FEBS Lett, 2004 Feb 27, 560(1-3), 173 - 7 Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon; Hohsaka T et al.; Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system . 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine . Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures . These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system. FEBS Lett, 2004 Feb 27, 560(1-3), 167 - 72 In vivo fragment complementation of a (beta/alpha)(8) barrel protein: generation of variability by recombination; Soberon X et al.; The high representation of the TIM barrel as a scaffold for enzymatic proteins makes it an interesting model for protein engineering . Based on previous reports of folding mechanisms of TIM barrels that suggest an independent folding unit formed by six (beta/alpha) subunits, we interrupted the gene of phosphoribosylanthranilate isomerase (PRAI) from Escherichia coli at three different positions to yield fragments with different combinations of (beta/alpha) subunits . When these constructions were expressed as polycistrons in a TrpF-E . coli strain, complementation of the function only occurred with fragments beta1-alpha4 and beta5-alpha8, demonstrating that (beta/alpha)(4) subunits are stable enough to survive in vivo conditions and to assemble to yield a functional enzyme . The expression of these fragments in a separated plasmid/phagemid system to complement the function gave a slower complementation in the TrpF-E . coli strain; this was overcome by introducing extra secondary elements to the structure that reinforce their interaction. FEBS Lett, 2004 Feb 27, 560(1-3), 158 - 66 A novel calcium-dependent soluble inorganic pyrophosphatase from the trypanosomatid Leishmania major; Gomez-Garcia MR et al.; A single-copy gene IPP encoding a putative soluble inorganic pyrophosphatase (LmsPPase, EC 3.6.1.1) was identified in the genome of the parasite protozoan Leishmania major . The full-length coding sequence (ca . 0.8 kb) was obtained from genomic DNA by polymerase chain reaction (PCR) and cloned into an Escherichia coli expression vector, and was overexpressed for functional protein purification and characterization . The recombinant LmsPPase, purified to electrophoretic homogeneity by a two-step chromatography procedure, exhibited a predicted molecular mass of ca . 30 kDa . The enzyme has an absolute requirement for divalent cations, exhibits a pH optimum of 7.5-8.0 and does not hydrolyze polyphosphates or adenosine triphosphate (ATP) . LmsPPase differs from previously studied soluble pyrophosphatases with respect to cation selectivity, Ca(2+) being far more effective than Mg(2+) . Comparisons to known sPPases show a short N-terminal extension predicted to be a mitochondrial transit peptide, and changes in active-site residues and the neighboring region . Subcellular fractionation of L . major promastigotes suggests a mitochondrial localization . Molecular phylogenetic analysis indicates that LmsPPase is a highly divergent eukaryotic Family I sPPase, perhaps an ancestral class of eukaryotic sPPases functionally adapted to a calcium-rich, probably mitochondrial, environment. FEBS Lett, 2004 Feb 27, 560(1-3), 91 - 7 Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity; van der Veen BA et al.; Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose . Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation . Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme . A convenient zero background expression cloning strategy was developed . Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme trade mark, followed by recombination of the PCR products by DNA shuffling . A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source . Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer . A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions) . Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants . A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced . This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes. FEBS Lett, 2004 Feb 27, 560(1-3), 75 - 80 The artificial zinc finger protein 'Blues' binds the enhancer of the fibroblast growth factor 4 and represses transcription; Libri V et al.; The design of novel genes encoding artificial transcription factors represents a powerful tool in biotechnology and medicine . We have engineered a new zinc finger-based transcription factor, named Blues, able to bind and possibly to modify the expression of fibroblast growth factor 4 (FGF-4, K-fgf), originally identified as an oncogene . Blues encodes a three zinc finger peptide and was constructed to target the 9 bp DNA sequence: 5'-GTT-TGG-ATG-3', internal to the murine FGF-4 enhancer, in proximity of Sox-2 and Oct-3 DNA binding sites . Our final aim is to generate a model system based on artificial zinc finger genes to study the biological role of FGF-4 during development and tumorigenesis. FEBS Lett, 2004 Feb 27, 560(1-3), 45 - 50 Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp . M128; Yaoi K et al.; A novel xyloglucan-specific endo-beta-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp . M128 . It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-beta-glucan . Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (-2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites . The full-length cDNA encoding XEG was cloned and sequenced . It consists of a 2436-bp open reading frame encoding a 776-amino acid protein . From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase . The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained. Biochimie, 2004 Jan, 86(1), 21 - 9 Functional idiosyncrasies of tRNA isoacceptors in cognate and noncognate aminoacylation systems; Fender A et al.; The specificity of transfer RNA aminoacylation by cognate aminoacyl-tRNA synthetase is a crucial step for synthesis of functional proteins . It is established that the aminoacylation identity of a single tRNA or of a family of tRNA isoacceptors is linked to the presence of positive signals (determinants) allowing recognition by cognate synthetases and negative signals (antideterminants) leading to rejection by the noncognate ones . The completion of identity sets was generally tested by transplantation of the corresponding nucleotides into one or several host tRNAs which acquire as a consequence the new aminoacylation specificities . Such transplantation experiments were also useful to detect peculiar structural refinements required for optimal expression of a given aminoacylation identity set within a host tRNA . This study explores expression of the defined yeast aspartate identity set into different tRNA scaffolds of a same specificity, namely the four yeast tRNA(Arg) isoacceptors . The goal was to investigate whether expression of the new identity is similar due to the unique specificity of the host tRNAs or whether it is differently expressed due to their peculiar sequences and structural features . In vitro transcribed native tRNA(Arg) isoacceptors and variants bearing the aspartate identity elements were prepared and their aminoacylation properties established . The four wild-type isoacceptors are active in arginylation with catalytic efficiencies in a 20-fold range and are inactive in aspartylation . While transplanted tRNA(1)(Arg) and tRNA(4)(Arg) are converted into highly efficient substrates for yeast aspartyl-tRNA synthetase, transplanted tRNA(2)(Arg) and tRNA(3)(Arg) remain poorly aspartylated . Search for antideterminants in these two tRNAs reveals idiosyncratic features . Conversion of the single base-pair C6-G67 into G6-C67, the pair present in tRNA(Asp), allows full expression of the aspartate identity in the transplanted tRNA(2)(Arg), but not in tRNA(3)(Arg) . It is concluded that the different isoacceptor tRNAs protect themselves from misaminoacylation by idiosyncratic pathways of antidetermination. Sports Med, 2004, 34(3), 141 - 50 Doping with artificial oxygen carriers: an update; Schumacher YO et al.; There is a long history of science seeking to develop artificial substitutes for body parts damaged by disease or trauma . While defective teeth and limbs are commonly replaced by imitations without major loss of functionality, the development of a substitute for red blood cells has proved elusive . There is a permanent shortage of donor blood in western societies . Nevertheless, despite whole blood transfusions carrying measurable risks due to immunogenicity and the transmission of blood-borne infectious diseases, red blood cells are still relatively inexpensive, well tolerated and widely available . Researchers seeking to develop products that are able to meet and perhaps exceed these criteria have responded to this difficult challenge by adopting many different approaches . Work has focussed on two classes of substances: modified haemoglobin solutions and perfluorocarbon emulsions . Other approaches include the creation of artificial red cells, where haemoglobin and supporting enzyme systems are encapsulated into liposomes . Haemoglobin is ideally suited to oxygen transport when encased by the red cell membrane; however, once removed, it rapidly dissociates into dimers and is cleared by the kidney . Therefore, it must be stabilised before it can be safely re-infused into humans . Modifications concomitantly alter the vascular half-life, oxygen affinity and hypertensive characteristics of raw haemoglobin, which can be sourced from outdated blood stores, genetically-engineered Escherichia coli or even bovine herds . In contrast, perfluorocarbons are entirely synthetic molecules that are capable of dissolving oxygen but biologically inert . Since they dissolve rather than bind oxygen, their capacity to serve as a blood substitute is determined principally by the oxygen pressure gradients in the lung and at the target tissue . Blood substitutes have important potential areas of clinical application including red cell replacement during surgery, emergency resuscitation of traumatic blood loss, oxygen therapeutic applications in radiography (oxygenation of tumour cells is beneficial to the effect of certain chemotherapeutic agents), other medical applications such as organ preservation, and finally to meet the requirements of patients who cannot receive donor blood because of religious beliefs . Given the elite athlete's historical propensity to experiment with novel doping strategies, it is likely that the burgeoning field of artificial oxygen carriers has already attracted their attention . Scientific data concerning the performance benefits associated with blood substitutes are virtually nonexistent; however, international sporting federations have been commendably proactive in adding this category to their banned substance lists . The current situation is vulnerable to exploitation by immoral athletes since there is still no accepted methodology to test for the presence of artificial oxygen carriers. Anal Chem, 2004 Mar 1, 76(5), 1403 - 10 Dual-labeled glucose binding protein for ratiometric measurements of glucose; Ge X et al.; Highly sensitive glucose monitoring has potential applications in conditions where the glucose levels are below the detection limit of currently available technology . Examples include bioprocess monitoring of bacterial cultures and measurement of minute amounts of human interstitial fluid extracted by iontophoresis . Here we describe a ratiometric glucose sensor capable of measuring micromolar levels of glucose . This sensor is based on an E . coli glucose binding protein (GBP) labeled with two fluorophores . The L255C mutant of GBP was labeled with the environment-sensitive fluorophore, acrylodan, at the cysteine mutation and a long-lived metal ligand complex of ruthenium (ruthenium bis(2,2'-bipyridyl)-1, 10-phenanthroline-9-isothiocyanate) at the N-terminal . The acrylodan emission is quenched in the presence of glucose while the ruthenium emission remained constant, thereby serving as a reference . The sensitivity of the sensor is in the micromolar range (K(d) = 0.4-1.4 microM) . Thus, it is possible to measure glucose concentrations at micromolar levels and higher (with dilution) . Calculations of the fluorescence energy-transfer efficiency between acrylodan and ruthenium gave an approximate distance of 25 A between the two fluorophores, consistent with X-ray crystallographic data . The effect of temperature on glucose binding was measured and analyzed . Maximum signal changes and apparent binding constants increase with temperature . The enthalpy change for glucose binding as calculated from the apparent binding constants is approximately 43.1 kJ/mol . In addition to ratiometric measurements, the presence of the long-lived ruthenium metal ligand complex allows for low-cost modulation-based sensing. Anal Chem, 2004 Mar 1, 76(5), 1359 - 65 One- and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection; Sluszny C et al.; Miniaturized electrophoresis was successfully coupled with native fluorescence detection for direct analysis of proteins in one- and two-dimensional separations . The detection setup was based on direct observation of the UV-induced fluorescence of proteins using a CCD camera and a Hg (Xe) lamp for sample