Biotechniques, 2002 Sep, 33(3), 592, 594, 596 - 8 passim Improved version of the red fluorescent protein (drFP583/DsRed/RFP); Knop M et al.; GFP has established itself as a highly useful tool throughout many areas of modern biology . Recently, the novel fluorescent protein drFP583, also termed DsRed or RFP, was clonedfrom a coral of the Discosoma genus . The protein is only weakly homologous to GFP and has a red emission spectrum, which makes drFP583 an attractive candidate for in vivo double labeling together with GFP variants . However, wildtype drFP583 has several drawbacks, including inefficient folding of the protein, extremely slow maturation of the chromophore, and tetramerization even in dilute solutions . Here we report on important improvements to this reporter that lead to higher levels of fluorescent drFP583 species in the cell . We further characterized our best mutant for applications in yeast and mammalian cell biology. Biotechniques, 2002 Sep, 33(3), 578, 580, 584 - 8 passim Affinity tag for protein purification and detection based on the disulfide-linked complex of InaD and NorpA; Kimple ME et al.; Affinity tags are not only used for the expression and purification of recombinant proteins but also for the detection of protein-protein interactions . Common problems with many affinity tags are excessive length, which may interfere with the structure and function of tagged proteins, and low affinity and/or specificity for primary detection and purification agents . Preliminary results suggest that the C-terminalfive residues of the Drosophila protein NorpA, based on the short, covalent interaction they make with the N-terminal PDZ domain (PDZI) of InaD, are useful as a general affinity tag . First, a PDZI-alkaline phosphatase fusion protein specifically detects both its physiological ligand and a heterologous protein expressing the NorpA C-terminal five residues . The interaction of PDZI with a NorpA-tagged protein is reversible by a reducing agent, which allows nitrocellulose membranes to be stripped completely and reused . In addition, a NorpA-tagged protein can specifically bind to immobilized PDZI resin, while other cellular proteins are washed through . After washing, the NorpA-tagged protein is eluted by a reducing buffer . The NorpA tag's short length makes it the smallest affinity tag available, and its specific and high-affinity interaction with PDZI could yield a powerful system that improves on currently available technology. J Biomol NMR, 2002 Jul, 23(3), 181 - 94 A comparison of methods for calculating NMR cross-relaxation rates (NOESY and ROESY intensities) in small peptides; Feenstra KA et al.; Three methods for calculating nuclear magnetic resonance cross-relaxation rates from molecular dynamics simulations of small flexible molecules have been compared in terms of their ability to reproduce relaxation data obtained experimentally and to produce consistent descriptions of the system . The importance of the accuracy of the simulation versus the amount of sampling of phase space has also been assessed by comparing different length simulations performed with different time step schemes . A nine-residue peptide from the protein HPr of E . coli was used as a test system . The work shows that, in this case, single conformations or a limited ensemble of configurations are insufficient to properly describe the behavior of the peptide and that different approaches to incorporate molecular motions lead to significant differences in the cross-relaxation rates calculated . The correlation between the cross-relaxation rates calculated from simulations performed with different time step schemes was high and increased with increasing simulation length indicating that the extent of sampling is more important than the details of the atomic motion. Inflammation, 2002 Oct, 26(5), 223 - 32 Endomorphin-2 modulates productions of TNF-alpha, IL-1beta, IL-10, and IL-12, and alters functions related to innate immune of macrophages; Azuma Y et al.; We evaluate immunological effects of opioid peptide endomorphin-2 on the production of cytokines related to inflammation and Th1/Th2 balance, and functions related to innate immune of rat peritoneal macrophages . Endomorphin-2 inhibited TNF-alpha, IL-10, and IL-12 productions, but potentiated IL-1beta production by macrophages . Moreover, endomorphin-2 potentiated macrophage adhesion to fibronectin, and the expression of adhesion molecule Mac-1 on macrophages . In contrast, endomorphin-2 suppressed phagocytosis of opsonized E . coli by macrophages, without affecting phagocytosis of non-opsonized E . coli . In addition, endomorphin-2 inhibited macrophage chemotaxis, and the production of superoxide anion by macrophages . These results suggest that endomorphin-2 may alter macrophage functions such as cytokine productions and functions related to innate immune. J Radiat Res (Tokyo), 2002 Jun, 43(2), 195 - 203 Characterization of spontaneous mutation in the delta soxR and SoxS overproducing strains of Escherichia coli; Yamamura E et al.; To examine the role of the soxRS regulon in mutagenesis, we characterized the spontaneous mutations occurring in the endogenous tonB gene in the delta soxR strain and the SoxS overproducing strain of Escherichia coli . Neither the delta soxR strain nor the SoxS overproducing strain led to an enhancement or diminishment of the spontaneous mutation frequency . By DNA sequencing, we determined 50 spontaneous mutants from the delta soxR strains, and found that 36% were both base substitutions and IS insertions, 14% frameshifts and 10% deletions . Among the base substitutions, G:C-->T:A transversions and G:C-->A:T transitions predominated, followed by A:T-->T:A transversions . We determined 54 spontaneous mutants from the SoxS overproducing strains, and found that 37% were IS insertions, 31% base substitutions, 17% frameshifts, 9% deletions and 6% duplications . Among the base substitutions, G:C-->T:A transversions dominated, followed by A:T-->T:A transversions and G:C-->A:T transitions . These results were similar to those from the soxRS+ strains . Thus, it is suggested that the soxRS-regulated genes do not play a significant role in the defense against spontaneous mutagenesis. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(1), 1 - 7 Expression of the atpE Gene Coding for the epsilon Subunit of Maize Chloroplast Coupling Factor; Shi J et al.; The entire atpE gene of the maize chloroplast coupling factor was inserted into the polylinker region of vectors pJLA505 and pWA to form recombinant plasmids pJLA505-atpE and pWA-atpE respectively . These expression plasmids were transformed into E . coli NM522 which induced at 42 degrees . By the analysis of SDS-PAGE, the expressed product of interest was observed to account fore more than 3o% of total E . coliproteins . The identification of the expressed product demonstrated that its immunological specificity was well retained . The antiserum cross-reacted with the expressed epsilon protein and CF(1)-epsilon protein of spinach and produced precipitin lines on Ouchterlony immunodiffusion test . The expressed product aggregated insolubly as the inclusion body and was purified to over 80% purity . The purified product had the same function as that of the native epsilon subunit. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(2), 177 - 186 The Expression of HCG Epitope Fused to Hepatits B Virus Core Antigen; Li GD et al.; The DNA fragments encoding HGC-beta-37-CTP was amplified by PCR and fused to the core gene of HBV at the position of amino Acid 1(N-terminal fusion, pCn-HCG), 154(C-terminal fusion, pCc-HCG), 75-83(internal fusion,pCm-HCG), and both 75-83 and 154 (pC-HCG2) respectively . The fused genes were expressed in E . coli, and the antigenicity of both HBcAg and HCG as well as the expression level were analyzed . In addition, the chimeric particular characteristics of the proteins and their immunogenicity were identified . It revealed that the fusion proteins pCm-HCG and pCc-HCG were able to form particles, and that the fusion protein pCm-HCG could induce antibody of anti-HCG of high titers in mice, suggesting that the position at 75-83 amino acid residue should be a relatively promising fusion site for HCG. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(2), 170 - 176 Cloning and Expression of the rpoH Gene from Escherichia coli and Its Product Purification; Chen YH et al.; The rpoH gene, encoding the heat shock protein transcription factor sigma(32), was obtained by PCR and cloned into plasmid pUHE with the tac promoter . Upon induction with IPTG, it directed the expression of sigma(32) tailed with six additional histidine residues at its C-terminus . The histidine-tagged sigma(32) was purified with nickel-iminodiacetic acid affinity chromatography . Using (35)S radiolabeling methods in vivo, we found that even at low temperature the expression of sigma(32)-6 His may markedly increase the synthesis of the heat shock proteins such as GroEL, Dnak, and HtpH. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(3), 316 - 320 Interaction of the Yeast PH02 Protein or Its Mutants with the PHO5 UAS in vitro; Yang J et al.; The GST gene fusion system was used to express PHO2 gene and its mutants in E . coli Gel retardation assays showed that PHO2 fusion protein can bind to the upstream activation sequence (UAS) of the acid phosphatase gene PHO5 . The homeodomain of PHO2 protein has such structure as alpha-helix 2-beta-turn-alpha-helix 3, which acts as the DNA binding domain of the transcriptional factor . The Mutation of lle 123 to Pro in helix 3 or the insertion of 4 amino acids (PDPD) between 112 and 113 in alpha-helix 2 led to the complete loss of DNA-binding activity of PHO2, while the mutation of Pro 117 to Ala in beta-turn did not affect the binding activity significantly . Deletions of the PHO80 homologous region, acidic region or C-terminal 132 residues had no great effect on the DNA-binding activity, although these mutants had lost the ability to activate PHO5 expression in vivo. Protein Sci, 2002 Oct, 11(10), 2464 - 70 Zinc-substituted Desulfovibrio gigas desulforedoxins: resolving subunit degeneracy with nonsymmetric pseudocontact shifts; Goodfellow BJ et al.; Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one {Fe-4S} center per monomer . Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli . The three forms of the protein, the two homodimers {Fe(III)/Fe(III)}Dx and {Zn(II)/Zn(II)}Dx, and the heterodimer {Fe(III)/Zn(II)}Dx, can be separated by ion exchange chromatography on the basis of their charge differences . Once separated, the desulforedoxins containing iron can be reduced with added dithionite . For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C . Spectral assignments were determined for {Fe(II)/Fe(II)}Dx and {Fe(II)/Zn(II)}Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for {Zn(II)/Zn(II)}Dx . Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment . Comparison of (1)H-(15)N HSQC spectra of {Zn(II)/Zn(II)}Dx, {Fe(II)/Fe(II)}Dx and {Fe(II)/Zn(II)}Dx revealed that the pseudocontact shifts in {Fe(II)/Zn(II)}Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for {Fe(II)/Fe(II)}Dx . The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx . The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits. Protein Sci, 2002 Oct, 11(10), 2456 - 63 Crystal structure of the ADP-dependent glucokinase from Pyrococcus horikoshii at 2.0-A resolution: a large conformational change in ADP-dependent glucokinase; Tsuge H et al.; Although ATP is the most common phosphoryl group donor for kinases, some kinases from certain hyperthermophilic archaea such as Pyrococcus horikoshii and Thermococcus litoralis use ADP as the phosphoryl donor . Those are ADP-dependent glucokinases (ADPGK) and phosphofructokinases in their glycolytic pathway . Here, we succeeded in gene cloning the ADPGK from P . horikoshii OT3 (phGK) in Escherichia coli,and in easy preparation of the enzyme, crystallization, and the structure determination of the apo enzyme . Recently, the three-dimensional structure of the ADPGK from T . litoralis (tlGK) in a complex with ADP was reported . The overall structure of two homologous enzymes (56.7%) was basically similar: This means that they consisted of large alpha/beta-domains and small domains . However, a marked adjustment of the two domains, which is a 10-A translation and a 20 degrees rotation from the conserved GG sequence located at the center of the hinge, was observed between the apo-phGK and ADP-tlGK structures . The ADP-binding loop (430-439) was disordered in the apo form . It is suggested that a large conformational change takes place during the enzymatic reaction. Protein Sci, 2002 Oct, 11(10), 2427 - 36 Site-specific DNA cleavage of synthetic NarL sites by an engineered Escherichia coli NarL protein-1,10-phenanthroline cleaving agent; Xiao G et al.; The NarL response regulatory protein of Escherichia coli has been engineered by covalent modification with 1,10-phenanthroline (OP) to create a set of site-specific DNA-cleaving agents . This was accomplished by introducing single cysteine amino acid replacements at selected locations within the carboxy-terminal DNA-binding domain in or nearby the helix 8 to helix 9 region of the NarL protein using site-directed mutagenesis . Of 18 modified NarL-OP proteins made, 13 retained the ability to bind DNA as evidenced by gel mobility assays, whereas 10 of the 1,10-phenanthroline-modified proteins also exhibited specific cleavage activity for a synthetic NarL recognition sequence . These DNA-cleaving agents were divided into two groups based on the location of the cleavage sites . The first class set