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| Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9539 - 44 Rapid GTP binding and hydrolysis by G(q) promoted by receptor and GTPase-activating proteins; Mukhopadhyay S et al.; Receptor-promoted GTP binding and GTPase-activating protein (GAP)-promoted GTP hydrolysis determine the onset and termination of G protein signaling; they coordinately control signal amplitude . The mechanisms whereby cells independently regulate signal kinetics and signal amplitude are therefore central to understanding G protein function . We have used quench-flow kinetic methods to measure the rates of the individual reactions of the agonist-stimulated GTPase cycle for G(q) during steady-state signaling . G(q) and m1 muscarinic cholinergic receptor were co-reconstituted into proteoliposomes with one of two GAPs: phospholipase C (PLC)-beta1, the major G(q)-regulated effector protein, and RGS4, a GAP commonly thought to be an inhibitor of G(q) signaling . In this system, the rate constant for GAP-stimulated hydrolysis of Galpha(q)-bound GTP at 30 degrees C was 9-12 s(-1) for PLC-beta1 and 22-27 s(-1) for RGS4 . These rates are 1,000- to 2,000-fold faster than in the absence of a GAP and far faster than measured previously . G(q) can thus hydrolyze bound GTP with deactivation half-times of 25-75 ms at 30 degrees C, commensurate with physiological rates of signal termination . GDP/GTP exchange, which reactivates G(q), was the principal rate-limiting step for the GTPase cycle and was also faster than previously thought . At physiological concentrations of GTP, exchange was limited by the rate of dissociation of GDP from the receptor-G(q) complex, with a maximal rate of 1.8 s(-1) at 30 degrees C . Comparison of activation and deactivation rates help explain how GDP/GTP exchange balance rapid GTP hydrolysis to maintain steady-state signal amplitude. J Clin Microbiol, 1999 Sep, 37(9), 2974 - 8 Phenotypic diversity of enterotoxigenic Escherichia coli strains from a community-based study of pediatric diarrhea in periurban Egypt; Peruski LF Jr et al.; No past studies of diarrhea in children of the Middle East have examined in detail the phenotypes of enterotoxigenic Escherichia coli (ETEC) strains, which are important pathogens in this setting . During a prospective study conducted from November 1993 to September 1995 with 242 children under 3 years of age with diarrhea living near Alexandria, Egypt, 125 episodes of diarrhea were positive for ETEC . ETEC strains were available for 98 of these episodes, from which 100 ETEC strains were selected and characterized on the basis of enterotoxins, colonization factors (CFs), and O:H serotypes . Of these representative isolates, 57 produced heat-stable toxin (ST) only, 34 produced heat-labile toxin (LT) only, and 9 produced both LT and ST . Twenty-three ETEC strains expressed a CF, with the specific factors being CF antigen IV (CFA/IV; 10 of 23; 43%), CFA/II (5 of 23; 22%), CFA/I (3 of 23; 13%), PCFO166 (3 of 23; 13%), and CS7 (2 of 23; 9%) . No ETEC strains appeared to express CFA/III, CS17, or PCFO159 . Among the 100 ETEC strains, 47 O groups and 20 H groups were represented, with 59 O:H serotypes . The most common O serogroups were O159 (13 strains) and O43 (10 strains) . O148 and O21 were each detected in five individual strains, O7 and O56 were each detected in four individual strains, O73, O20, O86, and O114 were each detected in three individual strains, and O23, O78, O91, O103, O128, and O132 were each detected in two individual strains . The most common H serogroups were H4 (16 strains), 12 of which were of serogroup O159; H2 (9 strains), all of which were O43; H18 (6 strains); H30 (6 strains); and H28 (5 strains); strains of the last three H serogroups were all O148 . Cumulatively, our results suggest a high degree of clonal diversity of disease-associated ETEC strains in this region . As a low percentage of these strains expressed a CF, it remains possible that other adhesins for which we either did not assay or that are as yet undiscovered are prevalent in this region . Our findings point out some potential barriers to effective immunization against ETEC diarrhea in this population and emphasize the need to identify additional protective antigens commonly expressed by ETEC for inclusion in future vaccine candidates. Curr Opin Struct Biol, 1999 Aug, 9(4), 448 - 54 'Feeling the pressure': structural insights into a gated mechanosensitive channel; Spencer RH et al.; The structure determination of the large-conductance mechanosensitive channel (MscL) from Mycobacterium tuberculosis has revealed the architecture of the first full-length, gated pentameric ion channel . This structure provides insights into the elements participating in the conductance and gating mechanisms of these channels. J Mol Biol, 1999 Aug 20, 291(3), 703 - 13 Unfolding and refolding of Escherichia coli chaperonin GroES is expressed by a three-state model; Higurashi T et al.; The guanidine-hydrochloride (Gdn-HCl) induced unfolding and refolding characteristics of the co-chaperonin GroES from Escherichia coli, a homoheptamer of subunit molecular mass 10,000 Da, were studied by using intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS) binding, and size-exclusion HPLC . When monitored by tyrosine fluorescence, the unfolding reaction of GroES consisted of a single transition, with a transition midpoint at around 1.0 M Gdn-HCl . Interestingly, however, ANS binding and size-exclusion HPLC experiments strongly suggested the existence of an intermediate state in the transition . In order to confirm the existence of an intermediate state between the native heptameric and unfolded monomeric states, a tryptophan residue was introduced into the interface of GroES subunits as a fluorescent probe . The unfolding reaction of GroES I48W as monitored by tryptophyl fluorescence showed a single transition curve with a transition midpoint at 0.5 M Gdn-HCl . This unfolding transition curve as well as the refolding kinetics were dependent on the concentration of GroES protein . CD spectrum and size-exclusion HPLC experiments demonstrated that the intermediates assumed a partially folded conformation at around 0.5 M Gdn-HCl . The refolding of GroES protein from 3 M Gdn-HCl was probed functionally by measuring the extent of inhibition of GroEL ATPase activity and the enhancement of lactate dehydrogenase refolding yields in the presence of GroEL and ADP . These results clearly demonstrated that the GroES heptamer first dissociated to monomers and then unfolded completely upon increasing the concentration of Gdn-HCl, and that both transitions were reversible . From the thermodynamic analysis of the dissociation reaction, it was found that the partially folded monomer was only marginally stable and that the stability of GroES protein is governed mostly by the association of the subunits . J Mol Biol, 1999 Aug 20, 291(3), 683 - 92 Proximity relationships between helices I and XI or XII in the lactose permease of Escherichia coli determined by site-directed thiol cross-linking; Wang Q et al.; The lactose permease of Escherichia coli was expressed in two fragments (split permease), each with a Cys residue, and cross-linking was studied . Split permease with a discontinuity in either loop II/III (N2C10permease) or loop VI/VII (N6C6permease) was used . Proximity of multiple pairs of Cys residues in helices I and XI or XII was examined by using three homobifunctional thiol-specific cross-linking reagents of different lengths and flexibilities (6 A, rigid; 10 A, rigid; 16 A, flexible) or iodine . Cys residues in the periplasmic half of helix I cross-link to Cys residues in the periplasmic half of helix XI . In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices I and XI . Therefore, the periplasmic halves of helices I and XI are in close proximity, and the helices tilt away from each other towards the cytoplasmic face of the membrane . Cross-linking is also found with paired Cys residues near the middle of helices I and XII, but not with paired Cys residues near either end of the helices . Thus, helices I and XII are in close proximity only in the approximate middle of the membrane . Based on the findings, a modified helix packing model is proposed . J Mol Biol, 1999 Aug 20, 291(3), 615 - 35 The structure of pyruvate kinase from Leishmania mexicana reveals details of the allosteric transition and unusual effector specificity; Rigden DJ et al.; Glycolysis occupies a central role in cellular metabolism, and is of particular importance for the catabolic production of ATP in protozoan parasites such as Leishmania and Trypanosoma . In these organisms pyruvate kinase plays a key regulatory role, and is unique in responding to fructose 2,6-bisphosphate as allosteric activator . The determination of the first eukaryotic pyruvate kinase crystal structure in the T-state is reported . A comparison of the leishmania and yeast R-state enzymes reveals fewer differences than the previous comparison of Escherichia coli T-state and rabbit muscle non-allosteric enzymes . Structural changes related to the allosteric transition can therefore be distinguished from those that are a consequence of the inherent wide structural divergence between bacterial and mammalian proteins . The allosteric transition involves significant changes in a tightly packed array of eight alpha helices at the interface near the catalytic site . At the other interface the allosteric transition appears to be accompanied by the bending of a ten-stranded intersubunit beta sheet adjacent to the effector site . Helix Calpha1 makes contacts to the N-terminal helical domain and bridges both interfaces . A comparison of the effector sites of the leishmania and yeast enzymes reveals the structural basis for the different effector specificity . Two loops comprising residues 443-453 and 480-489 adopt very different conformations in the two enzymes, and Lys453 and His480 that are a feature of trypanosomatid enzymes provide probable ligands for the 2-phospho group of the effector molecule . These differences offer an opportunity for the design of drugs that would bind to the trypanosomatid enzymes but not to those of the mammalian host . Plant Cell, 1999 Aug, 11(8), 1591 - 602 Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex; Pan S et al.; The 14-3-3 family of multifunctional proteins is highly conserved among animals, plants, and yeast . Several studies have shown that these proteins are associated with a G-box DNA binding complex and are present in the nucleus in several plant and animal species . In this study, 14-3-3 proteins are shown to bind the TATA box binding protein (TBP), transcription factor IIB (TFIIB), and the human TBP-associated factor hTAF(II)32 in vitro but not hTAF(II)55 . The interactions with TBP and TFIIB were highly specific, requiring amino acid residues in the box 1 domain of the 14-3-3 protein . These interactions do not require formation of the 14-3-3 dimer and are not dependent on known 14-3-3 recognition motifs containing phosphoserine . The 14-3-3-TFIIB interaction appears to occur within the same domain of TFIIB that binds the human herpes simplex virus transcriptional activator VP16, because VP16 and 14-3-3 were able to compete for interaction with TFIIB in vitro . In a plant transient expression system, 14-3-3 was able to activate GAL4-dependent beta-glucuronidase reporter gene expression at low levels when translationally fused with the GAL4 DNA binding domain . The in vitro binding with general transcription factors TBP and TFIIB together with its nuclear location provide evidence supporting a role for 14-3-3 proteins as transcriptional activators or coactivators when part of a DNA binding complex. Plant Cell, 1999 Aug, 11(8), 1553 - 64 Insertion of leader peptidase into the thylakoid membrane during synthesis in a chloroplast translation system Houben E, de Gier JW, van Wijk KJ. The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood . In this study, we have used a translation system isolated from chloroplasts to begin to investigate these mechanisms . The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well understood for Escherichia coli and for the endoplasmic reticulum . Lep could thus provide insight into the functional homologies between the different membrane systems . Lep was efficiently expressed in the chloroplast translation system, and the protein could be inserted into thylakoid membranes with the same topology as in E . coli cytoplasmic membranes, following the positive-inside rule . Insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity . Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if thylakoid membranes were present during translation reactions but not when thylakoid membranes were added after translation reactions were terminated . To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribosome nascent chain complexes . These complexes showed a strong, salt-resistant affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep . Our study supports a functional homology for the insertion of Lep into the thylakoid membrane and the E . coli cytoplasmic membrane. Plant Cell, 1999 Aug, 11(8), 1485 - 98 S-methylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyltransferase; Bourgis F et al.; All flowering plants produce S-methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met . The functions of SMM and the reasons for its interconversion with Met are not known . In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that l-SMM is a major constituent of the phloem sap moving to wheat ears . The SMM level in the phloem ( approximately 2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis . Similarly, l-SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given l-(35)S-Met . cDNA clones for the enzyme that catalyzes SMM synthesis (S-adenosylMet:Met S-methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora, maize, and Arabidopsis . The deduced amino acid sequences revealed the expected methyltransferase domain ( approximately 300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes . These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event . The resulting bipartite enzyme is unlike any other known methyltransferase. EMBO J, 1999 Aug 16, 18(16), 4579 - 89 SsrA-mediated peptide tagging caused by rare codons and tRNA scarcity; Roche ED et al.; SsrA RNA mediates the addition of a C-terminal peptide tag (AANDENYALAA) to bacterial proteins translated from mRNAs without in-frame stop codons . This process involves both tRNA- and mRNA-like functions of SsrA and targets the tagged proteins for degradation . By designing an SsrA variant that adds a peptide tag (AANDENYALDD) that does not result in rapid degradation, we show that tagging of a model protein synthesized from an mRNA without stop codons can be detected both in vivo and in vitro . We also use this assay to demonstrate that ribosome stalling at clusters of rare arginine codons in mRNA is sufficient to recruit and activate the SsrA peptide tagging system . An essential requirement for tagging at rare AGA codons is a scarcity of the cognate tRNA; supplemental tRNA(AGA) suppresses tagging, and depleting the available pool of tRNA(AGA) enhances tagging and reveals tagging caused by single rare AGA codons . Protein tagging at sites corresponding to rare codons appears to involve SsrA action at an internal mRNA site rather than at the 3' end of a cleaved mRNA. EMBO J, 1999 Aug 16, 18(16), 4513 - 22 X-ray structure of aminopeptidase A from Escherichia coli and a model for the nucleoprotein complex in Xer site-specific recombination; Strater N et al.; The structure of aminopeptidase A (PepA), which functions as a DNA-binding protein in Xer site-specific recombination and in transcriptional control of the carAB operon in Escherichia coli, has been determined at 2.5 A resolution . In Xer recombination at cer, PepA and the arginine repressor (ArgR) serve as accessory proteins, ensuring that recombination is exclusively intramolecular . In contrast, PepA homologues from other species have no known DNA-binding activity and are not implicated in transcriptional regulation or control of site-specific recombination . PepA comprises two domains, which have similar folds to the two domains of bovine lens leucine aminopeptidase (LAP) . However, the N-terminal domain of PepA, which probably plays a significant role in DNA binding, is rotated by 19 degrees compared with its position in LAP . PepA is a homohexamer of 32 symmetry . A groove that runs from one trimer face across the 2-fold molecular axis to the other trimer face is proposed to be the DNA-binding site . Molecular modelling supports a structure of the Xer complex in which PepA, ArgR and a second PepA molecule are sandwiched along their 3-fold molecular axes, and the accessory sequences of the two recombination sites wrap around the accessory proteins as a right-handed superhelix such that three negative supercoils are trapped. Curr Opin Cell Biol, 1999 Aug, 11(4), 496 - 502 Structure and function of facilitative sugar transporters; Barrett MP et al.; Sugar transporters from one group of the major facilitator superfamily of membrane transporters . A conserved common central pore structure lies at the heart of these transporters and diverse functionality is brought about by alterations to this pore or regions associated with it . Recent mutagenesis studies of sugar transporters within the framework of tenable models for the distantly related lactose permease argue that this model is a good paradigm for other members of the major facilitator superfamily. Microbiol Immunol, 1999, 43(5), 481 - 4 Identification of copper-zinc superoxide dismutase gene from enteroaggregative Escherichia coli; Kumar R et al.; We describe here the identification of sodC gene from enteroaggregative Escherichia coli (EAggEC) . A 294 bp gene-specific fragment was amplified from the organism by DNA as well as RT-PCR using primers from bacterial sodC sequences . The metal co-factor present in the protein was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide . However, the nonpathogenic E . coli possesses the gene but does not express it . Thus, the presence of copper-zinc superoxide dismutase encoded by sodC was demonstrated for the first time in EAggEC, which means it could be a novel candidate for a virulence marker. Microbiol Immunol, 1999, 43(5), 419 - 24 Conservation of putative promoter sequences located upstream of chlamydial major sigma factor gene, sigA among Chlamydia spp; Ochiai Y et al.; A highly conserved 40-nucleotide sequence was identified . Two completely conserved sequences, TAGATT and TAAACT, separated by 17 nucleotides resemble the consensus sequence recognized by the Escherichia coli major sigma factor and sequence found in other chlamydial promoters . In addition, the adenine-rich sequence present in many chlamydial promoters was also conserved upstream of the putative -35 element . These findings suggest that the conserved sequence may play a role in the regulatory function at the transcriptional level . Multiple ATG codons were found at the 5'-terminal region of the chlamydial sigA ORFs except for Chlamydia pneumoniae, although the putative Shine-Dargarno sequence was absent. J Bioenerg Biomembr, 1999 Apr, 31(2), 95 - 104 The role of the amino-terminal beta-barrel domain of the alpha and beta subunits in the yeast F1-ATPase; Yao B et al.; The crystal structure of mitochondrial F1-ATPase indicates that the alpha and beta subunits fold into a structure defined by three domains: the top beta-barrel domain, the middle nucleotide-binding domain, and the C-terminal alpha-helix bundle domain (Abrahams et al., 1994); Bianchet et al., 1998) . The beta-barrel domains of the alpha and beta subunits form a crown structure at the top of F1, which was suggested to stabilize it (Abrahams et al . 1994) . In this study, the role of the beta-barrel domain in the alpha and beta subunits of the yeast Saccharomyces cerevisiae F1, with regard to its folding and assembly, was investigated . The beta-barrel domains of yeast F1alpha and beta subunits were expressed individually and together in Escherichia coli . When expressed separately, the beta-barrel domain of the beta subunit formed a large aggregate structure, while the domain of the alpha subunit was predominately a monomer or dimer . However, coexpression of the beta-barrel domain of alpha subunit with the beta-barrel domain of beta subunit, greatly reduced the aggregation of the beta subunit domain . Furthermore, the two domains copurified in complexes with the major portion of the complex found in a small molecular weight form . These results indicate that the beta-barrel domain of the alpha and beta subunits interact specifically with each other and that these interactions prevent the aggregation of the beta-barrel domain of the beta subunit . These results mimic in vivo results and suggest that the interactions of the beta-barrel domains may be critical during the folding and assembly of F1. Prog Mol Subcell Biol, 1999, 23, 183 - 95 Inorganic polyphosphate regulates responses of Escherichia coli to nutritional stringencies, environmental stresses and survival in the stationary phase; Rao NN et al.; The molecular mechanisms responsible for polyP accumulation in E . coli remain largely obscure . Based on the available data, a tentative model is proposed (Fig . 1; Ault-Riche et al . 1998) . Inhibition by (p)ppGpp of PPX interrupts the dynamic balance between the synthesis of polyP by PPK and its hydrolysis by PPX, accounting for polyP accumulation . However, mutants lacking PhoB, the response regulator of the Pho regulon, fail to accumulate polyP even in the face of high levels of (p)ppGpp . Clearly, PhoB is required in some undefined manner . With regard to osmotic stress, the pathway to polyP accumulation is also distinct from the one identified with the activation of envZ and the associated changes in membrane functions . A tentative scheme attempting to describe the metabolic turnover of polyP is given in Fig . 4 . {figure: see text} In adaptations to stress, cells must coordinate major changes in the rates of transcription, translation, and replication as well as make choices in the genes expressed (Kolter et al . 1993) . PolyP could provide activated phosphates or coordinate an adaptive response by binding metals and/or specific proteins . Accumulation of polyP in E . coli and other organisms is commonly assumed to provide a reservoir of energy convertible to ATP . This seems implausible because of the turnover of ATP which consumes only a fraction of a second (Chapman and Atkinson 1977) . Thus, other functions for polyP need to be considered, among them a regulatory role . PolyP, even at very low levels, is essential in E . coli for adaptations in stationary phase and for survival (Rao and Kornberg 1996) . As a polyanionic polymer, polyP has chemical similarities to DNA and RNA in interactions with basic domains of proteins . Further investigation of the cellular location of polyP, its state of metabolic availability and identification of its binding partners are needed . In view of the ubiquity of polyP in eukaryotic cells (including dynamic turnover in the nuclei of some mammalian cells), studies similar to those undertaken in E . coli may reveal comparable functions. Biochem Biophys Res Commun, 1999 Aug 19, 262(1), 297 - 301 Tryptophanless recombinant horseradish peroxidase: stability and catalytic properties; Gazaryan IG et al.; The tryptophanless mutant of horseradish peroxidase, W117F, has been constructed and expressed in Escherichia coli . The mutation affects enzyme folding and stability . The optimum composition of the refolding medium requires the presence of ammonium sulfate . The yield of mutant is ca . 8000 U per liter of the optimized refolding medium with the specific activity of 1100-1500 U/mg (compared to 25, 000 U per liter and 2000 U/mg for the recombinant wild-type enzyme) . The mutant is more stable in acid media, in the reaction course and toward irradiation . The effect of hydrogen peroxide pretreatment on radiation-induced inactivation of the wild-type and mutant enzyme indirectly indicates participation of Trp-117 in electron transfer pathways through the enzyme molecule . This is in agreement with the steady-state kinetic data interpreted in terms of Trp-117 participation in electron transfer within the Michaelis complex . Biochem Biophys Res Commun, 1999 Aug 19, 262(1), 44 - 9 Characterization of Acholeplasma laidlawii ftsZ gene and its gene product; Kukekova AV et al.; The ftsZ gene was found among representatives of all bacterial groups . FtsZ protein is an essential component of cell division ring . Contraction of this cytoskeleton-like ring is believed to be the universal way of bacterial division . Acholeplasma laidlawii possesses all features of the minimal mycoplasma cell and some traits of cell-wall bacteria and seems to be a promising object for study of basic principles of the bacterial division process . We cloned an A . laidlawii chromosomal fragment containing ftsZ gene and two flanking orf which also were identified . A . laidlawii FtsZ protein has been determined with polyclonal antibodies raised in rabbit . It was demonstrated that ftsZ gene of A . laidlawii could be expressed in E . coli cells . We also revealed that A . laidlawii FtsZ had a low similarity to proteins of Mycoplasma genitalium and M . pneumoniae . The comparison of FtsZ structures may be used for investigation of bacterial phylogenetic relations . Biochem Biophys Res Commun, 1999 Aug 19, 262(1), 20 - 4 Resveratrol is a selective human cytochrome P450 1A1 inhibitor; Chun YJ et al.; Resveratrol (trans-3,4',5-trihydroxystilbene) is a phytoalexin compound found in juice and wine produced from dark-skinned grape cultivars and reported to have anti-inflammatory and anticarcinogenic activities . To investigate the mechanism of anticarcinogenic activities of resveratrol, the effects on cytochrome P450 (P450) were determined in human liver microsomes and Escherichia coli membranes coexpressing human P450 1A1 or P450 1A2 with human NADPH-P450 reductase (bicistronic expression system) . Resveratrol slightly inhibited ethoxyresorufin O-deethylation (EROD) activity in human liver microsomes with an IC(50) of 1.1 mM . Interestingly, resveratrol exhibited potent inhibition of human P450 1A1 in a dose-dependent manner with IC(50) of 23 microM for EROD and IC(50) of 11 microM for methoxyresorufin O-demethylation (MROD) . However, the inhibition of human P450 1A2 by resveratrol was not so strong (IC(50) 1.2 mM for EROD and 580 microM for MROD) . Resveratrol showed over 50-fold selectivity for P450 1A1 over P450 1A2 . The activities of human NADPH-P450 reductase were not significantly changed by resveratrol . In a human P450 1A1/reductase bicistronic expression system, resveratrol inhibited human P450 1A1 activity in a mixed-type inhibition (competitive-noncompetitive) with a K(i) values of 9 and 89 microM . These results suggest that resveratrol is a selective human P450 1A1 inhibitor, and may be considered for use as a strong cancer chemopreventive agent in humans . Immunology, 1999 Jul, 97(3), 515 - 21 A recombinant BCG vaccine generates a Th1-like response and inhibits IgE synthesis in BALB/c mice; Kumar M et al.; The tubercle vaccine, bacille Calmette-Guerin (BCG), is a strong inducer of T-helper type 1 (Th1) responsiveness, and it has been suggested that recombinant BCG (rBCG), which produces and secretes antigens, may be used to prevent allergic diseases . The effects of rBCG vaccination on allergic responses in a murine model were examined in this study . A BCG-Escherichia coli shuttle vector was developed with the promoter and signal sequence of the alpha-antigen of Mycobacterium bovis, and the vector was tested using E . coli beta-galactosidase as the model antigen and allergen . This vector enabled the expression of the E . coli beta-galactosidase gene in BCG, which was detected in its protein extract by immunoblotting analysis . Vaccination of mice with a single dose of 106 recombinant BCG generated a beta-galactosidase-specific antibody response . The splenocytes of vaccinated mice compared with controls produced significantly higher amounts of interferon-gamma (IFN-gamma) (P<0 . 