excitation . Protein mixtures were readily separated by size on a 1-cm segment of the one-dimensional gel in 8 min, and a detection limit of 0.04 ng per band was achieved . The dynamic range of the system was larger than 2 orders of magnitude . Miniaturized slab gel electrophoresis was performed on a special holder designed to couple isoelectric focusing with SDS-PAGE . Two-dimensional separation, including rehydration of IEF strip and fluorescence detection was completed in 2.5 h . Approximately 200 protein spots from Escherichia coli were detected on a 1 cm(2) area . A detection limit of 0.1 microg of total protein was achieved . The operation should be amenable to total automation. Amyloid, 2003 Dec, 10(4), 240 - 9 Splenic ellipsoids: an early target for deposition of AA amyloid induced in mink; Wien TN et al.; The spleen is the primary target for spontaneous as well as experimental AA amyloidosis in animals such as mice and mink, and is therefore a valuable organ for study of the initial phases of amyloid fibrillogenesis and deposition . We have investigated splenic amyloid AA deposits induced in the mink, and we demonstrate a novel target for AA, namely the splenic ellipsoids . We show presence of amyloid P component (AP), glycosaminoglycans (GAGs) and apolipoprotein E (apoE), all well-known common elements of amyloid, co-localizing with AA . In addition, apolipoprotein AI (apoAI) was seen co-localized to the AA deposits in the ellipsoids . We hypothesize that the ellipsoids may be important splenic structures for initial AA formation . The apoAI in the ellipsoids could displace SAA from acute phase HDL at this site, thereby making SAA available for amyloid formation and deposition. Yi Chuan Xue Bao, 2003 Dec, 30(12), 1161 - 6 {Isolation and characterization of a BRCA1-interacting protein}; Yan JH et al.; BRCA1 (breast cancer susceptibility gene-1) plays important roles in DNA damage repair, cell checkpoint regulation, gene transcription, chromosome stability, and apoptosis . At the C-terminus of BRCA1 is the activation domain with a number of acidic amino acid residues that includes two tandem repeats of BRCT(BRCT1 and BRCT2) . In this study, to identify proteins that interact with the BRCT2 domain of BRCA1, the standard yeast two-hybird screen was performed . FHL2 was isolated from a human ovary library, with the BRCT2 domain of BRCA1 as bait . Furthermore, the specific interaction of FHL2 with the BRCT2 domain of BRCA1, but not with the BRCT1 domain of BRCA1 and the BRCT domain of Rap1, was verified by yeast mating . To confirm the interaction between BRCA1 and FHL2 in vitro, the GST pull-down assay was performed, the coding sequences of BRCT1 and BRCT2 domains were fused in-frame with the coding region of GST in the pGEX-2T vector, generating the pGST-BRCT1 and pGST-BRCT2 recombinant plasmids the fusion proteins GST-BRCT1 and GST-BRCT2 were expressed in E . coli DH5 alpha . The purified fusion proteins were obtained by GST-Sepharose 4B affinity chromatography . The purified fusion proteins were incubated with in vitro translated 35S-methinine-labeled FHL2 . Consistent with the two-hybird results, FHL2 could specifically bind to the BRCT2 domain, but not BRCT1 in vitro . To further assess the binding specificity of FHL2 to the BRCT2 domain of BRCA1 in vivo, pFLAG-FHL2 and pHABRCT1/pHA-BRCT2 recombinant plasmids were cotransfected into 293T cells . Then the coimmunoprecipitation assay were performed . The results also showed that FHL2 specifically interacted with the BRCT2 domain in vivo . Furthermore, the coimmunoprecipitation assay demonstrated that FHL2 could interact with endogenous BRCA1 in vivo . These findings lay solid foundations for study on the function of BRCA1 and FHL2 in cancer development and progression. Eur J Clin Microbiol Infect Dis, 2004 Mar, 23(3), 208 - 11 Epub 2004 Feb 19. Enzyme-linked immunosorbent assay for detection of Shiga toxin-producing Escherichia coli infection by antibodies to Escherichia coli secreted protein B in children with hemolytic uremic syndrome; Sjogren AC et al.; In order to detect immunoglobulin (Ig)A and IgG antibodies to Escherichia coli-secreted protein B in sera of children infected with Shiga toxin-producing Escherichia coli, an enzyme-linked immunosorbent assay was developed . The assay was tested using acute sera from 40 children with diarrhea-associated hemolytic uremic syndrome compared with 238 sera obtained from pediatric controls . Two cut-off values were used for children <5 (n=27) or > or =5 (n=13) years of age . Among the younger patients, 24 of 27 had IgA antibodies to Escherichia coli-secreted protein B (sensitivity, 89%; specificity, 98%) and 22 of 27 had IgG antibodies (sensitivity, 82%; specificity, 94%) . Among the older patients, 13 of 13 had IgA antibodies (sensitivity, 100%; specificity, 96%) and 11 of 13 had IgG antibodies (sensitivity, 85%; specificity, 96%) . This enzyme-linked immunosorbent assay detects Shiga-toxin-producing Escherichia coli independent of serogroup and could serve as a complementary assay for detection of infection. Pediatr Nephrol, 2004 Apr, 19(4), 419 - 25 Epub 2004 Feb 24. Clinico-pathological findings in diarrhoea-negative haemolytic uraemic syndrome; Taylor CM et al.; This is a retrospective, national clinico-pathological study of past and current patients with haemolytic uraemic syndrome not associated with diarrhoea (D- HUS) . Thirty-four patients were analysed and notified by members of the British Association for Paediatric Nephrology in 1998-1999 . There was a 2:1 excess of males . Ten presented in infancy . The aetiology included 5 patients with complement abnormalities, 2 patients with complications of pneumococcal infection, and 2 with malignancies . Parental consanguinity was noted in 6 patients . Five children died, 9 developed chronic renal failure, and 10 end-stage renal failure . Only 7 made full recoveries . With a single exception, the pathological findings were unlike the previously reported glomerular thrombosis that is characteristic of diarrhoea-associated HUS, or HUS complicating verocytotoxin-producing Escherichia coli infection . Early and late glomerulopathy could be distinguished . Arteriolar and arterial disease was observed in 8 and 7 patients, respectively . Arterial disease correlated with a poor outcome . The pathology of D- HUS is of prognostic value, but this study was not powered to identify specific aetiological/pathological correlations. J Mol Med, 2004 May, 82(5), 280 - 97 Epub 2004 Feb 24. Novel aspects of macromolecular repair and relationship to human disease; Krokan HE et al.; Cellular and humoral defence mechanisms are essential for the survival of individuals and species . Thus, DNA repair prevents mutations and cytotoxicity from DNA damage, thereby reducing the risks of inappropriate cell death, developmental defects, premature ageing and cancer . Similarly, antigen-dependent acquired immune responses prevent infections and also have a role in cancer prevention . DNA repair is highly complex and functions in an intricate network that also involves transcription, replication, cell cycle regulation, and the immune system . DNA damage is repaired by at least four major mechanisms, each requiring many different proteins . In addition there are "subpathways", and back-up mechanisms both within and between pathways . Various defects in DNA repair result in different forms of cancer, e.g . the rare syndrome Xeroderma pigmentosum and the more common diseases early-onset breast cancer and hereditary non-polyposis colon cancer . Surprisingly, recent research has revealed molecular interactions between the ancient DNA repair mechanisms and the much younger acquired immune system . Thus, the classical base excision enzyme uracil-DNA glycosylase encoded by the UNG gene is also involved in somatic hypermutation and class switch recombination, e.g . from IgM antibodies to IgG, yielding secreted high affinity antibodies . Mutations in both alleles of UNG result in a hyper-IgM syndrome with life-threatening infections . Furthermore, it has recently become clear that not only DNA, but also RNA and proteins are repaired . Thus, certain aberrant methylations in RNA are repaired by oxidative demethylation in one step restoring the normal base, and at least in a bacterial model system this increases survival several-fold after exposure to methylating agents . Proteins are repaired both at the peptide amino acid level and at the structural level . RNA and protein repair are likely to be important to prevent the formation of cytotoxic protein aggregates of the types known to cause neurodegenerative diseases e.g . Alzheimer's, Parkinson's and Huntington's diseases, and other diseases as well . In conclusion, recent research has demonstrated an unexpected complexity of cellular defence mechanisms that function in intricate networks, rather than as independent mechanisms . The new knowledge opens for interventions that are based on a deeper understanding of the mechanisms of defence. Nature, 2004 Feb 26, 427(6977), 862 - 5 Visualization of release factor 3 on the ribosome during termination of protein synthesis; Klaholz BP et al.; Termination of protein synthesis by the ribosome requires two release factor (RF) classes . The class II RF3 is a GTPase that removes class I RFs (RF1 or RF2) from the ribosome after release of the nascent polypeptide . RF3 in the GDP state binds to the ribosomal class I RF complex, followed by an exchange of GDP for GTP and release of the class I RF . As GTP hydrolysis triggers release of RF3 (ref . 4), we trapped RF3 on Escherichia coli ribosomes using a nonhydrolysable GTP analogue . Here we show by cryo-electron microscopy that the complex can adopt two different conformational states . In 'state 1', RF3 is pre-bound to the ribosome, whereas in 'state 2' RF3 contacts the ribosome GTPase centre . The transfer RNA molecule translocates from the peptidyl site in state 1 to the exit site in state 2 . This translocation is associated with a large conformational rearrangement of the ribosome . Because state 1 seems able to accommodate simultaneously both RF3 and RF2, whose position is known from previous studies, we can infer the release mechanism of class I RFs. Genes Immun, 2004 May, 5(3), 176 - 82 Mechanisms by which transcription can regulate somatic hypermutation; Wright BE et al.; Mechanisms for somatic hypermutation (SHM) have proven elusive . An actively transcribed substrate was analyzed to elucidate the role of stem-loop structures (SLSs) in SHM . Analysis with a new computer algorithm indicates that the location and mutability of a base are regulated by: (a) the extent to which it is unpaired, (b) the degree to which it is exposed by stabilization of SLSs containing and flanking it, and (c) the level of transcription that drives supercoiling, which creates and stabilizes SLSs containing unpaired bases vulnerable to mutation . New mechanisms are described by which transcription can differentially stabilize SLSs harboring targeted bases and establish specific base exposure patterns . Assuming that transcription levels correlate with the magnitude of superhelicity induced and the lengths of ssDNA forming SLSs, this analysis accounts for the location of all mutable bases during SHM. Protein Eng Des Sel, 2004 Jan, 17(1), 77 - 83 A cell-based screen for function of the four-helix bundle protein Rop: a new tool for combinatorial experiments in biophysics; Magliery TJ et al.; Combinatorial methodologies have revolutionized studies in biomolecular function, but they have so far proven less useful for understanding macromolecular structure and stability . This is largely because of the difficulty of screening libraries of molecules for biophysical properties, and the difficulty of interpreting structural effects in complicated molecules . Here, we report a novel, robust, cell-based screen for function of the four-helix bundle protein, Rop . By expression of green fluorescent protein from a ColE1 plasmid, the screen reports the copy number of the plasmid, which is modulated in Escherichia coli by Rop . We have engineered the screen so that the fluorescent phenotype can correspond to either Rop activity or lack thereof . We have used the screen to demonstrate with systematically constructed Rop core variants that not all molecules that bind small stem-loop RNAs in vitro are active in vivo . Rop is well understood from structural work and systematic mutations, which makes it possible to construct rational, targeted libraries . This screen makes it possible to rapidly interrogate such libraries effectively for proper protein folding and stability . In addition to its intended utility for combinatorial experiments in biophysics, the screen will allow further dissection of the mechanism of Rop-mediated plasmid copy number regulation in vivo. Protein Eng Des Sel, 2004 Jan, 17(1), 67 - 75 Enzyme-like proteins from an unselected library of designed amino acid sequences; Wei Y et al.; Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins with interesting and important activities . However, since arbitrary sequences rarely fold into well ordered protein-like