J Biol Chem, 2000 Jul 7, 275(27), 20540 - 4 Identification and characterization of human endometase (Matrix metalloproteinase-26) from endometrial tumor; Park HI et al.; We report the discovery, cloning, and characterization of a novel human matrix metalloproteinase 26 (MMP-26) (matrixin) gene, endometase, an endometrial tumor-derived metalloproteinase . Among more than three million expressed sequence tags sequenced, the endometase gene was only obtained from human endometrial tumor cDNA library . Endometase mRNA was expressed specifically in human uterus, not in other tissues/cells tested, e.g . testis, heart, brain, lungs, liver, thymus, and melanoma G361 . Endometase protein has a signal peptide, a propeptide domain, and a catalytic domain with a unique "cysteine switch" propeptide sequence, PHCGVPDGSD, and a zinc-binding motif, VATHEIGHSLGLQH . Endometase is 43, 41, 41, and 39% identical to human metalloelastase, stromelysin, collagenase-3, and matrilysin, respectively . The zymogen was expressed and isolated from Escherichia coli as inclusion bodies with a molecular mass of 28 kDa . The identity and homogeneity of the recombinant protein was confirmed by protein N-terminal sequencing, silver stain, and immunoblot analyses . The pro-enzyme was partially activated during the folding process . Endometase selectively cleaved type I gelatin and alpha(1)-proteinase inhibitor; however, it did not digest collagens, laminin, elastin, beta-casein, plasminogen, soybean trypsin inhibitor, or Bowman-Birk inhibitor . It hydrolyzed peptide substrates of matrixins and tumor necrosis factor-alpha converting enzyme . Endometase may selectively cleave extracellular matrix proteins, inactivate serpins, and process cytokines. J Biol Chem, 2000 Jul 14, 275(28), 21107 - 13 Size-dependent disaggregation of stable protein aggregates by the DnaK chaperone machinery; Diamant S et al.; Classic in vitro studies show that the Hsp70 chaperone system from Escherichia coli (DnaK-DnaJ-GrpE, the DnaK system) can bind to proteins, prevent aggregation, and promote the correct refolding of chaperone-bound polypeptides into native proteins . However, little is known about how the DnaK system handles proteins that have already aggregated . In this study, glucose-6-phosphate dehydrogenase was used as a model system to generate stable populations of protein aggregates comprising controlled ranges of particle sizes . The DnaK system recognized the glucose-6-phosphate dehydrogenase aggregates as authentic substrates and specifically solubilized and refolded the protein into a native enzyme . The efficiency of disaggregation by the DnaK system was high with small aggregates, but the efficiency decreased as the size of the aggregates increased . High folding efficiency was restored by either excess DnaK or substoichiometric amounts of the chaperone ClpB . We suggest a mechanism whereby the DnaK system can readily solubilize small aggregates and refold them into active proteins . With large aggregates, however, the binding sites for the DnaK system had to be dynamically exposed with excess DnaK or the catalytic action of ClpB and ATP . Disaggregation by the DnaK machinery in the cell can solubilize early aggregates that formed accidentally during chaperone-assisted protein folding or that escaped the protection of "holding" chaperones during stress. Structure Fold Des, 2000 May 15, 8(5), 527 - 40 Crystal structure of the ffh and EF-G binding sites in the conserved domain IV of Escherichia coli 4.5S RNA; Jovine L et al.; BACKGROUND: Bacterial signal recognition particle (SRP), consisting of 4.5S RNA and Ffh protein, plays an essential role in targeting signal-peptide-containing proteins to the secretory apparatus in the cell membrane . The 4.5S RNA increases the affinity of Ffh for signal peptides and is essential for the interaction between SRP and its receptor, protein FtsY . The 4.5S RNA also interacts with elongation factor G (EF-G) in the ribosome and this interaction is required for efficient translation . RESULTS: We have determined by multiple anomalous dispersion (MAD) with Lu(3+) the 2.7 A crystal structure of a 4.5S RNA fragment containing binding sites for both Ffh and EF-G . This fragment consists of three helices connected by a symmetric and an asymmetric internal loop . In contrast to NMR-derived structures reported previously, the symmetric loop is entirely constituted by non-canonical base pairs . These pairs continuously stack and project unusual sets of hydrogen-bond donors and acceptors into the shallow minor groove . The structure can therefore be regarded as two double helical rods hinged by the asymmetric loop that protrudes from one strand . CONCLUSIONS: Based on our crystal structure and results of chemical protection experiments reported previously, we predicted that Ffh binds to the minor groove of the symmetric loop . An identical decanucleotide sequence is found in the EF-G binding sites of both 4.5S RNA and 23S rRNA . The decanucleotide structure in the 4.5S RNA and the ribosomal protein L11-RNA complex crystals suggests how 4.5S RNA and 23S rRNA might interact with EF-G and function in translating ribosomes. Structure Fold Des, 2000 May 15, 8(5), 505 - 14 Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site; Walsh MA et al.; BACKGROUND: Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide . In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate . The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs . The enzyme shows no significant amino acid sequence homology with other proteins . RESULTS: We have determined the crystal structure of cyanase at 1.65 A resolution using the multiwavelength anomalous diffraction (MAD) method . Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit . Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing . Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site . CONCLUSIONS: The cyanase monomer is composed of two domains . The N-terminal domain shows structural similarity to the DNA-binding alpha-helix bundle motif . The C-terminal domain has an 'open fold' with no structural homology to other proteins . The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level . The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers . The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer . The structural data allow a conceivable reaction mechanism to be proposed. Chem Biol, 2000 May, 7(5), 313 - 21 Chemically induced dimerization of dihydrofolate reductase by a homobifunctional dimer of methotrexate; Kopytek SJ et al.; BACKGROUND: Chemically induced dimerization (CID) can be used to manipulate cellular regulatory pathways from signal transduction to transcription, and to create model systems for study of the specific interactions between proteins and small-molecule chemical ligands . However, few CID systems are currently available . The properties of, and interactions between, Escherichia coli dihydrofolate reductase (DHFR) and the ligand methotrexate (MTX) meet many of the desired criteria for the development of a new CID system . RESULTS: BisMTX, a homobifunctional version of MTX, was synthesized and tested for its ability to induce dimerization of DHFR . Gel-filtration analysis of purified DHFR confirmed that, in vitro, the protein was a monomer in the absence of dimerizer drug; in the presence of bisMTX, a complex of twice the monomeric molecular weight was observed . Furthermore, the off-rate was found to be 0.0002 s(-1), approximately 100 times slower than that reported for DHFR-MTX . Interestingly, the addition of excess bisMTX did not result in formation of the binary complex (1 protein:1 dimerizer) over the ternary complex (2 proteins:1 dimerizer), which suggests cooperative binding interactions (affinity modulation) between the two DHFR molecules in the bisMTX:DHFR(2) ternary complex . CONCLUSIONS: The combination of DHFR and bisMTX provides a new CID system with properties that could be useful for applications in vivo . Formation of the bisMTX:DHFR(2) ternary complex in vitro is promoted over a wide range of dimerizer concentrations, consistent with the idea that formation of the ternary complex recruits energetically favorable interactions between the DHFR monomers in the complex. Curr Opin Cell Biol, 2000 Jun, 12(3), 280 - 5 ATPase switches controlling DNA replication initiation; Lee DG et al.; Proteins that bind and hydrolyze ATP are frequently involved in the early steps of DNA replication . Recent studies of Saccharomyces cerevisiae suggest that two members of the AAA+ ATPase family--the origin recognition complex and Cdc6p--have separable roles for ATP binding and ATP hydrolysis during eukaryotic DNA replication . Intriguingly, the proposed regulation of these eukaryotic replication proteins by ATP has functional similarities to the ATP-dependent control of the DnaA and DnaC initiation factors from Escherichia coli . Comparison of the ATP regulation of these factors suggests that ATP binding and hydrolysis acts as a molecular switch that couples key events during initiation of replication . This switch results in a significant change in protein function. Curr Biol, 2000 May 4, 10(9), R328 - 30 Organelle division: Self-assembling GTPase caught in the middle; Margolin W; Recent findings indicate that the dynamin GTPase helps to divide animal and fungal mitochondria, and that the tubulin-like FtsZ GTPase is involved in division of, not only most bacteria, but also chloroplasts and probably mitochondria of unicellular eukaryotes. Curr Biol, 2000 Apr 20, 10(8), R318 - 20 Towards a circuit engineering discipline; McAdams HH et al.; Genetic circuits can now be engineered that perform moderately complicated switching functions or exhibit predictable dynamical behavior . These 'forward engineering' techniques may have to be combined with directed evolution techniques to produce robustness comparable with naturally occurring circuits. J Mol Biol, 2000 May 19, 298(5), 917 - 26 Stabilization of GroEL minichaperones by core and surface mutations; Wang Q et al.; We report the crystal structures of two hexa-substituted mutants of a GroEL minichaperone that are more stable than wild-type by 7.0 and 6.1 kcal mol(-1) . Their structures imply that the increased stability results from multiple factors including improved hydrophobic packing, optimised hydrogen bonding and favourable structural rearrangements . It is commonly believed that protein core residues are immutable and generally optimized for energy, while on the contrary, surface residues are variable and hence unimportant for stability . But, it is now becoming clear that mutations of both core and surface residues can increase protein stability, and that protein cores are more flexible and thus more tolerant to mutation than expected . Sequence comparison of homologous proteins has provided a way to pinpoint the residues that contribute constructively to stability and to guide the engineering of protein stability . Stabilizing mutations identified by this approach are most frequently located at protein surfaces but with a few found in protein cores . In the latter case, local flexibility in the hydrophobic core is the key factor that allows the energetically favourable burial of larger hydrophobic side-chains without undue energetic penalties from steric clashes . J Mol Biol, 2000 May 19, 298(5), 795 - 805 The phenotype of mutations of the base-pair C2658.G2663 that closes the tetraloop in the sarcin/ricin domain of Escherichia coli 23 S ribosomal RNA; Chan YL et al.; The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA is a part of the site for the association of elongation factors with ribosomes and for that reason is critical for the binding of aminoacyl-tRNA and for translocation during the reiterative elongation reactions of protein synthesis . The SRD has a GAGA tetraloop that is shut off by a Watson-Crick C2658 x G2663 pair . The contribution of this pair to the function of the ribosome has been evaluated by constructing mutations in the nucleotides and determining their phenotype . Constitutive expression of a plasmid-encoded rrnB operon with a G2663C transversion mutation that disrupts the Watson-Crick pair was lethal . Double transversion mutations, C2658G x G2663C and C2658A x G2663U, that reverse the polarity of the pyrimidine and the purine but restore the potential to form a canonical pair, were also lethal . Induction of transcription of 23 S rRNA with the same mutations, but encoded in a plasmid with a lambdaP(L) promoter and expressed at a lower level, retarded growth . The sedimentation profiles of ribosomes with transversion mutations in C2658 and/or G2663 are altered; the ratio of 50 S subunits to 30 S particles is changed and polysomes are reduced . Ribosomes with a G2663C, a C2658G x G2663C, or a C2658A x G2663U mutation in 23 S rRNA were not active in protein synthesis, indeed, they appeared to inhibit the activity of ribosomes with wild-type 23 S rRNA . Transversion mutations in the analogs of C2658 and G2663 decreased binding of EF-G to SRD oligoribonucleotides; the same mutations in 23 S rRNA decreased binding of the factor to intact ribosomes . The most severe phenotype, in growth, in protein synthesis, and in the binding of EF-G, was associated with a C2658G x G2663C mutation; it is surprising that this