Escherichia coli

Plant Mol Biol, 2004 May, 55(1), 97 - 107
Maize recombinant non-C4 NADP-malic enzyme: a novel dimeric malic enzyme with high specific activity; Saigo M et al.; Among the different isoforms of NADP-malic enzyme (NADP-ME) involved in a wide range of metabolic pathways in plants, the NADP-ME that participates in C(4)-photosynthesis is the most studied . In the present work, the expression in E . coli of a cDNA encoding for a maize non-photosynthetic NADP-ME is presented . The recombinant NADP-ME thus obtained presents kinetic and structural properties different from the enzyme previously purified from etiolated leaves and roots . Moreover, the recombinant non-photosynthetic NADP-ME presents very high intrinsic NADP-ME activity, which is unexpected for a non-C( 4) NADP-ME . Using antibodies against this recombinant enzyme, an immunoreactive band of 66 kDa is detected in different maize tissues indicating that the 66 kDa-NADP-ME is in fact a protein expressed in vivo . The recombinant NADP-ME assembles as a dimer, although the results obtained indicate that a higher molecular mass oligomeric state of the enzyme is found in maize roots in vivo . In this way, maize presents at least three NADP-ME isoforms: a 72 kDa constitutive form (previously characterized); the novel non-photosynthetic 66 kDa isoform characterized in this work (which is the product of the ZmChlMe2 gene and the likely precursor to the evolution of the photosynthetic C(4) NADP-ME) and the 62 kDa isoform (implicated in C(4) photosynthesis) . The contribution of the present work anticipates further studies concerning the equilibrium between the oligomeric states of the NADP-ME isoforms and the evolution towards the C(4) isoenzyme in maize.

Nucleic Acids Res, 2004 Dec 16, 32(22), 6643 - 9 Print 2004.
An extended transcriptional regulatory network of Escherichia coli and analysis of its hierarchical structure and network motifs; Ma HW et al.; Recent studies of genome-wide transcriptional regulatory network (TRN) revealed several intriguing structural and dynamic features of gene expression at a system level . Unfortunately, the network under study is often far from complete . A critical question is thus how much the network is incomplete and to what extent this would affect the results of analysis . Here we compare the Escherichia coli TRN built by Shen-Orr et al . (Nature Genet., 31, 64-68) with two TRNs reconstructed from RegulonDB and Ecocyc respectively and present an extended E.coli TRN by integrating information from these databases and literature . The scale of the extended TRN is about twice as large as the previous ones . The new network preserves the multi-layer hierarchical structure which we recently reported but has more layers . More global regulators are inferred . While the feed forward loop (FFL) is confirmed to be highly representative in the network, the distribution of the different types of FFLs is different from that based on the incomplete network . In contrast to the notion of motif aggregation and formation of homologous motif clusters, we found that most FFLs interact and form a giant motif cluster . Furthermore, we show that only a small portion of the genes is solely regulated by only one FFL . Many genes are regulated by two or more interacting FFLs or other more complicated network motifs together with transcriptional factors not belonging to any network motifs, thereby forming complex regulatory circuits . Overall, the extended TRN represents a more solid basis for structural and functional analysis of genome-wide gene regulation in E.coli.

J Gen Virol, 2005 Jan, 86(Pt 1), 225 - 9
Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein; Isogai M et al.; The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed . MP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin-UTP-labelled RNA probe and gel-retardation analysis . The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA) . When analysed for NaCl dependence of the RNA-binding activity, the majority of the MP could bind ssRNA even in binding buffer with 1 M NaCl . Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity . MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis . Amino acid residues 82-126 and 127-287 potentially contain two independently active, single-stranded nucleic acid-binding domains.

Science, 2004 Dec 17, 306(5704), 2101 - 5
Phosphorylation of proteins by inositol pyrophosphates; Saiardi A et al.; The inositol pyrophosphates IP7 and IP8 contain highly energetic pyrophosphate bonds . Although implicated in various biologic functions, their molecular sites of action have not been clarified . Using radiolabeled IP7, we detected phosphorylation of multiple eukaryotic proteins . We also observed phosphorylation of endogenous proteins by endogenous IP7 in yeast . Phosphorylation by IP7 is nonenzymatic and may represent a novel intracellular signaling mechanism.

Cancer Res, 2004 Dec 15, 64(24), 8876 - 81
Myh deficiency enhances intestinal tumorigenesis in multiple intestinal neoplasia (ApcMin/+) mice; Sieber OM et al.; Monoallelic APC and biallelic MYH (homolog of Escherichia coli mutY) germ-line mutations are independently associated with a strong predisposition to colorectal adenomas and carcinoma in humans . Whereas mice heterozygous for mutant Apc develop intestinal tumors, mice homozygous for mutant Myh do not show increased tumor susceptibility . We analyzed the phenotype of Apc(Min/+)/Myh(-/-) mice and found that they developed significantly more adenomas in the small intestine than did Apc(Min/+)/Myh(+/+) or Apc(Min/+)/Myh(+/-) mice (median 231 versus 151 versus 152) . In the large bowel, Apc(Min/+)/Myh(-/-) mice showed significant increases in the number of aberrant crypt foci . In addition, Apc(Min/+)/Myh(-/-) mice developed an increased number of mammary tumors . Molecular analyses suggested that at least 19% of intestinal tumors from Apc(Min/+)/Myh(-/-) mice had acquired intragenic Apc mutations rather than allelic loss . Consistent with a defect in base excision repair, three intragenic Apc mutations in polyps without allelic loss from Apc(Min/+)/Myh(-/-) mice were shown to be G:C to T:A transversions which resulted in termination codons; no such mutations were found in polyps from Apc(Min/+)/Myh(+/+) or Apc(Min/+)/Myh(+/-) mice . Tumors from Apc(Min/+)/Myh(+/-) mice harbored neither somatic mutations nor allelic loss at Myh . Thus, homozygous, but not heterozygous, Myh deficiency enhanced intestinal tumorigenesis in Apc(Min/+) mice . The excess small-bowel adenomas in Apc(Min/+)/Myh(-/-) mice, therefore, appear to be a model of MYH-associated polyposis in humans.

J Immunol Methods, 2004 Nov, 294(1-2), 81 - 8
Binding of a monoclonal antibody positively correlates with bioactivity of the F4 fimbrial adhesin FaeG associated with post-weaning diarrhoea in piglets; Verdonck F et al.; Piglets are susceptible to F4 (K88)+ enterotoxigenic Escherichia coli (ETEC)-induced neonatal and post-weaning diarrhoea . The F4 fimbriae are composed of some minor subunits and the major subunit FaeG that also constitutes the adhesin . Parenteral vaccination of sows with an F4-containing vaccine protects the suckling piglets against neonatal F4+ ETEC-induced diarrhoea, but no commercial (mucosal) vaccine exists against F4+ ETEC-induced weaning diarrhoea . To develop a vaccine, a bioactive F4-receptor (F4R) binding FaeG molecule is required that binds to the F4R following oral immunization and induces a FaeG-specific immune response . The present study reports the altered binding of the FaeG-specific monoclonal antibody IMM01 with bioactive versus non-bioactive F4 fimbrial adhesin FaeG . The correlation of altered IMM01 binding with altered FaeG bioactivity permits the use of an IMM01-based ELISA as a fast, specific and sensitive in vitro selection for potent F4 or (recombinant) FaeG antigen formulations, useful in an F4+ ETEC vaccine.

Vaccine, 2005 Jan 4, 23(7), 946 - 50
Improved immune responses to influenza vaccination in the elderly using an immunostimulant patch; Frech SA et al.; The elderly have greater morbidity and mortality due to influenza, and respond poorly to influenza vaccination compared to younger adults . This study was designed to determine if the adjuvant heat-labile enterotoxin from Escherichia coli (LT), administered as an immunostimulant (IS) patch on the skin with influenza vaccination, improves influenza immune responses in the elderly . Three weeks following vaccination, hemagglutination inhibition (HAI) responses in LT IS patch recipients showed improvement over those of elderly receiving vaccine alone, as demonstrated by significance or trends in fold rise {A/Panama (P = 0.004), A/New Caledonia (P = 0.09)}, seroconversion {A/New Caledonia (63% versus 40%, P = 0.01), A/Panama (54% versus 36%, P = 0.08)} and seroprotection {26%, 20% and 16% greater for the patch group for A/New Caledonia, A/Panama and B/Shandong strains, respectively} . The data suggest that an LT IS patch may further enhance influenza vaccine immune responses in the elderly.

BMC Bioinformatics . 2004 Dec 16;5(1):199 {Epub ahead of print}
Hierarchical structure and modules in the Escherichia coli transcriptional regulatory network revealed by a new top-down approach; Ma HW et al.; BACKGROUND: Cellular functions are coordinately carried out by groups of genes forming functional modules . Identifying such modules in the transcriptional regulatory network (TRN) of organisms is important for understanding the structure and function of these fundamental cellular networks and essential for the emerging modular biology . So far, the global connectivity structure of TRN has not been well studied and consequently not applied for the identification of functional modules . Moreover, network motifs such as feed forward loop are recently proposed to be basic building blocks of TRN . However, their relationship to functional modules is not clear . RESULTS: In this work we proposed a top-down approach to identify modules in the TRN of E . coli . By studying the global connectivity structure of the regulatory network, we first revealed a five-layer hierarchical structure in which all the regulatory relationships are downward . Based on this regulatory hierarchy, we developed a new method to decompose the regulatory network into functional modules and to identify global regulators governing multiple modules . As a result, 10 global regulators and 39 modules were identified and shown to have well defined functions . We then investigated the distribution and composition of the two basic network motifs (feed forward loop and bi-fan motif) in the hierarchical structure of TRN . We found that most of these network motifs include global regulators, indicating that these motifs are not basic building blocks of modules since modules should not contain global regulators . CONCLUSION: The transcriptional regulatory network of E . coli possesses a multi-layer hierarchical modular structure without feedback regulation at transcription level . This hierarchical structure builds the basis for a new and simple decomposition method which is suitable for the identification of functional modules and global regulators in the transcriptional regulatory network of E . coli . Analysis of the distribution of feed forward loops and bi-fan motifs in the hierarchical structure suggests that these network motifs are not elementary building blocks of functional modules in the transcriptional regulatory network of E . coli.

Mol Cell Biol, 2005 Jan, 25(1), 241 - 9
The core histone N-terminal tail domains negatively regulate binding of transcription factor IIIA to a nucleosome containing a 5S RNA gene via a novel mechanism; Yang Z et al.; Reconstitution of a DNA fragment containing a 5S RNA gene from Xenopus borealis into a nucleosome greatly restricts binding of the primary 5S transcription factor, TFIIIA . Consistent with transcription experiments using reconstituted templates, removal of the histone tail domains stimulates TFIIIA binding to the 5S nucleosome greater than 100-fold . However, we show that tail removal increases the probability of 5S DNA unwrapping from the core histone surface by only approximately fivefold . Moreover, using site-specific histone-to-DNA cross-linking, we show that TFIIIA binding neither induces nor requires nucleosome movement . Binding studies with COOH-terminal deletion mutants of TFIIIA and 5S nucleosomes reconstituted with native and tailless core histones indicate that the core histone tail domains play a direct role in restricting the binding of TFIIIA . Deletion of only the COOH-terminal transcription activation domain dramatically stimulates TFIIIA binding to the native nucleosome, while further C-terminal deletions or removal of the tail domains does not lead to further increases in TFIIIA binding . We conclude that the unmodified core histone tail domains directly negatively influence TFIIIA binding to the nucleosome in a manner that requires the C-terminal transcription activation domain of TFIIIA . Our data suggest an additional mechanism by which the core histone tail domains regulate the binding of trans-acting factors in chromatin.

J Bacteriol, 2005 Jan, 187(1), 388 - 91
The Mrp Na+/H+ antiporter increases the activity of the malate:quinone oxidoreductase of an Escherichia coli respiratory mutant; Swartz TH et al.; Mrp catalyzes secondary Na+/H+ antiport and was hypothesized to have an additional primary energization mode . Mrp-dependent complementation of nonfermentative growth of an Escherichia coli respiratory mutant supported this hypothesis but is shown here to be related to increased expression of host malate:quinone oxidoreductase, not to catalytic activity of Mrp.

J Bacteriol, 2005 Jan, 187(1), 358 - 65
New temperature-sensitive alleles of ftsZ in Escherichia coli; Addinall SG et al.; We isolated five new temperature-sensitive alleles of the essential cell division gene ftsZ in Escherichia coli, using P1-mediated, localized mutagenesis . The five resulting single amino acid changes (Gly109-->Ser109 for ftsZ6460, Ala129-->Thr129 for ftsZ972, Val157-->Met157 for ftsZ2066, Pro203-->Leu203 for ftsZ9124, and Ala239-->Val239 for ftsZ2863) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42 degrees C . In each case the division block is associated with loss of Z-ring formation such that fewer than 2% of cells show Z rings at 42 degrees C . The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30 degrees C and therefore cause significant defects in FtsZ polymerization, even at the permissive temperature . Neither purified FtsZ9124 nor purified FtsZ6460 exhibited polymerization when it was assayed by light scattering or electron microscopy, even in the presence of calcium or DEAE-dextran . Hence, both mutations also cause defects in FtsZ polymerization in vitro . Interestingly, FtsZ9124 has detectable GTPase activity, although the activity is significantly reduced compared to that of the wild-type FtsZ protein . We demonstrate here that unlike expression of ftsZ84, multicopy expression of the ftsZ6460, ftsZ972, and ftsZ9124 alleles does not complement the respective lethalities at the nonpermissive temperature . In addition, all five new mutant FtsZ proteins are stable at 42 degrees C . Therefore, the novel isolates carrying single ftsZ(Ts) point mutations, which are the only such strains obtained since isolation of the classical ftsZ84 mutation, offer significant opportunities for further genetic characterization of FtsZ and its role in cell division.

J Bacteriol, 2005 Jan, 187(1), 349 - 57
Ler is a negative autoregulator of the LEE1 operon in enteropathogenic Escherichia coli; Berdichevsky T et al.; Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children . Essential for colonization of the host intestine is the LEE pathogenicity island, which comprises a cluster of operons encoding a type III secretion system and related proteins . The LEE1 operon encodes Ler, which positively regulates many EPEC virulence genes in the LEE region and elsewhere in the chromosome . We found that Ler acts as a specific autorepressor of LEE1 transcription . We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro . A comparison of the Ler affinities to different DNA regions suggests that the autoregulation mechanism limits the steady-state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters . This mechanism may reflect the need of EPEC to balance maximizing the colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression . In addition, we found that the autoregulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression . Our findings point to a new negative regulatory circuit that suppresses the noise and optimizes the expression levels of ler and other LEE1 genes.

J Bacteriol, 2005 Jan, 187(1), 320 - 8
The transmembrane helix of the Escherichia coli division protein FtsI localizes to the septal ring; Wissel MC et al.; FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of about a dozen division proteins that localize to the septal ring . FtsI comprises a short amino-terminal cytoplasmic domain, a single transmembrane helix (TMH), and a large periplasmic domain that encodes the catalytic (transpeptidase) activity . We show here that a 26-amino-acid fragment of FtsI is sufficient to direct green fluorescent protein to the septal ring in cells depleted of wild-type FtsI . This fragment extends from W22 to V47 and corresponds to the TMH . This is a remarkable finding because it is usual for a TMH to target a protein to a site more specific than the membrane . Alanine-scanning mutagenesis of the TMH identified several residues important for septal localization . These residues cluster on one side of an alpha-helix, which we propose interacts directly with another division protein to recruit FtsI to the septal ring.

J Bacteriol, 2005 Jan, 187(1), 304 - 19
pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12; Maurer LM et al.; Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH . Cultures were grown to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7 . For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E . coli arrays . Analysis of variance with an alpha level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression . Normalized expression indices were calculated for each gene and intergenic region (IG) . Differential expression among the three pH classes was observed for 763 genes and 353 IGs . Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes) . Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient . High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II . By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7 . These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell . Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress . All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH . Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins.

J Bacteriol, 2005 Jan, 187(1), 231 - 7
Methionine sulfoxide reduction and assimilation in Escherichia coli: new role for the biotin sulfoxide reductase BisC; Ezraty B et al.; Methionine ranks among the amino acids most sensitive to oxidation, which converts it to a racemic mixture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO) . The methionine sulfoxide reductases MsrA and MsrB reduce free and protein-bound MetSO, MsrA being specific for Met-S-SO and MsrB for Met-R-SO . In the present study, we report that an Escherichia coli metB1 auxotroph lacking both msrA and msrB is still able to use either of the two MetSO enantiomers . This indicates that additional methionine sulfoxide reductase activities occur in E . coli . BisC, a poorly characterized biotin sulfoxide reductase, was identified as one of these new methionine sulfoxide reductases . BisC was purified and found to exhibit reductase activity with free Met-S-SO but not with free Met-R-SO as a substrate . Moreover, a metB1 msrA msrB bisC strain of E . coli was unable to use Met-S-SO for growth, but it retained the ability to use Met-R-SO . Mass spectrometric analyses indicated that BisC is unable to reduce protein-bound Met-S-SO . Hence, this study shows that BisC has an essential role in assimilation of oxidized methionines . Moreover, this work provides the first example of an enzyme that reduces free MetSO while having no activity on peptide-bound MetSO residues.

J Bacteriol, 2005 Jan, 187(1), 193 - 201
Genetic analysis of the HAMP domain of the Aer aerotaxis sensor localizes flavin adenine dinucleotide-binding determinants to the AS-2 helix; Ma Q et al.; HAMP domains are signal transduction domains typically located between the membrane anchor and cytoplasmic signaling domain of the proteins in which they occur . The prototypical structure consists of two helical amphipathic sequences (AS-1 and AS-2) connected by a region of undetermined structure . The Escherichia coli aerotaxis receptor, Aer, has a HAMP domain and a PAS domain with a flavin adenine dinucleotide (FAD) cofactor that senses the intracellular energy level . Previous studies reported mutations in the HAMP domain that abolished FAD binding to the PAS domain . In this study, using random and site-directed mutagenesis, we identified the distal helix, AS-2, as the component of the HAMP domain that stabilizes FAD binding . AS-2 in Aer is not amphipathic and is predicted to be buried . Mutations in the sequence coding for the contiguous proximal signaling domain altered signaling by Aer but did not affect FAD binding . The V264M residue replacement in this region resulted in an inverted response in which E . coli cells expressing the mutant Aer protein were repelled by oxygen . Bioinformatics analysis of aligned HAMP domains indicated that the proximal signaling domain is conserved in other HAMP domains that are not involved in chemotaxis or aerotaxis . Only one null mutation was found in the coding sequence for the HAMP AS-1 and connector regions, suggesting that these are not active signal transduction sites . We consider a model in which the signal from FAD is transmitted across a PAS-HAMP interface to AS-2 or the proximal signaling domain.

J Bacteriol, 2005 Jan, 187(1), 45 - 53
Simulated diffusion of phosphorylated CheY through the cytoplasm of Escherichia coli; Lipkow K et al.; We describe the use of a computational model to study the effects of cellular architecture and macromolecular crowding on signal transduction in Escherichia coli chemotaxis . A newly developed program, Smoldyn, allows the movement and interaction of a large number of individual molecules in a structured environment to be simulated (S . S . Andrews and D . Bray, Phys . Biol., in press) . With Smoldyn, we constructed a three-dimensional model of an E . coli cell and examined the diffusion of CheYp from the cluster of receptors to the flagellar motors under control conditions and in response to attractant and repellent stimuli . Our simulations agree well with experimental observations of cell swimming responses and are consistent with the diffusive behavior expected in wild-type and mutant cells . The high resolution available to us in the new program allows us to calculate the loci of individual CheYp molecules in a cell and the distribution of their lifetimes under different cellular conditions . We find that the time delay between stimulus and response differs for flagellar motors located at different positions in the cell . We explore different possible locations for the phosphatase CheZ and show conditions under which a gradient of CheYp exists in the cell . The introduction of inert blocks into the cytoplasm, representing impenetrable structures such as the nucleoid and large protein complexes, produces a fall in the apparent diffusion coefficient of CheYp and enhances the differences between motors . These and other results are left as predictions for future experiments.

Malar J . 2004 Dec 15;3(1):50 {Epub ahead of print}
Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli; Flick K et al.; BACKGROUND: The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic . High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process . Therefore, the majority of P . falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells . METHODS: Several domains of PfEMP1 genes obtained from different P . falciparum strains were expressed in E . coli as GST-fusion proteins . Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage . RESULTS AND CONCLUSIONS: When expressed in E . coli recombinant proteins derived from P . falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies . The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation . Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality . Furthermore, these proteins were shown to be functionally active . Other factors such as temperature, pH, bacterial proteases or the codon optimization for E . coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct . In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons.

Phys Rev Lett . 2004 Nov 26;93(22):228103 . Epub 2004 Nov 23.
Pattern formation within Escherichia coli: diffusion, membrane attachment, and self-interaction of MinD molecules; Kulkarni RV et al.; In E . coli, accurate cell division depends upon the oscillation of Min proteins from pole to pole . We provide a model for the polar localization of MinD based only on diffusion, a delay for nucleotide exchange, and different rates of attachment to the bare membrane and the occupied membrane . We derive analytically the probability density, and correspondingly the length scale, for MinD attachment zones . Our simple analytical model illustrates the processes giving rise to the observed localization of cellular MinD zones.

J Am Chem Soc, 2004 Dec 22, 126(50), 16608 - 20
Magic angle spinning solid-state NMR spectroscopy for structural studies of protein interfaces . resonance assignments of differentially enriched Escherichia coli thioredoxin reassembled by fragment complementation; Marulanda D et al.; De novo site-specific backbone and side-chain resonance assignments are presented for U-15N(1-73)/U-13C,15N(74-108) reassembly of Escherichia coli thioredoxin by fragment complementation, determined using solid-state magic angle spinning NMR spectroscopy at 17.6 T . Backbone dihedral angles and secondary structure predicted from the statistical analysis of 13C and 15N chemical shifts are in general agreement with solution values for the intact full-length thioredoxin, confirming that the secondary structure is retained in the reassembled complex prepared as a poly(ethylene glycol) precipitate . The differential labeling of complementary thioredoxin fragments introduced in this work is expected to be beneficial for high-resolution structural studies of protein interfaces formed by protein assemblies by solid-state NMR spectroscopy.

Sichuan Da Xue Xue Bao Yi Xue Ban, 2003 Jan, 34(1), 5 - 8
{Gene therapy for ovarian cancer by adenovirus-mediated transduction of cytosine deaminase gene in vitro}; Li Q et al.; OBJECTIVE: To study the value of adenovirus mediated expression of the Escherichia coli cytosine deaminase (CD) gene with 5-fluorocytosine (5-FC) in the gene therapy of ovarian cancer cells . METHODS: Ovarian cancer cells were transduced CD gene by adenovirus vector and exposed to varying concentration of 5-FC . After 5 days of incubation, MTT assays were performed to measure the cell viability . RESULTS: It was shown that tumor cells transduced with CD gene had a high sensitivity to 5-FC . Mixed cellular assay revealed that CD-postive cells had a neighbor cell killing effect on CD-negative cells when exposed to 5-FC . 20% CD positive cells killed more than 80% mixed cells . CONCLUSION: CD/5-FC gene therapy approach is an effective anti-tumor strategy in the treatment of ovarian cancer.

Arch Insect Biochem Physiol . 2004 Dec 14;58(1):39-46 {Epub ahead of print}
Expression of the Helicoverpa cathepsin b-like proteinase during embryonic development; Zhao XF et al.; Cathepsin B-like proteinase from Helicoverpa armigera (HCB) was proposed as being involved in the degradation of yolk proteins during embryonic development . Recombinant HCB was expressed as a fusion protein with GST in Escherichia coli BL21 on the basis of its cDNA and purified to homogeneity . The fusion protein was cleaved with thrombin to generate a soluble protease with a mass of 37 kDa . A polyclonal antiserum against this recombinant protein, raised in the rabbit, recognized three isoforms of HCB in an ovary homogenate of this insect . Expression of this enzyme during embryonic development was studied using immunoblotting, immunohistochemistry and activity assay . It was found that HCB was expressed during embryonic development and that its proteolytic activity was detected from embryonic developmental eggs . The fact that HCB activity is observed in ovaries and developing eggs suggested that the enzyme had already been activated before embryonic development . Immunohistochemistry indicated that the enzyme was located in follicular cells, the sphere of yolk granules, and the fat bodies of female adult . These lines of evidence suggested strongly that HCB takes part in the degradation of yolk proteins during the development of embryo . Arch . Insect Biochem . Physiol . 58:39-46, 2005 . (c) 2004 Wiley-Liss, Inc.

J Microbiol Immunol Infect, 2004 Dec, 37(6), 327 - 34
Genetic detection of diarrheagenic Escherichia coli isolated from children with sporadic diarrhea; Teng LJ et al.; Escherichia coli strains are among the major bacterial causes of diarrheal illness . At least 5 categories of diarrheagenic E . coli (DEC) are recognized, namely enterotoxigenic E . coli (ETEC), enteropathogenic E . coli (EPEC), enteroinvasive E . coli (EIEC), enteroaggregative E . coli (EAEC), and enterohemorrhagic E . coli (EHEC) . Due to the need for costly and labor-intensive diagnostic procedures, identification of DEC is difficult at standard laboratories . Therefore, the epidemiology of DEC infections remains obscure in Taiwan . Recently, polymerase chain reaction (PCR) or dot blot has been used for genetic detection of DEC . In this study, we analyzed 150 E . coli isolates from diarrheal stools of children under 5 years old . The PCR tests detected 5 ETEC (3.3%), 6 EPEC (4%), 4 EIEC (2.7%), and 13 EAEC (8.7%) isolates . No EHEC was detected . Dot blot and sequence analysis were used to confirm the results of PCR . The cellular fatty acid (CFA) profiles from E . coli isolates were also analyzed . Comparison of CFA composition revealed minor variation in the percentage of each fatty acid detected among DEC isolates of ETEC, EPEC, EIEC and EAEC, but did not provide enough evidence for differentiating between categories of DEC by CFA profiles alone.

Genes Genet Syst, 2004 Oct, 79(5), 283 - 91
Cloning and characterization of a novel radish protein kinase which is homologous to fungal cot-I like and animal Ndr protein kinases; Imai T et al.; According to the similarity of the amino acid sequences in their catalytic domains, eukaryotic protein kinases have been classified into the five main groups: 'AGC', 'CaMK', 'CMGC', 'PTK' and 'other' . The AGC group, represented by the cyclic nucleotide-dependent kinases (PKA and PKG), the calcium-phospholipid-dependent kinases (PKC) and the ribosomal S6 protein kinases, are poorly characterized in plants except for a few cases . In this study, in order to gain a better understanding of plant protein kinases in the AGC group, three cDNAs encoding novel protein kinases, RsNdr1 and RsNdr2a/b, were cloned from radish and characterized by molecular and biochemical methods . The deduced amino acid sequences of RsNdr1 and RsNdr2a/b contained all 12 conserved catalytic subdomains which are characteristic of the eukaryotic Ser/Thr protein kinases . A cell lysate from E . coli overexpressing RsNdr1 fusion protein had protein kinase activity toward a conventional protein substrate (myelin basic protein), whereas that from E . coli harboring a fusion plasmid encoding kinase-dead RsNdr1 or RsNdr2 did not show any protein kinase activity . A phylogenetic tree for 17 protein kinases from various organisms showed that the RsNdrs are more closely related to the protein kinases in a particular subgroup of the 'AGC' (fungal cot1-like and animal Ndr kinases) than to the authentic 'AGC' protein kinases, such as PKA, PKC or ribosomal S6 kinase.

Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Apr, 30(2), 234 - 8
{The construction and screening of genomic library based on transformable artificial chromosome (TAC) vector in Lotus japonicus}; Guo XZ et al.; Many vectors, especially artificial chromosome vectors, have been developed for genome-scale mapping and sequencing . As the first artificial chromosome, YAC has been extensively used in the genomic library construction and mapping . Shizuya et al . (1992) devised the bacterial artificial chromosome (BAC) originated from F factor of E.coli, which is much easy to handle and isolated with large harboring capacity . Liu et al . (1999) designed the transformation-competent artificial chromosome (TAC) vector, derived from PAC vector . TAC could shuttle a large-scale DNA fragment between bacteria and Agrobaceria . Further, it could integrate a large targetted DNA fragment into plant genome, as being documented in Arabidopsis and rice genome research (Liu et al . 2000, 2002) . The time-saving virtue of TAC should be significant in genomics research in Lotus japonicus, a model plant of legume . Using a nuclei-based method of Liu and Whittier, high molecular weight DNA was isolated from Lotus japonicus (Gifu ecotype) . The DNA was digested partially with Hind III and size-fractionated in the 10- to 20-kb size range as described (Liu and Whittier 1994) . The partially digested and size selected DNA fragments were ligated with Hind III-digested pYLTAC7 and then used for transformation of E . coli DH10B by electroporation . Transformants carrying inserts were selected on LB agar plates containing 25 mg/L kanamycin and 5% sucrose . The library, 6 haploid genome equivalents, was pooled in 12 96-well microtiter plates at about 150 transformants per well . The TAC library was then arrayed in nylon membranes and subjected to screening . The probe, a homolog fragment of CEN gene controlling the structure of inflorescence Antirrhinum, was used for screening . 0.5 muL solution from the positive pool was titered and the secondary screening was conducted in the same way . Finally, these positive clonies were confirmed by Southern blot . These data showed that this genomic library was reliable for further molecular research in Lotus japonicus.

J Biochem (Tokyo), 2004 Sep, 136(3), 359 - 62
Increased A:T->C:G Mutations in the mutT Strain upon 8-Hydroxy-dGTP Treatment: Direct Evidence for MutT Involvement in the Prevention of Mutations by Oxidized dGTP; Kamiya H et al.; The Escherichia coli MutT protein hydrolyzes 8-hydroxy-dGTP (8-OH-dGTP) in vitro, and mutT gene deficiencies cause increased spontaneous A:T-->C:G mutations . However, no direct evidence exists for enhanced mutagenicity of 8-OH-dGTP in mutT cells . In this study, 8-OH-dGTP was introduced into wild type and mutT E . coli cells, and mutations of a chromosomal gene were monitored . 8-OH-dGTP induced mutations of the rpoB gene, the degree of the mutation induction in the mutT strain being approximately 6-fold higher than that in the wild type strain . On the other hand, 2-hydroxy-dATP, which is not a substrate of the MutT protein, increased the mutation to similar degrees in the two strains . These results constitute the first evidence that the MutT protein suppresses mutation by 8-OH-dGTP in vivo.

J Biochem (Tokyo), 2004 Sep, 136(3), 335 - 42
Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin; Saiki K et al.; Cytolethal distending toxin (CDT) is one of the exotoxins produced by Actinobacillus actinomycetemcomitans, an agent of localized aggressive periodontitis . We constructed N-terminal deletion mutants of CdtA using an Escherichia coli expression system and found that ADelta(19-47), with a deletion from Asn-19 to Pro-47, showed comparable CDT activity but no apparent heterogeneity of CdtA . The wild-type CDT (wtCDT) and the mutant CDT (A(Delta)(19-47)CDT) were purified to homogeneity by introducing a histidine tag into the C-terminal end of CdtB . Both purified wtCDT and purified A(Delta)(19-47)CDT showed strong CDT activity and a tripartite structure composed of CdtA (subunit A), 31 kDa CdtB (subunit B), and 18.5 kDa CdtC (subunit C) in nearly a 1:1:1 stoichiometry . Importantly, subunit A was identified as heterogeneous with three CdtA variants in wtCDT, but homogeneous in A(Delta)(19-47)CDT . Purified CDTs also showed high stability that was absolutely dependent on the presence of sucrose in the buffer . In conclusion, the region from the Asn-19 to Pro-47 of CdtA contributes to the heterogeneous production of CdtA, but is dispensable for the toxin activity . Furthermore, this study describes an effective protocol for the purification of a native rather than reconstituted CDT, and clarifies the subunit composition of the active CDT holotoxin.

J Biochem (Tokyo), 2004 Sep, 136(3), 301 - 12
Relationship between Intron 4b Splicing of the Rat Geranylgeranyl Diphosphate Synthase Gene and the Active Enzyme Expression Level; Matsumura Y et al.; Geranylgeranyl diphosphate synthase (GGPS) is a branch point enzyme in the mevalonate pathway that catalyzes the synthesis of geranylgeranyl diphosphate used for the geranylgeranylation of Rho, Rac and Rab proteins . The current study showed the production of multiple forms of GGPS mRNA from a single GGPS gene in rat . The mRNAs resulted from combinations of multiple alternative introns and two poly(A) sites in the 3'-translated and 3'-untranslated regions . These are classified into 1a-type and 1b-type mRNAs, based on the splicing of intron 4b resulting in the difference in deduced amino acid sequence between the C-terminal regions . The 1a-type and 1b-type proteins expressed in both Escherichia coli and HeLa cells were active and inactive, respectively . In the case of HeLa cells, the latter protein expression level was about 10% relative to the former one . This was also observed for Cos-7 and 293 cells . When fusions of beta-galactosidase with C-terminal regions differing between the 1a-type and 1b-type proteins were expressed in HeLa cells, the expressed fusion proteins were both found to be active but the latter fusion protein expression level was considerably low compared with the former one . The expression level of 1a-type mRNA was higher than that of 1b-type mRNA in brain, liver, heart, and thymus, but the two expression levels were the same in testis and ovary . During testis development the total GGPS mRNA expression level increased, accompanied by an increase in 1b-type mRNA, the expression level of 1a-type mRNA encoding active GGPS remaining kept unchanged . These results indicate that the expression level of rat active GGPS is at least regulated through the splicing of intron 4b of its gene.

Nucleic Acids Res, 2004 Dec 14, 32(22), 6548 - 56 Print 2004.
A 16S rRNA-tRNA product containing a nucleotide phototrimer and specific for tRNA in the P/E hybrid state in the Escherichia coli ribosome; Huggins W et al.; Ribosome complexes containing deacyl-tRNA1(Val) or biotinylvalyl-tRNA1(Val) and an mRNA analog have been irradiated with wavelengths specific for activation of the cmo5U nucleoside at position 34 in the tRNA1(Val) anticodon loop . The major product for both types of tRNA is the cross-link between 16S rRNA (C1400) and the tRNA (cmo5U34) characterized already by Ofengand and his collaborators {Prince et al . (1982) Proc . Natl Acad . Sci . USA, 79, 5450-5454} . However, in complexes containing deacyl-tRNA1(Val), an additional product is separated by denaturing PAGE and this is shown to involve C1400 and m5C967 of 16S rRNA and cmo5U34 of the tRNA . Puromycin treatment of the biotinylvalyl-tRNA1(Val) -70S complex followed by irradiation, results in the appearance of the unusual photoproduct, which indicates an immediate change in the tRNA interaction with the ribosome after peptide transfer . These results indicate an altered interaction between the tRNA anticodon and the 30S subunit for the tRNA in the P/E hybrid state compared with its interaction in the classic P/P state.

Cytokine, 2005 Jan 21, 29(2), 77 - 83
Cloning and expression of feline interleukin 15; Dean GA et al.; A cDNA encoding feline interleukin 15 (IL15) was cloned from the lymph node of a cat infected with feline infectious peritonitis virus . The cDNA is 486bp in length and encodes a protein of 162 amino acids . Recombinant protein was readily expressed as a GST fusion in Escherichia coli and purified by glutathione affinity chromatography . Expression of recombinant protein in mammalian cells was only accomplished by eliminating the 5' and 3' UTR, replacing the IL15 signal peptide with the tissue plasminogen activator signal peptide, and adding 3' sequence to disrupt presumptive secondary structure of the mRNA . Biologically active feline IL15 was expressed in HEK293T cells and was shown to sustain primary feline lymphocytes, a feline T cell line, and mouse CTLL-2 cells . Proliferation of CTLL-2 cells was induced by the recombinant protein in a dose-dependent manner . Monoclonal and polyclonal antibodies against human IL15 recognized feline IL15 in immunofluorescence and Western blot assays . Additionally, feline IL15 was detectable using a commercially available human IL15 ELISA kit.

Cytokine, 2005 Jan 21, 29(2), 56 - 66
Kinetics of development and characteristics of antibodies induced in cancer patients against yeast expressed rDNA derived granulocyte macrophage colony stimulating factor (GM-CSF); Rini B et al.; We have determined the presence and kinetics of granulocyte macrophage colony stimulating factor (GM-CSF) antibodies induced after repeated administration of a yeast expressed GM-CSF product in prostate cancer patients with minimal recurrent disease using a panel of assays for detection and characterization of antibodies . Results showed that all 15 prostate cancer patients treated with GM-CSF developed GM-CSF reactive antibodies during the course of therapy . Most patients (87%) developed GM-CSF reactive antibodies within 3 months while in other patients (13%), these antibodies were induced after additional cycles of GM-CSF treatment . For most patients, the timing of occurrence of these antibodies was the same regardless of whether the ELISA or surface plasmon resonance (SPR) assays were used for detection . However, in two patients, the recognition of GM-CSF reactive antibodies by SPR assays preceded their detection by ELISA . A significant number of patients (n=9, 60%) developed GM-CSF antibodies which neutralized the biological activity of GM-CSF in vitro in a cell-line based bioassay . These antibodies also recognized GM-CSF protein from different expression systems including the non-glycosylated protein from E . coli indicating that the antibody response is directed towards the amino acid backbone of the protein . A significant effect of GM-CSF antibodies on PSA modulation was not observed in this small cohort of patients despite an alteration in PSA levels in some treated patients . The study design used here did not allow conclusions regarding the relationship between neutralizing antibodies and the PSA levels which were used as a marker for clinical outcome . Implementation of a clinical strategy which permits monitoring for antibody development and for levels of a relevant pre-determined clinical marker at appropriate time-points is necessary for assessing the impact of antibody development on the therapeutic efficacy of the protein.

