGE and the TOXCAT genetic assay to reexamine the homodimerization of the synaptobrevin transmembrane domain in detergents and the Escherichia coli inner membrane, respectively . To gauge the magnitude of synaptobrevin homodimerization, we used the well-characterized glycophorin A homodimer as a positive standard . In contrast to previous studies, we found synaptobrevin homodimerization in E . coli is very weak when compared to glycophorin A . Recombinant synaptobrevin forms a small amount of dimer and higher order oligomers in detergents that are highly dependent on solublization conditions . We estimate a dissociation constant of 10 mM for synaptobrevin dimerization in detergent . Thus, the dimerization of synaptobrevin in membranes is very weak, questioning any possible functional role for this association in vivo.

Biochemistry, 2002 Dec 31, 41(52), 15803 - 9
Pre-steady-state kinetic studies on the Glu171Gln active site mutant of adenosylcobalamin-dependent glutamate mutase; Madhavapeddi P et al.; Glutamate-171 is involved in recognizing the amino group of the substrate in glutamate mutase . The effect of mutating this residue to glutamine on the ability of the enzyme to catalyze the homolysis of adenosylcobalamin has been investigated using UV-visible stopped-flow spectroscopy . Although Glu171 does not contact the coenzyme, the mutation results in the apparent rate constants for substrate-induced homolysis of the coenzyme that are slower by 7-fold and 13-fold with glutamate and methylaspartate, respectively, than those measured for the wild-type enzyme; furthermore, it weakens the binding of these substrates by approximately 50-fold and approximately 400-fold, respectively . These observations lend support to the idea that the enzyme may use substrate binding energy to accelerate homolysis of the coenzyme . The mutation also results in isotope effects on coenzyme homolysis that are much smaller than the very large effects observed when the wild-type enzyme is reacted with deuterated substrates . This observation is consistent with adenosylcobalamin homolysis being slowed relative to hydrogen abstraction from the substrate.

Biochemistry, 2002 Dec 31, 41(52), 15780 - 94
Expression and characterization of ferredoxin and flavin adenine dinucleotide binding domains of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath); Blazyk JL et al.; Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) catalyzes the selective oxidation of methane to methanol, the first step in the primary catabolic pathway of methanotrophic bacteria . A reductase (MMOR) mediates electron transfer from NADH through its FAD and {2Fe-2S} cofactors to the dinuclear non-heme iron sites housed in a hydroxylase (MMOH) . The structurally distinct {2Fe-2S}, FAD, and NADH binding domains of MMOR facilitated division of the protein into its functional ferredoxin (MMOR-Fd) and FAD/NADH (MMOR-FAD) component domains . The 10.9 kDa MMOR-Fd (MMOR residues 1-98) and 27.6 kDa MMOR-FAD (MMOR residues 99-348) were expressed and purified from recombinant Escherichia coli systems . The Fd and FAD domains have absorbance spectral features identical to those of the {2Fe-2S} and flavin components, respectively, of MMOR . Redox potentials, determined by reductive titrations that included indicator dyes, for the {2Fe-2S} and FAD cofactors in the domains are as follows: -205.2 +/- 1.3 mV for {2Fe-2S}(ox/red), -172.4 +/- 2.0 mV for FAD(ox/sq), and -266.4 +/- 3.5 mV for FAD(sq/hq) . Kinetic and spectral properties of intermediates observed in the reaction of oxidized MMOR-FAD (FAD(ox)) with NADH at 4 degrees C were established with stopped-flow UV-visible spectroscopy . Analysis of the influence of pH on MMOR-FAD optical spectra, redox potentials, and NADH reaction kinetics afforded pK(a) values for the semiquinone (FAD(sq)) and hydroquinone (FAD(hq)) MMOR-FAD species and two protonatable groups near the flavin cofactor . Electron transfer from MMOR-FAD(hq) to oxidized MMOR-Fd is extremely slow (k = 1500 M(-1) s(-1) at 25 degrees C, compared to 90 s(-1) at 4 degrees C for internal electron transfer between cofactors in MMOR), indicating that cofactor proximity is essential for efficient interdomain electron transfer.

Biochemistry, 2002 Dec 31, 41(52), 15566 - 77
Interactions of the products, 8-oxo-dGMP, dGMP, and pyrophosphate with the MutT nucleoside triphosphate pyrophosphohydrolase; Saraswat V et al.; The MutT enzyme from E . coli, in the presence of a divalent cation, catalyzes the hydrolysis of nucleoside- and deoxynucleoside-triphosphate (NTP) substrates by nucleophilic substitution at Pbeta, to yield a nucleotide (NMP) and PPi . The best substrate of MutT is believed to be the mutagenic nucleotide 8-oxo-dGTP, on the basis of its 10(3.4)-fold lower K(m) than that of dGTP (Maki, H., and Sekiguchi, M . (1992) Nature 355, 273-275) . To determine the true affinity of MutT for an 8-oxo-nucleotide and to elucidate the kinetic scheme, product inhibition by 8-oxo-dGMP and dGMP and direct binding of these nucleotides to MutT were studied . With Mg(2+)-activated dGTP hydrolysis, 8-oxo-dGMP is a noncompetitive inhibitor with K(I)(sl)(o)(pe) = 49 nM, which is 10(4.6)-fold lower than the K(I)(sl)(o)(pe)of dGMP (1.7 mM) . Similarly, the K(I)(intercept) of 8-oxo-dGMP is 10(4.0)-fold lower than that of dGMP . PPi is a linear uncompetitive inhibitor, suggesting that it dissociates first from the product complex, followed by the nucleotide . Noncompetitive inhibition by dGMP and 8-oxo-dGMP indicates an "iso" mechanism in which the nucleotide product leaves an altered form of the enzyme which slowly reverts to the form which binds substrate . Consistent with this kinetic scheme, (1)H-(15)N HSQC titration of MutT with dGMP reveals weak binding and fast exchange from one site with a K(D) = 1.8 mM, in agreement with its K(I)(sl)(o)(pe) . With 8-oxo-dGMP, tight binding and slow exchange (n = 1.0 +/- 0.1, K(D) < 0.25 mM) are found . Isothermal calorimetric titration of MutT with 8-oxo-dGMP yields a K(D) of 52 nM, in agreement with its K(I)(sl)(o)(pe) . Changing the metal activator from Mg(2+) to Mn(2+) had little effect on the K(I)(sl)(o)(pe) of dGMP or of 8-oxo-dGMP, consistent with the second-sphere enzyme-M(2+)-H(2)O-NTP-M(2+) complex found by NMR (Lin, J., Abeygunawardana, C., Frick, D . N., Bessman, M . J., and Mildvan, A . S . (1997) Biochemistry 36, 1199-1211), but it decreased the K(I) of PPi 12-fold, suggesting direct coordination of the PPi product by the enzyme-bound divalent cation . The tight binding of 8-oxo-dGMP to MutT (DeltaG degrees = -9.8 kcal/mol) is driven by a highly favorable enthalpy (<DeltaH(binding)> = -32 +/- 7 kcal/mol), with an unfavorable entropy (<-TDeltaS(o)(binding)> = +22 +/- 7 kcal/mol), as determined by van't Hoff analysis of the effect of temperature on the K(I)(sl)(o)(pe) and by isothermal titration calorimetry in two buffer systems . The binding of 8-oxo-dGMP to MutT induces changes in backbone (15)N and NH chemical shifts of 62 residues widely distributed throughout the protein, while dGMP binding induces smaller changes in only 22 residues surrounding the nucleotide binding site, suggesting that the unusually high affinity of MutT for 8-oxo-nucleotides is due not only to interactions with the altered 8-oxo or 7-NH positions on guanine, but results primarily from diffuse structural changes which tighten the protein structure around the 8-oxo-nucleotide.

Biochemistry, 2002 Dec 31, 41(52), 15459 - 67
Histidine 407, a phantom residue in the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex, activates reductive acetylation of lipoamide on the E2 subunit . An explanation for conservation of active sites between the E1 subunit and transketolase; Nemeria N et al.; Least squares alignment of the E . coli pyruvate dehydrogenase multienzyme complex E1 subunit and yeast transketolase crystal structures indicates a general structural similarity between the two enzymes and provides a plausible location for a short-loop region in the E1 structure that was unobserved due to disorder . The residue H407, located in this region, is shown to be able to penetrate the active site . Suggested by this comparison, the H407A E1 variant was created, and H407 was shown to participate in the reductive acetylation of both an independently expressed lipoyl domain and the intact 1-lipoyl E2 subunit . While the H407A substitution only modestly affected the reaction through pyruvate decarboxylation (ca . 14% activity compared to parental E1), the overall complex has a much impaired activity, at most 0.15% compared to parental E1 . Isothermal titration calorimetry measurements show that the binding of the lipoyl domain to the H407A E1 variant is much weaker than that to parental E1 . At the same time, mass spectrometric measurements clearly demonstrate much impaired reductive acetylation of the independently expressed lipoyl domain and of the intact 1-lipoyl E2 by the H407A variant compared to the parental E1 . A proposal is presented to explain the remarkable conservation of the three-dimensional structure at the active centers of the E . coli E1 subunit and transketolase on the basis of the parallels in the ligation-type reactions carried out and the need to protonate a very weak acid, a dithiolane sulfur atom in the former, and a carbonyl oxygen atom in the latter.

Mol Biol (Mosk), 2002 Nov-Dec, 36(6), 1062 - 7
{Mutational analysis of the conserved motif of the Arda anti-restriction protein encoded by self-transmissible IncI plasmid ColIb-p9}; Del'ver EP et al.; A study was made of the functional role of the ArdA antirestriction motif (130-LLADVPETVALYFD-143) conserved among all known Ard (alleviation of restriction of DNA) proteins, which are encoded by self-transmissible plasmids and specifically inhibit type I restriction-modification systems . Conserved residues of the motif were individually changed, and the resulting mutants tested for in vivo activity . Hydrophobic L130, L131, and V138 were substituted with negatively charged E; negatively charged D133, E136, and D143 substituted with hydrophobic V; and D127, D150, and D154 neighboring the antirestriction motif substituted with V . Four substitutions (L130E, L131E, V138E, and D143V) substantially (25-1000 times) reduced the ArdA activity . The other substitutions within or beyond the motif had no appreciable effect . Substitutions L130A and L131A each reduced the ArdA activity 10- to 20-fold, indicating that high hydrophobicity of L130 and L131 is important for the ArdA function . Thus, the antirestriction role of ArdA is indeed due to its conserved motif.

Mol Biol (Mosk), 2002 Nov-Dec, 36(6), 1055 - 61
{The correcting role of autonomous 3'-->5' exonucleases in mammalian multienzyme DNA polymerase complexes}; Shevelev IV et al.; A study was made of the correcting role of autonomous 3'-->5' exonucleases (AE) contained in multienzyme DNA polymerase complexes of rat hepatocytes or calf thymocytes . DNA was synthesized on phage psi X174 amber3 or M13mp2 primer-templates, and used to transfect Escherichia coli spheroplasts . Frequencies were estimated for direct and reverse mutations resulting from mistakes made in the course of in vitro DNA synthesis . The mistake rate of the hepatocytic complex was estimated at 3 x 10(-6) with equimolar dNTP, and increased tenfold when proteins accounting for 70% of the total 3'-->5' exonuclease activity of the complex were removed . The fidelity of DNA synthesis was completely restored in the presence of exogenous AE (epsilon subunit of E . coli DNA polymerase III) . Nuclear (Pol delta n) and cytosolic (Pol delta c) forms of DNA polymerase delta were isolated from calf thymocytes . The former was shown to contain an AE (TREX2) absent from the latter . As compared with Pol delta c, Pol delta n had a 20-fold higher exo/pol ratio and allowed 4-5 times higher fidelity of DNA synthesis . The mistake rate of DNA polymerase complexes changed when dNTP were used in nonequimolar amounts.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 174 - 6 Epub 2002 Dec 19.
Crystallization and preliminary X-ray diffraction studies of a novel alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix; Guy JE et al.; A novel alcohol dehydrogenase enzyme has been cloned from the hyperthermophilic archaeon Aeropyrum pernix and overexpressed in Escherichia coli . This zinc-containing enzyme has been crystallized by the sitting-drop vapour-diffusion method using PEG 600 as precipitant . The crystals diffract to 1.5 A resolution and belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 100.7, b = 103.2, c = 67.5 A . The asymmetric unit contains two enzyme monomers . Two synchrotron data sets have been collected: one at a wavelength near the absorption edge of zinc and one at a remote wavelength . Three strong zinc-ion positions were visible in the anomalous Patterson map . Two additional weaker zinc ions have been identified by anomalous Fourier synthesis.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 171 - 3 Epub 2002 Dec 19.
Crystallization and preliminary X-ray diffraction studies of Hje, a HolliDay junction resolving enzyme from Sulfolobus solfataricus; Middleton CL et al.; HolliDay junction endonuclease (Hje) from Sulfolobus solfataricus is a resolving enzyme involved in cleaving specific sites on either side of recombinant four-way HolliDay junctions . The HJE gene from S . solfataricus was cloned from genomic DNA into the pET19b Escherichia coli expression vector and recombinant protein was expressed to high levels . Hje was purified using heat treatment, cation exchange and gel filtration . Hanging-drop crystallization trials yielded primitive hexagonal crystals which diffract to 2.4 A on a laboratory source . Systematic absences (only 00l = 6n present) and poor scaling in P622 indicate that the space group is P6(1) or its enantiomer . Failed attempts at molecular replacement using models of a related archaeal resolving enzyme, Hjc, raise the possibility of a difference in quaternary structure between Hjc and Hje, which may be responsible for differences in their activities.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 168 - 70 Epub 2002 Dec 19.
SseA, a 3-mercaptopyruvate sulfurtransferase from Escherichia coli: crystallization and preliminary crystallographic data; Spallarossa A et al.; SseA, the translation product of the Escherichia coli sseA gene, is a 31 kDa protein endowed with 3-mercaptopyruvate:cyanide sulfurtransferase activity in vitro . As such, SseA is the prototype of a sulfurtransferase subfamily distinguished from the better known rhodanese sulfurtransferases, which display thiosulfate:cyanide sulfurtransferase activity . The physiological role of the two homologous enzyme families, whose catalytic activity is centred on a reactive invariant cysteine, is a matter of debate . In this framework, the forthcoming crystal structure analysis of SseA will be based on the tetragonal crystal form (space group P4(1) or P4(3)) reported here, with unit-cell parameters a = b = 150.2, c = 37.9 A.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 161 - 2 Epub 2002 Dec 19.
Jel44 monoclonal Fab fragment specific for HPr of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and the complex of Jel44 Fab fragment with HPr: preparation, crystallization and preliminary crystallographic analysis; Gulyaeva-Tcherkassova J et al.; Jel44 is a mouse monoclonal antibody specific for the histidine-containing phosphocarrier protein (HPr), a component of a sugar-transport system in Escherichia coli . Because Jel44 binding to HPr is dependent upon ionic strength and the enthalpic and entropic contributions do not vary over the temperature range 277-310 K, the complex is of great interest . A single crystal of the Jel44 Fab fragment was obtained and diffracted X-rays to a maximum resolution of 4.6 A on an in-house X-ray source . The crystal belongs to space group P2(1), with unit-cell parameters a = 68.6, b = 67.7, c = 105.5 A, beta = 96 degrees . Although crystals of the complex of Jel44 Fab fragment with HPr could not be fully characterized owing to suspected crystal twinning, it was encouraging that they diffracted X-rays to 2.5 A on an in-house X-ray source . It is thus foreseen that improvement of crystal quality will allow the complete solution of this novel structure.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 136 - 8 Epub 2002 Dec 19.
Crystals of trp repressor suitable for high-resolution neutron Laue diffraction studies; Daniels BV et al.; Crystallization and preliminary neutron-diffraction measurements of wild-type variant Val58-->Ile of the Escherichia coli trp repressor are reported . A vapor-diffusion chamber suitable for initial protein-solution Volumes in the range 0.2-0.5 ml was used to grow cube-shaped crystals with edge dimensions in the range 0.8-1.4 mm . Neutron Laue measurements to a nominal resolution of 2.1 A were recorded from a D(2)O-exchanged crystal using the LADI instrument at ILL . These results demonstrate that it will be possible for the first time to obtain a full-atom neutron structural model of a DNA-binding protein plus its associated solvent . Direct observation of hydrogen bonding between protein and solvent should enhance understanding of the role of solvent in protein-DNA recognition.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 73 - 6 Epub 2002 Dec 19.
Structure of Escherichia coli uridine phosphorylase at 2.0 A; Burling FT et al.; The 2.0 A crystal structure has been determined for Escherichia coli uridine phosphorylase (UP), an essential enzyme in nucleotide biosynthesis that catalyzes the phosphorolytic cleavage of the C-N glycosidic bond of uridine to ribose-1-phosphate and uracil . The structure determination of two independent monomers in the asymmetric unit revealed the residue composition and atomic details of the apo configurations of each active site . The native hexameric UP enzyme was revealed by applying threefold crystallographic symmetry to the contents of the asymmetric unit . The 2.0 A model reveals a closer structural relationship to other nucleotide phosphorylase enzymes than was previously appreciated.

J Biol Chem, 2003 Mar 21, 278(12), 10691 - 6 Epub 2002 Dec 22.
Mapping CD55 function . The structure of two pathogen-binding domains at 1.7 A; Williams P et al.; Decay-accelerating factor (CD55), a regulator of the alternative and classical pathways of complement activation, is expressed on all serum-exposed cells . It is used by pathogens, including many enteroviruses and uropathogenic Escherichia coli, as a receptor prior to infection . We describe the x-ray structure of a pathogen-binding fragment of human CD55 at 1.7 A resolution containing two of the three domains required for regulation of human complement . We have used mutagenesis to map biological functions onto the molecule; decay-accelerating activity maps to a single face of the molecule, whereas bacterial and viral pathogens recognize a variety of different sites on CD55.

Antimicrob Agents Chemother, 2003 Jan, 47(1), 188 - 95
Exploring the structure and function of the mycobacterial KatG protein using trans-dominant mutants; DeVito JA et al.; In order to probe the structure and function of the mycobacterial catalase-peroxidase enzyme (KatG), we employed a genetic approach using dominant-negative analysis of katG merodiploids . Transformation of Mycobacterium bovis BCG with various katG point mutants (expressed from low-copy-number plasmids) resulted in reductions in peroxidase and catalase activities as measured in cell extracts . These reductions in enzymatic activity usually correlated with increased resistance to the antituberculosis drug isoniazid (INH) . However, for the N138S trans-dominant mutant, the catalase-peroxidase activity was significantly decreased while the sensitivity to INH was retained . trans-dominance required katG expression from multicopy plasmids and could not be demonstrated with katG mutants integrated elsewhere on the wild-type M . bovis BCG chromosome . Reversal of the mutant phenotype through plasmid exchange suggested the catalase-peroxidase deficiency occurred at the protein level and that INH resistance was not due to a second site mutation(s) . Electrophoretic analysis of KatG proteins from the trans-dominant mutants showed a reduction in KatG dimers compared to WT and formation of heterodimers with reduced activity . The mutants responsible for these defects cluster around proposed active site residues: N138S, T275P, S315T, and D381G . In an attempt to identify mutants that might delimit the region(s) of KatG involved in subunit interactions, C-terminal truncations were constructed (with and without the D381G dominant-negative mutation) . None of the C-terminal deletions were able to complement a DeltakatG strain, nor could they cause a dominant-negative effect on the WT . Taken together, these results suggest an intricate association between the amino- and carboxy-terminal regions of KatG and may be consistent with a domain-swapping mechanism for KatG dimer formation.