structures, randomly generated libraries will yield functional proteins only very rarely . To enhance the likelihood of finding functional de novo proteins, we use binary patterning of polar and non-polar amino acids to design focused libraries of sequences that are predisposed to fold into ordered structures . Proteins isolated from binary patterned libraries have been shown previously to fold into well ordered and native-like three-dimensional structures . To probe the potential of such libraries to also yield proteins with enzyme-like activity, we measured the esterase activity of S-824, a de novo binary patterned protein whose alpha-helical three-dimensional structure was reported recently . Protein S-824 displayed a rate enhancement (k(cat)/k(uncat)) of 8700 . The observed activity is similar to, or better than, that observed for several esterases designed previously using rational design or automated computational methods . Moreover, the observed activity rivals those of the first catalytic antibodies . To assess whether the activity of S-824 is representative of other proteins in binary patterned libraries, we measured the esterase activity of six additional proteins from two libraries . These libraries were 'naive' in that they were neither designed to bind substrate, nor subjected to high throughput screens for activity . All six of the additional proteins displayed esterase activity significantly above background . These findings demonstrate that novel proteins with enzyme-like properties are surprisingly common in focused libraries designed by binary patterning . Moreover, the activity of these unselected proteins provides a reference state for the levels of activity that have been obtained by selection and/or computational design. Protein Eng Des Sel, 2004 Jan, 17(1), 57 - 66 Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity? Trabbic-Carlson K, Meyer DE, Liu L, Piervincenzi R, Nath N, LaBean T, Chilkoti A. The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing . We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T(t)) . Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T(t) such that purification conditions (T(t)) may be predicted a priori for new recombinant proteins . We report here the effect that fusing six different proteins has on the T(t) of an ELP . A negative correlation between T(t) and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure . The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendamistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T(t) by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly. Protein Eng Des Sel, 2004 Jan, 17(1), 49 - 55 Directed enzyme evolution guided by multidimensional analysis of substrate-activity space; Larsson AK et al.; The directed evolution of protein function frequently involves identification of mutants with improved properties from a population of variants obtained by mutagenesis . The selection of clones to parent the subsequent generation is crucial to the continued creation of superior progeny . In the present study, multivariate analysis guided the evolution of human glutathione transferase (GST) T1-1 to 65-fold enhanced alkyltransferase activity . Six alternative substrates monitored the substrate-activity space that characterized a mutant library of enzymes, obtained by recombination of DNA and heterologous expression in Escherichia coli . A subset of mutants was identified by their proximity in the targeted region of six-dimensional factor space . DNA from these mutants was recombined to create a new generation of GST variants from which an improved enzyme was isolated . The multidimensional cluster analysis is applicable to quantitative properties in any population of molecules undergoing evolution and can guide the tailoring of proteins, nucleic acids and other chemical structures to novel and improved functions. J Biol Chem, 2004 May 14, 279(20), 21177 - 82 Epub 2004 Feb 25. Two motifs within a transmembrane domain, one for homodimerization and the other for heterodimerization; Gerber D et al.; Protein assembly is a critical process involved in a wide range of cellular events and occurs through extracellular and/or transmembrane domains (TMs) . Previous studies demonstrated that a GXXXG motif is crucial for homodimer formation . Here we selected the TMs of ErbB1 and ErbB2 as a model since these receptors function both as homodimers and as heterodimers . Both TMs contain two GXXXG-like motifs located at the C and N termini . The C-terminal motifs were implicated previously in homodimer formation, but the role of the N-terminal motifs was not clear . We used the ToxR system and expressed the TMs of both ErbB1 and ErbB2 containing only the N-terminal GXXXG motifs . The data revealed that the ErbB2 but not the ErbB1 construct formed homodimers . Importantly, a synthetic ErbB1 TM peptide was able to form a heterodimer with ErbB2, by displacing the ErbB2 TM homodimer . The specificity of the interaction was demonstrated by using three controls: (i) Two single mutations within the GXXXG-like motif of the ErbB1 peptide reduced or preserved its activity, in agreement with similar mutations in glycophorin A . (ii) A TM peptide of the bacterial Tar receptor did not assemble with the ErbB2 construct . (iii) The ErbB1 peptide had no effect on the dimerization of a construct containing the TM-1 domain of the Tar receptor . Fluorescence microscopy demonstrated that all the peptides localized on the membrane . Furthermore, incubation with the peptides had no effect on bacterial growth and protein expression levels . Our results suggest that the N-terminal GXXXG-like motif of the ErbB1 TM plays a role in heterodimerization with the ErbB2 transmembrane domain . To our knowledge, this is the first demonstration of a transmembrane domain with two distinct recognition motifs, one for homodimerization and the other for heterodimerization. Biochem Biophys Res Commun, 2004 Mar 19, 315(4), 1140 - 6 Prion proteins and ECTO-NOX proteins exhibit similar oscillating redox activities; Kim C et al.; Both recombinant full-length mouse prion protein expressed in Escherichia coli and native prion protein (PrPsc) from mouse brain exhibited NADH oxidase and protein disulfide-thiol interchange activities similar to those formerly thought to be properties exclusive to the growth-related, cell surface ECTO-NOX proteins . The two activities exhibited the complex 2+3 pattern of oscillations characteristic of ECTO-NOX proteins where the two activities alternate to generate a period length of 24 min . The oscillations were augmented by copper and diminished by addition of the copper chelator bathocuproene . That the activity might be attributable to a contaminating protein was ruled out by experiments where the purified recombinant prion-containing extracts were resolved by SDS-PAGE and the activity was restricted to a single band corresponding to the predicted Mr of the recombinant prion as verified by Western blot analyses. Biochem Biophys Res Commun, 2004 Mar 19, 315(4), 1039 - 44 Functional identification of the SecB homologue in Methanococcus jannaschii and direct interaction of SecB with trigger factor; Ha SC et al.; In this study Mj0357 protein, a hypothetical protein from Methanococcus jannaschii which shows an 18% sequence identity with SecB from E . coli, has been identified as a functional homologue of SecB in M . jannaschii through a number of biochemical and biophysical examinations . It is composed mostly of beta-strands and exists as a homotetramer in solution . Mj0357 protein exhibits in vitro chaperone-like activity, suppressing thermal aggregation of citrate synthase and binding to partially folded maltose-binding protein . Upon binding to a peptide ligand, the protein undergoes a conformational change to expose a hydrophobic patch on the protein surface . All these physicochemical properties are highly similar to those of E . coli SecB . In addition, E . coli trigger factor (TF) has been shown here for the first time to bind E . coli SecB and Mj0357 protein with low micromolar affinities, indicating that the TF could interact directly along the SecB-dependent translocation pathway . These results indicate that the translocation pathway is conserved and functionally homologous in at least one of the archaeal organisms. Biochem Biophys Res Commun, 2004 Mar 19, 315(4), 998 - 1003 Interaction of the CDK2-associated protein-1, p12(DOC-1/CDK2AP1), with its homolog, p14(DOC-1R); Buajeeb W et al.; Human DOC-1/CDK2AP1 gene encodes a growth suppressor protein of 12kDa (p12(DOC-1/CDK2AP1)) . Recently, p12(DOC-1/CDK2AP1) has been shown to associate with cell cycle proteins including CDK2 and DNA polymerase alpha/primase . It negatively regulates CDK2 activities and suppresses DNA replication . Therefore, identification of other p12(DOC-1/CDK2AP1) interacting proteins might clarify its role in the cell cycle regulation and carcinogenesis . The purpose of this study was to identify additional p12(DOC-1/CDK2AP1) interacting proteins using the yeast two-hybrid system . Using human p12(DOC-1/CDK2AP1) as a bait in a liver cDNA library screening, cDNA clones identical to human DOC-1R transcript were identified . The interaction between p12(DOC-1/CDK2AP1) and p14(DOC-1R) was verified in vitro and in cells . GST pull-down assay and immunoprecipitation experiments confirmed the interaction between the two proteins . The critical region for p12(DOC-1/CDK2AP1)'s interaction with p14(DOC-1R) was defined to amino acids 20-25 by using a series of deletion mutants as baits in the yeast two-hybrid system . Our data indicated that p12(DOC-1/CDK2AP1) could associate with its homologous protein, p14(DOC-1R). Biochem Biophys Res Commun, 2004 Mar 19, 315(4), 830 - 5 Effect of bismuth subgallate on nitric oxide and prostaglandin E2 production by macrophages; Lin CY et al.; Bismuth subgallate (BSG) is used widely in clinics, including Vincent's angina, syphilis, and adenotonsillectomy . This study examined the effects of BSG on nitric oxide (NO) and prostaglandin E2 (PGE2) production in activated RAW 264.7 cells . BSG suppressed production of NO and PGE2 in a dose-dependent manner . BSG could increase TGF-beta1 production, which in turn might promote degradation of iNOS mRNA, thus inhibiting NO production . Additionally, BSG inhibited mPGES protein expression and COX-2 activity in activated RAW 264.7 cells . Exogenous addition of SNP reversed the inhibition effect of PGE2 production by BSG . This behavior indicates that PGE2 inhibition by BSG exerts an indirect effect through NO inhibition. Biochem Biophys Res Commun, 2004 Mar 19, 315(4), 815 - 22 A novel microperoxidase activity: methyl viologen-linked nitrite reducing activity of microperoxidase; Suruga K et al.; To investigate the nitrite reducing activity of microperoxidases (mps) in the presence of methyl viologen and dithionite, the fragments C14-K22 (mp9), V11-L32 (mp22), and G1-M65 (mp65) containing heme were prepared by enzymatic hydrolysis of commercially equine heart cytochrome c (Cyt c), in which His is axially coordinated to heme iron, and acts as its fifth ligand . The nitrite reducing activity of mps was measured under anaerobic condition, and the nitrite reducing activity of mps increased with the cutting of the peptide chain . The activity of the shortest nonapeptide mp9 was approximately 120-fold that of Cyt c (104 amino acid residues) and 3.2-fold that of nitrite reductase (EC 1.7.7.1) from Escherichia coli . In the nitrite reduction by mp, nitrite was completely reduced to ammonia . We presumed that ferrous mps reduced NO2- to NO by donating one electron, the NO was completely reduced to NH4+ under anaerobic condition via ferrous-NO complexes as a reaction intermediate using visible spectra and ESR spectra, and this overall reaction was a 6-electron and 8-proton reduction . Sepharose-immobilized mp9 had a nitrite reducing activity similar to that of mp9 in solution, and the resin retained the activity after five uses and even 1-year storage . The mp will be able to use as a substitute for nitrite reductase. Biochem J, 2004 Jun 1, 380(Pt 2), 449 - 54 Determination of cellular nicotinic acid-adenine dinucleotide phosphate (NAADP) levels; Churamani D et al.; Nicotinic acid-adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger . In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously {Patel, Churchill and Galione (2000) Biochem . J . 352, 725-729} . We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor-ligand interaction . Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown . Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells. Int J Artif Organs, 2004 Jan, 27(1), 24 - 8 Super high flux hemodialysis at high dialysate flows: an ex vivo assessment; Lee WC et al.; BACKGROUND AND OBJECTIVES: The removal of cytokines by standard hemofiltration is limited . Super high flux membranes may significantly improve removal even when used in dialysis mode . We sought to measure cytokine clearance using a large surface super high-flux membrane and a standard hemodialysis setting . SETTING: ICU laboratory of a tertiary institution . SUBJECTS: Six healthy volunteers . METHODS: Blood form healthy volunteers was incubated for 4 hours with E . coli endotoxin to stimulate cytokine production . Cytokine containing blood was then circulated through a dialysis circuit at 3 different dialysate flow rates . Blood and dialysate were sampled for cytokine and albumin measurements and calculation of clearances . RESULTS: Super high-flux dialysis achieved high median cytokine clearances (IL-1 clearance of 106 ml/min, IL-6 clearance of 66.8 ml/min, IL-8 clearance of 61.7 ml/min and TNF clearance of 36.1 ml/min) . Increasing dialysate flow rate from 300 to 500 ml/min did not significantly increase cytokine clearances . Albumin clearances however were between 2.7 and 5.4 ml/min . CONCLUSIONS: Cytokine dialysis is feasible at high dialysate flow rates yielding high cytokine clearances . Albumin loss, however, is appreciable and may require separate supplementation in the clinical setting. Cell Stress Chaperones, 2003 Fall, 8(3), 272 - 86 Stress-induced extracellular Hsp72 is a functionally significant danger signal to the immune system; Campisi J et al.; Extracellular heat-shock proteins (eHsp) such as those belonging to the 70-kDa family of Hsp (eg, Hsp72) have been hypothesized to act as a "danger signal" to immune cells, promote immune responses, and improve host defense . The current study tested this hypothesis . Adult male F344 rats were exposed to an acute laboratory stressor (100, 5-second, 1.6-mA inescapable tail shocks) and challenged with Escherichia coli . The number of colony-forming units (CFU) of bacteria at the site of injection, the levels of eHsp72, the immune response to eHsp72 and E . coli-derived lipopolysaccharide (LPS), and the amount of time required to recover from in vivo bacterial challenge were measured . CFUs were reduced 2, 4, and 6 hours after injection of E . coli in rats exposed to stress . Rats exposed to stress had elevated eHsp72 that was elevated rapidly (25 minutes) and remained elevated in the circulation and at the inflammatory site (2 hours after stressor termination) . Both stressor exposure and eHsp72 administration in the absence of stress resulted in a facilitated pattern of recovery after bacterial inflammation induced by subcutaneous E . coli injection . Rats exposed to acute restraint (100 minutes) did not demonstrate elevated circulating eHsp72 or a facilitated pattern of recovery after bacterial challenge . In vitro stimulation of rat splenocytes and macrophages with eHsp72 elevated nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, and this effect was specific to eHsp72 because it was not diminished by polymyxin B and was reduced by earlier heat-denature treatment . Stimulation of cells with eHsp72 combined with LPS resulted in a greater NO and cytokine response than that observed after stimulation with eHsp72 or LPS alone . In vivo, at the inflammatory site, the bacterial-induced NO response was potentiated by stress, and NO inhibition (L-NIO) reduced the stress-induced facilitation but had no effect on the control kinetics of bacterial inflammation recovery . Thus, these results lend support to the hypothesis that intense stressor exposure increases eHsp72, which acts as a danger signal to potentiate the NO response to bacterial challenge and facilitate recovery from bacterial inflammation. Cell Stress Chaperones, 2003 Fall, 8(3), 218 - 24 GrpE, a nucleotide exchange factor for DnaK; Harrison C; The cochaperone GrpE functions as a nucleotide exchange factor to promote dissociation of adenosine 5'-diphosphate (ADP) from the nucleotide-binding cleft of DnaK . GrpE and the DnaJ cochaperone act in concert to control the flux of unfolded polypeptides into and out of the substrate-binding domain of DnaK by regulating the nucleotide-bound state of DnaK . DnaJ stimulates nucleotide hydrolysis, and GrpE promotes the exchange of ADP for adenosine triphosphate (ATP) and also augments peptide release from the DnaK substrate-binding domain in an ATP-independent manner . The eukaryotic cytosol does not contain GrpE per se because GrpE-like function is provided by the BAG1 protein, which acts as a nucleotide exchange factor for cytosolic Hsp70s . GrpE, which plays a prominent role in mitochondria, chloroplasts, and bacterial cytoplasms, is a fascinating molecule with an unusual quaternary structure . The long alpha-helices of GrpE have been hypothesized to act as a thermosensor and to be involved in the decrease in GrpE-dependent nucleotide exchange that is observed in vitro at temperatures relevant to heat shock . This review describes the molecular biology of GrpE and focuses on the structural and kinetic aspects of nucleotide exchange, peptide release, and the thermosensor hypothesis. Cell Mol Biol (Noisy-le-grand), 2003 Dec, 49(8), 1261 - 7 The chemorepellent semaphorin is expressed in the horseshoe crab, Limulus polyphemus; Cao Z et al.; Semaphorins are a family of soluble and membrane-bound proteins that play a critical role in axonal guidance and other processes of neuronal development . Currently, more than twenty semaphorins have been identified, all of which share a conserved 500 amino acid domain near the amino terminus . Semaphorins are divided into eight classes according to species of origin and structural similarities . Classes 1 and 2 are found in invertebrates, classes 3 through 7 are present in vertebrates and viruses encode class V semaphorin . Microarray analysis of Limulus CNS RNA revealed the presence of a semaphorin-like gene in Limulus polyphemus . Based on these data, we aligned 31 different sequences and designed degenerate primers for the consensus domains (WTT/SFLKA) and (DPY/VCA/GW) . RT-PCR products were generated using 6 forward primers and 4 reverse primers . The expected size PCR products (750 bp) was obtained and then ligated with pCR II TOPO vector and transferred into E . coli Top 10 . Five partial semaphorin cDNAs were found in Limulus: semaphorins 1a, 1b, 2a, 2b and F (now known as 5) were partially cloned . Subsequent Northern blot analyses using these Limulus specific-probes revealed hybridization with total RNAs purified from six different tissues. Dtsch Tierarztl Wochenschr, 2004 Jan, 111(1), 28 - 35 Effect of endotoxins on contractility of smooth muscle preparations from the bovine abomasal antrum; Kaze C et al.; The effects of endotoxin (ET) on spontaneous contractility and of carbachol- and alpha-methyl-5-hydroxytryptamine-(alpha-M-5-HT; 5-HT2 receptor agonist) induced contractions of smooth muscle preparations from the bovine abomasal antrum were investigated in vitro . Preparations from the abomasal antrum of freshly slaughtered healthy dairy cows were cut parallel to the longitudinal and circular fibres, suspended in isolated organ baths, and contractility was recorded and analyzed, using digitalized data . The traits maximum amplitude, time till maximum amplitude, frequency, basal tone, and area under curve were calculated . The contractile effect of Carbachol (CH) was concentration dependent . Repeated administration of CH (3.75 x 10(-6) M), each time interrupted by flushing of the organ baths, did not reveal any significant effect on contractility traits of CH-induced contractions . Endotoxin (10 micrograms/ml; lipopolysaccharide from E . coli, O26:B6) significantly reduced some of the spontaneously occurring contractility traits and of carbachol-(3.75 x 10(-6) M) and alpha-M-5-HT-induced (2.14 x 10(-5) M) contractions . The effects of higher and lower concentrations of ET occurred less consistently . The inhibitory effect of endotoxin was more pronounced after 6 hours as compared to 2 hours of incubation . The results of the present study (i) support the hypothesis of a possible role of endotoxin in reducing motility of the abomasum during the development of spontaneous abomasal displacement in dairy cows, and (ii) may serve as the basis for the development of an in vitro model of abomasal displacement with endotoxemia for future studies on the effect of motility modulating drugs. Protein Eng, 2003 Dec, 16(12), 875 - 9 A backbone-reversed all-beta polypeptide (retro-CspA) folds and assembles into amyloid nanofibres; Shukla A et al.; The backbone-reversed or 'retro', form of a model all-beta-sheet protein, Escherichia coli CspA, was produced from a synthetic gene in E.coli in fusion with an N-terminal affinity tag . Following purification under denaturing conditions and dialysis-based removal of urea, the protein was found to fold into a soluble, poorly structured multimer . Upon concentration, this state readily transformed into amyloid nanofibres . Congo Red-binding amorphous forms were also observed . Since a beta-sheet-forming sequence is expected to retain high beta-sheet-forming propensity even after backbone reversal and given the fact that folding of retro-CspA occurs only to a poorly structured form, we conclude that the increase effected in protein concentration may be responsible for the formation of intermolecular beta-sheets, facilitating the bleeding away of the protein's conformational equilibrium into aggregates that generate well-formed fibres . Since every molecule in these fibres contains a peptide tag for binding Ni(2+), the fibres may provide a template for deposition of nickel to generate novel materials. Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2299 - 304 Design of artificial cell-cell communication using gene and metabolic networks; Bulter T et al.; Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria . Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication . To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable . Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell-cell communication . This circuit uses a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogen-regulation two-component system . As one application of the cell-cell communication circuit we created an artificial quorum sensor . Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability . In these cells the circuit induced gene expression in response to a threshold cell density . This threshold can be tuned effectively by controlling DeltapH over the cell membrane, which determines the partition of acetate between medium and cells . Mutagenesis of the enhancer sequence of the glnAp2 promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components . The behavior of the circuit shows remarkable predictability based on a mathematical design model. Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2275 - 80 Heterodimeric interactions among the 1-amino-cyclopropane-1-carboxylate synthase polypeptides encoded by the Arabidopsis gene family; Tsuchisaka A et al.; The pyridoxal phosphate-dependent enzyme, 1-aminocyclopropane-1-carboxylate synthase (ACS; EC 4.4.1.14), catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants . The Arabidopsis genome encodes nine ACS polypeptides that form eight functional (ACS2, ACS4-9, ACS11) and one nonfunctional (ACS1) homodimers . Because the enzyme is a homodimer with shared active sites, the question arises whether the various polypeptides can form functional heterodimers . Intermolecular complementation experiments in Escherichia coli by coexpressing the K278A and Y92A mutants of different polypeptides show that all of them have the capacity to heterodimerize . However, functional heterodimers are formed only among gene family members that belong to one or the other of the two phylogenetic branches . ACS7 is an exception to this rule, which forms functional heterodimers with some members of both branches when it provides the wt K278 residue . ACS1, the nonfunctional polypeptide as a homodimer, can also form functional heterodimers with members of its phylogenetic branch when its partners provide the wt K278 residue . The ACS gene family products can potentially form 45 homo- and heterodimers of which 25 are functional . Bimolecular fluorescence complementation and biochemical coaffinity purification assays show that the inactivity of certain heterodimers is not due to the absence of heterodimerization but rather to structural restraint(s) that prevents the shared active sites from being functional . We propose that functional heterodimerization enhances the isozyme diversity of the ACS gene family and provides physiological versatility by being able to operate in a broad gradient of S-adenosylmethionine concentration in various cells/tissues during plant growth and development . Nonfunctional heterodimerization may also play a regulatory role during the plant life cycle. Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2259 - 64 Acetylation of p53 augments its site-specific DNA binding both in vitro and in vivo; Luo J et al.; p53 promotes tumor suppression through its ability to function as a transcriptional factor and is activated by posttranslational modifications that include acetylation . Our earlier study demonstrated that p53 acetylation can enhance its sequence-specific DNA binding in vitro, and this notion was later confirmed in several other studies . However, a recent study has reported that in vitro acetylation of p53 fails to stimulate its DNA binding to large DNA fragments, raising an important issue that requires further investigation . Here, we show that unacetylated p53 is able to bind weakly to its consensus site within the context of large DNA fragments, although it completely fails to bind the same site within short oligonucleotide probes . Strikingly, by using highly purified and fully acetylated p53 proteins obtained from cells, we show that acetylation of the C-terminal domain can dramatically enhance site-specific DNA binding on both short oligonucleotide probes and long DNA fragments . Moreover, endogenous p53 apparently can be fully acetylated in response to DNA damage when both histone deacetylase complex 1 (HDAC1)- and Sir2-mediated deacetylation are inhibited, indicating dynamic p53 acetylation and deacetylation events during the DNA damage response . Finally, we also show that acetylation of endogenous p53 indeed significantly augments its ability to bind an endogenous target gene and that p53 acetylation levels correlate well with p53-mediated transcriptional activation in vivo . Thus, our results clarify some of the confusion surrounding acetylation-mediated effects on p53 binding to DNA and suggest that acetylation of p53 in vivo may contribute, at least in part, to its transcriptional activation functions. Ann Pharmacother, 2004 Apr, 38(4), 602 - 5 Epub 2004 Feb 24. Possible gatifloxacin-induced hyperglycemia; Donaldson AR et al.; OBJECTIVE: To report a case of possible gatifloxacin-induced hyperglycemia in a nondiabetic middle-aged woman . CASE SUMMARY: A 64-year-old Indian woman with an extensive cardiovascular history was admitted for urosepsis . On admission, her blood glucose was 117 mg/dL . She was empirically started on gatifloxacin 400 mg/day; after 3 days of gatifloxacin therapy, her blood glucose was 607 mg/dL . On day 4, therapy was changed to cefazolin for sensitive Escherichia coli and her blood glucose levels began to return to normal . DISCUSSION: Although gatifloxacin has been previously reported as a potential cause of both hyper- and hypoglycemia, the exact mechanism is unknown . Several factors that may have been involved in our patient's hyperglycemia are discussed . She experienced hyperglycemic changes more rapidly than did the typical patients of previous reports . The Naranjo probability scale suggests a possible drug-related event . CONCLUSIONS: The temporal relationship between gatifloxacin administration and the patient's hyperglycemia suggests an iatrogenic cause . Based on our experience and the product labeling, clinicians should be more aware of the blood glucose-altering effects of gatifloxacin. J Leukoc Biol, 2004 Jun, 75(6), 982 - 94 Epub 2004 Feb 24. Defective macrophage function in neonates and its impact on unresponsiveness of neonates to polysaccharide antigens; Chelvarajan RL et al.; Neonates do not respond to thymus-independent (TI) antigens (Ag), making them vulnerable to infection with encapsulated bacteria . The antibody (Ab) response of adult and neonatal B cells to TI Ag requires certain cytokines, which are provided by T cells or macrophages (MPhi) . Lipopolysaccharide (LPS) failed to induce neonatal MPhi to produce interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha) mRNA and to secrete IL-1beta, IL-12, and TNF-alpha . However, LPS induced neonates to secrete some IL-6 and three- to fivefold more IL-10 than adults . Accordingly, adding adult but not neonatal MPhi could restore the response of purified adult B cells to trinitrophenol (TNP)-LPS, a TI Ag . Increased IL-10 is causally related to decreased IL-1beta and IL-6 production, as IL-10(-/-) neonatal MPhi responded to LPS by secreting more IL-1beta and IL-6 than wild-type (WT) neonatal MPhi . When cultures were supplemented with a neutralizing Ab to IL-10, WT neonatal MPhi secreted increased amounts of IL-6 and allowed neonatal MPhi to promote adult B cells to mount an Ab response against TNP-LPS . Thus, neonates do not respond to TI Ag as a result of the inability of neonatal MPhi to secrete cytokines, such as IL-1beta and IL-6, probably as a result of an excess production of IL-10 . This dysregulated cytokine secretion by neonatal MPhi may be a result of a reduction in expression of Toll-like receptor-2 (TLR-2) and TLR-4 and CD14. Antimicrob Agents Chemother, 2004 Mar, 48(3), 1017 - 20 Quantitative analysis of a parasitic antiviral strategy; Kim H et al.; We extended a computer simulation of viral intracellular growth to study a parasitic antiviral strategy that diverts the viral replicase toward parasite growth . This strategy inhibited virus growth over a wide range of conditions, while minimizing host cell perturbations . Such parasitic strategies may inhibit the development of drug-resistant virus strains. Antimicrob Agents Chemother, 2004 Mar, 48(3), 897 - 902 Fluorescence detection-based functional assay for high-throughput screening for MraY; Stachyra T et al.; We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan . This was accomplished by using UDP-MurNAc-N(epsilon)-dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain . Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium . The latter assay was validated with a set of natural and synthetic MraY inhibitors. Mol Microbiol, 2004 Mar, 51(5), 1471 - 81 Translocation of Escherichia coli RNA polymerase against a protein roadblock in vivo highlights a passive sliding mechanism for transcript elongation; Mosrin-Huaman C et al.; Current models for transcription elongation infer that RNA polymerase (RNAP) moves along the template by a passive sliding mechanism that takes advantage of random lateral oscillations in which single basepair sliding movements interconvert the elongation complex between pre- and post-translocated states . Such passive translocational equilibrium was tested in vivo by a systematic change in the templated NTP that is to be incorporated by RNAP, which is temporarily roadblocked by the lac repressor . Our results show that, under these conditions that hinder the forward movement of the polymerase, the elongation complex is able to extend its RNA chain one nucleotide further when the incoming NTP is a kinetically favoured substrate (i.e . low K(m)) . The addition of an extra nucleotide destabilizes the repressor-operator roadblock leading to an increase in transcriptional readthrough . Similar results are obtained when the incoming NTPs are less kinetically favoured substrates (i.e . high K(m)s) by specifically increasing their intracellular concentrations . Altogether, these in vivo data are consistent with a passive sliding model in which RNAP forward translocation is favoured by NTP binding . They also suggest that fluctuations in the intracellular NTP pools may play a key role in gene regulation at the transcript elongation level. Mol Microbiol, 2004 Mar, 51(5), 1439 - 46 A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles; Tjaden J et al.; The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles . Hydrogenosomes, i.e . ATP- and hydrogen-generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria . Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial-type AACs . Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis . Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs . However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial-type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far . Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently. Mol Microbiol, 2004 Mar, 51(5), 1401 - 12 Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli; Soksawatmaekhin W et al.; The functions of the putative cadaverine transport protein CadB were studied in Escherichia coli . CadB had both cadaverine uptake activity, dependent on proton motive force, and cadaverine excretion activity, acting as a cadaverine-lysine antiporter . The Km values for uptake and excretion of cadaverine were 20.8 and 303 microM respectively . Both cadaverine uptake and cadaverine-lysine antiporter activities of CadB were functional in cells . Cell growth of a polyamine-requiring mutant was stimulated slightly at neutral pH by the cadaverine uptake activity and greatly at acidic pH by the cadaverine-lysine antiporter activity . At acidic pH, the operon containing cadB and cadA, encoding lysine decarboxylase, was induced in the presence of lysine . This caused neutralization of the extracellular medium and made possible the production of CO(2) and cadaverine and aminopropylcadaverine instead of putrescine and spermidine . The induction of the cadBA operon also generated a proton motive force . When the cadBA operon was not induced, the expression of the speF-potE operon, encoding inducible ornithine decarboxylase and a putrescine-ornithine antiporter, was increased . The results indicate that the cadBA operon plays important roles in cellular regulation at acidic pH. Mol Microbiol, 2004 Mar, 51(5), 1347 - 59 Escherichia coli prereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaA; Ryan VT et al.; Initiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre-RC) that unwind the Escherichia coli chromosome's origin of replication, oriC . Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E . coli . Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiled oriC templates . Low levels of Fis inhibited oriC unwinding by blocking both IHF and DnaA binding to low affinity sites . As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites on oriC . This behaviour in vitro is analogous to observed assembly of pre-RC in synchronized E . coli . We propose that as new DnaA is synthesized in E . coli, opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony. Mol Microbiol, 2004 Mar, 51(5), 1311 - 20 Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with the C-terminal domain of the RNA polymerase alpha subunit; Grainger DC et al.; We have investigated the role of the RNA polymerase alpha subunit during MelR-dependent activation of transcription at the Escherichia coli melAB promoter . To do this, we used a simplified melAB promoter derivative that is dependent on MelR binding at two 18 bp sites, located from position -34 to -51 and from position -54 to -71, upstream of the transcription start site . Results from experiments with hydroxyl radical footprinting, and with RNA polymerase, carrying alpha subunits that were tagged with a chemical nuclease, show that the C-terminal domains of the RNA polymerase alpha subunits are located near position -52 and near position -72 during transcription activation . We demonstrate that the C-terminal domain of the RNA polymerase alpha subunit is needed for open complex formation, and we describe two experiments showing that the RNA polymerase alpha subunit can interact with MelR . Finally, we used alanine scanning to identify determinants in the C-terminal domain of the RNA polymerase alpha subunit that are important for MelR-dependent activation of the melAB promoter. Mol Microbiol, 2004 Mar, 51(5), 1297 - 309 Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase sigma subunit; Grainger DC et al.; Activation of transcription initiation at the Escherichia coli melAB promoter is dependent on MelR, a transcription factor belonging to the AraC family . MelR binds to 18 bp target sites using two helix-turn-helix (HTH) motifs that are both located in its C-terminal domain . The melAB promoter contains four target sites for MelR . Previously, we showed that occupation of two of these sites, centred at positions -42.5 and -62.5 upstream of the melAB transcription start point, is sufficient for activation . We showed that MelR binds as a direct repeat to these sites, and we proposed a model to describe how the two HTH motifs are positioned . Here, we have used suppression genetics to confirm this model and to show that MelR residue 273, which is in HTH 2, interacts with basepair 13 of each target site . As our model for DNA-bound MelR suggests that HTH 2 must be adjacent to the melAB promoter -35 element, we searched this part of MelR for amino acid side-chains that might be able to interact with sigma . We describe genetic evidence to show that MelR residue 261 is close to residues 596 and 599 of the RNA polymerase sigma(70) subunit, and that they can interact . Similarly, MelR residue 265 is shown to be able to interact with residue 596 of sigma(70) . In the final part of the work, we describe experiments in which the MelR binding site at position -42.5 was improved . We show that this is detrimental to MelR-dependent transcription activation because bound MelR is mispositioned so that it is unable to make 'correct' interactions with sigma. Mol Microbiol, 2004 Mar, 51(5), 1279 - 95 Chromosomal fragmentation in dUTPase-deficient mutants of Escherichia coli and its recombinational repair; Kouzminova EA et al.; Recent findings suggest that DNA nicks stimulate homologous recombination by being converted into double-strand breaks, which are mended by RecA-catalysed recombinational repair and are lethal if not repaired . Hyper-rec mutants, in which DNA nicks become detectable, are synthetic-lethal with recA inactivation, substantiating the idea . Escherichia coli dut mutants are the only known hyper-recs in which presumed nicks in DNA do not cause inviability with recA, suggesting that nicks stimulate