was more severe than an analogous C2658A x G2663U mutation . A double transition mutation, C2658U x G2663A, which is not known to have occurred in nature, had no effect on the growth of cells or on the function of ribosomes . The lethal phenotype of transversion mutations in C2658 and G2663 appears to derive from a loss of the capacity of ribosomes to bind EF-G and by indirection the EF-Tu ternary complex . J Mol Biol, 2000 May 19, 298(5), 779 - 93 tRNA leucine identity and recognition sets; Tocchini-Valentini G et al.; Transfer RNAs (tRNAs) are grouped into two classes based on the structure of their variable loop . In Escherichia coli, tRNAs from three isoaccepting groups are classified as type II . Leucine tRNAs comprise one such group . We used both in vivo and in vitro approaches to determine the nucleotides that are required for tRNA(Leu) function . In addition, to investigate the role of the tRNA fold, we compared the in vivo and in vitro characteristics of type I tRNA(Leu) variants with their type II counterparts.A minimum of six conserved tRNA(Leu) nucleotides were required to change the amino acid identity and recognition of a type II tRNA(Ser) amber suppressor from a serine to a leucine residue . Five of these nucleotides affect tRNA tertiary structure; the G15-C48 tertiary "Levitt base-pair" in tRNA(Ser) was changed to A15-U48; the number of nucleotides in the alpha and beta regions of the D-loop was changed to achieve the positioning of G18 and G19 that is found in all tRNA(Leu); a base was inserted at position 47n between the base-paired extra stem and the T-stem; in addition the G73 "discriminator" base of tRNA(Ser) was changed to A73 . This minimally altered tRNA(Ser) exclusively inserted leucine residues and was an excellent in vitro substrate for LeuRS . In a parallel experiment, nucleotide substitutions were made in a glutamine-inserting type I tRNA (RNA(SerDelta); an amber suppressor in which the tRNA(Ser) type II extra-stem-loop is replaced by a consensus type I loop) . This "type I" swap experiment was successful both in vivo and in vitro but required more nucleotide substitutions than did the type II swap . The type I and II swaps revealed differences in the contributions of the tRNA(Leu) acceptor stem base-pairs to tRNA(Leu) function: in the type I, but not the type II fold, leucine specificity was contingent on the presence of the tRNA(Leu) acceptor stem sequence . The type I and II tRNAs used in this study differed only in the sequence and structure of the variable loop . By altering this loop, and thereby possibly introducing subtle changes into the overall tRNA fold, it became possible to detect otherwise cryptic contributions of the acceptor stem sequence to recognition by LeuRS . Possible reasons for this effect are discussed . Biochemistry, 2000 May 16, 39(19), 5868 - 75 Modulation of the activities of catalase-peroxidase HPI of Escherichia coli by site-directed mutagenesis; Hillar A et al.; Catalase-peroxidases have a predominant catalatic activity but differ from monofunctional catalases in exhibiting a substantial peroxidatic reaction which has been implicated in the activation of the antitubercular drug isoniazid in Mycobacterium tuberculosis . Hydroperoxidase I of Escherichia coli encoded by katG is a catalase-peroxidase, and residues in its putative active site have been the target of a site directed-mutagenesis study . Variants of residues R102 and H106, on the distal side of the heme, and H267, the proximal side ligand, were constructed, all of which substantially reduced the catalatic activity and, to a lesser extent, the peroxidatic activity . In addition, the heme content of the variants was reduced relative to the wild-type enzyme . The relative ease of heme loss from HPI and a mixture of tetrameric enzymes with 2, 3, and 4 hemes was revealed by mass spectrometry analysis . Conversion of W105 to either an aromatic (F) or aliphatic (I) residue caused a 4-5-fold increase in peroxidatic activity, coupled with a >99% inhibition of catalatic activity . The peroxidatic-to-catalatic ratio of the W105F variant was increased 2800-fold such that compound I could be identified by both electronic and EPR spectroscopy as being similar to the porphyrin cation radical formed in other catalases and peroxidases . Compound I, when generated by a single addition of H(2)O(2), decayed back to the native or resting state within 1 min . When H(2)O(2) was generated enzymatically in situ at low levels, active compound I was evident for up to 2 h . However, such prolonged treatment resulted in conversion of compound I to a reversibly inactivated and, eventually, to an irreversibly inactivated species, both of which were spectrally similar to compound I. Biochemistry, 2000 May 16, 39(19), 5653 - 61 Distances between DNA and ATP binding sites in the TyrR-DNA complex; Sawyer WH et al.; The Escherichia coli regulatory protein TyrR controls the expression of eight transcription units that encode proteins involved in the biosynthesis and transport of aromatic amino acids . It binds to DNA as a homodimer with a subunit molecular mass of 57 640 Da, each of which has a single site for the binding of ATP within a central structural domain . This paper reports distances between four sites on the DNA and the ATP binding site as determined by fluorescence resonance energy transfer . The DNA was a 30mer containing a centrally located binding site for TyrR . Replacement of a thymidine residue with an aminouridine residue at positions -9, -7, -3, and 2 of the palindromic oligonucleotide sequence enabled the placement of a single fluorescein group along the major groove of the DNA . The energy transfer acceptor was ATP labeled with a rhodamine group through positions 2' and 3' of the ribose, positions that are known to cause minimal interference with the binding of ATP to protein . The dissociation constant for the binding of rhodamine-ATP to TyrR was 300 nM as determined by steady-state fluorescence anisotropy titrations . The energy transfer efficiencies were determined by measuring the level of quenching of donor fluorescence on binding rhodamine-ATP to the TyrR-DNA complex . The experimental transfer efficiencies were compared to theoretical values calculated for a model of the DNA-TyrR complex in which the position of the ATP binding site was allowed to vary over the surface of the monomer unit . Theory was written to account for the transfer from one donor to two acceptors, one on each monomer unit of the TyrR dimer . The results indicate that the ATP binding site is about 40-45 A from the nearest point on the DNA and distant from the DNA helix-turn-helix binding domain . The effects of ATP binding of (i) increasing the TyrR binding affinity by a factor of 4-5 and (ii) permitting the binding of the tyrosine corepressor must therefore occur because of a significant allosteric change in the conformation of the protein. J Biol Chem, 2000 Jul 21, 275(29), 22082 - 9 Identification and molecular characterization of the first alpha -xylosidase from an archaeon; Moracci M et al.; We here report the first molecular characterization of an alpha-xylosidase (XylS) from an Archaeon . Sulfolobus solfataricus is able to grow at temperatures higher than 80 degrees C on several carbohydrates at acidic pH . The isolated xylS gene encodes a monomeric enzyme homologous to alpha-glucosidases, alpha-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31 . xylS belongs to a cluster of four genes in the S . solfataricus genome, including a beta-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function . The alpha-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90 degrees C and a half-life at this temperature of 38 h . The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides . Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the alpha-xylosidase and the beta-glycosidase from S . solfataricus . The analysis of the total S . solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed. Am J Physiol Gastrointest Liver Physiol, 2000 May, 278(5), G811 - 9 Escherichia coli Shiga toxins induce apoptosis in epithelial cells that is regulated by the Bcl-2 family; Jones NL et al.; Human intestinal cells lack globotriaosylceramide (Gb(3)), the receptor for Shiga toxin-1 (Stx1) and Shiga toxin-2 (Stx2) . Therefore, the role of these toxins in mediating intestinal disease during infection with Shiga toxin-producing Escherichia coli is unclear . The aims of this study were to determine whether Stx1 and Stx2 induce apoptosis in epithelial cells expressing (HEp-2, Caco-2) or lacking (T84) Gb(3) and to characterize the role of the Bcl-2 family . Stx1 (12.5 ng/ml) induced apoptosis in both HEp-2 (21.9 +/- 7.9% vs . 0.8 +/- 0.3%, P = 0.01) and Caco-2 (10.1 +/- 1.2% vs . 3.1 +/- 0.4%, P = 0.006) cells but not in Gb(3)-deficient T84 cells . Toxin-mediated apoptosis of HEp-2 cells was associated with enhanced expression of the proapoptotic protein Bax . Inhibition of caspase activation prevented toxin-stimulated apoptosis . In addition, overexpression of Bcl-2 by transient transfection blocked Stx1-stimulated cell death . These findings indicate that Shiga toxins produced by E . coli signal Gb(3)-expressing epithelial cells to undergo apoptosis in association with enhanced Bax expression, thereby resulting in activation of the caspase cascade. Nature, 2000 Apr 27, 404(6781), 999 - 1003 Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases; Kawabuchi M et al.; The Src family of protein tyrosine kinases (Src-PTKs) is important in the regulation of growth and differentiation of eukaryotic cells . The activity of Src-PTKs in cells of different types is negatively controlled by Csk, which specifically phosphorylates a conserved regulatory tyrosine residue at the carboxy-terminal tail of the Src-PTKs . Csk is mainly cytoplasmic and Src-PTKs are predominantly membrane-associated . This raises a question about the mechanism of interaction between these enzymes . Here we present Cbp--a transmembrane phosphoprotein that is ubiquitously expressed and binds specifically to the SH2 domain of Csk . Cbp is involved in the membrane localization of Csk and in the Csk-mediated inhibition of c-Src . In the plasma membrane Cbp is exclusively localized in the GM1 ganglioside-enriched detergent-insoluble membrane domain, which is important in receptor-mediated signalling . These findings reveal Cbp as a new component of the regulatory mechanism controlling the activity of membrane-associated Src-PTKs. Growth Factors, 2000, 17(4), 287 - 300 A phase I and pharmacokinetic study of subcutaneously-administered recombinant human interleukin-4 (rhuIL-4) in patients with advanced cancer; Davis ID et al.; PURPOSE: To investigate the pharmacokinetics and tolerability of recombinant human interleukin-4 (rhuIL-4), administered by daily subcutaneous injection, in patients with advanced cancer . PATIENTS AND METHODS: Fourteen patients with advanced cancer treated with rhuIL-4 at escalating dose levels of 0.25, 1.0 and 5.0 microg/kg/day, on days 1, 8-17, and 28-57 . The primary endpoints of the study were toxicity of rhuIL-4 and the determination of the pharmacokinetics of rhuIL-4 when given by subcutaneous injection . Secondary endpoints included effects on blood counts, hematopoietic cell precursors, and various immunologic parameters . RESULTS: rhuIL-4 was well tolerated at all three dose levels . Detectable serum levels of IL-4 were found in patients at the 1.0 and 5.0 microg/kg/day dose levels . Peak serum IL-4 levels were achieved about 2 h after injection and IL-4 was still detectable 8 h after injection . No grade 4 toxicities were observed and grade 3 toxicities were confined to fever, headache and raised hepatic alkaline phosphatase . No consistent hematological or immunologic effects were observed . Although therapeutic efficacy was not an endpoint, one complete response (Hodgkin's disease) was observed . One patient with chronic lymphocytic leukemia progressed on therapy . CONCLUSION: rhuIL-4 up to 5.0 microg/kg/day is well tolerated when given by subcutaneous injection . Biologically relevant serum IL-4 levels can be achieved and sustained for at least 8 h after a single injection. Methods Enzymol, 2000, 316, 671 - 87 Expression and characterization of peripherin/rds-rom-1 complexes and mutants implicated in retinal degenerative diseases; Goldberg AF et al.; Nearly 40 disease-linked mutations have been reported for peripherin/rds to date; heterologous expression in tissue culture cells offers a valuable means of efficiently characterizing the biochemical properties of the various mutants . Peripherin/rds is proposed to act as an essential structural element in outer segment disk morphogenesis, and a present transgenic mice offer the sole tractable system in which recombinant peripherin/rds may be examined functionally in situ . Because the generation and characterization of transgenic animals are both expensive and time consuming, heterologous expression in cultured cells offers an important and complementary means of addressing protein structure and function . The immunopurification and detection of the peripherin/rds-rom-1 complex are performed using specific immunochemical reagents, monoclonal and polyclonal antibodies, that are not commonly available . Several laboratories