Chem Biol, 2002 Dec, 9(12), 1337 - 46
Insights into trehalose synthesis provided by the structure of the retaining glucosyltransferase OtsA; Gibson RP et al.; Trehalose is a nonreducing disaccharide that plays a major role in many organisms, most notably in survival and stress responses . In Mycobacterium tuberculosis, it plays a central role as the carbohydrate core of numerous immunogenic glycolipids including "cord factor" (trehalose 6,6'-dimycolate) . The classical pathway for trehalose synthesis involves the condensation of UDP-glucose and glucose-6-phosphate to afford trehalose-6-phosphate, catalyzed by the retaining glycosyltransferase OtsA . The configurations of two anomeric positions are set simultaneously, resulting in the formation of a double glycoside . The three-dimensional structure of the Escherichia coli OtsA, in complex with both UDP and glucose-6-phosphate, reveals the active site at the interface of two beta/alpha/beta domains . The overall structure and the intimate details of the catalytic machinery reveal a striking similarity to glycogen phosphorylase, indicating a strong evolutionary link and suggesting a common catalytic mechanism.

Chem Biol, 2002 Dec, 9(12), 1297 - 303
Trapping distinct structural states of a protein/DNA interaction through disulfide crosslinking; He C et al.; The N-terminal domain of the Escherichia coli Ada protein (N-Ada) repairs methyl phosphotriesters in DNA through a zinc-mediated transfer to Cys38 of the protein . Methylation of Cys38 enhances the sequence-specific DNA affinity of N-Ada by approximately 1000-fold, thereby enabling the protein to activate the genes of a methylation-resistance regulon . It is of interest to understand the structural basis for metalloactivated methyl transfer and methylation-dependent enhancement of DNA binding activity . Although recent progress has been made on the structural front, efforts to develop a complete picture of N-Ada structure/function have been hampered by the inability to prepare homogeneous protein/DNA complexes representing different states of the unmethylated protein . Here, we describe the development of an approach to trap both sequence-specific and nonsequence-specific DNA recognition complexes of N-Ada through formation of an intermolecular disulfide crosslink between the protein and DNA.

J Mol Biol, 2003 Jan 17, 325(3), 461 - 70
Structure of the filamentous phage pIV multimer by cryo-electron microscopy; Opalka N et al.; The homo-multimeric pIV protein constitutes a channel required for the assembly and export of filamentous phage across the outer membrane of Escherichia coli . We present a 22 A-resolution three-dimensional reconstruction of detergent-solubilized pIV by cryo-electron microscopy associated with image analysis . The structure reveals a barrel-like complex, 13.5 nm in diameter and 24 nm in length, with D14 point-group symmetry, consisting of a dimer of unit multimers . Side views of each unit multimer exhibit three cylindrical domains named the N-ring, the M-ring and the C-ring . Gold labeling of pIV engineered to contain a single cysteine residue near the N or C terminus unambiguously identified the N-terminal region as the N-ring, and the C-terminal region was inferred to make up the C-ring . A large pore, ranging in inner diameter from 6.0 nm to 8.8 nm, runs through the middle of the multimer, but a central domain, the pore gate, blocks it . Moreover, the pore diameter at the N-ring is smaller than the phage particle . We therefore propose that the pIV multimer undergoes a large conformational change during phage transport, with reorganization of the central domain to open the pore, and widening at the N-ring in order to accommodate the 6.5 nm diameter phage particle .

Curr Biol, 2002 Dec 23, 12(24), R846 - 8
DNA repair: bioinformatics helps reverse methylation damage; Jiricny J; Recent work has uncovered a novel DNA repair enzyme: the AlkB protein of Escherichia coli, which oxidises the methyl groups of 1-methyladenine and 3-methylcytosine to hydroxymethyl moieties; the oxidised groups are subsequently released as formaldehyde, regenerating the unmodified bases.

Chromosome Res, 2002, 10(7), 561 - 70
Transient CENP-E-like kinetochore proteins in plants; ten Hoopen R et al.; Derived from candidate sequences of a barley EST database two proteins with homology to the coiled coil region of the human kinetochore protein (KP) CENP-E were generated and classified as centromere protein E-like 1 and 2 (Cpell and Cpe12) . Specific antibodies produced against recombinant Cpe11 and Cpe12 proteins labeled the centromere on mitotic chromosomes of barley and field bean and recognized specifically proteins from nuclear/chromosomal protein extracts on immunoblots . No function was predicted for homologues of Cpe11 within the databases for Arabidopsis and rice genomes . However, the centromeric location of Cpe11 and Cpe12 suggests they may have a function within the kinetochore . Plant homologues to barley Cpe12 are N-type kinesins, suggesting that Cpe12 is functionally homologous to human CENP-E.

Hum Mutat . 2003 Jan;21(1):99.
Mutations spectrum of GNE in hereditary inclusion body myopathy sparing the quadriceps; Eisenberg I et al.; Hereditary Inclusion Body Myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult onset and a typical muscle pathology . We have recently identified the gene encoding for a bifunctional enzyme, UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamine kinase (GNE), as the mutated gene in the prototype form of the disease presenting quadriceps sparing, particularly common in Middle Eastern Jews . Interestingly, we have identified the homozygous M712T Middle Eastern Jewish mutation also in two unrelated Middle Eastern Moslem families . We have also evaluated the involvement of GNE in several families from worldwide non-Jewish ethnic origins presenting symptoms similar to the Middle Eastern HIBM prototype . A total of 14 GNE mutations were identified (one nonsense and 13 missense), of which six are novel: an homozygous missense mutation in a consanguineous family from Italy and in a non consanguineous family from USA, and distinct compound heterozygotes in families from Germany, Italy, Ireland, Bahamas, USA and East India . This study brings to 17 the number of reported GNE mutations in quadriceps sparing myopathy, occurring either in the epimerase or the kinase domain of the enzyme . The mechanism leading to this unique phenotype still remains to be elucidated .

Nat Genet, 2003 Jan, 33(1), 23 - 4 Epub 2002 Dec 23.
Human mitochondrial transcription factor B1 methylates ribosomal RNA at a conserved stem-loop; Seidel-Rogol BL et al.; Human mitochondrial transcription factor B1 (h-mtTFB1) has an unprecedented relationship to RNA methyltransferases . Here, we show that this protein methylates a conserved stem-loop in bacterial 16S rRNA and that the homologous sequence in the human mitochondrial 12S molecule is similarly modified . Thus, h-mtTFB1 appears to be dual-function protein, acting both as a transcription factor and an rRNA-modification enzyme.

J Immunol, 2003 Jan 1, 170(1), 508 - 19
Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR "homotolerance" versus "heterotolerance" on NF-kappa B signaling pathway components; Dobrovolskaia MA et al.; In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-kappaB activation . Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-alpha secretion, IL-1R-associated kinase-1, and IkappaB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-kappaB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 "homotolerance," respectively) . Despite inhibited NF-kappaB DNA binding, increased levels of nuclear NF-kappaB were detected in agonist-pretreated macrophages . For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity . IKK kinase activity was unperturbed in heterotolerance . TNF-alpha secretion was also suppressed in P . gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-alpha . Experiments designed to elucidate novel mechanisms of NF-kappaB inhibition in tolerized cells revealed the potential contribution of IkappaBepsilon and IkappaBxi inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression . Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-kappaB signaling pathway.

J Immunol, 2003 Jan 1, 170(1), 373 - 83
High affinity xenoreactive TCR:MHC interaction recruits CD8 in absence of binding to MHC; Buslepp J et al.; The TCR from a xenoreactive murine cytotoxic T lymphocyte clone, AHIII 12.2, recognizes murine H-2D(b) complexed with peptide p1058 (FAPGFFPYL) as well as human HLA-A2.1 complexed with human self-peptide p1049 (ALWGFFPVL) . To understand more about T cell biology and cross-reactivity, the ectodomains of the AHIII 12.2 TCR have been produced in E . coli as inclusion bodies and the protein folded to its native conformation . Flow cytometric and surface plasmon resonance analyses indicate that human p1049/A2 has a significantly greater affinity for the murine AHIII 12.2 TCR than does murine p1058/D(b) . Yet, T cell binding and cytolytic activity are independent of CD8 when stimulated with human p1049/A2 as demonstrated with anti-CD8 Abs that block CD8 association with MHC . Even in the absence of direct CD8 binding, stimulation of AHIII 12.2 T cells with "CD8-independent" p1049/A2 produces p56(lck) activation and calcium flux . Confocal fluorescence microscopy and fluorescence resonance energy transfer flow cytometry demonstrate CD8 is recruited to the site of TCR:peptide MHC binding . Taken together, these results indicate that there exists another mechanism for recruitment of CD8 during high affinity TCR:peptide MHC engagement.

J Biol Chem, 2003 Feb 28, 278(9), 7215 - 9 Epub 2002 Dec 19.
Chromophore environment provides clue to "kindling fluorescent protein" riddle; Chudakov DM et al.; asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm . The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation . In this work, we used asCP mutants to investigate the mechanism underlying kindling . Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr(66) in the chromophore are crucial for kindling . We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization . Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins . Remarkably, most kindling mutants were capable of both reversible and irreversible kindling . Also, we obtained novel variants that kindled upon blue light irradiation . The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.

J Biol Chem, 2003 Mar 14, 278(11), 9042 - 51 Epub 2002 Dec 19.
Apoptin induces tumor-specific apoptosis as a globular multimer; Leliveld SR et al.; The chicken anemia virus-derived Apoptin protein induces tumor-specific apoptosis . Here, we show that recombinant Apoptin protein spontaneously forms non-covalent globular aggregates comprising 30 to 40 subunits in vitro . This multimerization is robust and virtually irreversible, and the globular aggregates are also stable in cell extracts, suggesting that they remain intact within the cell . Furthermore, studies of Apoptin expressed in living cells confirm that Apoptin indeed exists in large complexes in vivo . We map the structural motifs responsible for multimerization in vitro and aggregation in vivo to the N-terminal half of the protein . Moreover, we show that covalently fixing the Apoptin monomers within the recombinant protein multimer by internal cross-linking does not affect the biological activity of Apoptin, as these fixed aggregates exhibit similar tumor-specific localization and apoptosis-inducing properties as non-cross-linked Apoptin . Taken together, our results imply that recombinant Apoptin protein is a multimer when inducing apoptosis, and we propose that this multimeric state is an essential feature of its ability to do so . Finally, we determine that Apoptin adopts little, if any, regular secondary structure within the aggregates . This surprising result would classify Apoptin as the first protein for which, rather than the formation of a well defined tertiary and quaternary structure, semi-random aggregation is sufficient for activity.

J Biol Chem, 2003 Feb 28, 278(9), 6765 - 70 Epub 2002 Dec 20.
Metabolism of homocysteine-thiolactone in plants; Jakubowski H et al.; Editing of the amino acid homocysteine (Hcy) by certain aminoacyl-tRNA synthetases results in the formation of an intramolecular thioester, Hcy-thiolactone . Here we show that the plant yellow lupin, Lupinus luteus, has the ability to synthesize Hcy-thiolactone . The inhibition of methylation of Hcy to methionine by the anitifolate drug aminopterin results in greatly enhanced synthesis of Hcy-thiolactone by L . luteus plants . Methionine inhibits the synthesis of Hcy-thiolactone in L . luteus, suggesting involvement of methionyl-tRNA synthetase . Consistent with this suggestion is our finding that the plant Oryza sativa methionyl-tRNA synthetase, expressed in Escherichia coli, catalyzes conversion of Hcy to Hcy-thiolactone . We also show that Hcy is a component of L . luteus proteins, most likely due to facile reaction of Hcy-thiolactone with protein amino groups . In addition, L . luteus possesses constitutively expressed, highly specific Hcy-thiolactone-hydrolyzing enzyme . Thus, Hcy-thiolactone and Hcy bound to protein by an amide (or peptide) linkage (Hcy-N-protein) are significant components of plant Hcy metabolism.

Infect Immun, 2003 Jan, 71(1), 536 - 40
Role of virulence factors in resistance of avian pathogenic Escherichia coli to serum and in pathogenicity; Mellata M et al.; In chickens, colibacillosis is caused by avian pathogenic Escherichia coli (APEC) via respiratory tract infection . Many virulence factors, including type 1 (F1A) and P (F11) fimbriae, curli, aerobactin, K1 capsule, and temperature-sensitive hemagglutinin (Tsh) and plasmid DNA regions have been associated with APEC . A strong correlation between serum resistance and virulence has been demonstrated, but roles of virulence factors in serum resistance have not been well elucidated . By using mutants of APEC strains TK3, MT78, and chi7122, which belong to serogroups O1, O2, and O78, respectively, we investigated the role of virulence factors in resistance to serum and pathogenicity in chickens . Our results showed that serum resistance is one of the pathogenicity mechanisms of APEC strains . Virulence factors that increased bacterial resistance to serum and colonization of internal organs of infected chickens were O78 lipopolysaccharide of E . coli chi7122 and the K1 capsule of E . coli MT78 . In contrast, curli, type 1, and P fimbriae did not appear to contribute to serum resistance . We also showed that the iss gene, which was previously demonstrated to increase resistance to serum in certain E . coli strains, is located on plasmid pAPEC-1 of E . coli chi7122 but does not play a major role in resistance to serum for strain chi7122.

Infect Immun, 2003 Jan, 71(1), 384 - 92
Interaction of Ler at the LEE5 (tir) operon of enteropathogenic Escherichia coli; Haack KR et al.; The genome of enteropathogenic Escherichia coli (EPEC) encodes a global regulator, Ler (locus of enterocyte effacement {LEE}-encoded regulator), which activates expression of several polycistronic operons within the 35.6-kb LEE pathogenicity island, including the LEE2-LEE3 divergent operon pair containing overlapping -10 regions and the LEE5 (tir) operon . Ler is a predicted 15-kDa protein that exhibits amino acid similarity with the nucleoid protein H-NS . In order to study Ler-mediated activation of virulence operons in EPEC, we used a molecular approach to characterize the interactions of purified Ler protein with the upstream regulatory sequences of the LEE5 operon . We determined the cis-acting DNA sequences necessary for Ler binding at LEE5 by mobility shift and DNase I protection assays, demonstrating that Ler acts directly at LEE5 by binding sequences between positions -190 and -73 in relation to the transcriptional start site . Based on the molecular weight of Ler, the similarity to H-NS, and the extended region of protection observed in a DNase I footprint at LEE5, we hypothesized that multiple Ler proteins bind upstream of the LEE5 promoter to increase transcriptional activity from a distance . Using an hns deletion strain, we demonstrated that like the LEE2-LEE3 operon pair, H-NS represses LEE5 transcription . We describe a model in which Ler activates transcription at both divergent overlapping paired and single promoters by displacing H-NS, which results in the disruption of a repressing nucleoprotein complex.

Infect Immun, 2003 Jan, 71(1), 327 - 34
Antiviral activity of shiga toxin requires enzymatic activity and is associated with increased permeability of the target cells; Basu I et al.; This study expanded our earlier finding that Shiga toxin type 1 (Stx1) has activity against bovine leukemia virus (BLV) (W . A . Ferens and C . J . Hovde, Infect . Immun . 68:4462-4469, 2000) . The Stx molecular motifs required for antiviral activity were identified, and a mechanism of Stx action on virally infected cells is suggested . Using inhibition of BLV-dependent spontaneous lymphocyte proliferation as a measure of antiviral activity, we showed that Stx2 had antiviral activity similar to that of Stx1 . Enzymatic and antiviral activities of three StxA1 chain mutants deficient in enzymatic activity or aspects of receptor-mediated cytotoxicity were compared . Using protein synthesis inhibition to measure enzymatic activity, the mutant E167D was 300-fold less catalytically active than wild-type StxA1, was minimally active in antiviral assays, and did not inhibit synthesis of viral proteins . Two StxA1 mutants, A231D-G234E and StxA(1)1 (enzymatically active but unable to kill cells via the classical receptor-mediated route), had undiminished antiviral activity . Although binding of radiolabeled StxA1 to bovine blood cells or to free virus was not detected, flow cytometric analysis showed that the number of BLV-expressing cells were specifically reduced in cultures treated with Stx . These unique and rare lymphocytes were highly permeable to 40- and 70-kDa fluorescent dextrans, indicating that direct absorption of toxins by virus-expressing cells is a potential mechanism of target cell intoxication . These results support the hypothesis that Stx-producing Escherichia coli colonization of the gastrointestinal tract may benefit ruminant hosts by the ability of Stxs to exert antiviral activity.

Infect Immun, 2003 Jan, 71(1), 181 - 6
Purification and characterization of a UDP-glucosyltransferase produced by Legionella pneumophila; Belyi I et al.; Legionella pneumophila is the agent of Legionnaires' disease . It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells . The molecular mechanisms of the Legionella interaction with target cells are not fully defined . In an attempt to discover novel virulence factors of L . pneumophila, we searched for bacterial enzymes with transferase activity . Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells . An approximately 60-kDa UDP-glucosyltransferase was purified from L . pneumophila and subjected to microsequencing . An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L . pneumophila genome database . The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase . We designated this gene ugt (UDP-glucosyltransferase) . The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function . Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L . pneumophila strains but was absent from the other Legionella species . This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L . pneumophila proliferation.

Infect Immun, 2003 Jan, 71(1), 13 - 21
Pathogenicity and immune response measured in mice following intranasal challenge with enterotoxigenic Escherichia coli strains H10407 and B7A; Byrd W et al.; The pathogenicity and immunogenicity induced in BALB/c mice by intranasal (i.n.) inoculation of enterotoxigenic Escherichia coli (ETEC) strains H10407 (O78:H11:CFA/I:LT(+):ST(+)) and B7A (O148:H28:CS6:LT(+):ST(+)) (two ETEC strains previously used in human challenge trials) were studied . The i.n . inoculation of BALB/c mice with large doses of ETEC strains H10407 and B7A caused illness and death . The H10407 strain was found to be consistently more virulent than the B7A strain . Following i.n . challenge with nonlethal doses of H10407 and B7A, the bacteria were cleared from the lungs of the mice at a steady rate over a 2-week period . Macrophages and neutrophils were observed in the alveoli and bronchioles, and lymphocytes were observed in the septa, around vessels, and in the pleura of the lungs in mice challenged with H10407 and B7A . In mice i.n . challenged with H10407, serum immunoglobulin G (IgG) and IgM antibodies were measured at high titers to the CFA/I and O78 lipopolysaccharide (LPS) antigens . In mice i.n . challenged with B7A, low serum IgG antibody titers were detected against CS6, and low serum IgG and IgM antibody titers were detected against O148 LPS . The serum IgG and IgM antibody titers against the heat-labile enterotoxin were equivalent in the H10407- and B7A-challenged mice . The CFA/I and O78 LPS antigens gave mixed T-helper cell 1-T-helper cell 2 (Th1-Th2) responses in which the Th2 response was greater than the Th1 response (i.e., stimulated primarily an antibody response) . These studies indicate that the i.n . challenge of BALB/c mice with ETEC strains may provide a useful animal model to better understand the immunogenicity and pathogenicity of ETEC and its virulence determinants . This model may also be useful in providing selection criteria for vaccine candidates for use in primate and human trials.