BMC Biotechnol . 2004 Dec 14;4(1):32 {Epub ahead of print}
Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression; Dyson MR et al.; BACKGROUND: In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein . Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX) . Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners . Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study . RESULTS: Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions . There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location . Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression . Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion . 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions . CONCLUSIONS: By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E . coli as soluble product and also which are best targeted for a eukaryotic expression system . In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E . coli.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Jun, 22(3), 173 - 5
{Cloning, sequencing and subcloning of cDNA coding for group I allergen of Dermatophagoides farinae}; Yang QG et al.; OBJECTIVE: To clone, sequence and subclone the cDNA coding for group 1 allergen of Dermatophagoides farinae (Der f 1) . METHODS: The cDNA of Der f 1 was amplified by RT-PCR and PCR . After purified, the gene fragment was cloned into a vector pMD-18T . The recombinant plasmid pMD-18T-Der f 1 was transformed into E . coli JM109 . Positive clones were screened and identified by PCR and digestion with restriction enzyme . The sequence of inserted Der f 1 gene fragment was also detected . Der f 1 was then subcloned into the vector of pET-32a(+) . RESULTS: The Der f 1 gene fragment of Dermatophagoides farinae was specifically amplified from RNA by RT-PCR and PCR . The recombinant plasmid pMD-18T-Der f 1 and pET-32a(+)-Der f 1 was constructed and digested by Bam H I and Sac I, the size of gene fragment was 646 bp and in accordance with the expected one . CONCLUSION: The pET-32a(+)-Der f 1 subcloning has been constructed successfully.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Jun, 22(3), 133 - 5
{Cloning and expression of aldolase encoding gene of Plasmodium falciparum FCC1/HN strain}; Zhang RJ et al.; OBJECTIVE: To clone, sequence, and express the aldolase (ALD) encoding gene of Plasmodium falciparum FCC1/HN strain . METHODS: The ALD encoding gene was amplified by PCR from genomic DNA of FCC1/HN strain . The positive clones were screened and identified by agarose gel electrophoresis and endonuclease . The recombinant plasmid was transformed into E . coli M15 . The fusion protein was expressed by IPTG induction and purified by Ni-NTA affinity chromatography and anion exchange column . RESULTS: The ALD gene of P . falciparum was amplified . Analysis of sequencing showed that the ALD gene of P . falciparum was identical with the sequence of other reported isolates . A Mr 41,000 fusion protein was induced by IPTG and was purified by chromatography . CONCLUSION: The ALD gene of P . falciparum FCC1/HN strain was identical to the other reported isolates . ALD fusion protein of P . falciparum was expressed and purified.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Jun, 22(3), 129 - 32
{Mucosal immunization of recombinant Schistosoma japonicum ferritin}; Chen LY et al.; OBJECTIVE: To subclone and express the gene encoding Schistosoma japonicum ferritin (SjFer) and study its immune protection against challenge infection in mice vaccinated intranasally . METHODS: The SjFer gene was amplified by PCR, and subcloned into the N-terminal of intein 2 of the pTWIN1 vector . The positive recombinant was screened by PCR, restriction enzyme digestion and sequence analysis . The positive recombinant was transformed into E . coli ER2566 . The soluble recombinant fusion protein (rSjFer-intein 2) was expressed in E . coli by induction of low IPTG concentration under low temperature, and analyzed by SDS-PAGE and Western blotting . Mice were immunized intranasally with rSjFer, using chitosan as adjuvant . Two weeks after the third vaccination, challenge infection with S . japonicum cercariae was carried out . Worms and eggs collected from the livers of mice were counted at 42 days after the challenge . Levels of specific antibodies were detected by ELISA before infection . RESULTS: SjFer was successfully subcloned into pTWIN1 vector and expressed in E . coli . In mice vaccinated intranasally with rSjFer and adjuvant chitosan, the worm reduction rate was 35.51% and the reduction rate of eggs per gram liver tissue (LEPG) was 52.17% . As compared with the control groups, levels of IgG, IgA in sera and SIgA in saliva increased significantly . CONCLUSION: The expressed rSjFer can induce partial protective immunity against S . japonicum infection in mice when they were vaccinated intranasally, with chitosan as adjuvant.

Biotechniques, 2004 Dec, 37(6), 948 - 52
Positive selection system for identification of recombinants using alpha-complementation plasmids; Reddy M; A number of selection systems have been developed for direct selection of recombinant plasmids in cloning experiments (positive selection) . In this study, the Commonly used LacZ-based alpha-complementation plasmid vectors have been used for designing a positive selection system for the selection of recombinants . The basis for the Strategy is the phenomenon of galactose sensitivity exhibited by galactose epimerase (galE) mutants of Escherichia coli . It is known that lacZ+ galE, but not lacZ- galE cells are killed upon addition of lactose due to the accumulation of a toxic intermediate, UDP-galactose, by hydrolysis of lactose . Using a galE mutant strain of E . coli that carries the lacZAM15 allele, various alpha-complementation plasmids that vary in their copy number were examined for their ability to be killed following addition of lactose . The results show that some plasmids that exhibit relatively high beta-galactosidase enzyme activity can be used effectively for positive selection . This selection would be extremely useful during primary cloning experiments such as construction of genomic or cDNA libraries and also in instances involving selection for rare recombinants.

Phytother Res, 2004 Nov, 18(11), 900 - 5
The effects of Pycnogenol on DNA damage in vitro and expression of superoxide dismutase and HP1 in Escherichia coli SOD and catalase deficient mutant cells; Kim YG et al.; The procyanidin-rich French maritime pine bark extract Pycnogenol (PYC) is believed to be an antioxidant . To access whether PYC protects DNA against Fenton reaction radicals, pUC 19 plasmid DNA was damaged by OH- radicals generated from Fe(II) plus H2O2 in the presence and absence of PYC . DNA damage was quantified by measuring decreases in supercoiled DNA (SC-DNA) using agarose gel electrophoresis . The results showed that PYC (50 microg/mL) did not inhibit DNA damage from Fenton reaction-generated oxyradicals, when the Fenton reaction was allowed to proceed . However, PYC did protect against DNA damage when added prior to the initiation of the Fenton reaction, suggesting that protection resulted from chelation of Fe rather than from the scavenging of radicals . Moreover, we unexpectedly observed PYC-associated DNA breakage, which was dose- and time- dependent . Other antioxidants such as desferrioxamine (DFO) including butylated hydroxyltoluene (BHT), curcumin and alpha-tocopherol exhibited protective effects against DNA damage caused by the Fenton reaction . Subsequently, we assessed the possible pro-oxidant function of PYC on DNA damage in E . coli SOD deficient mutants under oxidative stress, after observing that PYC can induce SOD under oxidative stress . Although, PYC did not enhance DNA damage, it likewise did not protect against oxidative stress-mediated DNA damage . All together, our data indicate that PYC under some circumstances can enhance oxidative stress-mediated DNA damage in vitro and in vivo .

Proc Natl Acad Sci U S A, 2004 Dec 21, 101(51), 17777 - 82 Epub 2004 Dec 13.
Association of RNA polymerase with transcribed regions in Escherichia coli; Wade JT et al.; We examine the association of the beta-, alpha-, and sigma(70)-subunits of Escherichia coli RNA polymerase (RNAP) and the NusA elongation factor with transcribed regions in vivo by using chromatin immunoprecipitation . RNAP preferentially associates with the promoter-proximal region of several operons, and this preference is particularly pronounced at the lexA-dinF promoter . When cells are grown in exponential phase, little or no sigma(70) is associated with RNAP during early elongation . However, during stationary phase, sigma(70) is retained in a fraction of elongating RNAP complexes throughout the melAB operon . In contrast, sigma(70) is not observed in elongating RNAP complexes at the lacZYA operon during stationary phase . At both operons, NusA associates with RNAP during early elongation, and this association is greatly reduced during stationary phase . These observations suggest that in vivo association of sigma(70) and NusA with elongating RNAP is regulated by growth conditions.

J Biol Chem . 2004 Dec 13; {Epub ahead of print}
Function of the SmpB tail in tmRNA translation revealed by a nucleus-encoded form; Jacob Y et al.; Stalled bacterial ribosomes are freed when they switch to the translation of tmRNA . This process requires the tmRNA-binding and ribosome-binding cofactor SmpB, a beta-barrel protein with a protruding C-terminal tail of unresolved structure . Some plastid genomes encode tmRNA, but smpB genes have only been reported from bacteria . Here we identify smpB in the nuclear genomes of both a diatom and a red alga, encoding a signal for import into the plastid, where mature SmpB could activate tmRNA . Diatom SmpB was active for tmRNA translation with bacterial components in vivo and in vitro, although less so than E . coli SmpB . Tail-truncated diatom SmpB, the hypothetical product of a misspliced mRNA, was inactive in vivo . Tail-truncated E . coli SmpB was likewise inactive for tmRNA translation, yet it was still able to bind ribosomes and its affinity for tmRNA was only slightly diminished . This work suggests that SmpB is a universal cofactor of tmRNA . It also reveals a tail-dependent role for SmpB in tmRNA translation that supersedes a simple role of linking tmRNA to the ribosome, which the SmpB body alone could provide.

Protein Expr Purif, 2005 Jan, 39(1), 97 - 106
High-level heterologous expression and properties of a novel lipase from Ralstonia sp . M1; Quyen DT et al.; The mature lipase LipA and its 56aa-truncated chaperone DeltaLipBhis (with 6xhis-tag) from Ralstonia sp . M1 were over-expressed in Escherichia coli BL21 under the control of T7 promoter with a high level of 70 and 12mg protein per gram of wet cells, respectively . The simply purified lipase LipA was effectively refolded by Ni-NTA purified chaperone DeltaLipBhis in molar ratio 1:1 at 4 degrees C for 24 hours in H(2)O . The in vitro refolded lipase LipA had an optimal activity in the temperature range of 50-55 degrees C and was stable up to 45 degrees C with more than 84% activity retention . The maximal activity was observed at pH 10.75 for hydrolysis of olive oil and found to be stable over alkaline pH range 8.0-10.5 with more than 52% activity retention . The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine (remaining activity 137-334%), but inhibited by 1-butanol and acetonitrile (40-86%) . Metal ions Cu(2+), Sn(2+), Mn(2+), Mg(2+), and Ca(2+) stimulated the lipase slightly with increase in activity by up to 22%, whereas Zn(2+) significantly inhibited the enzyme with the residual activity of 30-65% and Fe(3+) to a lesser degree (activity retention of 77-86%) . Tween 80, Tween 60, and Tween 40 induced the activation of the lipase LipA (222-330%) and 0.2-1% (w/v) of Triton X-100, X-45, and SDS increased the lipase activity by up to 52% . However, 5% (w/v) of Triton X-100, X-45, and SDS inhibited strongly the activity by 31-89% . The inhibitors including DEPC, EDTA, PMSF, and 2-mercaptoethanol (0.1-10mM) inhibited moderately the lipase with remaining activity of 57-105% . The lipase LipA hydrolyzed a wide range of triglycerides, but preferentially short length acyl chains (C4 and C6) . In contrast to the triglycerides, medium length acyl chains (C8 and C14) of p-nitrophenyl (p-NP) esters were preferential substrates of this lipase . The enzyme preferentially catalyzed the hydrolysis of cottonseed oil (317%), cornoil (227%), palm oil (222%), and wheatgerm oil (210%) in comparison to olive oil (100%).

Protein Expr Purif, 2005 Jan, 39(1), 18 - 26
Expression, refolding, and purification of recombinant human granzyme B; Lorentsen RH et al.; Granzyme B (GrB) is a member of a family of serine proteases involved in cytotoxic T-lymphocyte-mediated killing of potentially harmful cells, where GrB induces apoptosis by cleavage of a limited number of substrates . To investigate the suitability of GrB as an enzyme for specific fusion protein cleavage, two derivatives of human GrB, one dependent on blood coagulation factor X(a) (FX(a)) cleavage for activation and one engineered to be self-activating, were recombinantly expressed in Escherichia coli . Both derivatives contain a hexa-histidine affinity tag fused to the C-terminus and expressed as inclusion bodies . These were isolated and solubilized in guanidiniumHCl, immobilized on a Ni(2+)-NTA agarose column, and refolded by application of a cyclic refolding protocol . The refolded pro-rGrB-H6 could be converted to a fully active form by cleavage with FX(a) or, for pro(IEPD)-rGrB-H6, by autocatalytic processing during the final purification step . A self-activating derivative in which the unpaired cysteine of human GrB was substituted with phenylalanine was also prepared . Both rGrB-H6 and the C228F mutant were found to be highly specific and efficient processing enzymes for the cleavage of fusion proteins, as demonstrated by cleavage of fusion proteins containing the IEPD recognition sequence of GrB.

Biochemistry, 2004 Dec 21, 43(50), 15929 - 35
Mechanism of frameshift (deletion) generated by acetylaminofluorene-derived DNA adducts in vitro; Shibutani S et al.; We have investigated the mechanism of frameshift (deletion) mutagenesis induced by acetylaminofluorene- (AAF-) derived DNA adducts . dG-AAF-modified oligodeoxynucleotides, with different bases positioned 5' to the lesion, were annealed to (32)P-labeled 13-mer primers and then used in primer extension reactions catalyzed by the 3'-->5' exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I . When the dNMP positioned opposite dG-AAF could pair with its complementary base at the 5' flanking position, single-base deletions were produced at high frequency . Similarly, when the complementary base was two positions 5' to the dG-AAF, two-base deletions occurred . The relative frequency of base insertions opposite dG-AAF followed the order dCMP > dAMP > dGMP > dTMP; the frequency of dNTP insertion opposite the lesion paralleled the formation of frameshift deletions . When a template designed to induce three-base deletions was used for translesion synthesis catalyzed by the exo(-) Klenow fragment, the expected three-base deletion was formed . When dG-AAF-modified templates containing iterated bases 5' to the lesion were annealed to primers with the complementary dNMP positioned opposite the lesion, the dNMP inserted opposite the dG-AAF tended to pair with the complementary base 5' to the lesion, thereby forming shorter deletions . Taken together, these results support the molecular mechanism for frameshift deletion proposed earlier by Shibutani and Grollman in which direct base insertion precedes misalignment {(1993) J . Biol . Chem . 268, 11703}.

Biochemistry, 2004 Dec 21, 43(50), 15922 - 8
Mutagenic potential of benzo{a}pyrene-derived DNA adducts positioned in codon 273 of the human P53 gene; Dong H et al.; Codon 273 ((5)(')CGT) of the human P53 gene is a mutational hot spot for the environmental carcinogen benzo{a}pyrene . We incorporated a single (+)- or (-)-trans-anti-benzo{a}pyrene diol epoxide (BPDE) DNA adduct at the second position of codon 273 of the human P53 gene and explored the mutagenic potential of this lesion in mammalian cells . Oligodeoxyribonucleotides ((5)(')GAGGTGCG(BPDE)TGTTTGT) modified with (+)- or (-)-trans-dG-N(2)-BPDE were incorporated into single-stranded shuttle vectors and transfected into simian kidney cells . Progeny plasmids were then used to transform Escherichia coli DH10B . Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and spectrum produced by the adducted base . We determined the mutational frequencies associated with (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE adduction to be 26.5% and 17.5%, respectively . The predominant mutations generated by both stereoisomers were G --> T transversions, with some G --> A transitions . When the cytosine 5' to dG-N(2)-BPDE was replaced by 5-methylcytosine, the mutational frequencies of (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE were reduced to 11.1% and 10.6%, respectively, while the mutational specificity remained unchanged . Thus, the mutational "hot spot" at codon 273 in P53 may reflect either sequence-specific reactivity of BPDE and/or inefficient repair of BPDE-DNA adducts positioned at this site.

J Proteome Res, 2004 Nov-Dec, 3(6), 1191 - 200
Large-scale quantitative proteomic study of PUMA-induced apoptosis using two-dimensional liquid chromatography-mass spectrometry coupled with amino acid-coded mass tagging; Gu S et al.; By coupling two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) with amino acid-coded mass tagging (AACT), we have greatly increased the analytical throughput and sequence coverage of MS-based methods for proteome-wide quantitation . The dynamic range and reproducibility of this 2D-LC-AACT quantitative approach were evaluated by profiling the mixtures with different ratios of E . coli cells grown in either regular or AACT medium . A SQL-based high thoughput MASCOT data analysis tool was developed for proteomic data sorting and mining . We investigated the early stage of apoptosis by inducing the p53 upregulated modulator of apoptosis (PUMA) through the analyses of the relative ratios of the pairwise isotope signals that were originated from the control and labeled PUMA-induced cells . In 20-hour 2D-LC-MS/MS run, 480 proteins were conclusively identified, and more than half of them were quantified . A noteworthy change in the quantitative profile was that histones and a ubiquitin conjugate protein UBC9, which are involved in DNA double-strand break (DSB) repair were significantly down-regulated in the PUMA-overexpressing apoptotic cells, suggesting the detection of DSB in the apoptotic process . The quantitative profiling efficiency of this approach was compared with the gel-based quantitative analysis scheme.

J Proteome Res, 2004 Nov-Dec, 3(6), 1155 - 63
Two-dimensional mass spectra generated from the analysis of 15N-labeled and unlabeled peptides for efficient protein identification and de novo peptide sequencing; Zhong H et al.; Protein identification has been greatly facilitated by database searches against protein sequences derived from product ion spectra of peptides . This approach is primarily based on the use of fragment ion mass information contained in a MS/MS spectrum . Unambiguous protein identification from a spectrum with low sequence coverage or poor spectral quality can be a major challenge . We present a two-dimensional (2D) mass spectrometric method in which the numbers of nitrogen atoms in the molecular ion and the fragment ions are used to provide additional discriminating power for much improved protein identification and de novo peptide sequencing . The nitrogen number is determined by analyzing the mass difference of corresponding peak pairs in overlaid spectra of (15)N-labeled and unlabeled peptides . These peptides are produced by enzymatic or chemical cleavage of proteins from cells grown in (15)N-enriched and normal media, respectively . It is demonstrated that, using 2D information, i.e., m/z and its associated nitrogen number, this method can, not only confirm protein identification results generated by MS/MS database searching, but also identify peptides that are not possible to identify by database searching alone . Examples are presented of analyzing Escherichia coli K12 extracts that yielded relatively poor MS/MS spectra, presumably from the digests of low abundance proteins, which can still give positive protein identification using this method . Additionally, this 2D MS method can facilitate spectral interpretation for de novo peptide sequencing and identification of posttranslational or other chemical modifications . We envision that this method should be particularly useful for proteome expression profiling of organelles or cells that can be grown in (15)N-enriched media.

J Mol Recognit . 2004 Dec 10; {Epub ahead of print}
Tetraplex formation of surface-immobilized human telomere sequence probed by surface plasmon resonance using single-stranded DNA binding protein; Zeng ZX et al.; Many sequences in genomic DNA are able to form unique tetraplex structures . Such structures are involved in a variety of important cellular processes and are emerging as a new class of therapeutic targets for cancers and other diseases . Screening for molecules targeting the tetraplex structure has been explored using such sequences immobilized on solid surfaces . Immobilized nucleic acids, in certain situations, may better resemble the molecules under in vivo conditions . In this report, we studied the formation of tetraplex structure of both the G-rich and C-rich strands of surface-immobilized human telomere sequence by surface plasmon resonance using the single-stranded DNA binding protein from Escherichia coli as probe . We demonstrate how the formation of G-quadruplex and i-motif could be probed under various conditions by this sequence-universal method . Our results also show that immobilization destabilized the tetraplex structure . Copyright (c) 2004 John Wiley & Sons, Ltd.

Proteins . 2004 Dec 8; {Epub ahead of print}
Crystal structure of the YgfY from Escherichia coli, a protein that may be involved in transcriptional regulation; Lim K et al.; No abstract.

Clin Pharmacol Ther, 2004 Dec, 76(6), 519 - 27
A novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo; Fukami T et al.; Cytochrome P450 (CYP) 2A6 is a major CYP responsible for the metabolism of nicotine and coumarin in humans . We identified a novel allele, designated CYP2A6*17 , which contains A51G (exon 1), C209T (intron 1), G1779A (exon 3), C4489T (intron 6), G5065A (V365M, exon 7), G5163A (intron 7), C5717T (exon 8), and A5825G (intron 8) . We developed a genotyping method by polymerase chain reaction-restriction fragment length polymorphism for the CYP2A6*17 allele, targeting the G5065A mutation . The allele frequency in black subjects (n = 96) was 9.4% (95% confidence interval {CI}, 5.3%-13.5%) . The allele was not found in white subjects (95% CI, 0%-0.9%; n = 163), Japanese subjects (95% CI, 0%-1.6%; n = 92), and Korean subjects (95% CI, 0%-0.7%; n = 209) . To examine the effects of the amino acid change in the CYP2A6*17 allele on the enzymatic activity, we expressed a wild-type or variant (V365M) CYP2A6 together with NADPH-CYP reductase in Escherichia coli . For coumarin 7-hydroxylation, the apparent Michaelis-Menten constant value of variant CYP2A6 (1.06 +/- 0.11 micromol/L) was significantly (P < .005) higher than that of wild type (0.60 +/- 0.05 micromol/L) . The maximum velocity values of the wild-type and variant CYP2A6 were 0.61 +/- 0.06 and 0.64 +/- 0.07 pmol . min -1 . pmol -1 CYP, respectively . For nicotine C -oxidation, the apparent Michaelis-Menten constant values of the wild-type or variant CYP2A6 were 31.6 +/- 2.9 micromol/L and 31.3 +/- 3.1 micromol/L, respectively . The maximum velocity value of variant CYP2A6 (0.72 +/- 0.21 pmol . min -1 . pmol -1 CYP) was significantly (P < .05) lower than that of the wild type (1.80 +/- 0.42 pmol . min -1 . pmol -1 CYP) . Thus the intrinsic clearance values for coumarin 7-hydroxylation and nicotine C -oxidation by the variant were both significantly (P < .05) decreased to 40% to 60% compared with the wild type . Furthermore, cotinine/nicotine ratios after 1 piece of nicotine gum was chewed, used as an index of in vivo nicotine metabolism, were significantly (P < .05) decreased in heterozygotes of the CYP2A6*17 allele (5.4 +/- 2.7, n = 12) compared with homozygotes of the wild type (11.5 +/- 10.5, n = 37) . A subject with CYP2A6*17 / CYP2A6*17 revealed the lowest cotinine/nicotine ratio (1.8) . We found a novel allele in black subjects that affects the nicotine metabolism in vitro and in vivo.

Korean J Parasitol, 2004 Dec, 42(4), 195 - 200
Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits; You MJ; We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H . longicornis P27/30 protein . The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates . Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H . longicornis, and identified P27/30 as a troponin I-like protein . In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H . longicornis P27/30 protein is a potential candidate antigen for a tick vaccine . These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H . longicornis.

J Dairy Sci, 2005 Jan, 88(1), 419 - 25
The association of herd milk production and management with a return-over-feed index in ontario dairy herds; McLaren CJ et al.; Associations of herd milk production and management variables to a return-over-feed (ROF) herd profit index were examined among 95 dairy farms . The ROF index is derived from 2 important determinants of profit on dairy farms: milk income and feed cost . All producers were participants in the Dairy Herd Improvement (DHI) ROF program in Ontario, Canada during 2002 . Nutrition, housing, health, and other management data were collected through a phone survey of herd managers . Herd milk production, milk component percentages, and somatic cell count data were obtained from the Ontario DHI database . The linear regression model accounting for significant variation in ROF with highest R(2) (0.66) included standardized milk production, milk protein percentage, milk fat percentage, and use of monensin in lactating cow rations . A 1-kg increase in standardized milk production (kg/d per cow) or a 0.1 percentage unit increase in milk protein was associated with $0.35/d per cow or $0.26/d per cow increase, respectively, in the ROF of the dairy herd . However, a 0.1 percentage unit increase in milk fat was associated with a $0.10/d per cow decrease in ROF, probably because of a negative association of milk fat with milk yield . Use of monensin in lactating cow rations was associated with a $0.39/d per cow increase in ROF . In a separate model (R(2) = 0.27) that examined management factors independent of production variables, herds using 3 times daily milking had a $1.25/d per cow higher ROF vs . herds using twice daily, whereas use of an Escherichia coli mastitis vaccine was associated with $0.59/d per cow higher ROF . Production-related variables accounted for more variation in the ROF index than management variables, and the latter, e.g., use of monensin, only marginally increased R(2) of production-based regression models.

J Biol Chem . 2004 Dec 8; {Epub ahead of print}
Catalytic mechanism of chlamydia trachomatis flavin dependent thymidylate synthase; Griffin J et al.; Here we report on a Chlamydia trachomatis gene that complements the growth defect of a thymidylate synthase deficient strain of Escherichia coli . The complementing gene encodes a 60.9 kDa protein which shows low level primary sequence homology to a new class of thymidylate synthesizing enzymes, termed folate dependent thymidylate synthases (FDTS) . Purified recombinant chlamydial FDTS (CTthyX) contains bound flavin . Results with site directed mutants indicate that highly conserved arginine residues are required for flavin binding . Kinetic characterization indicates that CTThyX is active as a tetramer with NADPH, CH2H4folate, and dUMP required as substrates, serving as source of reducing equivalents, methyl donor, and methyl acceptor, respectively . dTMP and H4folate are products of the reaction . Production of H4folate rather than H2folate, as in the classical thymidylate synthase reaction, eliminates the need for dihydrofolate reductase explaining the trimethoprim resistant phenotype displayed by thyA- E.coli expressing CTThyX . In contrast to the extensively characterized thyA encoded thymidylate synthases, which form a ternary complex with substrates dUMP and CH2H4folate and follow an ordered sequential mechanism, CTThyX follows a ping-pong kinetic mechanism involving a methyl enzyme intermediate . Mass spectrometry was used to localize the methyl group to a highly conserved arginine and site directed mutagenesis showed this arginine to be critical for thymidylate synthesizing activity . These differentiating characteristics clearly distinguish FDTS from ThyA, making this class of enzymes attractive targets for rational drug design.

Mol Biol Evol . 2004 Dec 8; {Epub ahead of print}
Comparison of the Accuracies of Several Phylogenetic Methods Using Protein and DNA Sequences; Hall BG; A biologically realistic method was used to simulate evolutionary trees . The method uses a real DNA coding sequence as the starting point, simulates mutation according to the mutational spectrum of Escherichia coli including base substitutions, insertions and deletions, and separates the processes of mutation and selection . Trees of 8, 16, 32 and 64 taxa were simulated with average branch lengths of 50, 100, 150, 200 and 250 changes per branch . The resulting sequences were aligned with ClustalX and trees were estimated by Neighbor Joining, Parsimony, Maximum Likelihood and Bayesian methods from both DNA sequences and the corresponding protein sequences . The estimated trees were compared with the true trees and both topological and branch length accuracies were scored . Over the variety of conditions tested Bayesian trees estimated from DNA sequences that had been aligned according to the alignment of the corresponding protein sequences were the most accurate, followed by Maximum Likelihood trees estimated from DNA sequences and Parsimony trees estimated from protein sequences.

Microbiol Mol Biol Rev, 2004 Dec, 68(4), 639 - 68
Control of rRNA synthesis in Escherichia coli: a systems biology approach; Dennis PP et al.; The first part of this review contains an overview of the various contributions and models relating to the control of rRNA synthesis reported over the last 45 years . The second part describes a systems biology approach to identify the factors and effectors that control the interactions between RNA polymerase and rRNA (rrn) promoters of Escherichia coli bacteria during exponential growth in different media . This analysis is based on measurements of absolute rrn promoter activities as transcripts per minute per promoter in bacterial strains either deficient or proficient in the synthesis of the factor Fis and/or the effector ppGpp . These absolute promoter activities are evaluated in terms of rrn promoter strength (V(max)/K(m)) and free RNA polymerase concentrations . Three major conclusions emerge from this evaluation . First, the rrn promoters are not saturated with RNA polymerase . As a consequence, changes in the concentration of free RNA polymerase contribute to changes in rrn promoter activities . Second, rrn P2 promoter strength is not specifically regulated during exponential growth at different rates; its activity changes only when the concentration of free RNA polymerase changes . Third, the effector ppGpp reduces the strength of the rrn P1 promoter both directly and indirectly by reducing synthesis of the stimulating factor Fis . This control of rrn P1 promoter strength forms part of a larger feedback loop that adjusts the synthesis of ribosomes to the availability of amino acids via amino acid-dependent control of ppGpp accumulation.

J Biol Chem . 2004 Dec 8; {Epub ahead of print}
A novel putrescine utilization pathway involves gamma-glutamylated intermediates of Escherichia coli K-12; Kurihara S et al.; A novel bacterial putrescine utilization pathway was discovered . Seven genes, the functions of whose products were not known, are involved in this novel pathway . Five of them encode enzymes which catabolize putrescine, one encodes a putrescine importer, and the other, a transcriptional regulator . This novel pathway involves six sequential steps: 1) import of putrescine, 2) ATP-dependent gamma-glutamylation of putrescine, 3) oxidization of gamma-glutamyl-putrescine (gamma-Glu-Put), 4) dehydrogenation of gamma-glutamyl-gamma-aminobutyraldehyde, 5) hydrolysis of the gamma-glutamyl linkage of gamma-glutamyl-gamma-aminobutyrate (gamma-Glu-GABA), and 6) transamination of gamma-aminobutyrate (GABA) to form the final product of this pathway, succinate semialdehyde, which is the precursor of succinate.

Curr Eye Res, 2004 Oct-Nov, 29(4-5), 337 - 47
Expression of calpain small subunit 2 in mammalian tissues; Ma H et al.; Purpose . The purpose of the current experiments was to more closely define the distribution and the function of calpain small subunit 2 (css2) . Css2 is a newly discovered regulatory protein for the calcium activated proteases, mu- and m-calpains . Methods . Tissues from rat, monkey, and man of various ages were used to determine expression patterns of css2 by relative quantitative RT-PCR using 18S rRNA as an endogenous standard . Recombinant css2 and the 80kDa catalytic subunit of m-calpain (80 kDa/css2) were co-expressed in Escherichia coli . Casein zymography was used to measure the enzymatic activity of 80 kDa/css2 proteins . Lens alpha-crystallin and betaB1-crystallin were used as substrates to determine proteolysis by 80 kDa/css2 . Computer-based homology modeling was used to predict interactions between the traditional small subunit (css1) or css2 with the 80kDa catalytic subunit . Results . Css2 appears to be a functional equivalent of css1 in vitro in that the calcium-dependent proteolytic activity of 80 kDa/css2 was similar to recombinant m-calpain (80 kDa/css1) . In rat and human lens, css2 transcripts increased with age, whereas css1 transcripts decreased with age . Human betaB1-crystallin and rat alphaA-crystallin were cleaved similarly by 80 kDa/css2 and 80 kDa/css1 . Interestingly, alphaA-insert crystallin was not hydrolyzed when css2 was substituted for css1 in the calpain dimer, suggesting that css2 may perform different functions from css1 in terms of proteolysis of lens crystallins during maturational growth of the lens . Css2 may also assist in the proper folding of the 80kDa subunit and regulate protease activity in the absence of calcium . Conclusions . The wide distribution of css2 transcripts in rat and monkey suggested that css2 is a second, widely distributed (rather than tissue-specific) calpain small subunit, in addition to the long-recognized css1 . Further studies at the protein level will indicate if css2 has unique functions apart from css1.

DNA Repair (Amst), 2005 Feb 3, 4(2), 221 - 229
Cellular response to horizontally transferred DNA in Escherichia coli is tuned by DNA repair systems; Delmas S et al.; We studied how DNA divergence between recombining DNAs and the mismatch repair system modulate the SOS response in Escherichia coli . The observed positive log-linear correlation between SOS induction and DNA divergence, and the negative correlation between SOS induction and frequency of recombination, suggest that the level of SOS induction precisely reflects the difficulty of RecA protein to initiate a productive strand exchange process . Our results suggest that the mismatch repair system could contribute to this SOS induction more by affecting the RecA-catalyzed homology search than by acting on mismatched recombination intermediates . The propensity of the recombination machinery to promote recombination between the blocks of sequences with the highest identity results in the increasing ratios of merodiploids (partial diploids) over genuine recombinants (homologous replacements) with increasing DNA divergence . We discuss the role of molecular mechanisms involved in the control of the recombination between diverged DNA sequences in the maintenance of genomic stability and genome evolution.

DNA Repair (Amst), 2005 Feb 3, 4(2), 163 - 70
The yeast Snm1 protein is a DNA 5'-exonuclease; Li X et al.; Interstrand cross-links (ICL) in DNA arise from bifunctional alkylating agents, including nitrogen mustards, mitomycin C and psoralens . Such adducts prevent normal transcription or replication and are mutagenic . Therefore, cellular mechanisms for removing ICL damage are needed to maintain genome stability . Normal ICL repair requires the action of a number of genes, some specific for such damage . The yeast Snm1 protein is one such protein, but its function has been unknown . Incision for ICL repair is normal in mutants lacking Snm1, so it appears to act after the earliest steps . We have used recombinant SNM1 constructs in an Escherichia coli (E . coli) expression system to demonstrate that the yeast gene encodes a 5'-exonuclease . The exonuclease activity is required for Snm1 to be functional in ICL repair.

Biosens Bioelectron, 2005 Jan 15, 20(7), 1380 - 7
Preparation of inert magnetic nano-particles for the directed immobilization of antibodies; Fuentes M et al.; Various activated supports (cyanogen bromide, glutaraldehyde, epoxy-chelates, primary amino) were evaluated for the immobilization of IgG anti-horseradish peroxidase . Cyanogen bromide and glutaraldehyde supports greatly reduced the recognition capacity of the antigen, probably due to the incorrect orientation of the antibody on the support . Hetero-functional epoxy-chelate and immobilization by the sugar chain on primary amino groups had little effect on high recognition of the antigen (near to the theoretically expected value) . However, the immobilization by the sugar chain resulted in a higher adsorption rate of horseradish peroxidase, possibly due to a favourable orientation on a flexible spacer arm) . Antibodies immobilized on aminated surfaces showed two major drawbacks . Firstly, the biological activity of the immobilized antibody sharply decreased over several days when stored at low ionic strength, although this effect could be partially reversed by incubation at high ionic strength . Secondly, a high level of non-specific proteins adsorption on the support surface was observed . Both problems could be successfully resolved by controlling the coating of the support with aldehyde-aspartic-dextran . We propose that the loss of biological activity was related to the ionic adsorption of the immobilized antibody on the support surface, leading to a blocking of the recognition areas . This optimized protocol was applied to the immobilization of IgG anti-horseradish peroxidase from rabbit on magnetic nano-particles . A 10mug preparation of nanoparticles was able to capture more than 75% of the 0.1 microgram of recombinant horseradish peroxidase present in 10L of crude protein extract (1g/L) from Escherichia coli.

FEBS Lett, 2004 Dec 17, 578(3), 262 - 8
Structural modeling of dual-affinity purified Pho84 phosphate transporter; Lagerstedt JO et al.; The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments . The three-dimensional (3D) organization of the Pho84 protein has not yet been determined . However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined . On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER . To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12 . Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84 . The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.

Adv Protein Chem, 2004, 69, 229 - 64
Properties and functions of Escherichia coli: Pol IV and Pol V; Fuchs RP et al.; Escherichia coli possesses two members of the newly discovered class of Y DNA polymerases (Ohmori et al., 2001): Pol IV (dinB) and Pol V (umuD'C) . Polymerases that belong to this family are often referred to as specialized or error-prone DNA polymerases to distinguish them from the previously discovered DNA polymerases (Pol I, II, and III) that are essentially involved in DNA replication or error-free DNA repair . Y-family DNA polymerases are characterized by their capacity to replicate DNA, through chemically damaged template bases, or to elongate mismatched primer termini . These properties stem from their capacity to accommodate and use distorted primer templates within their active site and from the lack of an associated exonuclease activity . Even though both belong to the Y-family, Pol IV and Pol V appear to perform distinct physiological functions . Although Pol V is clearly the major lesion bypass polymerase involved in damage-induced mutagenesis, the role of Pol IV remains enigmatic . Indeed, compared to a wild-type strain, a dinB mutant exhibits no clear phenotype with respect to survival or mutagenesis following treatment with DNA-damaging agents . Subtler dinB phenotypes will be discussed below . Moreover, despite the fact that both dinB and umuDC loci are controlled by the SOS response, their constitutive and induced levels of expression are dramatically different . In noninduced cells, Pol V is undetectable by Western analysis . In contrast, it is estimated that there are about 250 copies of Pol IV per cell . On SOS induction, it is believed that only about 15 molecules of Pol V are assembled per cell (S . Sommer, personal communication), whereas Pol IV levels reach approximately 2500 molecules . In fact, despite extensive knowledge of the individual enzymatic properties of all five E . coli DNA polymerases, much more work is needed to understand how their activities are orchestrated within a living cell.

J Mol Biol, 2005 Jan 28, 345(4), 827 - 36
Three-state kinetic folding mechanism of the H2A/H2B histone heterodimer: the N-terminal tails affect the transition state between a dimeric intermediate and the native dimer; Placek BJ et al.; The H2A/H2B heterodimer is a component of the nucleosome core particle, the fundamental repeating unit of chromatin in all eukaryotic cells . The kinetic folding mechanism for the H2A/H2B dimer has been determined from unfolding and refolding kinetics as a function of urea using stopped-flow, circular dichroism and fluorescence methods . The kinetic data are consistent with a three-state mechanism: two unfolded monomers associate to form a dimeric intermediate in the dead-time of the SF instrument (approximately 5 ms); this intermediate is then converted to the native dimer by a slower, first-order reaction . Analysis of the burst-phase amplitudes as a function of denaturant indicates that the dimeric kinetic intermediate possesses approximately 50% of the secondary structure and approximately 60% of the surface area burial of the native dimer . The stability of the dimeric intermediate is approximately 30% of that of the native dimer at the monomer concentrations employed in the SF experiments . Folding-to-unfolding double-jump experiments were performed to monitor the formation of the native dimer as a function of folding delay times . The double-jump data demonstrate that the dimeric intermediate is on-pathway and obligatory . Formation of a transient dimeric burst-phase intermediate has been observed in the kinetic mechanism of other intertwined, segment-swapped, alpha-helical, DNA-binding dimers, such as the H3-H4 histone dimer, Escherichia coli factor for inversion stimulation and E.coli Trp repressor . The common feature of a dimeric intermediate in these folding mechanisms suggests that this intermediate may accelerate protein folding, when compared to the folding of archael histones, which do not populate a transient dimeric species and fold more slowly.