01) and interleukin-2 (IL-2) (P<0.05) and lower amounts of IL-5 (P<0 . 01) . Mice vaccinated with rBCG had significantly less (P<0.01) serum IgE compared with controls . These results together demonstrate that rBCG secreting antigens or allergens may be utilized for the induction of a Th1-like response and the down-regulation of IgE antibody response. Biochim Biophys Acta, 1999 Aug 18, 1439(3), 415 - 23 Rat sn-glycerol-3-phosphate acyltransferase: molecular cloning and characterization of the cDNA and expressed protein; Ganesh Bhat B et al.; Rat mitochondrial glycerol-3-phosphate acyltransferase (GPAT) cDNA was cloned and characterized . We identified a cDNA containing an open reading frame of 828 amino acids that had an 89% homology with the coding region of the previously characterized mouse mitochondrial GPAT and a predicted amino acid sequence that was 96% identical . The rat 5' UTR was only 159 nucleotides, in contrast to the 926 nucleotide 5' UTR of the mouse cDNA and had an internal deletion of 167 nucleotides . GPAT was expressed in Sf21 insect cells, and specific inhibitors strongly suggest that, like the Escherichia coli GPAT, the recombinant mitochondrial GPAT and the mitochondrial GPAT isoform in rat liver contain critical serine, histidine, and arginine residues. Mol Microbiol, 1999 Aug, 33(4), 846 - 57 sbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants; Bidnenko V et al.; We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork . Annealing of the newly synthesized strands creates a double-stranded end adjacent to a Holliday junction . The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double-stranded tail . Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth . We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination . This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination-dependent replication restart . However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains . We also show that ruvAB inactivation suppresses the lethality and the formation of double-stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants . This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates. Mol Microbiol, 1999 Aug, 33(4), 741 - 52 A novel chromosomal locus of enteropathogenic Escherichia coli (EPEC), which encodes a bfpT-regulated chaperone-like protein, TrcA, involved in microcolony formation by EPEC; Tobe T et al.; The bfpTVW operon, also known as the per operon, of enteropathogenic Escherichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle-forming pili (BFP) . An immobilized T7-tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to 'fish out' from a promoter library other EPEC chromosomal fragments that are bound by the BfpT protein . After screening for promoters exhibiting bfpTVW-dependent expression, one was identified that was positively regulated by bfpTVW and that is not present in the chromosomes of two non-virulent E . coli laboratory strains, DH5alpha and HB101 . Further analysis of this positively regulated promoter in EPEC showed that it resided within a 4.9 kb sequence that is not present in E . coli K12 . This locus, located downstream of the potB gene, was found to contain four open reading frames (ORFs): bfpTVW-activated promoter was localized upstream of ORF1 . An ORF1 knockout mutant produced less of the BFP structural subunit (BfpA) and formed smaller than normal adherent microcolonies on cultured epithelial cells; however, this mutation did not affect bfp transcription . An ORF1-His6 fusion protein specifically bound the preprocessed and mature forms of the BfpA protein and thus appears to stabilize the former within the cytoplasmic compartment . ORF1 therefore is a newly isolated EPEC chromosomal gene that encodes a chaperone-like protein involved in the production of BFP . Hence, ORF1 was designated trcA (bfpT-regulated chaperone-like protein gene) . The TrcA protein also specifically bound 39 kDa and 90 kDa proteins that are expressed by EPEC but not by E . coli K12 . The 90 kDa protein was revealed to be intimin, a protein product of the eae gene, which is required for the EPEC attaching/effacing phenotype, suggesting a direct interaction of TrcA with intimin in the cytoplasmic compartment. Mol Microbiol, 1999 Aug, 33(4), 732 - 40 Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA-dependent error-prone DNA replication activity; Al Mamun AA et al.; A base substitution mutation (mutA) in the Escherichia coli glyV tRNA gene potentiates asp --> gly mistranslation and confers a strong mutator phenotype that is SOS independent, but requires recA, recB and recC genes . Here, we demonstrate that mutA cells express an error-prone DNA polymerase by using an in vitro experimental system based on the conversion of phage M13 single-stranded viral DNA bearing a model mutagenic lesion to the double-stranded replicative form . Amplification of the newly synthesized strand followed by multiplex DNA sequence analysis revealed that mutation fixation at 3, N4-ethenocytosine (varepsilonC) was approximately 3% when the DNA was replicated by normal cell extracts, approximately 48% when replicated by mutA cell extracts and approximately 3% when replicated by mutA recA double mutant cell extracts, in complete agreement with previous in vivo results . Mutagenesis at undamaged DNA sites was significantly elevated by mutA cell-free extracts in the M13 lacZ(alpha) forward mutagenesis system . Neither polA (DNA polymerase I) nor polB (DNA polymerase II) genes are required for the mutA phenotype, suggesting that the phenotype is mediated through a modification of DNA polymerase III or the activation of a previously unidentified DNA polymerase . These findings define the major features of a novel mutagenic pathway and imply the existence of previously unrecognized links between translation, recombination and replication. Mol Microbiol, 1999 Aug, 33(4), 721 - 31 Co-ordination of legionella pneumophila virulence with entry into stationary phase by ppGpp; Hammer BK et al.; Legionella pneumophila survives in aquatic environments, but replicates within amoebae or the alveolar macrophages of immunocompromised individuals . Here, the signal transduction pathway that co-ordinates L . pneumophila virulence expression in response to amino acid depletion was investigated . To facilitate kinetic and genetic studies, a phenotypic reporter of virulence was engineered by fusing flaA promoter sequences to a gene encoding green fluorescent protein . When subjected to amino acid depletion, L . pneumophila accumulated ppGpp and converted from a replicative to a virulent state, as judged by motility and sodium sensitivity . ppGpp appeared to initiate this response, as L . pneumophila induced to express the Escherichia coli RelA ppGpp synthetase independently of nutrient depletion accumulated ppGpp, exited the exponential growth phase and expressed flaAgfp, motility, sodium sensitivity, cytotoxicity and infectivity, five traits correlated with virulence . Although coincident with the stationary phase, L . pneumophila virulence expression appeared to require an additional factor: mutant Lp120 accumulated ppGpp and acquired two stationary phase traits but none of six virulence phenotypes analysed . We propose that, when nutrients are limiting, ppGpp acts as an alarmone, triggering the expression of multiple traits that enable L . pneumophila to escape its spent host, to survive and disperse in the environment and to re-establish a protected intracellular replication niche. J Vet Pharmacol Ther, 1999 Jun, 22(3), 202 - 8 Norfloxacin pharmacokinetics in lactating cows with sub-clinical and clinical mastitis; Gips M et al.; Single-dose pharmacokinetics of norfloxacin after intravenous administration of norfloxacin nicotinate at 10 mg norfloxacin/kg body weight was investigated in cows with healthy udders and in cows with chronic subclinical and postacute clinical mastitis . An HPLC method was used to determine the norfloxacin concentrations in serum and milk . Significant differences were observed in norfloxacin pharmacokinetics when administered to cows with infected udder quarters . The clearance (Cl) values were 10.4+/-2.5, 13.2+/-1.9 and 14.2+/-2.1 mL/min/kg (mean +/-SD) in the control (healthy udder) cows and in cows with subclinical and postacute clinical mastitis, respectively . There appeared to be a trend of increasing clearance according to severity of the disease . The volume of distribution at steady state (Vss) in the respective groups was 3.1+/-0.7, 2.2+/-0.6 and 1.3+/-0.2 L/kg . The volume of distribution was significantly decreased in the cows with postacute clinical mastitis . The half-lives (t1/2) and mean residence times (MRT) of norfloxacin were 353, 206 and 115 min (harmonic means) and 306+/-76, 168+/-39 and 95+/-9 min in control cows or in cows with subclinical and postacute clinical mastitis, respectively . The half-lives in the clinical mastitis group were significantly shorter than in the control group and the mean residence times were significantly shorter in the two mastitis groups when compared to the control group . Norfloxacin concentrations in milk were extremely high when compared to the respective serum concentrations . The area under the concentration vs . time curve (AUC) of norfloxacin in milk was 23899+/-6206 mg/L x min in the control cow group . The AUC in milk was significantly lower in the infected udder quarters of the mastitis groups (5075+/-1887 mg/L x min and 7484+/-4645 mg/L x min in the subclinical and the clinical group) . The AUC values were significantly lower in milk from the infected udder quarters of the cows with chronic subclinical and postacute clinical mastitis when compared to the values in milk from the healthy quarters of the same udder . Norfloxacin was marginally bound to serum protein . The binding was concentration dependent and was 19, 13 and 6% at 0.2, 1.0 and 8.4 mg/L, respectively . Binding to milk protein was 46-51% and concentration independent . An in vitro dialysis model was used to simulate drug transport between serum and milk as a function of protein binding . The results showed that the rate of norfloxacin disposition from milk to serum was slower than from serum to milk, which was in agreement with the findings obtained in the pharmacokinetic study . Norfloxacin was poorly soluble in organic solvents and our results suggest that changes in the degree of ionization of the drug in different body fluids considerably affect its disposition. Eur J Biochem, 1999 Aug, 264(1), 242 - 9 Properties of the methylcobalamin:H4folate methyltransferase involved in chloromethane utilization by Methylobacterium sp . strain CM4; Studer A et al.; Methylobacterium sp . strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source . Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate . The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) {Vannelli et al . (1999) Proc . Natl Acad . Sci . USA 96, 4615-4620} . We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II . This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1 . The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1 . The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme . Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II . Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II. Eur J Biochem, 1999 Aug, 264(1), 223 - 32 Asparaginyl endopeptidase (VmPE-1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH-EP); Okamoto T et al.; A vacuolar cysteine proteinase, designated SH-EP, is synthesized in cotyledons of germinated Vigna mungo seeds and is responsible for degradation of the seed proteins accumulated in protein bodies (protein storage vacuoles) . SH-EP belongs to the papain proteinase family and has a large N-terminal prosegment consisting of 104 amino acid residues and a C-terminal prosegment of 10 amino acid residues . It has been suggested that an asparaginyl endopeptidase, V . mungo processing enzyme 1 (VmPE-1), is involved in the N-terminal post-translational processing of SH-EP . The recombinant proform of SH-EP (rSH-EP) was produced in Escherichia coli cells, purified to homogeneity and refolded by stepwise dialysis . 31P-NMR analysis of intact germinated cotyledons revealed that the vacuolar pH of cotyledonary cells changes from 6.04 to 5.47 during seed germination and early seedling growth . rSH-EP was converted in vitro to the mature form through autocatalytic processing at a pH mimicking the vacuolar pH at the mid and late stages of seed germination, but not at the pH of the early stage . VmPE-1 accelerated the rate of processing of rSH-EP in vitro at the pH equivalent to the vacuolar pH at the early and mid stages of germination . In addition, the cleavage sites of the in vitro processed intermediates and the mature form of SH-EP were identical to those of SH-EP purified from germinated cotyledons of V . mungo . We propose that the asparaginyl endopeptidase (VmPE-1)-mediated processing mainly functions in the activation of proSH-EP at the early stage of seed germination, and both VmPE-1-mediated and autocatalytic processings function synergistically in the activation of proSH-EP in cotyledons at the mid and late stages. Eur J Biochem, 1999 Aug, 264(1), 161 - 7 Class II DNA photolyase from Arabidopsis thaliana contains FAD as a cofactor; Kleiner O et al.; The major UV-B photoproduct in DNA is the cyclobutane pyrimidine dimer (CPD) . CPD-photolyases repair this DNA damage by a light-driven electron transfer . The chromophores of the class II CPD-photolyase from Arabidopsis thaliana, which was cloned recently {Taylor, R., Tobin, A . & Bray, C . (1996) Plant Physiol . 