Biophys J, 2002 Dec, 83(6), 3482 - 9
FTIR spectroscopy of the M photointermediate in pharaonis rhoborhodopsin; Furutani Y et al.; pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis . During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate (ppR(M)) . The present FTIR spectroscopy of ppR(M) revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR) . In addition, the C==O stretching vibrations of Asn-105 were assigned for ppR and ppR(M) . The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate) . These results implicate similar protein structural changes between ppR and BR . However, BR(M) decays to BR(N) accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure . In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M(N)) at alkaline pH . In ppR, such an N-like (M(N)-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.

Biophys J, 2002 Dec, 83(6), 3113 - 25
Addition of missing loops and domains to protein models by x-ray solution scattering; Petoukhov MV et al.; Inherent flexibility and conformational heterogeneity in proteins can often result in the absence of loops and even entire domains in structures determined by x-ray crystallographic or NMR methods . X-ray solution scattering offers the possibility of obtaining complementary information regarding the structures of these disordered protein regions . Methods are presented for adding missing loops or domains by fixing a known structure and building the unknown regions to fit the experimental scattering data obtained from the entire particle . Simulated annealing was used to minimize a scoring function containing the discrepancy between the experimental and calculated patterns and the relevant penalty terms . In low-resolution models where interface location between known and unknown parts is not available, a gas of dummy residues represents the missing domain . In high-resolution models where the interface is known, loops or domains are represented as interconnected chains (or ensembles of residues with spring forces between the C(alpha) atoms), attached to known position(s) in the available structure . Native-like folds of missing fragments can be obtained by imposing residue-specific constraints . After validation in simulated examples, the methods have been applied to add missing loops or domains to several proteins where partial structures were available.

Biophys J, 2002 Dec, 83(6), 2987 - 3000
Gauging of the PhoE channel by a single freely diffusing proton; Bransburg-Zabary S et al.; In the present study we combined a continuum approximation with a detailed mapping of the electrostatic potential inside an ionic channel to define the most probable trajectory for proton propagation through the channel (propagation along a structure-supported trajectory (PSST)) . The conversion of the three-dimensional diffusion space into propagation along a one-dimensional pathway permits reconstruction of an ion motion by a short calculation (a few seconds on a state-of-the-art workstation) rather than a laborious, time-consuming random walk simulations . The experimental system selected for testing the accuracy of this concept was the reversible dissociation of a proton from a single pyranine molecule (8-hydroxypyrene-1,2,3-trisulfonate) bound by electrostatic forces inside the PhoE ionic channel of the Escherichia coli outer membrane . The crystal structure coordinates were used for calculation of the intra-cavity electrostatic potential, and the reconstruction of the observed fluorescence decay curve was carried out using the dielectric constant of the intra-cavity space as an adjustable parameter . The fitting of past experimental observations (Shimoni, E., Y . Tsfadia, E . Nachliel, and M . Gutman . 1993 . Biophys . J . 64:472-479) was carried out by a modified version of the Agmon geminate recombination program (Krissinel, E . B., and N . Agmon . 1996 . J . Comp . Chem . 17:1085-1098), where the gradient of the electrostatic potential and the entropic terms were calculated by the PSST program . The best-fitted reconstruction of the observed dynamics was attained when the water in the cavity was assigned epsilon </= 55, corroborating the theoretical estimation of Sansom (Breed, J . R., I . D . Kerr, and M . S . P . Sansom . 1996 . Biophys . J . 70:1643-1661) . The dielectric constant calculated for reversed micelles of comparable size (Cohen, B., D . Huppert, K . M . Solntsev, Y . Tsfadia, E . Nachliel, and M . Gutman . 2002 . JACS . 124:7539-7547) allows us to set a margin of epsilon = 50 +/- 5.

J Hosp Infect, 2003 Jan, 53(1), 46 - 57
Device-related sources of bacteraemia in English hospitals--opportunities for the prevention of hospital-acquired bacteraemia; Coello R et al.; Between 1997 and 2001, 17 teaching and 56 non-teaching acute English hospitals conducted hospital-wide surveillance of hospital-acquired bacteraemia (HAB) using a standard protocol drawn up by the Nosocomial Infection National Surveillance Scheme (NINSS) . The sources of organisms, the incidence of device-related HAB, and the distribution of HABs from individual device-related sources by specialty and type of hospital were determined for 6,956 HABs in order to identify where resources should best be targeted to reduce these infections . The overall incidence of HAB was higher in teaching than in non-teaching hospitals: 5.39 and 2.83 HABs per 1,000 patients at risk, respectively (P<0.001) . Device-related sources were responsible for 52.4 and 43.2% of all HABs in teaching and non-teaching hospitals, respectively (P<0.001), and central lines were the commonest source, causing 38.3% of HABs in teaching versus 22.3% in non-teaching hospitals (P<0.001) . In teaching hospitals, general intensive care units (ICUs), haematology, special care baby units (SCBUs), nephrology, and oncology accounted for only 6.1% of the population surveyed, but had the highest incidence of HAB, and contributed 47.8% of 2091 HABs and 56.9% of 1,095 device-related bacteraemias . Of 623 device-related bacteraemias in these high-risk specialties, 554 (88.9%) were from central lines . Thus, in teaching hospitals, resources should be targeted primarily at the prevention of central line-related bacteraemia in these five high-risk specialties, and the surveillance should include data on central line use . In non-teaching hospitals, nearly two thirds (63.3%) of 4,865 HABs and 60.7% of 2,103 device-related bacteraemias were from a few specialties with a low incidence of bacteraemia, but large numbers of patients, namely general medicine, general surgery, geriatric medicine and urology . These specialties accounted for 50.5% of the population surveyed . Central lines were the most common source of bacteraemia in general medicine and surgery, and together accounted for 23.3% of all device-related bacteraemias . However, in geriatric medicine and urology, central line sources were infrequent, accounting for only 1.7% of all device-related bacteraemias . On the other hand, bacteraemia from catheter-associated UTI were common in all these four specialties accounting for 20.9% of all device-related bacteraemias . Thus, in non-teaching hospitals, resources should be targeted primarily at these low-risk specialties and surveillance should include, at least, bacteraemia from central lines and from catheter-associated UTI . Further benefit can be obtained by including central line-related bacteraemias from general ICU and haematology patients, as they contributed 17.0% of all device-related bacteraemias in non-teaching hospitals.

Microb Pathog, 2002 Dec, 33(6), 251 - 64
Bovine lymphocytes express functional receptors for Escherichia coli Shiga toxin 1; Stamm I et al.; Interactions of Shiga toxins (Stxs) and immune cells contribute to the pathogenesis of diseases due to Stx-producing Escherichia coli (STEC) infections in humans and facilitate the persistence of infection in asymptomatically infected cattle . Our recent findings that bovine B and T lymphocytes express Gb(3)/CD77, the human Stx-receptor, prompted us to determine whether the bovine homologue also mediates binding and internalization of Stx1 . In fact, Stx1 holotoxin and recombinant B subunit (rStxB1) bound to stimulated bovine peripheral blood mononuclear cells, especially to those subpopulations (B cells, BoCD8(+) T cells) that are highly sensitive to Stx1 . Competition and HPTLC-binding studies confirmed that Stx1 binds to bovine Gb(3), but different receptor isoforms with varying affinities for rStxB1 were expressed during the course of lymphocyte activation . At least one of these isoforms mediated toxin uptake . An anti-StxB1 mouse monoclonal antibody, used as a model for bovine serum antibodies specific for Stx1, modulated rather than generally prevented rStxB1 binding to and internalization by the receptors . The presence of functional Stx1-receptors on bovine lymphocytes explains the immunomodulatory effect of Stx1 observed in cattle at a molecular level . Furthermore, expression of such receptors by bovine but not human T cells enlightens the background for the differential outcome of STEC infections in cattle and man, i.e., persistent infection and development of disease, respectively.

J Pharm Pharmacol, 2002 Nov, 54(11), 1471 - 9
Enhanced anaerobic degradation of polymeric azo compounds by Escherichia coli in the presence of low-molecular-weight redox mediators; Rau J et al.; The effects of the redox mediator lawsone (2-hydroxy-1,4-naphthoquinone) on the ability of Escherichia coli to reduce anaerobically polymeric azo compounds were analysed . Two types of polymeric azo compounds were tested, that have been proposed as putative tools for the site-specific targeting of drugs to the colon . The first group of polymers consisted basically of linear chains of polymethacrylic acid or polymethylmethacrylate which were interrupted by subunits of 4,4'-bis(methacryloylamino)azobenzene . These polymers differed significantly in their hydrophilicity according to the relative proportion of polymethacrylic acid used for the polymerization procedure . The second group of polymers consisted of almost water-insoluble poly(ether-ester)azo polymers that were composed of 4-(6-hydroxyhexyl)oxy-phenylazobenzoate and 16-hydroxyhexadecanoate . The addition of lawsone to the anaerobically incubated cultures of E . coli resulted in a pronounced increase in the reduction rates of the water-soluble poly(methacrylate-co-4,4'-bis(methacryloylamino)azobenzene) and in a much smaller, but significant, increase in the reduction rates of the hydrophobic poly(ether-ester)azo polymers . An increase in the amount of azo groups resulted, for the hydrophobic poly(ether-ester)azo polymers, in an increased reduction rate in the presence of the redox mediator lawsone.

Biochemistry (Mosc), 2002 Nov, 67(11), 1235 - 9
Recombinant human peroxiredoxin VI: preparation and protective properties in vitro; Merkulova MI et al.; cDNA of human peroxiredoxin VI, one of the recently discovered novel antioxidant proteins, was expressed in Escherichia coli cells . The expression product was obtained in water-soluble form and purified by a two-step chromatographic procedure using DEAE-Sepharose and Sephacryl S-200 . According to CD data, the polypeptide chain of the recombinant human peroxiredoxin VI contains approximately 40% alpha-helical region and 30% beta-structure, which is the same as for native rat peroxiredoxin VI . The protective properties of the recombinant protein determined as its ability to prevent the inactivation of glutamine synthetase from E . coli in a model oxidation system were comparable with the protective properties of native rat peroxiredoxin VI.

Inorg Chem, 2002 Dec 30, 41(26), 7159 - 69
Thermally inert metal ammines as light-inducible DNA-targeted agents . Synthesis, photochemistry, and photobiology of a prototypical rhodium(III)-intercalator conjugate; Barry CG et al.; The recent discovery of the promising tumor cell kill by a novel platinum-acridine conjugate {Martins, E . T.; et al . J . Med . Chem . 2001, 44, 4492} has prompted us to explore the utility of analogous light-activatable rhodium(III) compounds as photocytotoxic agents . Here, the design and synthesis of {Rh(NH(3))(5)L}(n)(+) complexes are described with L = 1,1,3,3-tetramethylthiourea (tmtu) or 1-{2-(acridin-9-ylamino)ethyl}-1,3,3-trimethylthiourea (2) . The intercalator-based DNA-affinic carrier ligand 2 was synthesized from N-acridin-9-yl-N'-methylethane-1,2-diamine and dimethylthiocarbamoyl chloride and isolated as the hydrotriflate salt 2(CF(3)SO(3)) . {Rh(NH(3))(5)(tmtu)}(3+) (1) and {Rh(NH(3))(5)(2)}(4+) (3) were obtained from the reactions of the trifluoromethanesulfonato complex {Rh(NH(3))(5)(OSO(2)CF(3))}(CF(3)SO(3))(2) with the appropriate thiourea in noncoordinating solvents . All compounds were characterized by (1)H NMR and UV-vis spectroscopies and by elemental analyses . The single-crystal X-ray structures of 1(CF(3)SO(3))(3) x 2MeOH, 2(CF(3)SO(3)), and 3(CF(3)SO(3))(4) x H(2)O have been determined . Ligand-field photolysis of thermally inert 1 (lambda(max) = 378 nm) resulted in the aquation of 2 equiv of ammine ligand without noticeable release of sulfur-bound tmtu ((1)H NMR spectroscopy, NH(3)-sensitive electrode measurements) . This was confirmed by (15)N{(1)H} NMR spectroscopy using (15)N-labeled {Rh((15)NH(3))(5)(tmtu)}(3+) (1), which also indicated photoisomerization of the {RhN(5)S} moiety . Despite greatly accelerated ligand exchange, rhodium in 1 and 3 did not show light-enhanced formation of covalent adducts in calf thymus DNA . "Dark binding" levels of 3 in native DNA were slightly higher than for nontargeted 1, but significantly lower than those observed for analogous platinum-acridine . Agarose gel electrophoresis revealed photocleavage of supercoiled pUC19 plasmid DNA in the presence of hybrid 3 and its individual constituents 1 and 2 . Simple 1 induced single-strand breaks while 3 produced complete degradation of the DNA after 24 h of continuous irradiation . Acridine 2 alone produced double-strand breaks . The extent of DNA damage observed for 1-3 correlates with the photocytotoxicity of the compounds in human leukemia cells, suggesting that DNA might be the cellular target of these agents.

J Med Entomol, 2002 Nov, 39(6), 931 - 4
The immune response of larvae and pupae of Calliphora vicina (Diptera: Calliphoridae), upon administered insult with Escherichia coli; Crowley LD et al.; Adult blowflies, Calliphora vicina Robineau-Desvoidy, are exposed to bacteria-laden decay as adults and larvae . They are protected from infections in such habitats by a family of lytic proteins called Cecropins . In this study, we explore the relationshipbetween the developmental stages of the blowfly and the strength of the immune response when insulted by an endogenous pathogen, Echerichia coli . Our data indicate that the protective function of Cecropin B varies with developmental stage . Pupae that were not fully tanned exhibited the greatest response to an insult by E coli . The expression of an increased immune response is due in part to an increased concentration of Cecropin B manufactured by this instar.

Avian Dis, 2002 Oct-Dec, 46(4), 816 - 25
Cloning and analysis of the gene for a major surface antigen of Mycoplasma gallisepticum; Spencer DL et al.; Myplasma gallisepticum infects a wide variety of gallineaceous birds including chickens, turkeys, and pheasants . Infection occurs both horizontally and vertically . Thus, control of the spread of M . gallisepticum to noninfected flocks is difficult . Continual monitoring is necessary to identify infected flocks even under the most stringent infectious control practices . Monitoring, however, is usually performed by measuring hemagglutination activity (HA) in serum, an insensitive and variable test . Variability in the HA test arises differences in agglutination antigen, changes in antigenic profiles of the M . gallisepticum strain, and variability in reading the agglutination reaction . Enzyme-linked immunosorbent assays (ELISAs) are the preferred method of testing because of the ease in obtaining sera and the sensitivity and reproducibility of the assays, but the ELISA suffers from a lack of standardization in the test antigen . The ELISA test will be more easily accepted once the test antigen has been standardized . To this end, we have identified, cloned, and characterized the gene for an antigen that has potential as a species-specific antigen for M . gallisepticum The gene codes for a 75-kD protein, P75, that is recognized during natural infections . Recombinant P75 is not recognized in immunoblots by convalescent sera produced in chickens infected with Mycoplasma synoviae, Mycoplasma gallinarum, and Mycoplasma gallinaceum or in turkeys infected with Mycoplasma meleagridis.

In Vivo, 2002 Nov-Dec, 16(6), 471 - 7
Monocyte/macrophage recruitment, activation and differentiation modulate interleukin-8 production: a paracrine role of tumor-associated macrophages in tumor angiogenesis; Varney ML et al.; Tumor-associated macrophages (TAM) have been shown to play an important role in tumor angiogenesis . The purpose of this study was to determine whether monocyte recruitment, activation and differentiation mediated by monocyte chemotactic protein-1 (MCP-1) and macrophage colony stimulating factor (M-CSF) modulate the expression of the angiogenic factor, Interleukin (IL)-8 . Isolated human peripheral blood monocytes secreted low basal levels of IL-8 . Incubation of monocytes with M-CSF or MCP-1 resulted in an up-regulation of IL-8 mRNA and protein expression . The differential expression of IL-8 by monocytes following MCP-1 and M-CSF treatments involved activation of the NFkB transcription factor . Further activation with lipopolysaccharide (LPS) caused an increase in IL-8 secretion in monocytes but not in monocyte-derived macrophages (MDM) . MDM-conditioned media significantly up-regulated IL-8 expression in human malignant melanoma cells in vitro . In summary, we demonstrated that MCP-1 and M-CSF, critical for monocyte recruitment, activation and differentiation, differentially regulate IL-8 expression and may play an important role in monocyte/macrophage-mediated tumor angiogenesis.

J Exp Bot, 2003 Jan, 54(381), 223 - 37
Expression of a cyanobacterial sucrose-phosphate synthase from Synechocystis sp . PCC 6803 in transgenic plants; Lunn JE et al.; Sucrose-phosphate synthase (SPS) from the cyanobacterium Synechocystis sp . PCC 6803 lacks all of the Ser residues known to be involved in the regulation of higher plant SPS by protein phosphorylation . The Synechocystis SPS is also not allosterically regulated by glucose 6-phosphate or orthophosphate . To investigate the effects of expressing a potentially unregulated SPS in plants, the Synechocystis sps gene was introduced into tobacco, rice and tomato under the control of constitutive promoters . The Synechocystis SPS protein was expressed at a high level in the plants, which should have been sufficient to increase overall SPS activity 2-8-fold in the leaves . However, SPS activities and carbon partitioning in leaves from transgenic and wild-type plants were not significantly different . The maximal light-saturated rates of photosynthesis in leaves from tomato plants expressing the Synechocystis SPS were the same as those from wild-type plants . Tomato plants expressing the maize SPS showed 2-3-fold increases in SPS activity, increased partitioning of photoassimilate to sucrose and up to 58% higher maximal rates of photosynthesis . To investigate the apparent inactivity of the Synechocystis SPS the enzyme was purified from transgenic tobacco and rice plants . Surprisingly, the purified enzyme was found to have full catalytic activity . It is proposed that some other protein in plant cells binds to the Synechocystis SPS resulting in inhibition of the enzyme.

J Exp Bot, 2003 Jan, 54(381), 203 - 11
Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus; Moyano E et al.; In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus . This gene codes for putrescine:SAM N-methyltransferase (PMT; EC . 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway . Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots . Both hyoscyamine and scopolamine production were improved in hairy root cultures of D . metel, whereas in H . muticus only hyoscyamine contents were increased by pmt gene overexpression . These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited . The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.