J Mol Biol, 2005 Jan 28, 345(4), 717 - 30
New non-detrimental DNA-binding mutants of the Escherichia coli initiator protein DnaA; Asklund M et al.; The initiator protein DnaA has several unique DNA-binding features . It binds with high affinity as a monomer to the nonamer DnaA box . In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin . We have carried out an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR . We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins . The selection gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized . The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino acid residues are located between positions 30 and 80 of the 94 residue domain . Residues where substitutions with non-closely related amino acids have very little effect on protein function are located primarily on the periphery of the 3D structure . By comparison of the effect of substitutions on the activity for initiation of replication with the activity for repression of the mioC promoter, we identified residues that might be involved specifically in the cooperative interaction with ATP-DnaA boxes.

J Mol Biol, 2005 Jan 28, 345(4), 659 - 66
Identification of an RNA-binding domain in human SRP72; Iakhiaeva E et al.; The signal recognition particle (SRP) is a ribonucleoprotein complex that plays a crucial role during the delivery of secretory proteins from the ribosome to the cell membrane . Among the six proteins of the eukaryotic SRP, the 72 kDa protein (SRP72) is the largest and least characterized . Polypeptides corresponding to various regions of the entire human SRP72 sequence were expressed in Escherichia coli, purified, and partially proteolyzed . Human SRP RNA bound with high affinity to a 63 amino acid residue region near the C terminus of SRP72 . Mild treatment of the fragment with chymotrypsin abolished its RNA-binding activity . A conserved sequence with the consensus PDPXRWLPXXER was identified within a 56 amino acid residue RNA-binding domain . Sucrose gradient centrifugation and filter-binding analysis using mutant SRP RNAs showed that SRP72 bound to the moderately conserved portion of SRP RNA helix 5 . Nine tetratricopeptide-like repeats (TPRs) poised to interact with other SRP or ribosomal proteins were predicted in the NH2-terminal region . These identifications assign two important functions to a large portion of SRP72 and demonstrate the RNA-binding capacity of the protein.

AIDS Res Hum Retroviruses, 2004 Nov, 20(11), 1269 - 81
Mucosal and systemic anti-HIV responses in rhesus macaques following combinations of intranasal and parenteral immunizations; Vajdy M et al.; There is an urgent need to develop vaccines that can elicit immunological memory responses against HIV . Using the rhesus macaque model and a combination of intranasal (IN) and parenteral immunizations with DNA or protein adsorbed to microparticles or mixed with mucosal adjuvants we sought to induce anti-HIV memory-type immune responses in both the mucosal and systemic compartments . Prime/boost immunizations were performed through five IN immunizations alone with HIV-env oligomeric gp140 (Ogp140) or HIV-gag-p24 mixed with Escherichia coli heat labile-derived mutant adjuvants or two parenteral immunizations with DNA encoding HIV-env or -gag adsorbed to microparticles followed by three IN immunizations with p24 gag protein and the mutant adjuvants . Both modes of immunizations induced anti-gp140 plasma and vaginal IgG and IgA as well as interferon (IFN)-gamma secreting peripheral blood mononuclear cells (PBMC) after HIV-env and -gag peptide restimulation . After a resting period of 4 months, when the levels of humoral and cellular responses had decreased, intramuscular (IM) booster immunizations with p55-gag protein adsorbed to microparticles and Ogp140 in MF59 oil in water emulsion significantly enhanced anti-HIV plasma and vaginal antibody, as well as peripheral blood IFN-gamma responses in all groups of vaccinated macaques . Importantly, plasma neutralization activity against both homologous and heterologous HIV strains was observed in all groups following the IM booster immunizations with protein . These findings show that IN priming alone or combinations of parenteral and IN immunizations followed by IM booster immunizations hold promise to significantly enhance mucosal and systemic memory-type immune responses against HIV-1 antigens.

Biochem J . 2004 Dec 9; {Epub ahead of print}
Ypr140wp, "the yeast tafazzin", displays a mitochondrial lyso-PC acyltransferase activity related to triglyceride and mitochondrial lipid synthesis; Testet E et al.; When the yeast protein Ypr140w was expressed in Escherichia coli, a lyso-PC acyltransferase activity was found associated with the membranes of the bacteria . To our knowledge, this is the first identification of a protein able to catalyse the acylation of lyso-PC molecules to form PC . Fluorescence microscopy analysis of living yeasts revealed that Ypr140w-GFP fusion protein is targeted to the mitochondria . Moreover, in contrast with wild type cells, in the absence of acyl-CoA, the yeast mutant deleted for the YPR140w gene has no lyso-PC acyltransferase activity associated with the mitochondrial fraction . When yeast cells were grown in the presence of lactate, the mutant synthesized two-fold more triglycerides than the wild type . Moreover its mitochondrial membranes contained a reduced amount of phosphatidylcholine and cardiolipin, and the fatty acid composition of these latter was greatly changed . These modifications were accompanied by a two-fold increase in the respiration rates (state 3 and state 4) of the mitochondria . The relationship between the deletion of the YPR140w gene and the lipid composition of the ypr140wDelta cells is discussed.

Physiol Res, 2004, 53(6), 669 - 74
Fluid secretory responses to enterotoxin STa and 8-bromo-cyclic GMP in fed and nutrionally-deprived gerbils: jejunum, ileum and colon in vivo; Al-Balool FY; Fluid transport was measured gravimetrically in vivo in the jejunum, ileum and colon of fed, fasting (four days) and undernourished (50 % of control food intake for 21 days) gerbils (Gerbillus cheesmani) . The effects of luminal enterotoxin Escherichia coli STa (50 ng/ml) and luminal 8-bromo-cyclic GMP (cGMP 1 mM) on fluid transport across jejunum, ileum and colon were also assessed . Fasting and undernourishment reversed the normal basal fluid absorption measured in fed ileum and colon into secretion . Neither fasting nor undernourishment had any effect on jejunal basal fluid absorption . In jejuna, ilea and colons of fed animals as well as in jejuna from fasting and undernourished gerbils STa (50 ng) reversed the normal absorptive "tone" to secretion but it had no significant effects on fluid secretion in either the ileum or colon from fasted gerbils . STa increased significantly the fluid secretion in ileum from undernourished gerbils . Luminal cGMP had no effect on basal absorptive tone in the jejunum of fed and fasted gerbils, but reversed absorption into secretion in the jejuna from undernourished gerbils . In the ilea taken from fed animals the small basal absorption was reversed to secretion by luminal cGMP . Although cGMP produced no significant changes in fluid secretion in the ilea taken from fasted gerbils, yet it caused a significant increase in those from undernourished gerbils . In the colon taken from fed animals cGMP decreased the basal fluid absorption significantly, but it had no significant effect on fluid secretion in the colon of fasted or undernourished gerbils . We conclude that fasting and undernourishment have no significant effects on fluid transport across the gerbil jejunum but reversed basal absorption in the fed ileum and colon into secretion . cGMP mimic the effects of STa in the jejunum taken from undernourished gerbils, in the ileum obtained under the three nutritional states and in the colon taken from fasting animals.

Transgenic Res, 2004 Oct, 13(5), 397 - 410
Development of a hybrid delta-endotoxin and its expression in tobacco and cotton for control of a polyphagous pest Spodoptera litura; Singh PK et al.; A hybrid delta-endotoxin protein was designed against a polyphagous lepidopteran insect pest Spodoptera litura, which is tolerant to most of the known delta-endotoxins . The hybrid delta-endotoxin was created by replacing amino acid residues 530-587 in a poorly active natural Cry1Ea protein, with a highly homologous 70 amino acid region of Cry1Ca in domain III . The truncated delta-endotoxins Cry1Ea, Cry1Ca and the hybrid protein Cry1EC accumulated in Escherichia coli to form inclusion bodies . The solubilised Cry1EC made from E . coli was 4- fold more toxic to the larvae of S . litura than Cry1Ca, the best known delta-endotoxin against Spodoptera sp . None of the two truncated toxins, solubilised from E . coli caused larval mortality . However, trypsinised Cry1Ca protoxin obtained from E . coli and solubilised from inclusion bodies caused mortality of S . litura with LC50 513 ng/ml semi synthetic diet . A synthetic gene coding for the hybrid delta-endotoxin Cry1EC was designed for high level expression in plants, taking into consideration several features found in the highly expressed plant genes . Transgenic, single copy plants of tobacco as well as cotton were developed . The selected lines expressed Cry1EC at 0.1-0.7% of soluble leaf protein . Such plants were completely resistant to S . litura and caused 100% mortality in all stages of larval development . Hence, unlike in E . coli, the hybrid delta-endotoxin folded into a functionally active conformation in both tobacco and cotton leaves . The truncated Cry1EC expressed in tobacco leaves was about 8-fold more toxic (LC50 58 ng/ml diet) compared to expression in E . coli.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Aug, 22(4), 231 - 4
{High efficiency expression and antigenicity analysis of the SAG2 gene from Toxoplasma gondii RH strain}; Lei MJ et al.; OBJECTIVE: To make high efficiency expression of the SAG2 gene from Toxoplasma gondii RH strain in E . coli and study the antigenicity of the expressed product . METHODS: The SAG2 gene fragment of T . gondii RH strain amplified by PCR method from genome DNA was cloned into the pMD-18T vector and transformed into E . coli DH5alpha . After nucleotide sequencing, the SAG2 gene fragment was subcloned into the expression vector pET23a with correct orientation and transformed into E . coli DH5alpha . The plasmid from the correct clone identified by PCR method and endonuclease digestion was transformed into E . coli BL21 (DE3) and induced for expression . The expressed product was studied by SDS-PAGE and Western blot . RESULTS: 502 bp gene fragment was amplified by PCR as anticipated . Nucleotide sequencing showed a 100% homology with that of the published sequence in GenBank . The molecular weight of the expressed protein was about Mr 19,000 . Western blotting indicated that the antigenicity of the protein was specific . CONCLUSION: The plasmid pET23a-SAG2 was constructed and a high efficiency expression of the SAG2 gene from T . gondii RH strain was made . The expressed product shows a specific antigenicity.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Aug, 22(4), 193 - 8
{Gene cloning, expression and serological evaluation of diagnostic antigen Em18 for alveolar echinococcosis}; Jiang L et al.; OBJECTIVE: To clone, express and serologically evaluate the Em18 antigen gene of Echinococcus multilocularis for diagnostic purpose . METHODS: Polymerase chain reaction (PCR) was employed for amplification of the target gene fragments which was then ligated with pET28a+ vector . The constructed plasmid was transferred into E . coli BL21 (DE3) for expression . The recombinant proteins were purified with Ni-NTA agarose by affinity chromatography . 237 sera were used for evaluating diagnostic value of the recombinant Em18 antigen . RESULTS: Two high-level expression clones (designated as ReEm18-1 and ReEm18-2) were obtained . ReEm18-1 showed the expected sequence, ReEm18-2 showed the same sequence but with 27 nucleotides deletion . The molecular weight of the two expression proteins was Mr 28,000 and 26,000, respectively . Serological evaluation by ELISA was carried out using sera from 101 patients with alveolar echinococcosis (AE), 47 with cystic echinococcosis (CE), 30 with cysticercosis (CC), 10 with hepatic cancer (HC), 9 with schistosomiasis (Sj) and 40 from healthy persons (NH) from both endemic and non-endemic areas . The results showed an overall sensitivity of 86.1% and 90.1% with ReEm18-1 and ReEm18-2 for AE sera, specificity 93.4% and 94.1%, positive predictive value 90.6% and 91.9%, negative predictive value 90.1% and 92.8% and efficiency 90.3% and 92.4%, respectively . The correlation analysis between the size of AE lesions and the serum absorbance reacted with recombinant Em18 antigens showed that there was a positive correlation between antibody level and the course of the disease . CONCLUSION: ReEm18 antigens are specific for AE diagnosis, and the serum antibody level displays a good correlation with the course of the disease at early stage . Similar results achieved by both ReEm18-1 and ReEm18-2 antigens.

J Vet Diagn Invest, 2004 Nov, 16(6), 576 - 8
Polymerase chain reaction analysis of eae gene subtypes present in attaching and effacing Escherichia coli isolated from pigs with diarrhea; Ha SK et al.; In this study the subtype of eae gene was determined by polymerase chain reaction for a total of 59 attaching and effacing Escherichia coli isolated from preweaned (38 isolates) and postweaned (21 isolates) pigs . The eae(beta) gene detected in 19 E . coli from preweaned pigs and 10 E . coli from postweaned pigs was found to be the most common subtype, followed by eae(gamma), eae(epsilon), and eae(zeta) genes . Subtypes were not determined for 7 E . coli isolates . No other subtype of the eae gene was detected in eae+ E . coli evaluated in this study.

Zhejiang Da Xue Xue Bao Yi Xue Ban, 2004 Nov, 33(6), 519 - 23
{Effects of lactose inducing on expression of Helicobacter pylori rUreB and rHpaA, and Escherichia coli rLTKA63 and rLTB}; Zhao SF et al.; OBJECTIVE: To determine the effects of lactose inducing on the expression of recombinant Helicobacter pylori rUreB and rhpaA, and Escherichia coli rLTB and rLTKA63 . METHODS: BIO-RAD gel image analysis system was applied to detect the outputs of the recombinant proteins . SDS-PAGE was performed to measure the target protein expression of recombinant genes at various periods of growth, different lactose concentrations, various inducing temperatures and times . The results of the target protein expression induced by lactose were compared to those by isopropyl-beta-D-thiogalactoside (IPTG) . RESULTS: Lactose showed higher efficiency to induce the expression of rHpaA, rUreB, rLTB and rLTKA63 than IPTG . The expression outputs of target recombinant proteins induced at 37 degrees C were remarkably higher than those at 28 degrees C . The optimal expression parameters were 0.8 of OD600 value, 50 g/L of lactose, 4 hours of inducing time for rHpaA, and 1.2 of OD600 value, 100 g/L of lactose, 5 hours of inducing time for both the rUreB and rLtB,and 0.8 of OD600 value, 100 g/L of lactose, 4 hours for rLTKA63 . CONCLUSION: Lactose, a sugar with non-toxicity and low cost, is able to induce the recombinant genes to express the target proteins with higher efficiency than IPTG.

Methods Mol Med, 2005, 109, 329 - 46
Producing bispecific and bifunctional antibodies; Das D et al.; Bispecific antibodies are artificially engineered monoclonal antibodies (MAbs) that consist of two distinct binding sites and are capable of binding two different antigens noncovalently . They can be produced by chemical cross-linkage, genetic engineering, or somatic hybridization . This chapter describes a rapid method using somatic fusion to generate hybrid hybridomas (quadromas) . Two fluorescence-labeled hybridoma cell lines were fused with polyethylene glycol (PEG) to generate the quadroma . Generation of a quadroma secreting bsMAb against biotin and HRPO is described, along with a benzhydroxamic acid-agarose affinity chromatography procedure to purify the bsMAb-HRPO complex . This bsMAb can be used for ultrasensitive ELISA detection of biotinylated antigens . Essentially a similar method can be used for fusing any two hybridomas for therapeutic applications . Bifunctional antibodies are colinear molecules with one or more paratopes linked with diagnostic or therapeutic molecules . There are some limitations of therapeutic monoclonal antibodies in the clinic that can be overcome by engineering smaller and more effective antibody fragments . Here we describe a stepwise procedure for developing a bifunctional ScFv (bfScFv) . We constructed a bfScFv from a hybridoma cell line using PCR strategies . The VL and VH gene segments are linked with a 45-bp linker and fused with a biotin mimic sequence at the 3' end . This engineered bifunctional antibody fragment gene could be expressed and the protein purified on a large scale in Escherichia coli as inclusion bodies . Such bifunctional antibody molecules could have useful applications in the area of immunodiagnostics and immunotherapy . Similar strategies can be used to incorporate a second single-chain antibody or any nonantibody entity such as a cytokine for therapeutic applications.

Methods Mol Med, 2005, 109, 137 - 54
Identification of Tumor-Associated Autoantigens With SEREX; Tureci O et al.; Serological analysis of tumor antigens by recombinant cDNA expression cloning (SEREX) allows the systematic cloning of tumor antigens recognized by the spontaneous autoantibody repertoire of cancer patients . For SEREX, cDNA expression libraries are constructed from fresh tumor specimens, packaged into lambda-phage vectors, and expressed recombinantly in Escherichia coli . Recombinant proteins expressed during the lytic infection of bacteria are transferred onto nitrocellulose membranes to be probed with diluted autologous patient serum for identification of clones reactive with high-titered IgG antibodies . This chapter describes the SEREX technology in detail.

J Immunol, 2004 Dec 15, 173(12), 7317 - 23
Plasmablast and plasma cell production and distribution in trout immune tissues; Bromage ES et al.; These studies describe the in vitro and ex vivo generation of plasmablasts and plasma cells in trout (Oncorhynchus mykiss) peripheral blood and splenic and anterior kidney tissues . Cells were derived either from naive trout and cultured with the polyclonal activator, Escherichia coli LPS, or from trout that had been immunized with trinitrophenyl-keyhole limpet hemocyanin . Hydroxyurea was used to resolve populations of replicating (plasmablast) and nonreplicating (plasma cell) Ab-secreting cells (ASC) . Complete inhibition of Ig secretion was only observed within the PBL . Both anterior kidney and splenic lymphocytes possessed a subset of ASCs that were hydroxyurea resistant . Thus, in vitro production of plasma cells appears to be restricted to the latter two tissues, whereas peripheral blood is exclusively restricted to the production of plasmablasts . After immunization with trinitrophenyl-keyhole limpet hemocyanin, specific ASC could be isolated from all immune organs; however, the anterior kidney contained 98% of all ASC . Late in the response (>10 wk), anterior kidney ASC secreted specific Ab for at least 15 days in culture, indicating that they were long-lived plasma cells . Cells from spleen and peripheral blood lost all capacity to secrete specific Ab in the absence of Ag . Late in the Ab response, high serum titer levels are solely the result of Ig secretion from anterior kidney plasma cells.

Proc Natl Acad Sci U S A, 2004 Dec 21, 101(51), 17675 - 80 Epub 2004 Dec 07.
Protein engineering of cytochrome b562 for quinone binding and light-induced electron transfer; Hay S et al.; The central photochemical reaction in photosystem II of green algae and plants and the reaction center of some photosynthetic bacteria involves a one-electron transfer from a light-activated chlorin complex to a bound quinone molecule . Through protein engineering, we have been able to modify a protein to mimic this reaction . A unique quinone-binding site was engineered into the Escherichia coli cytochrome b(562) by introducing a cysteine within the hydrophobic interior of the protein . Various quinones, such as p-benzoquinone and 2,3-dimethoxy-5-methyl-1,4-benzoquinone, were then covalently attached to the protein through a cysteine sulfur addition reaction to the quinone ring . The cysteine placement was designed to bind the quinone approximately 10 A from the edge of the bound porphyrin . Fluorescence measurements confirmed that the bound hydroquinone is incorporated toward the protein's hydrophobic interior and is partially solvent-shielded . The bound quinones remain redox-active and can be oxidized and rereduced in a two-electron process at neutral pH . The semiquinone can be generated at high pH by a one-electron reduction, and the midpoint potential of this can be adjusted by approximately 500 mV by binding different quinones to the protein . The heme-binding site of the modified cytochrome was then reconstituted with the chlorophyll analogue zinc chlorin e(6) . By using EPR and fast optical techniques, we show that, in the various chlorin-protein-quinone complexes, light-induced electron transfer can occur from the chlorin to the bound oxidized quinone but not the hydroquinone, with electron transfer rates in the order of 10(8) s(-1).

J Am Chem Soc, 2004 Dec 15, 126(49), 16242 - 8
Autocatalytic formation of green heme: evidence for H2O2-dependent formation of a covalent methionine-heme linkage in ascorbate peroxidase; Metcalfe CL et al.; The mammalian heme peroxidases are distinguished from their plant and fungal counterparts by the fact that the heme group is covalently bound to the protein through ester links from glutamate and aspartate residues to the heme 1- and 5-methyl groups and, in the case of myeloperoxidase, through an additional sulfonium link from the Cbeta of the 2-vinyl group to a methionine residue . To duplicate the sulfonium link in myeloperoxidase and to obtain information on its mechanism of formation, we have engineered a methionine residue close to the 2-vinyl group in recombinant pea cytosolic ascorbate peroxidase (rpAPX) by replacement of Ser160 by Met (S160M variant) . The S160M variant is isolated from Escherichia coli as apo-protein . Reconstitution of apo-S160M with exogenous heme gives a red protein (S160M(R)) which has UV-visible (lambda(max)/nm = 407, 511, 633) and steady-state kinetic (kcat = 156 +/- 7 s(-1), KM = 102 +/- 15 microM) properties that are analogous to those of rpAPX . The reaction of S160M(R) with H2O2 gives a green protein (S160M(G)) . Electronic spectroscopy, mass spectrometry, and HPLC analyses are consistent with the formation of a covalent linkage between the methionine residue and the heme vinyl group in S160M(G) . Single-wavelength and photodiode array stopped-flow kinetic analyses identify a transient Compound I species as a reaction intermediate . The results provide the first direct evidence that covalent heme linkage formation occurs as an H2O2-dependent process that involves Compound I formation . A mechanism that is consistent with the data is presented.

J Am Chem Soc, 2004 Dec 15, 126(49), 15984 - 9
In situ chemical aminoacylation with amino acid thioesters linked to a peptide nucleic acid; Ninomiya K et al.; tRNA-specific chemical aminoacylation was achieved with nonnatural amino acids . A nonnatural amino acid was activated as a thioester derivative, and the latter was linked through a spacer to the N-terminal of a 9-mer peptide nucleic acid that is complementary to the 3'-terminal region of yeast phenylalanine tRNA . Efficient aminoacylation was observed when the amino acid thioester-spacer-PNA conjugate was mixed with the tRNA . The PNA-assisted aminoacylation was also successful in an Escherichia coli in vitro protein synthesizing system that contained an orthogonalized tRNA . The in situ aminoacylation/in vitro translation gave a mutant protein in which the nonnatural amino acid was incorporated into the position directed by a CGGG 4-base codon/anticodon pair.

J Chromatogr A, 2004 Nov 19, 1057(1-2), 115 - 24
Integrated process for purification of plasmid DNA using aqueous two-phase systems combined with membrane filtration and lid bead chromatography; Kepka C et al.; An integrated process for purifying a 6.1 kilo base pair (kbp) plasmid from a clarified Escherichia coli cell lysate based on an ultra/diafiltration step combined with polymer/polymer aqueous two-phase system and a new type of chromatography is described . The process starts with a volume reduction (ultrafiltration) and buffer exchange (diafiltration) of the clarified lysate using a hollow fibre membrane system . The concentrated and desalted plasmid solution is then extracted in a thermoseparating aqueous two-phase system, where the contaminants (RNA and proteins) to a large extent are removed . While the buffer exchange (diafiltration) is necessary in order to extract the plasmid DNA exclusively to the top phase, experiments showed that the ultrafiltration step increased the productivity of the aqueous two-phase system by a factor of more than 10 . The thermoseparated water phase was then subjected to a polishing step using lid bead chromatography . Lid beads are a new type of restricted access chromatography beads, here with a positively charged inner core that adsorbed the remaining RNA while its inert surface layer prevented adsorption of the plasmid DNA thus passing in the flow-through of the column . Differently-sized plasmid DNA in the range of 2.7-20.5 kbp were also partitioned in the aqueous two-phase system . Within this size range, all plasmid DNA was exclusively extracted to the top phase . The complete process is free of additives and easy scalable for use in large scale production of plasmid DNA . The overall process yield for plasmid DNA was 69%.

Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2004 Dec, 21(6), 548 - 51
{Cloning, ligation and expression of the variable region genes of the monoclonal antibody against human HnRNPA2/B1.}; Wang X et al.; OBJECTIVE: To clone the variable region genes of the monoclonal antibody (McAb) against human heterogeneous nuclear ribonucleoprotein A2/B1 (HnRNPA2/B1), ligate them to assemble single chain Fv (ScFv) gene and express in Escherichia coli . METHODS: The specificity of the anti-HnRNPA2/B1 McAb 3E8 to synthetic HnRNPA2/B1 peptide, HnRNPA2/B1 protein in lung cancer cells were examined by dot-immunobinding assay, Western blot and immunohistochemistry . The variable region genes of heavy chain (VH) and light chain (VL) were amplified from hybridoma cell by reverse transcription-polymerase chain reaction(RT-PCR), and then were linked by a linker peptide using SOE-PCR (splicing by overlap extension-PCR) to construct recombination ScFv gene . The latter was cloned into the expression vector pET28 (a+) and expressed in E coli BL21 . The expressed product was identified by SDS-PAGE and competitive ELISA inhibition test . RESULTS: It was shown that the McAb combined specifically with synthetic HnRNPA2/B1 peptide and HnRNPA2/B1 protein in three lung cancer cells . The cloned VH gene and VL gene were 345 bp and 309 bp respectively and were linked successfully to obtain ScFv gene . The ScFv protein was expressed in the form of inclusion body, with molecular weight of 28,000 and immunoreactivity to HnRNPA2/B1 . CONCLUSION: VH gene, VL gene and ScFv gene of anti-HnRNPA2/B1 antibody were cloned, constructed and functionally expressed in E coli . These results provide the experimental basis for elucidating the role of HnRNPA2/B1 in lung cancer.

Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2399 - 402 Epub 2004 Dec.
Crystallography of the integral membrane protein EmrE from Escherichia coli; Ma C et al.; Crystals of the EmrE membrane-protein imposed several technical challenges for X-ray crystallography, including high mosaicity, poor diffraction and a relatively large number of heavy atoms . Consequently, the heavy-atom substructure solution was difficult to obtain . By removing the histidine tag for protein purification, the mosaicity and the diffraction quality were greatly improved . The direct-methods Shake-and-Bake program SnB was successful in locating the heavy-atom sites from a mutant of EmrE which lacks a cysteine and therefore has a reduction in the number of heavy-atom sites . The substructure solution was solved from data with anomalous difference at a resolution of 5.5 A and the structure was determined to 3.8 A.

Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2389 - 90 Epub 2004 Dec.
Escherichia coli stress protein YciF: expression, crystallization and preliminary crystallographic analysis; Liu D et al.; The Escherichia coli stress protein YciF was overexpressed and purified by three chromatographic steps . Crystals were obtained using ammonium sulfate as a precipitant . The YciF protein crystals diffracted beyond 2.25 A resolution using a rotating-anode X-ray source . The lattice type is rhombohedral, with unit-cell parameters a = b = 80.0, c = 131.03 A, alpha = beta = 90.0, gamma = 120.0 degrees . The crystal belongs to space group R32.

Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2387 - 8 Epub 2004 Dec.
Crystallization and preliminary crystallographic studies of human coactosin-like protein (CLP); Li X et al.; The human coactosin-like protein (CLP) belongs to the actin-depolymerizing factor (ADF) family of actin-binding proteins . CLP interacts with 5-lipoxygenase (5LO) and filamentous actin (F-actin) via different binding sites . The full-length CLP comprising of 142 amino acids has been overexpressed in Escherichia coli . Crystals of CLP were obtained using the hanging-drop vapour-diffusion technique with ammonium sulfate as precipitant at pH 8.5 . Diffraction data to 1.9 A resolution were collected from a crystal belonging to space group P2(1), with unit-cell parameters a = 25.6, b = 55.2, c = 37.4 A, beta = 96.0 degrees . There is one molecule per asymmetric unit.

Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2380 - 2 Epub 2004 Dec.
Crystallization and preliminary X-ray diffraction analysis of the unliganded human growth hormone receptor; McKinstry WJ et al.; The crystal structure of the extracellular domain of growth hormone receptor complexed to its ligand, growth hormone, has been known since 1992 . However, no information exists for the unliganded form of the receptor . The human growth hormone receptor's extracellular ligand-binding domain, encompassing amino-acid residues 1-238, has been expressed in Escherichia coli, purified by anion ion-exchange chromatography and crystallized in its unliganded state by the hanging-drop vapour-diffusion method in 100 mM HEPES pH 7.0 containing 27.5%(w/v) PEG 5000 monomethyl ether and 200 mM ammonium sulfate as the co-precipitants . The crystals belong to the othorhombic space group C222(1), have unit-cell parameters a = 99.7, b = 112.2, c = 93.2 A and diffract to 2.5 A resolution using synchrotron radiation . The crystal structure will shed light on the nature of any conformation changes that occur upon ligand binding and will provide information to develop potential low-molecular-weight agonists/antagonists to treat clinical diseases in which the growth hormone receptor is implicated.

J Clin Microbiol, 2004 Dec, 42(12), 5904 - 8
Characterization of hemolytic Escherichia coli strains in ferrets: recognition of candidate virulence factor CNF1; Marini RP et al.; Diseases associated with Escherichia coli infection are the subject of renewed interest due to emerging conditions such as hemolytic uremia syndrome . A collection of 15 strains of beta-hemolytic E . coli was isolated from diarrheic feces and diseased tissues of ferrets . All 15 strains were positive in specific PCR assays for the presence of hlyA, pap1, and cnf1 . Seven of the cnf1-positive isolates were tested and shown to have a cytopathic effect on HeLa cell monolayers . The pathogenesis of these strains warrants future study.

J Clin Microbiol, 2004 Dec, 42(12), 5502 - 11
Exploration of biases that affect the interpretation of restriction fragment patterns produced by pulsed-field gel electrophoresis; Singer RS et al.; Pulsed-field gel electrophoresis (PFGE) has been used extensively in epidemiological investigations of bacteria, especially during food-borne outbreaks or nosocomial infections . The relationship between similarities in PFGE patterns and true genetic relatedness is poorly understood . In this study, computer-simulated populations of Escherichia coli isolates were created by mutating the sequence of E . coli K-12 strain MG1655 . The simulated populations of isolates were then digested, again through simulation, with different restriction enzymes and were analyzed for their relatedness by different techniques . Errors associated with band determination and band matching were incorporated into the analyses, as both of these error types have been shown to affect PFGE interpretations . These errors increased the apparent similarities of the isolates . The use of multiple enzymes improved the fidelity between the results of PFGE analyses and the true sequence similarities . These findings, when they are combined with results from laboratory studies, emphasize the need for the inclusion of multiple enzymes and additional epidemiological data in order to make more accurate interpretations.

Microbiology, 2004 Dec, 150(Pt 12), 4171 - 80
Characterization of a temperature-sensitive DNA ligase from Escherichia coli; Lavesa-Curto M et al.; DNA ligases are essential enzymes in cells due to their ability to join DNA strand breaks formed during DNA replication . Several temperature-sensitive mutant strains of Escherichia coli, including strain GR501, have been described which can be complemented by functional DNA ligases . Here, it is shown that the ligA251 mutation in E . coli GR501 strain is a cytosine to thymine transition at base 43, which results in a substitution of leucine by phenylalanine at residue 15 . The protein product of this gene (LigA251) is accumulated to a similar level at permissive and non-permissive temperatures . Compared to wild-type LigA, at 20 degrees C purified LigA251 has 20-fold lower ligation activity in vitro, and its activity is reduced further at 42 degrees C, resulting in 60-fold lower ligation activity than wild-type LigA . It is proposed that the mutation in LigA251 affects the structure of the N-terminal region of LigA . The resulting decrease in DNA ligase activity at the non-permissive temperature is likely to occur as the result of a conformational change that reduces the rate of adenylation of the ligase.

Microbiology, 2004 Dec, 150(Pt 12), 4137 - 45
Transcriptional and translational analysis of the ccaR gene from Streptomyces clavuligerus; Wang L et al.; CcaR is a positive-acting transcriptional regulator involved in cephamycin C and clavulanic acid biosynthesis in Streptomyces clavuligerus . Previous sequence analyses of the ccaR gene revealed two possible start codons, an ATG, and a GTG located in-frame 18 bp downstream of the ATG . To determine the true start codon, ccaR was expressed, either from the GTG or ATG codon, in Escherichia coli . A protein product was only obtained from the ATG construct . Similarly, ccaR constructs originating from ATG or GTG and designed for expression from a glycerol-regulated promoter in Streptomyces species were prepared and used to complement a S . clavuligerus ccaR mutant . Bioassays showed that only the ATG construct could complement the ccaR mutant to restore cephamycin C production, and Western analysis confirmed the presence of CcaR in the mutant complemented with the ATG construct only . To ensure that expression of ccaR from its native promoter also initiated at the ATG rather than GTG, a conservative point mutation was introduced into ccaR, converting the GTG to GTC . The GTC construct still fully complemented a ccaR mutant, confirming that ATG is the true start codon . Inspection of the region upstream of ccaR by S1 nuclease protection and primer extension analyses indicated the presence of two transcript start points that mapped to residues located 74 and 173 bp upstream of the ATG codon.

J Biol Chem . 2004 Dec 6; {Epub ahead of print}
Involvement of a flavosemiquinone in the enzymatic oxidation of nitroalkanes catalyzed by 2-nitropropane dioxygenase; Francis K et al.; 2-Nitropropane dioxygenase (E.C . 1.13.11.32) catalyzes the oxidation of nitroalkanes into their corresponding carbonyl compounds and nitrite . In this study, the recombinant enzyme expressed in Escherichia coli was characterized in its biochemical and mechanistic properties . With neutral nitroalkanes and anionic nitronates other then propyl-nitronates, for which a non-enzymatic free radical reaction involving superoxide was established using superoxide dismutase, substrate oxidation occurs within the enzyme active site . The enzyme was more specific for nitronates than nitroalkanes, as suggested by the second order rate constant kcat/Km determined with 2-nitropropane and primary nitroalkanes with alkyl chain lengths between 2 and 6 carbons . The steady state kinetic mechanism with 2-nitropropane, nitroethane, nitrobutane, and nitrohexane, in either the neutral or anionic form, was determined to be sequential, consistent with oxygen reacting with a reduced form of enzyme before release of the product . Enzyme monitored turnover with ethylnitronate indicated that the catalytically relevant reduced form of enzyme is an anionic flavin semiquinone, whose formation requires the substrate, but not O2, as suggested by anaerobic substrate reduction with nitroethane or ethylnitronate . Substrate deuterium kinetic isotope effects with 1,2-{2H4}-nitroethane and 1,1,2-{2H3}-ethylnitronate at pH 8 yielded normal and inverse effects on the kcat/Km value, respectively, and were negligible on the kcat value . The kcat/Km and kcat pH profiles with anionic nitronates showed the requirement of a Lewis acid, whereas those for neutral nitroalkanes were consistent with the involvement of both a Lewis acid and a base in catalysis . The kinetic data are consistent with an oxidase-style catalytic mechanism for 2-nitropropane dioxygenase, in which the flavin-mediated oxidation of the substrate and the subsequent oxidation of the enzyme-bound flavin occur in two independent steps, and with the participation of a flavosemiquinone in catalysis . A chemical mechanism for the oxidation of both anionic nitronates and neutral nitroalkanes catalyzed by 2-nitropropane dioxygenase is discussed.

J Virol Methods, 2005 Jan, 123(1), 41 - 8
A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor; Chou CF et al.; Severe acute respiratory syndrome (SARS), a life-threatening disease, is caused by the newly identified virus SARS coronavirus (SARS-CoV) . In order to study the spike (S) protein of this highly contagious virus, we established a clonal cell-line, CHO-SG, from the Chinese hamster ovary cells that stably expresses C-terminally EGFP-tagged SARS-CoV S protein (S-EGFP) . The ectodomain of the S glycoprotein is localized on the surface of CHO-SG cells with N-acetyl-glucosamine-terminated carbohydrate structure . CHO-SG cells associated tightly with Vero E6 cells, a SARS-CoV receptor (ACE2) expressing cell-line, and the interaction remained stable under highly stringent condition (1M NaCl) . This interaction could be blocked by either the serum from a SARS convalescent patient or a goat anti-ACE2 antibody, indicating that the interaction is specific . A binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit anti-sera raised against five denatured recombinant S protein fragments expressed in Escherichia coli . One of the sera obtained from the fragment encompassing amino acids 48-358 significantly blocked the interaction between CHO-SG and Vero E6 cells . The region is useful for studying neutralizing antibodies in future vaccine development . This paper describes an easy and safe cell-based assay suitable for studying the binding between SARS-CoV S protein and its receptor.

Comp Immunol Microbiol Infect Dis, 2005 Mar, 28(2), 103 - 20
Expression and characterization of the recombinant swine interleukin-6; Nuntaprasert A et al.; The swine interleukin-6 (SwIL-6) cDNA was cloned by RT-PCR and each expression system of recombinant SwIL-6 in Escherichia coli, insect cells, and mammalian cells was developed . Recombinant SwIL-6 produced in bacteria was applied for generation of the polyclonal antibodies . The rSwIL-6 was purified from supernatant of insect cells with a Q-sepharose or anti-SwIL-6 monoclonal antibody based immunoaffinity column . The antibodies showed that the molecular weight of rSwIL-6 was approximately 26kDa in E . coli, 25, 26, 30kDa in insect cells, and 26 and 30kDa in mammalian cells . These variations of molecular weight were probably due to the different modifications of glycosylation . All these recombinant proteins retained the antigenicity and biological activity on 7TD1 mouse cells.

Virology, 2005 Jan 5, 331(1), 128 - 35
Immune responses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine; Zhao P et al.; The nucleocapsid (N) protein of SARS-coronavirus (SARS-CoV) is the key protein for the formation of the helical nucleocapsid during virion assembly . This protein is believed to be more conserved than other proteins of the virus, such as spike and membrane glycoprotein . In this study, the N protein of SARS-CoV was expressed in Escherichia coli DH5alpha and identified with pooled sera from patients in the convalescence phase of SARS . A plasmid pCI-N, encoding the full-length N gene of SARS-CoV, was constructed . Expression of the N protein was observed in COS1 cells following transfection with pCI-N . The immune responses induced by intramuscular immunization with pCI-N were evaluated in a murine model . Serum anti-N immunoglobulins and splenocytes proliferative responses against N protein were observed in immunized BALB/c mice . The major immunoglobulin G subclass recognizing N protein was immunoglobulin G2a, and stimulated splenocytes secreted high levels of gamma interferon and IL-2 in response to N protein . More importantly, the immunized mice produced strong delayed-type hypersensitivity (DTH) and CD8(+) CTL responses to N protein . The study shows that N protein of SARS-CoV not only is an important B cell immunogen, but also can elicit broad-based cellular immune responses . The results indicate that the N protein may be of potential value in vaccine development for specific prophylaxis and treatment against SARS.