112, 862; Ahmad, M., Jarillo, J.A., Klimczak, L.J., Landry, L.G., Peng, T., Last, R.L . & Cashmore, A.R . (1997) Plant Cell 9, 199-207}, have not been characterized so far . Here we report on the overexpression of the Arabidopsis CPD photolyase in Escherichia coli as a 6 x His-tag fusion protein, its purification and the analysis of the chromophore composition and enzymatic activity . Like class I photolyase, the Arabidopsis enzyme contains FAD but a second chromophore was not detectable . Despite the lack of a second chromophore the purified enzyme has photoreactivating activity. Cancer Chemother Pharmacol, 1999, 44(4), 343 - 8 Comparative cytotoxicity and pharmacokinetics of antimelanoma immunotoxins containing either natural or recombinant gelonin; Rosenblum MG et al.; Immunotoxins are a class of targeted therapeutic agents under development by various research groups . The murine monoclonal antibody designated ZME-018 recognizes a high molecular weight glycoprotein present on most human melanoma cells and biopsy specimens and has been utilized for clinical imaging studies in patients with melanoma . The plant toxin gelonin is a ribosome-inactivating protein (RIP) with n-glycosidase activity similar to that of ricin A chain . In previous studies by our group, the gelonin toxin was sequenced, cloned and expressed in E . coli . The purified recombinant gelonin (RG) was found to have identical protein synthesis inhibitory activity to that of natural gelonin (NG) . For comparative purposes, chemical conjugates of antibody ZME and either RG or NG were produced using the heterobifunctional crosslinking reagents SPDP and SMPT . The ZME-NG and ZME-RG immunotoxins were found to be 10(4)- to 10(5)-fold more cytotoxic to antigen-positive human melanoma cells than free toxin . NG toxin alone was cytotoxic to intact cells (IC(50) = 100 nM) while RG was nontoxic to cells at doses up to 1 microM . Both ZME-NG and ZME-RG immunoconjugates were nontoxic to antigen-negative (Me-180) cells . ZME-RG immunotoxins constructed with the more stable SMPT reagent were slightly more effective in culture than conjugates made with SPDP . Tissue distribution studies in tumor-bearing nude mice demonstrated that tumor uptake of the ZME-RG immunotoxin was similar to that of the intact ZME antibody with reduced distribution to normal organs compared to an immunoconjugate produced with NG . Pharmacokinetic studies showed that the terminal-phase plasma half-life of ZME-RG was similar to that of ZME itself (42 h vs 50 h) and almost threefold higher than that of ZME-NG (11.5 h) . The area under the concentration curve (Cxt) for ZME-RG was 50% lower than that for ZME due to an increased apparent volume of distribution (Vd(a)) but was almost tenfold higher than the Cxt for ZME-NG . These studies suggest that immunoconjugates comprising RG demonstrate identical in vitro cytotoxic effects to immunoconjugates produced with NG and immunotoxins with RG display improved in vivo pharmacodynamics and tissue distribution compared to immunotoxins containing NG. Cancer Res, 1999 Aug 1, 59(15), 3621 - 6 Elevated mutant frequencies in lymphoid tissues persist throughout plasmacytoma development in BALB/c.lambdaLIZ mice; Felix K et al.; Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, the mean mutant frequencies in the target gene, lacI, were found to be significantly increased in lymphoid tissues of congenic BALB/c.lambdaLIZ N5 mice in the terminal stage of a plasmacytoma induction experiment, 213-280 days after the first i.p . injection of the plasmacytomagenic agent pristane (2,6,10,14-tetramethylpentadecane) . In plasmacytoma-bearing mice (n = 7), mutant frequencies in the spleens and mesenteric lymph nodes were elevated 2.46-fold and 5.35-fold, respectively, when compared with age-matched controls . In plasmacytoma-negative mice (n = 11), mutant frequencies were increased 2.30-fold (spleens) and 3.48-fold (mesenteric nodes) . These results, interpreted in conjunction with our previous findings (K . Felix et al., Cancer Res., 58: 1616-1619, 1998) of approximately 3-fold elevations in pristane-induced splenic mutagenesis on day 42 postpristane, indicate that increased mutant levels in lymphoid tissues persist throughout plasmacytomagenesis in genetically susceptible BALB/c mice. Hum Gene Ther, 1999 Jul 20, 10(11), 1853 - 66 Intramuscular grafts of myoblasts genetically modified to secrete glial cell line-derived neurotrophic factor prevent motoneuron loss and disease progression in a mouse model of familial amyotrophic lateral sclerosis; Mohajeri MH et al.; Effects of ex vivo GDNF gene delivery on the degeneration of motoneurons were studied in the G1H transgenic mouse model of familial ALS carrying a human superoxide dismutase (SOD1) with a Gly93Ala mutation (Gurney et al., 1994) . Retroviral vectors were made to produce human GDNF or E . coli beta-galactosidase (beta-Gal) by transient transfection of the Phoenix cell line and used to infect primary mouse myoblasts . In 6-week-old G1H mice, 50,000 myoblasts per muscle were injected bilaterally into two hindlimb muscles . Untreated G1H and wild-type mice served as additional controls . At 17 weeks of age, 1 week before sacrifice, these muscles were injected with fluorogold (FG) to retrogradely label spinal motoneurons that maintained axonal projections to the muscles . There were significantly more large FG-labeled alpha motoneurons at 18 weeks in GDNF-treated G1H mice than in untreated and beta-Gal-treated G1H mice . A morphometric study of motoneuron size distribution showed that GDNF shifted the size distribution of motoneurons toward larger cells compared with control G1H mice, although the average size and number of large motoneurons in GDNF-treated mice were less than that in wild-type mice . GDNF also prolonged the onset of disease, delayed the deterioration of performance in tests of motor behavior, and slowed muscle atrophy . Quantitative, real-time RT-PCR and PCR showed persistence of transgene mRNA and DNA in muscle for up to 12 weeks postgrafting . These observations demonstrate that ex vivo GDNF gene therapy in a mouse model of FALS promotes the survival of functional motoneurons, suggesting that a similar approach might delay the progression of neurodegeneration in ALS. Parasitology, 1999 Jul, 119 ( Pt 1), 81 - 93 Cloning and analysis of a Trichinella britovi gene encoding a cytoplasmic heat shock protein of 72 kDa; Vayssier M et al.; A gene encoding a protein of 646 amino acid residues with a molecular mass of 71.3 kDa showing homology to the cytoplasmic form of the 70 kDa heat shock protein was cloned and sequenced from the nematode parasite Trichinella britovi (Tb) . The gene was expressed in vitro as a protein of 71 kDa that was immunoprecipitated by a Trichinella-infected rabbit serum . Monospecific polyclonal antibodies raised against the recombinant Tb Hsp70 expressed in Escherichia coli, recognized a protein of 70 kDa by Western blot analysis of Tb soluble antigen (muscular stage) . Tb Hsp70 was located in the nuclei of the muscle larvae as determined by the indirect immunofluorescent pattern on cross-sections of the worm . The expression of this protein was not detected in adult worm nuclei suggesting a differential expression of Hsp70 between the 2 stages of Trichinella. Biochim Biophys Acta, 1999 Aug 12, 1451(1), 196 - 205 RGD-CAP ((beta)ig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin alpha(1)beta(1); Ohno S et al.; In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human (beta)ig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-beta . In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes . When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or beta-galactosidase-coated dishes) . The effect of RGD-CAP on the cell spreading required divalent cations (Mg(2+) or Mn(2+)), and was reduced by EDTA . Monoclonal antibodies (mAbs) to the human integrin alpha(1) or beta(1) subunit, but not to the alpha(2), alpha(3), alpha(5) or beta(2) subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts . In a parallel experiment, the mAb to the alpha(5) subunit, but not the mAb to the alpha(1) subunit, suppressed fibronectin-induced spreading of these cells . These findings suggest that RGD-CAP is a novel ligand for integrin alpha(1)beta(1) that dose not bind to the RGD motif . Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading. Biochim Biophys Acta, 1999 Aug 17, 1433(1-2), 343 - 9 Molecular cloning of a cold-shock domain protein, zfY1, in zebrafish embryo(1); Chang BE et al.; Cold-shock domain proteins in vertebrates contain a highly conserved domain which is related to the Escherichia coli cold-shock proteins . Here we report the cloning of a cold-shock domain protein from zebrafish embryo . Using the combination of PCR techniques with degenerate primers, 5'RACE and 3'RACE, the full length cDNA of a cold-shock domain protein in the zebrafish embryo was successfully cloned without constructing and screening a library . Determined from the deduced amino acid sequence, this protein is most similar to Xenopus, FRGY1, and this newly cloned zebrafish gene was therefore designated as zfY1. Biochim Biophys Acta, 1999 Aug 17, 1433(1-2), 170 - 87 The reaction mechanism of ribonuclease II and its interaction with nucleic acid secondary structures; Cannistraro VJ et al.; Ribonuclease II is a processive 3'- to 5'-exoribonuclease in Escherichia coli with two binding sites: a catalytic site associated with the first few 3'-nucleotides and an anchor site binding nucleotides approximately 15 to 25 from the 3'-end . When RNase II degrades single-stranded helical poly(C), the enzyme-substrate complex dissociates at discrete intervals of 12 nucleotides . RNase II stalled at the last rC of single-stranded 3'-(rC)(n)(dC)(m) oligonucleotides . The more residues released, the faster the stalled complex dissociated and the less it inhibited RNase II activity, i.e . the enzyme-substrate association weakened progressively . Using phosphodiesterase I (PDE I) as a probe, a method was developed to identify cytidine residues in (32)P-oligonucleotides interacting with a protein . PAGE bands corresponding to nucleotides 1-6 from the 3'-end were consistent with interaction at the catalytic site, and following a gap, bands approximately 15 to 25 from the 3'-end, with anchor site association . Both 3' and 5' binding were necessary to maintain the complex . Of most significance, the original anchor site nucleotides remained fixed at the anchor site while the 3'-end was pulled, or threaded, through the catalytic site, i.e . the substrate did not 'slide' through the enzyme . DNA oligonucleotides with double-stranded stem-loops were good competitive inhibitors of RNase II . A 3'-single-stranded arm was essential, while optimal binding required both 5'- and 3'-arms . PDE I probing indicated that the nucleotides at the anchor site were specified by the spatial distance from the catalytic site, and on only one of the duplex strands . When degradation of a structured RNA paused or stopped, the RNase II-product commenced cycles of dissociation-reassociation . Duplex strand binding by RNase II made complex DNA or RNA structures accessible to degradation by other nucleases and further verified the PDE I footprinting method. Biochim Biophys Acta, 1999 Aug 17, 1433(1-2), 103 - 9 Autonomous folding of a C-terminal inhibitory fragment of Escherichia coli isoleucine-tRNA synthetase; Michaels JE et al.; We previously reported that C-terminal fragments of Escherichia coli Ile-tRNA synthetase, a monomeric enzyme of 939 amino acids, act as dominant negative inhibitors of the wild-type enzyme in vivo and in vitro . Our experiments suggested that it is possible to block the functional assembly of a monomeric protein by interfering with the folding pathway . We postulated that the inhibitory C-terminal fragments fold autonomously, and in the presence of full-length Ile-tRNA synthetase, trap the N-terminal portion of polypeptide in an unproductive complex . Here, we report the results of experiments aimed at understanding the mechanism of dominant negative inhibition . We have carried out biophysical experiments on fragment 585-939 of Ile-tRNA synthetase, which we previously determined to be the minimal inhibitory unit . Circular dichroism and fluorescence spectroscopy indicate that this fragment forms a compact and stable structure in solution . The secondary structure of this fragment is predominantly alpha-helical, consistent with the crystal structure of Ile-tRNA synthetase from another organism . The C-terminal fragment is capable of forming native-like secondary and tertiary structure after refolding from guanidine HCl . Taken together, the results are consistent with the hypothesis that the inhibitory fragment of Ile-tRNA synthetase forms an independent folding unit. Biochim Biophys Acta, 1999 Aug 20, 1420(1-2), 63 - 72 Melibiose carrier of Escherichia coli: use of cysteine mutagenesis to identify the amino acids on the hydrophilic face of transmembrane helix 2; Matsuzaki S et al.; The melibiose carrier from Escherichia coli is a galactoside-cation symporter . Based on both experimental evidence and hydropathy analysis, 12 transmembrane helices have been assigned to this integral membrane protein . Transmembrane helix 2 contains several charged and polar amino acids that have been shown to be essential for the cation-coupled transport of melibiose . Starting with the cysteine-less melibiose carrier, we have individually substituted cysteine for amino acids 39-66, which includes the proposed transmembrane helix 2 . In the resulting derivative carriers, we measured the transport of melibiose, determined the effect of the hydrophilic sulfhydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), on transport in intact cells and inside out vesicles, and examined the ability of melibiose to protect the carrier from inactivation by the sulfhydryl reagent . We found a set of seven positions in which the reaction with the sulfhydryl reagent caused partial or complete loss of carrier function measured in intact cells or inside-out vesicles . The presence of melibiose protected five of these positions from reaction with PCMBS . The reaction of two additional positions with PCMBS resulted in the partial loss of transport function only in inside-out vesicles . Melibiose protected these two positions from reaction with the reagent . Together, the PCMBS-sensitive sites and charged residues assigned to helix 2 form a cluster of amino acids that map in three rows with each row comprised of every fourth residue . This is the pattern expected of residues that are part of an alpha-helical structure and thus the rows are tilted at an angle of 25 degrees to the helical axis . We suggest that these residues line the path of melibiose and its associated cation through the carrier. Mech Dev, 1999 Aug, 86(1-2), 113 - 23 Drosophila terminalia as an appendage-like structure; Gorfinkiel N et al.; In Drosophila, the homeotic gene Distal-less (Dll) has a fundamental role in the establishment of the identity of ventral appendages such as the leg and antenna . This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp) . During the development of the two components of the anal primordium - the hindgut and the analia - only the latter is dependent on Dll and hedgehog (hh) functions . The hindgut is defined by the expression of the homeobox gene even-skipped . The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain . Meanwhile targeted ectopic Dll represses eve expression and hindgut formation . The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia . In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream to Dpp . These results suggest that the terminalia show similar behaviour to the leg disc or the antennal part of the eye-antennal disc consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage. Nucleic Acids Res . 