J Exp Bot, 2003 Jan, 54(381), 191 - 201
Molecular characterization of XVSAP1, a stress-responsive gene from the resurrection plant Xerophyta viscosa Baker; Garwe D et al.; The strategy of 'complementation by functional sufficiency' was used to isolate a cDNA designated XVSAP1 from a cDNA library constructed from dehydrated Xerophyta viscosa Baker leaves . Analysis of the cDNA sequence indicated a highly hydrophobic protein with six transmembrane regions . Southern blot analysis revealed that there are at least two copies of XVSAP1 in X . viscosa . The deduced amino acid sequence showed 49% identity to WCOR413, a low-temperature-regulated protein from wheat . The protein also showed between 25% to 56% identity to WCOR413-like proteins from Arabidopsis thaliana . Expression of XVSAP1 in Escherichia coli (srl::Tn10) conferred osmotic stress tolerance when the cells were grown in 1 M sorbitol . Analysis of gene expression using semi-quantitative RT-PCR indicated that XVSAP1 is induced by dehydration, salt stress (100 mM), both low (4 degrees C) and high temperature (42 degrees C) and high light treatment (1500 micromol m(-2) s(-1)) . These results suggest that XVSAP1 may have a significant role to play in the response of X . viscosa to abiotic stresses.

Protein Sci, 2003 Jan, 12(1), 185 - 91
Partial NMR assignments and secondary structure mapping of the isolated alpha subunit of Escherichia coli tryptophan synthase, a 29-kD TIM barrel protein; Vadrevu R et al.; The alpha subunit of tryptophan synthase (alphaTS) from S . typhimurium belongs to the triosephosphate isomerase (TIM) or the (beta/alpha)(8) barrel fold, one of the most common structures in biology . To test the conservation of the global fold in the isolated Escherichia coli homolog, we have obtained a majority of the backbone assignments for the 29-kD alphaTS by using standard heteronuclear multidimensional NMR methods on uniformly (15)N- and (15)N/(13)C-labeled protein and on protein selectively (15)N-labeled at key hydrophobic residues . The secondary structure mapped by chemical shift index, nuclear Overhauser enhancements (NOEs), and hydrogen-deuterium (H-D) exchange, and several abnormal chemical shifts are consistent with the conservation of the global TIM barrel fold of the isolated E . coli alphaTS . Because most of the amide protons that are slow to exchange with solvent correspond to the beta-sheet residues, the beta-barrel is likely to play an important role in stabilizing the previously detected folding intermediates for E . coli alphaTS . A similar combination of uniform and selective labeling can be extended to other TIM barrel proteins to obtain insight into the role of the motif in stabilizing what appear to be common partially folded forms.

Protein Sci, 2003 Jan, 12(1), 124 - 34
Screening for functional expression and overexpression of a family of diiron-containing interfacial membrane proteins using the univector recombination system; Berthold DA et al.; The large number of uncharacterized genes emerging from genome sequencing projects has resulted in a need for quick and reliable screening methods for protein expression parameters . We have utilized the univector plasmid recombination system (as previously reported) to develop a series of vectors for rapid screening for expression in Escherichia coli . A high level of recombinant protein expression is a requirement for purification of protein for structural determination and other purposes . In other applications, successful complementation of a missing enzyme activity in E . coli, as well as directed evolution studies and metabolic engineering, often require a much lower level of protein expression . In this report we describe the construction of a number of new pHOST vectors that can be screened for both low- and high-level expression . We isolated a mutant vector for MBP fusions that exhibited a more optimal level of expression for complementation of aerobic respiration in hemA(-) E . coli, our functional assay for the alternative oxidase . We then demonstrated the use of our system to rapidly screen for both optimal functional expression and optimal overexpression of the alternative oxidase as well as two other members of a family of membrane-bound diiron carboxylate proteins, the plastid terminal oxidase and 5-demethoxyquinone hydroxylase.

J Biol Chem, 2003 Feb 28, 278(9), 7131 - 4 Epub 2002 Dec 18.
Structure-based rational design of streptavidin mutants with pseudo-catalytic activity; Pazy Y et al.; Introduction of enzymatic activity into proteins or other types of polymers by rational design is a major objective in the life sciences . To date, relatively low levels of enzymatic activity could be introduced into antibodies by using transition-state analogues of haptens . In the present study, we identify the structural elements that contribute to the observed hydrolytic activity in egg white avidin, which promote the cleavage of active biotin esters (notably biotinyl p-nitrophenyl ester) . The latter elements were then incorporated into bacterial streptavidin via genetic engineering . The streptavidin molecule was thus converted from a protector to an enhancer of hydrolysis of biotin esters . The conversion was accomplished by the combined replacement of a "lid-like loop" (L3,4) and a leucine-to-arginine point mutation in streptavidin . Interestingly, neither of these elements play a direct role in the hydrolytic reaction . The latter features were thus shown to be responsible for enhanced substrate hydrolysis . This work indicates that structural and non-catalytic elements of a protein can be modified to promote the induced fit of a substrate for subsequent interaction with either a catalytic residue or water molecules . This approach complements the conventional design of active sites that involves direct modifications of catalytic residues.

J Biol Chem, 2003 Feb 28, 278(9), 7034 - 42 Epub 2002 Dec 19.
Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum; Ngosuwan J et al.; Hsp70 molecular chaperones and their co-chaperones work together in various cellular compartments to guide the folding of proteins and to aid the translocation of proteins across membranes . Hsp70s stimulate protein folding by binding exposed hydrophobic sequences thereby preventing irreversible aggregation . Hsp40s stimulate the ATPase activity of Hsp70s and target unfolded proteins to Hsp70s . Genetic and biochemical evidence supports a role for cytosolic Hsp70s and Hsp40s in the post-translational translocation of precursor proteins into endoplasmic reticulum and mitochondria . To gain mechanistic insight, we measured the effects of Saccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p (Hsp40) on the translocation of histidine-tagged prepro-alpha-factor (ppalphaF6H) into microsomes . Radiolabeled ppalphaF6H was affinity purified from wheat germ translation reactions (or Escherichia coli) to remove endogenous chaperones . We demonstrated that either Ssa1p or Ydj1p stimulates post-translational translocation by preventing ppalphaF6H aggregation . The binding and/or hydrolysis of ATP by Ssa1p were required to maintain the translocation competence of ppalphaF6H . To clarify the contributions of membrane-bound and cytosolic Ydj1p, we compared the efficiency of chaperone-dependent translocation into wild-type and Ydj1p-deficient microsomes . Neither soluble nor membrane-bound Ydj1p was essential for post-translational protein translocation . The ability of Ssa1p, Ydj1p, or both chaperones to restore the translocation competence of aggregated ppalphaF6H was negligible.

Crit Care, 2002 Dec, 6(6), 536 - 9 Epub 2002 Sep 25.
Bovine colostrum in oral treatment of enterogenic endotoxaemia in rats; Dohler JR et al.; INTRODUCTION: Under conditions of shock, bacteria and endotoxins in the intestines can traverse the mucosal barrier by translocation and enter the blood and lymphatic system . Immunoglobulins and lactoferrin have been reported to neutralize endotoxins and bacteria . We studied the essential therapeutic factors of colostrum products in an animal experiment . METHOD: We simulated endotoxaemia by per-oral administration of a suspension of Escherichia coli and antibiotics into the duodenum of anaesthetized rats after giving intraperitoneal carrageenan . At the same time, pure bovine colostrum or lactoferrin-enriched bovine colostrum was given . Therapeutic effects were studied by examining plasma endotoxin activity and bacterial contamination of mesenterial lymph nodes and peritoneal lavages . Albumin was used in a control group . RESULTS: The most effective bovine colostrum was able to reduce the maximum plasma endotoxin value by 67% as compared with the albumin group . The combination of this colostrum with lactoferrin brought about a reduction by 80% . The reduction in bacterial contamination of lymph nodes and peritoneal lavages was also evident . CONCLUSION: Both gammaglobulin and lactoferrin may help to eliminate endotoxins when bovine colostrum is administered into the gut in conditions of septic shock.

Mol Microbiol, 2003 Jan, 47(1), 209 - 21
CesT is a bivalent enteropathogenic Escherichia coli chaperone required for translocation of both Tir and Map; Creasey EA et al.; Map is an enteropathogenic Escherichia coli (EPEC) protein that is translocated into eukaryotic cells by a type III secretion system . Although not required for the induction of attaching and effacing (A/E) lesion formation characteristic of EPEC infection, translocated Map is suggested to disrupt mitochondrial membrane potential, which may impact upon subsequent functions of the organelle such as control of cell death . Before secretion, many effector proteins are maintained in the bacterial cytosol by association with a specific chaperone . In EPEC, chaperones have been identified for the effector proteins translocated intimin receptor (Tir) and EspF, and for the translocator proteins EspB and EspD . In this study, we present evidence that the Tir-specific chaperone, CesT, also performs a chaperone function for Map . Using a combination of biochemical approaches, we demonstrate specific interaction between CesT and Map . Similar to other chaperone-effector pairings, binding is apparent at the amino-terminus of Map and is indicated to proceed by a similar mechanism to CesT:Tir interaction . Map secretion from a cesT mutant strain (SE884) is shown to be reduced and, importantly, its translocation from this strain after infection of HEp-2 cells is almost totally abrogated . Although other chaperones are reported to have a bivalent binding specificity, CesT is the first member of its family that chaperones more than one protein for translocation.

Mol Microbiol, 2003 Jan, 47(1), 183 - 94
Two different modes of transcription repression of the Escherichia coli acetate operon by IclR; Yamamoto K et al.; IclR is a repressor for the Escherichia coli aceBAK operon, which encodes isocitrate lyase (aceB), malate synthase (aceA) and isocitrate dehydroge-nase kinase/phosphorylase (aceK) in the glyoxylate bypass . IclR also represses the expression of iclR in an autogenous manner . DNase I footprinting and in vitro transcription assays indicated that IclR binds to an IclR box (-21 to +14), which overlaps the iclR promoter and thus competes with the RNA polymerase for DNA binding, leading to transcription repression . In the case of the aceBAK operon, IclR binds to IclR box II between -52 and -19 of the aceB promoter and interferes with binding of the RNA polymerase to this promoter . A secondary IclR binding site (IclR box I) was identified between -125 and -99 of the aceB promoter . IclR binds to this IclR box I even after formation of the aceB promoter open complex and, moreover, induces disassembly of the open complex, leading to repression of aceB transcription . In parallel, the location of the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) on DNA is shifted close to the IclR box I, indicating that direct interaction between the alphaCTD and the IclR box I-associated IclR caused the repression.

Mol Microbiol, 2003 Jan, 47(1), 75 - 88
Ectopic RNase E sites promote bypass of 5'-end-dependent mRNA decay in Escherichia coli; Baker KE et al.; In Escherichia coli, 5'-terminal stem-loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear . A synthetic 5'-terminal hairpin stabilizes the rpsT mRNA sixfold . This stabilization is dependent on efficient translational initiation and ribosome transit through at least two-thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA . Insertion of a 12-15 residue 'ectopic' RNase E cleavage site from either the rne leader or 9S pre-rRNA into the 5'-non-coding region of the rpsT mRNA significantly reduces the stabilizing effect of the terminal stem-loop, dependent on RNase E . A similar insertion into the rpsT coding sequence is partially destabilizing . These findings demonstrate that RNase E can bypass an interaction with the 5'-terminus, and exploit an alternative 'internal entry' pathway . We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5'-termini, the use of internal entry for bypass of barriers to decay, 'ectopic sites' and the role of translating ribosomes.

Plant J, 2002 Dec, 32(6), 997 - 1009
Poplar potassium transporters capable of controlling K+ homeostasis and K+-dependent xylogenesis; Langer K et al.; The cambial K+ content of poplar increases during the growth period in a K+ supply dependent manner . Upon K+ starvation or application of tetraethylammoniumchloride (TEA+), a K+ channel blocker, the average vessel lumen and expansion zone area were significantly reduced . In search for the molecular basis of potassium-dependent xylogenesis in poplar, K+ transporters homologous to those of known function in Arabidopis phloem- and xylem-physiology were isolated from a poplar wood EST library . The expression profile of three distinct K+ channel types and one K+ transporter, Populus tremula K+ uptake transporter 1 (PtKUP1), was analysed by quantitative RT-PCR . Thereby, we found P . tremula outward rectifying K+ channel (PTORK) and P . tremula K+ channel 2 (PTK2) correlated with the seasonal wood production . K+ transporter P . tremula 1 (KPT1) was predominantly found in guard cells . Following the heterologous expression in Xenopus oocytes the biophysical properties of the different channels were determined . PTORK, upon membrane de-polarization mediates potassium release . PTK2 is almost voltage independent, carrying inward K+ flux at hyperpolarized potential and K+ release upon de-polarization . PtKUP1 was expressed in a K+ uptake-deficient Escherichia coli strain, where this K+ transporter rescued K+-dependent growth . In order to link the different K+ transporters to the cambial activity and wood production, we compared the expression profiles to seasonal changes in the K+ content of the bark as well as xylem vessel diameter . Thereby, we found PTORK and PTK2 transcripts to follow the annual K+ variations in poplar branches . PtKUP1 was expressed at a low level throughout the year, suggesting a housekeeping function . From these data, we conclude that K+ channels are involved in the regulation of K+-dependent wood production.

Eur J Biochem, 2003 Jan, 270(1), 146 - 54
A hydrophobic segment within the C-terminal domain is essential for both client-binding and dimer formation of the HSP90-family molecular chaperone; Yamada S et al.; The alpha isoform of human 90-kDa heat shock protein (HSP90alpha) is composed of three domains: the N-terminal (residues 1-400); middle (residues 401-615) and C-terminal (residues 621-732) . The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90 . Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90 . The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions . A bacterial two-hybrid system revealed that residues 650-697 of HSP90alpha were essential for the binding to the middle domain . An almost identical region (residues 657-720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein . Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662-678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein . The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein . Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90alpha and the 94-kDa glucose-regulated protein through their middle-C-terminal domains . Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins.

Eur J Biochem, 2003 Jan, 270(1), 129 - 36
Characterization of native and recombinant A4 glyceraldehyde 3-phosphate dehydrogenase . Kinetic evidence for confromation changes upon association with the small protein CP12; Graciet E et al.; A4 glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purified from the green alga Chlamydomonas reinhardtii and was also overexpressed in Escherichia coli . Both purified A4 tetramers of recombinant and native GAPDH were characterized for the first time . The pH optimum for both native and recombinant enzymes was close to 7.8 . The pKs of the residues involved in catalysis indicate that a cysteine and a histidine may take part in catalysis by chloroplast GAPDH, as is the case for glycolytic GAPDH . Native and recombinant GAPDH show Michaelis-Menten kinetics with respect to their cofactors, NADH and NADPH, with greater specificity for NADPH . The kinetic parameters are similar to those of the heterotetrameric A2B2 spinach chloroplast GAPDH . Native C . reinhardtii and recombinant GAPDHs exhibit a cooperative behavior towards the substrate 1,3-bisphosphoglycerate (BPGA) . This positive cooperativity is specific to the C . reinhardtii enzyme, as higher plant A2B2 GAPDHs show Michaelis-Menten kinetics . Native GAPDH has twofold lower catalytic constant and K0.5 for BPGA than recombinant GAPDH . Mass spectrometry analysis of native GAPDH shows that it is a complex of GAPDH and the small protein CP12 . In vitro reconstitution assays indicate that the kinetic differences are the result conformation changes of GAPDH upon association with CP12.

J Protein Chem, 2002 Aug, 21(6), 407 - 11
Identification of phospholipase D from cabbage as N-terminally acetylated PLD2; Schops R et al.; Recently, the genes of two isoenzymes of phospholipase D from white cabbage (PLD1 and PLD2) with molecular masses of 91.7 and 91.9 kDa, respectively, have been sequenced and expressed in Escherichia coli {Schaffner, I., Rucknagel, K.-P., Mansfeld, J., and Ulbrich-Hofmann, R . (2002) . Eur . J . Lipid Sci . Technol . 104: 79-87} . Both enzymes are highly homologous (91% identity) and behave very similarly . Phospholipase D purified from white cabbage leaves (PLDcab) is compared with the two recombinant enzymes in sodium dodecylsulfate and native polyacrylamide gel electrophoresis, isoelectric focusing, N-terminal sequencing, and mass spectrometry after tryptic digestion . As a result, PLDcab clearly can be assigned to PLD2 . In contrast to recombinant PLD2, however, PLDcab is N-terminally acetylated.

J Protein Chem, 2002 Aug, 21(6), 393 - 400
Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase; Jabalquinto AM et al.; Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn2+ as the activating metal ion . The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase . Comparison with the crystalline structure of homologous E . coli PEP carboxykinase {Tari, L . W., Matte, A., Goldie, H., and Delbaere, L . T . J . (1997) . Nature Struct . Biol . 4, 990-994} suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions . In this work, these residues were individually changed to Gln (His225) or Asn . The mutated enzymes present 3-6 orders of magnitude lower values of Vmax/Km, indicating high catalytic relevance for these residues . The His225Gln mutant showed increased Km values for Mn2+ and PEP as compared with wild-type enzyme, suggesting a role of His225 in Mn2+ and PEP binding . From 1.5-1.6 Kcal/mol lower affinity for the 3'(2')-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A . succiniciproducens PEP carboxykinases, implying a role of His225 and Asp263 in nucleotide binding.

Angew Chem Int Ed Engl, 2002 Apr 2, 41(7), 1098 - 113
Molecular chaperones--cellular machines for protein folding; Walter S et al.; Proteins are linear polymers synthesized by ribosomes from activated amino acids . The product of this biosynthetic process is a polypeptide chain, which has to adopt the unique three-dimensional structure required for its function in the cell . In 1972, Christian Anfinsen was awarded the Nobel Prize for Chemistry for showing that this folding process is autonomous in that it does not require any additional factors or input of energy . Based on in vitro experiments with purified proteins, it was suggested that the correct three-dimensional structure can form spontaneously in vivo once the newly synthesized protein leaves the ribosome . Furthermore, proteins were assumed to maintain their native conformation until they were degraded by specific enzymes . In the last decade this view of cellular protein folding has changed considerably . It has become clear that a complicated and sophisticated machinery of proteins exists which assists protein folding and allows the functional state of proteins to be maintained under conditions in which they would normally unfold and aggregate . These proteins are collectively called molecular chaperones, because, like their human counterparts, they prevent unwanted interactions between their immature clients . In this review, we discuss the principal features of this peculiar class of proteins, their structure-function relationships, and the underlying molecular mechanisms.

J Neurovirol, 2002 Dec, 8 Suppl 2, 134 - 7
Conditional virus replication as an approach to a safe live attenuated human immunodeficiency virus vaccine; Berkhout B et al.; Despite intensive efforts, no safe and effective vaccine has been developed for the prophylaxis of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) . Studies with the simian immunodeficiency virus (SIV)/macaque model demonstrated that live attenuated viruses are the most effective vaccines tested thus far . However, due to ongoing low-level replication of the attenuated virus and the error-prone replication machinery, the attenuated virus may regain replication capacity and become pathogenic . We therefore designed a novel vaccine strategy with an HIV-1 virus that replicates exclusively in the presence of the nontoxic effector doxycycline (dox) . This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression . This designer HIV-rtTA virus replicates in a strictly dox-dependent manner and may represent an improved vaccine strain because its replication can be turned on and off at will . Spontaneous virus evolution resulted in optimization of the components of the Tet system for their new function to support virus replication in human cells . The optimised Tet system may be of particular use in other applications such as inducible expression of gene therapy vectors in the brain.