cleaved the DNA nearby the center of a synthetic 7-2-7 sequence composed of two NarL heptamer sites separated by a 2-bp spacer element . The second class cut the DNA at the periphery of the 7-2-7 sequence . The cleavage data are consistent with the ability of two NarL monomers to recognize and bind to the DNA in a head-to-head orientation . A second set of DNA-cleaving agents was constructed using the carboxy-terminal domain of NarL called NarL(C) . Similar cleavage patterns were observed whether full-length NarL or NarL(C) was used . The availability of 1,10-phenanthroline-modified NarL and NarL(C) proteins opens up the possibility to explore the position, orientation, and number of NarL recognition sites at E . coli promoters predicted to contain multiple and complex arrangements of NarL-binding sites. Protein Sci, 2002 Oct, 11(10), 2403 - 16 Molecular modeling-based analysis of interactions in the RFC-dependent clamp-loading process; Venclovas C et al.; Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions . The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved . In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint complex, using computationally derived molecular models . Combining the modeled structures for each RFC subunit with known structural, biochemical, and genetic data, we propose detailed models of how two of the RFC subunits, RFC1 and RFC3, interact with the C-terminal regions of PCNA . RFC1 is predicted to bind PCNA similarly to the p21-PCNA interaction, while the RFC3-PCNA binding is proposed to be similar to the E . coli delta-beta interaction . Additional sequence and structure analysis, supported by experimental data, suggests that RFC5 might be the third clamp loader subunit to bind the equivalent PCNA region . We discuss functional implications stemming from the proposed model of the RFC1-PCNA interaction and compare putative clamp-interacting regions in RFC1 and its paralogs, Rad17 and Ctf18 . Based on the individual intermolecular interactions, we propose RFC and PCNA arrangement that places three RFC subunits in association with each of the three C-terminal regions in PCNA . The two other RFC subunits are positioned at the two PCNA interfaces, with the third PCNA interface left unobstructed . In addition, we map interactions at the level of individual subunits between the alternative clamp loader/clamp system, Rad17-RFC(2-5)/Rad9-Rad1-Hus1 . The proposed models of interaction between two clamp/clamp loader pairs provide both structural framework for interpretation of existing experimental data and a number of specific findings that can be subjected to direct experimental testing. J Gen Virol, 2002 Oct, 83(Pt 10), 2385 - 92 Characterization of a novel envelope protein (VP281) of shrimp white spot syndrome virus by mass spectrometry; Huang C et al.; The primary structure of a novel envelope protein from shrimp white spot syndrome virus (WSSV) was characterized using a combination of SDS-PAGE and mass spectrometry . The resulting amino acid sequence matched an open reading frame (ORF), ORF1050, of the WSSV genome ORF database . ORF1050 contained 843 nt, encoding 281 aa, and was termed the vp281 gene . Computer-assisted analysis showed that both the vp281 gene and its product shared no significant homology with other known viruses . However, they shared striking identity/similarity with another WSSV structural protein, VP292, at both the nucleotide and amino acid sequence level, suggesting that vp281 and vp292 might have evolved by gene duplication from a common ancestral gene . WSSV VP281 cDNA was cloned into a pET32a(+) expression vector containing a T7 RNA polymerase promoter to produce (His)(6)-tagged fusion proteins in Escherichia coli strain BL21 . Specific mouse antibodies were raised using the purified fusion protein (His)(6)-VP281 . Western blot analysis showed that the mouse anti-(His)(6)-VP281 antibodies bound specifically to VP281 of WSSV, without cross-reactivity with VP292 . The transmission electron microscope immunogold-labelling method was used to localize VP281 in the WSSV virion as an envelope protein . The cell attachment 'Arg-Gly-Asp' motif in VP281 indicated that this protein might play an important role in mediating WSSV infectivity. J Gen Virol, 2002 Oct, 83(Pt 10), 2367 - 76 A DNA vaccine containing an infectious Marek's disease virus genome can confer protection against tumorigenic Marek's disease in chickens; Tischer BK et al.; A DNA vaccine containing the infectious BAC20 clone of serotype 1 Marek's disease virus (MDV) was tested for its potential to protect against Marek's disease (MD) . Chickens were immunized at 1 day old with BAC20 DNA suspended either in PBS, as calcium phosphate precipitates, incorporated into chitosan nanoparticles, in Escherichia coli DH10B cells, or bound to gold particles for gene-gun delivery . Challenge infection with MDV strain EU1 was performed at 12 days old, and four out of seven birds immunized with BAC20 DNA in saline by the intramuscular route remained free of MD until day 77 after challenge infection . A delay in the development of the disease could be observed in some animals vaccinated with other BAC20 DNA formulations, but clinical MD and tumour formation were evident in all but one bird . Five out of seven animals immunized with the vaccine virus CVI988 were protected against MD, but none out of seven birds survived EU1 challenge infection after injection of negative-control plasmid DNA . In a second animal experiment, five out of 12 chickens immunized with BAC20 DNA and six out of eight birds immunized with virus reconstituted from BAC20 DNA remained free of MD after challenge infection . In contrast, none out of 12 chickens survived challenge infection after immunization with BAC20 DNA lacking the essential gE gene or with gE-negative BAC20 virus . The results suggested that an MDV BAC DNA vaccine has potential to protect chickens against MD, but that in vivo reconstitution of vaccine virus is a prerequisite for protection. Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12795 - 800 Epub 2002 Sep 17. Intrinsic and extrinsic contributions to stochasticity in gene expression; Swain PS et al.; Gene expression is a stochastic, or "noisy," process . This noise comes about in two ways . The inherent stochasticity of biochemical processes such as transcription and translation generates "intrinsic" noise . In addition, fluctuations in the amounts or states of other cellular components lead indirectly to variation in the expression of a particular gene and thus represent "extrinsic" noise . Here, we show how the total variation in the level of expression of a given gene can be decomposed into its intrinsic and extrinsic components . We demonstrate theoretically that simultaneous measurement of two identical genes per cell enables discrimination of these two types of noise . Analytic expressions for intrinsic noise are given for a model that involves all the major steps in transcription and translation . These expressions give the sensitivity to various parameters, quantify the deviation from Poisson statistics, and provide a way of fitting experiment . Transcription dominates the intrinsic noise when the average number of proteins made per mRNA transcript is greater than approximately 2 . Below this number, translational effects also become important . Gene replication and cell division, included in the model, cause protein numbers to tend to a limit cycle . We calculate a general form for the extrinsic noise and illustrate it with the particular case of a single fluctuating extrinsic variable-a repressor protein, which acts on the gene of interest . All results are confirmed by stochastic simulation using plausible parameters for Escherichia coli. J Biol Chem, 2002 Nov 22, 277(47), 45510 - 7 Epub 2002 Sep 16. Functional analysis of the TFIID-specific yeast TAF4 (yTAF(II)48) reveals an unexpected organization of its histone-fold domain; Thuault S et al.; Yeast TFIID comprises the TATA binding protein and 14 TBP-associated factors (TAF(II)s), nine of which contain histone-fold domains (HFDs) . The C-terminal region of the TFIID-specific yTAF4 (yTAF(II)48) containing the HFD shares strong sequence similarity with Drosophila (d)TAF4 (dTAF(II)110) and human TAF4 (hTAF(II)135) . A structure/function analysis of yTAF4 demonstrates that the HFD, a short conserved C-terminal domain (CCTD), and the region separating them are all required for yTAF4 function . Temperature-sensitive mutations in the yTAF4 HFD alpha2 helix or the CCTD can be suppressed upon overexpression of yTAF12 (yTAF(II)68) . Moreover, coexpression in Escherichia coli indicates direct yTAF4-yTAF12 heterodimerization optimally requires both the yTAF4 HFD and CCTD . The x-ray crystal structure of the orthologous hTAF4-hTAF12 histone-like heterodimer indicates that the alpha3 region within the predicted TAF4 HFD is unstructured and does not correspond to the bona fide alpha3 helix . Our functional and biochemical analysis of yTAF4, rather provides strong evidence that the HFD alpha3 helix of the TAF4 family lies within the CCTD . These results reveal an unexpected and novel HFD organization in which the alpha3 helix is separated from the alpha2 helix by an extended loop containing a conserved functional domain. J Inorg Biochem, 2002 Sep 20, 91(4), 644 - 54 Deletions in the loop surrounding the iron-sulfur cluster of adrenodoxin severely affect the interactions with its native redox partners adrenodoxin reductase and cytochrome P450(scc) (CYP11A1); Zollner A et al.; The redox active iron-sulfur center of bovine adrenodoxin is coordinated by four cysteine residues in positions 46, 52, 55 and 92 and is covered by a loop containing the residues Glu-47, Gly-48, Thr-49, Leu-50 and Ala-51 . In plant-type {2Fe-2S} ferredoxins, the corresponding loop consists of only four amino acids . The loop is positioned at the surface of the proteins and forms a boundary separating the {2Fe-2S} cluster from solvent . In order to analyze the biological function of the five amino acids of the loop in adrenodoxin (Adx) for this electron transfer protein each residue was deleted by site-directed mutagenesis . The resulting five recombinant Adx variants show dramatic differences among each other regarding their spectroscopic characteristics and functional properties . The redox potential is affected differently depending on the position of the conducted deletion . In contrast, all mutations in the protein loop influence the binding to the redox partners adrenodoxin reductase (AdR) and cytochrome P450(scc) (CYP11A1) indicating the importance of this loop for the physiological function of this iron--sulfur protein. J Inorg Biochem, 2002 Sep 20, 91(4), 635 - 43 Mesopone cytochrome c peroxidase: functional model of heme oxygenated oxidases; Immoos CE et al.; The effect of heme ring oxygenation on enzyme structure and function has been examined in a reconstituted cytochrome c peroxidase . Oxochlorin derivatives were formed by OsO(4) treatment of mesoporphyrin followed by acid-catalyzed pinacol rearrangement . The northern oxochlorin isomers were isolated by chromatography, and the regio-isomers assignments determined by 2D COSY and NOE 1H NMR . The major isomer, 4-mesoporphyrinone (Mp), was metallated with FeCl(2) and reconstituted into cytochrome c peroxidase (CcP) forming a hybrid green protein, MpCcP . The heme-altered enzyme has 99% wild-type peroxidase activity with cytochrome c . EPR spectroscopy of MpCcP intermediate compound I verifies the formation of the Trp(191) radical similar to wild-type CcP in the reaction cycle . Peroxidase activity with small molecules is varied: guaiacol turnover increases approximately five-fold while that with ferrocyanide is approximately 85% of native . The electron-withdrawing oxo-substitutents on the cofactor cause a approximately 60-mV increase in Fe(III)/Fe(II) reduction potential . The present investigation represents the first structural characterization of an oxochlorin protein with X-ray intensity data collected to 1.70 A . Although a mixture of R- and S-mesopone isomers of the FeMP cofactor was used during heme incorporation into the apo-protein, only the S-isomer is found in the crystallized protein. J Inorg Biochem, 2002 Sep 20, 91(4), 607 - 17 Intermolecular electron transfer in cytochrome P450cam covalently bound with Tris(2,2'-bipyridyl)ruthenium(II): structural changes detected by FTIR spectroscopy; Contzen J et al.; Using Fourier transform infrared spectroscopy (FTIR) we have monitored the changes in the protein structure following photoinduced electron transfer from Ru(bpy)(3)(2+) covalently attached to cysteine 334 on the surface of cytochrome P450cam (CYP101) . The FTIR difference spectra between the oxidized and reduced form indicate changes in a salt link and the secondary structure (alpha-helix and turn regions) . Photoreduction was carried out in the presence of carbon monoxide in order to prove the reduction of the heme iron by means of the appearance of the characteristic CO stretch vibration infrared band at 1940 cm(-1) for the camphor-bound protein . This infrared band has also been used to estimate electron transfer rates . The observed rates depend on the protein concentration, indicating that intermolecular electron transfer occurs between the labeled molecules. J Inorg Biochem, 2002 Sep 20, 91(4), 515 - 26 Catalytically functional flavocytochrome chimeras of P450 BM3 and nitric oxide synthase; Fuziwara S et al.; P450 BM3 and the nitric oxide synthases are related classes of flavocytochrome mono-oxygenase enzymes, containing NADPH-dependent FAD- and FMN-containing oxidoreductase modules fused to heme b-containing