have developed antibodies to peripherin/rds and rom-1 in rabbits and mice, using a variety of immunogens: purified ROS membranes, purified E . coli fusion proteins, and synthetic peptides coupled to proteins . The C-terminal regions appear to be most highly antigenic, although antibodies have been generated to other regions as well . Regardless of their source, antibodies must be thoroughly characterized; specificity is often a function of solution conditions and must be determined empirically . The approach as described here has provided explanations for several instances of peripherin/rds-associated disease, including digenic RP linked to as L185P mutation, and adRP associated with C118/119del and C214S mutations . In addition, the R172W mutation, linked to macular dystrophy and preferential loss in cone function, is shown to behave normally with respect to biosynthesis and subunit assembly; it likely involves a more subtle functional defect that remains to be described . Finally, the methodology reported here has suggested the existence of a novel (homotetrameric) form of peripherin/rds in individuals lacking rom-1; this hypothesis has been confirmed in rom-1 knockout mice . The information obtained thus far demonstrates the utility of using heterologously expressed peripherin/rds and rom-1 to investigate the consequences of disease-linked mutations in these polypeptides . Heterologous cell expression coupled with transgenic mouse methodologies should continue to provide a more detailed understanding of molecular mechanisms underlying inherited retinal degenerative diseases. Biotechnol Bioeng, 2000 Jun 20, 68(6), 689 - 96 Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression; Aris A et al.; A recombinant, multifunctional protein has been designed for optimized, cell-targeted DNA delivery and gene expression in mammalian cells . This hybrid construct comprises a viral peptide ligand for integrin alpha(V)beta(3) binding, a DNA-condensing poly-L-lysine domain, and a complete, functional beta-galactosidase protein that serves simultaneously as purification tag and DNA-shielding agent . This recombinant protein is stable; it has been produced successfully in Escherichia coli and can be purified in a single step by affinity chromatography . At optimal molar ratios, mixtures of this vector and a luciferase-reporter plasmid form stable complexes that transfect cultured cells . After exposure to these cell-targeted complexes, steady levels of gene expression are observed for more than 3 days after transfection, representing between 20 and 40% of those achieved with untargeted, lipid-based DNA-condensing agents . The principle to include viral motifs for cell infection in single polypeptide recombinant proteins represents a promising approach towards the design of non-viral modular DNA transfer vectors that conserve the cell-target- ing specificity of native viruses and that do not need further processing after bioproduction in a recombinant host . J Immunol, 2000 May 15, 164(10), 5362 - 8 Pivotal role of the CC chemokine, macrophage-derived chemokine, in the innate immune response; Matsukawa A et al.; Macrophage-derived chemokine (MDC), a recently identified CC chemokine, has been regarded to be involved in chronic inflammation and dendritic cell and lymphocyte homing . In this study, we demonstrate a pivotal role for MDC during experimental sepsis induced by cecal ligation and puncture (CLP) . Intraperitoneal administration of MDC (1 microg/mouse) protected mice from CLP-induced lethality . The survival was accompanied by increased number of peritoneal macrophages and decreased recovery of viable bacteria from the peritoneum and peripheral blood . In addition, mice treated with an i.p . injection of MDC cleared bacteria more effectively than those in the control when 3 x 108 CFU live Escherichia coli was i.p . inoculated . Endogenous MDC was detected in the peritoneum after CLP, and neutralization of the MDC with anti-MDC Abs decreased CLP-induced recruitment of peritoneal macrophages and increased the recovery of viable bacteria from the peritoneum and peripheral blood . MDC blockade was deleterious in the survival of mice after CLP . In vitro, MDC enhanced the phagocytic and killing activities of peritoneal macrophages to E . coli and induced both a respiratory burst and the release of lysozomal enzyme from macrophages . Furthermore, MDC dramatically ameliorated CLP-induced systemic tissue inflammation as well as tissue dysfunction, which were associated in part with decreased levels of TNF-alpha, macrophage inflammatory proteins-1alpha and -2, and KC in specific tissues . Collectively, these results indicate novel regulatory activities of MDC in innate immunity during sepsis and suggest that MDC may aid in an adjunct therapy in sepsis. FEMS Immunol Med Microbiol, 2000 Jun, 28(2), 133 - 7 Determination of the immunodominant part in the O-antigenic polysaccharide from Escherichia coli O128 by ELISA-inhibition study; Sengupta P et al.; The immunodominant part in the O-antigenic polysaccharide from Escherichia coli O128 was immunologically characterized by an enzyme-linked immunosorbent assay (ELISA) . The antibody specificity was determined by the inhibitory effects of the methyl glycosides of constituent mono- and oligosaccharides synthesized related to the O-antigenic polysaccharide from E . coli O128 . It was found that methyl alpha-L-fucopyranoside was the most effective inhibitor amongst the monosaccharides while the highest antibody specificity was directed towards the trisaccharide with the structure: beta-D-GalpNAc-(1-->6)-{alpha-L-Fucp-(1-->2)}-beta-D-Galp-1-->OMe suggesting that the monospecific antibody has the extended combining site. FEMS Immunol Med Microbiol, 2000 Jun, 28(2), 97 - 104 Characterization of the interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn calves; Al-Majali AM et al.; Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa) . We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves . We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine . This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains. J Biol Chem, 2000 May 12, 275(19), 14659 - 66 Cytochrome P450 CYP79A2 from Arabidopsis thaliana L . Catalyzes the conversion of L-phenylalanine to phenylacetaldoxime in the biosynthesis of benzylglucosinolate; Wittstock U et al.; Glucosinolates are natural plant products gaining increasing interest as cancer-preventing agents and crop protectants . Similar to cyanogenic glucosides, glucosinolates are derived from amino acids and have aldoximes as intermediates . We report cloning and characterization of cytochrome P450 CYP79A2 involved in aldoxime formation in the glucosinolate-producing Arabidopsis thaliana L . The CYP79A2 cDNA was cloned by polymerase chain reaction, and CYP79A2 was functionally expressed in Escherichia coli . Characterization of the recombinant protein shows that CYP79A2 is an N-hydroxylase converting L-phenylalanine into phenylacetaldoxime, the precursor of benzylglucosinolate . Transgenic A . thaliana constitutively expressing CYP79A2 accumulate high levels of benzylglucosinolate . CYP79A2 expressed in E . coli has a K(m) of 6.7 micromol liter(-1) for L-phenylalanine . Neither L-tyrosine, L-tryptophan, L-methionine, nor DL-homophenylalanine are metabolized by CYP79A2, indicating that the enzyme has a narrow substrate specificity . CYP79A2 is the first enzyme shown to catalyze the conversion of an amino acid to the aldoxime in the biosynthesis of glucosinolates . Our data provide the first conclusive evidence that evolutionarily conserved cytochromes P450 catalyze this step common for the biosynthetic pathways of glucosinolates and cyanogenic glucosides . This strongly indicates that the biosynthesis of glucosinolates has evolved based on a cyanogenic predisposition. J Biol Chem, 2000 May 12, 275(19), 14642 - 8 Characterization of recombinant phosphatidylinositol 4-kinase beta reveals auto- and heterophosphorylation of the enzyme; Zhao XH et al.; Phosphatidylinositol (PI) 4-kinases catalyze the synthesis of PI 4-phosphate, an important intermediate for the synthesis of membrane polyphosphoinositides, regulators of multiple cellular functions . Two mammalian PI 4-kinases have been cloned, a 230-kDa enzyme (alpha-form) and a 110-kDa (beta-form), both of which are inhibited by >0.1 microm concentrations of the PI 3-kinase inhibitor, wortmannin (WT) . In the present study, we created a glutathione S-transferase-PI4Kbeta fusion protein for expression in Escherichia coli . The purified protein was biologically active and phosphorylated PI in its 4-position with WT sensitivity and kinetic parameters that were identical to those of purified bovine brain PI4Kbeta . In addition to its lipid kinase activity, the enzyme exhibited autophosphorylation that was enhanced by Mn(2+) ions and inhibited by WT and another PI 3-kinase inhibitor, LY 294002 . The recombinant protein was unable to transphosphorylate, but its isolated C-terminal catalytic domain still displayed autophosphorylation, suggesting that the autophosphorylation site resides within the C-terminal catalytic domain of the protein and is held in position by intramolecular interactions . Autophosphorylation inhibited subsequent lipid kinase activity, which was reversed upon dephosphorylation, by protein phosphatases, PP1 and PP2A(1), suggesting that it may represent a regulatory mechanism for the enzyme . Phosphorylation of endogenous or overexpressed PI4Kbeta was also observed in COS-7 cells; however, the in vivo phosphorylation of the expressed protein was only partially inhibited by WT and also occurred in a catalytically inactive form of the enzyme, indicating the presence of additional phosphorylation site(s) . Successful bacterial expression of PI4Kbeta should aid research on the structure-function relationships of this protein as well as of other, structurally related enzymes. J Biol Chem, 2000 May 12, 275(19), 14457 - 65 A 56-kDa selenium-binding protein participates in intra-Golgi protein transport; Porat A et al.; Transport of proteins between intracellular membrane compartments is a highly regulated process that depends on several cytosolic factors . By using the well characterized intra-Golgi cell-free transport assay, we purified from bovine brain cytosol a 56-kDa protein that shows a significant transport activity . Partial sequencing of four tryptic peptides obtained from the 56-kDa protein revealed its identity to a cytosolic protein previously characterized as a selenium-binding protein, SBP56 . Recombinant SBP56 expressed in Escherichia coli exhibited transport activity when added to the cell-free intra-Golgi transport . Affinity purified anti-SBP56 polyclonal antibodies specifically inhibited intra-Golgi transport in vitro . Although SBP56 is predominantly localized in the cytosol, a significant amount is associated with membranes . Subcellular fractionation showed that this protein is peripherally associated with the Golgi membrane . The experiments presented in this study indicate that SBP56 participates in late stages of intra-Golgi protein transport. J Biol Chem, 2000 May 12, 275(19), 14432 - 9 A map of protein-rRNA distribution in the 70 S Escherichia coli ribosome; Svergun DI et al.; Neutron scattering exploits the enormous scattering difference between protons and deuterons . A set of 42 x-ray and neutron solution scattering curves from hybrid Escherichia coli ribosomes was obtained, where the proteins and rRNA moieties in the subunits were either protonated or deuterated in all possible combinations . This extensive data set is analyzed using a novel method . The volume defined by the cryoelectron microscopic model of Frank and co-workers (Frank, J., Zhu, J., Penczek, P., Li, Y . H., Srivastava, S., Verschoor, A., Radermacher, M., Grassucci, R., Lata, R . K., and Agrawal, R . K . (1995) Nature 376, 441-444) is divided into 7890 densely packed spheres of radius 0.5 nm . Simulated annealing is employed to assign each sphere to solvent, protein, or rRNA moieties to simultaneously fit all scattering curves . Twelve independent reconstructions starting from random approximations yielded reproducible results . The resulting model at a resolution of 3 nm represents the volumes occupied by rRNA and protein moieties at 95% probability threshold and displays 15 and 20 protein subvolumes in the 30 S and 50 S, respectively, connected by rRNA . 17 proteins with known atomic structure can be tentatively positioned into the protein subvolumes within the ribosome in agreement with the results from other methods . The protein-rRNA map enlarges the basis for the models of the rRNA folding and can further help to localize proteins in high-resolution crystallographic density maps. J Biol Chem, 2000 May 12, 275(19), 14273 - 80 Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin; Pethe K et al.; The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end . Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding . Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes . Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein . Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface . The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells . Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm . Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA . The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis. J Biol Chem, 2000 May 12, 275(19), 14264 - 72 The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase; Milon L et al.; We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M . L . (1997) Hum . Genet . 99, 550-557) . Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting . The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity . X-ray crystallographic analysis was performed on active truncated Nm23-H4 . Like other eukaryotic NDP kinases, it is a hexamer . Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase . The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions . Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized . Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells . Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity . Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes. J Biol Chem, 2000 May 12, 275(19), 14102 - 6 A new SUMO-1-specific protease, SUSP1, that is highly expressed in reproductive organs; Kim KI et al.; A full-length cDNA encoding a SUMO-1-specific protease, named SUSP1, was identified and cloned for the first time from the human brain . Nucleotide sequence analysis of the cDNA containing an open reading frame of 3336 base pairs revealed that the protease consists of 1112 amino acids with a calculated molecular mass of 126,116 Da . Like yeast Ulp1, SUSP1 is a cysteine protease containing the well conserved His/Asp/Cys catalytic triad . SUSP1 expressed in Escherichia coli cells efficiently released SUMO-1 from SUMO-1 . beta-galactosidase fusion but not from other ubiquitin-like protein fusions, including Smt3.beta-galactosidase, suggesting its role in the generation of matured SUMO-1 specifically from its precursors . Interestingly, reproductive organs, such as testis, ovary, and prostate, contained much higher amounts of SUSP1 mRNA than colon and peripheral blood leukocyte, whereas other tissues, such as heart and spleen, had little or none . In addition, confocal microscopy using green fluorescent protein.SUSP1 fusion showed that SUSP1 is exclusively localized to the cytoplasm of NIH3T3 and HeLa cells . These results suggest that SUSP1 may play a role in the regulation of SUMO-1-mediated cellular processes particularly related to reproduction. J Biol Chem, 2000 May 12, 275(19), 14046 - 55 Membrane type 4 matrix metalloproteinase (MMP17) has tumor necrosis factor-alpha convertase activity but does not activate pro-MMP2; English WR et al.; Membrane type 4 matrix metalloproteinase (MT4-MMP) shows the least sequence homology to the other MT-MMPs, suggesting a distinct function for this protein . We have isolated a complete cDNA corresponding to the mouse homologue which includes the signal peptide and a complete pro-domain, features that were lacking from the human form originally isolated . Mouse MT4-MMP (mMT4-MMP) expressed in COS-7 cells is located at the cell surface but does not show ability to activate pro-MMP2 . The pro-catalytic domain was expressed in Escherichia coli as insoluble inclusions and active enzyme recovered after refolding . Activity of the isolated catalytic domain against synthetic peptides commonly used for MMP enzyme assays could be inhibited by TIMP1, -2, and -3 . The recombinant mMT4-MMP catalytic domain was also unable to activate pro-MMP2 and was very poor at hydrolyzing components of the extracellular matrix with the exception of fibrinogen and fibrin . mMT4-MMP was able to hydrolyze efficiently a peptide consisting of the pro-tumor necrosis factor alpha (TNFalpha) cleavage site, a glutathione S-transferase-pro-TNFalpha fusion protein, and was found to shed pro-TNFalpha when co-transfected in COS-7 cells . MT4-MMP was detected by Western blot in monocyte/macrophage cell lines which in combination with its fibrinolytic and TNFalpha-converting activity suggests a role in inflammation. Clin Diagn Lab Immunol, 2000 May, 7(3), 501 - 3 Intestinal secretory immunoglobulin A response to enteroaggregative Escherichia coli in travelers with diarrhea; Sutjita M et al.; We examined stool samples from travelers for secretory immunoglobulin A (sIgA) to enteroaggregative Escherichia coli (EAEC) during episodes of acute diarrhea . Ten paired samples from 10 patients with diarrhea caused by EAEC were examined for the presence of specific sIgA by dot blot and Western blot immunoassays . Five samples were positive by dot blotting, and two samples were positive by Western blotting. Biochem Biophys Res Commun, 2000 May 10, 271(2), 518 - 25 ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation; Howell LD et al.; Residues 417-830 of the cystic fibrosis transmembrane conductance regulator (CFTR) were expressed as a glutathione-S-transferase fusion protein . This fusion protein, NBD1/R/GST, contains the regulatory and first nucleotide binding domains of CFTR . NBD1/R/GST hydrolyzed ATP with a K(M) (60 microM) and V(max) (330 nmol/min/mg) that differed from those reported for CFTR and for a peptide containing CFTR residues 433-589 . The ATPase inhibitor profile of NBD1/R/GST indicates that CFTR resembles P-glycoprotein with respect to the NBD1 ATPase catalytic mechanism . ATP hydrolysis by NBD1/R/GST was unaffected by genistein, glybenclamide, and other agents known to affect CFTR's chloride channel function, suggesting that these agents do not act by directly influencing the ATPase function of NBD1 . The disease-causing mutation, G551D, reduced ATP hydrolysis by NBD1/R/GST by increasing the K(M) for ATP fourfold . This suggests that when G551D occurs in patients with cystic fibrosis, it affects CFTR function by reducing the affinity of NBD1 for ATP . Biochem Biophys Res Commun, 2000 May 10, 271(2), 292 - 8 Stable interdomain interaction within the cytoplasmic domain of CD45 increases enzyme stability; Felberg J et al.; CD45 is a leukocyte-specific, two domain transmembrane tyrosine phosphatase . Co-purification of a recombinant protein containing the first phosphatase domain of CD45 (6His-D1) with a recombinant protein containing the second phosphatase domain (GST-D2) from E . coli indicated a stable interaction which resulted in increased stability of the active phosphatase domain present in 6His-D1 . This interaction was not dependent on the acidic region unique to CD45 domain 2, but was affected by a destabilizing point mutation (Q1180G) in GST-D2 . CD45 domain 2 enhanced phosphatase activity of the first domain in the full length cytoplasmic domain protein, whereas a chimeric protein with the SH2 domain of p56(lck) in place of the CD45 C-terminal region did not . Thus the C-terminal domain of CD45 associates with the N-terminal domain and this stabilizes the active phosphatase domain . A single destabilizing point mutation in the second domain is sufficient to attenuate this effect . Microb Pathog, 2000 May, 28(5), 291 - 300 Presence of the LEE (locus of enterocyte effacement) in pig attaching and effacing Escherichia coli and characterization of eae, espA, espB and espD genes of PEPEC (pig EPEC) strain 1390; An H et al.; In the present study, attaching and effacing Escherichia coli (AEEC) O45 isolates from post-weaning pigs with diarrhoea were examined for the presence of the LEE (locus of enterocyte effacement) using various DNA probes derived from the LEE of human enteropathogenic E . coli (EPEC) strain E2348/69 . The LEE fragment was conserved among the eae -positive pig isolates . The attaching and effacing activity of PEPEC (pig EPEC) O45 isolates is highly correlated with the presence of the LEE . Nevertheless, for some PEPEC isolates, the insertion site of the LEE is different or has diverged during evolution . The presence of the LEE fragment in PEPEC isolates provides further evidence that the LEE region is conserved among AEEC of different animal origins . In addition, the nucleotide sequence of the region containing the eae gene and esp genes of a pig AEEC isolate, strain 1390, was determined . Among examined Eae proteins, Eae of strain 1390 showed the highest similarity with Eae belonging to the beta intimin group such as the Eae of rabbit AEEC . Moreover, all pig strains that produced attaching and effacing lesions in piglets and pig ileal explants belonged to the beta intimin group . The deduced amino acid sequences of the EspA, EspB and EspD proteins of strain 1390 showed particularly strong homology to those of AEEC strains presenting a beta intimin allele . Thus, pig AEEC possess the LEE sequences, and for the strain 1390, sequences of the eae and esp regions are related to those of other AEEC, in particular, strains presenting a beta intimin allele, such as the rabbit AEEC . Invest Ophthalmol Vis Sci, 2000 May, 41(6), 1392 - 401 Herpes simplex virus-mediated gene delivery to the rodent visual system; Spencer B et al.; PURPOSE: To determine the types of cells in the visual system of the mouse and rat that can express a transgene delivered by an attenuated replication competent Herpes simplex virus-1 (HSV-1) vector . METHODS: C57/BL6 x BALB/C mice and Albino rats were treated with 1 x 10(7) pfu of the HSV-1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene . The hrR3 virus was delivered by topical application to the cornea, intravitreal (IV) injection, intracameral injection (IC), or stereotactic injection into the visual cortex (VC) . At specified times postinfection, animals were killed and tissues were removed, fixed, sectioned, and stained with X-gal or hematoxylin and eosin for histochemical and histopathologic examination . RESULTS: Topical delivery after corneal scarification in both mouse and rat resulted in lacZ expression in 25% of the corneal epithelial cells and 25% of the retinal pigment epithelium (RPE) cells . Topical application without concurrent scarification also resulted in transgene delivery to 20% of the RPE cells of the rat but not the mouse . IV injection resulted in expression primarily in RPE cells, with up to 100% of the cells expressing lacZ in the mouse and rat . Other cells expressing the transgene included ciliary body (CB) and optic nerve cells . Up to 25% of the retinal ganglion cells in the rat expressed lacZ, but only rarely in mice . IC delivery in rats resulted in expression in trabecular meshwork, CB cells, RPE, and iris epithelium . Injection into area 17 of the rat VC resulted in efficient labeling of the VC neurons and neurons in the lateral geniculate nucleus without any evident pathology or inflammation . Neither inflammation nor disease pathology was observed in either the mouse or rat after any route of delivery . CONCLUSIONS: It was demonstrated that the hrR3 HSV-1 virus can deliver a functioning gene to several cell types in the eye and neurons in the VC and that the virus can move via retrograde transport to nuclei that project to the VC. Plant Mol Biol, 2000 Feb, 42(3), 415 - 27 Plant homologue of flap endonuclease-1: molecular cloning, characterization, and evidence of expression in meristematic tissues; Kimura S et al.; Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair . We isolated a 1.4 kb cDNA from rice (Oryza sativa), termed OsFEN-1, encoding a protein which shows homology with the eukaryotic FEN-1 proteins . OsFEN-1 protein was overexpressed in Escherichia coli and purified to near homogeneity . DNA cleavage analysis using different branched DNA structures indicated that OsFEN-1 protein possesses both 5'-flap endonuclease and 5' to 3' double-stranded DNA exonuclease activities . OsFEN-1 protein incises a 5'-flap and 5'-pseudo Y structure one base 3' of the branched point in the duplex region . The enzymatic properties indicated that we succeeded in obtaining the gene and the protein of a plant counterpart of FEN-1 . OsFEN-1 transcripts were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively . No expression was detected in mature leaves although the leaves were exposed to UV . We analyzed the spatial distribution pattern of OsFEN-1 transcripts by in situ hybridization . In the shoot apex, OsFEN-1 mRNA was abundant in the shoot apical meristem, tiller bud, leaf primordia, ligule primordia and marginal meristem of young leaves . In the roots, the transcript accumulated to high levels in the root apical meristem . Our results indicate that OsFEN-1 is expressed in tissues rich in proliferating cells, and its expression may be required for cell growth and organ formation. J Med Microbiol, 2000 May, 49(5), 409 - 13 Altered expression of oligopeptide-binding protein (OppA) and aminoglycoside resistance in laboratory and clinical Escherichia coli strains; Acosta MB et al.; Oligopeptide-binding protein (OppA) is the periplasmic component of the major oligopeptide transport system of enteric bacteria . Genetic and biochemical evidence suggests that OppA plays a role in the uptake of aminoglycoside antibiotics in Escherichia coli K-12 . Forty-six (82%) of 56 aminoglycoside-resistant mutants of E . coli K-12 selected in vitro had reduced or undetectable OppA levels, as compared with their parent strain . Moreover, nine (36%) of 25 aminoglycoside-resistant clinical isolates of E . coli expressed reduced or undetectable levels of OppA . No decrease in OppA expression was observed among aminoglycoside-sensitive E . coli strains from patients . Twenty-three (42%) of 56 