Arch Toxicol, 2003 Jan, 77(1), 42 - 7 Epub 2002 Nov 12.
Hibiscus protocatechuic acid inhibits lipopolysaccharide-induced rat hepatic damage; Lin WL et al.; Hibiscus protocatechuic acid (PCA), a phenolic compound found in the dried flowers of Hibiscus sabdariffa L . (Malvaceae), was demonstrated to have an antioxidant effect in vitro and in vivo, and an antitumor property in our previous study . In the present study, we used lipopolysaccharide (LPS, an endotoxin) to induce rat liver inducible nitric oxide synthase (iNOS), and found that pretreatment with PCA decreased the liver iNOS and the serum total nitrite induced by LPS . Our investigation showed that pretreatment of rats with PCA (0.2 and 0.5 mmol/kg dosed by gavage) for 5 days significantly decreased the serum levels of the hepatic enzyme markers alanine- and aspartate aminotransferase (ALT, alanine aminotransferase; AST, aspartate aminotransferase) induced by the 6-h treatment with LPS (i.p.; 5 mg/kg) . Histopathological evaluation of the rat livers revealed that PCA reduced the incidence of liver lesions induced by LPS, including neutrophil infiltration, congestion, and liver cell swelling induced by LPS in rats . We conclude that PCA, an antioxidant, presents an inhibitory potential on iNOS and hepatic damage induced by LPS.

Nucleic Acids Res . 2002 Dec 15;30(24):e139.
Random DNA fragmentation with endonuclease V: application to DNA shuffling; Miyazaki K; The enzyme endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3' to uracil sites . I applied the enzyme to random fragmentation of DNA to revise the complex DNA shuffling protocol . The merit of using endonuclease V is that cleavage occurs at random sites and the length of the fragments can easily be adjusted by varying the concentration of dUTP in the polymerase chain reaction . Unlike the conventional method using DNase I, no partial digestion or gel separation of fragments is required . Therefore, labor is dramatically reduced and reproducibility ensured . I applied this method to recombine two truncated green fluorescent protein (GFP) genes and demonstrated successful DNA shuffling by the appearance of the fluorescent full-length GFP genes.

Nucleic Acids Res, 2002 Dec 15, 30(24), 5376 - 81
A hairpin near the 5' end stabilises the DNA gyrase mRNA in Mycobacterium smegmatis; Unniraman S et al.; RNA is amongst the most labile macromolecules present in the cells . The steady-state levels of mRNA are regulated both at the stages of synthesis and degradation . Recent work in Escherichia coli suggests that controlling the rate of degradation is as important as the process of synthesis . The stability of mRNA is probably more important in slow- growing organisms like mycobacteria . Here, we present our analysis of the cis elements that determine the stability of the DNA gyrase message in Mycobacterium smegmatis . The message appears to be stabilised by a structure close to its 5' end . The effect is especially pronounced in a nutrient-depleted state . These results largely parallel the model proposed in E.coli for mRNA degradation/ stability with subtle differences . Furthermore, these results suggest that the slow-growing organisms might use stable mRNAs as a method to reduce the load of transcription on the cell.

Nucleic Acids Res, 2002 Dec 15, 30(24), 5360 - 8
Interaction of C5 protein with RNA aptamers selected by SELEX; Lee JH et al.; RNA aptamers binding to C5 protein, the protein component of Escherichia coli RNase P, were selected and characterized as an initial step in elucidating the mechanism of action of C5 protein as an RNA-binding protein . Sequence analyses of the RNA aptamers suggest that C5 protein binds various RNA molecules with dissociation constants comparable to that of M1 RNA, the RNA component of RNase P . The dominant sequence, W2, was chosen for further study . Interactions between W2 and C5 protein were independent of Mg2+, in contrast to the Mg2+ dependency of M1 RNA-C5 protein interactions . The affinity of W2 for C5 protein increased with increasing concentration of monovalent NH4+, suggesting interactions via hydrophobic attraction . W2 forms a fairly stable complex with C5 protein, although the stability of this complex is lower than that of the complex of M1 RNA with C5 protein . The core RNA motif essential for interaction with C5 protein was identified as a stem-loop structure, comprising a 5 bp stem and a 20 nt loop . Our results strongly imply that C5 protein is an interacting partner protein of some cellular RNA species apart from M1 RNA.

Bioinformatics, 2002 Dec, 18(12), 1609 - 16
Calculation of the minimum number of replicate spots required for detection of significant gene expression fold change in microarray experiments; Black MA et al.; MOTIVATION: We present statistical methods for determining the number of per gene replicate spots required in microarray experiments . The purpose of these methods is to obtain an estimate of the sampling variability present in microarray data, and to determine the number of replicate spots required to achieve a high probability of detecting a significant fold change in gene expression, while maintaining a low error rate . Our approach is based on data from control microarrays, and involves the use of standard statistical estimation techniques . RESULTS: After analyzing two experimental data sets containing control array data, we were able to determine the statistical power available for the detection of significant differential expression given differing levels of replication . The inclusion of replicate spots on microarrays not only allows more accurate estimation of the variability present in an experiment, but more importantly increases the probability of detecting genes undergoing significant fold changes in expression, while substantially decreasing the probability of observing fold changes due to chance rather than true differential expression.

DNA Cell Biol, 2002 Nov, 21(11), 847 - 53
Cellular gene dose and kinetics of gene expression in mouse livers transfected by high-volume tail-vein injection of naked DNA; Rossmanith W et al.; Direct gene transfer to mammalian tissues has significant potential for biomedical research and gene therapy . Recently, the efficient transfer of naked plasmid DNA to the mouse liver by a rapid high-volume tail-vein injection was reported . We carried out a systematic analysis of the dose and time dependence of the expression of the Escherichia coli beta-galactosidase gene transferred by this technique . Surprisingly, the DNA concentration of the administered solution determined primarily the cellular gene dose and, hence, the expression of the transgene in individual hepatocytes, while the number of transfected cells was largely independent of the supplied plasmid mass . Transgene expression was transient: after a rapid onset and a peak at 8 h past injection, it gradually declined and was no longer detectable 4 weeks later . Although gene transfer was accompanied by tissue damage and subsequent regenerative proliferation, the decline in transgene expression was not due to increased hepatocyte turnover or to promoter downregulation, but instead cells apparently lost the plasmid DNA . Furthermore, we show that "nakedness" of the injected DNA is indeed a prerequisite for efficient transfer by the hydrodynamics-based procedure . Our data provide important clues for the successful use of this gene transfer technique, and may point directions for studies on the underlying mechanisms.

DNA Cell Biol, 2002 Nov, 21(11), 839 - 45
Endonucleases that may recognize the ssDNA-backbone structure: purification from Sf9 cells and characterization; Akaboshi E; Endonucleases that cleave ssDNA under conditions that minimize the formation of secondary structures were detected in Spodoptera frugiperda Sf9 cells . One endonuclease was purified from Sf9 cells and another from Sf9 cells infected by baculoviruses . Polynucleotides containing an A- or T-tract, or a CAP binding region were digested in the presence of ATP at low Mg ions with these two nucleases . ATP could be replaced by citrate but not by EDTA . The cleavage patterns were different from those obtained with endonuclease I and exonuclease VII of Escherichia coli . The cleavages were dependent on the sequence of the polynucleotides but not associated with specific bases . EndoSfV cleaved mainly AT-rich regions.

Biochem J, 2003 Apr 15, 371(Pt 2), 351 - 60
Purification and kinetic characterization of the magnesium protoporphyrin IX methyltransferase from Synechocystis PCC6803; Shepherd M et al.; Magnesium protoporphyrin IX methyltransferase (ChlM), catalyses the methylation of magnesium protoporphyrin IX (MgP) at the C(6) propionate side chain to form magnesium protoporphyrin IX monomethylester (MgPME) . Threading methods biased by sequence similarity and predicted secondary structure have been used to assign this enzyme to a particular class of S-adenosyl-L-methionine (SAM)-binding proteins . These searches suggest that ChlM contains a seven-stranded beta-sheet, common among small-molecule methyltransferases . Steady-state kinetic assays were performed using magnesium deuteroporphyrin IX (MgD), a more water-soluble substrate analogue of MgP . Initial rate studies showed that the reaction proceeds via a ternary complex . Product (S-adenosyl-L-homocysteine; SAH) inhibition was used to investigate the kinetic mechanism further . SAH was shown to exhibit competitive inhibition with respect to SAM, and mixed inhibition with respect to MgD . This is indicative of a random binding mechanism, whereby SAH may bind productively to either free enzyme or a ChlM-MgD complex . Our results provide an overview of the steady-state kinetics for this enzyme, which are significant given the role of MgP and MgPME in plastid-to-nucleus signalling and their likely critical role in the regulation of this biosynthetic pathway.

J Vet Med A Physiol Pathol Clin Med, 2002 Nov, 49(9), 470 - 2
Haemolytic-uraemic syndrome in a dog; Chantrey J et al.; An 11-year-old female German Shepherd dog presented with lethargy and anorexia, which progressed to haemorrhagic vomiting, diarrhoea and seizures . Serum biochemistry and haematology results showed azotaemia and mild thrombocytopaenia . Euthanasia was elected and the dog was submitted for necropsy examination . There were widespread serosal and mucosal petechial and ecchymotic haemorrhages within the abdomen, with ascites and multiple renal infarcts . The renal infarcts were associated with fibrinoid necrosis and thrombosis of inter-lobular arteries and arterioles . These arterial lesions and clinical signs are consistent with haemolytic-uraemic syndrome, which has not previously been reported in dogs in Europe.

J Vet Med B Infect Dis Vet Public Health, 2002 Nov, 49(9), 429 - 37
Effects of various applications of lipopolysaccharides on blood parameters of pigs; Muller G et al.; In five experiments, lipopolysaccharides (LPS) of Escherichia coli O26:B6 and O111:B4 were applied intravenously, intramuscularly, subcutaneously or intrabronchially in doses of 5000-15,000 U/kg body mass to a total of 47 weaner pigs and compared with the application of sodium chloride . Different parameters of blood cells were investigated, including cell numbers, in vivo interleukin secretion, radical formation, phagocytosis capacity and IL-6 as well as TNFalpha formation ex vivo . Non-specific effects and dependencies on the type of application and LPS dose are discussed.

Environ Mol Mutagen, 2002, 40(4), 251 - 7
Transmission of mutations in the lacI transgene to the offspring of ENU-treated Big Blue male mice; Barnett LB et al.; The purpose of the study was to determine: 1) if male germ cells of Big Blue mice carrying newly induced mutations in the lacI transgene were effective in fertilization; 2) if offspring arising from such mutant sperm had the mutation in germ cells and multiple somatic tissues; and 3) how the frequency of mutants induced in the lacI transgene compared to the frequency induced in endogenous genes traditionally employed to study germ cell mutagenesis in mice . Male B6C3F(1) mice hemizygous for the lambda/lacI transgene were treated weekly with 100 mg/kg body weight of the mammalian germ cell mutagen N-ethyl-N-nitrosourea (ENU) . The cumulative dose for each treated animal was 300 mg ENU/kg body weight . Ten weeks later the treated mice were mated to T stock females and the resulting offspring were screened for specific-locus mutations at six loci affecting external appearance, as well as for mutations in the lacI transgene in multiple somatic tissues and germ cells . Five offspring carrying recessive specific-locus mutations were observed among 597 offspring screened (mutant frequency = 139.6 x 10(-5) per locus) . Four offspring carrying lacI mutations were observed among 280 offspring screened (mutant frequency = 35.7 x 10(-5) per locus (assuming 40 target loci)) . Each of the four lacI mutant offspring carried a different mutation . Three of the mutations were A:T-->G:C transitions and one a G:C-->A:T transition . Consistent with the expectation that a mutation induced in a parental germ cell and transmitted to a conceptus would exist in every cell of the offspring, each mutant mouse had identical mutations in all somatic tissues sampled, as well as in its germ cells . These data provide preliminary evidence for the biological validity of assessing induced, heritable mutations using transgenic mice, without the need for generating an F(1) generation .

Cancer Gene Ther, 2003 Jan, 10(1), 23 - 9
Antitumor activity of 2-fluoro-2'-deoxyadenosine against tumors that express Escherichia coli purine nucleoside phosphorylase; Parker WB et al.; The selective expression of Escherichia coli purine nucleoside phosphorylase (PNP) in solid tumors has been successfully used to activate two purine nucleoside analogs {9-(2-deoxy-beta-D-ribofuranosyl)-6-methylpurine (MeP-dR) and 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-araA)} resulting in lasting tumor regressions and cures . E . coli PNP also cleaves 2-fluoro-2'-deoxyadenosine (F-dAdo) to 2-F-adenine, which is the toxic purine analog liberated from F-araA that has high bystander activity and is active against nonproliferating tumor cells . As F-dAdo is 3000 times better than F-araA as a substrate for E . coli PNP, we have evaluated its antitumor activity against D54 gliomas that express E . coli PNP and have characterized its in vivo metabolism in order to better understand its mechanism of action with respect to the other two agents . Like MeP-dR and F-araA-5'-monophosphate (F-araAMP, a prodrug of F-araA), treatment of mice bearing D54 tumors that express E . coli PNP with F-dAdo resulted in excellent antitumor activity . Although F-dAdo was as active as MeP-dR and better than F-araAMP, it was not dramatically better than either compound because of its short plasma half-life and the limited activation of F-adenine to toxic metabolites . Regardless, these results indicated that F-dAdo was also an excellent prodrug for use with gene vectors that deliver E . coli PNP to tumor cells.

J Clin Gastroenterol, 2003 Jan, 36(1), 44 - 6
Verocytotoxin-producing Escherichia coli EH 0157:H7 colitis; Morandi E et al.; 0157:H7 is a known etiologic agent of hemorrhagic colitis . The clinical and histologic picture of colitis is largely similar to that of ischemic colitis, with areas of submucosal hemorrhage and edema, erosions, and ulcerations . We present a case report and review of the literature . A 52-year-old HIV-positive man, in apparently good immunologic condition, developed severe hemorrhagic colitis characterized by the onset of multiple colonic perforations and an unfavorable outcome . The diagnosis of 0157:H7 colitis should therefore be considered in all patients with indeterminate hematic diarrhea . Further studies are warranted to verify whether HIV infection may play a determinant role in the clinical course of 0157:H7 infection.

J Biomed Biotechnol, 2001, 1(2), 79 - 84
Response of Escherichia coli Containing Mycobacterial Carotene Genes to UV Radiation; Houssaini-Iraqui M et al.; The plasmid pC5, which encodes biogenesis of lycopene in Mycobacterium aurum A(+), was partially digested by restriction endonucleases and generated fragments were cloned . After transformation of Escherichia coli (colorless bacteria) with the plasmids so constructed, seven orange clones were detected and found to carry the same recombinant plasmid (pC51) . E . coli cells containing this plasmid synthesize neurosporene and lycopene, and were more resistant to ultraviolet irradiation than non pigmented strain.

J Biomed Biotechnol, 2002, 2(2), 66 - 74
Answering the Call: Coping with DNA Damage at the Most Inopportune Time; Crowley DJ et al.; DNA damage incurred during the process of chromosomal replication has a particularly high possibility of resulting in mutagenesis or lethality for the cell . The SOS response of Escherichia coli appears to be well adapted for this particular situation and involves the coordinated up-regulation of genes whose products center upon the tasks of maintaining the integrity of the replication fork when it encounters DNA damage, delaying the replication process (a DNA damage checkpoint), repairing the DNA lesions or allowing replication to occur over these DNA lesions, and then restoring processive replication before the SOS response itself is turned off . Recent advances in the fields of genomics and biochemistry has given a much more comprehensive picture of the timing and coordination of events which allow cells to deal with potentially lethal or mutagenic DNA lesions at the time of chromosomal replication.

Mol Cell Proteomics, 2002 Nov, 1(11), 896 - 903
Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis: identification of more than 800 Escherichia coli proteins; Gevaert K et al.; A novel gel-free proteomic technology was used to identify more than 800 proteins from 50 million Escherichia coli K12 cells in a single analysis . A peptide mixture is first obtained from a total unfractionated cell lysate, and only the methionine-containing peptides are isolated and identified by mass spectrometry and database searching . The sorting procedure is based on the concept of diagonal chromatography but adapted for highly complex mixtures . Statistical analysis predicts that we have identified more than 40% of the expressed proteome, including soluble and membrane-bound proteins . Next to highly abundant proteins, we also detected low copy number components such as the E . coli lactose operon repressor, illustrating the high dynamic range . The method is about 100 times more sensitive than two-dimensional gel-based methods and is fully automated . The strongest point, however, is the flexibility in the peptide sorting chemistry, which may target the technique toward quantitative proteomics of virtually every class of peptides containing modifiable amino acids, such as phosphopeptides, amino-terminal peptides, etc., adding a new dimension to future proteome research.

J Biol Chem, 2003 Feb 28, 278(9), 7206 - 14 Epub 2002 Dec 17.
Myristoyl-CoA:protein N-myristoyltransferase, an essential enzyme and potential drug target in kinetoplastid parasites; Price HP et al.; Co-translational modification of eukaryotic proteins by N-myristoylation aids subcellular targeting and protein-protein interactions . The enzyme that catalyzes this process, N-myristoyltransferase (NMT), has been characterized in the kinetoplastid protozoan parasites, Leishmania and Trypanosoma brucei . In Leishmania major, the single copy NMT gene is constitutively expressed in all parasite stages as a 48.5-kDa protein that localizes to both membrane and cytoplasmic fractions . Leishmania NMT myristoylates the target acylated Leishmania protein, HASPA, when both are co-expressed in Escherichia coli . Gene targeting experiments have shown that NMT activity is essential for viability in Leishmania . In addition, overexpression of NMT causes gross changes in parasite morphology, including the subcellular accumulation of lipids, leading to cell death . This phenotype is more extreme than that observed in Saccharomyces cerevisiae, in which overexpression of NMT activity has no obvious effects on growth kinetics or cell morphology . RNA interference assays in T . brucei have confirmed that NMT is also an essential protein in both life cycle stages of this second kinetoplastid species, suggesting that this enzyme may be an appropriate target for the development of anti-parasitic agents.

J Biol Chem, 2003 Mar 14, 278(11), 9481 - 8 Epub 2002 Dec 17.
Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis; Hayashi H et al.; Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates . Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis . To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe . The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme . When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system . The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme . However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value . The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value . Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.

J Biol Chem, 2003 Feb 21, 278(8), 5584 - 96 Epub 2002 Dec 17.
RAG1-DNA binding in V(D)J recombination . Specificity and DNA-induced conformational changes revealed by fluorescence and CD spectroscopy; Ciubotaru M et al.; The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination . RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex . To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria . The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nM) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K(D) = 41 nM) to the RSS . Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution . Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule . Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS . Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment . RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.