oxygenase domains . Domain-swap hybrids of these two multi-domain enzymes were created by genetic engineering of different segments of reductase and heme domains from neuronal nitric oxide synthase and P450 BM3, as a means of investigating the catalytic competence and substrate-binding properties of the fusions and the influence of tetrahydrpbiopterin and calmodulin binding regions on the electron transfer kinetics of the chimeras . Despite marked differences in hybrid stability and solubility, four catalytically functional chimeras were created that retained good reductase activity and which could be expressed successfully in Escherichia coli and purified . All of the BM3 reductase domain chimeras (chimeras I-III) exhibited inefficient flavin-to-heme inter-domain electron transfer, diminishing their oxygenase activity . However, the chimera containing the neuronal nitric oxide synthase reductase domain (chimera IV) showed good oxygenase domain activity, indicating that the flavin-to-heme electron transfer reaction is relatively efficient in this case . The data reinforce the importance of the nature of inter-domain linker constitution in multi-domain enzymes, and the difficulties posed in attempts to create chimeric enzymes with enhanced catalytic properties. Biochem Biophys Res Commun, 2002 Sep 20, 297(2), 309 - 16 C-terminal lysine truncation increases thermostability and enhances chaperone-like function of porcine alphaB-crystallin; Liao JH et al.; The carboxyl-terminal segment of alpha-crystallin, a major lens protein of all vertebrates, has a short and flexible peptide extension of about 20 amino acid residues that are very susceptible to proteolytic truncation and modifications under physiological conditions . To investigate its role in crystallin aggregation and chaperone-like activity, we constructed a mutant of porcine alphaB-crystallin with C-terminal lysine truncated end, which unexpectedly showed better chaperone-like function than wild-type alphaB-crystallin . From circular dichroism (CD) spectra, we show that the mutant possesses similar secondary and tertiary structures to those of native purified and recombinant alphaB-crystallins . Analytical ultracentrifugation revealed that the truncated mutant was smaller than wild-type alphaB-crystallin in aggregation size and mass . The observed higher thermostability and anti-thermal aggregation propensity of the truncated alphaB-crystallin mutant than wild-type alphaB-crystallin are in contrast to the prevailing notion that mutations at the C-terminal lysines of alphaB-crystallin result in substantial loss of chaperone-like activity, despite the overall preservation of secondary structure . The detailed characterization of the C-terminal deletion mutants may provide some deeper insight into the chaperoning mechanism of the structurally related small heat-shock protein family. Biochem Biophys Res Commun, 2002 Sep 20, 297(2), 275 - 81 Characteristics of the end-joining of DNA double-strand breaks by the ataxia-telangiectasia nuclear extract; Tachibana A et al.; A double-strand break was introduced in plasmid pZErO-2 at a specific site within the ccdB gene that is lethal to Escherichia coli cells and treated with nuclear extracts from human cells . The efficiency of rejoining was monitored by Southern blot analysis and the fidelity of rejoining was measured by expressing the ccdB gene after bacterial transformation . The efficiency of rejoining in the nuclear extract from an ataxia-telangiectasia (A-T) cell line was comparable to that from a control cell line . However, the accuracy of rejoining was much lower for the A-T cell extract than for the control cell extract . All mutations were deletions, most of which contained short direct repeats at the breakpoint junctions . The deletion spectrum caused by the A-T nuclear extract was distinct from that of the control extract . These results indicate that the ccdB gene is useful for analysis of mis-rejoining and that A-T cells have certain deficiencies in end-joining of double-strand breaks in DNA. Bioconjug Chem, 2002 Sep-Oct, 13(5), 1163 - 70 A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies; Ratner V et al.; Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins . This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins . Some applications such as the steady state or time-resolved fluorescence resonance energy transfer (FRET) detection of the kinetics of protein folding require relatively large quantities (approximately 10-100 mg) of site-specific doubly labeled protein samples . Engineered cysteine residues are common targets for labeling of proteins . The challenge here is to develop methods for selective modification of one of two reactive sulfhydryl groups in a protein molecule . A general systematic procedure for selective labeling of each of two cysteine residues in a protein molecule was developed, using Escherichia coli adenylate kinase (AKe) as a model protein . Potential sites for insertion of cysteine residues were selected by examination of the crystal structure of the protein . A series of single-cysteine mutants was prepared, and the rates of the reaction of each engineered cysteine residue with a reference reagent {5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)} were determined . Two-cysteine mutants were prepared by selection of pairs of sites for which the ratio of this reaction rate constant was high (>80) . The conditions for the selective labeling reaction were optimized . In a first cycle of labeling, the more reactive cysteine residue was labeled with a fluorescent probe (donor) . The second probe was attached to the less reactive site under unfolding conditions in the second cycle of labeling . The doubly and singly labeled mutants retained full enzymatic activity and the capacity for a reversible folding-unfolding transition . High yields (70-90%) of the preparation of the pure, site-specific doubly labeled AK mutant were obtained . The procedure described herein is a general outline of procedures, which can meet the double challenge of both site specificity and large-scale preparation of doubly labeled proteins. Bioconjug Chem, 2002 Sep-Oct, 13(5), 1119 - 23 Development of annexin V mutants suitable for labeling with Tc(i)-carbonyl complex; Tait JF et al.; (99m)Tc-annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection . We investigated whether the novel Tc-carbonyl labeling method would be suitable for annexin V . Two mutant molecules of annexin V, called annexin V-122 and annexin V-123, were constructed with N-terminal extensions containing either three or six histidine residues . These molecules were expressed cytoplasmically in E . coli and purified with a final yield of 33 mg of protein/L of culture . Analysis by SDS-PAGE, isoelectric focusing, gel filtration chromatography, and mass spectrometry confirmed the purity and homogeneity of the protein preparations . Both mutant proteins retained full binding affinity for cell membranes with exposed phosphatidylserine . Using the Tc-carbonyl reagent, both proteins could be labeled with (99m)Tc to specific activities of at least 10-20 microCi/microg with full retention of bioactivity . The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro, and there was no transchelation of label to serum proteins during in vitro incubation . In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for (99m)Tc-carbonyl without altering its high affinity for cell membranes. Yakugaku Zasshi, 2002 Sep, 122(9), 607 - 14 {The transport mechanism of polycationic compounds across intestinal and renal cell membrane}; Kobayashi M; This article reviewed the transport mechanism of polycationic compounds across rat intestinal and renal cell membranes . The inside-negative diffusion potential stimulated the initial uptake of dicationic compounds into intestinal brush-border membrane vesicles, and a good correlation was observed between lipophilicity and the amount of diffusion potential-dependent transport of the dications . On the other hand, tri- and tetracationic compounds were not affected by the diffusion potential because of their much lower lipophilicity . The membrane surface potential affected to the transport of polycationic compounds, similar to monocationic compounds . Therefore it appears that the membrane surface potential plays a common role in the transport of mono- and polycationic compounds across cell membranes . On the intestinal basolateral membrane, it was found that there was a Na+/putrescine symporter . This recognized dicationic compounds and transported them from the blood into intestinal cells . This transporter did not recognize spermine and spermidine . Furthermore, we found a novel transport system, a Na+/spermine antiporter, on the rat renal brush-border membrane . This transporter recognized aliphatic polycation, which has more than four amino groups, and actively secreted spermine and trientine into the renal proximal tubules in vitro and in vivo . However, this transporter did not recognize trientine-copper complex . These results are useful for the prediction of the intestinal absorption and renal excretion of polyamine derivatives. Wiad Lek, 2002, 55(5-6), 332 - 7 {Molecular basis of the classical type of Ehlers-Danlos syndrome}; Wieczorek P et al.; Classical type of Ehlers-Danlos syndrome (EDS) is the most common variant of the disease, however, its molecular background is still unknown . The paper presents the latest findings concerning this type of EDS . In most cases, mutations in genes encoding type V collagen were found . In some cases, the mutations were excluded indicating the genetic heterogeneity of the disease. J Biol Chem, 2002 Nov 8, 277(45), 43089 - 95 Epub 2002 Aug 30. Structural conservation between the actin monomer-binding sites of twinfilin and actin-depolymerizing factor (ADF)/cofilin; Paavilainen VO et al.; Twinfilin is an evolutionarily conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals . It is composed of two actin-depolymerization factor homology (ADF-H) domains that show approximately 20% sequence identity to ADF/cofilin proteins . In contrast to ADF/cofilins, which bind both G-actin and F-actin and promote filament depolymerization, twinfilin interacts only with G-actin . To elucidate the molecular mechanisms of twinfilin-actin monomer interaction, we determined the crystal structure of the N-terminal ADF-H domain of twinfilin and mapped its actin-binding site by site-directed mutagenesis . This domain has similar overall structure to ADF/cofilins, and the regions important for actin monomer binding in ADF/cofilins are especially well conserved in twinfilin . Mutagenesis studies show that the N-terminal ADF-H domain of twinfilin and ADF/cofilins also interact with actin monomers through similar interfaces, although the binding surface is slightly extended in twinfilin . In contrast, the regions important for actin-filament interactions in ADF/cofilins are structurally different in twinfilin . This explains the differences in actin-interactions (monomer versus filament binding) between twinfilin and ADF/cofilins . Taken together, our data show that the ADF-H domain is a structurally conserved actin-binding motif and that relatively small structural differences at the actin interfaces of this domain are responsible for the functional variation between the different classes of ADF-H domain proteins. J Biol Chem, 2002 Nov 8, 277(45), 43536 - 43 Epub 2002 Aug 31. Autocatalytic processing of gamma-glutamyltranspeptidase; Suzuki H et al.; gamma-Glutamyltranspeptidase is the key enzyme in glutathione metabolism, and we previously presented evidence suggesting that it belongs to the N-terminal nucleophile hydrolase superfamily . Enzymatically active gamma-glutamyltranspeptidase, which consists of one large subunit and one small subunit, is generated from an inactive common precursor through post-translational proteolytic processing . The processing mechanism for gamma-glutamyltranspeptidase of Escherichia coli K-12 has been analyzed by means of in vitro studies using purified precursors . Here we show that the processing of a precursor of gamma-glutamyltranspeptidase is an intramolecular autocatalytic event and that the catalytic nucleophile for the processing reaction is the oxygen atom of the side chain of Thr-391 (N-terminal residue of the small (beta) subunit), which is also the nucleophile for the enzymatic reaction. J Biol Chem, 2002 Nov 8, 277(45), 42463 - 70 Epub 2002 Aug 30. Evidence for regulation of the tumor necrosis factor alpha-convertase (TACE) by protein-tyrosine phosphatase PTPH1; Zheng Y et al.; Tumor necrosis factor alpha-convertase (TACE) is a metalloprotease-disintegrin involved in the ectodomain shedding of several proteins and is critical for proper murine development . TACE-mediated ectodomain shedding is regulated, and the cytoplasmic domain of TACE contains several potential signaling motifs, suggesting that this domain may play a role in regulating the metalloprotease activity . Here we report that the protein-tyrosine phosphatase PTPH1, which contains both a band 4.1 domain and a single PDZ domain, can interact with the cytoplasmic domain of TACE . The interaction was initially observed in a yeast two-hybrid screen and was confirmed using an in vitro binding assay and co-immunoprecipitations from eukaryotic cell extracts . The interaction is mediated via binding of the PDZ domain of PTPH1 to the COOH terminus of TACE . The latter represents a novel group I PDZ binding sequence characterized by a terminal cysteine residue . In co-expression experiments, significantly lower levels of TACE were observed in the presence of catalytically active forms of PTPH1 compared with catalytically inactive forms of PTPH1 . Furthermore, phorbol ester-stimulated shedding of the TACE substrate tumor necrosis factor-alpha was decreased in cells expressing catalytically active PTPH1 compared with inactive PTPH1 . Taken together, these results suggest that PTPH1 may be a negative regulator of TACE levels and function, and thus provide the first evidence for the regulation of TACE through a cytoplasmic protein. J Biol Chem, 2002 Nov 8, 277(45), 42645 - 53 Epub 2002 Aug 30. Structure-function analysis of Escherichia coli DNA helicase I reveals non-overlapping transesterase and helicase domains; Byrd DR et al.; TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer . The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient . Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively . Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309 . Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity . Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity . The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity . Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare . Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively . The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs . This region is required for helicase activity. J Biol Chem, 2002 Nov 1, 277(44), 41865 - 71 Epub 2002 Aug 30. Electron transfer from the Rieske iron-sulfur protein (ISP) to cytochrome f in vitro . Is a guided trajectory of the ISP necessary for competent docking? Soriano GM, Guo LW, De Vitry C, Kallas T, Cramer WA. The time course of electron transfer in vitro between soluble domains of the Rieske iron-sulfur protein (ISP) and cytochrome f subunits of the cytochrome b(6)f complex of oxygenic photosynthesis was measured by stopped-flow mixing . The domains were derived from Chlamydomonas reinhardtii and expressed in Escherichia coli . The expressed 142-residue soluble ISP apoprotein was reconstituted with the {2Fe-2S} cluster . The second-order rate constant, k(2)((ISP-f)) = 1.5 x 10(6) m(-1) s(-1), for ISP to cytochrome f electron transfer was <10(-2) of the rate constant at low ionic strength, k(2)((f-PC))(> 200 x 10(6) m(-1) s(-1)), for the reduction of plastocyanin by cytochrome f, and approximately 1/30 of k(2)((f-PC)) at the ionic strength estimated for the thylakoid interior . In contrast to k(2)((f-PC)), k(2)((ISP-f)) was independent of pH and ionic strength, implying no significant role of electrostatic interactions . Effective pK values of 6.2 and 8.3, respectively, of oxidized and reduced ISP were derived from the pH dependence of the amplitude of cytochrome f reduction . The first-order rate constant, k(1)((ISP-f)), predicted from k(2)((ISP-f)) is approximately 10 and approximately 150 times smaller than the millisecond and microsecond phases of cytochrome f reduction observed in vivo . It is proposed that in the absence of electrostatic guidance, a productive docking geometry for fast electron transfer is imposed by the guided trajectory of the ISP extrinsic domain . The requirement of a specific electrically neutral docking configuration for ISP electron transfer is consistent with structure data for the related cytochrome bc(1) complex. J Biol Chem, 2002 Nov 8, 277(45), 42748 - 54 Epub 2002 Aug 30. Cloning and characterization of mitochondrial 5-formyltetrahydrofolate cycloligase from higher plants; Roje S et al.; 5-Formyltetrahydrofolate cycloligase (5-FCL) catalyzes the conversion of 5-formyltetrahydrofolate (5-CHO-H(4)PteGlu(n)) to 5,10-methenyltetrahydrofolate and is considered to be the main means whereby 5-CHO-H(4)PteGlu(n) is metabolized in mammals, yeast, and bacteria . 5-CHO-H(4)PteGlu(n) is known to occur in plants and to be highly abundant in leaf mitochondria . Genomics-based approaches identified Arabidopsis and tomato cDNAs encoding proteins homologous to 5-FCLs of other organisms but containing N-terminal extensions with the features of mitochondrial targeting peptides . These homologs were shown to have 5-FCL activity by characterizing recombinant enzymes produced in Escherichia coli and by functional complementation of a yeast fau1 mutation with the Arabidopsis 5-FCL cDNA . The recombinant Arabidopsis enzyme is active as a monomer, prefers the penta- to the monoglutamyl form of 5-CHO-H(4)PteGlu(n), and has kinetic properties broadly similar to those of 5-FCLs from other organisms . Enzyme assays and immunoblot analyses indicated that 5-FCL is located predominantly if not exclusively in plant mitochondria and that the mature, active enzyme lacks the putative targeting sequence . Serine hydroxymethyltransferase (SHMT) from plant mitochondria was shown to be inhibited by 5-CHO-H(4)PteGlu(n) as are SHMTs from other organisms . Since mitochondrial SHMT is crucial to photorespiration, 5-FCL may help prevent 5-CHO-H(4)PteGlu(n) from reaching levels that would inhibit this process . Consistent with this possibility, 5-FCL activity was far higher in leaf mitochondria than root mitochondria. Nucleic Acids Res . 2002 Sep 15;30(18):e95. Rapid gene cloning using terminator primers and modular vectors; Donahue WF et al.; We seek to create useful biological diversity by exploiting the modular nature of genetic information . In this report we describe experiments that focus on the modular nature of plasmid cloning vectors . Bacterial plasmids are modular entities composed of origins of replication, selectable markers and other components . We describe a new ligation-independent cloning method that allows for rapid and seamless assembly of vectors from component modules . We further demonstrate that gene cloning can be accomplished simultaneously with assembly of a modular vector . This approach provides considerable flexibility as it allows for 'menu driven' cloning of genes into custom assembled modular vectors. Nucleic Acids Res, 2002 Sep 15, 30(18), 4094 - 101 Binding affinity of Escherichia coli RNA polymerase*sigma54 holoenzyme for the glnAp2, nifH and nifL promoters; Vogel SK et al.; Escherichia coli RNA polymerase associated with the sigma54 factor (RNAP*sigma54) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase*sigma70 com plex . Promoters for RNAP*sigma54 vary in their overall 'strength' and show differences in their response to the presence of DNA curvature between enhancer and promoter . In order to examine whether these effects are related to the promoter affinity, we have determined the equilibrium dissociation constant K(d) for the binding of RNAP*sigma54 to the three promoters glnAp2, nifH and nifL . Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP*sigma54 to carboxyrhodamine-labeled DNA duplexes . For the glnAp2 and nifH promoters similar values of K(d) = 0.94 +/- 0.55 nM and K(d) = 0.85 +/- 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with K(d) = 8.5 +/- 1.9 nM . The logarithmic dependence of K(d) on the ionic strength I was -Deltalog(K(d))/Deltalog(I) = 6.1 +/- 0.5 for the glnAp2, 5.2 +/- 1.2 for the nifH and 2.1 +/- 0.1 for the nifL promoter . This suggests that the polymerase can form fewer ion pairs with the nifL promoter, which would account for its weaker binding affinity. Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12944 - 8 Epub 2002 Sep 16. Acceleration of genomic evolution caused by enhanced mutation rate in endocellular symbionts; Itoh T et al.; Endosymbionts, which are widely observed in nature, have undergone reductive genome evolution because of their long-term intracellular lifestyle . Here we compared the complete genome sequences of two different endosymbionts, Buchnera and a protist mitochondrion, with their close relatives to study the evolutionary rates of functional genes in endosymbionts . The results indicate that the rate of amino acid substitution is two times higher in symbionts than in their relatives . This rate increase was observed uniformly among different functional classes of genes, although strong purifying selection may have counterbalanced the rate increase in a few cases . Our data suggest that, contrary to current views, neither the Muller's ratchet effect nor the slightly deleterious mutation theory sufficiently accounts for the elevated evolutionary rate . Rather, the elevated evolutionary rate appears to be mainly due to enhanced mutation rate, although the possibility of relaxation of purifying selection cannot be ruled out. J Pharmacol Exp Ther, 2002 Oct, 303(1), 265 - 72 Dual mechanisms for ethanol-induced inhibition of monocyte chemotactic protein-3 mRNA expression in activated glial cells; Ren L et al.; The differential display of mRNA technique was used to screen the expressed genes in control and 50 mM chronic ethanol-treated rat C6 glial cells, with and without activation by lipopolysaccharide (LPS) combined with phorbol 12-myristate 13-acetate (PMA) . One differentially expressed transcript was identified as that corresponding to the chemokine monocyte chemotactic protein (MCP)-3 . MCP-3 is a broadly active chemokine that functions in chemoattraction and activation of monocytes, T lymphocytes, eosinophils, basophils, natural killer cells, and dendritic cells . Steady-state MCP-3 mRNA levels were elevated 6-fold after 24-h stimulation of control cells but less than 3-fold after stimulation of 9-day chronic ethanol-exposed cells . One- and 5-day exposures to 50 mM ethanol were not effective at reducing steady-state MCP-3 mRNA levels in stimulated cells, whereas 1-day exposure to >150 mM ethanol was effective . Stimulation with tumor necrosis factor-alpha elevated MCP-3 mRNA in C6 glial cells to a lesser extent than with LPS plus PMA, but the effects of ethanol were consistent . To gain insight into possible mechanisms for ethanol-induced reductions in steady-state MCP-3 mRNA, additional studies examined nuclear MCP-3 RNA levels and MCP-3 mRNA degradation . MCP-3 RNA content was greatly reduced in isolated nuclei from acute and chronic ethanol-exposed cells, suggesting transcriptional inhibition . On the other hand, acute ethanol exposure enhanced degradation of preexisting MCP-3 mRNA, indicating message destabilization . Thus, the results are consistent with a dual mechanism for ethanol-induced reductions in steady-state MCP-3 mRNA levels. J Exp Med, 2002 Sep 16, 196(6), 805 - 16 Major histocompatibility complex class I allele-specific cooperative and competitive interactions between immune evasion proteins of cytomegalovirus; Wagner M et al.; Cytomegaloviruses (CMVs) deploy a set of genes for interference with antigen presentation in the major histocompatibility complex (MHC) class I pathway . In murine CMV (MCMV), three genes were identified so far: m04/gp34, m06/gp48, and m152/gp40 . While their function as immunoevasins was originally defined after their selective expression, this may not necessarily reflect their biological role during infection . The three immunoevasins might act synergistically, but they might also compete for their common substrate, the MHC class I complexes . To approach this question in a systematic manner, we have generated a complete set of mutant viruses with deletions of the three genes in all seven possible combinations . Surface expression of a set of MHC class I molecules specified by haplotypes H-2(d) (K(d), D(d), and L(d)) and H-2(b) (K(b) and D(b)) was the parameter for evaluation of the interference with class I trafficking . The data show the following: first, there exists no additional MCMV gene of major influence on MHC class I surface expression; second, the strength of the inhibitory effect of immunoevasins shows an allele-specific hierarchy; and third, the immunoevasins act not only synergistically but can, in certain combinations, interact antagonistically . In essence, this work highlights the importance of studying the immunosubversive mechanisms of cytomegaloviruses in the context of gene expression during the viral replicative cycle in infected cells. J Biol Chem, 2002 Nov 29, 277(48), 46753 - 62 Epub 2002 Sep 15. Crystal structure of the heterodimeric complex of the adaptor, ClpS, with the N-domain of the AAA+ chaperone, ClpA; Guo F et al.; Substrate selectivity and proteolytic activity for the E . coli ATP-dependent protease, ClpAP, is modulated by an adaptor protein, ClpS . ClpS binds to ClpA, the regulatory component of the ClpAP complex . We report the crystal structure of ClpS in complex with the isolated N-terminal domain of ClpA in two different crystal forms at 2.3- and 3.3-A resolution . The ClpS structure forms an alpha/beta-sandwich and is topologically analogous to the C-terminal domain of the ribosomal protein L7/L12 . ClpS contacts two surfaces on the N-terminal domain in both crystal forms; the more extensive interface was shown to be favored in solution by protease protection experiments . The N-terminal 20 residues of ClpS are not visible in the crystal structures; the removal of the first 17 residues produces ClpSDeltaN, which binds to the ClpA N-domain but no longer inhibits ClpA activity . A zinc binding site involving two His and one Glu residue was identified crystallographically in the N-terminal domain of ClpA . In a model of ClpS bound to hexameric ClpA, ClpS is oriented with its N terminus directed toward the distal surface of ClpA, suggesting that the N-terminal region of ClpS may affect productive substrate interactions at the apical surface or substrate entry into the ClpA translocation channel. J Biol Chem, 2002 Dec 6, 277(49), 46959 - 65 Epub 2002 Sep 13. A novel sterol 14alpha-demethylase/ferredoxin fusion protein (MCCYP51FX) from Methylococcus capsulatus represents a new class of the cytochrome P450 superfamily; Jackson CJ et al.; Sterol 14alpha-demethylase encoded by CYP51 is a member