aminoglycoside-resistant mutants of E . coli K-12 and six (24%) of 25 clinical isolates also were deficient for expression of ornithine or arginine decarboxylases, or both, and these deficiencies might negatively affect OppA expression by reducing polyamine synthesis . These results support the view that reduced OppA expression is associated with aminoglycoside resistance in E . coli strains. DNA Cell Biol, 2000 Apr, 19(4), 235 - 41 A triplex-mediated knot between separated polypurine-polypyrimidine tracts in circular DNA blocks transcription by Escherichia coli RNA polymerase; Ashley C et al.; Polypurine-polypyrimidine tracts are overrepresented in eukaryotes and many have the potential to form triplex DNA . Transmolecular triplexes form between separated but complementary polypurine-polypyrimidine tracts in duplex DNA . Transmolecular triplexes (T-loops) were studied previously using a circular plasmid containing a pair of separated polypurine-polypyrimidine tracts designed to able to form a triplex with each other . T-Loops formed when the nicked plasmid was incubated at low pH in the presence of spermine . When the pH was raised to 8, the T-loops were constrained by a hydrogen-bonded knot composed of multistranded and single-stranded regions . The present experiments used T-loops as a model system to investigate the influence of transmolecular triplex formation on transcription . T-Loops and control open circular, linear, and supercoiled plasmid forms were isolated from bands on agarose gels . Transcription assays were carried out with the isolated plasmid forms and Escherichia coli RNA polymerase holoenzyme and the core enzyme, which lacked sigma70 . Transcription was significantly inhibited in T-loop forms compared with control plasmid forms . There was no evidence that the single-stranded regions of T-loops facilitated nonspecific initiation of transcription . Instead, the multistranded component of the hydrogen-bonded knot at the root of the T-loop structure inhibited transcription. Neuron, 2000 Apr, 26(1), 143 - 54 Structure of the Homer EVH1 domain-peptide complex reveals a new twist in polyproline recognition; Beneken J et al.; Homer EVH1 (Ena/VASP Homology 1) domains interact with proline-rich motifs in the cytoplasmic regions of group 1 metabotropic glutamate receptors (mGluRs), inositol-1,4,5-trisphosphate receptors (IP3Rs), and Shank proteins . We have determined the crystal structure of the Homer EVH1 domain complexed with a peptide from mGluR (TPPSPF) . In contrast to other EVH1 domains, the bound mGluR ligand assumes an unusual conformation in which the side chains of the Ser-Pro tandem are oriented away from the Homer surface, and the Phe forms a unique contact . This unusual binding mode rationalizes conserved features of both Homer and Homer ligands that are not shared by other EVH1 domains . Site-directed mutagenesis confirms the importance of specific Homer residues for ligand binding . These results establish a molecular basis for understanding the biological properties of Homer-ligand complexes. J Pept Res, 2000 Apr, 55(4), 318 - 24 Isolation of leptin-binding peptides from a random peptide phage library; Chan KW et al.; Leptin plays a role in regulating the body weight in mice . Injection of recombinant mouse leptin expressed in Escherichia coli reduced the food intake and body weight in normal, ob/ob and diet-induced obesity mice . Hyperglycemia, hyperinsulinemia and hypothermia can also be corrected in ob/ob mice after leptin injection . Leptin is a 16-kDa secretory protein comprising 167 amino acids produced in adipose tissue and is secreted to blood stream . In this study, a recombinant mouse leptin was generated and purified from a baculovirus expression system . This protein was used to identify putative ligands using a phage library of random peptides . Three leptin-binding phage clones were found, which were characterized by DNA sequencing and ELISA methods . The amino acid sequences of the reactive peptides are: LAYCSDPVRCLVWWY, MFWISAVSFVDHALV and LVLVLSAFLCCGVG . All three clones bound to recombinant human and mouse leptins . These peptides may be useful tools to study leptin-receptor interaction, food intake and body weight regulation. J Clin Lab Anal, 2000, 14(3), 97 - 100 Availability of Nagarse for DNA analysis as a substitute for proteinase K; Yokota M et al.; The availability of Nagarse, a protease, as a substitute for proteinase K for digestion of leukocytic or bacterial DNAs was studied . The amount and purity of DNAs extracted from leukocytes and several bacterial strains with Nagarse were compared with those of DNAs treated with proteinase . Nagarse exhibited the same behavior as proteinase K in digesting leukocytes, and it could also be used for bacterial digestion for physical mapping of genomic DNA by biased sinusoid field gel electrophoresis . Nagarse was thus comparable to proteinase K for use in biochemical experiments . The principal advantage of Nagarse is that it is inexpensive, unlike proteinase K, and our findings indicated that Nagarse is very useful as a substitute for proteinase K for the DNA study . J Med Virol, 2000 Jun, 61(2), 266 - 74 Antigenic properties and diagnostic potential of recombinant dobrava virus nucleocapsid protein; Kallio-Kokko H et al.; Dobrava hantavirus (DOBV) causes severe hemorrhagic fever with renal syndrome in the Balkan region and has been detected recently also in Russia, Estonia, and Germany . DOBV nucleocapsid protein (N) was produced in insect cells, using the baculovirus expression system (bac-DOBV-N), and in E . coli as a truncated (aa 1-165) glutathione-S transferase fusion protein (DOBV-dN-GST) . The antigenic properties of bac-DOBV-N were found identical to native DOBV-N when examined by a panel of hantavirus-specific monoclonal antibodies . Enzyme immunoassays for detection of IgM and IgG antibodies were set up using DOBV recombinant N proteins and compared with those based on recombinant Hantaan and Puumala virus N, using panels of sera collected from DOBV, Hantaan and Puumala virus-infected patients . Full-length N protein (bac-DOBV-N) was found to be a more sensitive antigen than DOBV-dN-GST . The sensitivity values for sera from DOBV-infected patients were 100% for bac-DOBV-N and 86% for DOBV-dN-GST by IgM assays, and 98% for bac-DOBV-N and 88% for DOBV-dN-GST by IgG assays . The specificity values were 100% for bac-DOBV-N and 99% for DOBV-dN-GST by IgM assays, and 100% for both antigens by IgG assays . J Med Virol, 2000 Jun, 61(2), 228 - 34 Expression of capsid {correction of caspid} protein VP1 for use as antigen for the diagnosis of enterovirus 71 infection; Shih SR et al.; To produce enterovirus 71 antigen for diagnostic purposes, the gene encoding the entire capsid protein VP1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and expressed in Escherichia coli as a poly-histidine fusion protein . Western blotting experiments with sera from patients with enterovirus 71 infection indicated that immunoglobulin G (IgG) and IgM antibodies bound to a single polypeptide VP1 . According to these results, IgM anti-VP1 appeared in sera of patients with a symptomatic enterovirus 71 acute infection, whereas IgG anti-VP1 was present in sera of past infection . This finding suggests that detecting IgG and IgM immune responses against linear epitopes of recombinant VP1 is an effective means of determining the different phases of enterovirus 71 infection . In addition, sera containing coxsackie virus 16 (CA16) antibodies did not cross-react with the recombinant VP1 of enterovirus 71, despite the homology between VP1 proteins of both viruses . Comparison with reference PCR and neutralization assays showed these antibody tests to be appropriate for the serodiagnosis of enterovirus 71 infection . Biotechnol Bioeng, 2000 Jun 5, 68(5), 576 - 83 Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography; Diogo MM et al.; The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of plasmid DNA according to defined specifications . A new method is described for the purification of a cystic fibrosis plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether . The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded plasmid DNA . RNA, denatured genomic and plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled plasmid, were retained and separated from nonbinding plasmid DNA in a 14-cm HIC column . Anion-exchange HPLC analysis proved that >70% of the loaded plasmid was recovered after HIC . RNA and denatured plasmid in the final plasmid preparation were undetectable by agarose electrophoresis . Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively) . The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold . Host proteins were not detected in the final preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining . Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments . The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns . Biomol Eng, 1999 Dec 31, 16(1-4), 119 - 25 In vivo enzymatic protein biotinylation; Chapman-Smith A et al.; Biotin is biologically active only when protein-bound and is covalently attached to a class of important metabolic enzymes, the biotin carboxylases and decarboxylases . Biotinylation is a relatively rare modification, with between one and five biotinylated protein species found in different organisms . We discuss the mechanism and structures involved in this extraordinarily specific protein modification and its exploitation in tagging recombinant proteins. Biomol Eng, 1999 Dec 31, 16(1-4), 87 - 92 Recombinant avidin and avidin-fusion proteins; Airenne KJ et al.; Both chicken egg-white avidin and its bacterial relative streptavidin are well known for their extraordinary high affinity with biotin (Kd approximately 10(-15) M) . They are widely used as tools in a number of affinity-based separations, in diagnostic assays and in a variety of other applications . These methods have collectively become known as (strept)avidin-biotin technology . Biotin can easily and effectively be attached to different molecules, termed binders and probes, without destroying their biological activity . The exceptional stability of the avidin-biotin complex and the wide range of commercially available reagents explain the popularity of this system . In order by genetic engineering to modify the unwanted properties of avidin and to further expand the existing avidin-biotin technology, production systems for recombinant avidin and avidin-fusion proteins have been established . This review article presents an overview of the current status of these systems . Future trends in the production and applications of recombinant avidin and avidin-fusion proteins are also discussed. Arch Microbiol, 2000 Feb, 173(2), 110 - 6 Analysis of the cleavage site specificity of the endopeptidase involved in the maturation of the large subunit of hydrogenase 3 from Escherichia coli; Theodoratou E et al.; The maturation of {NiFe}-hydrogenases is a catalysed process in which the activities of at least seven proteins are involved . The last step consists of the endoproteolytic cleavage of the precursor of the large subunit after the {NiFe}-metal centre has been assembled . The amino acid sequence requirements for the endopeptidase HycI involved in the C-terminal processing of HycE, the large subunit of the hydrogenase 3 from Escherichia coli, were investigated . Mutational alteration of the amino acid residues neighbouring the cleavage site showed that proteolysis still occurred when chemically similar amino acids were exchanged . Processing was blocked, however, in a variant in which the methionine at the C-terminal side was replaced by a glutamate residue . Truncation of the precursor from the C-terminal end rendered variants amenable to maturation even when two-thirds of the extension were removed but abolished proteolysis upon further deletion of a cluster of six basic amino acids . A construct in which the C-terminal extension from the large subunit of the hydrogenase 2 was fused to the mature part of the large subunit of hydrogenase 3 was neither processed by HycI nor by HybD, the endopeptidase specific for the large subunit of hydrogenase 2 . The maturation endopeptidase, therefore, exhibits a relaxed sequence constraint in recognition of its cleavage site and does not require the entire C-terminal extension . The results point to an interaction of the C-terminus with some domain of the large subunit, rendering a conformation amenable to recognition by the endopeptidase. Arch Virol, 2000, 145(3), 641 - 50 Molecular characterization and coat protein serology of watermelon leaf mottle virus (Potyvirus); De Sa PB et al.; A cDNA library was generated from purified RNA of watermelon leaf mottle virus (WLMV) (Genus Potyvirus) . Two overlapping clones totaling 2,316 nucleotides at the 3' terminus of the virus were identified by immunoscreening with coat protein antiserum . The sequence analyses of the clones indicated an open reading frame (ORF) of 2,050 nucleotides which encoded part of the replicase and the coat protein, a 243-nucleotide non-coding region (3'UTR), and 23 adenine residues of the poly (A) tail . The taxonomic status of WLMV was determined by comparisons of the sequence of the cloned coat protein gene and 3'UTR with potyvirus sequences