J Biol Chem, 2003 Feb 21, 278(8), 5894 - 901 Epub 2002 Dec 16.
The proline-rich domain of dynamin-2 is responsible for dynamin-dependent in vitro potentiation of endothelial nitric-oxide synthase activity via selective effects on reductase domain function; Cao S et al.; The GTPase dynamin-2 (dyn-2) binds and positively regulates the nitric oxide-generating enzyme, endothelial nitric-oxide synthase (eNOS) (Cao, S., Yao, Y., McCabe, T., Yao, Q., Katusic, Z., Sessa, W., and Shah, V . (2001) J . Biol . Chem . 276, 14249-14256) . Here we demonstrate, using purified proteins, that this occurs through a selective influence of the dyn-2 proline-rich domain (dyn-2 PRD) on the eNOS reductase domain . In vitro studies demonstrate that dyn-2 PRD fused with glutathione S-transferase (GST) binds recombinant eNOS protein specifically and with binding kinetics comparable with that observed between dyn-2 full-length and eNOS . Additionally, GST-dyn-2 PRD binds the in vitro transcribed (35)S-eNOS reductase domain but not the (35)S-eNOS oxygenase domain . Furthermore GST-dyn-2 PRD binds a (35)S-labeled eNOS reductase domain fragment (amino acids 645-850) that partially overlaps with the FAD binding domain of eNOS . A recombinant form of the SH3-containing protein Fyn competes the binding of recombinant eNOS protein with dyn-2 PRD, thereby implicating the SH3-like region contained within this reductase domain fragment as the dyn-2 binding region . Mammalian two-hybrid screen corroborates these interactions in cells as well . Functional studies demonstrate that dyn-2 PRD selectively potentiates eNOS activity in a concentration-dependent manner in an order of magnitude similar to that observed with dyn-2 full-length and in a manner that requires calmodulin . Although dyn-2 PRD does not influence eNOS oxygenase domain function or ferricyanide reduction, it does potentiate the ability of recombinant eNOS to reduce cytochrome c, supporting an influence of dyn-2 PRD on electron transfer between FAD and FMN . (These data indicate that the binding domains of dyn-2 and eNOS reside within the dyn-2 PRD domain and the FAD binding region of the eNOS reductase domains, respectively, and that dyn-2 PRD is sufficient to mediate dyn-2-dependent potentiation of eNOS activity, at least in part, by potentiating electron transfer.)

J Mol Biol, 2003 Jan 10, 325(2), 285 - 97
Tyr212: a key residue involved in strand discrimination by the DNA mismatch repair endonuclease MutH; Friedhoff P et al.; The molecular mechanism of how the dam-methylation status of the DNA is recognized during DNA mismatch repair by the strand discrimination endonuclease MutH is not known . A comparison of the crystal structure of MutH with those of co-crystal structures of several restriction endonucleases, together with a multiple sequence alignment of MutH and related proteins suggested that Phe94, Arg184 and Tyr212 could be involved in discrimination between a methylated or unmethylated adenine in the d(GATC) sequence . A mutational analysis revealed that the variants R184A and Y212S, but not F94A, were substantially reduced in their ability to complement a mismatch repair deficiency in a mutH(-) Escherichia coli strain . In vitro, R184A displayed a strongly reduced endonuclease activity, whereas the Y212S variant has almost completely lost its preference for cleaving the unmethylated strand at hemimethylated d(GATC) sites . Furthermore, the Y212 variant can cleave fully methlyated d(GATC) sites at a comparable rate to unmethylated d(GATC) sites . This demonstrates that Tyr212 is an important, if not the only amino acid residue in MutH for sensing the methylation status of the DNA.

J Mol Biol, 2003 Jan 10, 325(2), 275 - 84
The N-terminal domain of the regulatory subunit is sufficient for complete activation of acetohydroxyacid synthase III from Escherichia coli; Mendel S et al.; We have previously proposed a model for the fold of the N-terminal domain of the small, regulatory subunit (SSU) of acetohydroxyacid synthase isozyme III . The fold is an alpha-beta sandwich with betaalphabetabetaalphabeta topology, structurally homologous to the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase . We suggested that the N-terminal domains of a pair of SSUs interact in the holoenzyme to form two binding sites for the feedback inhibitor valine in the interface between them . The model was supported by mutational analysis and other evidence . We have now examined the role of the C-terminal portion of the SSU by construction of truncated polypeptides (lacking 35, 48, 80, 95, or 112 amino acid residues from the C terminus) and examining the properties of holoenzymes reconstituted using these constructs . The Delta35, Delta48, and Delta80 constructs all lead to essentially complete activation of the catalytic subunits . The Delta80 construct, corresponding to the putative N-terminal domain, has the highest level of affinity for the catalytic subunits and leads to a reconstituted enzyme with k(cat)/K(M) about twice that of the wild-type enzyme . On the other hand, none of these constructs binds valine or leads to a valine-sensitive enzyme on reconstitution . The enzyme reconstituted with the Delta80 construct does not bind valine, either . The N-terminal portion (about 80 amino acid residues) of the SSU is thus necessary and sufficient for recognition and activation of the catalytic subunits, but the C-terminal half of the SSU is required for valine binding and response . We suggest that the C-terminal region of the SSU contributes to monomer-monomer interactions, and provide additional experimental evidence for this suggestion.

Biochim Biophys Acta, 2002 Dec 23, 1567(1-2), 41 - 8
Viability of Escherichia coli after combined osmotic and thermal treatment: a plasma membrane implication; Mille Y et al.; This study investigates the influence of temperature (T) and osmotic pressure (Pi) on the viability of Escherichia coli K12 during an osmotic treatment . Osmotic shock (dehydration and rehydration within 1 s) in liquid media at different temperatures (4, 10, 30 and 37 degrees C) and different levels of osmotic pressure (26, 30, 35, 40, 82 and 133 MPa) were realized.Results show that a sudden dehydration, below 40 MPa, destroyed up to 80% of the bacterial population for each tested temperature, whereas viability was greater than 90% for an osmotic pressure less than 26 MPa . The influence of T and Pi on the membrane's physical structure is finally considered to explain the results in light of FTIR and electron microscopy study of the influence of temperature and osmotic pressure on E . coli membrane phospholipids conformation.

J Am Chem Soc, 2002 Dec 25, 124(51), 15217 - 24
Synthesis and application of a fluorescent substrate analogue to study ligand interactions for undecaprenyl pyrophosphate synthase; Chen AP et al.; Farnesyl pyrophosphate (FPP) serves as a common substrate for many prenyltransferases involved in the biosynthesis of isoprenoid compounds . Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the chain elongation of FPP to C(55) undecaprenyl pyrophosphate (UPP) which acts as a lipid carrier in bacterial peptidoglycan synthesis . In this study, 7-(2,6-dimethyl-8-diphospho-2,6-octadienyloxy)-8-methyl-4-trifluoromethyl-chromen-2-one geranyl pyrophosphate, a fluorescent analogue of FPP, was prepared and utilized to study ligand interactions with E . coli UPPs . This compound displays an absorbance maximum at 336 nm and emission maximum at 460 nm without interference from protein autofluorescence . It is a competitive inhibitor with respect to FPP (K(i) = 0.57 microM) and also serves as an alternative substrate (K(m) = 0.69 microM and k(cat) = 0.02 s(-)(1)), but mainly reacts with one isopentenyl pyrophosphate (IPP) probably due to unfavorable product translocation . Fluorescence intensity of this compound is reduced when bound to the enzyme (1:1 stoichiometry), and is recovered by FPP replacement . Using stopped-flow apparatus, the interaction of enzyme with the compound was measured (k(on) = 55.3 microM(-)(1) s(-)(1) and k(off) = 31.6 s(-)(1)) . The product dissociation rate constant (0.5 s(-)(1)) determined from the competition experiments is consistent with our previous prediction from kinetic simulation . Unlike several other prenyltransferase reactions in which FPP dissociates slowly, UPPs binds FPP in a rapid equilibrium manner with a fast release rate constant of 30 s(-)(1) . The fluorescent analogue of FPP presented here may provide a tool to investigate the ligand interactions for a broad class of FPP-binding proteins.

J Dairy Sci, 2002 Nov, 85(11), 2859 - 68
Functional maturation during bovine granulopoiesis; Van Merris V et al.; Granulocytic precursor cells undergo morphologic changes in the nucleus and the cytoplasm during the process of granulopoiesis, which takes place in the bone marrow . These changes are associated with the development of stage-specific proteins necessary for the highly specialized roles of polymorphonuclear leukocytes in phagocytosis, bacterial killing, and in mediating the inflammatory process . The objective of the current study was to sequence the various events that occur upon functional development of granulocytic bone marrow cells in the bovine species . Cells were obtained from the bone marrow of clinically healthy cows and separated into different stages of maturation using density gradient centrifugation . Three cellular fractions were obtained that were enriched for either early immature, late immature or mature granulocytic cells . Functions and receptor expressions assessed in the three maturation stages were:Fc-IgG2 receptor and CD11b expression, phagocytosis of Escherichia coli, respiratory burst activity, and cellular myeloperoxidase activity . Immature cells expressed already Fc-IgG2 receptor and CD11b on their cytoplasma membrane . Phagocytic ability was acquired in the myelocytic stage, but only the more mature forms were readily capable of phagocytosis . Promyelocytes, myelocytes and metamyelocytes showed no respiratory burst activity . Only band and segmented cells produced reactive oxygen species . Myeloperoxidase was present at all stages of maturity . Thus, each of the maturation stages was characterized by a selective expression of one or more functions and receptors . Therefore, sequential biochemical maturation is postulated during bovine granulopoiesis.

Extremophiles, 2002 Dec, 6(6), 469 - 77 Epub 2002 Jul 30.
Biochemical characterization of an ATP-dependent DNA ligase from the hyperthermophilic crenarchaeon Sulfolobus shibatae; Lai X et al.; A gene encoding a putative ATP-dependent DNA ligase was identified in the genome of the hyperthermophilic archaeon Sulfolobus shibatae and expressed in Escherichia coli . The 601 amino acid recombinant polypeptide was a monomeric protein capable of strand joining on a singly nicked DNA substrate in the presence of ATP ( K(m)=34 micro mu) and a divalent cation (Mn(2+), Mg(2+), or Ca(2+)) . dATP was partially active in supporting ligation catalyzed by the protein, but GTP, CTP, UTP, dGTP, dCTP, dTTP, and NAD(+) were inactive . The cloned Ssh ligase showed an unusual metal cofactor requirement; it was significantly more active in the presence of Mn(2+) than in the presence of Mg(2+) or Ca(2+) . Unexpectedly, the native Ssh ligase preferred Mg(2+) and Ca(2+) rather than Mn(2+) . Both native and recombinant enzymes displayed optimal nick-joining activity at 60-80 degrees C . Ssh ligase discriminated against substrates containing mismatches on the 3'-side of nick junction and was more tolerant of mismatches at the 5'-end than of those at the penultimate 5'-end . The enzyme showed little activity on a 1-nucleotide gapped substrate . This is the first biochemical study of a DNA ligase from the crenarchaeotal branch of the archaea domain.

Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16660 - 5 Epub 2002 Dec 16.
Reversal of DNA alkylation damage by two human dioxygenases; Duncan T et al.; The Escherichia coli AlkB protein protects against the cytotoxicity of methylating agents by repair of the DNA lesions 1-methyladenine and 3-methylcytosine, which are generated in single-stranded stretches of DNA . AlkB is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase that oxidizes the relevant methyl groups and releases them as formaldehyde . Here, we identify two human AlkB homologs, ABH2 and ABH3, by sequence and fold similarity, functional assays, and complementation of the E . coli alkB mutant phenotype . The levels of their mRNAs do not appear to correlate with cell proliferation but tissue distributions are different . Both enzymes remove 1-methyladenine and 3-methylcytosine from methylated polynucleotides in an alpha-ketoglutarate-dependent reaction, and act by direct damage reversal with the regeneration of the unsubstituted bases . AlkB, ABH2, and ABH3 can also repair 1-ethyladenine residues in DNA with the release of acetaldehyde.

J Bacteriol, 2003 Jan, 185(1), 317 - 24
Interdomain linkers of homologous response regulators determine their mechanism of action; Walthers D et al.; OmpR and PhoB are response regulators that contain an N-terminal phosphorylation domain and a C-terminal DNA binding effector domain connected by a flexible interdomain linker . Phosphorylation of the N terminus results in an increase in affinity for specific DNA and the subsequent regulation of gene expression . Despite their sequence and structural similarity, OmpR and PhoB employ different mechanisms to regulate their effector domains . Phosphorylation of OmpR in the N terminus stimulates the DNA binding affinity of the C terminus, whereas phosphorylation of the PhoB N terminus relieves inhibition of the C terminus, enabling it to bind to DNA . Chimeras between OmpR and PhoB containing either interdomain linker were constructed to explore the basis of the differences in their activation mechanisms . Our results indicate that effector domain regulation by either N terminus requires its cognate interdomain linker . In addition, our findings suggest that the isolated C terminus of OmpR is not sufficient for a productive interaction with RNA polymerase.

J Bacteriol, 2003 Jan, 185(1), 221 - 30
Contrasting sensitivities of Escherichia coli aconitases A and B to oxidation and iron depletion; Varghese S et al.; Superoxide damages dehydratases that contain catalytic {4Fe-4S}(2+) clusters . Aconitases are members of that enzyme family, and previous work showed that most aconitase activity is lost when Escherichia coli is exposed to superoxide stress . More recently it was determined that E . coli synthesizes at least two isozymes of aconitase, AcnA and AcnB . Synthesis of AcnA, the less-abundant enzyme, is positively controlled by SoxS, a protein that is activated in the presence of superoxide-generating chemicals . We have determined that this arrangement exists because AcnA is resistant to superoxide in vivo . Surprisingly, purified AcnA is extremely sensitive to superoxide and other chemical oxidants unless it is combined with an uncharacterized factor that is present in cell extracts . In contrast, AcnB is highly sensitive to a variety of chemical oxidants in vivo, in extracts, and in its purified form . Thus, the induction of AcnA during oxidative stress provides a mechanism to circumvent a block in the tricarboxylic acid cycle . AcnA appears to be as catalytically competent as AcnB, so the retention of the latter as the primary housekeeping enzyme must provide some other advantage . We observed that the {4Fe-4S} cluster of AcnB is in dynamic equilibrium with the surrounding iron pool, so that AcnB is rapidly demetallated when intracellular iron pools drop . AcnA and other dehydratases do not show this trait . Demetallated AcnB is known to bind its cognate mRNA . The absence of AcnB activity also causes the accumulation and excretion of citrate, an iron chelator for which E . coli synthesizes a transport system . Thus, AcnB may be retained as the primary aconitase because the lability of its exposed cluster allows E . coli to sense and respond to iron depletion.

J Bacteriol, 2003 Jan, 185(1), 204 - 9
Requirement of ArcA for redox regulation in Escherichia coli under microaerobic but not anaerobic or aerobic conditions; Alexeeva S et al.; In Escherichia coli, the two-component regulatory ArcAB system functions as a major control system for the regulation of expression of genes encoding enzymes involved in both aerobic and anaerobic catabolic pathways . Previously, we have described the physiological response of wild-type E . coli to changes in oxygen availability through the complete range from anaerobiosis to full aerobiosis (S . Alexeeva, B . de Kort, G . Sawers, K . J . Hellingwerf, and M . J . Teixeira de Mattos, J . Bacteriol . 182:4934-4940, 2000, and S . Alexeeva, K . J . Hellingwerf, and M . J . Teixeira de Mattos, J . Bacteriol . 184:1402-1406, 2002) . Here, we address the question of the contribution of the ArcAB-dependent transcriptional regulation to this response . Wild-type E . coli and a mutant lacking the ArcA regulator were grown in glucose-limited chemostat cultures at controlled levels of oxygen availability ranging from full aerobiosis to complete anaerobiosis . A flux analysis of the distribution of catabolic fluxes over parallel pathways was carried out, and the intracellular redox state (as reflected by the NADH/NAD ratio) was monitored for all steady states . Deletion of ArcA neither significantly altered the in vivo activity of the pyruvate dehydrogenase complex and pyruvate formate lyase nor significantly affected catabolism under fully aerobic and fully anaerobic conditions . In contrast, profound effects of the absence of ArcA were seen under conditions of oxygen-restricted growth: increased respiration, an altered electron flux distribution over the cytochrome o- and d-terminal oxidases, and a significant change in the intracellular redox state were observed . Thus, the ArcA regulator was found to exert major control on flux distribution, and it is concluded that the ArcAB system should be considered a microaerobic redox regulator.

J Bacteriol, 2003 Jan, 185(1), 196 - 203
Recruitment of MinC, an inhibitor of Z-ring formation, to the membrane in Escherichia coli: role of MinD and MinE; Hu Z et al.; In Escherichia coli, the min system prevents division away from midcell through topological regulation of MinC, an inhibitor of Z-ring formation . The topological regulation involves oscillation of MinC between the poles of the cell under the direction of the MinDE oscillator . Since the mechanism of MinC involvement in the oscillation is unknown, we investigated the interaction of MinC with the other Min proteins . We observed that MinD dimerized in the presence of ATP and interacted with MinC . In the presence of a phospholipid bilayer, MinD bound to the bilayer and recruited MinC in an ATP-dependent manner . Addition of MinE to the MinCD-bilayer complex resulted in release of both MinC and MinD . The release of MinC did not require ATP hydrolysis, indicating that MinE could displace MinC from the MinD-bilayer complex . In contrast, MinC was unable to displace MinE bound to the MinD-bilayer complex . These results suggest that MinE induces a conformational change in MinD bound to the bilayer that results in the release of MinC . Also, it is argued that binding of MinD to the membrane activates MinC.

J Bacteriol, 2003 Jan, 185(1), 175 - 83
Biochemical and mutational characterization of the heme chaperone CcmE reveals a heme binding site; Enggist E et al.; CcmE is a heme chaperone that binds heme transiently in the periplasm of Escherichia coli and delivers it to newly synthesized and exported c-type cytochromes . The chemical nature of the covalent bond between heme and H130 is not known . We have purified soluble histidine-tagged CcmE and present its spectroscopic characteristics in the visible range . Alanine scanning mutagenesis of conserved amino acids revealed that H130 is the only residue found to be strictly required for heme binding and delivery . Mutation of the hydrophobic amino acids F37, F103, L127, and Y134 to alanine affected CcmE more than mutation of charged and polar residues . Our data are in agreement with the recently solved nuclear magnetic resonance structure of apo-CcmE (PDB code 1LIZ) and suggest that heme is bound to a hydrophobic platform at the surface of the protein and then attached to H130 by a covalent bond . Replacement of H130 with cysteine led to the formation of a covalent bond between heme and C130 at a low level . However, the H130C mutant CcmE was not active in cytochrome c maturation . Isolation and characterization of the heme-binding peptides obtained after a tryptic digest of wild-type and H130C CcmE support the hypothesis that heme is bound covalently at a vinyl group.