1999 Sep 1;27(17):e16. Simplified generation of targeting constructs using ET recombination; Angrand PO et al.; ET recombination is a way to engineer DNA in Escherichia coli using homologous recombination . Here we develop the potential of ET recombination in two ways relevant to complex engineering exercises such as building gene targeting constructs . First, a targeting construct was made in a single step . Second, ET recombination was used to place two unique restriction sites at precise positions in a large genomic clone . Subsequently a complex targeting construct was created by ligation with a multifunctional cassette. Nucleic Acids Res . 1999 Sep 1;27(17):e13. Use of a linear multicopy vector based on the mini-replicon of temperate coliphage N15 for cloning DNA with abnormal secondary structures; Ravin NV et al.; A new cloning vector pN15L is described . It is a linear 13.8 kb plasmid based on the coliphage N15 mini-replicon . The vector capacity exceeds 50 kb and the copy number is 250 per Escherichia coli chromosome . We show that some artificial and natural palindromes and approximately 5% of human DNA Bgl II fragments can be cloned effectively in linear vector pN15L, whereas they either sharply reduce the copy number of circular vector pUC19 or cannot be cloned at all . We conclude that pN15L may be usefully employed to clone large imperfect palindromes and some abnormal sequences of human DNA. Nucleic Acids Res, 1999 Sep 1, 27(17), 3583 - 8 Magnesium-dependent alternative foldings of active and inactive Escherichia coli tRNA(Glu) revealed by chemical probing; Madore E et al.; A stable conformer of Escherichia coli tRNA(Glu), obtained in the absence of Mg(2+), is inactive in the aminoacylation reaction . Probing it with diethylpyrocarbonate, dimethyl sulfate and ribonuclease V1 revealed that it has a hairpin structure with two internal loops; the helical segments at both extremities have the same structure as the acceptor stem and the anticodon arm of the native conformer of tRNA(Glu)and the middle helix is formed of nucleotides from the D-loop (G15-C20:2) and parts of the T-loop and stem (G51-C56), with G19 bulging out . This model is consistent with other known properties of this inactive conformer, including its capacity to dimerize . Therefore, this tRNA requires magnesium to acquire a conformation that can be aminoacylated, as others require a post-transcriptional modification to reach this active conformation. Nucleic Acids Res, 1999 Sep 1, 27(17), 3518 - 26 Characterization of the DNA binding and bending HMG domain of the yeast hypoxic repressor Rox1; Deckert J et al.; The yeast Rox1 hypoxic transcriptional repressor protein binds to and bends a specific DNA sequence through an HMG domain located at the N-terminus . To better understand the structure of Rox1 and how it interacts with DNA, 38 missense mutations in the HMG domain were isolated through a combination of random and site-directed mutageneses, the latter directed to two Ile residues that play an important role in DNA recognition and bending by HMG domains . The mutants were characterized in terms of their ability to repress the hypoxic gene ANB1 and the auto-repressed ROX1 gene in vivo . The mutant HMG domains were fused to maltose binding protein and expressed in and purified from Escherichia coli and their relative affinities for DNA and ability to bend DNA were determined . A model of the structure of the Rox1 HMG domain was derived using sequence similarities between Rox1 and the human protein SRY, the structure of which has been determined . The results of the mutational analysis are interpreted in terms of the model structure of Rox1. Nucleic Acids Res, 1999 Sep 1, 27(17), 3487 - 92 Differential effects of single-stranded DNA binding proteins (SSBs) on uracil DNA glycosylases (UDGs) from Escherichia coli and mycobacteria; Purnapatre K et al.; Deamination of cytosines results in accumulation of uracil residues in DNA, which unless repaired lead to GC-->AT transition mutations . Uracil DNA glyco-sylase excises uracil residues from DNA and initiates the base excision repair pathway to safeguard the genomic integrity . In this study, we have investigated the effect of single-stranded DNA binding proteins (SSBs) from Escherichia coli (Eco SSB) and Mycobacterium tuberculosis (Mtu SSB) on uracil excision from synthetic substrates by uracil DNA glycosylases (UDGs) from E . coli, Mycobacterium smegmatis and M.tuberculosis (referred to as Eco -, Msm - and Mtu UDGs respectively) . Presence of SSBs with all the three UDGs resulted in decreased efficiency of uracil excision from a single-stranded 'unstructured' oligonucleo-tide, SS-U9 . On the other hand, addition of Eco SSB to Eco UDG, or Mtu SSB to Mtu UDG reactions resulted in increased efficiency of uracil excision from a hairpin oligonucleotide containing dU at the second position in a tetraloop (Loop-U2) . Interestingly, the efficiency of uracil excision by Msm UDG from the same substrate was decreased in the presence of either Eco- or Mtu SSBs . Furthermore, Mtu SSB also decreased uracil excision from Loop-U2 by Eco UDG . Our studies using surface plasmon resonance technique demonstrated interactions between the homologous combinations of SSBs and UDGs . Heterologous combinations either did not show detectable interaction (Eco SSB with Mtu UDG) or showed a relatively weaker interaction (Mtu SSB with Eco UDG) . Taken together, our studies suggest differential interactions between the two groups (SSBs and UDGs) of the highly conserved proteins . Such studies may provide important clues to design selective inhibitors against this important class of DNA repair enzymes. Nucleic Acids Res, 1999 Sep 1, 27(17), 3481 - 6 Deletion errors generated during replication of CAG repeats; Kroutil LC et al.; Triplet repeat sequence instability is associated with hereditary neurological diseases and with certain types of cancer . Here we study one form of this instability, deletion of triplet repeats during replication of template (CAG)(n)sequences by DNA polymerases . To monitor loss of triplet codons, we inserted (CAG)(9)and (CAG)(17)repeats into the lacZ sequence in M13mp2 and changed one repeat to a TAG codon to yield DNA substrates with colorless plaque phenotypes . Templates containing these inserts within gaps were copied and errors were scored as blue plaque Lac revertants whose DNA was sequenced to determine if loss of the TAG codon resulted from substitutions or deletions . DNA synthesis by either DNA polymerase beta or exonuclease-deficient T7 DNA polymerase produced deletions involving loss of from 1 to 8 of 9 or 15 of 17 repeats . Thus, these polymerases utilize misaligned template-primers containing from 3 to 45 extra template strand nucleotides . Deletion frequencies were much higher than substitution frequencies at the TAG codon in certain repeats, indicating that triplet repeats are at high risk for mutation in the absence of error correction . Proofreading-proficient T7 DNA polymerase generated deletions at 2- to 10-fold lower frequencies than did its exonuclease-deficient derivative . This suggests that misaligned triplet repeat sequences are subject to proofreading, but at reduced efficiency compared to editing of single-base mismatches. Nucleic Acids Res, 1999 Sep 1, 27(17), 3474 - 80 Isolation of altered specificity mutants of the single-chain 434 repressor that recognize asymmetric DNA sequences containing the TTAA and TTAC subsites; Simoncsits A et al.; A novel single-chain (sc) protein framework containing covalently dimerized DNA-binding domains (DBD) of the phage 434 repressor was used to construct combinatorial mutant libraries in order to isolate mutant DBDs with altered specificities . The library members contain one wild-type DBD and one mutant domain with randomized amino acids in the DNA-contacting region . Based on previous studies, the mutant sc derivatives are expected to recognize a general ACAA-6 bp-NNNN sequence, where ACAA is contacted by the wild-type and NNNN by the mutant domain . In principle, any sequence can stand for NNNN and serve as a selection target . Here an in vivo library screening method was used to isolate mutant sc repressors that interact with an asymmetric operator containing the TTAA target . Several mutants showed high affinity in vitro binding to operators containing the target and strong (up to 80-fold) preference for the TTAA target over the wild-type TTGT . Specificity studies revealed that certain mutants bound with substantially higher affinities (K(d) approximately 10(-11)M) to operators containing the TTAC sequence, a close homolog of the TTAA target . Thus, we have fortuitously isolated mutant sc repressors that show up to a several hundred-fold preference for TTAC over TTGT. J Biol Chem, 1999 Aug 20, 274(34), 24308 - 15 An analysis of the binding of repressor protein ModE to modABCD (molybdate transport) operator/promoter DNA of Escherichia coli; Grunden AM et al.; Expression of the modABCD operon in Escherichia coli, which codes for a molybdate-specific transporter, is repressed by ModE in vivo in a molybdate-dependent fashion . In vitro DNase I-footprinting experiments identified three distinct regions of protection by ModE-molybdate on the modA operator/promoter DNA, GTTATATT (-15 to -8; region 1), GCCTACAT (-4 to +4; region 2), and GTTACAT (+8 to +14; region 3) . Within the three regions of the protected DNA, a pentamer sequence, TAYAT (Y = C or T), can be identified . DNA-electrophoretic mobility experiments showed that the protected regions 1 and 2 are essential for binding of ModE-molybdate to DNA, whereas the protected region 3 increases the affinity of the DNA to the repressor . The stoichiometry of this interaction was found to be two ModE-molybdate per modA operator DNA . ModE-molybdate at 5 nM completely protected the modABCD operator/promoter DNA from DNase I-catalyzed hydrolysis, whereas ModE alone failed to protect the DNA even at 100 nM . The apparent K(d) for the interaction between the modA operator DNA and ModE-molybdate was 0.3 nM, and the K(d) increased to 8 nM in the absence of molybdate . Among the various oxyanions tested, only tungstate replaced molybdate in the repression of modA by ModE, but the affinity of ModE-tungstate for modABCD operator DNA was 6 times lower than with ModE-molybdate . A mutant ModE(T125I) protein, which repressed modA-lac even in the absence of molybdate, protected the same region of modA operator DNA in the absence of molybdate . The apparent K(d) for the interaction between modA operator DNA and ModE(T125I) was 3 nM in the presence of molybdate and 4 nM without molybdate . The binding of molybdate to ModE resulted in a decrease in fluorescence emission, indicating a conformational change of the protein upon molybdate binding . The fluorescence emission spectra of mutant ModE proteins, ModE(T125I) and ModE(Q216*), were unaffected by molybdate . The molybdate-independent mutant ModE proteins apparently mimic in its conformation the native ModE-molybdate complex, which binds to a DNA sequence motif of TATAT-7bp-TAYAT. J Biol Chem, 1999 Aug 20, 274(34), 24195 - 201 Aspzincin, a family of metalloendopeptidases with a new zinc-binding motif . Identification of new zinc-binding sites (His(128), His(132), and Asp(164)) and three catalytically crucial residues (Glu(129), Asp(143), and Tyr(106)) of deuterolysin from Aspergillus oryzae by site-directed mutagenesis; Fushimi N et al.; Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da . Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis . Substitutions of His(128) and His(132) with Arg, of Glu(129) with Gln or Asp, of Asp(143) with Asn or Glu, of Asp(164) with Asn, and of Tyr(106) with Phe resulted in almost complete loss of the activity of the mutant enzymes . It can be concluded that His(128), His(132), and Asp(164) provide the Zn(2+) ligands of the enzyme according to a (65)Zn binding assay . Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu(129), Asp(143), and Tyr(106) are catalytically crucial residues in the enzyme . Glu(129) may be implicated in a central role in the catalytic function . We conclude that deuterolysin is a member of a family of Zn(2+) metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand. J Biol Chem, 1999 Aug 20, 274(34), 24137 - 41 Structural and functional consequences of the mutation of a conserved arginine residue in alphaA and alphaB crystallins; Kumar LV et al.; A point mutation of a highly conserved arginine residue in alphaA and alphaB crystallins was shown to cause autosomal dominant congenital cataract and desmin-related myopathy, respectively, in humans . To study the structural and functional consequences of this mutation, human alphaA and alphaB crystallin genes were cloned and the conserved arginine residue (Arg-116 in alphaA crystallin and Arg-120 in alphaB crystallin) mutated to Cys and Gly, respectively, by site-directed mutagenesis . The recombinant wild-type and mutant proteins were expressed in Escherichia coli and purified . The mutant and wild-type proteins were characterized by SDS-polyacrylamide gel electrophoresis, Western immunoblotting, gel permeation chromatography, fluorescence, and circular dichroism spectroscopy . Biophysical studies reveal significant differences between the wild-type and mutant proteins . The chaperone-like activity was studied by analyzing the ability of the recombinant proteins to prevent dithiothreitol-induced aggregation of insulin . The mutations R116C in alphaA crystallin and R120G in alphaB crystallin reduce the chaperone-like activity of these proteins significantly . Near UV circular dichroism and intrinsic fluorescence spectra indicate a change in tertiary structure of the mutants . Far UV circular dichroism spectra suggest altered packing of the secondary structural elements . Gel permeation chromatography reveals polydispersity for both of the mutant proteins . An appreciable increase in the molecular mass of the mutant alphaA crystallin is also observed . However, the change in oligomer size of the alphaB mutant is less significant . These results suggest that the conserved arginine of the alpha-crystallin domain of the small heat shock proteins is essential for their structural integrity and subsequent in vivo function. J Biol Chem, 1999 Aug 20, 274(34), 23893 - 900 Characterization of two recombinant Drosophila calpains . CALPA and a novel homolog, CALPB; Jekely G et al.; We have sequenced the cDNA of a novel Ca(2+)-activated cysteine proteinase (calpain) from the fruit fly, Drosophila melanogaster . The predicted protein, designated as CALPB, shows high similarity to the previously identified Drosophila calpain, CALPA . The two proteins were expressed in Escherichia coli and purified to homogeneity by metal-chelate affinity chromatography either from inclusion bodies or from the bacterial cytosol . Both enzymes were Ca(2+)-dependent proteinases and attained half-maximal activation in the presence of millimolar Ca(2+) . The activity and the rate of activation of CALPA, but not CALPB, could be activated by phosphatidylinositol 4,5-diphosphate, phosphatidylinositol 4-monophosphate, phosphatidylinositol, and phosphatidic acid . A truncated form of CALPA, lacking the CALPA-specific unique insertion region, has also been expressed and characterized . Although it lacked the 16-amino acid long putative membrane-anchoring segment, its activation by phospholipids was similar to that of the full-length CALPA protein . The enzymes undergo N-terminal autolysis in a Ca(2+)-dependent manner which was shown with CALPB to run parallel with enzyme activation . Moreover, fully autolyzed CALPB lacked the characteristic activation phase indicating the requirement for autolysis upon activation of this calpain form in vitro . The analysis of the mechanism of activation in Drosophila calpains seems to corroborate the autolysis model of calpain activation. J Biol Chem, 1999 Aug 20, 274(34), 23883 - 6 The L1Tc, long interspersed nucleotide element from Trypanosoma cruzi, encodes a protein with 3'-phosphatase and 3'-phosphodiesterase enzymatic activities; Olivares M et al.; The presence of a long interspersed nucleotide element, named L1Tc, which is actively transcribed in the parasite Trypanosoma cruzi, has been recently described . The open reading frame 1 of this element encodes the NL1Tc protein, which has apurinic/apyrimidinic endonuclease activity and is probably implicated in the first stage of the transposition of the element . In the present paper we show that NL1Tc effectively removes 3'-blocking groups (3'-phosphate and 3'-phosphoglycolate) from damaged DNA substrates . Thus, both 3'-phosphatase and 3'-phosphodiesterase activities are present in NL1Tc . We propose that these enzymatic activities would allow the 3'-blocking ends to function as targets for the insertion of L1Tc element, in addition to the apurinic/apyrimidinic sites previously described . The potential biological function of the NL1Tc protein has also been evidenced by its ability to repair the DNA damage induced by the methyl methanesulfonate alkylating or oxidative agents such as hydrogen peroxide and t-butyl hydroperoxide in Escherichia coli (xth and xth, nfo) mutants. J Biol Chem, 1999 Aug 20, 274(34), 23868 - 74 A single amino acid substitution in SecY stabilizes the interaction with SecA; Manting EH et al.; The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane . It binds the peripheral ATPase SecA to form the translocase . When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence . SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP . The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY . These data imply an important role for SecY transmembrane segment 7 in SecA binding . As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression. J Biol Chem, 1999 Aug 20, 274(34), 23841 - 3 Activity of yeast orotidine-5'-phosphate decarboxylase in the absence of metals; Miller BG et al.; Yeast orotidine-5'-phosphate decarboxylase was recently shown to contain zinc and to be inhibited by zinc-complexing agents . When the gene for the yeast enzyme was expressed in Escherichia coli, the gene product was devoid of metal atoms but exhibited a specific activity and molecular mass similar to those of the enzyme obtained directly from yeast . This invalidates the hypothesis that zinc is involved in substrate decarboxylation . The zinc-free enzyme undergoes thermal inactivation at a somewhat lower temperature than does the zinc-containing enzyme isolated from yeast. J Biol Chem, 1999 Aug 20, 274(34), 23814 - 9 Cloning and characterization of the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster; Johansson M et al.; A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L . (1998) J . Biol . Chem . 273, 3926-3931) . The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases . We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs . The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed . Dm-dNK is closely related to human and herpes simplex virus deoxyribonucleoside kinases . The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates . The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer. Mem Inst Oswaldo Cruz, 1999 Jul-Aug, 94(4), 513 - 8 A preliminary investigation on the chemical composition of the cell surface of five enteropathogenic Escherichia coli serotypes; Mangia AH et al.; The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy . Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins . Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues . The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100 degree C for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon . Phosphate, total sugar and protein contents were determined . Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose . Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction . Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction. RNA, 1999 Aug, 5(8), 1034 - 41 Negative in vitro selection identifies the rRNA recognition motif for ErmE methyltransferase; Nielsen AK et al.; Erm methyltransferases modify bacterial 23S ribosomal RNA at adenosine 2058 (A2058, Escherichia coli numbering) conferring resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics . The motif that is recognized by Erm methyltransferases is contained within helix 73 of 23S rRNA and the adjacent single-stranded region around A2058 . An RNA transcript of 72 nt that displays this motif functions as an efficient substrate for the ErmE methyltransferase . Pools of degenerate RNAs were formed by doping 34-nt positions that extend over and beyond the putative Erm recognition motif within the 72-mer RNA . The RNAs were passed through a series of rounds of methylation with ErmE . After each round, RNAs were selected that had partially or completely lost their ability to be methylated . After several rounds of methylation/selection, 187 subclones were analyzed . Forty-three of the subclones contained substitutions at single sites, and these are confined to 12 nucleotide positions . These nucleotides, corresponding to A2051-A2060, C2611, and A2614 in 23S rRNA, presumably comprise the RNA recognition motif for ErmE methyltransferase . The structure formed by these nucleotides is highly conserved throughout bacterial rRNAs, and is proposed to constitute the motif that is recognized by all the Erm methyltransferases. RNA, 1999 Aug, 5(8), 1021 - 33 Multiple binding modes of substrate to the catalytic RNA subunit of RNase P from Escherichia coli; Pomeranz Krummel DA et al.; M1 RNA that contained 4'-thiouridine was photochemically cross-linked to different substrates and to a product of the reaction it governs . The locations of the cross-links in these photochemically induced complexes were identified . The cross-links indicated that different substrates share some contacts but have distinct binding modes to M1 RNA . The binding of some substrates also results in a substrate-dependent conformational change in the enzymatic RNA, as evidenced by the appearance of an M1 RNA intramolecular cross-link . The identification of the cross-links between M1 RNA and product indicate that they are shared with only one of the three cross-linked E-S complexes that were identified, an indication of noncompetitive inhibition by the product . We also examined whether the cross-linked complexes between M1 RNA and substrate(s) or product are altered in the presence of the enzyme's protein cofactor (C5 protein) and in the presence of different concentrations of divalent metal ions . C5 protein enhanced the yield of certain M1 RNA-substrate cross-linked complexes for both wild-type M1 RNA and a deletion mutant of M1 RNA (delta{273-281}), but not for the M1 RNA-product complex . High concentrations of Mg2+ increased the yield of all M1 RNA-substrate complexes but not the M1 RNA-product complex. J Enzyme Inhib, 1999, 14(2), 167 - 74 Interaction of cystatin C variants with papain and human cathepsins B, H and L; Cimerman N et al.; Recombinant human cystatin C and two of its mutants were expressed in Escherichia coli . The recombinant inhibitor was found to be identical to authentic cystatin C as judged by isoelectric focusing (pI 9.2) and kinetics of inhibition of papain and human cathepsins B, H and L . N-terminal truncation of 8 residues resulted in a decrease of isoelectric point (pI 7.8), but the inhibitory properties were similar to those of recombinant cystatin C, suggesting that Leu9 is a critical residue for the inhibition . The mutation of Trp106 to Ser, however, resulted in a decreased affinity of the inhibitor for the enzymes tested, with the largest effect on cathepsin B inhibition (approximately 100-fold increase in Ki). J Periodontal Res, 1999 May, 34(4), 197 - 202 Porphyromonas gingivalis lipopolysaccharide delays human polymorphonuclear leukocyte apoptosis in vitro; Preshaw PM et al.; Apoptosis (programmed cell death) is a mechanism by which superfluous or damaged cells undergo changes that lead to selective removal from organ systems by phagocytic cells . Certain bacterial products delay apoptosis in neutrophils (PMNs) . In this study, PMNs were incubated for up to 8 h with varying concentrations of lipopolysaccharide (LPS), lipid A or capsular polysaccharide isolated from 3 strains of Porphyromonas gingivalis (Pg) (strains HG-184, A7A1-28 and 381) . Assay runs included controls containing cells and medium but no bacterial products . Fluorescence microscopy was used to evaluate apoptotic changes . PMNs exhibited a time-dependent increase in the number of apoptotic cells . When cells were cultured in the presence of LPS from any of the 3 Pg strains, apoptosis was delayed in a dose-dependent fashion (p < 0.05) . The effects of these LPS preparations were similar to each other and to Escherichia coli 0111:B4 LPS . Lipid A from the 3 Pg strains also delayed apoptosis (p < 0.05), but was less potent than LPS or synthetic lipid A . Capsular polysaccharide had no significant effect on apoptosis (p > 0.05) . Thus, LPS and lipid A from P . gingivalis appear to modulate the functional lifespan of PMNs . This could potentiate the inflammatory and destructive components of periodontal diseases. Genes Dev, 1999 Aug 1, 13(15), 2005 - 16 A novel property of the RecA nucleoprotein filament: activation of double- stranded DNA for strand exchange in trans; Mazin AV et al.; RecA protein catalyzes DNA strand exchange, a basic step of homologous recombination . Upon binding to single-stranded DNA (ssDNA), RecA protein forms a helical nucleoprotein filament . Normally, this nucleoprotein filament binds double-stranded DNA (dsDNA) and promotes exchange of base pairs between this dsDNA and the homologous ssDNA that is contained within this filament . Here, we demonstrate that this bound dsDNA can be activated by interaction with a heterologous RecA nucleoprotein filament for a novel type of strand exchange with homologous ssDNA that is external to, and, therefore, not within, the filament . We refer to this novel DNA strand exchange as being in trans . Thus, the RecA nucleoprotein filament is a protein scaffold that activates dsDNA for strand exchange with ssDNA either within the filament or external to it . This new property demonstrates that the RecA nucleoprotein filament makes dsDNA receptive for DNA strand exchange, and it defines an early step of the homology recognition mechanism. Am J Physiol, 1999 Aug, 277(2 Pt 1), C225 - 32 Functional changes in troponin T by a splice donor site mutation that causes hypertrophic cardiomyopathy; Nakaura H et al.; A splice donor site mutation in intron 15 of the cardiac troponin T (TnT) gene has been shown to cause familial hypertrophic cardiomyopathy (HCM) . In this study, two truncated human cardiac TnTs expected to be produced by this mutation were expressed in Escherichia coli and partially (50-55%) exchanged into rabbit permeabilized cardiac muscle fibers . The fibers into which a short truncated TnT, which lacked the COOH-terminal 21 amino acids because of the replacement of 28 amino acids with 7 novel residues, had been exchanged generated a Ca(2+)-activated maximum force that was slightly, but statistically significantly, lower than that generated by fibers into which wild-type TnT had been exchanged when troponin I (TnI) was phosphorylated by cAMP-dependent protein kinase . A long truncated TnT simply lacking the COOH-terminal 14 amino acids had no significant effect on the maximum force-generating capability in the fibers with either phosphorylated or dephosphorylated TnI . Both these two truncated TnTs conferred a lower cooperativity and a higher Ca(2+) sensitivity on the Ca(2+)-activated force generation than did wild-type TnT, independent of the phosphorylation of TnI by cAMP-dependent protein kinase . The results demonstrate that the splice donor site mutation in the cardiac TnT gene impairs the regulatory function of the TnT molecule, leading to an increase in the Ca(2+) sensitivity, and a decrease in the cooperativity, of cardiac muscle contraction, which might be involved in the pathogenesis of HCM. Br J Haematol, 1999 Jul, 106(1), 248 - 51 Prestorage and bedside leucofiltration of whole blood modulates storage-time-dependent suppression of in vitro TNFalpha release; Mynster T et al.; Immunosuppression after transfusion may be related to the content of leucocytes in the transfused blood . Therefore we studied the effects of prestorage and bedside leucodepletion by filtration on the suppression by whole blood of in vitro stimulated tumour necrosis factor alpha (TNFalpha) release . Nine units of whole blood were leucofiltered prestorage and stored for 35 d . 