Bioorg Med Chem, 2005 Jan 3, 13(1), 69 - 75
Synthesis and aminoacyl-tRNA synthetase inhibitory activity of aspartyl adenylate analogs; Bernier S et al.; Three nonhydrolyzable aspartyl adenylate analogs have been prepared and tested as inhibitors of E . coli aspartyl-tRNA synthetase . 5'-O-{N-(L-Aspartyl)sulfamoyl}adenosine is a potent competitive inhibitor (K(i) = 15 nM) whereas L-aspartol adenylate is a weaker inhibitor (K(i) = 45 microM) with respect to aspartic acid . The corresponding ketomethylphosphonate (a novel isosteric replacement) is also a strong inhibitor (K(i) = 123 nM).

Bioorg Med Chem Lett, 2005 Jan 3, 15(1), 89 - 92
Characterization of L-glutamine:2-deoxy-scyllo-inosose aminotransferase (tbmB) from Streptomyces tenebrarius; Kharel MK et al.; 2-Deoxystreptamine (DOS)-containing aminoglycoside-aminocyclitol (AmAc) antibiotics represent the majority of clinically important AmAcs . Biosynthetic investigations of formation of DOS in actinomycetes are limited to the characterization of 2-deoxy-scyllo-inosose synthase, the first step enzyme of the DOS biosynthetic pathway . A gene encoding L-glutamine:2-deoxy-scyllo-inosose aminotransferase (tbmB) from the tobramycin producer Streptomyces tenebrarius was expressed heterologously in Escherichia coli . The conversions of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and scyllo-inosose to scyllo-inosamine with the activity of TbmB were determined in vitro . The results indicate that tbmB catalyzes the second step of the DOS biosynthetic pathway during the biosynthesis of 2-deoxystreptamine, a subunit of tobramycin, in S . tenebrarius.

Comp Biochem Physiol B Biochem Mol Biol, 2004 Dec, 139(4), 681 - 93
Molecular cloning and expression analysis of phospholipase Cdelta from mud loach, Misgurnus mizolepis; Kim MS et al.; A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCdelta, was cloned from mud loach (Misgurnus mizolepis) liver . A complete cDNA encoding ML-PLCdelta was isolated by screening the cDNA library of mud loach liver and using the 5'-rapid amplification of cDNA ends (RACE) method . The full-length ML-PLCdelta gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector . It contains all of the characteristic domains found in mammalian PLCdelta isozymes (PH domain, EF-hands, X-Y catalytic region, and a C2 domain) . A homology search revealed that ML-PLCdelta shares relatively high sequence identity with mammalian PLCdelta1 (51-52%) and catfish PLCdelta (64%) . The recombinant ML-PLCdelta protein expressed as a histidine-tagged fusion protein in E . coli was purified to apparent homogeneity by Ni(2+)-NTA affinity chromatography . The recombinant ML-PLCdelta showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP(2)) and its activity was Ca(2+)-dependent, which was similar to mammalian PLCdelta isozymes.

Arch Biochem Biophys, 2005 Jan 15, 433(2), 447 - 53
Key NAD+-binding residues in human 15-hydroxyprostaglandin dehydrogenase; Cho H et al.; NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins, and is believed to be the key enzyme responsible for the biological inactivation of these biologically potent eicosanoids . The enzyme utilizes NAD(+) specifically as a coenzyme . Potential amino acid residues involved in binding NAD(+) and facilitating enzyme catalysis have been partially identified . In this report, we propose that three more residues in 15-PGDH, Ile-17, Asn-91, and Val-186, are also involved in the interaction with NAD(+) . Site-directed mutagenesis was used to examine their roles in binding NAD(+) . Several mutants (I17A, I17V, I17L, I17E, I17K, N91A, N91D, N91K, V186A, V186I, V186D, and V186K) were prepared, expressed as glutathione S-transferase (GST) fusion enzymes in Escherichia coli, and purified by GSH-agarose affinity chromatography . Mutants I17E, I17K, N91L, N91K, and V186D were found to be inactive . Mutants N91A, N91D, V186A, and V186K exhibited comparable activities to the wild type enzyme . However, mutants I17A, I17V, I17L, and V186I had higher activity than the wild type . Especially, the activities of I17L and V186I were increased nearly 4- and 5-fold, respectively . The k(cat)/K(m) ratios of all active mutants for PGE(2) were similar to that of the wild type enzyme . However, the k(cat)/K(m) ratios of mutants I17A and N91A for NAD(+) were decreased 5- and 10-fold, respectively, whereas the k(cat)/K(m) ratios of mutants I17V, N91D, V186I, and V186K for NAD(+) were comparable to that of the wild type enzyme . The k(cat)/K(m) ratios of mutants I17L and V186A for NAD(+) were increased over nearly 2-fold . These results suggest that Ile-17, Asn-91, and Val-186 are involved in the interaction with NAD(+) and contribute to the full catalytic activity of 15-PGDH.

Arch Biochem Biophys, 2005 Jan 1, 433(1), 322 - 34
The roles of the essential Asp-48 and highly conserved His-43 elucidated by the pH dependence of the pseudouridine synthase TruB; Hamilton CS et al.; All known pseudouridine synthases have a conserved aspartic acid residue that is essential for catalysis, Asp-48 in Escherichia coli TruB . To probe the role of this residue, inactive D48C TruB was oxidized to generate the sulfinic acid cognate of aspartic acid . The oxidation restored significant but reduced catalytic activity, consistent with the proposed roles of Asp-48 as a nucleophile and general base . The family of pseudouridine synthases including TruB also has a nearly invariant histidine residue, His-43 in the E . coli enzyme . To examine the role of this conserved residue, site-directed mutagenesis was used to generate H43Q, H43N, H43A, H43G, and H43F TruB . Except for phenylalanine, the substitutions seriously impaired the enzyme, but all of the altered TruB retained significant activity . To examine the roles of Asp-48 and His-43 more fully, the pH dependences of wild-type, oxidized D48C, and H43A TruB were determined . The wild-type enzyme displays a typical bell-shaped profile . With oxidized D48C TruB, logk(cat) varies linearly with pH, suggesting the participation of specific rather than general base catalysis . Substitution of His-43 perturbs the pH profile, but it remains bell-shaped . The ascending limb of the pH profile is assigned to Asp-48, and the descending limb is tentatively ascribed to an active site tyrosine residue, the bound substrate uridine, or the bound product pseudouridine.

Biochemistry, 2004 Dec 14, 43(49), 15463 - 71
A soluble C1b protein and its regulation of soluble type 7 adenylyl cyclase; Beeler JA et al.; Adenylyl cyclase (AC) is a prototypical cell-signaling molecule expressed in virtually all organisms from bacteria to man . While C1b, a poorly conserved region within mammalian AC, has been implicated in numerous isoform-specific regulatory properties, no one has purified the C1b region as a functional protein to homogeneity in order to study its role in enzyme function . We hypothesize that C1b is an internal regulatory subunit . To pursue this hypothesis, we constructed several soluble C1b proteins from type VII AC, arriving at one, 7C1b-S, which can be expressed and purified from Escherichia coli . 7C1b-S is relatively stable, as demonstrated by limited proteolytic analysis, circular dichroism, and UV Raman spectroscopy . Using size-exclusion chromatography and co-immunoprecipitation we demonstrate that 7C1b-S interacts with a cardinal activator of AC (Gsalpha) and with the conserved first catalytic domain (C1a) of type VII AC . We show that 7C1b-S inhibits Gsalpha-stimulated and Gsalpha-forskolin stimulated activity in our soluble ACVII model system . On the basis of these results, we suggest that 7C1b-S meets basic criteria to serve as a model protein for the C1b region and may be used as a prototype to develop other isoform C1b soluble model proteins to further investigate the role of this domain in isoform-specific regulation of adenylyl cyclase.

Plant Physiol, 2005 Jan, 137(1), 220 - 30 Epub 2004 Dec 03.
Characterization and expression analysis of a serine acetyltransferase gene family involved in a key step of the sulfur assimilation pathway in Arabidopsis; Kawashima CG et al.; Ser acetyltransferase (SATase; EC 2.3.1.30) catalyzes the formation of O-acetyl-Ser from l-Ser and acetyl-CoA, leading to synthesis of Cys . According to its position at the decisive junction of the pathways of sulfur assimilation and amino acid metabolism, SATases are subject to regulatory mechanisms to control the flux of Cys synthesis . In Arabidopsis (Arabidopsis thaliana) there are five genes encoding SATase-like proteins . Two isoforms, Serat3;1 and Serat3;2, were characterized with respect to their enzymatic properties, feedback inhibition by l-Cys, and subcellular localization . Functional identity of Serat3;1 and Serat3;2 was established by complementation of a SATase-deficient mutant of Escherichia coli . Cytosolic localization of Serat3;1 and Serat3;2 was confirmed by using fusion construct with the green fluorescent protein . Recombinant Serat3;1 was not inhibited by l-Cys, while Serat3;2 was a strongly feedback-inhibited isoform . Quantification of expression patterns indicated that Serat2;1 is the dominant form expressed in most tissues examined, followed by Serat1;1 and Serat2;2 . Although Serat3;1 and Serat3;2 were expressed weakly in most tissues, Serat3;2 expression was significantly induced under sulfur deficiency and cadmium stress as well as during generative developmental stages, implying that Serat3;1 and Serat3;2 have specific roles when plants are subjected to distinct conditions . Transgenic Arabidopsis plants expressing the green fluorescent protein under the control of the five promoters indicated that, in all Serat genes, the expression was predominantly localized in the vascular system, notably in the phloem . These results demonstrate that Arabidopsis employs a complex array of compartment-specific SATase isoforms with distinct enzymatic properties and expression patterns to ensure the provision of Cys in response to developmental and environmental changes.

BMC Microbiol . 2004 Dec 06;4(1):46.
P80, the HinT interacting membrane protein, is a secreted antigen of Mycoplasma hominis; Hopfe M et al.; BACKGROUND: Mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins . In Mycoplasma hominis, the genes encoding the surface-localized membrane complex P60/P80 are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic Histidine triad motif (HinT) . HinT is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes . Immuno-precipitation and BIACore analysis revealed an interaction between HinT and the P80 domain of the membrane complex . As the membrane anchored P80 carries an N-terminal uncleaved signal peptide we have proposed that the N-terminus extends into the cytoplasm and interacts with the cytosolic HinT . RESULTS: Further characterization of P80 suggested that the 4.7 kDa signal peptide is protected from cleavage only in the membrane bound form . We found several proteins were released into the supernatant of a logarithmic phase mycoplasma culture, including P80, which was reduced in size by 10 kDa . Western blot analysis of recombinant P80 mutants expressed in E . coli and differing in the N-terminal region revealed that mutation of the +1 position of the mature protein (Asn to Pro) which is important for signal peptidase I recognition resulted in reduced P80 secretion . All other P80 variants were released into the supernatant, in general as a 74 kDa protein encompassing the helical part of P80 . Incubation of M . hominis cells in phosphate buffered saline supplemented with divalent cations revealed that the release of mycoplasma proteins into the supernatant was inhibited by high concentrations of calciumions . CONCLUSIONS: Our model for secretion of the P80 protein of M . hominis implies a two-step process . In general the P80 protein is transported across the membrane and remains complexed to P60, surface-exposed and membrane anchored via the uncleaved signal sequence . Loss of the 4.7 kDa signal peptide seems to be a pre-requisite for P80 secretion, which is followed by a proteolytic process leading to a helical 74 kDa product . We propose that this novel form of two-step secretion is one of the solutions to a life with a reduced gene set.

J Biol Inorg Chem, 2005 Jan, 10(1), 41 - 50 Epub 2004 Dec 01.
Characterization of the active site and insight into the binding mode of the anti-angiogenesis agent fumagillin to the manganese(II)-loaded methionyl aminopeptidase from Escherichia coli; D'souza VM et al.; EPR spectra were recorded for methionine aminopeptidase from Escherichia coli (EcMetAP-I) samples (~2.5 mM) to which one and two equivalents of Mn(II) were added (the latter is referred to as {MnMn(EcMetAP-I)}) . The spectra for each sample were indistinguishable except that the spectrum of {MnMn(EcMetAP-I)} was twice as intense . The EPR spectrum of {MnMn(EcMetAP-I)} exhibited the characteristic six-line g approximately 2 EPR signal of mononuclear Mn(II) with A(av)((55)Mn)=9.3 mT (93 G) and exhibited Curie-law temperature dependence . This signal is typical of Mn(II) in a ligand sphere comprising oxygen and/or nitrogen atoms . Other features in the spectrum were observed only as the temperature was raised from that of liquid helium . The temperature dependences of these features are consistent with their assignment to excited state transitions in the S=1, 2 .. . 5 non-Kramer's doublets, due to two antiferromagnetically coupled Mn(II) ions with an S=0 ground state . This assignment is supported by the observation of a characteristic 4.5 mT hyperfine pattern, and by the presence of signals in the parallel mode consistent with a non-Kramers' spin ladder . Upon the addition of the anti-angiogenesis agent fumagillin to {MnMn(EcMetAP-I)}, very small changes were observed in the EPR spectrum . MALDI-TOF mass spectrometry indicated that fumagillin was, however, covalently coordinated to EcMetAP-I . Therefore, the inhibitory action of this anti-angiogenesis agent on EcMetAP-I appears to involve covalent binding to a polypeptide component at or near the active site rather than direct binding to the metal ions.

J Allergy Clin Immunol, 2004 Dec, 114(6), 1410 - 7
Ara h 8, a Bet v 1-homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy; Mittag D et al.; BACKGROUND: We recently described patients with soybean allergy mainly mediated by cross-reactivity to birch pollen allergens . A majority of those patients were reported to have peanut allergy . OBJECTIVE: We sought to study the occurrence of peanut allergy in patients allergic to birch pollen and characterized the Bet v 1-homologous peanut allergen Ara h 8 . METHODS: Recombinant Ara h 8 was cloned with degenerated primers and expressed in Escherichia coli . Nine Swiss and 11 Dutch patients with peanut and birch pollen allergy and a positive double-blind, placebo-controlled food challenge result to peanut were investigated for IgE reactivity to birch pollen and purified peanut allergens and cross-reactivity between birch and peanut . Ara h 8 stability against digestion and roasting was assessed by means of RAST inhibition . The IgE cross-linking potency of Ara h 8 was tested on the basis of basophil histamine release . RESULTS: During double-blind, placebo-controlled food challenge, all patients experienced symptoms in the oral cavity, progressing to more severe symptoms in 40% of patients . CAP-FEIA detected recombinant (r) Ara h 8-specific IgE in 85% . IgE binding to Ara h 8 was inhibited by Bet v 1 in peanut extract immunoblotting and in RAST inhibition . In EAST inhibition recombinant rAra h 8 inhibited IgE binding to peanut in 4 of 7 tested patient sera . Antipeanut response was dominated by Ara h 8 in 12 of 17 tested patients . Furthermore, our results demonstrate a low stability of Ara h 8 to roasting and no stability to gastric digestion . Basophil histamine release with rAra h 8 was more than 20% in 5 of 7 tested sera . CONCLUSIONS: Peanut allergy might be mediated in a subgroup of our patients by means of cross-reaction of Bet v 1 with the homologous peanut allergen Ara h 8.

Biol Pharm Bull, 2004 Dec, 27(12), 1979 - 85
cDNA isolation and functional expression of myrcene synthase from Perilla frutescens; Hosoi M et al.; cDNA cloning of a monoterpene synthase from Perilla frutescens whose steam-distilled oil contains 92.9% perillaketone, was performed by the PCR method using primers designed based on limonene synthase . The full-length nucleotide sequence of this cDNA consisted of 1978 bp including a 1827-bp translational region encoding a deduced protein of 608 amino acids, which was similar to that of limonene synthase from P . frutescens (85% identity) . Functional expression of this clone in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate into 53.8% myrcene, 20.9% sabinene, 19.8% linalool and 5.5% limonene . As for the extraction of reaction products, we performed SPME (solid phase micro extraction) as well as conventional solvent extraction, and compared these two extraction methods.

Methods Mol Biol, 2004, 296, 219 - 36
Methods for preparation of proteins and protein complexes that regulate the eukaryotic cell cycle for structural studies; Welburn J et al.; The determination of structures for proteins that control the eukaryotic cell cycle by nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography has made a significant contribution to our understanding of the molecular mechanisms that control cell cycle progression . CDK2 has proved particularly tractable to structural analysis, and CDK2 in complex with various regulatory proteins and in different phosphorylation states provides a paradigm for the control of this important kinase family . This chapter describes a number of protocols that can be used to prepare CDKs and selected CDK binding proteins suitable for structural studies by heterologous expression in either E . coli or insect cells.

J Bacteriol, 2004 Dec, 186(24), 8542 - 6
Activation of the gab operon in an RpoS-dependent manner by mutations that truncate the inner core of lipopolysaccharide in Escherichia coli; Joloba ML et al.; The gab operon (gabDTPC) in Escherichia coli functions in the conversion of gamma-aminobutyrate to succinate . One component of gab operon regulation involves the RpoS sigma factor, which mediates activation at high cell density . Transposon mutagenesis was used to identify new genes that regulate gab operon expression in rich media . A Tn5tmp insertion in the hldD (formerly rfaD) gene increased gabT::lacZ expression 12-fold . The hldD gene product, an ADP-L-glycerol-D-mannoheptose-6-epimerase, catalyzes the conversion of ADP-D-glycerol-D-mannoheptose to ADP-L-glycerol-D-mannoheptose, a precursor for the synthesis of inner-core lipopolysaccharide (LPS) . Defined mutations in hldE, required for heptose synthesis, and waaF, required for the addition of the second heptose to the inner core, also resulted in high-level gabT::lacZ expression . The hldD, hldE, and waaF mutants exhibited a mucoid colony phenotype due to production of a colanic acid capsule . However, in the hldD::cat background, the high-level expression of gabT::lacZ was independent of the regulatory components for colanic acid synthesis (rcsA, rcsB, and rcsC) and also independent of manC (cpsB), a structural gene for colanic acid synthesis . Activation of gabT::lacZ in the hldD::cat background was dependent on the RpoS sigma factor . The hldD::cat mutation resulted in a sixfold increase in the levels of a translational RpoS-LacZ fusion and had a marginal effect on a transcriptional fusion . This study reveals a stress-induced pathway, mediated by loss of the LPS inner core, that increases RpoS translation and gab operon expression in E . coli.

J Bacteriol, 2004 Dec, 186(24), 8537 - 41
Influence of L-leucine and L-alanine on Lrp regulation of foo, coding for F1651, a Pap homologue; Berthiaume F et al.; The foo operon encodes F165 1 fimbriae that belong to the P-regulatory family and are synthesized by septicemic Escherichia coli . Using an Lrp-deficient host and the lrp gene cloned under the arabinose pBAD promoter, we demonstrated that foo was transcribed proportionally to the amount of Lrp synthesized . L-leucine and L-alanine decreased drastically the steady-state transcription of foo and modified phase variation, independently of the presence of FooI . Specific mutations in the C-terminal region of Lrp reduced or abolished the repressive effect of these amino acids, indicating that they modulate F165 1 by affecting Lrp.

J Bacteriol, 2004 Dec, 186(24), 8499 - 507
RpoS-regulated genes of Escherichia coli identified by random lacZ fusion mutagenesis; Vijayakumar SR et al.; RpoS is a conserved alternative sigma factor that regulates the expression of many stress response genes in Escherichia coli . The RpoS regulon is large but has not yet been completely characterized . In this study, we report the identification of over 100 RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant and wild-type backgrounds . Forty-eight independent gene fusions were identified, including several in well-characterized RpoS-regulated genes, such as osmY, katE, and otsA . Many of the other fusions mapped to genes of unknown function or to genes that were not previously known to be under RpoS control . Based on the homology to other known bacterial genes, some of the RpoS-regulated genes of unknown functions are likely important in nutrient scavenging.

J Bacteriol, 2004 Dec, 186(24), 8453 - 62
Characterization of soluble enzyme II complexes of the Escherichia coli phosphotransferase system; Aboulwafa M et al.; Plasmid-encoded His-tagged glucose permease of Escherichia coli, the enzyme IIBCGlc (IIGlc), exists in two physical forms, a membrane-integrated oligomeric form and a soluble monomeric form, which separate from each other on a gel filtration column (peaks 1 and 2, respectively) . Western blot analyses using anti-His tag monoclonal antibodies revealed that although IIGlc from the two fractions migrated similarly in sodium dodecyl sulfate gels, the two fractions migrated differently on native gels both before and after Triton X-100 treatment . Peak 1 IIGlc migrated much more slowly than peak 2 IIGlc . Both preparations exhibited both phosphoenolpyruvate-dependent sugar phosphorylation activity and sugar phosphate-dependent sugar transphosphorylation activity . The kinetics of the transphosphorylation reaction catalyzed by the two IIGlc fractions were different: peak 1 activity was subject to substrate inhibition, while peak 2 activity was not . Moreover, the pH optima for the phosphoenolpyruvate-dependent activities differed for the two fractions . The results provide direct evidence that the two forms of IIGlc differ with respect to their physical states and their catalytic activities . These general conclusions appear to be applicable to the His-tagged mannose permease of E . coli . Thus, both phosphoenolpyruvate-dependent phosphotransferase system enzymes exist in soluble and membrane-integrated forms that exhibit dissimilar physical and kinetic properties.

J Bacteriol, 2004 Dec, 186(24), 8317 - 25
Negative regulation of DNA repair gene (ung) expression by the CpxR/CpxA two-component system in Escherichia coli K-12 and induction of mutations by increased expression of CpxR; Ogasawara H et al.; In Escherichia coli K-12 overexpressing CpxR, transcription of the ung gene for uracil-DNA glycosylase was repressed, ultimately leading to the induction of mutation . Gel shift, DNase I footprinting, and in vitro transcription assays all indicated negative regulation of ung transcription by phosphorylated CpxR . Based on the accumulated results, we conclude that ung gene expression is negatively regulated by the two-component system of CpxR/CpxA signal transduction.

J Bacteriol, 2004 Dec, 186(24), 8276 - 86
Comparative genomics of the T4-Like Escherichia coli phage JS98: implications for the evolution of T4 phages; Chibani-Chennoufi S et al.; About 130 kb of sequence information was obtained from the coliphage JS98 isolated from the stool of a pediatric diarrhea patient in Bangladesh . The DNA shared up to 81% base pair identity with phage T4 . The most conserved regions between JS98 and T4 were the structural genes, but their degree of conservation was not uniform . The head genes showed the highest sequence conservation, followed by the tail, baseplate, and tail fiber genes . Many tail fiber genes shared only protein sequence identity . Except for the insertion of endonuclease genes in T4 and gene 24 duplication in JS98, the structural gene maps of the two phages were colinear . The receptor-recognizing tail fiber proteins gp37 and gp38 were only distantly related to T4, but shared up to 83% amino acid identity to other T6-like phages, suggesting lateral gene transfer . A greater degree of variability was seen between JS98 and T4 over DNA replication and DNA transaction genes . While most of these genes came in the same order and shared up to 76% protein sequence identity, a few rearrangements, insertions, and replacements of genes were observed . Many putative gene insertions in the DNA replication module of T4 were flanked by intron-related endonuclease genes, suggesting mobile DNA elements . A hotspot of genome diversification was located downstream of the DNA polymerase gene 43 and the DNA binding gene 32 . Comparative genomics of 100-kb genome sequence revealed that T4-like phages diversify more by the accumulation of point mutations and occasional gene duplication events than by modular exchanges.

J Bacteriol, 2004 Dec, 186(24), 8248 - 53
Identification of the Escherichia coli K-12 ybhE gene as pgl, encoding 6-phosphogluconolactonase; Thomason LC et al.; We report identification of the Escherichia coli ybhE gene as the pgl gene that encodes 6-phosphogluconolactonase . A tentative identification was first made based on the known approximate location of the pgl gene and the similarity of the presumptive ybhE-encoded protein sequence to a known Pgl enzyme . To test this notion, the ybhE gene was deleted and replaced with a drug marker . Like previously characterized pgl mutants, the ybhE deletion mutant had a Blu- phenotype (dark-blue staining with iodine due to accumulation of starch after growth on minimal maltose) and demonstrated impaired growth on minimal glucose medium when combined with a pgi mutation . Biochemical assay of crude extracts for 6-phosphogluconolactonase enzymatic activity showed that ybhE encodes this activity . The ybhE gene was transferred from the E . coli chromosome to an expression vector . This ybhE clone complemented both the precise deletion of the ybhE gene and a larger deletion, pglDelta8, for the Blu- phenotype and for phosphogluconolactonase activity, confirming that ybhE is the pgl gene . A newly observed phenotype of pgl strains is a lowered frequency of appearance of Bgl+ mutants that can utilize the beta-glucoside salicin . This is likely due to poor growth of Bgl+ pgl strains on salicin due to the accumulation of 6-phosphogluconolactone.

J Bacteriol, 2004 Dec, 186(24), 8207 - 12
Role and regulation of sigma s in general resistance conferred by low-shear simulated microgravity in Escherichia coli; Lynch SV et al.; Life on Earth evolved in the presence of gravity, and thus it is of interest from the perspective of space exploration to determine if diminished gravity affects biological processes . Cultivation of Escherichia coli under low-shear simulated microgravity (SMG) conditions resulted in enhanced stress resistance in both exponential- and stationary-phase cells, making the latter superresistant . Given that microgravity of space and SMG also compromise human immune response, this phenomenon constitutes a potential threat to astronauts . As low-shear environments are encountered by pathogens on Earth as well, SMG-conferred resistance is also relevant to controlling infectious disease on this planet . The SMG effect resembles the general stress response on Earth, which makes bacteria resistant to multiple stresses; this response is sigma s dependent, irrespective of the growth phase . However, SMG-induced increased resistance was dependent on sigma s only in stationary phase, being independent of this sigma factor in exponential phase . sigma s concentration was some 30% lower in exponential-phase SMG cells than in normal gravity cells but was twofold higher in stationary-phase SMG cells . While SMG affected sigma s synthesis at all levels of control, the main reasons for the differential effect of this gravity condition on sigma s levels were that it rendered the sigma protein less stable in exponential phase and increased rpoS mRNA translational efficiency . Since sigma s regulatory processes are influenced by mRNA and protein-folding patterns, the data suggest that SMG may affect these configurations.

Nucleic Acids Res, 2004, 32(21), 6378 - 87 Print 2004.
PriA helicase and SSB interact physically and functionally; Cadman CJ et al.; PriA helicase is the major DNA replication restart initiator in Escherichia coli and acts to reload the replicative helicase DnaB back onto the chromosome at repaired replication forks and D-loops formed by recombination . We have discovered that PriA-catalysed unwinding of branched DNA substrates is stimulated specifically by contact with the single-strand DNA binding protein of E.coli, SSB . This stimulation requires binding of SSB to the initial DNA substrate and is effected via a physical interaction between PriA and the C-terminus of SSB . Stimulation of PriA by the SSB C-terminus may act to ensure that efficient PriA-catalysed reloading of DnaB occurs only onto the lagging strand template of repaired forks and D-loops . Correlation between the DNA repair and recombination defects of strains harbouring an SSB C-terminal mutation with inhibition of this SSB-PriA interaction in vitro suggests that SSB plays a critical role in facilitating PriA-directed replication restart . Taken together with previous data, these findings indicate that protein-protein interactions involving SSB may coordinate replication fork reloading from start to finish.

Nucleic Acids Res, 2004, 32(21), 6358 - 66 Print 2004.
Change of RNase P RNA function by single base mutation correlates with perturbation of metal ion binding in P4 as determined by NMR spectroscopy; Schmitz M; The solution structures of two 27 nt RNA hairpins and their complexes with cobalt(III)-hexammine {Co(NH(3))(6)(3+)} were determined by NMR spectroscopy . The RNA hairpins are variants of the P4 region from Escherichia coli RNase P RNA: a U-to-A mutant changing the identity of the bulged nucleotide, and a U-to-C, C-to-U double mutant changing only the bulge position . Structures calculated from NMR constraints show that the RNA hairpins adopt different conformations . In the U-to-C, C-to-U double mutant, the conserved bulged uridine in the P4 wild-type stem is found to be shifted in the 3'-direction by one nucleotide when compared with the wild-type structure . Co(NH(3))(6)(3+) is used as a spectroscopic probe for Mg(H(2)O)(6)(2+) binding sites because both complexes have octahedral symmetry and have similar radii . Intermolecular NOE crosspeaks between Co(NH(3))(6)(3+) and RNA protons were used to locate the site of Co(NH(3))(6)(3+) binding to both RNA hairpins . The metal ion binds in the major groove near a bulge loop in both mutants, but is shifted 3' by about one base pair in the double mutant . The change of the metal ion binding site is compared with results obtained on corresponding mutant RNase P RNA molecules as reported by Harris and co-workers (RNA, 1, 210-218).

Protein Sci, 2005 Jan, 14(1), 193 - 201 Epub 2004 Dec 02.
Identifying natural substrates for chaperonins using a sequence-based approach; Stan G et al.; The Escherichia coli chaperonin machinery, GroEL, assists the folding of a number of proteins . We describe a sequence-based approach to identify the natural substrate proteins (SPs) for GroEL . Our method is based on the hypothesis that natural SPs are those that contain patterns of residues similar to those found in either GroES mobile loop and/or strongly binding peptide in complex with GroEL . The method is validated by comparing the predicted results with experimentally determined natural SPs for GroEL . We have searched for such patterns in five genomes . In the E . coli genome, we identify 1422 (about one-third) sequences that are putative natural SPs . In Saccharomyces cerevisiae, 2885 (32%) of sequences can be natural substrates for Hsp60, which is the analog of GroEL . The precise number of natural SPs is shown to be a function of the number of contacts an SP makes with the apical domain (N(C)) and the number of binding sites (N(B)) in the oligomer with which it interacts . For known SPs for GroEL, we find approximately 4 < N(C) < 5 and 2 <or= N(B) <or= 4 . A limited analysis of the predicted binding sequences shows that they do not adopt any preferred secondary structure . Our method also predicts the putative binding regions in the identified SPs . The results of our study show that a variety of SPs, associated with diverse functions, can interact with GroEL.

Protein Sci, 2005 Jan, 14(1), 140 - 7 Epub 2004 Dec 02.
Ser95, Asn97, and Thr78 are important for the catalytic function of porcine NADP-dependent isocitrate dehydrogenase; Kim TK et al.; The mammalian mitochondrial NADP-dependent isocitrate dehydrogenase is a citric acid cycle enzyme and an important contributor to cellular defense against oxidative stress . The Mn(2+)-isocitrate complex of the porcine enzyme was recently crystallized; its structure indicates that Ser(95), Asn(97), and Thr(78) are within hydrogen-bonding distance of the gamma-carboxylate of enzyme-bound isocitrate . We used site-directed mutagenesis to replace each of these residues by Ala and Asp . The wild-type and mutant enzymes were expressed in Escherichia coli and purified to homogeneity . All the enzymes retain their native dimeric structures and secondary structures as monitored by native gel electrophoresis and circular dichroism, respectively . V(max) of the three alanine mutants is decreased to 24%-38% that of wild-type enzyme, with further decreases in the aspartate mutants . For T78A and S95A mutants, the major changes are the 10- to 100-fold increase in the K(m) values for isocitrate and Mn(2+) . The results suggest that Thr(78) and Ser(95) function to strengthen the enzyme's affinity for Mn(2+)-isocitrate by hydrogen bonding to the gamma-carboxylate of isocitrate . For the Asn(97) mutants, the K(m) values are much less affected . The major change in the N97A mutant is the increase in pK(a) of the ionizable metal-liganded hydroxyl of enzyme-bound isocitrate from 5.23 in wild type to 6.23 in the mutant enzyme . The hydrogen bond between Asn(97) and the gamma-carboxylate of isocitrate may position the substrate to promote a favorable lowering of the pK of the enzyme-isocitrate complex . Thus, Thr(78), Ser(95), and Asn(97) perform important but distinguishable roles in catalysis by porcine NADP-specific isocitrate dehydrogenase.

Nucleic Acids Res, 2004, 32(21), 6226 - 39 Print 2004.
EFICAz: a comprehensive approach for accurate genome-scale enzyme function inference; Tian W et al.; EFICAz (Enzyme Function Inference by Combined Approach) is an automatic engine for large-scale enzyme function inference that combines predictions from four different methods developed and optimized to achieve high prediction accuracy: (i) recognition of functionally discriminating residues (FDRs) in enzyme families obtained by a Conservation-controlled HMM Iterative procedure for Enzyme Family classification (CHIEFc), (ii) pairwise sequence comparison using a family specific Sequence Identity Threshold, (iii) recognition of FDRs in Multiple Pfam enzyme families, and (iv) recognition of multiple Prosite patterns of high specificity . For FDR (i.e . conserved positions in an enzyme family that discriminate between true and false members of the family) identification, we have developed an Evolutionary Footprinting method that uses evolutionary information from homofunctional and heterofunctional multiple sequence alignments associated with an enzyme family . The FDRs show a significant correlation with annotated active site residues . In a jackknife test, EFICAz shows high accuracy (92%) and sensitivity (82%) for predicting four EC digits in testing sequences that are <40% identical to any member of the corresponding training set . Applied to Escherichia coli genome, EFICAz assigns more detailed enzymatic function than KEGG, and generates numerous novel predictions.

Nucleic Acids Res, 2004, 32(21), 6200 - 11 Print 2004.
Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells; Kohrer C et al.; We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair . We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells . Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm . Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes . The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA . We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.

Biol Chem, 2004 Nov, 385(11), 1113 - 9
Lipopolysaccharide binding of an exchangeable apolipoprotein, apolipophorin III, from Galleria mellonella; Pratt CC et al.; A new role of apolipophorin III (apoLp-III) as an immune activator has emerged recently . To gain insight into this novel function, the interaction of apoLp-III with lipopoly-saccharide (LPS) was investigated . ApoLp-III from Galleria mellonella was incubated with LPS from Escherichia coli O55:B5, and analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE) . Protein staining showed that apoLp-III mobility was significantly reduced . In addition, silver and LPS fluorescent staining demonstrated that LPS mobility was increased upon incubation with apoLp-III . This result suggests association of apoLp-III with LPS . Sodium dodecyl sulfate (SDS) PAGE analysis showed decreased apoLp-III mobility upon LPS addition, indicative of LPS apoLp-III interaction in the presence of SDS . The unique tyrosine residue that resides in apoLp-III was used to provide additional evidence for LPS binding interaction . In the absence of LPS, apoLp-III tyrosine fluorescence was relatively low . However, LPS addition resulted in a progressive increase in the fluorescence intensity, indicating tertiary rearrangement in the environment of tyrosine 142 upon LPS interaction . Other well-characterized apoLp-IIIs were also examined for LPS binding . Manduca sexta , Bombyx mori and Locusta migratoria apoLp-III were all able to interact with LPS . The ability of apoLp-III to form complexes with LPS supports the proposed role of apoLp-III in innate immunity.

Biol Chem, 2004 Nov, 385(11), 1105 - 11
Thermoplasma acidophilum TAA43 is an archaeal member of the eukaryotic meiotic branch of AAA ATPases; Santos L et al.; Sequencing of the Thermoplasma acidophilum genome revealed a new gene, taa43 , which codes for a 43-kDa protein containing one AAA domain; we therefore termed it Thermoplasma AAA ATPase of 43 kDa (TAA43) . Close homologs of TAA43 are found only in related Thermoplasmales, e.g . T . volcanium and Ferroplasma acidarmanus , but not in other Archaea . A detailed phylogenetic analysis showed that TAA43 and its homologs belong to the 'meiotic' branch of the AAA family . Although AAA proteins usually assemble into high-molecular-weight complexes, native TAA43 is predominantly dimeric except for a minor fraction eluting in the void volume of a sizing column . Wild-type and mutant TAA43 proteins were overexpressed in Escherichia coli , purified as dimers and characterized functionally . Since the canonical proteasome activating nucleotidase is not present in Thermoplasmales, TAA43 was tested for stimulation of proteasome activity, which was, however, not detected . Interestingly, immunoprecipitation analysis with TAA43 specific antibodies found a fraction of native TAA43 associated with Thermoplasma ribosomal proteins.

Genome Biol . 2004;5(12):252 . Epub 2004.
Chromatin architecture and gene expression in Escherichia coli; Willenbrock H et al.; Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli.