J Bacteriol, 2003 Jan, 185(1), 126 - 34
Metal ion dependence of recombinant Escherichia coli allantoinase; Mulrooney SB et al.; Allantoinase is a suspected dinuclear metalloenzyme that catalyzes the hydrolytic cleavage of the five-member ring of allantoin (5-ureidohydantoin) to form allantoic acid . Recombinant Escherichia coli allantoinase purified from overproducing cultures amended with 2.5 mM zinc, 1 mM cobalt, or 1 mM nickel ions was found to possess approximately 1.4 Zn, 0.0 Co, 0.0 Ni, and 0.4 Fe; 0.1 Zn, 1.0 Co, 0.0 Ni, and 0.2 Fe; and 0.0 Zn, 0.0 Co, 0.6 Ni, and 0.1 Fe per subunit, respectively, whereas protein obtained from nonamended cultures contains near stoichiometric levels of iron . We conclude that allantoinase is incompletely activated in the recombinant cells, perhaps due to an insufficiency of a needed accessory protein . Enzyme isolated from nonsupplemented cultures possesses very low activity (k(cat) = 34.7 min(-1)) compared to the zinc-, cobalt-, and nickel-containing forms of allantoinase (k(cat) values of 5,000 and 28,200 min(-1) and 200 min(-1), respectively) . These rates and corresponding K(m) values (17.0, 19.5, and 80 mM, respectively) are significantly greater than those that have been reported previously . Absorbance spectroscopy of the cobalt species reveals a band centered at 570 nm consistent with five-coordinate geometry . Dithiothreitol is a competitive inhibitor of the enzyme, with significant K(i) differences for the zinc and cobalt species (237 and 795 micro M, respectively) . Circular dichroism spectroscopy revealed that the zinc enzyme utilizes only the S isomer of allantoin, whereas the cobalt allantoinase prefers the S isomer, but also hydrolyzes the R isomer at about 1/10 the rate . This is the first report for metal content of allantoinase from any source.

J Bacteriol, 2003 Jan, 185(1), 115 - 25
Global role for ClpP-containing proteases in stationary-phase adaptation of Escherichia coli; Weichart D et al.; To elucidate the involvement of proteolysis in the regulation of stationary-phase adaptation, the clpA, clpX, and clpP protease mutants of Escherichia coli were subjected to proteome analysis during growth and during carbon starvation . For most of the growth-phase-regulated proteins detected on our gels, the clpA, clpX, or clpP mutant failed to mount the growth-phase regulation found in the wild type . For example, in the clpP and clpA mutant cultures, the Dps protein, the WrbA protein, and the periplasmic lysine-arginine-ornithine binding protein ArgT did not display the induction typical for late-stationary-phase wild-type cells . On the other hand, in the protease mutants, a number of proteins accumulated to a higher degree than in the wild type, especially in late stationary phase . The proteins affected in this manner include the LeuA, TrxB, GdhA, GlnA, and MetK proteins and alkyl hydroperoxide reductase (AhpC) . These proteins may be directly degraded by ClpAP or ClpXP, respectively, or their expression could be modulated by a protease-dependent mechanism . From our data we conclude that the levels of most major growth-phase-regulated proteins in E . coli are at some point controlled by the activity of at least one of the ClpP, ClpA, and ClpX proteins . Cultures of the strains lacking functional ClpP or ClpX also displayed a more rapid loss of viability during extended stationary phase than the wild type . Therefore, regulation by proteolysis seems to be more important, especially in resting cells, than previously suspected.

J Bacteriol, 2003 Jan, 185(1), 89 - 97
Mutational analysis of a conserved signal-transducing element: the HAMP linker of the Escherichia coli nitrate sensor NarX; Appleman JA et al.; The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains . HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector . The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation . We generated missense mutations at these three positions and throughout the HAMP linker in the Escherichia coli nitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate . Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function . We also scanned the narX HAMP linker with an overlapping set of seven-residue deletions . Deletions in AS-1 and the connector resulted in constitutive phenotypes . Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for the narX(+) strain . These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic alpha-helices have different roles in signal transduction.

J Bacteriol, 2003 Jan, 185(1), 28 - 34
Regulation of the Escherichia coli rrnB P2 promoter; Murray HD et al.; The seven rRNA operons in Escherichia coli each contain two promoters, rrn P1 and rrn P2 . Most previous studies have focused on the rrn P1 promoters . Here we report a systematic analysis of the activity and regulation of the rrnB P2 promoter in order to define the intrinsic properties of rrn P2 promoters and to understand better their contributions to rRNA synthesis when they are in their natural setting downstream of rrn P1 promoters . In contrast to the conclusions reached in some previous studies, we find that rrnB P2 is regulated: it displays clear responses to amino acid availability (stringent control), rRNA gene dose (feedback control), and changes in growth rate (growth rate-dependent control) . Stringent control of rrnB P2 requires the alarmone ppGpp, but growth rate-dependent control of rrnB P2 does not require ppGpp . The rrnB P2 core promoter sequence (-37 to +7) is sufficient to serve as the target for growth rate-dependent regulation.

J Bacteriol, 2003 Jan, 185(1), 13 - 9
Cadaverine inhibition of porin plays a role in cell survival at acidic pH; Samartzidou H et al.; When grown at acidic pH, Escherichia coli cells secrete cadaverine, a polyamine known to inhibit porin-mediated outer membrane permeability . In order to understand the physiological significance of cadaverine excretion and the inhibition of porins, we isolated an OmpC mutant that showed resistance to spermine during growth and polyamine-resistant porin-mediated fluxes . Here, we show that the addition of exogenous cadaverine allows wild-type cells to survive a 30-min exposure to pH 3.6 better than cells expressing the cadaverine-insensitive OmpC porin . Competition experiments between strains expressing either wild-type or mutant OmpC showed that the lack of sensitivity of the porin to cadaverine confers a survival disadvantage to the mutant cells at reduced pH . On the basis of these results, we propose that the inhibition of porins by excreted cadaverine represents a novel mechanism that provides bacterial cells with the ability to survive acid stress.

J Biol Chem, 2003 Feb 28, 278(9), 7573 - 9 Epub 2002 Dec 13.
The prefusogenic intermediate of HIV-1 gp41 contains exposed C-peptide regions; Koshiba T et al.; The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is composed of a complex between the surface subunit gp120, which binds to cellular receptors, and the transmembrane subunit gp41 . Upon activation of the envelope glycoprotein by cellular receptors, gp41 undergoes conformational changes that mediate fusion of the viral and cellular membranes . Prior to formation of a fusogenic "trimer-of-hairpins" structure, gp41 transiently adopts a prefusogenic conformation whose structural features are poorly understood . An important approach toward understanding structural conformations of gp41 during HIV-1 entry has been to analyze the structural targets of gp41 inhibitors . We have constructed epitope-tagged versions of 5-Helix, a designed protein that binds to the C-peptide region of gp41 and inhibits HIV-1 membrane fusion . Using these 5-Helix variants, we examined which conformation of gp41 is the target of 5-Helix . We find that although 5-Helix binds poorly to native gp41, it binds strongly to gp41 activated by interaction of the envelope protein with either soluble CD4 or membrane-bound cellular receptors . This preferential interaction with activated gp41 results in the accumulation of 5-Helix on the surface of activated cells . These results strongly suggest that the gp41 prefusogenic intermediate is the target of 5-Helix and that this intermediate has a remarkably "open" structure, with exposed C-peptide regions . These results provide important structural information about this intermediate that should facilitate the development of HIV-1 entry inhibitors and may lead to new vaccine strategies.

J Biol Chem, 2003 Mar 14, 278(11), 9683 - 90 Epub 2002 Dec 13.
Yeast tRNA(Asp) charging accuracy is threatened by the N-terminal extension of aspartyl-tRNA synthetase; Ryckelynck M et al.; This study evaluates the role of the N-terminal extension from yeast aspartyl-tRNA synthetase in tRNA aspartylation . The presence of an RNA-binding motif in this extension, conserved in eukaryotic class IIb aminoacyl-tRNA synthetases, provides nonspecific tRNA binding properties to this enzyme . Here, it is assumed that the additional contacts the 70 amino acid-long appendix of aspartyl-tRNA synthetase makes with tRNA could be important in expression of aspartate identity in yeast . Using in vitro transcripts mutated at identity positions, it is demonstrated that the extension grants better aminoacylation efficiency but reduced specificity to the synthetase, increasing considerably the risk of noncognate tRNA mischarging . Yeast tRNA(Glu(UUC)) and tRNA(Asn(GUU)) were identified as the most easily mischarged tRNA species . Both have a G at the discriminator position, and their anticodon differs only by one change from the GUC aspartate anticodon.

EMBO J, 2002 Dec 16, 21(24), 6944 - 53
Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase; Trautinger BW et al.; The RuvABC and RecBCD proteins promote rescue of stalled or broken DNA replication forks in Escherichia coli . Strains lacking these proteins cope poorly with DNA damage and have problems with chromosome segregation and cell division . We show how these difficulties are overcome to varying degrees by a sub-class of RNA polymerase mutations selected for their stringent phenotype . Thirty-five mutations were sequenced . All but one change single amino acids in RpoB or RpoC that lie on or near the path taken by DNA through the enzyme, indicating they may affect the stability of transcription complexes . Four mutant enzymes are shown to form unstable open complexes at the lambdacro promoter . At least one may also release stalled complexes or limit their formation, as it reduces the need for reactivation of transcription by GreA or GreB, and for transcription-coupled DNA repair of UV damage by Mfd . The results shed light on the interplay between DNA replication and transcription and suggest ways in which conflicts between these two vital cellular processes are avoided or resolved.

EMBO J, 2002 Dec 16, 21(24), 6925 - 34
The function of SECIS RNA in translational control of gene expression in Escherichia coli; Thanbichler M et al.; The incorporation of selenocysteine into proteins is directed by specific UGA codons and mRNA secondary structures, designated SECIS elements . In bacteria, these elements are positioned within the reading frame of selenoprotein mRNAs immediately downstream of the triplet coding for selenocysteine, and they tether a complex of the selenocysteine-specific elongation factor SelB, GTP and selenocysteyl-tRNA(Sec) to the site of UGA decoding . A SECIS-like structure was identified in the 5' non-translated region of the selAB transcript, encoding selenocysteine synthase and SelB . It specifically binds to SelB and the formation of a SelB.GTP.selenocysteyl-tRNA(Sec) complex on the SECIS-like element represses expression of the downstream gene . This effect is abolished by mutations preventing formation of the complex . The regulatory pattern observed correlated with the levels of sel gene products . As quaternary complex formation on the SECIS-like element did not influence the transcription rate and only slightly reduced the level of selAB mRNA, it was concluded that the structure is involved in regulating translation initiation efficiency, thereby coupling selenocysteine biosynthesis to the availability of the trace element selenium.

EMBO J, 2002 Dec 16, 21(24), 6874 - 81
An inserted region of leucyl-tRNA synthetase plays a critical role in group I intron splicing; Rho SB et al.; Yeast mitochondrial leucyl-tRNA synthetase (LeuRS) binds to the bI4 intron and collaborates with the bI4 maturase to aid excision of the group I intron . Deletion analysis isolated the inserted LeuRS CP1 domain as a critical factor in the protein's splicing activity . Protein fragments comprised of just the LeuRS CP1 region rescued complementation of a yeast strain that expressed a splicing-defective LeuRS . Three-hybrid analysis determined that these CP1-containing LeuRS fragments, ranging from 214 to 375 amino acids, bound to the bI4 intron . In each case, interactions with only the LeuRS protein fragment specifically stimulated bI4 intron splicing activity . Substitution of a homologous CP1 domain from isoleucyl-tRNA synthetase or mutation within the LeuRS CP1 region of the smallest protein fragment abolished RNA binding and splicing activity . The CP1 domain is best known for its amino acid editing activity . However, these results suggest that elements within the LeuRS CP1 domain also play a novel role, independent of the full-length tRNA synthetase, in binding the bI4 group I intron and facilitating its self-splicing activity.

EMBO J, 2002 Dec 16, 21(24), 6673 - 80
Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum; Tahar R et al.; Deciphering molecular interactions between the malaria parasite and its mosquito vector is an emerging area of research that will be greatly facilitated by the recent sequencing of the genomes of Anopheles gambiae mosquito and of various Plasmodium species . So far, most such studies have focused on Plasmodium berghei, a parasite species that infects rodents and is more amenable to studies . Here, we analysed the expression pattern of nine An.gambiae genes involved in immune surveillance during development of the human malaria parasite P.falciparum in mosquitoes fed on parasite-containing blood from patients in Cameroon . We found that P.falciparum ingestion triggers a midgut-associated, as well as a systemic, response in the mosquito, with three genes, NOS, defensin and GNBP, being regulated by ingestion of gametocytes, the infectious stage of the parasite . Surprisingly, we found a different pattern of expression of these genes in the An.gambiae-P.berghei model . Therefore, differences in mosquito reaction against various Plasmodium species may exist, which stresses the need to validate the main conclusions suggested by the P.berghei-An.gambiae model in the P.falciparum-An.gambiae system.

Biosens Bioelectron, 2003 Mar, 18(2-3), 295 - 302
Electrochemistry of immobilized CuZnSOD and FeSOD and their interaction with superoxide radicals; Ge B et al.; Copper, zinc superoxide dismutase (CuZnSOD) from bovine erythrocytes and iron superoxide dismutase from Escherichia coli (FeSOD) were immobilized on 3-mercaptopropionic acid (MPA)-modified gold electrodes, respectively . The characterization of the SOD electrodes showed a quasi-reversible, electrochemical redox behavior with a formal potential of 47+/-4 mV and -154+/-5 mV (vs . Ag/AgCl, 1 M KCl) for surface adsorbed CuZnSOD and FeSOD, respectively . The heterogeneous electron transfer rate constants were determined to be about 65 and 35/s, respectively . Covalent fixation of both SODs was also feasible with only slight changes in the formal potential . The interaction of superoxide radicals (O(2)(-)) with the SOD electrode was investigated . No catalytic current could be observed . However, due to the fast cyclic redox reaction of SOD with superoxide, the communication of the protein with the electrode was strongly influenced . The amperometric detection of superoxide radicals is discussed.

Biomol Eng, 2003 Jan, 20(1), 1 - 5
Synthesis of 32P-labelled protein probes using a modified thioredoxin fusion protein expression system in Escherichia coli; Delucchi AB et al.; The thioredoxin fusion protein expression system from invitrogen was modified so that 32P-labelled recombinant proteins can be easily obtained in large quantities for functional studies . Proteins that are prone to form the inclusion bodies can be functionally expressed as thioredoxin fusion proteins in Escherichia coli . After expression, the recombinant proteins can be easily phosphorylated with 32P-gamma ATP and the 32P-labelled protein can be obtained functionally via a mild proteolytic digestion to cleave off the thioredoxin moiety . A deletion construct of the Ah receptor nuclear translocator protein was used as an example to illustrate how this protein expression system works.

Arch Biochem Biophys, 2002 Dec 1, 408(1), 131 - 6
Effects of proline analog binding on the spectroscopic and redox properties of PutA; Zhu W et al.; The PutA flavoprotein regulates proline metabolism in Escherichia coli by performing two distinct functions . First, in the cytoplasm, PutA represses transcription of the put (proline utilization) regulon . Second, PutA associates with the membrane to oxidize proline to glutamate using discrete proline dehydrogenase and Delta(1)-pyrroline-5-carboxylate dehydrogenase domains . Here, we identify a proline analog that will be useful for testing the role substrate binding has in regulating PutA functions . L-Tetrahydro-2-furoic acid (L-THFA) was found to display simple competitive inhibition of proline dehydrogenase activity in PutA (apparent K(i)=0.2mM) and to perturb the flavin adenine dinucleotide (FAD) absorbance spectrum upon complexation to PutA . At pH 7.5, a reduction potential (E(m)) of -0.089V for the FAD/FADH(2) couple in L-THFA-complexed PutA was determined by potentiometric titrations . The E(m) value for L-THFA-complexed PutA is 12mV more negative than the E(m) for uncomplexed PutA (E(m)=-0.077V, pH 7.5) and corresponds to just a twofold increase in the dissociation constant of L-THFA with PutA upon reduction of FAD.

Bioorg Chem, 2002 Oct, 30(5), 332 - 49
Making AppDNA using T4 DNA ligase; Chiuman W et al.; 5('),5(')-Adenylyl pyrophosphoryl DNA (AppDNA) contains a high-energy pyrophosphate linkage and can be exploited as an activated DNA substrate to derive new DNA enzymes for carrying out various DNA modification reactions . For this reason, enzymatic synthesis of AppDNA is highly desirable . AppDNA is a known intermediate in DNA ligase mediated DNA ligation reactions, but rarely accumulates under normal reaction conditions . Here we report that T4 DNA ligase can quantitatively convert 5(')-phosphoryl DNA donor into AppDNA in the absence of acceptor DNA but in the presence of a template DNA that contains at least one unpaired nucleotide opposite the 5(')-phosphoryl DNA donor site . This adenylylation behavior of T4 DNA ligase is not observed with Thermus aquaticus (Taq) and Escherichia coli DNA ligases . We further found that a donor-template duplex of 11-bp in length is required by T4 DNA ligase for the formation of AppDNA.

J Vet Med B Infect Dis Vet Public Health, 2002 Dec, 49(10), 469 - 75
Effects of thymomimetic drugs and zinc supplementation on the cellular immune response in hydrocortisone-suppressed mice; Obminska-Domoradzka B et al.; The studies were carried out on Balb/c mice (5-6 weeks of age) exposed to immunosuppression by a single intraperitoneal dose (125 mg/kg) of hydrocortisone . Prior to hydrocortisone injection the mice were treated with diethyldithiocarbamate (DTC) intra-peritoneally at a dose of 20 mg/kg, five times at 48 h intervals or calf thymus extract (TFX) at a dose of 10 mg/kg, 10 times at 24 h intervals . The two drugs were used per se or in zinc ions interactions, by adding zinc ions (as sulphate salt) to drinking water at a dose of 72 microg/mouse per day . The results obtained in the study show that hydrocortisone injection drastically decreases the number of thymocytes and splenocytes, which is also accompanied by a decreasing weight ratio of the thymus and spleen . The decreasing number of thymic and spleen cells corresponds to a decreasing percentage of CD4+, CD8+ and CD19+ splenocytes and double positive CD4+CD8+ thymocytes . Changes in the number of thymic cells affect their activity, which is expressed in a decreased proliferative response of thymocytes stimulated in vitro with concanavalin A (Con A) and phytohaemagglutinin (PHA) . It has also been found that a single hydrocortisone dose decreases interleukin (IL)-1 production by murine intraperitoneal macrophages stimulated in vitro with lipopolysaccharide (LPS) from Escherichia coli . TFX or DTC counteract hydrocortisone-induced immunosuppression, which is expressed in partial normalization of the total number of thymic and spleen cells, accelerated regeneration of the two lymphatic organs, shorter suppressive action of hydrocortisone on the percentage of CD4+, CD8+ splenocytes and double positive (CD4+CD8+) and CD4+ thymocytes . Furthermore, total counteraction against the suppressive action of hydrocortisone to proliferative activity of thymocytes stimulated in vitro with Con A and PHA was observed . TFX administered prior to hydrocortisone injection partially prevented the suppressive action of the drug on IL-1 production by intraperitoneal macrophages, but such an effect was not observed with DTC . The immunorestorative effect of TFX and DTC was augmented by zinc supplementation . The results obtained in the study show that neither TFX nor DTC administration per se and in interaction with zinc supplementation were able to change the suppressive effect of hydrocortisone on the percentage of B splenocytes (CD19+ cells).