of the cytochrome P450 (CYP) superfamily of enzymes and has been shown to have an essential role in sterol biosynthesis in eukaryotes, with orthologues recently being described in some bacteria . Examination of the genome sequence data for the proteobacterium Methylococcus capsulatus, a bacterial species known to produce sterol, revealed the presence of a single CYP with strong homology to CYP51, particularly to a form in Mycobacterium tuberculosis . This M . capsulatus CYP51 protein represents a new class of CYP consisting of the CYP domain naturally fused to a ferredoxin domain at the C terminus via an alanine-rich linker . Expression of the M . capsulatus MCCYP51FX fusion in Escherichia coli yielded a P450, which, when purified to homogeneity, had the predicted molecular mass approximately 62 kDa on SDS/PAGE and bound lanosterol as a putative substrate . Sterol 14alpha-demethylase activity was shown (0.24 nmol of lanosterol metabolized per minute per nanomole of MCCYP51FX fusion) by gas chromatography/mass spectrometry with the activity dependent upon the presence of ferredoxin reductase and NADPH . Our unique findings describe a new class of naturally existing cytochrome P450, which will provide pivotal information for CYP structure/function in general. Circulation, 2002 Sep 17, 106(12), 1460 - 4 High doses of vitamin C reverse Escherichia coli endotoxin-induced hyporeactivity to acetylcholine in the human forearm; Pleiner J et al.; BACKGROUND: Acute inflammation causes endothelial vasodilator dysfunction that may be mediated by oxidative stress . METHODS AND RESULTS: In this randomized, double-blind, crossover study, forearm blood flow responses to acetylcholine (ACh) (endothelium-dependent dilator) and glyceryl-trinitrate (GTN) (endothelium-independent dilator) were assessed before and after induction of acute systemic inflammation by low doses of Escherichia coli endotoxin (LPS) (20 IU/kg IV) in 8 healthy volunteers . The acute effect of intra-arterial vitamin C (24 mg/min) or placebo was studied 4 hours after LPS, respectively . Vitamin C alone was administered in control experiments . LPS administration caused systemic vasodilation, increased white blood count, elevated body temperature, and reduced vitamin C plasma concentrations . LPS decreased the responses of forearm blood flow to ACh by 30% (P<0.05) but not to GTN . Vitamin C completely restored the response to ACh, which was comparable with that observed under baseline conditions . Vitamin C had no effect on basal blood flow or ACh- or GTN-induced vasodilation in control subjects . CONCLUSIONS: Our data demonstrate that impaired endothelial vasodilation caused by E coli endotoxemia can be counteracted by high doses of antioxidants in vivo . Oxidative stress may play an important role in the pathogenesis of endothelial dysfunction during inflammation. EMBO J, 2002 Sep 16, 21(18), 4774 - 84 The disulfide bond isomerase DsbC is activated by an immunoglobulin-fold thiol oxidoreductase: crystal structure of the DsbC-DsbDalpha complex; Haebel PW et al.; The Escherichia coli disulfide bond isomerase DsbC rearranges incorrect disulfide bonds during oxidative protein folding . It is specifically activated by the periplasmic N-terminal domain (DsbDalpha) of the transmembrane electron transporter DsbD . An intermediate of the electron transport reaction was trapped, yielding a covalent DsbC-DsbDalpha complex . The 2.3 A crystal structure of the complex shows for the first time the specific interactions between two thiol oxidoreductases . DsbDalpha is a novel thiol oxidoreductase with the active site cysteines embedded in an immunoglobulin fold . It binds into the central cleft of the V-shaped DsbC dimer, which assumes a closed conformation on complex formation . Comparison of the complex with oxidized DsbDalpha reveals major conformational changes in a cap structure that regulates the accessibility of the DsbDalpha active site . Our results explain how DsbC is selectively activated by DsbD using electrons derived from the cytoplasm. Antimicrob Agents Chemother, 2002 Oct, 46(10), 3265 - 7 -11 Mutation in the ampC promoter increasing resistance to beta-lactams in a clinical Escherichia coli strain; Corvec S et al.; A mutation was discovered in the Pribnow box of the ampC promoter in a clinical Escherichia coli strain . This -11 C-to-T transition created a perfect homology with the -10 consensus sequence . The new promoter was cloned upstream of the cat gene of pKK232-8 and induced a sixfold increase in promoter strength. Gene, 2002 Jul 10, 294(1-2), 177 - 85 G350 of Escherichia coli RNase P RNA contributes to Mg2+ binding near the active site of the enzyme; Rasmussen TA et al.; G350 of Escherichia coli RNase P RNA is a highly conserved residue among all bacteria and lies near the known magnesium binding site for the RNase P ribozyme, helix P4 . Mutations at G350 have a dramatic effect on substrate cleavage activity for both RNA alone and holoenzyme; the G350C mutation has the most severe phenotype . The G350C mutation also inhibits growth of cells that express the mutant RNA in vivo under conditions of magnesium starvation . The results suggest that G350 contributes to Mg(2+) binding at helix P4 of RNase P RNA. Gene, 2002 Jul 10, 294(1-2), 157 - 66 Molecular cloning and expression of the cDNAs encoding luciferin-regenerating enzyme from Luciola cruciata and Luciola lateralis; Gomi K et al.; In the firefly light organ, oxyluciferin, a product of the light-emitting reaction of firefly luciferase, is thought to be converted into luciferin . Previously, we isolated the luciferin-regenerating enzyme (LRE) from Photinus pyralis . LRE plays an important role in the recycling of oxyluciferin into luciferin . We have cloned two cDNAs encoding LRE, G-LRE and H-LRE, from poly(A)+ RNA of the lanterns of Luciola cruciata and Luciola lateralis, using reverse transcription-polymerase chain reaction, 5'-RACE (5'-rapid amplification of cDNA ends) and 3'-RACE . The putative translation products have molecular masses of 33,804 and 34,285 Da, corresponding to 309 and 307 amino acids, respectively . The deduced amino acid sequence of G-LRE shows 57 and 56% identity with H-LRE and A-LRE (P . pyralis), respectively . LRE (G-LRE, H-LRE, A-LRE) shows at most 39% amino acid sequence identity with insect anterior fat protein (AFP) and mammalian senescence marker protein-30 (SMP30) . G-LRE and H-LRE were successfully expressed under the control of the lac promoter in Escherichia coli. Gene, 2002 Jul 10, 294(1-2), 109 - 17 Mouse cytosolic and mitochondrial deoxyribonucleotidases: cDNA cloning of the mitochondrial enzyme, gene structures, chromosomal mapping and comparison with the human orthologs; Rampazzo C et al.; Two of the five known mammalian 5'-nucleotidases show a preference for the dephosphorylation of deoxynucleoside-5'-phosphates . One is a cytoplasmic enzyme (dNT-1), the other occurs in mitochondria (dNT-2) . The human mitochondrial enzyme, recently discovered and cloned by us, is encoded by a nuclear gene located on chromosome 17 p11.2 in the critical region deleted in the Smith-Magenis syndrome (SMS), a genetic disease of unknown etiology . Looking for a model system to study the possible involvement of dNT-2 in the disease, we have cloned the cDNA of the mouse ortholog . The deduced protein sequence is 84% identical to the human ortholog, has a very basic NH(2)-terminus, a very high calculated probability of being imported into mitochondria and contains the DXDXT/V motif conserved among nucleotidases . Expression in Escherichia coli of the predicted processed form of the protein produced an active deoxyribonucleotidase . We also identified in genomic sequences present in the data base the structures of the murine genes for the cytosolic and mitochondrial deoxyribonucleotidases (Nt5c and Nt5m) . PAC clones for the two loci were isolated from a library and used for chromosomal localization by fluorescent in situ hybridization . Both genes map on chromosome 11: Nt5c at 11E and Nt5m at 11B, demonstrating the presence of the dNT-2 locus in the mouse shaker-2 critical region, the murine counterpart of the human SMS region . We performed pair-wise dot-plot and PIP (percent identity plot) analyses of mouse and human deoxyribonucleotidase genes, and found a strong conservation that extends also to some intronic sequences of possible regulatory significance. Arch Biochem Biophys, 2002 Oct 1, 406(1), 116 - 26 Electron acceptor specificity of ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli; Wan JT et al.; Reduced flavodoxin I (Fld1) is required in Escherichia coli for reductive radical generation in AdoMet-dependent radical enzymes and reductive activation of cobalamin-dependent methionine synthase . Ferredoxin (Fd) and flavodoxin II (Fld2) are also present, although their precise roles have not been ascertained . Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) was discovered in E . coli as an NADPH-dependent reductant of Fld1 that facilitated generation of active methionine synthase in vitro; FNR and Fld1 will also supply electrons for the reductive cleavage of AdoMet essential for generating protein or substrate radicals in pyruvate formate-lyase, class III ribonucleotide reductase, biotin synthase, and, potentially, lipoyl synthase . As part of ongoing efforts to understand the various redox pathways that will support AdoMet-dependent radical enzymes in E . coli, we have examined the relative specificity of E . coli FNR for Fd, Fld1, and Fld2 . While FNR will reduce all three proteins, Fd is the kinetically and thermodynamically preferred partner . Fd binds to FNR with high affinity (K(d)<or=0.5 microM) and is reduced under single-turnover conditions with k(obs)=2.3s(-1) and under steady state conditions with k(cat)=0.15s(-1) . Fld1 and Fld2 behave similarly with respect to FNR, with affinities approximately 4- to 7-fold weaker and reduction rates that are 10- to 100-fold slower than those for Fd . Surprisingly we find that Fld1 and Fld2 can obtain electrons from reduced Fd at rates that are comparable to those obtained with reduced FNR . Thus we propose that the primary electron acceptor for E . coli FNR is Fd, while Fld1 can obtain electrons slowly either from FNR or via Fd as a mediator. Arch Biochem Biophys, 2002 Oct 1, 406(1), 21 - 32 Enzymatic properties and regulation of ZPU1, the maize pullulanase-type starch debranching enzyme; Wu C et al.; Starch debranching enzymes (DBE) are required for mobilization of carbohydrate reserves and for the normal structural organization of storage glucan polymers . Two isoforms, the pullulanase-type DBEs and the isoamylase-type DBEs, are both highly conserved in plants . To address DBE functions in starch assembly and breakdown, this study characterized the biochemical activity of ZPU1, a pullulanase-type DBE that is the product of the maize Zpu1 gene . Assays showed directly that recombinant ZPU1 (ZPU1r) expressed in Escherichia coli functions as a pullulanase-type enzyme, and 1H-NMR spectroscopy demonstrated that ZPU1r specifically hydrolyzes alpha(1-->6) branch linkages . Preferred substrates for ZPU1r hydrolytic activity were determined, as were pH, temperature, and thermal stability optima . Kinetic properties of ZPU1r with respect to two substrates, beta-limit dextrin and pullulan, were determined . ZPU1 activity was increased by incubation with thioredoxin h, and native activity was decreased in mutants that accumulate soluble sugars, suggesting potential regulatory mechanisms. Anal Biochem, 2002 Sep 1, 308(1), 112 - 9 The determination of homocysteine-thiolactone in biological samples; Jakubowski H; Homocysteine-thiolactone, a cyclic thioester of homocysteine, is synthesized by methionyl-tRNA synthetase in all cell types . A new assay for the determination of homocysteine-thiolactone in biological samples is described . The assay involves separation of homocysteine-thiolactone from macromolecules by ultrafiltration . Homocysteine-thiolactone is further purified and quantified by high-pressure liquid chromatography either on a reverse phase or a cation exchange micro-bore column . The detection and quantitation are obtained by monitoring the absorbance at 240 nm, a maximum in a UV spectrum of homocysteine-thiolactone . The sensitivity of detection is 5 pmol . This assay has been applied to bacteria (Escherichia coli and Mycobacterium smegmatis), the yeast Saccharomyces cerevisiae, cultured human vascular endothelial cells, and human plasma . The data support the conclusion that homocysteine-thiolactone is a ubiquitous metabolite whose levels are directly related to homocysteine levels. Anal Biochem, 2002 Sep 1, 308(1), 18 - 25 Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions; Rusinova E et al.; The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution . We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and protein-nucleic acid interactions by fluorescence anisotropy . Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching . Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site . The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed . The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated. Pediatr Transplant, 2002 Aug, 6(4), 352 - 5 Fulminant recurrence of atypical hemolytic uremic syndrome during a calcineurin inhibitor-free immunosuppression regimen; Florman S et al.; Recurrence of hemolytic uremic syndrome (HUS) after kidney transplantation is frequent, occurring almost exclusively in patients with atypical HUS, which is not caused by Escherichia coli gastroenteritis and in which diarrhea is absent . Calcineurin inhibitors are associated with recurrence of HUS . In two children who underwent living donor kidney transplantation for atypical HUS, we pre-emptively employed sirolimus in a calcineurin inhibitor-free immunosuppression regimen . Both children had excellent early graft function, yet both developed severe recurrent disease and subsequently lost their grafts . Avoidance of calcineurin inhibitors did not prevent recurrence of severe HUS and graft loss . Transplantation for severe atypical HUS remains problematic. Biochemistry, 2002 Sep 24, 41(38), 11512 - 5 Regulation of lipid composition in Acholeplasma laidlawii and Escherichia coli membranes: NMR studies of lipid lateral diffusion at different growth temperatures; Lindblom G et al.; Lipid lateral diffusion coefficients have been directly determined by pulsed field gradient NMR spectroscopy on macroscopically aligned, fully hydrated lamellar phases containing dimyristoylphosphatidylcholine and total lipid extracts from Acholeplasma laidlawii and Escherichia coli . The temperature dependence of the diffusion coefficient was of the Arrhenius type in the temperature interval studied . The sharp increase in the diffusion coefficient at the growth temperature of E . coli obtained by FRAP measurements, using a fluorescent probe molecule (Jin, A . J., Edidin, M., Nossal, R., and Gershfeld, N . L . (1999) Biochemistry 38, 13275-13278), was not observed . Thus, we conclude that the lipid structural properties (i.e., those affecting the lipid phase behavior), rather than the lipid dynamics, are involved in the adjustment of the membrane lipid composition . Further support for this conclusion is given by the finding that lipid extracts from A . laidlawii grown at different temperatures have about the same diffusion coefficients . Finally, the lipid lateral diffusion in bilayers of phospholipids was found to be much faster than that in bilayers of mainly glucolipids, which can be understood in terms of a free volume theory for the diffusion process. Biochemistry, 2002 Sep 24, 41(38), 11472 - 8 Site-specific incorporation of (aminooxy)acetic acid into proteins; Eisenhauer BM et al.; By employing a general biosynthetic method for the elaboration of proteins containing unnatural amino acid analogues, we incorporated (aminooxy)acetic acid into positions 10 and 27 of Escherichia coli dihydrofolate reductase . Introduction of the modified amino acid into DHFR was accomplished in an in vitro protein biosynthesizing system by readthrough of a nonsense (UAG) codon with a suppressor tRNA that had been activated with (aminooxy)acetic acid . Incorporation of the amino acid proceeded with reasonable efficiency at codon position 10 but less well at position 27 . (Aminooxy)acetic acid was also incorporated into position 72 of DNA polymerase beta . Peptides containing (aminooxy)acetic acid have been shown to adopt a preferred conformation involving an eight-membered ring that resembles a gamma-turn . Accordingly, the present study may facilitate the elaboration of proteins containing conformationally biased peptidomimetic motifs at predetermined sites . The present results further extend the examples of ribosomally mediated formation of peptide bond analogues of altered connectivity and provide a conformationally biased linkage at a predetermined site . It has also been shown that the elaborated protein can be cleaved chemically at the site containing the modified amino acid. Biochemistry, 2002 Sep 24, 41(38), 11362 - 71 Systematic site-directed mutagenesis of human protein SRP54: interactions with signal recognition particle RNA and modes of signal peptide recognition; Huang Q et al.; The amino acid residues of human protein SRP54 which are required for binding to SRP RNA were identified by generating 40 nonoverlapping tri-alanine alterations within its methionine-rich M-domain (SRP54M) . The mutant polypeptides were expressed in Escherichia coli, and their ability to bind to human and Methanococcus jannaschii SRP RNA were determined in vitro . Residues at positions 379-387, 394-396, 400-405, and 409-411 of human SRP54 were within the predicted RNA binding site, and their alteration abolished the binding activities of the mutant polypeptides as expected . Changes at positions 418-423 had intermediate effects . Polypeptides containing mutations of 328-TLR-330 were inactive although these residues were far away from the presumed RNA binding site in the crystal structure of the free protein . Using the structures of the E . coli Ffh/4.5S core and of the human SRP54m dimer as templates, a molecular model of the complex between human SRP RNA helix 8 and a single SRP54M molecule was constructed in which Leucine 329 was positioned in closer proximity to the RNA binding domain . This representation was supported by studies of the SRP54m monomer/dimer ratio using gel filtration . The results were consistent with a change in the shape of the signal peptide binding groove upon binding of SRP54 to SRP RNA . We propose that the SRP RNA and a small region centered at a bulky nonpolar amino acid residue at position 329 of protein SRP54 play a critical role in the SRP-dependent binding and release of signal peptides. Physiol Res, 2002, 51(3), 291 - 8 Comparative study of several lymphocyte functions in two strains of mice with different models of endotoxic shock; Victor VM et al.; Previously, the changes in phagocyte functions such as adherence, chemotaxis or TNFalpha production were found to be associated with oxidative stress in endotoxin-induced septic shock . However, in this type of oxidative stress the lymphocyte involvement has rarely been studied . In the present report, we analyzed the above functions in peritoneal lymphocytes from male and female BALB/c mice with a lethal endotoxic shock caused by intraperitoneal injection of E . coli lipopolysaccharide (LPS) (100 mg/kg), male and female Swiss mice with lethal endotoxic shock caused by intraperitoneal injection of LPS (150 and 250 mg/kg, respectively) or non-lethal endotoxic shock (100 mg/kg) . In peritoneal lymphocytes obtained at 0, 2, 4, 12 or 24 h after LPS injection, the first two functions of these cells in the immune response, i.e . adherence to tissues and directed migration (chemotaxis), were studied . At 0, 0.5, 1, 1.5, 2, 4, 12 and 24 h after LPS injection, TNFalpha released by lymphocytes was also analyzed . The results show that endotoxic shock increases the adherence and TNFalpha release, and decreases the chemotaxis of peritoneal lymphocytes . These changes were more significant in mice with lethal than with non-lethal endotoxic shock, a fact that confirms the important role of lymphocytes during endotoxic shock. Biochem Cell Biol, 2002, 80(4), 435 - 43 Differential effects of a molybdopterin synthase sulfurylase (moeB) mutation on Escherichia coli molybdoenzyme maturation; Sambasivarao D et al.; We have generated a chromosomal mutant of moeB (moeBA228T) that demonstrates limited molybdenum cofactor (molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD)) availability in Escherichia coli and have characterized its effect on the maturation and physiological function of two well-characterized respiratory molybdoenzymes: the membrane-bound dimethylsulfoxide (DMSO) reductase (DmsABC) and the membrane-bound nitrate reductase A (NarGHI) . In the moeBA228T mutant strain, E . coli F36, anaerobic respiratory growth is possible on nitrate but not on DMSO, indicating that cofactor insertion occurs into NarGHI but not into DmsABC . Fluorescence analyses of cofactor availability indicate little detectable cofactor in the moeBA228T mutant compared with the wild-type, suggesting that NarGHI is able to scavenge limiting cofactor, whereas DmsABC is not . MoeB functions to sulfurylate MoaD, and in the structure of the MoeB-MoaD complex, Ala-228 is located in the interface region between the two proteins . This suggests that the moeBA228T mutation disrupts the interaction between MoeB and MoaD . In the case of DmsABC, despite the absence of cofactor, the twin-arginine signal sequence of DmsA is cleaved in the moeBA228T mutant, indicating that maturation of the holoenzyme is not cofactor-insertion dependent. Res Microbiol, 2002 Jul-Aug, 153(6), 379 - 83 Destabilized green fluorescent protein for monitoring transient changes in mycobacterial gene expression; Triccas JA et al.; The green fluorescent protein (GFP) is a useful reporter for the study of gene expression and protein localisation within living cells . The stability of GFP permits its intracellular accumulation and detection, but renders it less useful for assessing transient changes in gene expression . We have developed a destabilized form of GFP for monitoring gene expression in mycobacteria . By fusing to the C-terminal end of GFP an 11 amino acid peptide encoded by the E . coli ssrA gene, we have developed a form of GFP that exhibits gradual, time-dependent degradation within the fast-growing species Mycobacterium smegmatis . This unstable variant of GFP detected transient changes in the activity of the stress-induced Mycobacterium tuberculosis sigE promoter; by contrast, unmodified GFP only detected a delayed 'switch-on' of this promoter upon exposure to acid stress . Both forms of the protein displayed equivalent stability in the slow-growing species Mycobacterium bovis bacille Calmette-Guerin (BCG), suggesting differing recognition of the ssrA-encoded peptides in slow- and fast-growing mycobacteria . This system will facilitate studies exploring dynamic changes in mycobacterial gene expression. Bioseparation, 2001, 10(4-5), 211 - 20 Studies on the retention of plasmid DNA and Escherichia coli nucleic acids by hydrophobic interaction chromatography; Diogo MM et al.; This work presents studies on the interactions of supercoiled plasmid DNA and Escherichia coli genomic DNA (gDNA) and RNA, with an hydrophobic interaction chromatography (HIC) gel, obtained by derivatisation of Sepharose CL-6B with 1,4-butanediol diglycidyl ether . Nucleic acids purified from E . coli were injected separately in the above HIC column and eluted with 1.5 M (NH4)2SO4 in the buffer . The column was able to separate single-stranded from double-stranded nucleic acids . RNA and denatured gDNA were retarded in a different way due to the interactions of the exposed hydrophobic bases with the ligands . Supercoiled plasmid DNA, on the contrary, eluted in the flowthrough. Bioseparation, 2001, 10(4-5), 203 - 10 Acoustic field assisted demixing of aqueous two-phase polymer systems; Srinivas ND et al.; Acoustic field assisted demixing was employed to decrease the demixing time in polymer-polymer (polyethylene glycol-maltodextrin) two-phase system . Application of acoustic field has decreased the demixing time in these systems up to 2-fold . Ultrasonication has induced mild circulation currents in the phase dispersion, which has enhanced the rate of droplet coalescence, eventually resulting in decreased demixing time . In polymer-polymer systems, phase demixing was found to depend greatly on which of the phases is continuous and viscosity of the continuous phase was observed to have a strong influence on the movement of the droplets and hence the phase demixing . Addition of NaCl increased the demixing time and presence of E . coli cells did not seem to have any influence on phase demixing. No Shinkei Geka, 2002 Sep, 30(9), 967 - 71 {Congenital dermal sinus tract of recurrent pyrexia: case report}; Minegishi K et al.; The authors present a case of congenital dermal sinus tract with epidermoid tumor . This 1-year-old boy was referred to the pediatric service of another hospital with recurrent pyrexia of unknown origin in April, 1999 . The pediatrician found two dimples, pigmentation, and coarse hairs on the midline in his sacral region . Computerized tomography (CT) scans revealed a spina bifida below the S1 level . Magnetic resonance (MR) imaging revealed a dermal sinus tract in the cranial direction to a cystic tumor at L2-4 levels . He was transferred to our hospital, and the tract and tumor were totally removed in June, 1999 . The histological findings and Escherichia coli in the smear culture of the tumor contents identified it as an infected congenital dermal sinus tract with epidermoid tumor . The patient received antibiotics for two weeks after surgery and there was no clinical or radiographic recurrence of either infection or tumor . The authors propose early diagnosis and radical treatment, because infected congenital dermal sinus tract often leads to a bad neurological prognosis. J Infect Dis, 2002 Oct 1, 186(7), 1039 - 42 Epub 2002 Sep 13. Are quinolone-resistant uropathogenic Escherichia coli less virulent? Vila J, Simon K, Ruiz J, Horcajada JP, Velasco M, Barranco M, Moreno A, Mensa J. The prevalence of hemolysin, type 1 fimbriae, P fimbriae, cytotoxic necrotizing factor-1 (CNF-1), aerobactin, and autotransporter toxin (sat) was analyzed by polymerase chain reaction and phenotypic assays of 42 epidemiologically unrelated Escherichia coli strains causing acute pyelonephritis in women (21 nalidixic acid-susceptible and 21 nalidixic acid-resistant strains) and 58 E . coli strains causing cystitis in women (29 nalidixic acid-susceptible and 29 nalidixic acid-resistant strains) . Hemolysin and CNF-1 were less prevalent (P<.05) in nalidixic acid-resistant than in nalidixic acid-susceptible E . coli strains from patients with either pyelonephritis (14.3% vs . 52.4%) or cystitis (0% vs . 31.0%) . Among E . coli strains causing cystitis, type 1 fimbriae expression was less prevalent (P<.05) in the nalidixic acid-resistant group (55.2%) than in the nalidixic acid-susceptible group (86.2%) . None of the nalidixic acid-resistant and 20.7% of the nalidixic acid-susceptible strains causing cystitis showed the proteolytic toxin Sat (P<.05) . These results suggest that resistance to quinolones may be associated with a decrease in the presence or the expression of some virulence factors in uropathogenic E . coli. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(4), 380 - 388 The Fusion Expression of HBV preS Epitopes and the Core Antigen; Li YC et al.; The DNA fragments encoding the preS epitopes of HBV surface antigen were fused to the HBc gene and expressed in E . coli under the control of the tac promoter . The products were analyzed by ELISA and Western blotting, which confirmed that the hybrid proteins were expressed as expected . Analysis by electron microscopy and CsCl density gradient ultracentrifugation revealed that all fusion proteins were able to form particles, only with a slightly lower density than the native multimeric HBc . Partially purified fusion particles were then used as immunogen to Balb/c mice and high titer antibody against the preS1(21-47) epitope was observed, which demonstrated that the immunogenictiy of preS1 (21-47) could be greatly improved when fused in the el loop in HBc protein. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(5), 531 - 539 A T7 Promoter-based Versatile Expression Vector System; Chen CZ et al.; A family of T7 promoter-based versatile expression plamides for E . coli Were constructed . These vectors were featured strong T7 promoter, translational start, stop elements, a multiple cloning site with eight unique restriction sites in all three reading frames and f1 phage origin which allows packaging of single stranded plasmid, mutagensis and gene sequencing without subcloning steps . With these vectors recombinant proteins can be expressed in mature or short fusion forms . Many heterolegous genes have been highly expressed using these vectors, most of them were expressed in soluble and active forms. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(5), 523 - 530 The Construction of T7 Promoter-based His(6)-tagging Vectors and the Single-step Purification of the Expression products; Chen CZ et al.; T7 promoter-based fusion expression vectors have been constructed that directed the synthesis of heterologous proteins in E . coli as fusions with a stretch of six consecutive histidine residues His(6) at N Terminus . The vectors were also featured with strong T7 promoter, terminator, translational start, multiple cloning sites with seven unique restriction sites in all three reading frames and the f1 phage origin which allows the packaging of single-stranded plasmid, mutagenesis and DNA sequencing without subcloning steps . In most cases, expressed fusion proteins are soluble . The His(6) tag allows the fusion proteins purified in one step by immobilized metal (Ni(2+)) chelation affinity chromatography in the denatured or native state . As an example of the general utility of these expression vectors, the His(6)-fused catalytic subunits of mouse cAMP-dependent protein kinase were expressed with high activity by using these vectors and could be purified to homogeneity in one step. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(5), 479 - 484 The Construction of GPRP-scu-PA(144-411) cDNA and the Study of Its Properties; Dong C et al.; A synthetic nucleic acids fragment encoding the GPRP peptides was linked with the scu-PA (144-411) cDNA at its 5' end, and was subsequently inserted into the expression vector pKK233-2 and expressed in E . coli with a level of 5% of the total bacterial proteins . The expressed product of GPRP-scu-PA (144-411) cDNA was purified by affinity chromatography . Its < italic>K(m) was 40 &mgr;M, with the clotlysis rate 2-3 times of that of LUK, and the fibrin affinity 6 times higher than that of LUK . At the same time it had a low affinity for fibrinogen . These results showed that the GPRP peptide fragment was able to improve the fibrin-specific affinity of urokinase. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(5), 458 - 463 A Structure-function Analysis of the Human Ciliary Neurotrophic Factor; He C et al.; The ciliary neurotrophic factor (CNTF) plays a very important role in the development and regeneration of the nervous system . In this study, the prediction of secondary structure and the hydrophobicity analysis of human CNTF were performed according to the amino acid sequence deduced from the nucleotide sequence of the cDNA . Based on the results of the prediction of structure, the human CNTF gene was modified by insertion and deletion mutagenesis . The various mutants were all highly expressed in E . coli . The recombinant proteins were purified from bacterial via DEAE A-50 and Sephacryl S-200 chromatography, and their survival-promoting activities were determined by using cultures of the dorsal root ganglion neurons of embryonic chick . The results showed that the alpha-helixes in CNTF were critical for the biological activity and the flexible C-terminus of human CNTF was not essential . Our data also indicated that the middle and the tail part of the D-helix might play crucial roles in the biological functions of CNTF. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(6), 647 - 652 Cloning of the LAC4 Promoter from K . lactis and Studies on the Function of its UASII; Bao WG et al.; The -661 - +21 bp region of the LAC4 gene form K . lactis CBS141 was obtained by PCR technique and fused with the lacZ gene from E . coli to construct and expression vector YFD 114 . It was then transformed into K . lactis Y167 . The function of the cloned LAC4 promoter was tested by induction with galactose, lactose, sorbitol or IPTG . The results revealed that galactose had higher inducing effect than lactose while sorbitol and IPTG had no inducing effect . The insertion of chemically synthesized LAC4 UASII at just the upstream of the cloned LAC4 promoter could increase its basic and induced expression level . The extents of increase were different with the orientation and the copy number of the inserted UASII. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(6), 606 - 615 Cloning and Expression of the cDNA Coding for Rat Peptidylglycine alpha-Amidating Monooxygenase; Jiang ZH et al.; The dDNA of genes encoding rat peptidylglycine alpha-aminating monooxygenase(rPAM) were cloned and their expression in E . coli studied . Three DNA fragments were isolated from the rat brain cDNA library using the methods of plaque hybridization and PCR . DNA sequencing showed that they contained the total coding sequence for rPAM-2 . By using the sited-directed mutation and PCR recombination methods, we obtained intact genes coding for the rPHM domain, the rPAL domain and rPAM, respectively . Different plasmids of these genes controlled under T(7) or P(L) promoter were constructed and transformed into E . coli . The high-level expression of rPAM-N260 in E . coli was first observed and its antiserum was prepared for the immunoassay of natural or recombinant PAMs . By the analysis with SDS-PAGE and Western blot, the products of rPHM and rPAM in E . coli were detected, and the amount of rPHM reached over 10% of the total bacterial proteins . It was found that low temperature and copper ion obviously increased the stability and solubility of the rPHM expressed in E . coli. Plant Physiol, 1994 Sep, 106(1), 203 - 209 Overproduction in Escherichia coli and Characterization of a Soybean Ferric Leghemoglobin Reductase; Ji L et al.; We previously cloned and sequenced a cDNA encoding soybean ferric leghemoglobin reductase (FLbR), an enzyme postulated to play an important role in maintaining leghemoglobin in a functional ferrous state in nitrogen-fixing root nodules . This cDNA was sub-cloned into an expression plasmid, pTrcHis C, and overexpressed in Escherichia coli . The recombinant FLbR protein, which was purified by two steps of column chromatography, was catalytically active and fully functional . The recombinant FLbR cross-reacted with antisera raised against native FLbR purified from soybean root nodules . The recombinant FLbR, the native FLbR purified from soybean (Glycine max L.) root nodules, and dihydrolipoamide dehydrogenases from pig heart and yeast had similar but not identical ultraviolet-visible absorption and fluorescence spectra, cofactor binding, and kinetic properties . FLbR shared common structural features in the active site and prosthetic group binding sites with other pyridine nucleotide-disulfide oxidoreductases such as dihydrolipoamide dehydrogenases, but displayed different microenvironments for the prosthetic groups. Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12721 - 6 Epub 2002 Sep 13. Sequence-specific DNA binding by the vnd/NK-2 homeodomain of Drosophila; Wang LH et al.; The ventral nervous system defective (vnd)/NK-2 homeodomain and some flanking amino acid residues were expressed in Escherichia coli, purified to homogeneity, and the protein was covalently coupled to Sepharose . Oligodeoxynucleotides that contained 16-bp random sequences were purified by vnd/NK-2 affinity column chromatography, cloned, and sequenced . The consensus nucleotide sequence of the vnd/NK-2 homeodomain binding site was shown to be T(T/C)AAGTG(G/C) . The apparent equilibrium dissociation constant (K(D)) of the vnd/NK-2 homeodomain for the consensus sequence is 1.9 x 10(-10) M . In addition, results of competition between oligodeoxynucleotides for binding to the vnd/NK-2 homeodomain and determination of the apparent K(D) values of oligodeoxynucleotides that differ from the consensus sequence by only a single base pair demonstrate that the four central nucleotides, AAGT, in this sequence play a major role in determining the affinity of binding. Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12669 - 74 Epub 2002 Sep 13. Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer; Sourjik V et al.; In Escherichia coli chemotaxis, signaling depends on modulation of the level of phosphorylation of CheY, a small protein that couples receptors and flagellar motors . Working in vivo, we used fluorescence resonance energy transfer (FRET) to measure the interaction of CheY approximately P with its target, FliM . Binding of CheY approximately P to FliM was found to be much less cooperative than motor switching; however, under the conditions of our experiment, most of the FliM appeared to be in the cytoplasm . We studied signal processing times in the chemotaxis pathway by measuring the changes in CheY approximately P binding to FliM on flash release of caged chemoeffectors . Following sudden addition of attractant, the amount of CheY approximately P bound to FliM decayed exponentially with a rate constant of about 2 s(-1) . Following sudden addition of repellent, FliM occupancy increased with a rate constant of about 20 s(-1) . Using these data, we were able to construct a simple model for the chemotactic pathway and to estimate values of rate constants for several key reactions. Plant Physiol, 1993 Apr, 101(4), 1181 - 1187 Porphyrin Accumulation and Export by Isolated Barley (Hordeum vulgare) Plastids (Effect of Diphenyl Ether Herbicides); Jacobs JM et al.; We have investigated the formation of porphyrin intermediates by isolated barley (Hordeum vulgare) plastids incubated for 40 min with the porphyrin precursor 5-aminolevulinate and in the presence and absence of a diphenylether herbicide that blocks protoporphyrinogen oxidase, the enzyme in chlorophyll and heme synthesis that oxidizes protoporphyrinogen IX to protoporphyrin IX . In the absence of herbicide, about 50% of the protoporphyrin IX formed was found in the extraplastidic medium, which was separated from intact plastids by centrifugation at the end of the incubation period . In contrast, uroporphyrinogen, an earlier intermediate, and magnesium protoporphyrin IX, a later intermediate, were located mainly within the plastid . When the incubation was carried out in the presence of a herbicide that inhibits protoporphyrinogen oxidase, protoporphyrin IX formation by the plastids was completely abolished, but large amounts of protoporphyrinogen accumulated in the extraplastidic medium . To detect extraplastidic protoporphyrinogen, it was necessary to first oxidize it to protoporphyrin IX with the use of a herbicide-resistant protoporphyrinogen oxidase enzyme present in Escherichia coli membranes . Protoporphyrinogen is not detected by some commonly used methods for porphyrin analysis unless it is first oxidized to protoporphyrin IX . Protoporphyrin IX and protoporphyrinogen found outside the plastid did not arise from plastid lysis, because the percentage of plastid lysis, measured with a stromal marker enzyme, was far less than the percentage of these porphyrins in the extraplastidic fraction . These findings suggest that of the tetrapyrrolic intermediates synthesized by the plastids, protoporphyrinogen and protoporphyrin IX, are the most likely to be exported from the plastid to the cytoplasm . These results help explain the extraplastidic accumulation of protoporphyrin IX in plants treated with photobleaching herbicides . In addition, these findings suggest that plastids may export protoporphyrinogen or protoporphyrin IX for mitochondrial heme synthesis. Acta Pharmacol Sin, 2002 Sep, 23(9), 782 - 6 Cloning and high level nonfusion expression of recombinant human basic fibroblast growth factor in Escherichia coli; Chen XJ et al.; AIM: To obtain high-level expression of nonfusion recombinant human basic fibroblast growth factor (rhbFGF) . METHODS: hbFGF cDNA was prepared from the total RNA of embryonic brain tissue . As a template, the obtained gene was used to clone nonfusion rhbFGF . New primers were employed to alter the translation initiation region (TIR) and reduce