Am J Transplant, 2004 Dec, 4(12), 1949 - 57
Over-expression of AIF-1 in liver allografts and peripheral blood correlates with acute rejection after transplantation in rats; Nagakawa Y et al.; Early and accurate detection of acute cellular rejection (ACR) is important in the management of liver allograft recipients . We hypothesized that expression of allograft inflammatory factor (AIF)-1 would be associated with liver allograft rejection as previous studies have shown that a relationship exists between kidney and heart transplantation . Indeed using rat orthotopic transplant models we found that the expression of allograft inflammatory factor-1 (AIF-1) can be detected in both allograft and peripheral blood leukocytes with peak levels detected 7 days following liver transplantation . Interestingly, AIF-1 expression increased 2-fold in acutely rejecting liver allografts compared to chronically accepted livers on days 5, 7 and 10 after transplantation . AIF-1 expression in peripheral blood leukocytes was also significantly greater in the rejection model than in the acceptance model . Flow cytometric analysis of peripheral blood leukocytes demonstrated that AIF-1 was expressed in ED2-positive cells, a marker for Kupffer cells . In vitro studies showed that AIF-1 expression in Kupffer cells was up-regulated by coculture with Th1 cytokines . However, neither LPS nor Escherichia coli (E . coli) administration had an affect on AIF-1 expression . These data indicate that high levels of AIF-1 expression reflect aggressive liver allograft rejection and suggest a role for monitoring AIF-1 in peripheral blood leukocytes as a monitor for increased immunosuppression.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1847 - 54
A recombinant glutamine-binding protein from Escherichia coli: effect of ligand-binding on protein conformational dynamics; Herman P et al.; We have investigated the effect of the binding of glutamine on the conformational dynamics of the recombinant glutamine binding protein (GlnBP) from Escherichia coli by steady-state and time-resolved fluorescence techniques . The structural stability of the protein was also studied by far-UV circular dichroism spectroscopy in the range of temperature between 25 and 80 degrees C . The results showed that the interaction of the protein with the ligand resulted in a marked change of the structural and conformational dynamics features of the protein . In particular, the fluorescence and circular dichroism data showed that the presence of glutamine resulted in a dramatic increase of the protein thermal stability of about 10 degrees C . In addition, the fluorescence time-resolved data pointed out that both in the absence and in the presence of glutamine the protein structure was highly rigid with small amplitude of segmental motion up to 65 degrees C and a low accessibility of the protein tryptophan residues to acrylamide . The obtained results on the structural properties of the recombinant glutamine-binding protein in the absence and in the presence of glutamine can contribute to a better understanding of the transport-related functions of the protein and structurally similar periplasmic transport proteins, as well as to the design and development of new biotechnological applications of this class of proteins.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1783 - 7
Direct refolding of inclusion bodies using reversed micelles; Sakono M et al.; The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT) . Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies . The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles . To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system . The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies . The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h . The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies . The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1749 - 56
Expression and characterization of a His-tagged 11S seed globulin from Amaranthus hypochondriacus in Escherichia coli; Medina-Godoy S et al.; DNA encoding a His-tagged 11S globulin from Amaranthus hypochondriacus (amarantin) was successfully expressed in Escherichia coli strains BL21 (DE3) and Origami (DE3) . The two strains produced different accumulation patterns . Whereas most of the proamarantin expressed in BL21 (DE3) was localized in inclusion bodies, that produced in Origami (DE3) was soluble (76 mg/L) . Sucrose density gradient ultracentrifugation analysis of the expressed soluble proamarantin revealed that the protein was assembled into trimers . Treatment of proamarantin trimers in vitro using purified asparaginyl endopeptidase resulted in the appearance of peptides of the sizes expected for acidic and basic chains . Because the proamarantin assembles into trimers with the expected sedimentation characteristics and is cleaved into acidic and basic chains rather than being degraded, the results suggest that the protein folding occurring in E . coli is similar to that taking place in seeds . The His-tagged proamarantin was purified in a single step by immobilized metal affinity chromatography with a final yield of 48 mg/L . The overexpression of proamarantin in E . coli, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1689 - 96
Expression of active murine granulocyte-macrophage colony-stimulating factor in an Escherichia coli cell-free system; Yang J et al.; Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine in the mammalian immune system . It has been expressed in Escherichia coli with the same biological activity as the native protein . Here, we report the synthesis of a murine recombinant GM-CSF in an E . coli cell-free protein synthesis system with a high yield . Since there are two disulfide bonds in the native structure of GM-CSF, an oxidizing redox potential of the reaction mixture was required . By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM-CSF was found in its oxidized form . However, the GM-CSF thus formed showed low activity because of poor folding . With the addition of DsbC, the periplasmic disulfide isomerase from E . coli, a high yield of active GM-CSF was produced in the cell-free reaction . Finally, successful folding of the cell-free synthesized GM-CSF-his6 was confirmed by its cell-proliferation activity after purification with a Ni2+ chelating column.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1623 - 33
Stimulation, monitoring, and analysis of pathway dynamics by metabolic profiling in the aromatic amino acid pathway; Oldiges M et al.; Using a concerted approach of biochemical standard preparation, analytical access via LC-MS/MS, glucose pulse, metabolic profiling, and statistical data analysis, the metabolism dynamics in the aromatic amino acid pathway has been stimulated, monitored, and analyzed in different tyrosine-auxotrophic L-phenylalanine-producing Escherichia coli strains . During the observation window from -4 s (before) up to 27 s after the glucose pulse, the dynamics of the first five enzymatic reactions in the aromatic amino acid pathway was observed by measuring intracellular concentrations of 3-deoxy-d-arabino-heptulosonate 7-phosphate DAH(P), 3-dehydroquinate (3-DHQ), 3-dehydroshikimate (3-DHS), shikimate 3-phosphate (S3P), and shikimate (SHI), together with the pathway precursors phosphoenolpyruvate (PEP) and P5P, the lumped pentose phosphate pool as an alternative to the nondetectable erythrose 4-phosphate (E4P) . Provided that a sufficient fortification of the carbon flux into the pathway of interest is ensured, respective metabolism dynamics can be observed . On the basis of the intracellular pool measurements, the standardized pool velocities were calculated, and a simple, data-driven criterion--called "pool efflux capacity" (PEC)--is derived . Despite its simplifying system description, the criterion managed to identify the well-known AroB limitation in the E . coli strain A (genotype delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) amp) and it also succeeded to identify AroL and AroA (in strain B, genotype delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) aroB amp) as promising metabolic engineering targets to alleviate respective flux control in subsequent L-Phe producing strains . Furthermore, using of a simple correlation analysis, the reconstruction of the metabolite sequence of the observed pathway was enabled . The results underline the necessity to extend the focus of glucose pulse experiments by studying not only the central metabolism but also anabolic pathways.

Appl Environ Microbiol, 2004 Dec, 70(12), 7342 - 7
In vivo 31P nuclear magnetic resonance investigation of tellurite toxicity in Escherichia coli; Lohmeier-Vogel EM et al.; Here we compare the physiological state of Escherichia coli exposed to tellurite or selenite by using the noninvasive technique of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy . We studied glucose-fed Escherichia coli HB101 cells containing either a normal pUC8 plasmid with no tellurite resistance determinants present or the pTWT100 plasmid which contains the resistance determinants tehAB . No differences could be observed in intracellular ATP levels, the presence or absence of a transmembrane pH gradient, or the levels of phosphorylated glycolytic intermediates when resistant cells were studied by 31P NMR in the presence or absence of tellurite . In the sensitive strain, we observed that the transmembrane pH gradient was dissipated and intracellular ATP levels were rapidly depleted upon exposure to tellurite . Only the level of phosphorylated glycolytic intermediates remained the same as observed with resistant cells . Upon exposure to selenite, no differences could be observed by 31P NMR between resistant and sensitive strains, suggesting that the routes for selenite and tellurite reduction within the cells differ significantly, since only tellurite is able to collapse the transmembrane pH gradient and lower ATP levels in sensitive cells . The presence of the resistance determinant tehAB, by an as yet unidentified detoxification event, protects the cells from uncoupling by tellurite.

Appl Environ Microbiol, 2004 Dec, 70(12), 7156 - 60
Restoration of gene function by homologous recombination: from PCR to gene expression in one step; Yosef I et al.; We have developed a simple method for single-step cloning of any PCR product into a plasmid . A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination . In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete lambdaPR or rrnA1 promoter regions . The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage lambda Red functions and the host plasmid . Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection . Recombinant plasmid vectors encoding beta-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems . This cloning strategy significantly reduces both the time and costs associated with cloning procedures.

Appl Environ Microbiol, 2004 Dec, 70(12), 7126 - 39
Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization; Yilmaz LS et al.; In fluorescent in situ hybridization (FISH), the efficiency of hybridization between the DNA probe and the rRNA has been related to the accessibility of the rRNA when ribosome content and cell permeability are not limiting . Published rRNA accessibility maps show that probe brightness is sensitive to the organism being hybridized and the exact location of the target site and, hence, it is highly unpredictable based on accessibility only . In this study, a model of FISH based on the thermodynamics of nucleic acid hybridization was developed . The model provides a mechanistic approach to calculate the affinity of the probe to the target site, which is defined as the overall Gibbs free energy change (DeltaG degrees overall) for a reaction scheme involving the DNA-rRNA and intramolecular DNA and rRNA interactions that take place during FISH . Probe data sets for the published accessibility maps and experiments targeting localized regions in the 16S rRNA of Escherichia coli were used to demonstrate that DeltaG degrees overall is a strong predictor of hybridization efficiency and superior to conventional estimates based on the dissociation temperature of the DNA/rRNA duplex . The use of the proposed model also allowed the development of mechanistic approaches to increase probe brightness, even in seemingly inaccessible regions of the 16S rRNA . Finally, a threshold DeltaG degrees overall of -13.0 kcal/mol was proposed as a goal in the design of FISH probes to maximize hybridization efficiency without compromising specificity.

Appl Environ Microbiol, 2004 Dec, 70(12), 7033 - 9
Broad-host-range plasmid pJB658 can be used for industrial-level production of a secreted host-toxic single-chain antibody fragment in Escherichia coli; Sletta H et al.; In industrial scale recombinant protein production it is often of interest to be able to translocate the product to reduce downstream costs, and heterologous proteins may require the oxidative environment outside of the cytoplasm for correct folding . High-level expression combined with translocation to the periplasm is often toxic to the host, and expression systems that can be used to fine-tune the production levels are therefore important . We previously constructed vector pJB658, which harbors the broad-host-range RK2 minireplicon and the inducible Pm/xylS promoter system, and we here explore the potential of this unique system to manipulate the expression and translocation of a host-toxic single-chain antibody variable fragment with affinity for hapten 2-phenyloxazol-5-one (phOx) (scFv-phOx) . Fine-tuning of scFv-phOx levels was achieved by varying the concentrations of inducers and the vector copy number and also different signal sequences . Our data show that periplasmic accumulation of scFv-phOx leads to cell lysis, and we demonstrate the importance of controlled and high expression rates to achieve high product yields . By optimizing such parameters we show that soluble scFv-phOx could be produced to a high volumetric yield (1.2 g/liter) in high-cell-density cultures of Escherichia coli.

Plant Cell Physiol, 2004 Nov, 45(11), 1586 - 94
Two distinct redox signaling pathways for cytosolic APX induction under photooxidative stress; Yabuta Y et al.; The cross-talk of two redox factors, H2O2 accumulation and redox status of photosynthetic electron transport (PET) in tobacco chloroplasts, on the expression of cytosolic ascorbate peroxidase (cAPX) was studied . Transgenic tobacco plants, which expressed respectively the Escherichia coli catalase and spinach thylakoid-membrane-bound APX in chloroplasts and showed increased tolerance to photooxidative stress, were used for evaluation of the relationship between H2O2 accumulation or change of redox status of PET and cAPX induction under photooxidative stress condition . There was no difference in the increase in the transcript level of cAPX in the first 1 h after irradiation with high-intensity light between the wild-type and either of the transgenic plants . The transcript level of cAPX in the wild-type showed a steady gradual increase during the next 5 h, while that in the transgenic plants during this period was stable . The H2O2 level of wild-type plants increased after 1 h, while those of transgenic plants remained constant . The decrease in q(p) value in all types of plants occurred earlier than the increase in H2O2 level . These results indicate that the induction of cAPX expression is caused by a redox change in PET, probably through the plastquinone pool at an early stage and thereafter by an increase in the cellular H2O2 level.

Hum Mol Genet, 2005 Jan 15, 14(2), 327 - 34 Epub 2004 Dec 01.
Ferrochelatase consisting of wild-type and mutated subunits from patients with a dominant-inherited disease, erythropoietic protoporphyria, is an active but unstable dimer; Ohgari Y et al.; Erythropoietic protoporphyria (EPP) is an autosomal inherited disease of heme biosynthesis caused by a partial deficiency of the enzyme ferrochelatase . Patients with EPP show only 20-30% normal activity because of mutations in one of the alleles of the ferrochelatase gene . To clarify the molecular mechanisms of this low level of activity, we co-expressed human ferrochelatase carrying His- and HA-tags in a tandem fashion in Escherichia coli . Purification of the His-tag-containing enzyme revealed that the His-enzyme forms an oligomer in association with the HA-enzyme, and analysis by gel-filtration confirmed that the enzyme is a dimer ( approximately 80 kDa) . Then we expressed homo- and heterodimers composed of the wild-type and engineered mutants of the enzyme (C395Delta, H157A, H263A, H388A) or mutants from EPP patients (I186T, M267I) . The levels of homodimeric enzymes produced were low, and the activities of the purified homodimeric mutants were abolished . On the other hand, the heterodimers with wild-type and mutated subunits exhibited potential, but weak, activities without a marked change of K(m) values for substrates . These results showed that heterodimers containing normal and mutated subunits retain the enzymic activity, which is inconsistent with the hypothesis that ferrochelatase is only active when the dimer contains two normal subunits . Pretreatment at 42 degrees C led to a rapid inactiviation of the heterodimeric mutants, indicating instability . Thus, we provide evidence that the instability of the heterodimer containing normal and mutated ferrochelatase as well as the low production levels due to the structural defect of the mutant protein, not the abolishment of the enzymic activity of the heterodimer, causes the weak activity in EPP patients.

J Biol Chem . 2004 Dec 1; {Epub ahead of print}
Convergent evolution of a 2-methylbutyryl-CoA dehydrogenase from isovaleryl-CoA dehydrogenase in solanum tuberosum; Goetzman ES et al.; The potato cDNAs St-IVD1 and St-IVD2 encode proteins that are 84% identical and 64% identical to human isovaleryl-CoA dehydrogenase (IVD) . St-IVD2 protein was previously partially purified from potato tubers and confirmed to be an IVD . The function of St-IVD1 is unknown . In the present experiments both proteins were expressed in Escherichia coli and purified as intact homotetramers . The substrate preference profile of the St-IVD2 protein was similar to that of human IVD . Recombinant St-IVD1, however, had maximal activity with 2-methylbutyryl-CoA, which in humans is dehydrogenated by short/branched-chain acyl-CoA dehydrogenase (SBCAD) . While molelcular modeling predicts that the 2MBCD and IVD substrate binding pockets are nearly identical, 2MBCD has amino acid substitutions at five residues that are invariant among all known and putative IVDs . Site-directed mutagenesis was used to match the human IVD active site to that of potato 2MBCD . The resulting mutant IVD had detectable activity with 2-methylbutyryl-CoA and no activity with isovaleryl-CoA . The 2MBCD active site was compared to that of human SBCAD using molecular modeling . Residues M361 and A365 of 2MBCD appear to partially substitute for the function of Y380 in human SBCAD, binding the methyl branch linked to C2 of 2-methylbutyryl-CoA, while residuesV88, V92 and V96 appear to bind the distal C4 methyl group . The presence of a 2MBCD in potato that is highly homologous to IVD is an example of convergent evolution within the ACD family, leading to the independent occurrence of two enzymes (SBCAD, 2MBCD) specific for 2-methylbutyryl-CoA.

Front Biosci, 2005 Jan 1, 10, 119 - 34 Print 2005 Jan 1.
Metabolism of vitamin D3 by cytochromes P450; Sakaki T et al.; The vitamin D3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) and 1alpha,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D3 metabolism . Using the heterologous expression in E . coli, enzymatic properties of the P450s were recently investigated in detail . Upon analyses of the metabolites of vitamin D3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha,25(OH)2D3 in addition to the major metabolite 25(OH)D3 . These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D3 metabolism . In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH)D3 and 24R,25(OH)2D3 . Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity . Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity . A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 supports the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen . CYP24A1 expressed in E . coli showed a remarkable metabolic processes of 25(OH)D3 and 1alpha,25(OH)2D3 . Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha,25(OH)2D3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway . In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha,25(OH)2D3-26,23-lactone . Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1 . The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans.

Mol Cell, 2004 Dec 3, 16(5), 799 - 805
Uniform binding of aminoacylated transfer RNAs to the ribosomal A and P sites; Fahlman RP et al.; The association and dissociation rate constants of eight different E . coli aminoacyl-tRNAs (aa-tRNAs) for E . coli ribosomes programmed with mRNAs of defined sequences were determined . Identical association and dissociation rate constants were observed for all eight aa-tRNAs in both the ribosomal A and P sites despite substantial differences in tRNA sequence, the type of esterified amino acid, and posttranscriptional modifications . These results indicate that the overall binding of all aa-tRNAs to the ribosome is uniform . However, when either the esterified amino acid or the tRNA modifications were removed, binding was no longer uniform . These results suggest that differences in tRNA sequences and tRNA modifications have evolved to offset differential thermodynamic contributions of the esterified amino acid and the codon-anticodon interaction so that ribosomal binding of all aa-tRNAs remains uniform.

Mol Cell, 2004 Dec 3, 16(5), 701 - 13
A biochemically defined system for mammalian nonhomologous DNA end joining; Ma Y et al.; Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs) . Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions . Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice . The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku . This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends . The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand . This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.

Commun Dis Intell, 2004, 28(3), 390 - 1
Laboratory surveillance of Shiga toxin producing Escherichia coli in South Australia and the Hunter Health Area, New South Wales, Australia; Doyle R et al.; To estimate the prevalence of Shiga toxin producing Escherichia coli in Australia, bloody stool samples from two Australian locations were screened for the presence of Shiga toxin genes, stx1 and stx2 . Four of 126 (3.2%) and 139 of 5,829 (2.4%) patients from the two locations had a positive polymerase chain reaction for Shiga toxin genes.

Zoolog Sci, 2004 Nov, 21(11), 1121 - 4
Effect of a glycine residue insertion into crustacean hyperglycemic hormone on hormonal activity; Katayama H et al.; Crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) have similar amino acid sequences and therefore comprise a peptide family referred to as the CHH family . All MIHs unexceptionally have an additional glycine residue at position 12, which is lacking in all CHHs . In order to understand the relevance of the absence of the glycine residue for hyperglycemic activity, a mutant CHH having a glycine residue insertion was prepared, and its hyperglycemic activity was assessed . This mutant CHH had the same disulfide bond arrangement as the recombinant CHH produced in Escherichia coli cells, and exhibited a similar circular dichroism spectrum to the recombinant CHH, indicating that the two CHHs possessed similar conformations . The mutant CHH showed a hyperglycemic effect weaker than the recombinant CHH by about one order of magnitude . These results suggest that the insertion of a glycine residue is one of the indices for structural and functional divergence of the CHH family peptides.

Mol Cell Biol, 2004 Dec, 24(24), 10742 - 56
Nuclear export of hnRNP Hrp1p and nuclear export of hnRNP Npl3p are linked and influenced by the methylation state of Npl3p; Xu C et al.; Eukaryotic mRNA processing and export are mediated by a series of complexes composed of heterogeneous nuclear ribonucleoproteins (hnRNPs) . Many of these hnRNPs are methylated at arginine residues within their RGG domains . Although cellular arginine methylation is required for the efficient nuclear export of several hnRNPs, its role in this process is unknown . To address this question, we replaced the methylated RGG tripeptides of two hnRNPs, Npl3p and Hrp1p, with KGG . We found that these substitutions specifically abolish their methylation but have different effects on their nuclear export activity . Although the efficient export of Hrp1p requires cellular methyltransferase activity, the modification of Hrp1p itself is dispensable . In contrast, we found that Npl3 arginine methylation not only facilitates its own export but also is required for Hrp1p to efficiently exit the nucleus . Consistent with this observation, we found that Npl3p and Hrp1p exist in a ribonucleoprotein complex . We provide the first evidence that the arginine methylation of a particular protein directly affects its activity . Efficient export does not require methylation per se, but unmethylated arginine residues lead to retention of hnRNPs . Thus, arginine methylation serves to mask the Npl3p RGG domain for efficient ribonucleoprotein export.

Proc Natl Acad Sci U S A, 2004 Dec 14, 101(50), 17480 - 5 Epub 2004 Nov 30.
Three-dimensional structure and organization of a receptor/signaling complex; Francis NR et al.; Transmembrane signaling in bacterial chemotaxis has become an important model system for experimental and theoretical studies . These studies have provided a wealth of detailed molecular structures, including the structures of CheA, CheW, and the cytoplasmic domain of the serine receptor Tsr . How these three proteins interact to form the receptor/signaling complex remains unknown . By using EM and single-particle image analysis, we present a three-dimensional reconstruction of the receptor/signaling complex . The complex contains CheA, CheW, and the cytoplasmic portion of the aspartate receptor Tar . We observe density consistent with a structure containing 24 aspartate-receptor monomers and additional density sufficient to house the expected four CheA monomers and six CheW monomers . Within this bipolar structure are four groups of three receptor dimers that are not threefold symmetric and are therefore unlike the symmetric trimers observed in the x-ray crystal structure of the cytoplasmic domain of the serine receptor . In the latter, the interdimer contacts occur in the signaling domains near the hairpin loop . In our structure, the signaling domains within trimers appear spaced apart by the presence of CheA and CheW . This structure argues against models where one CheA and one CheW bind to the outer face of each of the dimers in the trimer . This structure of the receptor/signaling complex provides an additional basis for understanding the architecture of the large arrays of chemotaxis receptors, CheA, and CheW found at the cell poles in motile bacteria.

J Mol Biol, 2005 Jan 14, 345(2), 415 - 23
The core TatABC complex of the twin-arginine translocase in Escherichia coli: TatC drives assembly whereas TatA is essential for stability; Mangels D et al.; Current models for the action of the twin-arginine translocation (Tat) system propose that substrates bind initially to the TatBC subunits, after which a separate TatA complex is recruited to form an active translocon . Here, we have studied the roles of individual subunits in the assembly and stability of the core TatBC-containing substrate-binding complex . Previous studies have shown that TatB and TatC are active when fused together; we show here that deletion of the entire TatB transmembrane span from this Tat(BC) fusion inactivates the Tat system but does not affect assembly of the core complex . In this mutated complex, TatA is present but more loosely bound, indicating a role for TatB in the correct binding of TatA . In the absence of TatA, the truncated TatBC fusion protein still assembles into a complex of the correct magnitude, demonstrating that the transmembrane spans of TatC are the only determinants within the membrane bilayer that specify assembly of this complex . Further studies on both the Tat(BC) construct and the wild-type TatBC subunits show that the TatBC complex is unstable in the absence of TatA, and we show that TatA stabilises the TatB subunit specifically within this complex . The results demonstrate a dual role and location for TatA: in the functioning/maintenance of the core complex, and as a separate homo-oligomeric complex.

J Mol Biol, 2005 Jan 14, 345(2), 387 - 400
Translocation of histone proteins across lipid bilayers and Mycoplasma membranes; Rosenbluh J et al.; We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) . In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles . Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system . Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin . Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes . The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules . Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers . The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them . Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes . The extent of this translocation was inversely related to the degree of membrane cholesterol . The addition of cholesterol also reduced the extent of histone penetration into the MLVs . Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them . Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).

J Mol Biol, 2005 Jan 14, 345(2), 351 - 61
Assembly of Acanthamoeba myosin-II minifilaments . Definition of C-terminal residues required to form coiled-coils, dimers, and octamers; Turbedsky K et al.; Acanthamoeba myosin-II forms bipolar octamers by three successive steps of dimerization of the C-terminal, coiled-coil tail . In this study, we generated N-terminal and C-terminal truncation constructs and point mutants of the Acanthamoeba myosin-II tail to delineate the structural requirements for assembly of bipolar mini-filaments . By the use of light-scattering, CD spectroscopy, analytical ultracentrifugation, and tryptophan fluorescence experiments, we determined that: (1) the C-terminal 14 heptad repeats plus most of the tailpiece (residues 1381-1509) are required to form antiparallel dimers of coiled-coils; (2) amino acid residues within heptads 23-32 (residues 1254-1325) are required to form tetramers; (3) the C-terminal 32 heptad repeats suffice to assemble octameric minifilaments; (4) A1378 is outside of the interaction interface; (5) the mutation L1475W inhibits dimerization; and (6) F1443 is involved in the dimerization interface but is exposed to the solvent . We propose that the tailpiece (residues 1483-1509) interacts with two heptads (13 and 14, residues 1381-1393), which are important for dimerization and coiled-coil formation . These results support a model in which hydrophobic as well as electrostatic interactions control the register between myosin-II coiled-coils and guide sequential steps of dimerization that generate stable, octameric mini-filaments.

J Mol Biol, 2005 Jan 14, 345(2), 315 - 24
Role of C-terminal residues in oligomerization and stability of lambda CII: implications for lysis-lysogeny decision of the phage; Datta AB et al.; A crucial element in the lysis-lysogeny decision of the temperate coliphage lambda is the phage protein CII, which has several interesting properties . It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN6TTGC at each . The three-dimensional structure of CII, a homo-tetramer of 97 residue subunits, is unknown . It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH) . This instability is essential for the function of CII in the lysis-lysogeny switch . From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo . From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation . Presumably, tetramerization is necessary for the latter functions . Based on these results, we propose a model for CII structure, in which protein-protein contacts for dimer and tetramer formation are different . The implications of tetrameric organization, essential for CII activity, on the recognition of the direct repeat sequence is discussed.

J Mol Biol, 2005 Jan 14, 345(2), 251 - 64
Cooperative binding of the leucine-responsive regulatory protein (Lrp) to DNA; Chen S et al.; The leucine-responsive regulatory protein (Lrp) of Escherichia coli activates expression of a number of operons and represses expression of others . For some members of the Lrp regulon, exogenous leucine mitigates the effect of Lrp, for some it potentiates the effect of Lrp, and for others it has no effect on Lrp action . For the ilvIH operon that we study, Lrp activates expression in vivo and mediates the repression of the operon by exogenous leucine . We studied Lrp-1, a leucine-insensitive variant, to investigate mechanisms by which leucine alters Lrp action as an activator of ilvIH expression . The Asp114Glu change did not have much effect on the amount of total Lrp-1 in cells but decreased the amount of free Lrp-1 two- to threefold . Lrp monomers associate to form octamers and hexadecamers (hexadecamer form predominates at micromolar concentrations; Kd=5.27x10(-8) M), and leucine promotes the dissociation of Lrp hexadecamer to a leucine-bound octamer . By contrast, Lrp-1 exists primarily as an octamer in solution (equilibrium dissociation constant 6.5x10(-5) M) and leucine had little effect on the equilibrium . Thus, the hexadecameric form that Lrp assumes in the absence of DNA is not required for activation of the ilvIH operon . Both leucine and the lrp-1 mutation reduced the apparent affinity of Lrp binding to ilvIH DNA (contains two groups of binding sites separated by 136 bp) but they have different effects on intrinsic binding affinity and binding cooperativity . Whereas leucine reduced intrinsic binding affinities and interactions of Lrps bound at upstream and downstream regions of ilvIH DNA, it increased cooperative dimer-dimer interactions of Lrps bound to two adjacent sites . By contrast, the lrp-1 mutation did not have much effect on intrinsic binding affinities but it decreased cooperative adjacent dimer-dimer interactions and enhanced interactions of Lrps bound at upstream and downstream regions of ilvIH DNA . Our analysis is consistent with the idea that leucine enhances dimer-dimer interactions that contribute to octamer formation, concomitantly reducing dimer-dimer interactions that contribute to the longer range interactions of Lrps that are required for activation of the ilvIH promoter.

BMC Microbiol . 2004 Nov 30;4(1):44.
A simulation model of Escherichia coli osmoregulatory switch using E-CELL system; Srividhya KV et al.; BACKGROUND: Bacterial signal transduction mechanism referred to as a "two component regulatory systems" contributes to the overall adaptability of the bacteria by regulating the gene expression . Osmoregulation is one of the well-studied two component regulatory systems comprising of the sensor, EnvZ and the cognate response regulator, OmpR, which together control the expression of OmpC and OmpF porins in response to the osmolyte concentration . RESULTS: A quantitative model of the osmoregulatory switch operative in Escherichia coli was constructed by integrating the enzyme rate equations using E-CELL system . Using the substance reactor logic of the E-CELL system, a total of 28 reactions were defined from the injection of osmolyte till the regulated expression of porins by employing the experimental kinetic constants as reported in literature . In the case of low osmolarity, steady state production of OmpF and repression of OmpC was significant . In this model we show that the steady state - production of OmpF is dramatically reduced in the high osmolarity medium . The rate of OmpC production increased after sucrose addition, which is comparable with literature results . The relative porin production seems to be unaltered with changes in cell volume changes, ATP, EnvZ and OmpR at low and high osmolarity conditions . But the reach of saturation was rapid at high and low osmolarity with altered levels of the above components . CONCLUSIONS: The E-CELL system allows us to perform virtual experiments on the bacterial osmoregulation model . This model does not take into account interaction with other networks in the cell . It suggests that the regulation of OmpF and OmpC is a direct consequence of the level of OmpRP in the cell and is dependent on the way in which OmpRP interacts with ompF and ompC regulatory regions . The preliminary simulation experiment indicates that both reaching steady state expression and saturation is delayed in the case of OmpC compared to OmpF . Experimental analysis will help improve the model . The model captures the basic features of the generally accepted view of EnvZ-OmpR signaling and is a reasonable starting point for building sophisticated models and explaining quantitative features of the system.

Anal Chem, 2004 Dec 1, 76(23), 7069 - 76
Development of a bioluminescence resonance energy-transfer assay for estrogen-like compound in vivo monitoring; Michelini E et al.; A new bioluminescence resonance energy transfer (BRET) homogeneous assay to evaluate the presence of estrogen-like compounds has been developed and optimized . The assay is based on the direct evaluation of estrogen alphareceptor (ERalpha) homodimerization as a result of estrogen-like compound binding . ERalpha monomer was genetically fused either to Renilla luciferase (Rluc) or to enhanced yellow fluorescent protein (EYFP) . In the presence of estrogens, ERalpha dimerization brings Rluc and EYFP molecules close enough for an energy transfer . An in vitro BRET assay was first developed using purified fusion proteins (ERalpha-Rluc and ERalpha-EYFP) expressed in Escherichia coli to evaluate and optimize the analytical performances of the assay in the presence of 17-beta estradiol . The "in vivo" BRET quantitative assay was then developed by coexpressing the two fusion proteins in live HepG2 cells . The assay can be performed in 96-well microplate format with a 30-min incubation and allows detection with adequate accuracy and precision of as low as 1 nM of 17-beta estradiol . This new "in vivo" BRET assay allows evaluating the estrogen-like activity and synthetic xenoestrogens from biological and environmental samples.

Anal Chem, 2004 Dec 1, 76(23), 6908 - 14
Electrokinetic bioprocessor for concentrating cells and molecules; Wong PK et al.; Bioprocessors for concentrating bioparticles, such as cells and molecules, are commonly needed in bioanalysis systems . In this microfluidic processor, a global flow field generated by ac electroosmosis transports the embedded particles to the regions near the electrode surface . The processor then utilizes electrophoretic and dielectrophoretic forces, which are effective in short range, to trap the target cells and molecules on the electrode surface . By optimizing the operating parameters, we have concentrated various biological objects in a large range of sizes, including Escherichia coli bacteria, lambda phage DNA, and single-stranded DNA fragments as small as 20 bases that have a radius of gyration of only 3 nm.

Nucleosides Nucleotides Nucleic Acids, 2004 Oct, 23(8-9), 1459 - 65
Cloning and expression of malarial pyrimidine enzymes; Christopherson RI et al.; We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from Plasmodium falciparum and sequence information from the Malarial Genome Project . Genes encoding dihydroorotase (reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) have been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C-terminus and the enzymes were expressed in Escherichia coli . To overcome the toxicity of malarial OMP decarboxylase when expressed in E . coli, and the unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low levels of T7 lysozyme to inhibit T7 RNA polymerase used for recombinant expression, and extra copies of rare tRNAs . Catalytically-active OMP decarboxylase has been purified in tens of milligrams by chromatography on Ni-NTA . The gene encoding orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N-terminus when compared with sequences for this enzyme from other organisms . We have found that other pyrimidine enzymes also contain unusual protein inserts . Milligram quantities of pure recombinant malarial enzymes from the pyrimidine pathway will provide targets for development of novel antimalarial drugs.

Acta Microbiol Immunol Hung, 2004, 51(3), 297 - 302
Cloning, expression and purification of SmpB from Mycobacterium tuberculosis; Kovacs L et al.; SmpB, a small tmRNA binding protein, is essential for trans-translation . 6His and FLAG tagged SmpB was cloned from Mycobacterium tuberculosis H37Rv . It was expressed in Escherichia coli using the T7 promoter-polymerase system . Anti-FLAG M2 agarose was used for its purification . Mycobacterial SmpB copurifies with other proteins . We identified elongation factor EF-Tu in the purified SmpB preparations.

Photochem Photobiol Sci, 2004 Nov-Dec, 3(11-12), 1011 - 6 Epub 2004 Nov-Dec.
Chromophore composition of a heterologously expressed BLUF-domain; Laan W et al.; Upon heterologous expression of the BLUF (for: Blue-Light sensing Using Flavin) domain from AppA, a transcriptional anti-repressor from Rhodobacter sphaeroides, in Escherichia coli, photoactive holo-protein is formed through non-covalent binding of a flavin . Whereas it is generally assumed that FAD is the physiological chromophore of this photo-perception domain in vivo, E . coli can (and does) insert, depending on the growth conditions, all naturally occurring flavins, i.e . riboflavin, FMN and FAD into this protein domain . The nature of the particular flavin bound affects the photochemical- and particularly the fluorescence properties of the N-terminal domain of this photosensory protein.

Nucleic Acids Res, 2004, 32(21), 6187 - 99 Print 2004.
Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification; Chan SH et al.; The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described . The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt . M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X . In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well . Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY) . Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII . Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected . Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites . Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C . Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification . Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.

Proc Natl Acad Sci U S A, 2004 Dec 7, 101(49), 17072 - 7 Epub 2004 Dec 7.
Single-cell FRET imaging of phosphatase activity in the Escherichia coli chemotaxis system; Vaknin A et al.; Two-component signaling systems, in which a receptor-coupled kinase is used to control the phosphorylation level of a response regulator, are commonly used in bacteria to sense their environment . In the chemotaxis system of Escherichia coli, the receptors, and thus the kinase, are clustered on the inner cell membrane . The phosphatase of this system also is recruited to receptor clusters, but the reason for this association is not clear . By using FRET imaging of single cells, we show that in vivo the phosphatase activity is substantially larger at the cluster, indicating that the signaling source (the kinase) and the signaling sink (the phosphatase) tend to be located at the same place in the cell . When this association is disrupted, a gradient in the concentration of the phosphorylated response regulator appears, and the chemotactic response is degraded . Such colocalization is inevitable in systems in which the activity of the kinase and the phosphatase are produced by the same enzyme . Evidently, this design enables a more rapid and spatially uniform response.

J Biol Chem . 2004 Nov 29; {Epub ahead of print}
A role for SlyD in the Escherichia coli hydrogenase biosynthetic pathway; Zhang JW et al.; The {NiFe} centers at the active sites of the Escherichia coli hydrogenase enzymes are assembled by a team of accessory proteins that includes the products of the hyp genes . To determine if any other proteins are involved in this process, the sequential peptide affinity (SPA) system was used . The analysis of proteins in a complex with HypB revealed the peptidyl-prolyl cis/trans-isomerase SlyD, a metal-binding protein that has not been previously linked to the hydrogenase biosynthetic pathway . The association between HypB and SlyD was confirmed by chemical cross-linking of purified proteins . Deletion of the slyD gene resulted in a marked reduction of the hydrogenase activity in cell extracts prepared from anaerobic cultures, and an in-gel assay was used to demonstrate diminished activities of both hydrogenase 1 and 2 . Western analysis revealed a decrease in the final proteolytic processing of the hydrogenase 3 HycE protein, indicating that the metal center was not assembled properly . These deficiencies were all rescued by growth in media containing excess nickel, but zinc did not have any phenotypic effect . Experiments with radioactive nickel demonstrated that less nickel accumulated in slyD cells compared to wild type, and overexpression of SlyD from an inducible promoter doubled the level of cellular nickel . These experiments demonstrate that SlyD has a role in the nickel insertion step of the hydrogenase maturation pathway, and the possible functions of SlyD are discussed.

Front Biosci, 2005 Jan 1, 10, 9 - 16 Print 2005 Jan 1.
Dual functional regulators coordinate DNA replication and gene expression in proliferating cells; Tye BK et al.; Gene products for cell growth must meet the pace of DNA replication and vice versa during the cell division cycle, therefore coordination of DNA replication and gene expression is vital to proliferating cells . During development in multicellular organisms when rapid cell divisions must be accompanied by the expression of particular gene sets in differentiating tissues, this coordination is even more crucial . Undoubtedly, multiple strategies are used to ensure the coordination of gene expression and DNA replication . In this review, we focus on the strategy that uses dual functional factors to serve both the functions of replication initiator and transcription regulator . Classical examples are the dual functional replication initiator/transcription regulators, DnaA of E . coli and T antigen of SV40, which bind replication origins and regulate their own synthesis . Emerging examples in eukaryotes are the growth responsive transcription factor E2f, the MADS domain combinatorial transcription factor Mcm1, and a subunit of the MCM2-7 helicase, Mcm7.

Zhonghua Yu Fang Yi Xue Za Zhi, 2004 Nov, 38(6), 369 - 73
{In vitro selection of specific aptamers against microcystin-LR.}; Gu KD et al.; OBJECTIVE: In vitro selection of specific RNA aptamers against microcystin-LR from a random RNA pool . METHODS: A RNA library with 40 randomized nucleotide positions was applied to select for specific aptamers to microcystin-LR covalently linked to Sepharose by using a standard in vitro selection protocol . RESULTS: The specific enriched RNA aptamer for microcystin-LR increased step by step from initial round to 11th round after which a plateau of the aptamer quantity was observed between 11th and 13th round . The enriched RNAs from last round were reverse transcribed, PCR amplified and cloned into E . coli DH10 b competent cells . Sixty colonies were sequenced from which 38 sequences were aligned and classified into 3 families and 5 duplicates and no conserved sequences were found among them . Eight representative clones from the groups were selected for further binding experiments comparing with original pool RNA . Four clone RNAs were identified with relatively high affinity to microcystin-LR, of which MC25 clone RNA could combine with microcystin-LR as lower as 0.5 micromol/L . CONCLUSION: Subpopulations of RNA molecules that bind specifically to microcystin-LR have been isolated from a population of random sequence RNA molecules, which might provide a new way for future application in environmental monitoring of microcystin.