homologous recombination directly . Here, we show that dut recA mutants are synthetic-lethal; specifically, dut mutants depend on the RecBC-RuvABC recombinational repair pathway that mends double-strand DNA breaks . Although induced for SOS, dut mutants are not rescued by full SOS induction if RecA is not available, suggesting that recombinational rather than regulatory functions of RecA are needed for their viability . We also detected chromosomal fragmentation in dut rec mutants, indicating double-strand DNA breaks . Both the synthetic lethality and chromosomal fragmentation of dut rec mutants are suppressed by preventing uracil excision via inactivation of uracil DNA-glycosylase or by preventing dUTP production via inactivation of dCTP deaminase . We suggest that nicks become substrates for recombinational repair after being converted into double-strand DNA breaks. Mol Microbiol, 2004 Mar, 51(5), 1267 - 77 Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress; Park SM et al.; We examined the biological function of cpmk1, which encodes a MAPK of Cryphonectria parasitica, and its regulation by mycovirus . Sequence comparisons revealed that cpmk1 had highest homology with osm1, a hog1-homologue from Magnaporthe grisea . A growth defect was observed in the cpmk1-null mutant under hyperosmotic conditions, indicating that cpmk1 functionally belongs to a hog1 subfamily . Immunoblot analyses indicated that the CpMK1 pathway was affected specifically in hyperosmotic conditions by the hypovirus CHV1-EP713 . Moreover, the virus-infected hypovirulent UEP1 strain also exhibited severe osmosensitivity compared to the virus-free isogenic strain EP155/2, thus providing additional evidence for viral regulation of cpmk1 in response to a hypertonic stress . Besides osmosensitivity, disruption of cpmk1 resulted in several, but not all, hypovirulence-associated changes, such as reduced pigmentation, conidiation, laccase production and cryparin expression . However, the cpmk1-null mutant exhibited an increased accumulation of pheromone gene transcripts . Virulence assays of the cpmk1-null mutant revealed reduced canker area, but not as severe as that of UEP1 . These results suggest that mycoviruses modulate the MAPK and thereby provoke the aberrant expression of target genes, some of which are likely to be implicated in viral symptom development. Acta Anaesthesiol Scand, 2004 Mar, 48(3), 317 - 21 Ketamine suppresses endotoxin-induced NF-kappaB activation and cytokines production in the intestine; Sun J et al.; BACKGROUND: Ketamine has been advocated for anesthesia in endotoxemic and other severely ill patients because it is a cardiovascular stimulant . However, ketamine also suppresses serum levels of endotoxin-induced tumor necrosis factor-alpha, and reduces mortality in mice in endotoxin shock . Our study was designed to investigate the protective effect of ketamine on the endotoxin-induced proinflammatory cytokines and nuclear factor kappa B (NF-kappaB) activation in vivo . METHODS: Adult male Wistar rats were randomly divided into six groups: saline controls; rats challenged with endotoxin (5 mg kg(-1)) and treated with saline; challenged with endotoxin (5 mg kg(-1)) and treated with ketamine (0.5 mg kg(-1)); challenged with endotoxin (5 mg kg(-1)) and treated with ketamine (5 mg kg(-1)); challenged with endotoxin (5 mg kg(-1)) and treated with ketamine (50 mg kg(-1)); and saline injected and treated with ketamine (50 mg kg(-1)) . TNF-alpha, IL-6 and NF-kappaB were investigated in the tissues of the intestine (jejunum) after 1, 4 and 6 h . RESULTS: Endotoxin caused transient production of TNF-alpha and IL-6 and activation of NF-kappaB in the intestine at peak times of 1, 4 and 1 h, respectively . Ketamine 0.5 mg kg(-1) suppressed endotoxin-induced TNF-alpha elevation and inhibited NF-kappaB activation in the intestine; a dose of 5 mg kg(-1) was required to inhibit IL-6 . CONCLUSION: Ketamine suppresses the production of proinflammatory cytokines such as TNF-alpha and IL-6 in the intestine, possibly via inhibition of NF-kappaB. Acta Anaesthesiol Scand, 2004 Mar, 48(3), 308 - 16 Renal transcription of high-affinity type-2 cationic amino acid transporter is up-regulated in LPS-stimulated rodents; Yang S et al.; OBJECTIVE: Sepsis stimulates renal nitric oxide (NO) biosynthesis through up-regulation of inducible NO synthase (iNOS) expression . Type-2 cationic amino acid transporter (CAT-2) mediation of trans-membrane L-arginine (L-Arg) transportation has been identified as one of the crucial regulatory mechanisms involved in the formation of NO by iNOS . We had previously shown that CAT-2B, a high-affinity alternative-spliced transcript of the CAT-2, is involved in induced NO biosynthesis by iNOS (Nitric Oxide, 2002) . In this present study, we sought to assess the effects of sepsis on the expression of CAT-2B in lipopolysaccharide (LPS)-stimulated rat kidney . METHODS: Forty rats were randomized to either a normal saline (N/S)-treated group or a LPS-treated group . Renal NO production was determined using chemiluminescence . Semi-quantitative RT-PCR was used to determine the mRNA concentrations of iNOS and L-Arg transporters (CAT-1, CAT-2 and CAT-2B) in kidney . RESULTS: Lipopolysaccharide-coinduced iNOS, CAT-2 and CAT-2B mRNA expression in kidney and caused renal NO overproduction . A significant linear regression relationship was defined between renal NO concentrations and iNOS, CAT-2 and CAT-2B, respectively . On the contrary, CAT-1 expression was not affected by LPS-stimulation . CONCLUSIONS: We provide the first evidence to illustrate that sepsis/septic shock induces the transcription of high-affinity CAT-2B in renal tissues . Transcription of iNOS, CAT-2 and CAT-2B correlates well with renal NO biosynthesis . Regulation of L-Arg uptake by modulating the expression regulation of induced CAT-2 and CAT-2B might be a potential target for therapies against renal pathologic conditions related to NO overproduction. Sichuan Da Xue Xue Bao Yi Xue Ban, 2004 Jan, 35(1), 8 - 10 {Construction of eukaryotic expression clone for human amelogenin}; Zhang JC et al.; OBJECTIVE: To construct-the eukaryotic expression clone for human amelogenin . METHODS: Total RNA was isolated from human fetal tooth buds . RT-PCR was used to amplify the amelogenin encoding region, and the amplified fragment for human amelogenin was inserted into eukaryotic expression vector PcDNA 3.1 . The positive clones were selected and analyzed by restriction endonuclease mapping and DNA sequencing . RESULTS: 570 bp fragment was produced by RT-PCR; it was of the same size as expected based on human ameloginin mRNA encoding area length . The sequence of the inserted fragment from the recombinant clone PcDNA 3.1-AMG was consistent with that of AMELX from GenBank with one mismatch on 485 from G to C, without affecting the amino acid sequence . CONCLUSION: The eukaryotic expression clone PcDNA 3.1-AMG was successfully constructed with the properly inserted DNA sequence encoding mature human amelogenin. J Med Virol, 2004 Apr, 72(4), 679 - 82 Antibody response to B19 parvovirus VP1 and VP2 linear epitopes in patients with haemophilic arthritis; Azzi A et al.; Parvovirus B19 infection occurs very frequently in patients with haemophilia on account of its transmission with plasma derivatives . In order to achieve a more defined serological pattern for the study of the role of B19 infection in haemophilic arthritis, 53 serum samples from 37 patients with haemophilic arthritis were investigated for the presence of IgG immune response against B19 VP2 and VP1 linear epitopes and VP2 conformational antigen compared to the serological reactivity against B19 NS1 and to the presence of B19 DNA in the synovial membranes . An IgG immune response against VP1 and VP2 linear epitopes was detected by immunoblot assay using recombinant proteins expressed in Escherichia coli . Specific IgG against VP2 and VP1 linear epitopes were present in 84.90 and 92.45% of haemophilic arthritis patients and in 28.0 and 64.0% of the controls (P<0.001) respectively . All 53 sera of the haemophiliacs (100%) and 66.0% of the controls (P<0.001) were IgG positive and IgM negative against VP2 structural epitopes . Specific IgG against VP2 linear epitopes, which are a serological marker of active or very recent B19 infection, proved to be significantly associated with the presence of anti-NS1 antibodies and with the presence of B19 DNA in synovial tissue in patients with haemophilic arthritis . In conclusion, in these patients the presence of B19 IgG anti-VP2 linear epitopes, in absence of IgM anti-VP2 structural antigens, can be a useful serological marker to diagnose active, recent or persistent B19 infection . Electrophoresis, 2004 Feb, 25(4-5), 692 - 6 Separation of capsular polysaccharide K4 and defructosylated K4 by high-performance capillary electrophoresis; Volpi N; A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product . The two polyanions, K4 and defructosylated K4, are separated and readily determined within 30 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm . A linear relationship was found for the two polysaccharides over a wide range of concentrations, from approximately 30 ng (0.5 microg/microL) to 210 ng (3.5 microg/microL) . The described method was used to evaluate the defructosylation process of K4 under drastic acid conditions. Biosci Biotechnol Biochem, 2004 Feb, 68(2), 458 - 61 Transcriptional analysis of the ostA/imp gene involved in organic solvent sensitivity in Escherichia coli; Ohtsu I et al.; Transcriptional initiation sites of the ostA gene involved in organic solvent sensitivity in Escherichia coli were found by primer extension analysis . Two transcriptional initiation sites were newly identified at -133 and -48 nucleotides from the initiation codon of ostA, but the previously reported sigmaE-dependent one at -227 could not be detected . No heat-inducible expression of ostA was observed by Northern blotting analysis, indicating that the contribution of sigmaE-dependent transcription was very small if any . SigmaD-dependent promoter-like sequences were found just upstream of the newly identified transcriptional initiation sites by computer-aided analysis . Deletion analysis of ostA-lacZ fusions demonstrated that these two promoters contributed almost equally to the constitutive expression of the ostA gene. Biosci Biotechnol Biochem, 2004 Feb, 68(2), 341 - 51 Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp . J-13-3; Okazaki K et al.; The gene encoding chitinase from Streptomyces sp . (strain J-13-3) was cloned and its nucleotide structure was analyzed . The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da . The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer . Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids) . The J-13-3 strain had a single chitinase gene . The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells . The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E . coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein . The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively . The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei. Biosci Biotechnol Biochem, 2004 Feb, 68(2), 324 - 32 Molecular cloning, functional expression, and mutagenesis of cDNA encoding class I chitinase from rye (Secale cereale) seeds; Ohnuma T et al.; A cDNA encoding rye seed chitinase-a (RSC-a) was cloned by rapid amplification of cDNA ends and PCR procedures . It consists of 1,191 nucleotides and encodes an open reading frame of 321 amino acid residues . Recombinant RSC-a (rRSC-a) was produced in the oxidative cytoplasm of Escherichia coli Origami(DE3) in a soluble form by inducing bacteria at a low temperature (20 degrees C) . Purified rRSC-a showed properties similar to the original enzyme from rye seeds in terms of chitinase activity toward a soluble substrate, glycolchitin, and an insoluble substrate, chitin beads, in chitin-binding ability to chitin, and in antifungal activity against Trichoderma sp . in vitro . rRSC-a mutants were subsequently produced and purified by the same procedures as those for rRSC-a . Mutation of Trp23 to Ala decreased the chitinase activity toward both substrates and impaired the chitin-binding ability . Furthermore, the antifungal activity of this mutant was weakened with increasing of the NaCl concentration in the culture medium . Complete abolishment of both activities was observed upon the mutation of Glu126 to Gln . The roles of these residues in both activities are discussed. Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 2758 - 63 Epub 2004 Feb 23. Global analysis of Escherichia coli RNA degradosome function using DNA microarrays; Bernstein JA et al.; RNase E, an essential endoribonuclease of Escherichia coli, interacts through its C-terminal region with multiple other proteins to form a complex termed the RNA degradosome . To investigate the degradosome's proposed role as an RNA decay machine, we used DNA microarrays to globally assess alterations in the steady-state abundance and decay of 4,289 E . coli mRNAs at single-gene resolution in bacteria carrying mutations in the degradosome constituents RNase E, polynucleotide phosphorylase, RhlB helicase, and enolase . Our results show that the functions of all four of these proteins are necessary for normal mRNA turnover . We identified specific transcripts and functionally distinguishable transcript classes whose half-life and abundance were affected congruently by multiple degradosome proteins, affected differentially by mutations in degradosome constituents, or not detectably altered by degradosome mutations . Our results, which argue that decay of some E . coli mRNAs in vivo depends on the action of assembled degradosomes, whereas others are acted on by degradosome proteins functioning independently of the complex, imply the existence of structural features or biochemical factors that target specific classes of mRNAs for decay by degradosomes. Mutagenesis, 2004 Mar, 19(2), 137 - 41 Effects of the order of exposure to a binary mixture of mutagens on the induced mutation spectra in the supF gene; McLuckie KI et al.; We have shown previously that UVC irradiation of benzo{a}pyrene diol epoxide (BPDE)-adducted DNA (BPDE/UVC) induces an increase in mutation frequency in the supF gene greater than the calculated additive value derived from either treatment alone, with a greater absolute increase in the level of BPDE signature transversions . Possible explanations were that (i) the BPDE adducts are photoactivated to a more mutagenic lesion or (ii) the presence of UV-induced DNA damage enhanced the mutagenicity of BPDE adducts elsewhere on the DNA . In the present study, to determine which of these mechanisms is responsible for the enhanced mutagenicity of the combined treatment, plasmid pSP189 containing supF was treated with UVC radiation before BPDE treatment (UVC/BPDE) . If BPDE adducts were being modified by UV irradiation to more mutagenic species, then reversing the order of exposure would be predicted to lower the mutation frequency and the number of transversions . Conversely, if merely the presence of UV damage influences the mutagenicity of BPDE adducts (or vice versa), the observed mutagenicity should be independent of the order of exposure . Previously, treatment with BPDE/UVC increased the mutation frequency by >400% over the calculated additive value derived from the individual BPDE and UV exposures . In the present study, treatment with UVC followed by BPDE increased the mutation frequency by only approximately 60%, compared with the corresponding calculated additive value . However, whilst this shows that the order of treatment affects the mutation frequency, there was little change in the percentage of base substitutions in the two spectra . Hence, whilst the change in mutation frequency is consistent with UVC directly enhancing the mutagenicity of the BPDE adducts, the similarity in the types of mutations induced by BPDE/UVC and UVC/BPDE suggests that the mechanism may not be that simple. J Biol Chem, 2004 May 7, 279(19), 19977 - 86 Epub 2004 Feb 23. Probing the active site loop motif of murine ferrochelatase by random mutagenesis; Shi Z et al.; Ferrochelatase catalyzes the terminal step of the heme biosynthetic pathway by inserting ferrous iron into protoporphyrin IX . A conserved loop motif was shown to form part of the active site and contact the bound porphyrin by molecular dynamics calculations and structural analysis . We applied a random mutagenesis approach and steady-state kinetic analysis to assess the role of the loop motif in murine ferrochelatase function, particularly with respect to porphyrin interaction . Functional substitutions in the 10 consecutive loop positions Gln(248)-Leu(257) were identified by genetic complementation in Escherichia coli strain Deltavis . Lys(250), Val(251), Pro(253), Val(254), and Pro(255) tolerated a variety of replacements including single substitutions and contained low informational content . Gln(248), Ser(249), Gly(252), Trp(256), and Leu(257) possessed high informational content, since permissible replacements were limited and only observed in multiply substituted mutants . Selected active loop variants exhibited k(cat) values comparable with or higher than that of wild-type murine ferrochelatase . The K(m) values for porphyrin increased, except for the single mutant V251L . Other than a moderate increase observed in the triple mutant S249A/K250Q/V251C, the K(m) values for Fe(2+) were lowered . The k(cat)/K(m) for porphyrin remained largely unchanged, with the exception of a 10-fold reduction in the triple mutant K250M/V251L/W256Y . The k(cat)/K(m) for Fe(2+) was improved . Molecular modeling of these active loop variants indicated that loop mutations resulted in alterations of the active site architecture . However, despite the plasticity of the loop primary structure, the relative spatial positioning of the loop in the active site appeared to be maintained in functional variants, supporting a role for the loop in ferrochelatase function. Hum Mol Genet, 2004 Mar 15, 13(6), 641 - 50 On the molecular pathology of neurodegeneration in IMPDH1-based retinitis pigmentosa; Aherne A et al.; Retinitis pigmentosa (RP), the hereditary degenerative disease of the photoreceptor neurons of the retina, probably represents the most prevalent cause of registered blindness amongst those of working age in developed countries . Mutations within the gene encoding inosine monophosphate dehydrogenase 1 (IMPDH1), the widely expressed rate-limiting enzyme of the de novo pathway of guanine nucleotide biosynthesis, have recently been shown to cause the RP10 form of autosomal dominant RP . We examined the expression of IMPDH1, IMPDH2 and HPRT transcripts, encoding enzymes of the de novo and salvage pathways of guanine nucleotide biosynthesis, respectively, in retinal sections of mice, the data indicating that the bulk of GTP within photoreceptors is generated by IMPDH1 . Impdh1(-/-) null mice are shown here to display a slowly progressive form of retinal degeneration in which visual transduction, analysed by electroretinographic wave functions, becomes gradually compromised, although at 12 months of age most photoreceptors remain structurally intact . In contrast, the human form of RP caused by mutations within the IMPDH1 gene is a severe autosomal dominant degenerative retinopathy in those families that have been examined to date . Expression of mutant IMPDH1 proteins in bacterial and mammalian cells, together with computational simulations, indicate that protein misfolding and aggregation, rather than reduced IMPDH1 enzyme activity, is the likely cause of the severe phenotype experienced by human subjects . Taken together, these findings suggest that RP10 may represent an attractive target for therapeutic intervention, based upon a strategy combining simultaneous suppression of transcripts from normal and mutant IMPDH1 alleles with supplementation of GTP within retinal tissues. Exp Neurol, 2004 Mar, 186(1), 6 - 19 Fate of multipotent neural precursor cells transplanted into mouse retina selectively depleted of retinal ganglion cells; Mellough CB et al.; In some parts of the CNS, depletion of a particular class of neuron might induce changes in the microenvironment that influence the differentiation of newly grafted neural precursor cells . This hypothesis was tested in the retina by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult female mice and examining whether intravitreally grafted male neural precursor cells (C17.2), a neural stem cell (NSC)-like clonal line, become incorporated into these selectively depleted retinae . In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection . Cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0-7 days after ON injury (adults) . Cells were also injected into non-RGC depleted neonatal and adult retinae . At 4 or 8 weeks, transplanted cells were identified using a Y-chromosome marker and in situ hybridisation or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal) . No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16) . Donor cells were however stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL) . These NSC-like cells were also present in intact retinae, but on average, there were fewer cells in GCL . In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin, a protein highly expressed by, but not specific to, developing RGCs . Targeted rapid RGC depletion thus increased cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype. Gene, 2004 Mar 3, 327(2), 171 - 83 CD109 represents a novel branch of the alpha2-macroglobulin/complement gene family; Solomon KR et al.; We report here the genomic organization and phylogenic relationships of CD109, a member of the the alpha2-macroglobulin/complement (AMCOM) gene family . CD109 is a GPI-linked glycoprotein expressed on endothelial cells, platelets, activated T-cells, and a wide variety of tumors . We cloned full-length CD109 cDNA from the mammalian U373 cell line by RT-PCR and performed analysis of its corresponding genomic sequence . The CD109 cDNA spans 128 kb of chromosome 6q with its 33 exons constituting approximately 3.3% of the total CD109 genomic sequence . Sequence analysis revealed that CD109 contains specific motifs in its N-terminus, that are highly conserved in all AMCOM members . CD109 also shares motifs with certain other AMCOM members including: (1) a thioester 'GCGEQ" motif, (2) a furin site of four positively charged amino acids, and (3) a double tyrosine near the C-terminus . Based on a phylogenic analysis of human CD109 with other human homologs as well as orthologs from other mammalian species, C . elegans (ZK337.1) and E . coli homologs, we propose CD109 represents a novel and independent branch of the alpha2-macroglobulin/complement gene family (AMCOM) and may be its oldest member. Bioorg Med Chem, 2004 Mar 1, 12(5), 969 - 77 First enzymatically activated Taxotere prodrugs designed for ADEPT and PMT; Bouvier E et al.; Described here are the syntheses and preliminary biological evaluations of the first two enzymatically activated prodrugs of docetaxel (Taxotere) reported to date . These prodrugs were designed as potential candidates for selective chemotherapy in ADEPT or PMT . They are constituted of a glucuronic acid moiety, a double spacer and the cytotoxic drug, differing only by the spacer substitution . The prodrugs were stable in a buffer, and the in vitro studies showed good detoxification and hydrolysis kinetics . As docetaxel was efficiently released in both cases, these compounds are very valuable candidates for further biological evaluations. Biochim Biophys Acta, 2004 Feb 24, 1670(3), 217 - 28 Determination of binding constant of transcription factor myc-max/max-max and E-box DNA: the effect of inhibitors on the binding; Park S et al.; The truncated myc and max proteins, only containing basic regions and helix-loop-helix/zipper (b/HLH/Zip) regions were over-expressed in E . coli and used for the determination of the binding constant and of the inhibitory mechanism on myc-max (or max-max)-DNA complex formation . The association kinetic constants (k(1) and k(-1)) of truncated max-max or myc-max dimer and DNA were determined as k(1)=(1.7+/-0.6)x10(5) M(-1) s(-1), k(-1)=(3.4+/-1.2)x10(-2) s(-1) for max-max and DNA or k(1)=(2.1+/-0.7)x10(5) M(-1) s(-1), k(-1)=(3.2+/-1.4)x10(-2) s(-1) for myc-max and DNA . The equilibrium binding constant (K(1)) was determined using these kinetic parameters {K(XXD)=(7.8+/-2.6)x10(6) M(-1) for max-max and DNA or K(XYD)=(6.9+/-2.2)x10(6) M(-1) for myc-max and DNA} . The binding constants of myc-max or max-max dimer formation were K(XX)=(2.6+/-0.9)x10(5) M(-1) or K(XY)=(1.3+/-0.4)x10(4) M(-1), respectively . When truncated proteins were used, the max-max dimer formation was easier than the myc-max dimer formation, contrary to the physiologically determined case . This leads us to deduce that domains other than b/HLH/Zip are very important for the transcriptional regulatory activity in physiological conditions . The truncated myc and max proteins, which were expressed in E . coli and contained only b/HLH/Zip regions were also used for the screening of inhibitors of myc-max-DNA complex formation . A synthesized curcuminoid, 1,7-bis(4-methyl-3-nitrophenyl)-1,6-heptadiene-3,5-dione (curcuminoid 004), showed the most potent inhibition out of the synthesized curcuminoids, in competition with DNA . The dissociation constant of max-max dimer and the inhibitor was 9 microM, when investigated using in vitro expressed b/HLH/Zip dimer proteins . The curcuminoid 004 showed an inhibitory effect on the binding of myc-max protein to the E-box element in SNU16 cells, and suppressed the expression of myc target genes including ornithine decarboxylase (ODC), cdc25a and c-myc in myc over-expressed human stomach cancer cell line SNU16. Redox Rep, 2003, 8(6), 379 - 83 Escherichia coli deltafur mutant displays low HPII catalase activity in stationary phase; Benov L et al.; Iron is among the most important micronutrients used by bacteria . As a partner of the Fenton reaction, however, iron potentiates oxygen toxicity . Strict regulation of iron metabolism, and its coupling with regulation of defenses against oxidative stress, is an essential factor