obtained from GenBank . The nucleotide sequence identities of WLMV compared with 17 other potyviruses ranged from 55.6 to 63.5% for the coat protein, and from 37.2 to 48.3% for the 3'UTR . Phylogenetic analyses of the coat protein region and the 3'UTR indicated that WLMV did not cluster with other potyviruses in a clade with high bootstrap support . The coat protein gene was expressed in Escherichia coli and a polyclonal antiserum was prepared to the expressed coat protein . In immunodiffusion tests, WLMV was found to be serologically distinct from papaya ringspot virus type W, watermelon mosaic virus 2, zucchini yellow mosaic virus, and Moroccan watermelon mosaic virus . In Western blots and ELISA, serological cross-reactivity with other cucurbit potyviruses was observed . Serological and sequence comparisons indicated that watermelon leaf mottle virus is a distinct member of the Potyvirus genus. Plant Cell Physiol, 2000 Feb, 41(2), 192 - 9 Overexpression, enzymatic properties and tissue localization of a ferrochelatase of cucumber; Suzuki T et al.; Ferrochelatase catalyzes the insertion of Fe2+ into protoporphyrin IX to generate protoheme . A putative mature region of a cucumber ferrochelatase cDNA (hemH) was overexpressed in Escherichia coli and purified to homogeneity (40 kDa) . The optimum pH was 7.7, and the apparent K(m) values for deuteroporphyrin IX and Fe2+ were 14.4 microM and 4.7 microM, respectively . The activity of the ferrochelatase was inhibited by N-methylprotoporphyrin IX (I50 = 4 nM) . Western blot analysis with a polyclonal antibody raised against the recombinant ferrochelatase showed that the antibody crossreacted with protein extracts from hypocotyls and roots of cucumber but not with that from cotyledons . The antibody did not crossreact with proteins of thylakoid membranes of chloroplasts in cucumber cotyledons, although the ferrochelatase activity was mainly associated with the thylakoid membranes . Northern blot analysis also indicated that the hemH gene was expressed mainly in hypocotyls and roots, but little in cotyledons, and the level of the hemH transcripts was not light-responsive . These results demonstrated that the cucumber hemH gene encodes a ferrochelatase which presumably functions for heme biosynthesis in non-photosynthetic tissues, such as hypocotyls and roots, and suggested the presence of other types of ferrochelatase in cucumber, one of which is located in thylakoid membranes of chloroplasts. Plant Cell Physiol, 2000 Feb, 41(2), 185 - 91 A cysteine protease from maize isolated in a complex with cystatin; Yamada T et al.; We recently purified a latent but SDS-activated protease complex (40, 15- or 13-kDa proteins) from maize {Yamada et al . (1998) Plant Cell Physiol . 39: 106} . Here, we revealed that the complex was composed of a cysteine protease (40 kDa) and a cystatin, cysteine protease inhibitor (15- or 13-kDa) . This is the first report on the isolation of a complex consisting of a cystatin and a target cysteine protease from plants . Cloning of the cysteine protease revealed that it had low homology (25-30%) to other maize cysteine proteases cloned to date but was highly homologous to other plant cysteine proteases such as rice oryzain alpha (84%) and the homologs (50-80%) . The cysteine protease expressed in Escherichia coli showed the same substrate and inhibitor specificities as the protease of the complex, demonstrating that the isolated cDNA clone exactly encodes the protease of the complex . The protease expressed in E . coli itself was active but not latent, probably because it was not bound to cystatin . It is most likely that in vitro activation of the protease complex by SDS is caused by the release of bound cystatin . The mRNA of protease was expressed in various tissues except for seeds. Clin Chem, 2000 May, 46(5), 658 - 66 Dual-label time-resolved immunofluorometric assay of free and total prostate-specific antigen based on recombinant Fab fragments; Eriksson S et al.; BACKGROUND: Recombinant Fab fragments are attractive as reagents for novel sandwich immunoassays, but no such assays have been described . We developed a dual-label two-site immunoassay based entirely on recombinant Fab fragments and compared it to the same assay with intact monoclonal antibodies . METHODS: The capture Fab fragment, which binds free prostate-specific antigen (PSA) and PSA in complex with alpha(1)-antichymotrypsin on an equimolar basis, is site-specifically biotinylated and attached to the solid phase in streptavidin-coated microtitration wells . The Fab fragment that detects only free PSA is site-specifically labeled with a fluorescent europium chelate, and the Fab fragment that detects both free and serpin-complexed PSA in an equimolar fashion is labeled with a fluorescent terbium chelate . Time-resolved fluorescence is used to measure both europium and terbium signals in one well . RESULTS: The detection limits of the assay (mean + 3 SD of zero calibrator) were 0.043 and 0.28 microgram/L, respectively, for free and total PSA . The within-run and day-to-day CVs were 2-11% and 4-10%, respectively . Mean recoveries were 93% and 98% in female and male sera, respectively . Compared with the commercial ProStatus PSA Free/Total Assay, the intercepts of the regression equations (r >0 . 99) were not significantly different from zero, and the slopes were 0.95-1.01 . In one female serum sample, PSA was falsely increased with the monoclonal assay but was undetectable with the recombinant assay . CONCLUSIONS: The performance of this novel assay based on recombinant components is comparable to a conventional assay based on monoclonal antibodies . The more complete control of the essential characteristics of site-specifically derivatized recombinant Fab fragments will be valuable for the design of miniaturized and multianalyte assay concepts where correct antibody orientation, density, and capacity as well as uncompromised binding affinity are required. Biochem J, 2000 May 15, 348 Pt 1, 167 - 72 Designing transthyretin mutants affecting tetrameric structure: implications in amyloidogenicity; Redondo C et al.; The molecular mechanisms that convert soluble transthyretin (TTR) tetramers into insoluble amyloid fibrils are still unknown; dissociation of the TTR tetramer is a pre-requisite for amyloid formation in vitro and involvement of monomers and/or dimers in fibril formation has been suggested by structural studies . We have designed four mutated proteins with the purpose of stabilizing {Ser(117)-->Cys (S117C) and Glu(92)-->Cys (E92C)} or destabilizing {Asp(18)-->Asn (D18N) and Leu(110)-->Ala (D110A)} the dimer/tetramer interactions in TTR, aiming at elucidating structural determinants in amyloidogenesis . The resistance of the mutated proteins to dissociation was analysed by HPLC studies of diluted TTR preparations . Both 'stabilized' mutants migrated as tetramers and, upon dilution, no other TTR species was observed, confirming the increased resistance to dissociation . For the 'destabilized' mutants, a mixture of tetrameric and monomeric forms co-existed at low dilution and the latter increased upon 10-fold dilution . Both of the destabilizing mutants formed amyloid in vitro when acidified . This result indicated that both the AB loop of TTR, destabilized in D18N, and the hydrophobic interactions affecting the dimer-dimer interfaces in L110A are implicated in the stability of the tetrameric structure . The stabilized mutants, which were dimeric in nature through disulphide bonding, were unable to polymerize into amyloid, even at pH 3.2 . When the amyloid formation assay was repeated in the presence of 2-mercaptoethanol, upon disruption of the S-S bridges of these stable dimers, amyloid fibril formation was observed . This experimental evidence suggests that monomers, rather than dimers, are the repeating structural subunit comprising the amyloid fibrils. Biochem J, 2000 May 15, 348 Pt 1, 145 - 50 Inhibition by long-chain acyl-CoAs of glucose 6-phosphate metabolism in plastids isolated from developing embryos of oilseed rape (Brassica napus L.); Johnson PE et al.; The effects of long-chain acyl-CoA (lcACoA) esters (both added exogenously and synthesized de novo) and acyl-CoA binding protein (ACBP) on plastidial glucose 6-phosphate (Glc6P) and pyruvate metabolism were examined using isolated plastids . The binding of lcACoA esters by ACBP stimulated the utilization of Glc6P for fatty acid synthesis, starch synthesis and reductant supply via the oxidative pentose phosphate (OPP) pathway . Stimulation occurred at low (1-10 microM) concentrations of ACBP . Pyruvate-dependent fatty acid synthesis was not directly affected by ACBP . However, addition of ACBP did increase the Glc6P-dependent stimulation of pyruvate utilization mediated through the OPP pathway . On the basis of these experiments, we conclude that lcACoA esters may inhibit Glc6P uptake into plastids, and that this inhibition is relieved by ACBP . We also suggest that utilization of other substrates for fatty acid synthesis may be affected by lcACoA/ACBP via their effects on the OPP pathway. Biochem J, 2000 May 15, 348 Pt 1, 119 - 28 Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms; Toutant M et al.; In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing . Two exons code for additional peptides of six and seven residues ('boxes' 6 and 7), located on either side of Tyr(397), which increase its autophosphorylation . Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons . The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells . Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr(397), a critical residue for the activation and function of FAK . The presence of boxes 6 and 7 increased autophosphorylation of Tyr(397) independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr) . Src-family kinases were also able to phosphorylate Tyr(397) in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both . Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr(397) . They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases. IUBMB Life, 1999 Aug, 48(2), 215 - 8 Involvement of ArcA and Fnr in expression of Escherichia coli thiol peroxidase gene; Kim SJ et al.; To explore the oxygen response regulators involved in thiol peroxidase gene (tpx) expression in Escherichia coli, we constructed a single-copy tpx-lacZ operon fusion and monitored tpx-lacZ expression in various genetic backgrounds . Expression of the tpx-lacZ fusion was increased 4-fold by aerobic growth . Anaerobic expression of tpx-lacZ in either (delta)arcA or delta(fnr) strains was 2.5-fold depressed compared with that of the wild-type strain . The results of immunoblotting experiments also demonstrated that ArcA and Fnr regulatory proteins repressed thiol peroxidase gene expression during anaerobic growth . Inspection of the tpx promoter region revealed putative binding sites for ArcA and Fnr . It thus appears that ArcA and Fnr function as repressors by blocking the binding of RNA polymerase to the tpx promoter in E . coli under anaerobic growth conditions. IUBMB Life, 1999 Aug, 48(2), 157 - 61 Superoxide and iron: partners in crime; Liochev SI et al.; Superoxide (O2-) poses multiple threats, which are diminished by a family of metalloenzymes, the superoxide dismutases . Among the damaging effects of O2- are direct oxidation of low-molecular-weight reductants; inactivation of a select group of enzymes; and reaction with NO to yield the strong oxidant, peroxynitrite . Of even greater import is the ability of O2- to univalently oxidize the {4 Fe-4 S} clusters of dehydratases, which causes release of iron . The "free" iron, which is kept reduced by cellular reductants, then reduces hydroperoxides to hydroxyl or alkoxyl radicals . Because the "free" iron will preferentially bind to anionic polymers, such as nucleic acids, or to anionic surfaces, such as cell membranes, these radicals will be generated adjacent to these vital targets and will preferentially attack them . O2- and iron can thus be viewed as partners in crime, and reciprocal regulatory effects between iron and O2- may be anticipated . These are discussed. Plant Mol Biol, 2000 Jan, 42(2), 377 - 86 Cloning of beta-1,3-glucanases expressed during Cichorium somatic embryogenesis; Helleboid S et al.; Three different beta-1,3-glucanase cDNA fragments, CG1, CG2 and CG3, were obtained by RT-PCR from RNA isolated from Cichorium hybrid '474' leaf fragments cultured for 11 days under somatic embryogenesis-inducing conditions . When expressed in Escherichia coli the proteins encoded by the three cDNAs were recognized by antibodies raised against 38 kDa extracellular beta-1,3-glucanases studied previously (Helleboid et al., Planta 205 (1998) 56-63) . The CG2 and CG3 cDNAs may represent expressed alleles of one gene because their sequences showed a very high identity (98.5%) and are only 70% identical with CG1 . Southern blot analysis revealed the presence of 3-4 genes coding for beta-1,3-glucanases in the Cichorium genome . Expression analysis of the genes corresponding to the three clones analysed by semi-quantitative RT-PCR indicated that CG1 mRNAs were only detectable in Cichorium hybrid '474' leaf fragments from day 3 of somatic embryogenesis induction, whereas CG2-CG3 mRNAs were already present in non-induced leaf tissue