Genes Cells, 2004 Dec, 9(12), 1175 - 87
In vitro transcription analysis by reconstituted cyanobacterial RNA polymerase: roles of group 1 and 2 sigma factors and a core subunit, RpoC2; Imamura S et al.; The RNA polymerase (RNAP) core enzyme of cyanobacterium Synechocystis sp . strain PCC 6803 was reconstituted with overproduced recombinant subunits and purified with C-terminal histidine-tagged RpoA . The core enzyme with purified a sigma factor, SigA/SigD or SigB, allowed specific in vitro transcription from the light-inducible psbA2 or the dark-/heat-inducible lrtA/hspA promoters, respectively . Further analysis using a mutant psbA2 promoter revealed that the -35 hexamer of the promoter was essential for SigA but not SigD . Similar but distinct patterns of psbA2 transcription were found for two types of RNAP, cyanobacterial (alpha2betabeta'gamma) and E . coli (alpha2betabeta') core enzymes . Specific binding of PCC 6803 RpoC2 (beta') to E . coli core enzyme and its contribution to efficient psbA2 transcription by RNAP-SigA/D suggest that this subunit could confer an important role on the cyanobactrial RNAP . Differences in affinity and specificity among cyanobacterial sigma factors for the core enzyme and promoters were discussed.

Genes Cells, 2004 Dec, 9(12), 1151 - 66
Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coli; Shimuta TR et al.; Escherichia coli DnaA protein initiates chromosomal replication and is an important regulatory target during the replication cycle . In this study, a suppressor mutation isolated by transposon mutagenesis was found to allow growth of the temperature-sensitive dnaA508 and dnaA167 mutants at 40 degrees C . The suppressor consists of a transposon insertion in a previously annotated ORF, here termed hspQ, a novel heat shock gene whose promoter is recognized by the major heat shock sigma factor sigma32 . Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 degrees C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ . Cellular DnaA508 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process . In contrast, DnaA46 protein is rapidly degraded in an hspQ-independent manner . Gel-filtration and chemical cross-linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers . Heat-shock induced proteases such as Clp contain homomultimers of subunit proteins . We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins.

Annu Rev Genet, 2004, 38, 749 - 70
rRNA transcription in Escherichia coli; Paul BJ et al.; Ribosomal RNA transcription is the rate-limiting step in ribosome synthesis in bacteria and has been investigated intensely for over half a century . Multiple mechanisms ensure that rRNA synthesis rates are appropriate for the cell's particular growth condition . Recently, important advances have been made in our understanding of rRNA transcription initiation in Escherichia coli . These include (a) a model at the atomic level of the network of protein-DNA and protein-protein interactions that recruit RNA polymerase to rRNA promoters, accounting for their extraordinary strength; (b) discovery of the nonredundant roles of two small molecule effectors, ppGpp and the initiating NTP, in regulation of rRNA transcription initiation; and (c) identification of a new component of the transcription machinery, DksA, that is absolutely required for regulation of rRNA promoter activity . Together, these advances provide clues important for our molecular understanding not only of rRNA transcription, but also of transcription in general.

Biochemistry, 2004 Dec 7, 43(48), 15103 - 10
Analysis of the role of intraprotein electron transfer in photoreactivation by DNA photolyase in vivo; Kavakli IH et al.; Escherichia coli DNA photolyase contains FADH(-) as the catalytic cofactor . The cofactor becomes oxidized to the FADH(*) blue neutral radical during purification . The E-FADH(*) form of the enzyme is catalytically inert but can be converted to the active E-FADH(-) form by a photoreduction reaction that involves intraprotein electron transfer from Trp306 . It is thought that the E-FADH(*) form is also transiently generated during pyrimidine dimer repair by photoinduced electron transfer, and it has been suggested that the FADH(*) that is generated after each round of catalysis must be photoreduced before the enzyme can engage in subsequent rounds of repair . In this study, we introduced the Trp306Phe mutation into the chromosomal gene and tested the non-photoreducible W306F mutant for photorepair in vivo . We find that both wild-type and W306F mutant photolyases carry out at least 25 rounds of photorepair at the same rate . We conclude that photoreduction by intraprotein electron transfer is not part of the photolyase photocycle under physiological conditions.

Nat Biotechnol, 2004 Dec, 22(12), 1583 - 7 Epub 2004 Dec.
Baculovirus expression system for heterologous multiprotein complexes; Berger I et al.; The discovery of large multiprotein complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function . Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein complexes comprising many subunits . Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites . The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition . This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated . Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure . Our system is useful for both recombinant multiprotein production and multigene transfer applications.

EMBO Rep, 2004 Dec, 5(12), 1159 - 64 Epub 2004 Dec.
NMR structure of the bovine prion protein isolated from healthy calf brains; Hornemann S et al.; NMR structures of recombinant prion proteins from various species expressed in Escherichia coli have been solved during the past years, but the fundamental question of the relevancy of these data relative to the naturally occurring forms of the prion protein has not been directly addressed . Here, we present a comparison of the cellular form of the bovine prion protein isolated and purified from healthy calf brains without use of detergents, so that it contains the two carbohydrate moieties and the part of the GPI anchor that is maintained after enzymatic cleavage of the glycerolipid moiety, with the recombinant bovine prion protein expressed in E . coli . We show by circular dichroism and (1)H-NMR spectroscopy that the three-dimensional structure and the thermal stability of the natural glycoprotein and the recombinant polypeptide are essentially identical . This result indicates possible functional roles of the glycosylation of prion proteins in healthy organisms, and provides a platform and validation for future work on the structural biology of prion proteins, which will have to rely primarily on the use of recombinant polypeptides.

EMBO Rep, 2004 Dec, 5(12), 1153 - 8
Electron and atomic force microscopy of the trimeric ammonium transporter AmtB; Conroy MJ et al.; Escherichia coli AmtB is an archetypal member of the ammonium transporter (Amt) family, a family of proteins that are conserved in all domains of life . Reconstitution of AmtB in the presence of lipids produced large, ordered two-dimensional crystals . From these, a 12 A resolution projection map was determined by cryoelectron microscopy, and high-resolution topographs were acquired using atomic force microscopy . Both techniques showed the trimeric structure of AmtB in which each monomer seems to have a pseudo-two-fold symmetry . This arrangement is likely to represent the in vivo structure . This work provides the first views of the structure of any member of the Amt family.

Virology, 2004 Dec 20, 330(2), 447 - 59
A complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions; Szajner P et al.; Early events in vaccinia virus (VAC) morphogenesis, particularly the formation of viral membranes and their association with viroplasm, are poorly understood . Recently, we showed that repression of A30 or G7 expression results in the accumulation of normal viral membranes that form empty-looking immature virions (IV), which are separated from large masses of electron-dense viroplasm . In addition, A30 and G7 physically and functionally interact with each other and with the F10 protein kinase . To identify other proteins involved in early morphogenesis, proteins from cells that had been infected with vaccinia virus expressing an epitope-tagged copy of F10 were purified by immunoaffinity chromatography and analyzed by gel electrophoresis . In addition to F10, A30, and G7, viral proteins A15, D2, D3, and J1 were identified by mass spectrometry of tryptic peptides . Further evidence for the complex was obtained by immunopurification of proteins associated with epitope-tagged A15, D2, and D3 . The previously unstudied A15, like other proteins in the complex, was expressed late in infection, associated with virus cores, and required for the stability and kinase activity of F10 . Biochemical and electron microscopic analyses indicated that mutants in which A15 or D2 expression was regulated by the Escherichia coli lac operator system exhibited phenotypes characterized by the presence of large numbers of empty immature virions, similar to the results obtained with inducible A30 and G7 mutants . Empty immature virions were also seen by electron microscopy of cells infected with temperature-sensitive mutants of D2 or D3, though the numbers of membrane forms were reduced perhaps due to additional effects of high temperature.

J Mol Biol, 2005 Jan 7, 345(1), 51 - 68
Structural analysis of the group II intron splicing factor CRS2 yields insights into its protein and RNA interaction surfaces; Ostheimer GJ et al.; Chloroplast RNA splicing 2 (CRS2) is a nuclear-encoded protein required for the splicing of nine group II introns in maize chloroplasts . CRS2 functions in the context of splicing complexes that include one of two CRS2-associated factors (CAF1 and CAF2) . The CRS2-CAF1 and CRS2-CAF2 complexes are required for the splicing of different subsets of CRS2-dependent introns, and they bind tightly and specifically to their genetically defined intron targets in vivo . The CRS2 amino acid sequence is closely related to those of bacterial peptidyl-tRNA hydrolases (PTHs) . To identify the structural differences between CRS2 and bacterial PTHs responsible for CRS2's gains of CAF binding and intron splicing functions, we determined the structure of CRS2 by X-ray crystallography . The fold of CRS2 is the same as that of Escherichia coli PTH, but CRS2 has two surfaces that differ from the corresponding surfaces in PTH . One of these is more hydrophobic in CRS2 than in PTH . Site-directed mutagenesis of this surface blocked CRS2-CAF complex formation, indicating that it is the CAF binding site . The CRS2 surface corresponding to the putative tRNA binding face of PTH is considerably more basic than in PTH, suggesting that CRS2 interacts with group II intron substrates via this surface . Both the sequence and the structural context of the amino acid residues essential for peptidyl-tRNA hydrolase activity are conserved in CRS2, yet expression of CRS2 is incapable of rescuing a pth(ts)E.coli strain.

J Microbiol Methods, 2005 Jan, 60(1), 93 - 105
Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli; Watterworth L et al.; A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes . Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E . coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E . coli (ETEC), adherence factor (EAF) of enteropathogenic E . coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E . coli (EIEC) . The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively . The multiplex primers and probes were tested for specificity against 31 pathogenic E . coli strains, nine nonpathogenic E . coli and non-E.coli enteric and environmental bacterial strains . The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains . The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E . coli.

Biochem Biophys Res Commun, 2005 Jan 7, 326(1), 254 - 9
Recombinant SEC14-like proteins (TAP) possess GTPase activity; Habermehl D et al.; The three human SEC14-like proteins TAP1, TAP2, and TAP3 were expressed in Escherichia coli and purified by means of an amino-terminal His-tag . The recombinant TAP proteins bound alpha-, beta-, gamma-, and delta-tocopherol, certain phospholipids, and squalene . Intriguingly, the TAP proteins showed considerable GTPase activity that was comparable to that of small GTP-binding proteins of the Rab family . Although the TAP proteins contain important motifs to provide GTPase activity, the surrounding secondary structure markedly differed from common G-protein domains . However, these motifs are located in close proximity in the TAP structure and may therefore form an active site for GTP-binding and hydrolysis.

Biochem Biophys Res Commun, 2005 Jan 7, 326(1), 100 - 7
Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes; Zheng CL et al.; Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs . To date, little is known about the process of vault assembly or about the stability of vault components . In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells . Using an immunoprecipitation assay, we found that it took more than 4h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5h . Once incorporated into the vault complex, both MVP and VPARP were very stable . Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology . The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles . This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles . In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly.

Infect Genet Evol, 2005 Jan, 5(1), 79 - 83
Distribution of virulence related genes among enteroaggregative Escherichia coli isolates: using multiplex PCR and hybridization; Bouzari S et al.; The importance of enteroaggregative Escherichia coli (EAEC) strains in public health around the world is becoming increasingly clear . EAEC diagnosis has long been problematic . In this study, the recently designed multiplex PCR based on three plasmid-borne genes (AA probe, aap, and aggR) and DNA hybridization assay with plasmid-derived DNA probes were used for detection of HEp-2 adherent strains . These were isolated from an epidemiologic study of diarrhea in Iran . Using AA and DA probes revealed that 32.4%, 16.2%, 23%, and 28.4% of these isolates were AA, DA, AA/DA, and non-AA/DA, respectively . However, employing multiplex PCR for detection of these isolates, showed that 51.3% of the strains were AA and the rest 48.7% were not AA . While presence of other genes (pet, shf, aggA, aafA) considered to be specific for EAEC were checked among these isolates . The data obtained revealed that except for AA, aap, and aggR, the rest of the virulence related genes are not specific for EAEC isolates and are randomly distributed among adherent isolates . Over all the results obtained here indicated that this multiplex PCR is specific and sensitive assay . Phenotypically adherent strains are divided into two main groups, by use of this multiplex PCR i.e., typical EAEC isolates that carry the three plasmid-borne genes all together and atypical EAEC isolates in which the three genes are not linked together.

Clin Diagn Virol, 1996 Aug, 6(2-3), 137 - 45
Diagnosis of hepatitis C Virus (HCV) infection by antigen-capturing ELISA; Cao C et al.; Background: Hepatitis C virus (HCV) is a major cause of non-A non-B hepatitis . Detection of circulating antibodies against HCV by enzyme-linked immunosorbent assay (ELISA) has provided the main approach for the diagnosis of HCV infection . Most ELISA kits use a mixture of core, NS3, NS4 and NS5 antigen as capture antigens and enzyme-labeled goat anti-human IgG as conjugate . Objectives: To establish an ELISA system based on the antigen-capturing principle, using a recombinant chimeric polyprotein containing four HCV antigenic components as antigen . Study design: HCV antigens were expressed in Escherichia coli as chimeric polyprotein either in inclusion bodies or in soluble form . Protein expressed in inclusion bodies was used as solid-phase antigen, and the antigen expressed in a soluble form was used as enzyme conjugate after being labeled with horseradish peroxidase (HRP) . Results: Genes coding HCV antigens were cloned and sequenced, chimeric polyproteins containing four immunodominant components (core, NS3, NS4 and NS5) were expressed in E . coli both in soluble and in inclusion body form . These two chimeric proteins retained the antigenicity of HCV antigens . Antibody-capturing ELISA using the chimeric antigens showed a sensitivity of 97% ( {Formula: see text} ) and a specificity of 98% ( {Formula: see text} ) using the reference panel from the National Institute for the Control of Pharmaceutic and Biological Products of China (NICPBC); the same assay showed a sensitivity of 97.9% ( {Formula: see text} ) and a specificity of 100% ( {Formula: see text} ) using the self-established reference panel . Antigen-capturing ELISA was set up using the antigen labeled with horseradish peroxidase as conjugate, and was shown to be as sensitive as (97.9%) and more specific than (100%) antibody-capturing ELISA using the reference panel in this work . The antigen-capturing ELISA also showed a high accordance (98.9%) with UBI HCV enzyme immunoassay (EIA) 4.0 kits (United Biomedical Inc . USA) . Conclusion: Antigen-capturing ELISA provided a convenient, sensitive and more specific approach for the diagnosis of hepatitis C virus infection.

Clin Diagn Virol, 1996 May, 5(2-3), 167 - 79
Artificial mosaic proteins as new immunodiagnostic reagents: the hepatitis E virus experience; Fields HA et al.; Background: Naturally occurring viral proteins derived from cell culture and recombinant proteins expressed in procaryotic systems have been used extensively as target proteins in the development of immunoassay methods for the detection of antibodies . However, immunoassays utilizing these proteins often yield false-positive reactions suggesting that it may be possible to identify and remove regions responsible for these non-specific reactions . Objective: In this paper we describe a new strategy for the construction of immunoreactive recombinant proteins designed to improve immunoassay specificity . Study design: A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligonucleotides by the polymerase chain reaction (PCR) . The polypeptide comprises a mosaic of three antigenically dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican strain . The mosaic protein was expressed in Escherichia coli as a chimera with glutathione-S-transferase or beta-galactosidase . Results: Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by immunoblot analysis and by enzyme immunoassay (EIA) the presence and accessibility of all HEV-specific antigenic epitopes designed into the mosaic protein . Both hybrid proteins were shown by immunoblot analysis using a panel of human anti-HEV-positive and -negative sera to be HEV-specific . A sensitive and specific EIA was developed to detect IgG anti-HEV activity in human sera . A neutralization test using individual synthetic peptides corresponding to the epitopes designed into the mosaic protein was also developed to confirm IgG anti-HEV activity by absorbing the specimen before retesting by EIA . Conclusion: An artificial mosaic protein composed of short linear HEV-specific antigenic epitopes was constructed from synthetic oligonucleotides by PCR and used to develop a sensitive and specific EIA for the detection of anti-HEV activity in human sera.

Clin Diagn Virol, 1995 Aug, 4(2), 175 - 82
Rapid detection of HSV with an enzyme-linked virus inducible system(trade mark) (ELVIS(trade mark)) employing a genetically modified cell line; Proffitt MR et al.; Background: Infections with herpes simplex viruses (HSV) are common and may cause severe disease in immunocompromised hosts and in neonates . Isolation of infectious HSV in tissue culture is the most sensitive method of detection, but is not the most rapid . Recently, however, an Enzyme-Linked Virus Inducible System(trade mark) (ELVIS(trade mark)) for rapid detection of HSV in culture has been developed . The system employs genetically engineered baby hamster kidney (BHK) cells (ELVIS(trade mark) cells) whose DNA bears and HSV inducible promoter gene chimerically linked to an E . coli LacZ "reporter" gene . Induction of the promoter by HSV leads to the production of LacZ product, beta-galactosidase, which is readily detected histochemically . Objective: To evaluate these ELVIS(trade mark) cells, as a test for HSV, in comparison with HSV detection in MRC-5 cells in shell vial cultures confirmed by staining with fluorescent antibodies . Study design: Over a period of one month, 167 specimens submitted to the laboratory for detection of HSV were evaluated . Specimens were inoculated onto MRC-5 cells growing on glass coverslips in each of two shell vials and into two wells of a 24-well cluster plate containing ELVIS(trade mark) cells . MRC-5 shell vial cultures were observed daily for cpe for up to 7 days . With the appearance of cpe, the coverslips were fixed and the cells were typed for HSV-1 and HSV-2 with monoclonal antibodies . Specimens inoculated onto ELVIS(trade mark) cells were incubated for 16-24 h, then substrate was added to stain for beta-galactosidase . ELVIS(trade mark) cells, induced by HSV infection to express beta-galactosidase, stained blue upon reaction with substrate . Results: Of 167 specimens inoculated onto MRC-5 cells, 13 were excluded because of contamination or toxicity . Among the remaining 154 specimens, 24 were positive for HSV in the MRC-5 shell vials . Of 166 specimens inoculated into the ELVIS(trade mark) cell, all were completed within 24 h . Twenty-three (23) of the 24 shell-vial-positive cultures also were positive on the ELVIS(trade mark) cells . All 23 specimens detected in the ELVIS(trade mark) cells were positive within 24 h, whereas only nine were positive within 24 hours in MRC-5 shell vial cultures . The remaining 15 became positive after 24 h . Specimens positive for viruses other than HSV-1 or HSV-2 were not positive on the ELVIS(trade mark) cells . Conclusions: The ELVIS(trade mark) assay for HSV is simple to perform, is rapid, sensitive, and specific . The assay detects both HSV-1 and HSV-2 . No antibodies are required unless typing, which can be done on the ELVIS(trade mark) cells, is necessary.

Clin Diagn Virol, 1994 Jan, 1(5-6), 279 - 87
Influence of HIV-infection on the phagocytic activity of monocytes/macrophages and granulocytes; Schafer V et al.; Because of the great importance of phagocytosis as a key process in host defence, the influence of HIV-infection on the phagocytic activity of monocytes/macrophages (M0/MAC) and granulocytes was investigated . Therefore, blood samples from the peripheral blood of 70 HIV-infected individuals were incubated with fluorescein isothiocyanate (FITC) labeled Escherichia coli . The uptake of the bacteria was monitored by flow cytometer analysis . A strong and significant increase in the relative number of phagocytic granulocytes was observed ranging from 12.8% in an uninfected control collective to over 30% in AIDS patients . This effect was obtained for all patients and independent of the stage of disease . For monocytes, only marginal changes were found in their phagocytic function . These data suggest that the high susceptibility of HIV patients for secondary infections is not linked to a loss of phagocytic ability of monocytes/macrophages and/or granulocytes.

Crit Care, 2004 Dec, 8(6), R451 - 8 Epub 2004 Dec.
Extravascular lung water assessed by transpulmonary single thermodilution and postmortem gravimetry in sheep; Kirov MY et al.; INTRODUCTION: Acute lung injury is associated with accumulation of extravascular lung water (EVLW) . The aim of the present study was to compare two methods for quantification of EVLW: transpulmonary single thermodilution (EVLWST) and postmortem gravimetric (EVLWG) . METHODS: Eighteen instrumented and awake sheep were randomly assigned to one of three groups . All groups received Ringer's lactate (5 ml/kg per hour intravenously) . To induce lung injury of different severities, sheep received Escherichia coli lipopolysaccharide 15 ng/kg per min intravenously for 6 hours (n = 7) or oleic acid 0.06 ml/kg intravenously over 30 min (n = 7) . A third group (n = 4) was subjected to sham operation . Haemodynamic variables, including EVLWST, were measured using a PiCCOplus monitor (Pulsion Medical Systems, Munich, Germany), and the last measurement of EVLWST was compared with EVLWG . RESULTS: At the end of experiment, values for EVLWST (mean +/- standard error) were 8.9 +/- 0.6, 11.8 +/- 1.0 and 18.2 +/- 0.9 ml/kg in the sham-operated, lipopolysaccharide and oleic acid groups, respectively (P < 0.05) . The corresponding values for EVLWIG were 6.2 +/- 0.3, 7.1 +/- 0.6 and 11.8 +/- 0.7 ml/kg (P < 0.05) . Ranges of EVLWIST and EVLWIG values were 7.5-21.0 and 4.9-14.5 ml/kg . Regression analysis between in vivo EVLWST and postmortem EVLWG yielded the following relation: EVLWST = 1.30 x EVLWG + 2.32 (n = 18, r = 0.85, P < 0.0001) . The mean bias +/- 2 standard deviations between EVLWST and EVLWG was 4.9 +/- 5.1 ml/kg (P < 0.001) . CONCLUSION: In sheep, EVLW determined using transpulmonary single thermodilution correlates closely with gravimetric measurements over a wide range of changes . However, transpulmonary single thermodilution overestimates EVLW as compared with postmortem gravimetry.

Clin Exp Pharmacol Physiol, 2004 Nov, 31(11), 811 - 6
Nitroreductase: a prodrug-activating enzyme for cancer gene therapy; Searle PF et al.; 1 . The prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) is activated by Escherichia coli nitroreductase (NTR) to a potent DNA-crosslinking agent . 2 . Virus-mediated expression of NTR in tumour cells sensitizes them to CB1954 in vitro and in vivo, providing the basis for a strategy of cancer gene therapy . 3 . A phase I trial of CB1954 in cancer patients has been completed, documenting the pharmacokinetics and establishing an acceptable dose . Subsequent trials of the replication-defective adenovirus CTL102 in patients with resectable tumours have documented expression of NTR in injected colorectal liver metastases, hepatocellular carcinoma, head and neck cancer and prostate cancer . Trials combining CTL102 and CB1954 are underway . 4 . An oncolytic (replication-competent) adenovirus vector allowed increased expression of NTR in vitro and in a mouse tumour model, resulting in a greater reduction in tumour growth when combined with CB1954 treatment . 5 . Alternative prodrugs may eventually prove superior to CB1954; a nitroaryl phosphoramide mustard prodrug activated by NTR shows a greater therapeutic index than CB1954 in a human ovarian carcinoma . 6 . The crystal structure of NTR provided the basis for site-directed mutagenesis, which has identified a number of mutants with improved kinetics of CB1954 activation . These can provide improved cell sensitization to CB1954 . Combinations of these are being tested . 7 . The basis for a positive selection for improved NTR variants has been demonstrated.

Cell Mol Biol (Noisy-le-grand), 2004 Jul, 50(5), 625 - 30
Characterization of the eicosapentaenoic acid biosynthesis gene cluster from Shewanella sp . strain SCRC-2738; Orikasa Y et al.; The 38 kb eicosapentaenoic acid (EPA) biosynthesis gene cluster of Shewanella sp . strain SCRC-2738 was cloned into the cosmid vector (pEPA) . A 27 kb nucleotide sequence of the XhoI to SpeI region of pEPA showed EPA production (6.3%) in E . coli JM109 . Among the nine open reading frames (ORFs) in this sequence, only five (ORFs 2 and 5-8) were essential for EPA production . High levels of production (16%-22%) were found in E . coli JM109 transformed with a multicopy pNEB vector carrying only the five essential ORFs and in that transformed with a pNEB vector that integrated ORFs 3, 5, 6, 7 and 8, and vector pSTV28 that integrated the ORF2 encoding phosphopantetheinyl transferase (PPTase) . Thus, production of EPA appears to be regulated by the presence of all the biosynthesis gene products and by the ratio of PPTase to the other gene products . The temperature -EPA production relationship in E . coli strain DH5alpha varied between constructs, suggesting that it is controlled not only by EPA biosynthesis enzymes but also by other factors in vivo . There was a strict upper temperature limit for EPA biosynthesis: no EPA was synthesized at 30 degrees C in E . coli transformants carrying any gene construct for EPA biosynthesis.

EMBO J . 2004 Nov 25; {Epub ahead of print}
UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli; Veaute X et al.; The roles of UvrD and Rep DNA helicases of Escherichia coli are not yet fully understood . In particular, the reason for rep uvrD double mutant lethality remains obscure . We reported earlier that mutations in recF, recO or recR genes suppress the lethality of uvrD rep, and proposed that an essential activity common to UvrD and Rep is either to participate in the removal of toxic recombination intermediates or to favour the proper progression of replication . Here, we show that UvrD, but not Rep, directly prevents homologous recombination in vivo . In addition to RecFOR, we provide evidence that RecA contributes to toxicity in the rep uvrD mutant . In vitro, UvrD dismantles the RecA nucleoprotein filament, while Rep has only a marginal activity . We conclude that UvrD and Rep do not share a common activity that is essential in vivo: while Rep appears to act at the replication stage, UvrD plays a role of RecA nucleoprotein filament remover . This activity of UvrD is similar to that of the yeast Srs2 helicase.

Biosci Biotechnol Biochem, 2004 Nov, 68(11), 2360 - 8
Cloning and characterization of farnesyl diphosphate synthase from the rubber-producing mushroom Lactarius chrysorrheus; Mekkriengkrai D et al.; Farnesyl diphosphate is involved in rubber biosynthesis as an initiating substrate for both polyprenol and mushroom rubber . So far, we have isolated the cDNA of a farnesyl diphosphate synthase (FPS) for the first time from a rare rubber-producing mushroom, Lactarius chrysorrheus, by the degenerate RT-PCR technique based on sequence information of FPS genes from fungi and yeasts . The open reading frame was clarified to encode a protein of 381 amino acid residues with a calculated molecular weight of 42.9 kDa . The deduced amino acid sequence of L . chrysorrheus FPS showed about 50% identity with those of other fungi and yeasts as well as plants . We expressed the cDNA of L . chrysorrheus FPS in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein . The purified obtained protein showed FPS activity in which geranyl diphosphate (GPP) served as primary substrate, with a 2.4-fold higher k(cat)/K(m) value for GPP than for dimethylallyl diphosphate (DMAPP).

Biosci Biotechnol Biochem, 2004 Nov, 68(11), 2289 - 98
Thermostable esterase from a thermoacidophilic archaeon: purification and characterization for enzymatic resolution of a chiral compound; Kim S et al.; Homolog to lipolytic enzymes having the consensus sequence Gly-X-Ser-X-Gly, from the Sulfolobus solfataricus P2 genome, were identified by multiple sequence alignments . Among three potential candidate sequences, one (Est3), which displayed higher activity than the other enzymes on the indicate plates, was characterized . The gene (est 3) was expressed in Escherichia coli, and the recombinant protein (Est3) was purified by chromatographic separation . The enzyme is a trimeric protein and has a molecular weight of 32 kDa in monomer form in its native structure . The optimal pH and temperature of the esterase were 7.4 and 80 degrees C respectively . The enzyme showed broad substrate specificities toward various p-nitrophenyl esters ranging from C2 to C16 . The catalytic activity of the Est3 esterase was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and diethyl p-nitrophenyl phosphate . Based on substrate specificity and the action of inhibitors, the Est3 enzyme was estimated to be a carboxylesterase (EC 3.1.1.1) . The enzyme with methyl (+/-)-2-(3-benzoylphenyl)propionate-hydrolyzing activity to (-)-2-(3-benzoylphenyl)propionic acid displayed a moderate degree of enantioselectivity . The product, (-)-2-(3-benzoylphenyl)propionic acid, rather than its methyl ester, was obtained in 80% enantiomeric excess (e.e.(p)) at 20% conversion at 60 degrees C after a 32-h reaction . This result indicates that S . solfataricus esterase can be used for application in the synthesis of chiral compounds.

Plant Cell Physiol, 2004 Oct, 45(10), 1390 - 5
The rbcX gene product promotes the production and assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase of Synechococcus sp . PCC7002 in Escherichia coli; Onizuka T et al.; The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp . PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order . Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp . PCC7002 and in Escherichia coli . To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli . The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein . The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.

Comp Immunol Microbiol Infect Dis, 2005 Jan, 28(1), 17 - 35
Expression and purification of recombinant swine interleukin-4; Nuntaprasert A et al.; The swine interleukin-4 (SwIL-4) cDNA was cloned by RT-PCR . It was expressed using an expression vector pQE30 in E . coli, a baculovirus AcNPV vector pVL1392 in insect cells, and a pCAGGS vector in mammalian cells . The rSwIL-4 proteins expressed from bacteria and insect cells were purified using a chelating affinity column and a mAb-coupled immunoaffinity column . The amount of the products and their bioactivities were compared . All recombinant cytokines were efficiently reacted with the specific antibodies and the molecular weight of rSwIL-4 was approximately 16 kDa in E . coli, 15 and 18 kDa in insect cells, and 15 and 20 kDa in mammalian cells . Variations of molecular weight observed in insect and mammalian cells were probably due to different modification ways of glycosylation . All these recombinant proteins retained their antigenicity and were biologically active in inducing human TF-1 cell proliferation in vitro . The simple purification method will make it possible to evaluate the in vitro and in vivo effects of IL-4 in pigs.

Comp Immunol Microbiol Infect Dis, 2005 Jan, 28(1), 1 - 15
Virulence characteristics of Escherichia coli isolates obtained from broiler breeders with salpingitis; Monroy MA et al.; Thirty isolates of Escherichia coli from broiler breeders with salpingitis were studied . Using the slide agglutination test, the isolates were found to belong to serogroups O1, O2, O5, O36, O45, O53 and O78 . Pathogenicity for day-old chicks was determined by air sac inoculation and isolates were categorized as having high, intermediate or low virulence . Growth on iron starvation medium was observed together with aerobactin production . Based on the results of in vitro adherence tests, attachment to oviduct epithelium from old birds was found to be superior to that observed using corresponding material from young birds . DNA hybridization testing for type 1, P, and S fimbriae revealed predominant expression of type 1, correlating with mannose-sensitive hemagglutination using guinea-pig erythrocytes . In this study, P and S fimbriae were not considered to be important adherence factors . Study findings would suggest that, as far as salpingitis is concerned, type 1 fimbriae can play an important role in E . coli infection in breeders . An interesting result to emerge from the study was the observation that E . coli isolates were completely resistant to serum from young breeders, whereas they were completely sensitive using serum from older breeders . Based on serogroups involved, pathogenicity for day-old chicks and virulence indicators, the salpingitis isolates were similar to those from cases of chronic respiratory disease.

Gene, 2004 Dec 8, 343, 127 - 32
CCC CGA is a weak translational recoding site in Escherichia coli; Shu P et al.; Previously published experiments had indicated unexpected expression of a control vector in which a beta-galactosidase reporter was in the +1 reading frame relative to the translation start . This control vector contained the codon pair CCC CGA in the zero reading frame, raising the possibility that ribosomes rephased on this sequence, with peptidyl-tRNA(Pro) pairing with CCC in the +1 frame . This putative rephasing might also be exacerbated by the rare CGA Arg codon in the second position due to increased vacancy of the ribosomal A-site . To test this hypothesis, a series of site-directed mutants was constructed, including mutations in both the first and second codons of this codon pair . The results show that interrupting the continuous run of C residues with synonymous codon changes essentially abolishes the frameshift . Further, changing the rare Arg codon to a common Arg codon also reduces the frequency of the frameshift . These results provide strong support for the hypothesis that CCC CGA in the zero frame is indeed a weak translational frameshift site in Escherichia coli, with a 1-2% efficiency . Because the vector sequence also contains another CCC triplet in the +1 reading frame starting within the next codon after the CGA, our data also support possible contribution to expression of a +7 nucleotide ribosome hop into the same +1 reading frame . We also confirm here a previous report that CCC UGA is a translational frameshift site, in these experiments, with about 5% efficiency.

Gene, 2004 Dec 8, 343, 99 - 106
Single-chain integration host factors as probes for high-precision nucleoprotein complex formation; Bao Q et al.; Integration host factor (IHF) is a heterodimeric, site-specific DNA-binding and DNA-bending protein from Escherichia coli . It is involved in high-precision DNA transactions where it serves as a key architectural component of specialized nucleoprotein structures (snups) . We described recently a novel approach for protein engineering using a single polypeptide chain IHF, termed scIHF2, as a first example . ScIHF2 is made up of the alpha subunit of IHF which was inserted into the beta subunit at peptide bond Q39/G40 via two short linkers . The monomer behaves very similarly to the heterodimeric, parental IHF in biochemical and functional assays . Here, we describe an extension of this approach in which we shortened either one or both linkers by one amino acid, thereby generating three new variants termed scIHF1, 3, and 4 . These variants exhibit distinct DNA-binding properties, different phenotypes in site-specific integrative and excisive recombination by phage lambda integrase in vitro, as well as in pSC101 replication assays in a DeltaIHF E . coli host . We also introduced a K45E substitution within the alpha domain of scIHF3 and based on electrophoretic mobility shift assays (EMSAs), argue that it significantly changes the DNA trajectory within the protein-DNA complex . Our results indicate that IHF's pleiotropic roles in DNA transactions inside E . coli require different types of high-precision DNA architectural activities . The scIHF variants described here will help to explore further how flexible these requirements are.

Plant Physiol, 2004 Dec, 136(4), 4048 - 60 Epub 2004 Nov 24.
Metabolic engineering of the chloroplast genome using the Echerichia coli ubiC gene reveals that chorismate is a readily abundant plant precursor for p-hydroxybenzoic acid biosynthesis; Viitanen PV et al.; p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers . In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5' untranslated region . CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate . The leaf content of pHBA glucose conjugates in fully mature T1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight . The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL . Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable . The highest CPL enzyme activity in pooled leaf material from adult T1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation . These experiments demonstrate that the current limitation for pHBA production in nuclear-transformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation . Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA . Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.

Proc Natl Acad Sci U S A, 2004 Dec 7, 101(49), 17090 - 5 Epub 2004 Dec 7.
The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli; Zheng L et al.; Ammonium is one of the most important nitrogen sources for bacteria, fungi, and plants, but it is toxic to animals . The ammonium transport proteins (methylamine permeases/ammonium transporters/rhesus) are present in all domains of life; however, functional studies with members of this family have yielded controversial results with respect to the chemical identity (NH(4)(+) or NH(3)) of the transported species . We have solved the structure of wild-type AmtB from Escherichia coli in two crystal forms at 1.8- and 2.1-A resolution, respectively . Substrate transport occurs through a narrow mainly hydrophobic pore located at the center of each monomer of the trimeric AmtB . At the periplasmic entry, a binding site for NH(4)(+) is observed . Two phenylalanine side chains (F107 and F215) block access into the pore from the periplasmic side . Further into the pore, the side chains of two highly conserved histidine residues (H168 and H318) bridged by a H-bond lie adjacent, with their edges pointing into the cavity . These histidine residues may facilitate the deprotonation of an ammonium ion entering the pore . Adiabatic free energy calculations support the hypothesis that an electrostatic barrier between H168 and H318 hinders the permeation of cations but not that of the uncharged NH(3.) The structural data and energetic considerations strongly indicate that the methylamine permeases/ammonium transporters/rhesus proteins are ammonia gas channels . Interestingly, at the cytoplasmic exit of the pore, two different conformational states are observed that might be related to the inactivation mechanism by its regulatory partner.

J Agric Food Chem, 2004 Dec 1, 52(24), 7297 - 9
Immunomodulatory activity of a new family of antioxidants obtained from grape polyphenols; Mitjans M et al.; We examined the potential antioxidant activity and the immunopharmacological activity of new epicatechin conjugates obtained by depolymerization of grape polymeric flavanols in the presence of cysteamine or cysteine and with or without gallate . The compounds studied were (-)-epicatechin (1), cysteinyl-epicatechin (2), cysteamine-epicatechin (3), (-)-epicatechin gallate (4), cysteinyl-epicatechin gallate (5), and cysteamine-epicatechin gallate (6) When incubated with an erythrocyte suspension, flavanols protected the erythrocyte membrane from hemolysis induced by 2,2'-azobis(2-amidinopropane) dihydrochloride, an azo free-radical initiator . All the epicatechin derivatives tested were more efficient as antioxidant than epicatechin . The most potent antioxidant was compound 6 . The compounds were tested for their capacity to modulate IL-1beta and IL-6, which are the main cytokine factors influencing the acute phase of the inflammatory response . (-)-Epicatechin and its related compounds inhibited the production of IL-1beta and IL-6 in whole blood incubated in the presence of Escherichia coli lipopolysaccharide . The most efficient inhibitor of cytokine formation was compound 3.

Bioorg Khim, 2004 Sep-Oct, 30(5), 481 - 6
{Recombinant thymosin alpha1}
{Cloning and expression of a homologue of human macrophage migration inhibitory factor from P . falciparum 3D7}
Han ZF, Shao DD, Wang H.

