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 o