Biochem J, 2003 Mar 15, 370(Pt 3), 963 - 70
beta-Glucosidase in cellulosome of the anaerobic fungus Piromyces sp . strain E2 is a family 3 glycoside hydrolase; Steenbakkers PJ et al.; The cellulosomes of anaerobic fungi convert crystalline cellulose solely into glucose, in contrast with bacterial cellulosomes which produce cellobiose . Previously, a beta-glucosidase was identified in the cellulosome of Piromyces sp . strain E2 by zymogram analysis, which represented approx . 25% of the extracellular beta-glucosidase activity . To identify the component in the fungal cellulosome responsible for the beta-glucosidase activity, immunoscreening with anti-cellulosome antibodies was used to isolate the corresponding gene . A 2737 bp immunoclone was isolated from a cDNA library . The clone encoded an extracellular protein containing a eukaryotic family 3 glycoside hydrolase domain homologue and was therefore named cel3A . The C-terminal end of the encoded Cel3A protein consisted of an auxiliary domain and three fungal dockerins, typical for cellulosome components . The Cel3A catalytic domain was expressed in Escherichia coli BL21 and purified . Biochemical analyses of the recombinant protein showed that the Cel3A catalytic domain was specific for beta-glucosidic bonds and functioned as an exoglucohydrolase on soluble substrates as well as cellulose . Comparison of the apparent K (m) and K (i) values of heterologous Cel3A and the fungal cellulosome for p -nitrophenyl-beta-D-glucopyranoside and D-glucono-1,5-delta-lactone respectively indicated that cel3A encodes the beta-glucosidase activity of the Piromyces sp . strain E2 cellulosome.

Biochemistry, 2002 Dec 24, 41(51), 15369 - 75
Engineering-specific pharmacological binding sites for peptidyl inhibitors of potassium channels into KcsA; Legros C et al.; The bacterial potassium channel, KcsA, can be modified to express a high-affinity receptor site for the scorpion toxin kaliotoxin (KTX) by substituting subregion I in the P region of KcsA with the one present in the human voltage-gated potassium channel Kv1.3 {Legros, C., Pollmann, V., Knaus, H . G., Farrell, A . M., Darbon, H., Bougis, P . E., Martin-Eauclaire, M . F., and Pongs, O . (2000) J . Biol . Chem . 275, 16918-16924} . This approach opened the way to investigate whether sequence differences in subregion I of Kv1 channels correlate with the distinct pharmacological profiles of peptide inhibitors . A panel of six chimeras between KcsA and human Kv1.1-6 were constructed, expressed in Escherichia coli, purified to homogeneity, and assessed in filter binding assays using either monoiodo-tyrosine-KTX ({(125)I}KTX) or monoiodo-tyrosine-hongotoxin(1)(A19Y/Y37F) ({(125)I}HgTX(1)(A19Y/Y37F)) . The KcsA-Kv1.X chimeras were found to have lower affinities for these ligands than the corresponding mammalian Kv1.X channels, indicating that other parts of the channels may contribute to binding or that subtle structural differences exist between these channels . The properties of the KcsA-Kv1.X chimeras were also characterized in surface plasmon resonance experiments . KcsA-Kv1.3 chimeras were immobilized on the surface of a sensor chip for determining, in real time, binding of the peptides . KTX binding properties to immobilized KcsA-Kv1.3 chimera were similar to those determined by filtration techniques . Taken together, our results demonstrate that the pharmacological profile of peptide toxins can be incorporated into KcsA-Kv1.X chimeras containing the subregion I of the corresponding mammalian Kv1.X channels . This innovative approach may facilitate the high-throughput screening of ligand libraries aimed at the discovery of novel potassium channel modulators.

Biochemistry, 2002 Dec 24, 41(51), 15334 - 41
RNA polymerase alters the mobility of an A-residue crucial to polymerase-induced melting of promoter DNA; Tsujikawa L et al.; Strand separation in promoter DNA induced by Escherichia coli RNA polymerase is likely initiated at a conserved A residue at position -11 of the nontemplate strand . Here we describe the use of fluorescence techniques to study the interaction of RNA polymerase with the -11 base . Forked DNA templates were employed, containing the fluorescent base, 2-aminopurine (2AP), substituted at the -11 position in a single-stranded tail comprising the nucleotides on the nontemplate strand at which base pairing is disrupted in an RNA polymerase-promoter complex . We demonstrate that the presence of 2AP instead of an A at position -11 has no major effect on the accessibility of DNA to DNase I or KMnO(4) in the presence or absence of RNA polymerase, thus justifying the use of templates containing the 2AP substitution in the fluorescence studies . A blue shift of the 2AP fluorescence emission maximum is observed in the presence of RNA polymerase . The results of fluorescence anisotropy decay studies indicate that about 60% of the 2AP residues at -11 are immobilized in an RNA polymerase complex . This value is in good agreement with the fraction of 2AP-substituted templates determined to be in a stable, heparin-resistant complex with RNA polymerase . These results are consistent with the residue at -11 being tightly bound in a hydrophobic pocket of the enzyme.

Biochemistry, 2002 Dec 24, 41(51), 15304 - 14
In vitro nucleotide misinsertion opposite the oxidized guanosine lesions spiroiminodihydantoin and guanidinohydantoin and DNA synthesis past the lesions using Escherichia coli DNA polymerase I (Klenow fragment); Kornyushyna O et al.; The low redox potential of 8-oxo-7,8-dihydroguanine (OG), a molecule regarded as a marker of oxidative damage in cells, makes it an easy target for further oxidation . Using a temperature-dependent method of synthesis, the oxidation products of OG, guanidinohydantoin (Gh) and/or its isomer iminoallantoin (Ia) as well as spiroiminodihydantoin (Sp), have been site-specifically incorporated into DNA oligomers . Single nucleotide insertion and primer extension experiments using Escherichia coli Kf exo(-) DNA polymerase were carried out under "standing start" and "running start" conditions in various sequence contexts . dAMP and dGMP were found to be inserted opposite these OG oxidation products . Steady-state kinetic studies show that the Gh/Ia.G base pair yields a lower K(m) value compared to the Sp.G pair or X.A (X = Gh/Ia or Sp) . Running start experiments using oxidized and unoxidized OG-containing templates showed enhanced full extension in the presence of all four dNTPs . A sequence preference for efficiency of extension was found when Gh/Ia and Sp are present in the DNA template, possibly leading to primer misalignment . Full extension is more efficient for the templates containing two Gs immediately 3' to the lesions compared to two As . Although these lesions cause a significant block for DNA elongation, results show that they are more easily bypassed by the polymerase when situated in the appropriate sequence context . UV melting studies carried out on duplexes mimicking the template/primer systems were used to characterize thermal stability of the duplexes . These experiments suggest that both Gh/Ia and Sp destabilize the duplex to a much greater extent than OG, with Sp being most severe.

Biochemistry, 2002 Dec 24, 41(51), 15173 - 80
Effect of phosphorylation on the interdomain interaction of the response regulator, NarL; Eldridge AM et al.; DNA binding by the effector domain (NarLC) of the response regulator, NarL, is modulated by the phosphorylation state of the receiver domain (NarLN) . The receiver domain appears to block the site of DNA binding in the nonphosphorylated state . Phosphorylation is proposed to disrupt this interaction, causing the effector domain to be released and free to bind DNA (Baikalov, I., Schroder, I., Kaczor-Grzeskowiak, M., Grzeskowiak, K., Gunsalus, R . P., and Dickerson, R . E . (1996) Biochemistry 35, 11053-61) . To better understand this modulation, we analyzed the interaction between the two domains in the absence of a polypeptide linkage . Using multidimensional NMR, we mapped chemical shift changes that occurred during a titration between the two isolated domains . Specific residues in NarLC exhibit large chemical shift changes upon the addition of NarLN . These residues are primarily at the interface between the two domains as seen in the crystal structure . Using the residues with the largest chemical shift changes, we observed a dissociation constant of 88 +/- 7 microM . In the presence of 10 mM MgCl(2), the affinity is reduced 4-fold to about 350 microM . This work shows that the domains interact in trans and that this interaction, while fairly weak, provides a way to monitor the energetics of domain-domain interaction in this system . Phosphorylation of NarLN by a small-molecule phosphate donor, phosphoramidate, decreases this interaction about 25-fold from the nonphosphorylated sample . The results support the model that the mechanism of activation of NarL involves a disruption of the interdomain interface and suggests that the linker is not necessary for the transmission of signal across the domain interface . The linker does play a role in increasing the local concentration of the domains and therefore increasing the amount of closed conformation with respect to the open conformation . We estimate the levels of open conformation to be low (about 1%) in the nonphosphorylated state in the absence of magnesium ion and much higher in the phospho state (near 50%) . This modulation of the open or active state via the interaction at the interface may also be applicable to other multidomain response regulator proteins.

Biochemistry, 2002 Dec 24, 41(51), 15130 - 4
The C-terminal domain of the epsilon subunit of the chloroplast ATP synthase is not required for ATP synthesis; Nowak KF et al.; The epsilon subunit of the ATP synthases from chloroplasts and Escherichia coli regulates the activity of the enzyme and is required for ATP synthesis . The epsilon subunit is not required for the binding of the catalytic portion of the chloroplast ATP synthase (CF1) to the membrane-embedded part (CFo) . Thylakoid membranes reconstituted with CF1 lacking its epsilon subunit (CF1-epsilon) have high ATPase activity and no ATP synthesis activity, at least in part because the membranes are very leaky to protons . Either native or recombinant epsilon subunit inhibits ATPase activity and restores low proton permeability and ATP synthesis . In this paper we show that recombinant epsilon subunit from which 45 amino acids were deleted from the C-terminus is as active as full-length epsilon subunit in restoring ATP synthesis to membranes containing CF1-epsilon . However, the truncated form of the epsilon subunit was significantly less effective as an inhibitor of the ATPase activity of CF1-epsilon, both in solution and bound to thylakoid membranes . Thus, the C-terminus of the epsilon subunit is more involved in regulation of activity, by inhibiting ATP hydrolysis, than in ATP synthesis.

J Am Soc Mass Spectrom, 2002 Dec, 13(12), 1432 - 42
Thermal dissociation of the protein homodimer ecotin in the gas phase; Felitsyn N et al.; The influence of charge on the thermal dissociation of gaseous, protonated, homodimeric, protein ecotin ions produced by nanoflow electrospray ionization (nanoES) was investigated using the blackbody infrared radiative dissociation technique . Dissociation of the protonated dimer, (E2 + nH)(n+) congruent to E2(n+) where n = 14-17, into pairs of monomer ions is the dominant reaction at temperatures from 126 to 175 degrees C . The monomer pair corresponding to the most symmetric charge distribution is preferred, although 50-60% of the monomer product ions correspond to an asymmetric partitioning of charge . The relative abundance of the different monomer ion pairs produced from E2(14+), E2(15+), and E2(16+) depends on reaction time, with the more symmetric charge distribution pair dominating at longer times . The relative yield of monomer ions observed late in the reaction is independent of temperature indicating that proton transfer between the monomers does not occur during dissociation and that the different monomer ion pairs are formed from dimer ions which differ in the distribution of charge between the monomers . For E2(17+), the yield of monomer ions is independent of reaction time but does exhibit slight temperature dependence, with higher temperatures favoring the monomers corresponding to most symmetric charge distribution . The charge distribution in the E2(15+) and E2(16+) dimer ions influences the dissociation kinetics, with the more asymmetric distribution resulting in greater reactivity . In contrast, the charge distribution has no measurable effect on the dissociation kinetics and energetics of the E2(17+) dimer.

J Gen Appl Microbiol, 2000 Jun, 46(3), 127 - 131
Ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibits the growth of Escherichia coli at alkaline pH; Arakawa Y et al.; The growth of Escherichia coli was inhibited by ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in a medium of initial pH 8.8 . The growth inhibition was reversed by the addition of CaCl(2) . E . coli could grow in the presence of EGTA at pH values below 8 . The concentration of free calcium ions increases with a decrease in medium pH because of a decrease in the calcium binding capacity of EGTA . So, although the results suggest that calcium ions are essential for the growth of E . coli, the minimum concentration required is very low.

J Gen Appl Microbiol, 2001 Jun, 47(3), 133 - 141
Efficient export of alkaline phosphatase overexpressed from a multicopy plasmid requires degP, a gene encoding a periplasmic protease of Escherichia coli; Kadokura H et al.; Escherichia coli DegP is an inducible serine protease which is involved in the breakdown of abberant proteins arising in the periplasmic compartment . Overexpression of alkaline phosphatase (PhoA) increased transcription of degP by twofold . To examine the significance of its induction, we overexpressed PhoA in a mutant strain deficient in the degP gene . Upon PhoA overexpression, the degP mutant produced a smaller amount of active PhoA, about one half of the enzymatic activity of its isogenic wild-type strain, and accumulated a larger amount of its precursor, indicating that degP is required for efficient export of overexpressed PhoA . Pulse-chase experiment showed that PhoA overexpression in the absence of degP causes a severe defect in the export of several proteins tested . Examination of the synthesis and the accumulation of the phoA gene products revealed that a part of them, synthesized in the wild-type strain, undergoes relatively rapid proteolysis and that degP is necessary for such a process . From these results, we discuss a possible role of DegP in facilitating protein export under stress conditions.

Oncogene, 2002 Dec 16, 21(58), 9008 - 21
RecQ family helicases: roles as tumor suppressor proteins; Nakayama H; RecQ family DNA helicases are defined as proteins sharing a homologous region with Escherichia coli RecQ and are basically regarded as enzymes involved in recombination . Humans have five RecQ family members, and deficiencies in three of them, BLM, WRN, and RTS, cause Bloom's, Werner's, and Rothmund-Thomson syndromes, respectively, each characterized by genomic instability and cancer predisposition . In this context, an important function of the RecQ homologs appears to be the unwinding of intermediates of recombination, thereby preventing its uncontrolled execution . As a consequence, their deficiencies give rise to elevated levels of recombination (the hyper-recombination phenotype), which result in chromosomal aberrations including loss of heterozygosity, a common chromosomal change associated with malignancies . Thus, those helicases qualify as caretaker-type tumor suppressor proteins . In addition, BLM and WRN deficiencies have been shown to attenuate p53-mediated apoptosis, suggesting that they also belong to the gatekeeper class of tumor suppressor proteins.

Oncogene, 2002 Dec 16, 21(58), 8957 - 66
How DNA lesions are turned into mutations within cells?
Pages V, Fuchs RP.
Genomes of all living organisms are constantly injured by endogenous and exogenous agents that modify the chemical integrity of DNA and in turn challenge its informational content . Despite the efficient action of numerous repair systems that remove lesions in DNA in an error-free manner, some lesions, that escape these repair mechanisms, are present when DNA is being replicated . Although replicative DNA polymerases are usually unable to copy past such lesions, it was recently discovered that cells are equipped with specialized DNA polymerases that will assist the replicative polymerase during the process of Translesion Synthesis (TLS) . These TLS polymerases exhibit relaxed fidelity that allows them to copy past lesions in DNA with an inherent risk of generating mutations at high frequency . We present recent aspects related to the genetics and biochemistry of TLS and highlight some of the remaining hot topics of this field.

Oncogene, 2002 Dec 16, 21(58), 8949 - 56
Subpathways of nucleotide excision repair and their regulation; Hanawalt PC; Nucleotide excision repair provides an important cellular defense against a large variety of structurally unrelated DNA alterations . Most of these alterations, if unrepaired, may contribute to mutagenesis, oncogenesis, and developmental abnormalities, as well as cellular lethality . There are two subpathways of nucleotide excision repair; global genomic repair (GGR) and transcription coupled repair (TCR), that is selective for the transcribed DNA strand in expressed genes . Some of the proteins involved in the recognition of DNA damage (including RNA polymerase) are also responsive to natural variations in the secondary structural features of DNA . Gratuitous repair events in undamaged DNA might then contribute to genomic instability . However, damage recognition enzymes for GGR are normally maintained at very low levels unless the cells are genomically stressed . GGR is controlled through the SOS stress response in E . coli and through the activated p53 tumor suppressor in human cells . These inducible responses in human cells are important, as they have been shown to operate upon chemical carcinogen DNA damage at levels to which humans are environmentally exposed . Interestingly, most rodent tissues are deficient in the p53-dependent GGR pathway . Since rodents are used as surrogates for environmental cancer risk assessment, it is essential that we understand how they differ from humans with respect to DNA repair and oncogenic responses to environmental genotoxins . In the case of terminally differentiated mammalian cells, a new paradigm has appeared in which GGR is attenuated but both strands of expressed genes are repaired efficiently.

Oncogene, 2002 Dec 16, 21(58), 8905 - 25
Enzymology of the repair of free radicals-induced DNA damage; Gros L et al.; A number of intrinsic and extrinsic mutagens induce structural damage in cellular DNA . These DNA damages are cytotoxic, miscoding or both and are believed to be at the origin of cell lethality, tissue degeneration, ageing and cancer . In order to counteract immediately the deleterious effects of such lesions, leading to genomic instability, cells have evolved a number of DNA repair mechanisms including the direct reversal of the lesion, sanitation of the dNTPs pools, mismatch repair and several DNA excision pathways including the base excision repair (BER) nucleotide excision repair (NER) and the nucleotide incision repair (NIR) . These repair pathways are universally present in living cells and extremely well conserved . This review is focused on the repair of lesions induced by free radicals and ionising radiation . The BER pathway removes most of these DNA lesions, although recently it was shown that other pathways would also be efficient in the removal of oxidised bases . In the BER pathway the process is initiated by a DNA glycosylase excising the modified and mismatched base by hydrolysis of the glycosidic bond between the base and the deoxyribose of the DNA, generating a free base and an abasic site (AP-site) which in turn is repaired since it is cytotoxic and mutagenic.

Oncogene, 2002 Dec 16, 21(58), 8895 - 904
Oxidative nucleotide damage: consequences and prevention; Sekiguchi M et al.; 8-Oxoguanine (8-oxo-7,8-dihydroguanine) is produced in DNA, as well as in nucleotide pools of cells, by reactive oxygen species normally formed during cellular metabolic processes . 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides with an almost equal efficiency, then transversion mutation ensues . MutT protein of Escherichia coli and related mammalian protein MTH1 specifically degrade 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing misincorporation of 8-oxoguanine into DNA . The bacterial and mammalian enzymes are close in their size and share a highly conserved region consisting of 23 residues with 14 identical amino acids . Following saturation mutagenesis of this region, most of these residues proved to be essential to exert 8-oxo-dGTPase activity . Gene targeting was done to establish MTH1-deficient cell lines and mice for study . When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1(-/-) mice, as compared with findings in wild-type mice . These proteins protect genetic information from untoward effects of threats of endogenous oxygen.

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