Molecular Parasitology Laboratory, Department of Etiology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, ChinaOBJECTIVE: To clone and express a homologue of human macrophage migration inhibitory factor (MIF) from P . falciparum 3D7--PfMIF . METHODS: The nucleotide sequence of PfMIF was found through blast P . falciparum genomic sequence databases with the amino acid sequence of human MIF (HuMIF) . RT-PCR, DNA sequencing, and bioinformatics analysis were used for the cloning of Pfmif gene . The recombinant protein was expressed in E . coli and purified through the affinity column . RESULTS: The full length of Pfmif gene was cloned and sequenced . It was composed of 351 nucleotides and encoded 116 amino acids with the typical characteristic of MIF family . The recombinant protein was successfully expressed and purified . CONCLUSIONS: The Pfmif gene and recombinant protein were successfully isolated and PfMIF was preliminarily identified as a novel member of MIF family.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4895 - 7
Chromosome-encoded CTX-M-3 from Kluyvera ascorbata: a possible origin of plasmid-borne CTX-M-1-derived cefotaximases; Rodriguez MM et al.; A gene identical to plasmid-borne bla(CTX-M-3) is present in the chromosome of one Kluyvera ascorbata strain . It is associated with a structure including an inverted repeat right and an open reading frame 477-like gene probably involved in the mobilization of bla(CTX-M-3) . Two other K . ascorbata strains rendered the previously described bla(KLUA-9) gene.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4889 - 91
Effects of a number of classes of 50S inhibitors on stop codon readthrough during protein synthesis; Thompson J et al.; The effect of a number of antibiotics on stop codon readthrough during protein synthesis in Escherichia coli was examined . Inhibitors which bind close to the entrance of the peptide exit tunnel on the 50S ribosomal subunit promote substantial levels of readthrough, presumably by disrupting the mechanism of peptide release.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4813 - 21
Novel nonnucleoside inhibitor of hepatitis C virus RNA-dependent RNA polymerase; Howe AY et al.; A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), {(1R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano{3,4-b}indol-1-yl} acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization . HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 microM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a . HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase . A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein . Multiple treatments with HCV-371 for 16 days led to a >3-log10 reduction in the HCV RNA level . In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log10 reduction of the viral RNA level . In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity . Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism . The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses . Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4778 - 83
Biochemical characterization of the THIN-B metallo-beta-lactamase of Janthinobacterium lividum; Docquier JD et al.; The THIN-B metallo-beta-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum, was overproduced in Escherichia coli by means of a T7-based expression system . The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis . The native THIN-B enzyme is a monomeric protein of 31 kDa . It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator . Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex.

J Biol Chem . 2004 Nov 23; {Epub ahead of print}
A Carboxypeptidase Inhibitor from the tick Rhipicephalus bursa . Isolation, cDNA cloning, recombinant expression, and characterization; Arolas JL et al.; A novel proteinaceous metallo-carboxypeptidase inhibitor, named TCI (tick carboxypeptidase inhibitor), was isolated from the ixodid tick Rhipicephalus bursa and N-terminally sequenced . The complete cDNA encoding this protein was cloned from tick mRNA by RT-PCR and rapid amplification of cDNA ends (RACE) techniques . The full-length TCI cDNA contains an open reading frame coding for a precursor protein of 97 amino acid residues that consists of a predicted signal peptide of 22 residues and of mature TCI, a 75-residue cysteine-rich protein (12 Cys) . The deduced amino acid sequence shows no homology to other known proteins; the C-terminus, however, resembles those of other protein metallo-carboxypeptidase inhibitors, suggesting a common mechanism of inhibition . Recombinant TCI expressed in Escherichia coli is fully functional and inhibits carboxypeptidases of the A/B subfamily with equilibrium dissociation constants in the nanomolar range . Structural analyses by circular dichroism and nuclear magnetic resonance indicate that TCI is a protein strongly constrained by disulfide bonds, unusually stable over a wide pH range and highly resistant to denaturing conditions . As a tight binding inhibitor of plasma carboxypeptidase B, also known as TAFIa, recombinant TCI stimulates fibrinolysis in vitro and thus may have potential for applications to prevent or treat thrombotic disorders.

J Mol Biol, 2004 Dec 10, 344(5), 1397 - 407
The outer membrane usher forms a twin-pore secretion complex; Li H et al.; The PapC usher is an outer membrane protein required for assembly and secretion of P pili in uropathogenic Escherichia coli . P pilus biogenesis occurs by the chaperone/usher pathway, a terminal branch of the general secretory pathway . Periplasmic chaperone-subunit complexes target to the PapC usher for fiber assembly and secretion through the usher to the cell surface . The molecular details of pilus biogenesis at the usher, and protein secretion across the outer membrane in general, are unclear . We studied the structure and oligomeric state of PapC by gel filtration, dynamic light scattering, and electron microscopy and image analysis . Two-dimensional crystals of wild-type PapC and a C-terminal deletion mutant of PapC were produced by reconstituting detergent purified usher into E.coli lipids . PapC formed a dimer both in detergent solution and in the phospholipid bilayer . Cryo-electron microscopy revealed that the usher forms a twin-pore complex . Removal of the C-terminal domain did not change the basic shape of the PapC molecule, but altered the dimeric association of the usher, suggesting that the C terminus forms part of the dimerization interface . The overall molecular size (11 nm), pore size (2 nm), and twin-pore configuration of PapC resemble that of the Tom40 complex, a mitochondrial outer membrane protein translocase.

J Mol Biol, 2004 Dec 10, 344(5), 1251 - 63
The heterodimeric primase of the hyperthermophilic archaeon Sulfolobus solfataricus possesses DNA and RNA primase, polymerase and 3'-terminal nucleotidyl transferase activities; Lao-Sirieix SH et al.; A eukaryotic-type primase was identified in the crenarchaeon Sulfolobus solfataricus . The two-subunit DNA-dependent primase, termed PriSL, was purified following co-expression of the subunits in Escherichia coli and its activity was characterised . PriSL was capable of utilising both ribonucleotides and deoxyribonucleotides for primer synthesis in the presence of natural, or synthetic, single-stranded DNA . A broad distribution of products was detected, ranging from dinucleotides to DNA molecules in excess of 7 kb and RNA up to 1 kb in length . However, PriSL had a significantly higher affinity for ribonucleotides than for deoxyribonucleotides . Using site-directed mutagenesis, two aspartate residues crucial for nucleic acid synthesis and residues important for the binding of free nucleotides were identified . In addition to the primase and polymerase activities, we reveal that the primase possesses a template-independent 3'-terminal nucleotidyl transferase activity.

Exp Cell Res, 2005 Jan 15, 302(2), 194 - 205
Galig, a novel cell death gene that encodes a mitochondrial protein promoting cytochrome c release; Duneau M et al.; Galectin-3 internal gene (Galig) was recently identified as an internal gene transcribed from the second intron of the human galectin-3 gene that is implicated in cell growth, cell differentiation, and cancer development . In this study, we show that galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death . These alterations were associated with extramitochondrial release of cytochrome c, a known cell death effector . Furthermore, Bcl-xL co-transfection significantly reduced the release of cytochrome c induced by galig expression, suggesting a common pathway between the cytotoxic activity of galig and the anti-apoptotic activity of Bcl-xL . This antagonism was not observed upon co-transfection of Bcl-2 and galig . Galig encodes a mitochondrial-targeted protein named mitogaligin . Structure-activity relationship studies showed that the mitochondrial addressing of mitogaligin relies on an internal sequence that is required and sufficient for the release of cytochrome c and cell death upon cell transfection . Moreover, incubation of isolated mitochondria with peptides derived from mitogaligin induces cytochrome c release . Altogether, these results show that galig is a novel cell death gene encoding mitogaligin, a protein promoting cytochrome c release upon direct interaction with the mitochondria.

Environ Microbiol, 2004 Dec, 6(12), 1210 - 9
Influence of form IA RubisCO and environmental dissolved inorganic carbon on the delta13C of the clam-chemoautotroph symbiosis Solemya velum; Scott KM et al.; Many nutritive symbioses between chemoautotrophic bacteria and invertebrates, such as Solemya velum, have delta(13)C values of approximately -30 to -35%, considerably more depleted than phytoplankton . Most of the chemoautotrophic symbionts fix carbon with a form IA ribulose 1,5-bisphosphate carboxylase (RubisCO) . We hypothesized that this form of RubisCO discriminates against (13)CO(2) to a greater extent than other forms . Solemya velum symbiont RubisCO was cloned and expressed in Escherichia coli, purified and characterized . Enzyme from this recombinant system fixed carbon most rapidly at pH 7.5 and 20-25 degrees C . Surprisingly, this RubisCO had an epsilon-value (proportional to the degree to which the enzyme discriminates against (13)CO(2)) of 24.4 per thousand, similar to form IB RubisCOs, and higher than form II RubisCOs . Samples of interstitial water from S . velum's habitat were collected to determine whether the dissolved inorganic carbon (DIC) could contribute to the negative delta(13)C values . Solemya velum habitat DIC was present at high concentrations (up to approximately 5 mM) and isotopically depleted, with delta(13)C values as low as approximately -6% . Thus environmental DIC, coupled with a high degree of isotopic fractionation by symbiont RubisCO likely contribute to the isotopically depleted delta(13)C values of S . velum biomass, highlighting the necessity of considering factors at all levels (from environmental to enzymatic) in interpreting stable isotope ratios.

Eur J Biochem, 2004 Nov, 271(22), 4582 - 93
Inhibition of pea ferredoxin-NADP(H) reductase by Zn-ferrocyanide; Dupuy DL et al.; Ferredoxin-NADP(H) reductases (FNRs) represent a prototype of enzymes involved in numerous metabolic pathways . We found that pea FNR ferricyanide diaphorase activity was inhibited by Zn2+ (Ki 1.57 microM) . Dichlorophenolindophenol diaphorase activity was also inhibited by Zn2+ (Ki 1.80 microM), but the addition of ferrocyanide was required, indicating that the inhibitor is an arrangement of both ions . Escherichia coli FNR was also inhibited by Zn-ferrocyanide, suggesting that inhibition is a consequence of common structural features of these flavoenzymes . The inhibitor behaves in a noncompetitive manner for NADPH and for artificial electron acceptors . Analysis of the oxidation state of the flavin during catalysis in the presence of the inhibitor suggests that the electron-transfer process between NADPH and the flavin is not significantly altered, and that the transfer between the flavin and the second substrate is mainly affected . Zn-ferrocyanide interacts with the reductase, probably increasing the accessibility of the prosthetic group to the solvent . Ferredoxin reduction was also inhibited by Zn-ferrocyanide in a noncompetitive manner, but the observed Ki was about nine times higher than those for the diaphorase reactions . The electron transfer to Anabaena flavodoxin was not affected by Zn-ferrocyanide . Binding of the apoflavodoxin to the reductase was sufficient to overcome the inhibition by Zn-ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partners may induce a conformational change in the reductase that alters or completely prevents the inhibitory effect.

Mol Biol Rep, 2004 Sep, 31(3), 177 - 89
The expression of three desaturase genes of Spirulina platensis in Escherichia coli DH5alpha . Heterologous expression of Spirulina-desaturase genes; Apiradee H et al.; The genes from a cyanobacterium--Spirulina platensis strain C1--that encode the acyl-lipid desaturases (desC, desA and desD) involved in gamma-linolenic (GLA) synthesis have been successfully expressed for the first time in Escherichia coli by employing a pTrcHisA expression system . In this report, the authors describe the expression of the three Spirulina N-terminal 6xHis-desaturases as well as the functional analysis of these recombinant proteins . The gene products of desC, desA and desD have approximate molecular masses of 37, 45, and 47 kDa, respectively . Enzymatic activity measurement of these products was carried out in vivo to demonstrate that (i) the expressed proteins are in functional form, and (ii) the cofactors of the host system can complement the system of Spirulina platensis . The study demonstrated that the gene products of desC and desA catalyzed the reactions in vivo where the enzyme substrates were provided in appropriate concentration . This indicates that the delta9 and delta12 desaturases were expressed in the heterologous host in their active form, and that these two reactions can be carried out in an E . coli host cell using its cofactors system . In contrast, delta6 desaturase activity can be detected only in vitro where electron carriers are provided . This suggests that while this enzyme is expressed in the heterologous host in its active form, its function in vivo is suppressed, as the electron carriers of the host system cannot complement the system of Spirulina platensis.

Nucleosides Nucleotides Nucleic Acids, 2004 Oct, 23(6-7), 895 - 906
Hybridization of antisense oligonucleotides with alpha-sarcin loop region of Escherichia coli 23S rRNA; Petyuk VA et al.; Binding of complementary oligonucleotides (ONs) with alpha-sarcin loop region (2638-2682) of Escherichia coli 23S rRNA was investigated . Four of the tested pentadecanucleotides efficiently bound to target sequences with association rate and equilibrium constants approximately 10(3) M(-1)s(-1) and 10(7) M(-1), respectively . ON S5 (CGAGAGGACCGGAGU) complementary to the sequence 2658-2672 displayed the highest affinity to the target . Activation energy for binding of ON S5 was measured to be 11 kcal/mol; this value corresponds to approximately 10% of the calculated enthalpy of the local RNA structure unfolding in the presence of this oligonucleotide . The activation energy value is evidence for the heteroduplex formation to occur via strand displacement pathway; the initiation of heteroduplex formation requires disruption of 1-2 base pairs in RNA hairpin.

J Basic Microbiol, 2004, 44(6), 424 - 9
Indirect identification of isoprenoid quinones in Escherichia coli by LC-MS with atmospheric pressure chemical ionization in negative mode; Gao M et al.; A novel analytical method was applied for identification of isoprenoid quinones in Escherichia coli by liquid chromatography atmospheric press chemical ionization mass spectrometry in negative mode (LC-NI-APCI-MS) . Extraction and clean-up of sample were carried out on Sep-Pak Plus Silica solid-phase extraction cartridges . Ubiquinone-7 (UQ-7), Ubiquinone-8 (UQ-8) and Mequinone-8 (MK-8) were determined directly using combined information on retention time, molecular ion mass, fragment ion masses and UV characteristic spectrometry without any standard reagent . It was found that UQ-8 was the major component of isoprenoid quinones in Escherichia coli under aerobic condition . Compared with UQ-8, the relative abundance of UQ-7 and MK-8 is only 15% and 14%, respectively . The average recoveries of UQ-6, UQ-10 and vitamin K(1) in Escherichia coli were investigated by standard spiking experiment . The recoveries were achieved in the range from 94 to 106%, and the relative standard deviations (RSD) of the triplicate analysis of the spiked samples (UQ-6, UQ-10 and vitamin K(1)) ranged from 3 to 8% . The detection limits of LC-NI-APCI-MS were estimated to be 5, 40 and 0.8 microg/g dry cell for UQ-6, UQ-10 and vitamin K(1), respectively . ((c) 2004 WILEY-VCH Verlag GmbH & Co . KGaA, Weinheim).

Biotechnol Bioeng, 2004 Dec 30, 88(7), 825 - 31
Substrate range of acetohydroxy acid synthase I from Escherichia coli in the stereoselective synthesis of alpha-hydroxy ketones; Engel S et al.; Acetohydroxy acid synthase I appears to be the most effective of the AHAS isozymes found in Escherichia coli in the chiral synthesis of phenylacetyl carbinol from pyruvate and benzaldehyde . We report here the exploration of a range of aldehydes as substrates for AHAS I and demonstrate that the enzyme can accept a wide variety of substituted benzaldehydes, as well as heterocyclic and heteroatomic aromatic aldehydes, to produce chiral carbinols . The active site of AHAS I does not appear to impose serious steric constraints on the acceptor substrate . The influence of electronic effects on the reaction has been probed using substituted benzaldehydes as substrates . The electrophilicity of the aldehyde acceptor substrates is most important to their reactivity, but the lipophilicity of substituents also affects their reactivity . AHAS I is an effective biosynthetic platform for production of a variety of alpha-hydroxy ketones, compounds with considerable potential as pharmacological precursors . 2004 Wiley Periodicals, Inc.

Mol Genet Genomics . 2004 Nov 19; {Epub ahead of print}
Microarray analysis of RpoS-mediated gene expression in Escherichia coli K-12; Patten CL et al.; The alternative sigma factor RpoS controls the expression of many stationary-phase genes in Escherichia coli and other bacteria . Though the RpoS regulon is a large, conserved system that is critical for adaptation to nutrient deprivation and other stresses, it remains incompletely characterized . In this study, we have used oligonucleotide arrays to delineate the transcriptome that is controlled by RpoS during entry into stationary phase of cultures growing in rich medium . The expression of known RpoS-dependent genes was confirmed to be regulated by RpoS, thus validating the use of microarrays for expression analysis . The total number of positively regulated stationary-phase genes was found to be greater than 100 . More than 45 new genes were identified as positively controlled by RpoS . Surprisingly, a similar number of genes were found to be negatively regulated by RpoS, and these included almost all genes required for flagellum biosynthesis, genes encoding enzymes of the TCA cycle, and a physically contiguous group of genes located in the Rac prophage region . Negative regulation by RpoS is thus much more extensive than has previously been recognized, and is likely to be an important contributing factor to the competitive growth advantage of rpoS mutants reported in previous studies.

Microbiol Immunol, 2004, 48(11), 865 - 74
Further Studies on the Hyperphosphorylated Form (p40) of the Rabies Virus Nominal Phosphoprotein (P); Toriumi H et al.; We investigated possible mechanisms involved in production of a hyperphosphorylated form (p40) of rabies virus P protein, to which two dimensional (2-D) gel electrophoresis was applied . The P gene products produced in Escherichia coli cells could be detected as a single spot of unphosphorylated 37-kDa form (termed as p37-0) in a 2-D gel . The 37-kDa proteins in the virus-infected cells are composed of some phosphorylated forms, including a major p37-1 and more phosphorylated minor forms (e.g., p37-2, p37-3, etc.), but little p37-0 is detected (Eriguchi et al., 2002) . When the E . coli -produced P protein analogues were incubated with BHK-21 cell lysates, heparin-sensitive phosphorylation occurred as described previously (Takamatsu et al., 1998), giving an additional 40-kDa spot . However, such a p40-like derivative displayed a little more basic pI value than that of the authentic p40 produced in the infected cells; hence, the former was termed p40-0 (pI=4.78), while the latter, p40-1 (pI=4.73) . In contrast, p40 produced in the P cDNAtransfected animal cell was detected at the p40-1 position . In addition, staurosporine did not affect the p40-1 production in virus-infected nor the P cDNA-transfected animal cells, while the agent reduced production of hyperphosphorylated forms of p37, resulting in accumulation of p37-1, but not of p37-0 . These results suggest that, although p37-0 may become a substrate for the heparin-sensitive protein kinase (PK) in vitro, only p37-1 is a substrate for p40 production catalyzed by heparin-sensitive PK in animal cells, and staurosporine-sensitive PK is involved in the production of more phosphorylated forms of p37, but not in p37-1 production from p37-0.

Infect Immun, 2004 Dec, 72(12), 7190 - 201
Evolutionary and functional relationships of colonization factor antigen i and other class 5 adhesive fimbriae of enterotoxigenic Escherichia coli; Anantha RP et al.; Colonization factor antigen I (CFA/I) is the archetype of eight genetically related fimbriae of enterotoxigenic Escherichia coli (ETEC) designated class 5 fimbriae . Assembled by the alternate chaperone pathway, these organelles comprise a rigid stalk of polymerized major subunits and an apparently tip-localized minor adhesive subunit . We examined the evolutionary relationships of class 5-specific structural proteins and correlated these with functional properties . We sequenced the gene clusters encoding coli surface antigen 4 (CS4), CS14, CS17, CS19, and putative colonization factor antigen O71 (PCFO71) and analyzed the deduced proteins and the published homologs of CFA/I, CS1, and CS2 . Multiple alignment and phylogenetic analysis of the proteins encoded by each operon define three subclasses, 5a (CFA/I, CS4, and CS14), 5b (CS1, CS17, CS19, and PCFO71), and 5c (CS2) . These share distant evolutionary relatedness to fimbrial systems of three other genera . Subclass divisions generally correlate with distinguishing in vitro adherence phenotypes of strains bearing the ETEC fimbriae . Phylogenetic comparisons of the individual structural proteins demonstrated greater intrasubclass conservation among the minor subunits than the major subunits . To correlate this with functional attributes, we made antibodies against CFA/I and CS17 whole fimbriae and maltose-binding protein fusions with the amino-terminal half of the corresponding minor subunits . Anti-minor subunit Fab preparations showed hemagglutination inhibition (HAI) of ETEC expressing homologous and intrasubclass heterologous colonization factors while anti-fimbrial Fab fractions showed HAI activity limited to colonization factor-homologous ETEC . These results were corroborated with similar results from the Caco-2 cell adherence assay . Our findings suggest that the minor subunits of class 5 fimbriae may be superior to whole fimbriae in inducing antiadhesive immunity.

Infect Immun, 2004 Dec, 72(12), 6764 - 72
Enteropathogenic Escherichia coli infection triggers host phospholipid metabolism perturbations; Wu Y et al.; Enteropathogenic Escherichia coli (EPEC) specifically recognizes phosphatidylethanolamine (PE) on the outer leaflet of host epithelial cells . EPEC also induces apoptosis in epithelial cells, which results in increased levels of outer leaflet PE and increased bacterial binding . Consequently, it is of interest to investigate whether EPEC infection perturbs host cell phospholipid metabolism and whether the changes play a role in the apoptotic signaling . Our findings indicate that EPEC infection results in a significant increase in the epithelial cell PE level and a corresponding decrease in the phosphatidylcholine (PC) level . PE synthesis via both the de novo pathway and the serine decarboxylation pathway was enhanced, and de novo synthesis of phosphatidylcholine via CDP-choline was reduced . The changes were transitory, and the maximum change was noted after 4 to 5 h of infection . Addition of exogenous PC or CDP-choline to epithelial cells prior to infection abrogated EPEC-induced apoptosis, suggesting that EPEC infection inhibits the CTP-phosphocholine cytidylyltransferase step in PC synthesis, which is reportedly inhibited during nonmicrobially induced apoptosis . On the other hand, incorporation of exogenous PE by the host cells enhanced EPEC-induced apoptosis and necrosis without increasing bacterial adhesion . This is the first report that pathogen-induced apoptosis is associated with significant changes in PE and PC metabolism, and the results suggest that EPEC adhesion to a host membrane phospholipid plays a role in disruption of host phospholipid metabolism.

Circulation, 2004 Dec 7, 110(23), 3560 - 6 Epub 2004 Dec 7.
Heat shock protein 70 confers cardiovascular protection during endotoxemia via inhibition of nuclear factor-kappaB activation and inducible nitric oxide synthase expression in the rostral ventrolateral medulla; Chan JY et al.; BACKGROUND: Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, plays a pivotal role in the manifestation of fatal cardiovascular depression during endotoxemia . The iNOS gene is regulated transcriptionally by nuclear factor-kappaB (NF-kappaB) activation . The present study tested the hypothesis that heat shock protein 70 (HSP70) may confer protection against sepsis-induced circulatory fatality via inhibition of iNOS gene expression in the RVLM through prevention of NF-kappaB activation . METHODS AND RESULTS: Adult male Sprague-Dawley rats subjected to a brief hyperthermic heat shock (42 degrees C for 15 minutes) exhibited significant upregulation of HSP70 in the RVLM . Brief heat shock preconditioning also significantly suppressed iNOS mRNA or protein surge and alleviated hypotension, bradycardia, and reduction in neurogenic sympathetic vasomotor activity manifested during experimental endotoxemia induced by intravenous administration of Escherichia coli lipopolysaccharide . An increase in DNA binding activity and nuclear translocation of transcription factor NF-kappaB were detected during endotoxemia . Heat shock preconditioning significantly decreased DNA binding activity of NF-kappaB, which was reversed by microinjection of an hsp70 antisense oligonucleotide bilaterally into the RVLM . Heat shock preconditioning also blocked inhibitory kappaB (IkappaB) kinase activity or degradation of IkappaB in the RVLM during endotoxemia . CONCLUSIONS: We conclude that HSP70 confers protection against sepsis-related circulatory fatality via inhibition of iNOS gene expression in the RVLM through prevention of NF-kappaB activation in cellular processes that include prevention of IkappaB kinase activation and inhibition of IkappaBalpha degradation.

Protein Sci, 2004 Dec, 13(12), 3274 - 84
Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion; Trabbic-Carlson K et al.; Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP . Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag . Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction . The total protein content and functional activity were quantified at each ITC purification step . For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion . For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography . Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.

Protein Sci, 2004 Dec, 13(12), 3264 - 73
Studies on recombinant single chain Jacalin lectin reveal reduced affinity for saccharides despite normal folding like native Jacalin; Sahasrabuddhe AA et al.; Sugar binding studies, inactivation, unfolding, and refolding of native Jacalin (nJacalin) from Artocarpus integrifolia and recombinant single-chain Jacalin (rJacalin) expressed in Escherichia coli were studied by intrinsic fluorescence and thermal and chemical denaturation approaches . Interestingly, rJacalin does not undergo any proteolytic processing in an E . coli environment . It has 100fold less affinity for methyl-alpha-galactose (Ka: 2.48 x 10(2)) in comparison to nJacalin (Ka: 1.58 x 10(4)), and it also binds Thomsen-Friedenreich (TF) disaccharide (Galbeta1-3GalNAc) with less affinity . Overall sugar binding characteristics of rJacalin are qualitatively similar to that of nJacalin (Gal<MealphaGal<MealphaTFdisaccharide) . Circular dichroism studies at near- and far-UV, thermal, and chemical denaturation studies reveal that the rJacalin behaves like nJacalin . Guanidine hydrochloride-induced denaturation, followed by renaturation, yielded total recovery of sugar binding activity of rJacalin in comparison to partial recovery for nJacalin . This signifies the minor changes in the refolding pathways between native and recombinant lectins . The stability of rJacalin is dramatically reduced in the extreme pH range unlike nJacalin . Both lectins do not bind 1-anilino-8-naphthalene sulfonic acid (ANS) in the pH range of 5 to 12 but they do in the pH range of 1-3 . Solute quenching studies of the lectin using acrylamide, KI, and CsCl indicated that the tryptophan residues have full accessibility to the neutral quencher and poor accessibility to ionic quenchers . In summary, biophysical and biochemical studies on the native versus recombinant Jacalin suggest that post-translational modification, i.e., the processing of Jacalin into two chains is probably not a prerequisite for sugar binding but may be required for higher affinity.

J Gen Virol, 2004 Dec, 85(Pt 12), 3493 - 500
Neutralizing human antibodies to varicella-zoster virus (VZV) derived from a VZV patient recombinant antibody library; Kausmally L et al.; Varicella-zoster virus (VZV), the causative agent of chickenpox and herpes zoster, can be life-threatening in prematurely born children and in children with immune defects or who are under immunosuppressive treatment . Therefore agents for passive immunization, such as VZV-specific immunoglobulin preparations (VZIG) derived from convalescent plasma, are crucial in the prophylaxis of VZV infection . This study describes the isolation of human VZV-neutralizing recombinant antibodies . A human single-chain variable fragment (scFv) phage display library was generated from RNA extracted from peripheral blood lymphocytes of a convalescent varicella patient . Specific phage antibodies were selected against VZV-infected human fibroblasts, and eight unique clones were further expressed as soluble scFv in Escherichia coli . They all showed binding characteristics to varicella antigens with affinities in the K(D) range 0.1-0.2 muM . Two of the scFv antibodies, VZV4 and VZV5, showed dose-dependent in vitro neutralization of VZV . VZV39 also showed a neutralizing effect as scFv, an effect that was increased 4000-fold by conversion into IgG and was further increased by the addition of complement . This is possibly the first time that monovalent scFv antibodies have been shown to neutralize VZV in vitro . This finding will have an impact on the production of new prophylactic antibodies, as such antibody fragments can be cost-effectively produced in E . coli . The antibodies isolated bind both complement-dependent and -independent epitopes for neutralization, thus they may prove useful tools for the study of VZV virulence mechanisms.

Plant Physiol, 2004 Dec, 136(4), 4246 - 55 Epub 2004 Nov 19.
Analysis in vitro of the enzyme CRTISO establishes a poly-cis-carotenoid biosynthesis pathway in plants; Isaacson T et al.; Most enzymes in the central pathway of carotenoid biosynthesis in plants have been identified and studied at the molecular level . However, the specificity and role of cis-trans-isomerization of carotenoids, which occurs in vivo during carotene biosynthesis, remained unresolved . We have previously cloned from tomato (Solanum lycopersicum) the CrtISO gene, which encodes a carotene cis-trans-isomerase . To study the biochemical properties of the enzyme, we developed an enzymatic in vitro assay in which a purified tomato CRTISO polypeptide overexpressed in Escherichia coli cells is active in the presence of an E . coli lysate that includes membranes . We show that CRTISO is an authentic carotene isomerase . Its catalytic activity of cis-to-trans isomerization requires redox-active components, suggesting that isomerization is achieved by a reversible redox reaction acting at specific double bonds . Our data demonstrate that CRTISO isomerizes adjacent cis-double bonds at C7 and C9 pairwise into the trans-configuration, but is incapable of isomerizing single cis-double bonds at C9 and C9' . We conclude that CRTISO functions in the carotenoid biosynthesis pathway in parallel with zeta-carotene desaturation, by converting 7,9,9'-tri-cis-neurosporene to 9'-cis-neurosporene and 7'9'-di-cis-lycopene into all-trans-lycopene . These results establish that in plants carotene desaturation to lycopene proceeds via cis-carotene intermediates.

Proc Natl Acad Sci U S A, 2004 Nov 30, 101(48), 16750 - 5 Epub 2004 Nov 30.
Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex; Kazmirski SL et al.; Clamp-loader complexes are heteropentameric AAA+ ATPases that load sliding clamps onto DNA . The structure of the nucleotide-free Escherichia coli clamp loader had been determined previously and led to the proposal that the clamp-loader cycles between an inactive state, in which the ATPase domains form a closed ring, and an active state that opens up to form a "C" shape . The crystal structure was interpreted as being closer to the active state than the inactive state . The crystal structure of a nucleotide-bound eukaryotic clamp loader {replication factor C (RFC)} revealed a different and more tightly packed spiral organization of the ATPase domains, raising questions about the significance of the conformation seen earlier for the bacterial clamp loader . We describe crystal structures of the E . coli clamp-loader complex bound to the ATP analog ATPgammaS (at a resolution of 3.5 A) and ADP (at a resolution of 4.1 A) . These structures are similar to that of the nucleotide-free clamp-loader complex . Only two of the three functional ATP-binding sites are occupied by ATPgammaS or ADP in these structures, and the bound nucleotides make no interfacial contacts in the complex . These results, along with data from isothermal titration calorimetry, molecular dynamics simulations, and comparison with the RFC structure, suggest that the more open form of the E . coli clamp loader described earlier and in the present work corresponds to a stable inactive state of the clamp loader in which the ATPase domains are prevented from engaging the clamp in the highly cooperative manner seen in the fully ATP-loaded RFC-clamp structure.

J Biol Chem . 2004 Nov 19; {Epub ahead of print}
The role of RuvA octamerisation for RuvAB function in vitro and in vivo; Privezentzev CV et al.; RuvA plays an essential role in branch migration of the Holliday junction by RuvAB, as part of the RuvABC pathway for processing Holliday junctions in Escherichia coli . Two types of RuvA-Holliday junction complexes have been characterised: complex I, containing a single RuvA tetramer, and complex II, in which the junction is sandwiched between two RuvA tetramers . The functional differences between the two forms are still not clear . To investigate the role of RuvA octamerisation we introduced 3 aminoacid substitutions designed to disrupt the E.coli RuvA tetramer-tetramer interface as identified by structural studies . The mutant RuvA was tetrameric and interacted with both RuvB and junction DNA but, as predicted, formed complex I only at protein concentrations up to 500 nM . We present biochemical and surface plasmon resonance evidence for functional and physical interactions of the mutant RuvA with RuvB and RuvC on synthetic junctions . The mutant RuvA with RuvB showed DNA helicase activity and could support branch migration of synthetic four-way and three-way junctions . However, junction binding and the efficiency of branch migration of four-way junctions were affected . The activity of the RuvA mutant was consistent with a RuvAB complex driven by one RuvB hexamer only and lead us to propose that one RuvA tetramer can only support the activity of one RuvB hexamer . Significantly, the mutant failed to complement the UV sensitivity of E . coli ruvA cells . The results of this investigation indicate strongly that RuvA octamerisation is essential for the full biological activity of the RuvABC system.

J Biol Chem . 2004 Nov 19; {Epub ahead of print}
Altered oxyanion selectivity in mutants of UhpT, the Pi-linked sugar phosphate carrier of escherichia coli; Hall JA et al.; In Escherichia coli, the UhpT transporter catalyzes the electroneutral accumulation of sugar 6-phosphate by exchange with internal inorganic phosphate (Pi) . The substrate specificity of UhpT is at least in part regulated by constituents of an Asp(388)-Lys(391) intrahelical salt bridge, and mutations that remove one, but not both, of these residues alter UhpT preference for organophosphate substrates . Using site-directed mutagenesis, we now examine the role played by these two positions in the selection of the oxyanion counter-substrate . We show that derivatives having aliphatic or polar residues at positions 388 and 391 are gain-of-function mutants capable of transporting SO(4) as well as Pi . These oxyanions share similar structures, but differ significantly in the presence of a proton(s) on Pi . Our findings therefore lead us to suggest that the Asp(388)-Lys(391) ion pair acts normally as a filter that prevents substrates lacking a donatable proton from occupying the UhpT active site.

FEBS Lett, 2004 Nov 19, 577(3), 555 - 62
Catalytic domains of tyrosine kinases determine the phosphorylation sites within c-Cbl; Grossmann AH et al.; Catalytic (SH1) domains of protein tyrosine kinases (PTKs) demonstrate specificity for peptide substrates . Whether SH1 domains differentiate between tyrosines in a physiological substrate has not been confirmed . Using purified proteins, we studied the ability of Syk, Fyn, and Abl to differentiate between tyrosines in a common PTK substrate, c-Cbl . We found that each kinase produced a distinct pattern of c-Cbl phosphorylation, which altered the phosphotyrosine-dependent interactions between c-Cbl and CrkL or phosphatidylinositol 3'-kinase (PI3-K) . Our data support the concept that SH1 domains determine the final sites of phosphorylation once PTKs reach their target proteins.

FEBS Lett, 2004 Nov 19, 577(3), 386 - 92
Probing the active site of homoserine trans-succinylase; Rosen R et al.; Homoserine trans-succinylase is the first enzyme in methionine biosynthesis of Escherichia coli and catalyzes the activation of homoserine via a succinylation reaction . The in vivo activity of this enzyme is subject to tight regulation by several mechanisms, including repression and activation of gene expression, feedback inhibition, temperature regulation and proteolysis . This complex regulation reflects the key role of this enzyme in bacterial metabolism . Here, we demonstrate--using proteomics and high-resolution mass spectrometry--that succinyl is covalently bound to one of the two adjacent lysine residues at positions 45 and 46 . Replacing these lysine residues by alanine abolished the enzymatic activity . These findings position the lysine residues, one of which is conserved, at the active site.

FEBS Lett, 2004 Nov 19, 577(3), 367 - 70
Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus; Lukacin R et al.; Caffeoyl-coenzyme A O-methyltransferase cDNA was cloned from dark-grown Ammi majus L . (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli . The translated polypeptide of 27.1-kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O-methyltransferase from parsley . For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal-affinity chromatography, although the recombinant enzyme did not contain any affinity tag . Based on sequence analysis and substrate specificity, the enzyme classifies as a cation-dependent O-methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg2+-ions . Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced by Mn2+ or Co2+ in the assays . This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O-methyltransferases.

FEBS Lett, 2004 Nov 19, 577(3), 315 - 21
The power of vanadate in crystallographic investigations of phosphoryl transfer enzymes; Davies DR et al.; The formation of transition state mimics of phosphoryl transfer reactions with the metal oxoanion vanadate is a powerful technique in macromolecular crystallography . The tendency of vanadate to form pentacovalent complexes exhibiting trigonal bipyramidal geometry makes this compound a close approximation of the transition state for such reactions . In many cases, vanadate complexes provide the most accurate visualization of the transition state that can be reasonably achieved . A survey of the Protein Data Bank reveals that a relatively small number of structures (39, representing 23 unique proteins) include vanadate, yet these structures represent four of the six E.C . categories of enzymes, and were obtained in crystals with pH values ranging from 5.0 to 7.8 . Vanadate has additional advantages over other compounds such as aluminum fluoride, beryllium fluoride and nitrate used for visualization of transition state mimics in that vanadate readily forms covalent bonds with a variety of ligands and has produced a wider variety of transition state mimics . Given the hundreds of crystal structures that have been solved for phosphoryl transfer enzymes, it is surprising that vanadate has not been used more frequently for visualization of transition state analogs . We propose that an opportunity exists for vanadate to become a more commonly utilized component of the macromolecular crystallographer's toolbox.

Anal Biochem, 2004 Dec 15, 335(2), 192 - 5
A method for coupled transcription and aminoacylation of cysteinyl-tRNA; Pavel I et al.; A novel method for coupled transcription and aminoacylation of transfer RNA was developed where Escherichia coli cysteine-specific tRNA (tRNA(cys)) was transcribed and aminoacylated in a single reaction . The cys-tRNA(cys) that was synthesized and aminoacylated using this method was functional in in vitro translation . The cys-tRNA(cys) was further modified with biotin (N-iodoacetyl-N-biotinhexylenediamine) to facilitate detection . The biotin-modified cys-tRNAs(cys) was also functional in in vitro translation, allowing the synthesis and detection of biotin-labeled protein.

Protein Expr Purif, 2004 Dec, 38(2), 237 - 47
Intracellular domains of the delta-subunits of Torpedo and rat acetylcholine receptors--expression, purification, and characterization; Kottwitz D et al.; There are quite detailed structural data on the extracellular ligand-binding domain and the intramembrane channel-forming domain of the nicotinic acetylcholine receptors (nAChR) . However, the structure of the intracellular domain, which has variable amino acid sequences in different nAChR subunits, remains unknown . We expressed in Escherichia coli the intracellular loops (between transmembrane fragments TM3 and TM4) of the delta-subunits from the Torpedo californica and Rattus norvegicus muscle nAChRs . To facilitate purification, (His)6-tags were attached with or without linkers, and the effects of protein truncations at C- or N-termini were examined . The proteins were purified from inclusion bodies under denaturing conditions by Ni-NTA-chromatography . Molecular weight and peptide mass fingerprint was determined by MALDI mass spectrometry . Size-exclusion chromatography revealed that the Torpedo intracellular delta-loop refolded in an aqueous buffer was present in solution as a dimer . Phosphorylation of this protein with protein kinase A and tyrosine kinase (Abl) occurred at the same serine and tyrosine residues as in the native receptor . According to CD spectra, the secondary structure was not sensitive to phosphorylation . The rat intracellular loops could be solubilized only in the presence of non-ionic detergents or lipids . CD spectra indicate that the Torpedo and rat proteins have differences in their secondary structure . In the presence of dodecylphosphocholine, high concentrations (up to 6 mg/ml) of the Torpedo and rat intracellular loops were achieved . The results suggest that the spatial structure of the intracellular loops is dependent on environment and species, but is not changed significantly upon enzymatic phosphorylation.