of both the embryogenic hybrid '474' and a non-embryogenic genotype . The level of CG1 mRNAs was particularly high when embryogenic cells were dividing to produce embryos, and when the amount of callose deposited in cell walls surrounding embryogenic cells and young embryos decreased . These results indicate that expression of the CG1 gene is correlated to the somatic embryogenesis process and that it encodes a 38 kDa beta-1,3-glucanase protein that may be involved in the degradation of callose localized around embryogenic cells and young embryos . A full-length CG1 cDNA clone was obtained using 3' and 5' RACE-PCR, and its sequence revealed that it encodes a beta-1,3-glucanase that is equally homologous to both class III and class IV plant beta-1,3-glucanases. Protein Sci, 2000 Apr, 9(4), 721 - 33 NMR solution structure of the theta subunit of DNA polymerase III from Escherichia coli; Keniry MA et al.; The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits (alpha, epsilon, and theta) . The theta subunit is the smallest, but the least understood of the three . As a first step in a program aimed at understanding its function, the structure of the theta subunit has been determined by triple-resonance multidimensional NMR spectroscopy . Although only a small protein, theta was difficult to assign fully because approximately one-third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange . The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3J(HN alpha) coupling constants and the consensus chemical shift index . The C-terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well-defined secondary structure and exists in a highly dynamic state . The N-terminal two-thirds has three helical segments (Gln10-Asp19, Glu38-Glu43, and His47-Glu54), one short extended segment (Pro34-Ala37), and a long loop (Ala20-Glu29), of which part may undergo intermediate conformational exchange . Solution of the three-dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of theta is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues . Preliminary chemical shift mapping experiments with a domain of the epsilon subunit have identified a loop region (Ala20-Glu29) in theta as the site of association with epsilon. Protein Sci, 2000 Apr, 9(4), 713 - 20 Slow conformational dynamics of an endonuclease persist in its complex with its natural protein inhibitor; Whittaker SB et al.; The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain . Double- and triple-resonance NMR spectra of the isolated DNase domain uniformly labeled with 13C/15N bound to unlabeled Im9 contain more signals than expected for a single DNase conformer, consistent with the bound DNase being present in more than one form . The presence of chemical exchange cross peaks in 750 MHz 15N-1H-15N HSQC-NOESY-HSQC spectra for backbone NH groups of Asp20, Lys21, Trp22, Leu23, Lys69, and Asn70 showed that the bound DNase was in dynamic exchange . The rate of exchange from the major to the minor form was determined to be 1.1 +/- 0.2 s(-1) at 298 K . Previous NMR studies have shown that the free DNase interchanges between two conformers with a forward rate constant of 1.61 +/- 0.11 s(-1) at 288 K, and that the bound Im9 is fixed in one conformation . The NMR studies of the bound DNase show that Im9 binds similarly to both conformers of the DNase and that the buried Trp22 is involved in the dynamic process . For the free DNase, all NH groups within a 9 A radius of any point of the Trp22 ring exhibit heterogeneity suggesting that a rearrangement of the position of this side chain is connected with the conformational interchange . The possible functional significance of this feature of the DNase is discussed. Theor Popul Biol, 2000 Mar, 57(2), 131 - 44 Competition by allelopathy proceeds in traveling waves: colicin-immune strain aids colicin-sensitive strain; Nakamaru M et al.; Producing toxic chemicals to suppress both the growth and survivorship of local competitors is called allelopathy; some strains of the bacteria Escherichia coli produce a toxin (named colicin) which may kill colicin-sensitive neighbors while they themselves are immune . In a previous paper, the competitive outcome between colicin-producing and colicin-sensitive strains was shown to differ between a spatially structured and a completely mixed population . In this paper, we analyze the role of a third, "colicin-immune," strain, which does not produce colicin but is immune to it . Without spatial structure, the colicin-immune strain suppresses the colicin-producing strain and enables the colicin-sensitive strain to win . In a spatially structured population, modeled as a reaction-diffusion system, we examine the speed of boundaries between areas dominated by different strains in traveling waves and the events after the collision of two such boundaries . The colicin-immune strain passes through the area dominated by the colicin-sensitive strain and drives the colicin-producing strain to extinction . Subsequently the colicin-sensitive strain occupies the whole population . Mol Microbiol, 2000 Apr, 36(2), 457 - 66 Effects of local transcription and H-NS on inversion of the fim switch of Escherichia coli; O'gara JP et al.; The fim switch of Escherichia coli is responsible for phase-variable expression of type 1 fimbriae . Switching in the ON-to-OFF and OFF-to-ON directions is promoted by the FimB recombinase, while the FimE recombinase directs switching predominantly in the ON-to-OFF direction . The effects of local promoter activity and the H-NS nucleoid-associated protein on inversion of the switch were assessed . In contrast to FimB-mediated inversion, inversion of the switch by the FimE recombinase was unaffected by the H-NS status of the cell . Transcription towards the switch from within a translationally inactivated fimE gene was found to bias the switch strongly in the OFF direction, creating a FimE+-like phenotype in the absence of the FimE protein . This biasing was H-NS dependent and was also contingent on transcription from within the switch . These data show that local transcription and a nucleoid-associated protein both contribute to the modulation of a site-specific recombination event on the bacterial chromosome. Lett Appl Microbiol, 2000 Apr, 30(4), 298 - 302 The solubility of the ribotoxin alpha-sarcin, produced as a recombinant protein in Escherichia coli, is increased in the presence of thioredoxin; Garcia-Ortega L et al.; The yield of purified recombinant alpha-sarcin increases approximately three- to fourfold when this toxin is co-expressed in Escherichia coli with thioredoxin . This increased production is attributed to the existence, in the presence of thioredoxin, of a reducing environment which allows rearrangement of incorrect disulphide bonds to produce the soluble native conformation . The protein thus produced retains the structural, spectroscopic and enzymatic features of the natural fungal alpha-sarcin. Clin Exp Immunol, 2000 May, 120(2), 384 - 91 Fine specificity of autoantibodies to calreticulin: epitope mapping and characterization; Eggleton P et al.; Extracellular calreticulin (CRT) as well as anti-CRT antibodies have been reported in patients with various autoimmune disorders and CRT has been implicated in 'epitope spreading' to other autoantigens such as the Ro/SS-A complex . In addition, antibodies against parasite forms of the endoplasmic reticulum chaperone, CRT, have been found in patients suffering from onchocerciasis and schistosomiasis . In this study, we screened sera for anti-CRT antibodies from patients with active and inactive systemic lupus ertythematosus (SLE) and primary or secondary Sjogren's syndrome . Approximately 40% of all SLE patients were positive for anti-CRT antibodies . The antigenic regions of CRT were determined using full length CRT and fragments of CRT prepared in yeast and Escherichia coli, respectively . Synthetic 15mer peptides corresponding to the major autoantigenic region of CRT (amino acids 1-289), each one overlapping by 12 amino acids, were used to map the B cell epitopes on the CRT protein recognized by autoimmune sera . Major antigenic epitopes were found to be associated with the N-terminal half of the protein in 69% of the SLE sera from active disease patients, while the C-domain was not antigenic . Major epitopes were found to be reactive with antibodies in sera from SLE patients with both active and inactive disease, spanning different regions of the N and P-domains . Sera from both healthy and disease controls and primary Sjogren's syndrome patients were non-reactive to these sequences . Limited proteolysis of CRT with two major leucocyte serine proteases, elastase and cathepsin G, demonstrated that an N-terminal region of CRT is resistant to digestion . Interestingly, some of the epitopes with the highest reactivity belong to the fragments of the protein which bind to C1q and inhibit complement activation . Whether C1q association with CRT is a pathological or protective interaction between these two proteins is currently under investigation. Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5657 - 62 A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution; Ostedgaard LS et al.; Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity . To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708-831 of CFTR . Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional . Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis . The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer . The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8 . These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity. Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5119 - 22 Misactivated amino acids translocate at similar rates across surface of a tRNA synthetase; Nomanbhoy TK et al.; Certain aminoacyl-tRNA synthetases have a second active site that destroys (by hydrolysis) errors of amino acid activation . For example, isoleucyl-tRNA synthetase misactivates valine (to produce valyl adenylate or Val-tRNA(Ile)) at its active site . The misactivated amino acid is then translocated to an editing site located >25 A away . The role of the misactivated amino acid in determining the rate of translocation is not known . Valyl-tRNA synthetase, a close homolog of isoleucyl-tRNA synthetase, misactivates threonine, alpha-aminobutyrate, and cysteine . In this paper, we use a recently developed fluorescence-energy-transfer assay to study translocation of misactivated threonine, alpha-aminobutyrate, and cysteine . Although their rates of misactivation are clearly distinct, their rates of translocation are similar . Thus, the rate of translocation is independent of the nature of the misactivated amino acid . This result suggests that the misactivated amino acid per se has little or no role in directing translocation. IUBMB Life, 1999 Jul, 48(1), 49 - 55 Identification of a novel cytosolic tocopherol-binding protein: structure, specificity, and tissue distribution; Stocker A et al.; Alpha-tocopherol plays an important role as a lipid-soluble antioxidant . It is present in all major mammalian cell types and shows tissue-specific distribution . This suggests the presence of specific proteins involved in intracellular distribution or metabolism of alpha-tocopherol . A diminution of tocopherol plasma concentrations contributes to the development of diseases such as vitamin E deficiency (AVED), atherosclerosis, and prostate cancer . Further evidence has been obtained for the existence of sites in cellular metabolism and signal transduction where alpha-tocopherol potentially plays a regulatory role . A signal transduction modulation specific for alpha-tocopherol has been described in several model systems . Using radioactively labeled alpha-tocopherol as tracer, we have isolated a new alpha-tocopherol-associated protein (TAP) from bovine liver . This protein has a molecular mass of 46 kDa and an isoelectric point of 8.1 . From its partial amino acid sequence, a human gene has been identified with high homology to the newly described protein . Sequence analysis has established that the new TAP has structural motifs suggesting its belonging to a family of hydrophobic ligand-binding proteins (RALBP, CRALBP, alpha-TTP, SEC 14, PTN 9, RSEC 45) . Human TAP has been cloned into Escherichia coli, and its tissue-specific expression has been assessed by Northern blot analysis. Int J Food Microbiol, 2000 Apr 10, 55(1-3), 245 - 7 Cellular activities in ultra-violet killed Escherichia coli; Villarino A et al.; In this work we analyze the physiological state of cells after lethal-UV dose disinfection using independent metabolic markers . Through the detection of some metabolic activities we proved that cell lysis does not immediately follow death in UV-irradiated Escherichia coli K12 cells. Int J Food Microbiol, 2000 Apr 10, 55(1-3), 3 - 9 The heat shock response of Escherichia coli; Arsene F et al.; A large variety of stress conditions including physicochemical factors induce the synthesis of more than 20 heat shock proteins (HSPs) . In E . coli, the heat shock response to temperature upshift from 30 to 42 degrees C consists of the rapid induction of these HSPs, followed by an adaptation period where the rate of HSP synthesis decreases to reach a new steady-state level . Major HSPs