for life in the presence of oxygen . In Escherichia coli, iron metabolism is regulated by the Fur protein . A Fur-deficient mutant, in stationary phase, displayed about 30y-fold lower HPII activity than the respective, Fur-proficient parental strain . Deletion of fur seems to affect HPII catalase specifically, since the mutant was capable of inducing HPI catalase when challenged with H(2)O(2) . Low HPII catalase activity appears to be among the reasons for hydrogen peroxide hypersensitivity of the deltafur mutant. Biochemistry, 2004 Mar 2, 43(8), 2309 - 13 The composition rather than position of polar residues (QxxS) drives aspartate receptor transmembrane domain dimerization in vivo; Sal-Man N et al.; Transmembrane (TM) helix association is an important process affecting the function of many integral membrane proteins . Consequently, aberrations in this process are associated with diseases . Unfortunately, our knowledge of the factors that control this oligomerization process in the membrane milieu is limited at best . Previous studies have shown a role for polar residues in the assembly of synthetic peptides in vitro and the association of de novo-designed TM helices in vivo . Here we examined, for the first time, the involvement of polar residues in the dimerization of a biological TM domain in its natural environment . We analyzed both the involvement of polar residues in the dimerization process and whether their influence is position-dependent . For this purpose, we used the TM domain of the Escherichia coli aspartate receptor (Tar) and 10 single and double mutants . Polar to nonpolar mutations in the sequence demonstrated the role of the QxxS motif in the dimerization of the Tar TM domain . Moreover, creating a GxxxG motif, instead of the polar motif, almost completely abolished dimerization . Swapping positions between two wild-type polar residues did not affect dimerization, implying a similar contribution from both positions . Interestingly, mutants that contain two identical strong polar residues, EE and QQ, demonstrated a substantially higher level of dimerization than a QE mutant, although all three TM domains contain two strong polar residues . This result suggests that, in addition to the polarity of the residues, the formation of symmetric bonds also plays a role in dimer stability . The results of this study may facilitate a rational modulation of membrane protein function for therapeutic purposes. Biochemistry, 2004 Mar 2, 43(8), 2288 - 96 Dioxygen reduction by bo-type quinol oxidase from Escherichia coli studied by submillisecond-resolved freeze-quench EPR spectroscopy; Matsuura K et al.; The mechanism of the dioxygen (O(2)) reduction conducted by cytochrome bo-type quinol oxidase was investigated using submillisecond-resolved freeze-quench EPR spectroscopy . The fully reduced form of the wild-type enzyme (WT) with the bound ubiquinone-8 at the high-affinity quinone-binding site was mixed with an O(2)-saturated solution, and the subsequent reaction was quenched at different time intervals from 0.2 to 50 ms . The EPR signals derived from the binuclear center and heme b were weak in the time domain from 0.2 to 0.5 ms . The signals derived from the ferric heme b and hydroxide-bound ferric heme o increased simultaneously after 1 ms, indicating that the oxidation of heme b is coupled to the formation of hydroxy heme o . In contrast, the enzyme without the bound ubiquinone-8 (Delta UbiA) showed the faster oxidation of heme b and the slower formation of hydroxy heme o than WT . It is interpreted that the F(I) intermediate possessing ferryl-oxo heme o, cupric Cu(B), and ferric heme b is converted to the F(II) intermediate within 0.2 ms by an electron transfer from the bound ubiquinonol-8 to ferric heme b . The conversion of the F(II) intermediate to the hydroxy intermediate occurred after 1 ms and was accompanied by the one-electron transfer from heme b to the binuclear center . Finally, it is suggested that the hydroxy intermediate possesses no bridging ligand between heme o and Cu(B) and is the final intermediate in the turnover cycle of cytochrome bo under steady-state conditions. Biochemistry, 2004 Mar 2, 43(8), 2272 - 8 Functional consequences of N- or C-terminal deletions of the delta subunit of chloroplast ATP synthase; Ni ZL et al.; Mutagenesis was used to generate seven truncation mutants of the spinach (Spinacia oleracea) chloroplast ATP synthase delta subunit lacking 5, 11, 17, or 35 amino acid residues from the N-terminus or 3, 9, or 15 residues from the C-terminus . Interactions between these mutants and all other subunits of the chloroplast ATPase were investigated by a yeast two-hybrid system . The results indicate that the N-terminal deletions mainly affected interactions between the delta subunit and the other part of CF(1), but did not significantly affect interactions with the CF(0) sector . In contrast, C-terminal truncations of the delta subunit mainly affected its interaction with the CF(0) sector and caused little impairment in interactions with the other part of CF(1) . The conformation of the delta subunit C-terminal domain seems to be more sensitive to the truncations, as shown by minimal expression driven by C-terminal deleted (nine residues) mutants . Further studies showed C-terminal truncations of the delta subunit greatly impaired its ability to restore cyclic photophosphorylation in NaBr vesicles, whereas N-terminal truncations had little effect on the ability of delta to plug the CF(0) channel . None of the mutants impaired ATP hydrolysis by CF(1). Epidemiol Infect, 2004 Jan, 132(1), 87 - 94 In vitro studies on the colonization of bovine colonic mucosa by Shiga-toxigenic Escherichia coli (STEC); Cobbold RN et al.; This study investigated host-related factors that influence intestinal colonization by Shiga-toxigenic E . coli (STEC) . A quantitative colonization assay was developed to comparatively measure attachment of STEC to bovine colonic tissues maintained in vitro . No differences were determined in colonization susceptibility between tissues derived from weaning calves and adult cattle, or for tissues from cattle fed grain and forage-based rations . Substrate conditions designed to represent various intra-enteric environments were tested for their effect on STEC/mucosal interaction . Under conditions corresponding to a well-fed ruminant (high volatile fatty acid and lactate concentrations, low pH), significantly less STEC colonized the mucosal surface of colonic biopsies . These results may help explain why fasted, poorly or intermittently fed cattle and pre-ruminant calves excrete STEC to a greater degree . Studies on the ecology of STEC within the ruminant gut help identify mechanisms to reduce their threat to public health. Epidemiol Infect, 2004 Jan, 132(1), 77 - 85 Verotoxins in commensal Escherichia coli in cattle: the effect of injectable subcutaneous oxytetracycline in addition to in-feed chlortetracycline on prevalence; O'Connor AM et al.; Using a self-paired observational study, the association between therapeutic oxytetracycline use and the prevalence of virulence genes in commensal Escherichia coli (E . coli) from cattle was examined . Faeces were collected from 39 yearling bulls prior to and after treatment with oxytetracycline and from 44 untreated animals . Between samplings all animals received in-feed chlortetracycline for 16 days . Five E . coli were isolated from each sample and tested by a polymerase chain reaction (PCR) capable of detecting all verotoxin (vt) genes . Positive isolates were further tested with a multiplex PCR to detect vt1, vt2, eaeA and hlyA . For vt, 23 animals were positive at both samplings, 26 negative at both samplings, 22 negative animals became positive and 12 positive animals became negative . Sixty-eight per cent of the discordant pairs changed from vt-negative to vt-positive (95% CI 48-80) suggesting pressure toward becoming vt-positive perhaps due to the transfer of genes due to mixing of cattle in the months between samplings or an effect of chlortetracycline. J Gene Med, 2004 Feb, 6(2), 238 - 45 Overexpression of human fibroblast growth factor 2 stimulates cell proliferation in an ex vivo model of articular chondrocyte transplantation; Madry H et al.; BACKGROUND: Genetically engineered chondrocytes could be used to enhance cartilage repair . Fibroblast growth factor 2 (FGF-2) is a mitogen for chondrocytes and may be a candidate for gene transfer approaches to stimulate chondrocyte proliferation . In the present study, we tested the hypothesis that human FGF-2 (hFGF-2) gene transfer into articular chondrocytes modulates cell proliferation in an ex vivo model of chondrocyte transplantation . METHODS: Transfection of articular chondrocytes with an expression plasmid vector carrying the cDNA for hFGF-2 under the control of the cytomegalovirus promoter/enhancer mediated transgene expression and synthesis of biologically relevant amounts of the recombinant hFGF-2 protein . Articular chondrocytes transfected with the Escherichia coli beta-galactosidase (lacZ) gene or a hFGF-2 cDNA were transplanted onto the surface of articular cartilage explants . RESULTS: The tissue formed by the chondrocytes expressing hFGF-2 was thicker and contained more cells than control cultures . Quantitative analysis of {(3)H}thymidine and {(35)S}sulfate incorporation in composite cultures revealed that hFGF-2 transfection stimulated mitogenic activity in the new tissue but did not augment matrix glycosaminoglycan synthesis . CONCLUSIONS: These data support the concept that chondrocytes overexpressing a hFGF-2 cDNA selectively modulate cell proliferation in an ex vivo model of chondrocyte transplantation . These results suggest that therapeutic hFGF-2 gene transfer may be applicable for the treatment of articular cartilage disorders, such as traumatic defects in which cellular repopulation is a therapeutic goal . J Gene Med, 2004 Feb, 6(2), 195 - 209 In vitro and in vivo delivery of intact BAC DNA -- comparison of different methods; Magin-Lachmann C et al.; BACKGROUND: The ability to deliver large (>100 kb) fragments of DNA to mammalian cells in vitro and in vivo is becoming increasingly important with the availability of BAC and PAC constructs for gene expression . Here we investigate in vitro and in vivo delivery of BACs up to 157 kb . METHODS: Different types of polyethylenimine (PEI) and Lipofectamine were used to deliver 150-kb BAC (bacterial artificial chromosome) DNA to mouse and human cell lines in tissue culture and the level of EGFP expression compared . To assess the intactness of the DNA delivered, a BAC carrying oriP/EBNA-1 was used to make stably transfected cell lines . Episomal DNA was then rescued into E . coli followed by analysis on a pulsed-field gel . Three different methods of in vivo delivery were also assessed for delivery of BAC DNA; intravenous injection of DNA/PEI particles, intramuscular injection with electroporation and high-volume injection into the tail vein . RESULTS: PEI22 (linear polymer form, 22 kDa) was found to be the most efficient method for delivery of 150-kb BAC DNA to both cell lines in tissue culture . However, Lipofectamine 2000 was found to give a higher proportion of intact DNA than PEI22 in stably transformed colonies and almost all the DNA delivered by Lipofectamine 2000 was intact . Intravenous injection of DNA/PEI particles was found to be inefficient for delivery of BAC DNA . Intramuscular injection with electroporation of pure BAC DNA was very efficient and expression was maintained for 105 days . High-volume injection of BAC DNA gave excellent expression in the liver and intact BAC DNA could be rescued 7 days after injection . CONCLUSIONS: These results demonstrate efficient delivery of intact, large (up to 157 kb) DNA constructs for in vitro gene expression and in vivo gene therapy applications . Protein Sci, 2004 Mar, 13(3), 652 - 8 The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics; Goda N et al.; A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion protein expression vector is described . The method, termed PRESAT-vector cloning, is based on a T-vector technique that does not require restriction endonuclease digestion of the PCR product . Subsequently, we applied a novel ORF selection method of the ligated plasmid products . This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies . To achieve this selection, we customized the 5'-sequence of the "rear" PCR primer corresponding to the C terminus of the protein to be expressed . The colonies harbored only the ligated products of the desired orientation at >90% efficiency . This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested. Protein Sci, 2004 Mar, 13(3), 567 - 74 Nucleotide-induced switch in oligomerization of the AAA+ ATPase ClpB; Akoev V et al.; ClpB is a member of the bacterial protein-disaggregating chaperone machinery and belongs to the AAA(+) superfamily of ATPases associated with various cellular activities . The mechanism of ClpB-assisted reactivation of strongly aggregated proteins is unknown and the oligomeric state of ClpB has been under discussion . Sedimentation equilibrium and sedi