Protein Expr Purif, 2004 Dec, 38(2), 228 - 36
Overexpression in Escherichia coli and purification of pteridine reductase (PTR1) from a clinical isolate of Leishmania donovani; Kumar P et al.; Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism . Its main function is to salvage pterins but a second one is to reduce folates . The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of developing specific inhibitors, which in combination with dihydrofolate reductase inhibitors could be highly effective against Leishmania . In order to increase our understanding of this putative important chemotherapeutic target, we present here the cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India . This recombinant enzyme will set the basis for inhibition studies as well as for structure-function relationships.

Biosystems, 2004 Dec, 78(1-3), 23 - 37
Analysis of two-component signal transduction by mathematical modeling using the KdpD/KdpE system of Escherichia coli; Kremling A et al.; A mathematical model for the KdpD/KdpE two-component system is presented and its dynamical behavior is analyzed . KdpD and KdpE regulate expression of the kdpFABC operon encoding the high affinity K+ uptake system KdpFABC of Escherichia coli . The model is validated in a two step procedure: (i) the elements of the signal transduction part are reconstructed in vitro . Experiments with the purified sensor kinase and response regulator in presence or absence of DNA fragments comprising the response regulator binding-site are performed . (ii) The mRNA and molecule number of KdpFABC are determined in vivo at various extracellular K+ concentrations . Based on the identified parameters for the in vitro system it is shown, that different time hierarchies appear which are used for model reduction . Then the model is transformed in such a way that a singular perturbation problem is formulated . The analysis of the in vivo system shows that the model can be separated into two parts (submodels which are called functional units) that are connected only in a unidirectional way . Hereby one submodel represents signal transduction while the second submodel describes the gene expression.

Mol Biochem Parasitol, 2004 Dec, 138(2), 195 - 203
Molecular characterization of peroxiredoxin from Entamoeba moshkovskii and a comparison with Entamoeba histolytica; Cheng XJ et al.; Peroxiredoxin of the pathogenic parasite, Entamoeba histolytica, is thought to be involved in protection from oxidative attack by host phagocytic cells and endogenously generated hydrogen peroxide . In this study, we cloned peroxiredoxin genes from the nonpathogenic ameba, Entamoeba moshkovskii, and characterized the peroxiredoxin protein . The open reading frame of three cloned cDNAs was demonstrated to encode a polypeptide of 218 or 217 amino acids . Identity of the amino acid sequence of peroxiredoxins between E . moshkovskii and E . histolytica was considerably high (77-81%), but the N-terminus portion of E . moshkovskii peroxiredoxin was shorter than that of E . histolytica . A recombinant peroxiredoxin of E . moshkovskii expressed in Escherichia coli exhibited hydrogen peroxidase activity . Its K(m) and V(max) values of 35 microM and 0.07 micromol/min/mg protein were approximately 1 and 1.5 times greater than E . histolytica peroxiredoxin, respectively . In addition, the protective effect of E . moshkovskii peroxiredoxin against oxidative-nicking of supercoiled plasmid DNA was shown to be greater than that of E . histolytica peroxiredoxin . Confocal laser scanning microscopy, using polyclonal antibody against the recombinant E . moshkovskii peroxiredoxin, demonstrated that this protein was localized in the nucleus and cytoplasm of trophozoites, supporting its function as a protectant against DNA damage . Southern blot and real-time reverse transcription PCR analyses of the E . moshkovskii peroxiredoxin gene demonstrated that it was a multi-copy gene and its expression was comparable to that of E . histolytica . These results suggest that the antioxidant peroxiredoxin is important for protection against endogenously generated hydrogen peroxide in the nonpathogenic ameba.

Biochem Biophys Res Commun, 2004 Dec 24, 325(4), 1487 - 94
Lysophospholipase I identified as a ghrelin deacylation enzyme in rat stomach; Shanado Y et al.; Ghrelin, discovered in rat stomach as an endogenous growth hormone secretagogue, is octanoylated at the Ser3 residue . Since this octanoylation is essential for the functions of ghrelin, the enzymes that catalyze acylation for ghrelin biosynthesis and deacylation (deactivation step) must be considered as important regulators . We found that rat stomach homogenate contained ghrelin deacylation activity, and we isolated the active fractions by column chromatography . After sequencing and expressing candidate proteins, the ghrelin deacylation enzyme in the stomach was identified as lysophospholipase I (LysoPLA I) . The enzyme properties were examined using recombinant rat LysoPLA I expressed in Escherichia coli . K(m) and V(max) values were determined as 6.5 microM and 2.3 micromol/min/mg for ghrelin and 2.2 x 10(2) microM and 0.5 micromol/min/mg for lysophosphatidylcholine (LysoPC), respectively . The deacylation of both substrates was inhibited by methyl arachidonyl fluorophosphonate (MAFP), which is known as an irreversible inhibitor of LysoPLA I . These results reveal that LysoPLA I catalyzes the removal of n-octanoic acid from ghrelin to form des-acyl ghrelin . Identification of the ghrelin deacylation enzyme in the stomach and a deacylation inhibitor will be helpful in investigating ghrelin biosynthesis.

Microbes Infect, 2004 Nov, 6(14), 1305 - 11
Oral immunization of mice with Japanese encephalitis virus envelope protein synthesized in Escherichia coli induces anti-viral antibodies; Rauthan M et al.; In order to evaluate the possibility of developing an oral vaccine against Japanese encephalitis virus (JEV), mice were fed with recombinant JEV envelope (E) protein synthesized in Escherichia coli . The protein was administered orally to mice with or without an immunostimulatory cytosine-phosphate-guanosine (CpG) motif containing synthetic oligodeoxynucleotide (ODN) as an adjuvant . The immunized mice made high-titered anti-E and anti-JEV antibodies . Mice immunized with JEV E protein along with the ODN adjuvant produced higher antibody titers and these were predominantly IgG2a type . These antibodies, however, failed to neutralize JEV activity in vitro, and the immunization did not protect the mice against lethal JEV challenge . Splenocytes from the immunized mice secreted large amounts of interferon (IFN)-gamma and showed proliferation in the presence of JEV E protein . Our results indicate that JEV E protein delivered orally to mice together with ODN generated both humoral and cellular immune responses to JEV, and these were of the Th1 type.

Zhonghua Liu Xing Bing Xue Za Zhi, 2004 Sep, 25(9), 783 - 6
{Cloning and expression of flagellin gene from a Chinese Borrelia burgdorferi PD91 strain.}; Lu B et al.; OBJECTIVE: To study the cloning and expression of flagellin gene from Chinese Borrelia burgdorferi, PD91 strain and to evaluate the feasibility of using recombinant protein as diagnostic antigen when comparing the gene sequence with flagellin gene from North American Borrelia burgdorferi B31 . METHODS: The piece of genes coding flagellin from Chinese Borrelia burgdorferi PD91 by polymerase chain reaction (PCR) method was obtained, and constructed recombinant plasmid, before transformed into E . coli BL21 strain, and induced . The recombinant plasmid was identified with enzyme cutoff and gene sequence comparison . Efficient expression strain was selected and the expression product was analyzed with sodium amplified polymorphic-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot method . RESULTS: The recombinant protein (r-flagellin) expressed in host bacteria was successful . By means of western-blot assay, the immunological response showed the same antigenicity between r-flagellin and PD91 flagellin . The piece of genes coding flagellin of PD91 was 1011 bp, but when comparing with that of North American Borrelia burgdorferi it showed 94.70% homology . Homology between the sequence of amino acid of the r-flagellin and that of B31 flagellin was 95.85% . CONCLUSION: Flagellin gene of Borrelia garinii of Chinese Lyme disease spirochete was successfully cloned and expressed for the first time . It was proved that the immunoreactivity of r-flagellin was the same as the natural flagellin.

Am J Gastroenterol, 2004 Nov, 99(11), 2235 - 41
Serologic testing with ANCA, ASCA, and anti-OmpC in children and young adults with Crohn's disease and ulcerative colitis: diagnostic value and correlation with disease phenotype; Zholudev A et al.; OBJECTIVES: Serologic testing is increasingly being utilized to evaluate children with suspected inflammatory bowel disease (IBD) . The aim of this paper was to evaluate the sensitivity and specificity of a currently available panel involving four antibodies: deoxyribonuclease (DNase)-sensitive perinuclear antineutrophil cytoplasmic antibody (DNase-sensitive pANCA), IgA and IgG antibodies to Saccharomyces cerevisiae (IgA and IgG ASCA), and antibody to Escherichia coli outer membrane porin (anti-OmpC) . We also wished to determine whether antibody levels correlated with disease activity, and whether a specific antibody pattern correlated with location and outcome of disease in children . METHODS: We studied sera from 81 children with Crohn's disease (CD), 54 with ulcerative colitis (UC), and 63 controls . Clinical data, disease activity, and disease diagnosis were gathered at the time of serum sampling, and charts were re-reviewed at time of the study to determine long-term outcome . Enzyme-linked immunosorbent assay was utilized to determine titers of antibodies to ASCA, DNase-sensitive pANCA, and anti-OmpC; the presence of perinuclear staining for ANCA was confirmed by immunofluorescence . RESULTS: We identified ASCA antibodies in 44% of CD patients, 0% of UC patients, and 1 control patient . DNase-sensitive pANCA antibodies were found in 70% of patients with UC, 18% of CD patients (predominantly Crohn's colitis), and 3% of controls . Anti-OmpC as an isolated assay had low sensitivity for both CD (24%) and UC (11%), and displayed a 5% false-positive rate . However, anti-OmpC did identify a small number of IBD patients not detected by the other assays . If any one or more of the four antibodies was positive, the overall sensitivity of the four antibody panel was 65% for CD and 76% for UC, with a specificity of 94% . Patients who were ASCA-positive were more likely to have disease of the ileum or ileum and right colon than patients who were ASCA-negative (58%vs 18%, p < 0.001) . Patients with ASCA-positive were also more likely to require ileocecal resection (36%vs 13%, p < 0.05) . CONCLUSIONS: A currently available commercial antibody panel has good sensitivity and excellent specificity for CD and UC . The ASCA antibodies, while highly specific for CD, identify predominantly the subset of children with disease of the ileum and ascending colon who may be at increased risk of surgery.

Mol Microbiol, 2004 Dec, 54(5), 1422 - 30
The RNase E of Escherichia coli has at least two binding sites for DEAD-box RNA helicases: functional replacement of RhlB by RhlE; Khemici V et al.; The non-catalytic region of Escherichia coli RNase E contains a protein scaffold that binds to the other components of the RNA degradosome . Alanine scanning yielded a mutation, R730A, that disrupts the interaction between RNase E and the DEAD-box RNA helicase, RhlB . We show that three other DEAD-box helicases, SrmB, RhlE and CsdA also bind to RNase E in vitro . Their binding differs from that of RhlB because it is not affected by the R730A mutation . Furthermore, the deletion of residues 791-843, which does not affect RhlB binding, disrupts the binding of SrmB, RhlE and CsdA . Therefore, RNase E has at least two RNA helicase binding sites . Reconstitution of a complex containing the protein scaffold of RNase E, PNPase and RhlE shows that RhlE can furnish an ATP-dependent activity that facilitates the degradation of structured RNA by PNPase . Thus, RhlE can replace the function of RhlB in vitro . The results in the accompanying article show that CsdA can also replace RhlB in vitro . Thus, RhlB, RhlE and CsdA are interchangeable in in vitro RNA degradation assays.

Mol Microbiol, 2004 Dec, 54(5), 1409 - 21
Physical and functional interactions among RNase E, polynucleotide phosphorylase and the cold-shock protein, CsdA: evidence for a 'cold shock degradosome'; Prud'homme-Genereux A et al.; Escherichia coli contains at least five ATP-dependent DEAD-box RNA helicases which may play important roles in macromolecular metabolism, especially in translation and mRNA decay . Here we demonstrate that one member of this family, CsdA, whose expression is induced by cold shock, interacts physically and functionally with RNase E . Three independent approaches show that after a shift of cultures to 15 degrees C, CsdA co-purifies with RNase E and other components of the RNA degradosome . Moreover, functional assays using reconstituted minimal degradosomes prepared from purified components in vitro show that CsdA can fully replace the resident RNA helicase of the RNA degradosome, RhlB . In addition, under these conditions, CsdA displays RNA-dependent ATPase activity . Taken together, our data are consistent with a model in which CsdA accumulates during the early stages of cold acclimatization and subsequently assembles into degradosomes with RNase E synthesized in cold-adapted cultures . These findings show that the RNA degradosome is a flexible macromolecular machine capable of adapting to altered environmental conditions.

Mol Microbiol, 2004 Dec, 54(5), 1352 - 63
A conservative amino acid change alters the function of BosR, the redox regulator of Borrelia burgdorferi; Seshu J et al.; Borrelia burgdorferi, the aetiologic agent of Lyme disease, modulates gene expression in response to changes imposed by its arthropod vector and mammalian hosts . As reactive oxygen species (ROS) are known to vary in these environments, we asked how B . burgdorferi responds to oxidative stress . The B . burgdorferi genome encodes a PerR homologue (recently designated BosR) that represses the oxidative stress response in other bacteria, suggesting a similar function in B . burgdorferi . When we tested the sensitivity of B . burgdorferi to ROS, one clonal non-infectious B . burgdorferi isolate exhibited hypersensitivity to t-butyl hydroperoxide when compared with infectious B . burgdorferi and other non-infectious isolates . Sequence analysis indicated that the hypersensitive non-infectious isolates bosR allele contained a single nucleotide substitution, converting an arginine to a lysine (bosRR39K) . Mutants in bosRR39K exhibited an increase in resistance to oxidative stressors when compared with the parental non-infectious strain, suggesting that BosRR39K functioned as a repressor . Complementation with bosRR39K and bosR resulted in differential sensitivity to t-butyl hydroperoxide, indicating that these alleles are functionally distinct . In contrast to BosR, BosRR39K did not activate transcription of a napA promoter-lacZ reporter in Escherichia coli nor bind the napA promoter/operator domain . However, we found that both BosR and BosRR39K bound to the putative promoter/operator region of superoxide dismutase (sodA) . In addition, we determined that cells lacking BosRR39K synthesized fourfold greater levels of the decorin binding adhesin DbpA suggesting that BosRR39K regulates genes unrelated to oxidative stress . Based on these data, we propose that the single amino acid substitution, R39K, dramatically alters the activity of BosR by altering its ability to bind DNA at target regulatory sequences.

Biochemistry, 2004 Nov 30, 43(47), 15086 - 94
Resolving complexity in the interactions of redox enzymes and their inhibitors: contrasting mechanisms for the inhibition of a cytochrome c nitrite reductase revealed by protein film voltammetry; Gwyer JD et al.; Cytochrome c nitrite reductase is a dimeric decaheme-containing enzyme that catalyzes the reduction of nitrite to ammonium . The contrasting effects of two inhibitors on the activity of this enzyme have been revealed, and defined, by protein film voltammetry (PFV) . Azide inhibition is rapid and reversible . Variation of the catalytic current magnitude describes mixed inhibition in which azide binds to the Michaelis complex (approximately 40 mM) with a lower affinity than to the enzyme alone (approximately 15 mM) and leads to complete inhibition of enzyme activity . The position of the catalytic wave reports tighter binding of azide when the active site is oxidized (approximately 39 microM) than when it is reduced . By contrast, binding and release of cyanide are sluggish . The higher affinity of cyanide for reduced versus oxidized forms of nitrite reductase is immediately revealed, as is the presence of two sites for cyanide binding and inhibition of the enzyme . Formation of the monocyano complex by reduction of the enzyme followed by a "rapid" scan to high potentials captures the activity-potential profile of this enzyme form and shows it to be distinct from that of the uninhibited enzyme . The biscyano complex is inactive . These studies demonstrate the complexity that can be associated with inhibitor binding to redox enzymes and illustrate how PFV readily captures and deconvolves this complexity through its impact on the catalytic properties of the enzyme.

Biochemistry, 2004 Nov 30, 43(47), 15022 - 36
Assembly of dimeric variants of coumermycins by tandem action of the four biosynthetic enzymes CouL, CouM, CouP, and NovN; Freel Meyers CL et al.; Coumermycin A(1) is a member of the aminocoumarin family of antibiotics . Unlike its structural relatives, novobiocin and clorobiocin, coumermycin A(1) is a dimer built on a 3-methyl-2,4-dicarboxypyrrole scaffold and bears two decorated noviose sugar components which are the putative target binding motifs for DNA gyrase . Starting with this scaffold, we have utilized the ligase CouL for mono- and bisamide formation with aminocoumarins to provide substrates for the glycosyltransferase CouM . CouM was subsequently shown to catalyze mono- and bisnoviosylation of the resulting CouL products . CouP was shown to possess 4'-O-methyltransferase activity on products from tandem CouL, CouM assays . A fourth enzyme, NovN, the 3'-O-carbamoyltransferase from the novobiocin operon, was then able to carbamoylate either or both arms of the CouP product . The tandem action of CouL, CouM, CouP, and NovN thus generates a biscarbamoyl analogue of the pseudodimer coumermycin A(1) . Starting from alternative dicarboxy scaffolds, these four enzymes can be utilized in tandem to create additional variants of dimeric aminocoumarin antibiotics.

Biochemistry, 2004 Nov 30, 43(47), 14979 - 86
Structural intermediate in the photocycle of a BLUF (sensor of blue light using FAD) protein Slr1694 in a Cyanobacterium Synechocystis sp . PCC6803; Hasegawa K et al.; Slr1694 in Synechocystis sp . PCC6803 is a family of blue-light photoreceptors based on flavin adenine dinucleotide (FAD) called BLUF (sensor of blue light using FAD) proteins, which include AppA from Rhodobacter sphaeroides and PAC from Euglena gracilis . Illumination of dark-state Slr1694 at 15 degrees C reversibly induced a signaling light state characterized by the red shift in the UV-visible spectrum and by the light-induced Fourier transform infrared (FTIR) difference spectrum for structural changes of a bound flavin and apo protein . Illumination at the medium-low temperature (-35 degrees C) led to the red shift in the UV-visible spectrum despite some small difference in the light-induced changes . In contrast, the -35 degrees C illumination resulted in a completely different light-induced FTIR spectrum, in which almost all of the bands were suppressed with the exception of the bands for the change of C4=O bonding of the FAD isoalloxazine ring . The C4=O bands were induced at -35 degrees C with almost the same intensity, but the band frequency for the light state was upshifted by 6 cm(-)(1) . The changes in frequency of the light-state C4=O band and in amplitude of other bands showed the same temperature dependence with a half-change temperature at approximately -20 degrees C . It was indicated that the light-induced structural changes of apo protein and FAD were inhibited at low temperature with the exception of the change in hydrogen bonding to the C4=O group . The light-induced formation of the FTIR bands was similarly inhibited by sample dehydration . We discussed the possibility that this constrained light state is a trapped intermediate state in the photocycle of Slr1694.

Biochemistry, 2004 Nov 30, 43(47), 14958 - 70
Quaternary structure of the severe acute respiratory syndrome (SARS) coronavirus main protease; Chou CY et al.; SARS (severe acute respiratory syndrome) has been one of the most severe viral infectious diseases last year and still remains as a highly risky public health problem around the world . Exploring the types of interactions responsible for structural stabilities of its component protein molecules constitutes one of the approaches to find a destabilization method for the virion particle . In this study, we performed a series of experiments to characterize the quaternary structure of the dimeric coronavirus main protease (M(pro), 3CL(pro)) . By using the analytical ultracentrifuge, we demonstrated that the dimeric SARS coronavirus main protease exists as the major form in solution at protein concentration as low as 0.10 mg/mL at neutral pH . The enzyme started to dissociate at acidic and alkali pH values . Ionic strength has profound effect on the dimer stability indicating that the major force involved in the subunit association is ionic interactions . The effect of ionic strength on the protease molecule was reflected by the drastic change of electrostatic potential contour of the enzyme in the presence of NaCl . Analysis of the crystal structures indicated that the interfacial ionic interaction was attributed to the Arg-4...Glu-290 ion pair between the subunits . Detailed examination of the dimer-monomer equilibrium at different pH values reveals apparent pK(a) values of 8.0 +/- 0.2 and 5.0 +/- 0.1 for the Arg-4 and Glu-290, respectively . Mutation at these two positions reduces the association affinity between subunits, and the Glu-290 mutants had diminished enzyme activity . This information is useful in searching for substances that can intervene in the subunit association, which is attractive as a target to neutralize the virulence of SARS coronavirus.

Biochemistry, 2004 Nov 30, 43(47), 14913 - 23
Comparative studies on the structure and stability of fluorescent proteins EGFP, zFP506, mRFP1, "dimer2", and DsRed1; Stepanenko OV et al.; To obtain more information about the structural properties and conformational stabilities of GFP-like fluorescent proteins, we have undertaken a systematic analysis of series of green and red fluorescent proteins with different association states . The list of studied proteins includes EGFP (green monomer), zFP506 (green tetramer), mRFP1 (red monomer), "dimer2" (red dimer), and DsRed1 (red tetramer) . Fluorescent and absorbance parameters, near-UV and visible CD spectra, the accessibility of the chromophores and tryptophans to acrylamide quenching, and the resistance of these proteins to the guanidine hydrochloride unfolding and kinetics of the approaching of the unfolding equilibrium have been compared . Tetrameric zFP506 was shown to be dramatically more stable than the EGFP monomer, assuming that association might contribute to the protein conformational stability . This assumption is most likely valid even though the sequences OF GFP and zPF506 are only approximately 25% identical . Interestingly, red FPs possessed comparable conformational stabilities, where monomeric mRFP1 was the most stable species under the equilibrium conditions, whereas the tetrameric DsRed1 possessed the slowest unfolding kinetics . Furthermore, EGFP is shown to be considerably less stable than mRFP1, whereas tetrameric zFP506 is the most stable species analyzed in this study . This means that the quaternary structure, being an important stabilizing factor, does not represent the only circumstance dictating the dramatic variations between fluorescent proteins in their conformational stabilities.

Biochemistry, 2004 Nov 30, 43(47), 14891 - 900
Kinetics and thermodynamics of the unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56; Dragan AI et al.; Temperature-induced reversible unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56, were studied by kinetic and thermodynamic methods, using CD spectroscopy, dynamic light scattering, and scanning calorimetry . It was found that both unfolding and refolding reactions of this protein in neutral solution in the presence of 100 mM NaCl are characterized by unusually slow kinetics, which permits detailed investigation of the mechanism of these reactions . Kinetic analyses show that the unfolding of this coiled coil represents a single-stage first-order reaction, while the refolding represents a single-stage third-order reaction . The activation enthalpy and entropy for unfolding do not depend noticeably on temperature and are both significantly greater than those for the folding reaction, which show a significant dependence on temperature . The activation heat capacity change for the unfolding reaction is close to zero, while it is quite significant for the folding reaction . The correlation between the activation and structural parameters obtained for the Lpp-56 coiled coil suggests that interhelical van der Waals interactions are disrupted in the transition state, which is nevertheless still compact, and water has not yet penetrated into the interface; the transition from the transient state to the unfolded state results in hydration of exposed apolar groups of the interface and the disruption of helices . The low propensity for the Lpp-56 strands to fold and associate is caused by the high number of charged groups at neutral pH . On one hand, these charges give rise to considerable repulsive forces destabilizing the helical conformation of the strands . On the other hand, they align the folded helices in parallel and in register so that the apolar sides face each other, and the oppositely charged groups may form salt links, which are important for the formation of the trimeric coiled coil . A decrease in pH, which eliminates the salt links, dramatically decreases the stability of Lpp-56; its structure becomes less rigid and unfolds much faster.

Biochemistry, 2004 Nov 30, 43(47), 14881 - 90
Light-induced structural changes of LOV domain-containing polypeptides from Arabidopsis phototropin 1 and 2 studied by small-angle X-ray scattering; Nakasako M et al.; Phototropin is a blue-light receptor of plants and comprises two light-receptive domains, LOV1 and LOV2, Ser/Thr kinase domain and one linker region connecting the LOV2 and the kinase domains . The LOV2 domain is thought to regulate predominantly the light-dependent autophosphorylation of the kinase domain, leading to cellular signaling cascades . In this study, we constructed recombinant LOV1, LOV2, and LOV2-linker polypeptides from phototropin 1 and phototropin 2 of Arabidopsis thaliana and studied their quaternary structures and light-dependent conformational changes by small-angle X-ray scattering . The molecular weights of the polypeptides determined from scattering intensities demonstrated the dimeric associations of LOV1 polypeptides of both isoforms . In contrast, while LOV2 and LOV2-linker polypeptides of phototropin 1 were homodimers, corresponding polypeptides of phototropin 2 existed as monomeric forms . Under blue-light irradiation, the LOV2-linker polypeptide of phototropin 1 displayed small but definite changes of the scattering profile . Through simulation of low-resolution molecular structures, the changes were likely explained as structural changes of the linker region and/or a movement of the region relative to the LOV2 domain . Light-induced profile changes were not observed in the Cys(512)Ala mutated LOV2-linker polypeptide of phototropin 1 losing the phototransformation capability . Thus, it was indicated that the photoreaction in the LOV2 domain probably caused the structural changes in the LOV2-linker polypeptide of phototropin 1 . On the basis of the results, the interdomain interactions in phototropin are discussed.

Biochemistry, 2004 Nov 30, 43(47), 14873 - 80
Crystal structure of the PH-BEACH domains of human LRBA/BGL; Gebauer D et al.; The beige and Chediak-Higashi syndrome (BEACH) domain defines a large family of eukaryotic proteins that have diverse cellular functions in vesicle trafficking, membrane dynamics, and receptor signaling . The domain is the only module that is highly conserved among all of these proteins, but the exact functions of this domain and the molecular basis for its actions are currently unknown . Our previous studies showed that the BEACH domain is preceded by a novel, weakly conserved pleckstrin homology (PH) domain . We report here the crystal structure at 2.4 A resolution of the PH-BEACH domain of human LRBA/BGL . The PH domain has the same backbone fold as canonical PH domains, despite sharing no sequence homology with them . However, our binding assays demonstrate that the PH domain in the BEACH proteins cannot bind phospholipids . The BEACH domain contains a core of several partially extended peptide segments that is flanked by helices on both sides . The structure suggests intimate association between the PH and the BEACH domains, and surface plasmon resonance studies confirm that the two domains of the protein FAN have high affinity for each other, with a K(d) of 120 nM.

Mol Biol (Mosk), 2004 Sep-Oct, 38(5), 937 - 44
{Escherichia coli ribosomes as the model to test new photoactivated tRNA analogues, containing 6-thioguanosine}
{New non-hydrolyzable substrate analogs for 8-oxoguanine-DNA glycosylases}
{No authors listed}

8-Oxoguanine-DNA glycosylases play a key role in the repair of oxidatively damaged DNA . The Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are DNA base excision repair enzymes that catalyze the removal of 7,8-dihydro-8-oxoguanine (oxoG) residue, and cleave DNA strand . Specific contacts between DNA phosphate groups and amino acids from active centers of these enzymes play a significant role in DNA-protein interactions . In order to design new non-hydrolyzable substrate analogs of Fpg and hOGG1 for structural studies modified DNA duplexes containing pyrophosphate or OEt-substituted pyrophosphate internucleotide (SPI) groups near the damage were tested . We showed that enzymes recognize and specifically bind to DNA duplexes obtained . The mechanism of incision of oxoG by the Fpg and hOGG1 was determined . We revealed that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the oxoG . In contrast, Fpg and hOGG1 effectively incise DNA duplex carrying analogous phosphate modifications 5' to the oxoG . Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or substituted pyrophosphate groups immediately 3' to the oxoG are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a oxoG-containing DNA bound to catalytically active wild-type enzymes as well as their pro- and eucaryotic homologs.

Mol Biol (Mosk), 2004 Sep-Oct, 38(5), 786 - 97
{Specificiety of DNA-protein interactions within transcription complexes of Escherichia coli}
{Preparation of recombinant human insulin--study of downstream process}
Yu R, Li X, Yang J, Wu W.

Department of Biopharmaceutics, West China School of Pharmacy, Sichuan University, Chengdu 610041, ChinaThis study was intended to establish a method of preparation of recombinant human insulin, with (His)6-Arg-Arg-human proinsulin (RRhPI) expressed by Escherichia coli . After DEAE-Sepharose Fast Flow ion-exchange chromatography, Sephadex G-25 chromatography and refolding, enzyme cleavage and Superdex 75 size exclusion chromatography,the RRhPI expressed by Escherichia coli in inclusion body form was converted to human insulin . The obtained recombinant human insulin was analyzed by SDS-PAGE, HPLC, amino acid composition analysis and bioidentity test (mouse convulsion test) . The results indicate that our obtained preparation is highly purified, active recombinant human insulin.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Oct, 21(5), 795 - 9
{Purification and partial characterization of hepatitis C virus (HCV) non-structural protein 5A (NS5A) expressed in Escherichia coli}; Luo H et al.; It has been suggested that non-structural protein 5A (NS5A) of hepatitis C virus (HCV) may have a regulatory role similar to other RNA viruses in RNA replication . In order to investigate the replication function of NS5A, we tried to purify recombinant His(6) tagged NS5A expressed in Escherichia coli by a denature-renaturing method . The native lysis buffer was used to remove most of the soluble non-specific proteins . His-NS5A protein was solublized with the denaturing lysis buffer containing 8 mol/L Urea, and then bound to Ni2+ -NTA resin . The protein bound resin was successively washed with buffer containing reducing concentrations of Urea in the presence of NaCl and DTT to renature the protein . The renatured His-NS5A protein was eluted from the resin and it was capable of interacting with glutathione S-transferase fused form NS5Bt (GST-NS5Bt) . The purified His-NS5A exhibited an inhibitory effect on RNA-dependent RNA Polymerase (RdRP) activity of GST-NS5Bt in vitro . Conclusively, in this study, we have established a purification method of bacterial recombinant HCV NS5A, and the results support the notion that NS5A may involve in the regulation of HCV replication through direct interaction with NS5B.

J Food Prot, 2004 Nov, 67(11), 2617 - 21
Demonstration of the applicability of the Weibull-log-logistic survival model to the isothermal and nonisothermal inactivation of Escherichia coli K-12 MG1655; Corradini MG et al.; Published isothermal semilogarithmic survival curves of Escherichia coli K-12 MG1655, in the range of 49.8 to 60.6 degrees C, all had noticeable downward concavity . They could be described by the model log S(t) = -b(T)t n, where S(t) = N(t)/N0, N(t) and N0 being the momentary and initial number of organisms, respectively; b(T) is a temperature-dependent rate parameter; and n is a constant found to be about 1.5 . The temperature dependence of b(T) could be described by the log-logistic model, b(T) = ln{1 + exp{k(T - Tc)}}, which had an almost perfect fit, with k = 0.88 degrees C(-1) and Tc = 60.5 degrees C . The constants, n, k, and Tc were considered the organism's survival parameters in the particular medium . They were incorporated into a rate equation on the assumption that in nonisothermal heating, the momentary inactivation rate is the isothermal rate at the momentary temperature at a time that corresponds to the momentary survival ratio . This model's estimates matched the actual survival curves obtained in the same work under two different nonisothermal heating profiles, lending support to the notion that the Weibull-log-logistic model combination can be used not only to describe isothermal inactivation mathematically, but also to predict survival patterns under nonisothermal conditions.

J Food Prot, 2004 Nov, 67(11), 2410 - 5
Juice irradiation with Taylor-Couette flow: UV inactivation of Escherichia coli; Forney LJ et al.; A novel reactor is described with flow characteristics that approach that of ideal plug flow but with a residence time that is uncoupled from the hydrodynamics or boundary layer characteristics . The design described consists of an inner cylinder that rotates within a stationary but larger outer cylinder . At low rotation rates, a laminar, hydrodynamic configuration called Taylor-Couette flow is established, which consists of a system of circumferential vortices within the annular fluid gap . The latter constitutes a spatially periodic flow that is the hydrodynamic equivalent to cross flow over a tube bank or lamp array . These vortices provide radial mixing, reduce the boundary layer thickness, and are independent of the axial flow rate and thus the fluid residence time . An additional feature of the rotating design is the repetitive exposure of the fluid parcels to a minimum number of lamps, which substantially reduces the maintenance requirements . Inactivation data for Escherichia coli (ATCC 15597) were recorded in commercial apple and grape juice that are relatively opaque to UV radiation . With initial E . coli concentrations of approximately 10(6) CFU/ml, Taylor-Couette flow was found to provide a 3- to 5-log improvement in the inactivation efficiency compared with simple channel flow between concentric cylinders.

Arzneimittelforschung, 2004, 54(10), 692 - 702
High resolution X-ray structure and potent anti-HIV activity of recombinant dianthin antiviral protein; Kurinov IV et al.; Dianthin antiviral protein (DAP) is a naturally occurring antiviral protein from the leaves of carnation (Dianthus caryophyllus) capable of depurinating HIV-1 RNA and inhibiting HIV-1 replication in human peripheral blood mononuclear cells . Escherichia coli-derived recombinant DAP (rDAP, amino acids 1-254) was purified to homogeneity for structural and functional studies . In the following paper the X-ray crystal structure of rDAP as well as its complexes with cyclic AMP and adenyl-guanosine (ApG) as substrate analogs at 1.7 A resolution are reported . Molecular modeling studies of the interactions of DAP and the structurally similar pokeweed antiviral protein (PAP) with a single-stranded RNA heptamer predicted a more potent anti-HIV activity for rDAP due to its unique surface topology and more favorable charge distribution in its 20 A-long RNA binding active center cleft . In accordance with the predictions of the modeling studies, rDAP was more potent than rPAP in depurinating HIV-1 RNA . To the knowledge of the authors, this is the first structural and functional characterization of recombinant DAP.

Biol Chem, 2004 Oct, 385(10), 935 - 42
X-ray structure of fumarylacetoacetate hydrolase family member homo sapiens FLJ36880; Manjasetty BA et al.; The human protein FLJ36880 belongs to the fumarylacetoacetate hydrolase family . The X-ray structure of FLJ36880 has been determined to 2.2 A resolution employing the semi-automated high-throughput structural genomics approach of the Protein Structure Factory . FLJ36880 adopts a mixed beta-sandwich roll fold and forms homodimers in crystals as well as in solution . One Mg2+ ion is bound to each subunit of the dimeric protein by coordination to three carboxylate oxygens and three water molecules . These metal binding sites are accessible from the same surface of the dimer, partly due to the disorder of the undecapeptide stretch D29 to L39 . The overall structure and metal binding site of FLJ36880 bear clear similarities to the C-terminal domain of the bifunctional enzyme HpcE from Escherichia coli C, fumarylacetoacetate hydrolase from Mus musculus and to YcgM (Apc5008) from E . coli 1262 . These similarities provide a framework for suggesting biochemical functions and evolutionary relationships of FLJ36880 . It appears highly probable that the metal binding sites are involved in an enzymatic activity related to the catabolism of aromatic amino acids . Two point mutations in the active-site of FAH, responsible for the metabolic disease hereditary tyrosinemia type I (HTI) in humans, affect residues that are structurally conserved in FLJ36880 and located in the putative catalytic site.

J Vet Med Educ, 2004 Winter, 31(4), 333 - 9
Emerging Challenges in Public Health Protection, Food Safety and Security: Veterinary Needs in the USDA's Food Safety and Inspection Service; Buntain BJ; Meeting the needs of public service practice is a responsibility of the veterinary profession . The United States Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) has undergone significant change since 1996, when the final rule on Pathogen Reduction and Hazard Analysis and Critical Control Point (HACCP) Systems and its regulations were published in response to food-borne illnesses and deaths due to E . coli 0157:H7 in undercooked hamburgers . As a result, the role of the veterinarian is changing from a focus on carcass inspection (reactive) to scientific-based systems analysis and enforcement (preventive) . With a large pool of veterinarians eligible to retire, a critical shortage of field veterinarians is predicted . The purpose of this article is to raise educators' awareness of this need, of the competencies required, and of the challenges and opportunities for veterinarians in the new public health-focused FSIS . An invitation to collaborate with the agency is offered to help meet emerging workforce requirements in public health practice.

J Infect Dis, 2004 Dec 15, 190(12), 2129 - 36 Epub 2004 Dec 15.
An interventional approach to block brain damage caused by Shiga toxin-producing Escherichia coli infection, by use of a combination of phosphodiesterase inhibitors; Okayama A et al.; We tested the combination of phosphodiesterase (PDE) 3 and PDE4 inhibitors as an interventional approach to prevent the development of brain damage after Shiga toxin (Stx)-producing Escherichia coli (STEC) infection, using mice with protein calorie malnutrition . The combination consisted of pentoxifylline and rolipram; the dose of each inhibitor was 7.5 mg/kg . Treatment with this combination, which was administered intraperitoneally twice daily at 12-h intervals, increased serum concentrations of each inhibitor to >2 microg/mL and afforded significant levels of protection when it was continued for 3 days, starting on day 2 (95% survival rate; P<.001) or day 3 (63% survival rate; P<.01) of infection . The treatment reduced plasma levels of Stx2; consequently, immunoreactions of Stx2 were not found in the brain, and survivors did not show neurologic symptoms . Protection was associated with decreased levels of tumor necrosis factor (TNF)- alpha and increased production of interleukin-10 in serum, the brain, and the cecum . Although the combination at doses >2 microg/mL reduced Gb3 content of and Stx2 binding to Caco-2 cells, its ability to suppress production of TNF- alpha seemed to be more important for the decrease in cell-bound Stx2 in intestinal epithelial cells . Therefore, the combination of PDE3 and PDE4 inhibitors might be used as an interventional approach to prevent brain damage caused by STEC infectionPublication Types:

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