are molecular chaperones, including DnaK, DnaJ and GrpE, and GroEL and GroES, and proteases . They constitute the two major chaperone systems of E . coli (15-20% of total protein at 46 degrees C) . They are important for cell survival, since they play a role in preventing aggregation and refolding proteins . The E . coli heat shock response is positively controlled at the transcriptional level by the product of the rpoH gene, the heat shock promoter-specific sigma32 subunit of RNA polymerase . Because of its rapid turn-over, the cellular concentration of sigma32 is very low under steady-state conditions (10-30 copies/cell at 30 degrees C) and is limiting for heat shock gene transcription . The heat shock response is induced as a consequence of a rapid increase in sigma32 levels and stimulation of sigma32 activity . The shut off of the response occurs as a consequence of declining sigma32 levels and inhibition of sigma32 activity . Stress-dependent changes in heat shock gene expression are mediated by the antagonistic action of sigma32 and negative modulators which act upon sigma32 . These modulators are the DnaK chaperone system which inactivate sigma32 by direct association and mediate its degradation by proteases . Degradation of sigma32 is mediated mainly by FtsH (HflB), an ATP-dependent metallo-protease associated with the inner membrane . There is increasing evidence that the sequestration of the DnaK chaperone system through binding to misfolded proteins is a direct determinant of the modulation of the heat shock genes expression . A central open question is the identity of the binding sites within sigma32 for DnaK, DnaJ, FtsH and the RNA polymerase, and the functional interplay between these sites . We have studied the role of two distinct regions of sigma32 in its activity and stability control: region C and the C-terminal part . Both regions are involved in RNA polymerase binding. EMBO J, 2000 May 2, 19(9), 2115 - 26 Rcl1p, the yeast protein similar to the RNA 3'-phosphate cyclase, associates with U3 snoRNP and is required for 18S rRNA biogenesis; Billy E et al.; RNA 3'-terminal phosphate cyclases are evolutionarily conserved enzymes catalysing conversion of the 3'-terminal phosphate in RNA to the 2',3'-cyclic phosphodiester . Their biological role remains unknown . The yeast Saccharomyces cerevisiae contains a gene encoding a protein with strong sequence similarity to the characterized cyclases from humans and Escherichia coli . The gene, named RCL1 (for RNA terminal phosphate cyclase like), is essential for growth, and its product, Rcl1p, is localized in the nucleolus . Depletion or inactivation of Rcl1p impairs pre-rRNA processing at sites A(0), A(1) and A(2), and leads to a strong decrease in 18S rRNA and 40S ribosomal subunit levels . Immunoprecipitations indicate that Rcl1p is specifically associated with the U3 snoRNP, although, based on gradient analyses, it is not its structural component . Most of Rcl1p sediments in association with the 70-80S pre-ribosomal particle and a 10S complex of unknown identity . Proteins similar to Rcl1p are encoded in genomes of all eukaryotes investigated and the mouse orthologue complements yeast strains depleted of Rcl1p . Possible functions of Rcl1p in pre-rRNA processing and its relationship to the RNA 3'-phosphate cyclase are discussed. EMBO J, 2000 May 2, 19(9), 2094 - 102 Measuring motion on DNA by the type I restriction endonuclease EcoR124I using triplex displacement; Firman K et al.; The type I restriction enzyme EcoR124I cleaves DNA following extensive linear translocation dependent upon ATP hydrolysis . Using protein-directed displacement of a DNA triplex, we have determined the kinetics of one-dimensional motion without the necessity of measuring DNA or ATP hydrolysis . The triplex was pre-formed specifically on linear DNA, 4370 bp from an EcoR124I site, and then incubated with endonuclease . Upon ATP addition, a distinct lag phase was observed before the triplex-forming oligonucleotide was displaced with exponential kinetics . As the distance between type I and triplex sites was shortened, the lag time decreased whilst the displacement reaction remained exponential . This is indicative of processive DNA translocation followed by collision with the triplex and oligonucleotide displacement . A linear relationship between lag duration and inter-site distance gives a translocation velocity of 400+/-32 bp/s at 20 degrees C . Furthermore, the data can only be explained by bi-directional translocation . An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation . The reaction is less processive, but can 'reset' in either direction whenever the DNA is released. Anal Biochem, 2000 May 1, 280(2), 301 - 7 Biotinylated phosphatidylinositol 3,4,5-trisphosphate as affinity ligand; Wang DS et al.; Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), a primary output signal of phosphoinositide (PI) 3-kinase, plays a crucial role in diverse cellular processes . Evidence indicates that PIP(3) exerts downstream signaling, in part, by recruiting effector proteins to plasma membranes . Consequently, identification of signaling enzymes with PIP(3)-binding motifs represents a viable approach to understand the mechanism by which specificity of the PI 3-kinase-mediated signaling network is maintained . To address this issue, we have developed biotinylated derivatives of PIP(3) as affinity probes for the purification and characterization of PIP(3)-binding proteins . Considering the relaxed requirement for the acyl moiety in PIP(3) recognition, these biotinylated PIP(3) analogues display two structural features . First, they contain short acyl side chains (C(4) and C(8)), allowing them to be soluble in aqueous milieu . This desirable feature avoids the formation of lipid aggregates, which minimizes nonspecific hydrophobic interactions with proteins . Second, the appended biotin is located at the terminus of the sn-1 acyl side chain, thereby maintaining the integrity of the phosphoinositol head group essential for selective recognition . The utility of these affinity ligands is validated by the purification of recombinant PIP(3)-binding proteins, expressed as GST fusion proteins, to homogeneity from bacterial lysates . These include the C-terminal SH2 domain of the p85 subunit of PI 3-kinase and the N-terminal PH domain of PLCgamma1 . The efficiency of biotinylated PIP(3) analogues in the purification of these recombinant proteins was approximately 20% of that of glutathione beads Microb Ecol, 2000 Jan, 39(1), 65 - 71 The Effect of Simulated Solar Radiation on Escherichia coli: The Relative Roles of UV-B, UV-A, and Photosynthetically Active Radiation; Muela A et al.; The relative role of components of solar radiation (UV-B, UV-A, and photosynthetically active radiation) as well as the effect of simulated sunlight upon the physiological state of Escherichia coli in fresh water were evaluated . Simulated solar radiation had a sublethal effect on E . coli populations in a short-time exposure by provoking loss of culturability and the formation of viable but nonculturable cells . Prolonged exposure increased the damage to cells but cellular integrity was never affected . However, important differences between the way the sunlight components acted were detected . After photosynthetically active radiation (PAR) exposure, cells remained metabolically active but only 10% of the cells were culturable . When cells were exposed to UV-A, the culturable fraction was similar to the one obtained after PAR irradiation, although formation of viable but nonculturable cells was not observed . For UV-B radiation short-time exposures (6 h) were enough to provoke loss of culturability and a reduction in activity similar to that of simulated sunlight exposed cells . The effect of simulated solar radiation on E . coli cells was mainly attributable to shorter wavelengths, but a synergistic interaction of the UV-B, UV-A and PAR components was detected . </hea Arch Insect Biochem Physiol, 2000 May, 44(1), 17 - 26 A juvenile hormone-repressible transferrin-like protein from the bean bug, Riptortus clavatus: cDNA sequence analysis and protein identification during diapause and vitellogenesis; Hirai M et al.; We found several juvenile hormone-responsive cDNAs in the bean bug, Riptortus clavatus, by using mRNA differential display (Hirai et al., 1998) . One of them, a juvenile hormone-repressible cDNA, JR-3, was cloned, sequenced, characterized and identified as a transferrin (RcTf) . RcTf cDNA encoded 652 amino acids with a calculated molecular weight of 71,453 Da . The deduced amino acid sequence showed significant homology with the transferin genes of several insects, Manduca sexta (43% identity), Blaberus discoidalis (43%), Aedes aegypti (43%), Drosophila melanogaster (36%), Sarcophaga peregrina (36%) and the human (25%) . Antiserum was prepared by using recombinant RcTf protein expressed in Escherichia coli as an antigen . The antiserum reacted specifically with both the recombinant protein and the native protein from the bugs, with sizes of 70 and 75 kDa, respectively . The 75 kDa protein was partially purified from hemolymph of diapausing female bugs and the first ten amino acids were found to be identical to that of RcTf cDNA, indicating that the 75 kDa protein is RcTf . The tissue distribution of RcTf in the bug was examined by Western blot analysis . In diapausing animals, RcTf was detected in the fat body, hemolymph and ovary but not in the gut . In the post-diapause stage, RcTf was also detected in eggs, in addition to the fat body and ovary . These results indicate that RcTf is incorporated into the oocytes during vitellogenesis, and suggest that it may provide iron for the developing embryos . J Clin Microbiol, 2000 May, 38(5), 2001 - 4 Multiplex PCRs for identification of Escherichia coli virulence genes; Pass MA et al.; PCRs were developed to detect 11 Escherichia coli virulence genes . Primers amplified the respective genes without cross-reaction with other genes . Specificity was maintained in multiplex reactions; excellent amplification of target genes was possible with a minimum of four multiplex reactions . These reactions successfully identified genes in E . coli from the feces of four dogs. J Clin Microbiol, 2000 May, 38(5), 1767 - 71 Identification of enterotoxigenic Escherichia coli harboring longus type IV pilus gene by DNA amplification; Gutierrez-Cazarez Z et al.; DNA amplification of lngA, the structural gene of longus type IV pilus produced by human enterotoxigenic Escherichia coli (ETEC) was achieved by the use of specific oligonucleotide primers designed from the nucleotide sequence of lngA . A 630-bp fragment representing the entire lngA gene was amplified in eight prototype strains previously characterized as longus positive . Five ETEC strains producing colonization factor antigen III (CFA III) (also a type IV pilus) were also positive by PCR, confirming the DNA homology between CFA III and longus . None of the non-ETEC and non-E . coli enteropathogens studied showed the 0.63-kbp amplicon . The procedure thus detected only ETEC strains harboring type IV pili genes with or without other colonization factors . Except for five lngA PCR-positive, probe-positive strains, all lngA PCR-positive strains produced the pilin as demonstrated by immunoblotting . To test the amplification procedure in a clinical setting, a collection of 264 fresh clinical E . coli strains isolated from 88 Mexican children with diarrhea was screened by PCR . Among 82 ETEC isolates found, 30 (36.5%) were lngA PCR-positive . Twenty-seven percent of the children shed ETEC that possessed lngA . In parallel with DNA probes or PCR protocols to detect enterotoxin genes, the lngA PCR method may prove useful for detection of ETEC harboring type IV pilus genes in epidemiological studies. Radiat Environ Biophys, 2000 Mar, 39(1), 41 - 5 Detection of radiation effects using recombinant bioluminescent Escherichia coli strains; Min J et al.; Effects of ionizing radiation (0.1-500 Gy) on recombinant Escherichia coli cells containing the stress promoters recA, grpE, or katG, fused to luxCDABE, were characterized by monitoring transcriptional responses reflected by the bioluminescent output . The minimum dose of gamma-irradiation detected by E . coli DPD2794 (recA::luxCDABE) was about 1.5 Gy, while the maximum response was obtained at 200 Gy . The amount of emitted bioluminescence increased proportionally with the gamma-ray doses which were found to elicit a DNA damage response in a range of 1-50 Gy . In addition, the cell growth rate was severely, but transiently, retarded by about 50 Gy . Quantification of the gamma-ray dose may be possible using the recA promoter fusion, since linear enhancement of the bioluminescence emission with increasing gamma-ray dose was observed . Other irradiated strains (50 Gy) responsive to either oxidative stress (DPD2511, katG::luxCDABE) or protein-damaging stress (TV1061, grpE::luxCDABE) did not display an increased bioluminescent output, while DPD2794 irradiated by the same dose of gamma-rays gave a significant bioluminescent output . This indicates that the recA promoter is the one most suitable for developing a biosensor for ionizing radiation. Surg Laparosc Endosc Percutan Tech, 2000 Apr, 10(2), 93 -