the G+C content through nucleotide change . Using pET-3C as vector, the cloned rhbFGF was expressed in BL21 (DE3) . RESULTS: rhbFGF was expressed in E coli up to 30 % of the total cellular protein . Cation exchange and heparin affinity chromatography were employed to purify the target protein from the supernatant of bacteria lysate . The bioactivity of the purified rhbFGF was identical with the standard bFGF . CONCLUSION: Modification of TIR is an effective means to increase nonfusion expression rate of recombinant proteins, such as rhbFGF, in E coli. J Endotoxin Res, 2002, 8(4), 295 - 305 A novel strategy for the synthesis of neoglycoconjugates from deacylated deep rough lipopolysaccharides; Muller-Loennies S et al.; We report a novel strategy for the preparation of neoglycoconjugates of oligosaccharides which are obtained after complete deacylation of bacterial deep rough lipopolysaccharides (LPS) isolated from recombinant Escherichia coli bacteria synthesizing a Kdo di-{alpha-Kdo-(2-->4)-alpha-Kdo-(2-->} and a Kdo trisaccharide {alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->} of Re-type and chlamydial LPS, respectively . Unlike acylated LPS, such oligosaccharides can be obtained in pure form and thus lead to well-defined neoglycoconjugates . Cleavage of the 1-phosphate of the lipid A moiety by alkaline phosphatase treatment leads to a free reducing glucosamine which can be further reacted with allylamine . After reductive amination, spacer elongation of the allyl group with cysteamine and activation with thiophosgene, the ligands were reacted with BSA . We have compared the immunological reactivity of such defined neoglycoconjugates obtained from natural sources with those obtained by chemical synthesis and report that such neoglycoconjugates are immunogenic and well suited as antigens for the study of epitope specificities of monoclonal antibodies . In addition, we have compared these conjugates with those in which ligands were coupled by glutardialdehyde to BSA . Our approach proved to be superior since the latter led upon immunization of mice to a relatively high percentage of antibodies that reacted with glutardialdehyde derivatized BSA without the carbohydrate ligand . This was not the case for cysteamine-spacered ligands coupled via their isothiocyanate-derivatives. J Endotoxin Res, 2002, 8(4), 285 - 93 The central role of thromboxane and platelet activating factor receptors in ex vivo regulation of endotoxin-induced monocyte tissue factor activity in human whole blood; Eilertsen KE et al.; Expression of tissue factor (TF) by activated monocytes may initiate thrombotic episodes associated with diseases, such as thrombosis and atherosclerosis . In this study, steps in the regulatory pathways of lipopolysaccharide (LPS)-induced monocyte TF activity and released TNF-alpha in human whole blood were probed for using an array of inhibitors, comprising specific inhibitors of cytosolic phospholipase A(2) (PLA(2)) (AACOCF(3)), secretory PLA(2) (SB-203347), protein kinase (PK) (staurosporine), PKC (GF-109203; BIM), and serine protease (Pefabloc SC), antagonists of thromboxane prostanoid (TP) receptor (R) (SQ-29548), platelet activating factor (PAF) R (BN-52021), leukotriene B(4) R (SC-41930), serotonin R (cyproheptadine), fibronectin/fibrinogen R (RGDS), and finally, creatine phosphate/creatine phosphokinase (CP/CPK) which removes ADP . Whereas when added alone neither of these agents significantly inhibited LPS-induced TF or TNF-alpha, when presented as a reference cocktail comprising all the agents, TF activity and TNF-alpha were reduced by 77% and 49%, respectively . By subsequently testing a series of incomplete inhibitory cocktails equal to the reference except for deleted single agents or combinations of two or three active agents, the inhibitory effect of the reference cocktail could be shown to depend on the presence of the protease inhibitor and the thromboxane A(2) and PAF antagonists. Eur J Biochem, 2002 Sep, 269(18), 4617 - 24 NblA from Anabaena sp . PCC 7120 is a mostly alpha-helical protein undergoing reversible trimerization in solution; Strauss H et al.; The nblA family of genes encodes for small proteins necessary for the ordered degradation of phycobilisomes under certain stress conditions, a process known as chlorosis . Genes homologous to nblA seem to occur in all phycobilisome-containing organisms . However, to date, no molecular mechanism is known for the action of NblA, nor have the gene products been characterized to understand the physical properties of the molecule and thus help elucidate the mechanism on a structural basis . In this study we report on the first characterization of an NblA-homologous gene product . The chromosomal gene from the cyanobacterium Anabaena sp . PCC 7120 was cloned, heterologously expressed in Escherichia coli and purified to apparent homogeneity . This allowed the protein to be characterized by analytical ultracentrifugation and CD spectroscopy . These experiments show that the NblA protein has a mostly alpha-helical structure, undergoing an association reaction of folded monomers to form trimers in solution . No dimers are detectable. Eur J Biochem, 2002 Sep, 269(18), 4516 - 23 Chaperone and antichaperone activities of trigger factor; Huang GC et al.; Reduced denatured lysozyme tends to aggregate at neutral pH and competition between productive folding and aggregation substantially reduces the efficiency of refolding . Trigger factor, a folding catalyst and chaperone can, depending on the concentration of trigger factor and the solution conditions, cause either a substantial increase (chaperone activity) or a substantial decrease (antichaperone activity) in the recovery of native lysozyme as compared with spontaneous refolding . When trigger factor is working as a chaperone, the reactivation rates of lysozyme are decelerated and aggregation decreases with increasing trigger factor concentrations . Under conditions where antichaperone activity of trigger factor dominates, the reactivation rates of lysozyme are accelerated and aggregation is increased . Trigger factor and lysozyme were both released from the aggregates on re-solubilization with urea indicating that trigger factor participates directly in aggregate formation and is incorporated into the aggregates . The apparently dual effect of trigger factor toward refolding of lysozyme is a consequence of the peptide binding ability and may be important in regulation of protein biosynthesis. Eur J Biochem, 2002 Sep, 269(18), 4505 - 15 Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I-domain III interactions in proton-translocating Escherichia coli transhydrogenase; Johansson C et al.; Membrane-bound transhydrogenases are conformationally driven proton-pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction) . This reaction is stimulated by an electrochemical proton gradient, Delta p, presumably through an increased release of NADPH . The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively . Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III . With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine-free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues beta A398, beta S404, beta I406, beta G408, beta M409 and beta V411 in loop D, and residue beta Y431 in loop E were selected . In addition, the previously made mutants betaD392C and betaT393C in loop D, and beta G430C and beta A432C in loop E, were included . All loop D and E mutants, especially beta I406C and beta G430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild-type enzyme . Determination of values indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E . In contrast, the cysteine-free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates . Most domain III mutants also showed a decreased affinity for domain I . The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III. Eur J Biochem, 2002 Sep, 269(18), 4468 - 75 Molecular cloning and functional expression of a gene encoding an antiarrhythmia peptide derived from the scorpion toxin; Peng F et al.; From a cDNA library of Chinese scorpion Buthus martensii Karsch, full-length cDNAs of 351 nucleotides encoding precursors (named BmKIM) that contain signal peptides of 21 amino acid residues, a mature toxin of 61 residues with four disulfide bridges, and an extra Gly-Lys-Lys tail, were isolated . The genomic sequence of BmKIM was cloned and sequenced; it consisted of two exons disrupted by an intron of 1622 bp, the largest known in scorpion toxin genomes, inserted in the region encoding the signal peptide . The cDNA was expressed in Escherichia coli . The recombinant BmKIM was toxic to both mammal and insects . This is the first report that a toxin with such high sequence homology with an insect-specific depressant toxin group exhibits toxicity to mammals . Using whole cell patch-clamp recording, it was discovered that the recombinant BmKIM inhibited the sodium current in rat dorsal root ganglion neurons and ventricular myocytes and protected against aconitine- induced cardiac arrhythmia. Eur J Biochem, 2002 Sep, 269(18), 4446 - 57 Isoprenoid biosynthesis via the methylerythritol phosphate pathway . Mechanistic investigations of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase; Hoeffler JF et al.; The 1-deoxyxylulose 5-phosphate reductoisomerase (DXR, EC 1.1.1.267) catalyzes the conversion of 1-deoxy-d-xylulose 5-phosphate (DXP) into 2-C-methyl-d-erythritol 4-phosphate (MEP) . This transformation is a two-step process involving a rearrangement of DXP into the putative intermediate 2-C-methyl-d-erythrose 4-phosphate followed by a NADPH-dependent reduction of the latter aldehyde . By using {1-(13)C}DXP as a substrate, the rearrangement of DXP into {5-(13)C}2-C-methyl-d-erythrose 4-phosphate was shown to be NADPH dependent, although it does not involve areduction step . The putative aldehyde intermediate, obtained by chemical synthesis, was converted into MEP by the DXR in the presence of NADPH and into DXP in the presence of NADP(+), indicating the reversibility of the reaction catalyzed by the DXR . This reversibility was confirmed by the conversion of MEP into DXP in the presence of NADP(+) . The equilibrium was, however, largely displaced in favour of the formation of MEP . The reduction step required the presence of a divalent cation such as Mg(2+) or Mn(2+). Biopolymers, 2002 Nov 5, 65(3), 190 - 201 Thermal stabilization of the DNA duplex by adducts of aflatoxin B1; Giri I et al.; The trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B(1) cationic guanine N7 adduct of aflatoxin B(1) thermally stabilizes the DNA duplex, as reflected in increased T(m) values upon adduction . The magnitude of the increased T(m) value is characteristically 2-3 degrees C . The major rotamer of the neutral guanine N7 adduct trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B(1) (the FAPY major adduct) exhibits a 15 degrees C increase in T(m) in 5'-d(CTAT(FAPY)GATTCA)-3'-5'-d(TGAATCATAG)-3' . Site-specific mutagenesis experiments reveal the FAPY major adduct induces G-->T mutations in Escherichia coli at a frequency six times higher than that of the cationic adduct (Smela, M . E.; Hamm, M . L.; Henderson, P . T.; Harris, C . M.; Harris, T . M.; Essigmann, J . M . Proc Natl Acad Sci USA, 99, 6655-6660) . Thus, the FAPY major lesion may account substantially for the genotoxicity of AFB(1) . Structural studies for cationic and FAPY adducts of aflatoxin B(1) suggest both adducts intercalate above the 5'-face of the modified deoxyguanosine and that in each instance the aflatoxin moiety spans the DNA helix . Intercalation of the aflatoxin moiety, accompanied by favorable stacking with the neighboring base pairs, is thought to account for the increased thermal stability of the aflatoxin cationic guanine N7 and the FAPY major adducts . However, the structural basis for the large increase in thermal stability of the FAPY major adduct in comparison to the cationic guanine N7 adduct of aflatoxin B(1) is not well understood . In light of the site-specific mutagenesis studies, it is of considerable interest . For both adducts, the intercalation structures are similar, although improved stacking with neighboring base pairs is observed for the FAPY major adduct . In addition, the presence of the formamido group in the aflatoxin B(1) FAPY major adduct may enhance duplex stability, perhaps via intrastrand sequence-specific hydrogen bonding interactions within the duplex . Science, 2002 Sep 13, 297(5588), 1864 - 7 Instruction of translating ribosome by nascent peptide; Gong F et al.; Expression of the tryptophanase operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination . An induction site activated by l-tryptophan is created in the translating ribosome during synthesis of TnaC, the 24-residue leader peptide . Replacing the tnaC stop codon with a tryptophan codon allows tryptophan-charged tryptophan transfer RNA to substitute for tryptophan as inducer . This suggests that the ribosomal A site occupied by the tryptophanyl moiety of the charged transfer RNA is the site of induction . The location of tryptophan-12 of nascent TnaC in the peptide exit tunnel was crucial for induction . These results show that a nascent peptide sequence can influence translation continuation and termination within a translating ribosome. Plant Physiol, 1995 Jul, 108(3), 1127 - 1132 Only the Mature Form of the Plastidic Chorismate Synthase Is Enzymatically Active; Henstrand JM et al.; Coding regions of a cDNA for precursor and mature chorismate synthase (CS), a plastidic enzyme, from Corydalis sempervirens were expressed in Escherichia coli as translational fusions to glutathione-S-tran