27 units, 3 x 9, were stored and leucofiltered at the bedside after 7, 21 and 35 d . Supernatants were collected from all units during storage and added to a whole blood assay of E . coli-LPS-stimulated TNFalpha release . The effects of storage were assessed and compared with supernatants collected immediately after donation as reference . TNFalpha release was storage time dependently suppressed to: 81%, 74% and 57% by supernatants from non-filtered blood stored for 7, 21 and 35 d, respectively . Prestorage leucofiltration almost eliminated this effect, but we still observed a storage-time-dependent suppression by bedside-leucofiltered blood to 88%, 78% and 65%, respectively . Prestorage leucofiltration appeared to reduce storage-time-dependent suppression of in vitro stimulated TNFalpha release induced by plasma from whole blood compared with non-filtered and bedside-leucofiltered whole blood. Plant Physiol, 1999 Aug, 120(4), 1147 - 56 The localization and expression of the class II starch synthases of wheat; Li Z et al.; The starch granules of hexaploid wheat (Triticum aestivum) contain a group of three proteins known as SGP-1 (starch granule protein-1) proteins, which have apparent molecular masses of 100, 108, and 115 kD . The nature and role of these proteins has not been defined previously . We demonstrate that these polypeptides are starch synthases that are present in both the starch granule and the soluble fraction at the early stages of wheat endosperm development, but that are exclusively granule bound at mid and late endosperm development . A partial cDNA clone encoding a fragment of the 100-kD protein was obtained by screening a wheat endosperm cDNA expression library using monoclonal antibodies . Three classes of cDNA were subsequently isolated from a wheat endosperm cDNA library by nucleic acid hybridization and were shown to encode the 100-, 108-, and 115-kD proteins . The cDNA sequences are highly homologous to class II starch synthases and have the highest homology with the maize SSIIa (starch synthase IIa) gene . mRNA for the SGP-1 proteins was detected in the leaf, pre-anthesis florets, and endosperm of wheat and is highly expressed in the leaf and in the grain during the early to mid stages of development . We discuss the roles of the SGP-1 proteins in starch biosynthesis in wheat. Free Radic Biol Med, 1999 Jul, 27(1-2), 193 - 202 Expression, purification, and biochemical characterization of SAG, a ring finger redox-sensitive protein; Swaroop M et al.; We recently reported the cloning and characterization of SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, that is redox responsive and protects mammalian cells from apoptosis . Here we report the expression, purification, and biochemical characterization of SAG . Bacterially expressed SAG is brown in color and dithiothreitol (DTT)-sensitive . SAG forms large oligomers without DTT that can be reduced into a monomer in the presence of DTT . These features help us to purify SAG using the chromatography with or without DTT . Likewise, purified SAG is redox sensitive . Upon H2O2 exposure, SAG forms oligomers as well as monomer doublets due to the formation of the inter- or intramolecular disulfide bonds, respectively . This process can be reversed by DTT or prevented by pretreatment with the alkylating reagent, N-ethylmaleimide (NEM) . Although SAG contains two putative heme-binding sites and a RING finger domain, the protein appears not to bind with heme and to lack transcription factor activity as determined in a Gal4-fusion/transactivation assay . Wildtype, but not RING finger domain-disrupted SAG mutants, prevents copper-induced lipid peroxidation . These results, along with our previous observations, suggest that SAG is an intracellular antioxidant molecule that may act as a redox sensor to buffer oxidative-stress induced damage. Free Radic Biol Med, 1999 Jul, 27(1-2), 90 - 4 Higher activity of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) coincides with lower background levels of 8-oxo-2'-deoxyguanosine in DNA of fetal compared with maternal mouse organs; Bialkowski K et al.; Mammalian homologues of Escherichia coli MutT, a protein having 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity, are thought to play the same role in preventing the incorporation of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) into DNA . One could thus expect that higher activity of 8-oxo-dGTPase should correlate with a lower background level of 8-oxo-dG in nuclear DNA . During transplacental carcinogenesis experiments, in control healthy Swiss mice on day 18 of gestation we found consistently lower levels of 8-oxo-dG in DNA in fetal livers and lungs (1.74+/-0.04 SE and 1.49+/-0.08 SE 8-oxo-dG/10(5) dG, respectively; pooled organs of fetuses of 8 dams) as compared with maternal organs (3.05+/-0.20 SE and 3.08+/-0.17 SE 8-oxo-dG/10(5) dG, respectively; n = 8) . The 8-oxo-dGTPase activity determination in the same organs revealed that the lower levels of 8-oxo-dG in fetal DNA did, indeed, coincide with higher 8-oxo-dGTPase activity (48.8+/-2.6 SE and 52.5+/-2.5 SE U/mg protein in livers and lungs, respectively); and vice versa, higher 8-oxo-dG levels in DNA of maternal organs were associated with lower levels of 8-oxo-dGTPase activity (24.3+/-1.3 SE and 4.7+/-0.6 SE U/mg protein, as above) . Without excluding other reasons for the relatively low 8-oxo-dG background in DNA of fetal tissues (e.g., higher level of antioxidants and antioxidative enzymes; more efficient DNA repair), this inverse relationship may support or at least does not contradict the concept of a guardian role of 8-oxo-dGTPase against 8-oxo-dGTP mutagenicity in mammalian cells. Biol Pharm Bull, 1999 Jul, 22(7), 734 - 7 Characterization of an antagonist monoclonal antibody, GHBP116, specific for human growth hormone receptors; Takagi K et al.; To obtain an antagonist antibody against human growth hormone receptors (hGHRs), we prepared monoclonal antibodies against the recombinant hGHR extracellular domain . One of the clones, GHBP116, exhibited binding activity to intact human IM-9 cells and effectively immunoprecipitated the receptors in cell lysate . GHBP116 competitively inhibited 125I-human growth hormone (hGH) binding to the cells . The antagonist activity of GHBP116 was assessed in terms of ligand-induced receptor internalization, degradation, and phosphorylation of signal transducer and activator of transcription (STAT) 5 . The antibody alone did not cause internalization or degradation of hGHRs, but a 1:25000 dilution of ascitic fluid almost completely inhibited ligand (1 nM hGH)-induced internalization and degradation of surface hGHRs . Moreover, GHBP116 alone did not stimulate the phosphorylation of STAT5, used as an indicator of Janus kinase (JAK)-STAT signaling, but almost completely inhibited hGH-induced phosphorylation of STAT5 . These results suggest that GHBP116 acts as a specific antagonist of hGH. Biophys Chem, 1999 Jun 28, 79(3), 187 - 92 Positioning proton-donating residues to the Schiff-base accelerates the M-decay of pharaonis phoborhodopsin expressed in Escherichia coli; Iwamoto M et al.; Phoborhodopsin (also called sensory rhodopsin II, sR-II) is a receptor for the negative phototaxis of Halobacterium salinarum (pR), and pharaonis phoborhodopsin (ppR) is the corresponding receptor of Natronobacterium pharaonis . pR and ppR are retinoid proteins and have a photocycle similar to that of bacteriorhodopsin (bR) . A major difference between the photocycle of the ion pump bR and the sensor pR or ppR is found in their turnover rates which are much faster for bR . A reason for this difference might be found in the lack of a proton-donating residue to the Schiff base which is formed between the lysine of the opsin and retinal . To reconstruct a bR-like photochemical behavior, we expressed ppR mutants in Escherichia coli in which proton-donating groups have been reintroduced into the cytoplasmic proton channel . In measurement of the photocycle it could be shown that the F86D mutant of ppR (Phe86 was substituted by Asp) showed a faster decay of M-intermediate than the wild-type, which was even accelerated in the F86D/L40T double mutant. Nitric Oxide, 1999 Jun, 3(3), 216 - 24 Inhibition of nitric oxide production rescues LPS-induced fetal abortion in mice; Athanassakis I et al.; In this report, we examined the involvement of the cytokines tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-4, and IL-10 as well as nitric oxide (NO) in the lipopolysaccharide (LPS)-induced experimental abortion model in BALB/c mice . Although in vivo administration of LPS in pregnant mice showed a 72% decrease of serum IL-10, no significant difference in serum TNF-alpha, IFN-gamma, and IL-4 levels, compared to controls, could be detected . At the same time, a correlation of fetal abortion and maternal splenomegaly with an important increase of NO synthesis in the serum was obtained . Simultaneous administration of LPS and aminoguanidine (AG; an inhibitor to NO synthase) rescued the LPS-induced fetal abortion, reduced maternal spleen weight to physiological levels, and decreased serum NO concentration to control levels . In vitro experiments showed that LPS directly induced NO production in primary placental cells and the TPOPHO-1 trophoblast cell line by stimulating the inducible isoform of NO synthase, which ultimately could be blocked by the NO synthase inhibitors AG and L-NAME . The results indicate that LPS, despite its beneficial involvement in intracellular infections, participates in inflammatory/autoimmune damage during pregnancy, leading to embryotoxicity, which is closely linked to the NO pathway. Curr Microbiol, 1999 Sep, 39(3), 123 - 8 Phylogenetic study of methanogens associated with rumen ciliates; Tokura M et al.; The phylogeny of methanogenic archaea associated with ciliate protozoa in a sheep rumen was investigated . Ruminal ciliate protozoa were exhaustively washed and mixtures of genomic DNA extracted . Archaea-specific nested PCR amplification was conducted with the ciliate genomic mixture . The resultant small subunit (16S) ribosomal RNA gene (ssu rDNA) was cloned into Escherichia coli JM 109 . Many methanogens were still observed on and/or in ciliate cells by fluorescent microscopy even after exhaustive washing with buffer . Partial sequences of ssu rDNA close to Methanobrevibacter smithii were dominant in the retrieved sequences . RFLP analyses on the retrieved sequences revealed the absence of Methanobrevibacter ruminantium in the protozoal preparation . The association of Methanobrevibacter spp . with ruminal ciliate protozoa was demonstrated by the isolation of archaeal ssu rDNA phylogenetically close to that of M . smithii. Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 676 - 81 Minithioredoxin: a folded and functional peptide fragment of thioredoxin; Ghoshal AK; A peptide fragment comprising the first 83 residues from the N-terminus of E . coli thioredoxin is purified by hydroxylamine cleavage of the intact protein . At physiological pH, the secondary and tertiary structure contents of the peptide are 70 and 35%, respectively, compared to the intact protein . Peptide 83 is able to display dual biological functions of thioredoxin, namely, a substrate for the enzyme E . coli thioredoxin-reductase and a processivity factor of T7 DNA polymerase . At present, peptide 83 represents the minimum functional and folding unit of thioredoxin . The highly conserved residue Phe 81 appears to play an important role in the folding of peptide 83, as judged from the packing analysis . Peptide 83 also mimics a particular kinetic folding intermediate of thioredoxin in terms of spectral properties and may serve as an equilibrium peptide model for the former . Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 669 - 75 Characterization of a novel allergen, a major IgE-binding protein from Aspergillus flavus, as an alkaline serine protease; Yu CJ et al.; Aspergillus species of fungi have been known to be one of the most prevalent aeroallergens . One important A . flavus allergen (Asp fl 1) was identified by means of immunoblotting with a serum pool of allergic patients on a two-dimensional electrophoretic gel . The cDNA coding for Asp fl 1 was cloned and sequenced . The clone encodes a full-length protein of 403 amino acid precursors of 42 kDa . After cleavage of a putative signal peptide of 21 amino acids and a prepeptide of 100 amino acids, a mature protein of 282 amino acids was obtained with a molecular mass of 33 kDa and a pI of 6.3 . A degree of identity was found in a range of 27 to 84% among related allergens derived from bacteria allergen subtilisin, mold allergen Pen c 1, and virulence factor of A . fumigatus . Recombinant Asp fl 1 (rAsp fl 1) was cloned into vector pQE-30 and expressed in E . coli M15 as a histidine-tag fusion protein and purified to homogeneity . The IgE binding capacity of rAsp fl 1 was tested by immunoblotting using a serum pool of Aspergillus-allergic patients . Recombinant allergen cross-reacted strongly with IgE specific for natural Asp fl 1 and Pen c 1, indicating that common IgE epitopes may exist between allergens of A . flavus and P . citrinum . Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 584 - 9 DNA sequence analysis of spontaneous tonB deletion mutations in a polA1 strain of Escherichia coli K12; Agemizu Y et al.; By DNA sequence analysis, we have determined a spectrum of 61 spontaneous mutations occurring in the endogenous tonB gene in the polA1 strain of Escherichia coli . The overall mutation frequency was approximately 2.4-fold higher in the polA1 strain and this was attributable to enhanced rates of deletion and frameshift mutations . Among 39 deletions, a hot spot (17 mutations) was detected: a 13-bp deletion presumably directed by a 3-bp repeated sequence at its end points . The remaining 22 were distributed among 19 different mutations either flanked (16/19) or not flanked (3/19) by repeated sequences . Single-base frameshifts accounted for 8 mutations of either repeated (3/8) or nonrepeated (5/8) bases among which 6 were minus one frameshift . In contrast to previous reports, we did not frequently observe a 5'-GTGG-3' sequence in the vicinity of the deletions and frameshifts . The results presented here indicated an anti-deletion and anti-frameshift role for DNA polymerase I . Arch Biochem Biophys, 1999 Aug 15, 368(2), 380 - 4 Characterization of zinc-depleted alanyl-tRNA synthetase from Escherichia coli: role of zinc; Sood SM et al.; To evaluate the role of zinc in Escherichia coli alanyl-tRNA synthetase, hydrodynamic measurements and circular dichroism spectra were obtained for the zinc-depleted protein and compared with those of the native enzyme . At a protein concentration of 5 mg ml(-1), pH 7.5, the sedimentation coefficient (s(20,w)) was 6.3 S and was virtually independent of temperature between 10 and 37 degrees C, similar to the results reported for the native form . However, the s(20,w) now decreased significantly as the concentration increased, indicative of a possible change in conformation . The s(20,w) value did not appear to change as the pH was increased to 9.5 . In standard buffer with 3.3 M added urea, a single peak with a s(20,w) of 3.6 S was obtained and with 6.6 M added urea, a peak with a s(20,w) of 2.7 S was seen . Added Gd-HCl (6 M) gave a single peak with s(20,w) of 2 . 0 S . Like the native form, laser light scattering studies indicated some heterogeneity and a radius of 6.4 nm which was virtually independent of concentration and temperature in the range of 10-37 degrees C . At 25 degrees C, a diffusion coefficient (D(20,w)) of 3.3 x 10(-7) cm(2) s(-1) was obtained . The combination of s(0)(20,w) and D(20,w) yielded a molecular mass of approximately 179 kDa, which is slightly less than that reported for the native dimeric form (186 kDa) . The intrinsic viscosity at 25 degrees C was extrapolated to 5 . 3 ml g(-1), a value significantly higher than that reported for the native form, which increased with temperature . These results indicate some conformational and flexibility changes from the native to the zinc-depleted form, which may explain differences in activity . Furthermore, urea denaturation experiments demonstrate the role of zinc in stabilization of AlaRS structure . Arch Biochem Biophys, 1999 Aug 15, 368(2), 347 - 53 Plant phospholipase Dalpha is an acidic phospholipase active at near-physiological Ca(2+) concentrations; Pappan K et al.; The conventional plant phospholipase D (PLD) requires Ca(2+) for activity; however, the most distinct and puzzling feature of this PLD is its in vitro need for 20 to 100 mM Ca(2+) . This noncytoplasmic Ca(2+) requirement has raised doubt about the role of Ca(2+) in regulating its function in vivo . Using the cloned conventional castor bean PLD, PLDalpha, expressed in Escherichia coli, this study demonstrates that this PLD is active at micromolar, near-physiological concentrations of Ca(2+), and this activity at low Ca(2+) requires an acidic pH (4.5-5.5) . By comparison, the newly cloned PLDbeta and -gamma were active only at neutral pH under the same Ca(2+) concentrations . This study also shows that PLDalpha activity at low Ca(2+) needs substrates presented as a mixture of membrane lipids . Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate are equally effective in stimulating the acidic PLDalpha activity, whereas phophatidylinositol is inactive . These results suggest that the conventional plant PLD in vivo is an acidic phospholipase that is active at near-physiological Ca(2+) concentrations . The possible physiological significance of these findings will be discussed . Arch Biochem Biophys, 1999 Aug 15, 368(2), 257 - 64 Kinetic studies on drug-resistant variants of Escherichia coli thymidylate synthase: functional effects of amino acid substitutions at residue 4; Mahdavian E et al.; A naturally occurring mutant of human thymidylate synthase (hTS) that contains a Tyr to His mutation at residue 33 was found to confer 4-fold resistance to 5-fluoro-2'-deoxyuridine (FdUrd), a prodrug of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) . The crystal structure of hTS implicated this Tyr residue in a drug resistance mechanistic role that may include both substrate binding and catalysis (Schiffer et al., Biochemistry, 34, 16279-16287, 1995) . Because of the existence of a defined kinetic scheme and the development of a bacterial expression vector for the overproduction of Escherichia coli TS (ecTS), we chose to initially study the corresponding residue in the bacterial enzyme, Tyr 4 of ecTS . Nine mutant ecTS enzymes that differed in sequence at position 4 were generated . Mutants with a charged or polar side chain (Ser, Cys, Asp, and Arg) and Gly precipitated in the cell paste, resulting in no catalytic activity in cell-free extracts . Although most of the His 4 mutant precipitated, sufficient amounts remained in the cell-free extract to permit isolation to near homogeneity . Wild-type ecTS and mutants with a hydrophobic side chain (Phe, Ile, and Val) were expressed at nearly 30% of the total cellular protein . The k(cat) values for the isolatable mutants were 2- to 10-fold lower than that of the wild-type enzyme, while the K(m) values for 2'-deoxyuridylate (dUMP) and 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were similar for all the mutants . Dissociation constants for binary complex formation determined by stopped-flow spectroscopy were similar for the wild-type and mutant enzymes for both dUMP and 2'-deoxythymidylate, indicating that this mutation does not significantly alter the binding of the natural nucleotide ligands . However, each mutant enzyme had three- to 5-fold lower affinity for FdUMP in the binary complex compared with the wild-type enzyme, and only His 4 showed a lower affinity for FdUMP in the ternary complex . Analysis of k(burst) showed that the initial binding of CH(2)H(4)folate is weaker for each mutant compared to the wild-type enzyme and that lower k(cat) values were due to compromised rates that govern the chemical transformation of bound substrates to bound products . Arch Biochem Biophys, 1999 Aug 15, 368(2), 232 - 43 cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis); Bohlmann J et al.; Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury . A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase . Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases . Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)) and geranylgeranyl diphosphate (C(20)), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (-)-camphene synthase, (-)-beta-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (-)-limonene and (-)-alpha-pinene . The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50-60 residues . cDNA truncation to delete the transit peptide allowed functional expression of the "pseudomature" forms of these enzymes, which exhibited no change in product outcome as a result of truncation . Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C(15)) synthases and diterpene (C(20)) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species . The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure-function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization-cyclization reaction . Biotechnol Prog, 1999 Jul-Aug, 15(4), 587 - 93 Effect of polyphosphate metabolism on the Escherichia coli phosphate-starvation response; Van Dien SJ et al.; A previously developed dynamic model of the Escherichia coli Pho regulon was extended to investigate the effect of polyphosphate synthesis and degradation on this control system . Differential equations for ATP and polyphosphate were formulated, and the model was applied to the growth of cells containing the ppk and ppx genes under control of separate, inducible promoters . In agreement with recent experimental observations, the degradation of polyphosphate by PPX during a period of phosphate limitation could repress the phosphate-starvation response . This is attributed to the release of phosphate from the cell into the periplasm, where it can be detected by the external phosphate sensor . A segregated model was then developed to account for differences in K(I), the dissociation constant for the repression complex, among cells of the population . Since K(I) is the key parameter in determining whether the Pho response is induced or repressed at a particular surface phosphate concentration, this permitted the induction of some cells while others remained repressed . The induction profiles resulting from the population-averaged values more closely matched experimental results than did those with the nonsegregated model. Hum Mol Genet, 1999 Sep, 8(9), 1647 - 55 Aberrant interactions of transcriptional repressor proteins with the Huntington's disease gene product, huntingtin; Boutell JM et al.; We detected an interaction of the N-terminus of huntingtin (htt171) with the C-terminal region of the nuclear receptor co-repressor (N-CoR) using the yeast two-hybrid system . This interaction was repeat length dependent and specific to htt171; the co-repressor did not interact with the repeat carrying a section of atrophin 1 nor with the androgen receptor or polyglutamine alone . The interaction was confirmed using His-tagged Escherichia coli -expressed C-terminal human and rat co-repressor protein which pulled full-length huntingtin out of homogenized rat brain and in pull-down assays . The N-CoR represses transcription from sequence-specific ligand-activated receptors such as the retinoid X-thyroid hormone receptor dimers and other nuclear receptors including Mad-Max receptor dimers . The mechanism of this repression appears to be through the formation of a complex of repressor proteins including the N-CoR, mSin3 and histone deacetylases . We have used N-CoR and mSin3A antibodies in immunohistochemical studies and find that in Huntington's disease (HD) cortex and caudate, the cellular localization of these proteins is exclusively cytoplasmic whilst in control brain they are localized in the nucleus as well as the cytoplasm; mSin3A immunoreactivity also occurred in a subset of huntingtin positive intranuclear inclusions . The relocalization of repressor proteins in HD brain may alter transcription and be involved in the pathology of the disease. Biochemistry, 1999 Aug 10, 38(32), 10594 - 605 Effect of serinate ligation at each of the iron sites of the {Fe4S4} cluster of Pyrococcus furiosus ferredoxin on the redox, spectroscopic, and biological properties; Brereton PS et al.; Pyrococcus furiosus ferredoxin (Fd) contains a single {Fe(4)S(4)} cluster coordinated by three cysteine (at positions 11, 17, and 56) and one aspartate ligand (at position 14) . In this study, the spectroscopic, redox, and functional consequences of D14C, D14C/C11S, D14S, D14C/C17S, and D14C/C56S mutations have been investigated . The four serine variants each contain a potential cluster coordination sphere of one serine and three cysteine residues, with serine ligation at each of the four Fe sites of the {Fe(4)S(4)} cluster . All five variants were expressed in Escherichia coli, and each contained a {Fe(4)S(4)}(2+,+) cluster as shown by UV-visible absorption and resonance Raman studies of the oxidized protein and EPR and variable-temperature magnetic circular dichroism (VTMCD) studies of the as-prepared, dithionite-reduced protein . Changes in both the absorption and resonance Raman spectra are consistent with changing from complete cysteinyl cluster ligation in the D14C variant to three cysteines and one oxygenic ligand in each of the four serine variants . EPR and VTMCD studies show distinctive ground and excited state properties for the paramagnetic {Fe(4)S(4)}(+) centers in each of these variant proteins, with the D14C and D14C/C11S variants having homogeneous S = (1)/(2) ground states and the D14S, D14C/C17S, and D14C/C56S variants having mixed-spin, S = (1)/(2) and (3)/(2) ground states . The midpoint potentials (pH 7.0, 23 degrees C) of the D14C/C11S and D14C/C17S variants were unchanged compared to that of the D14C variant (E(m) = -427 mV) within experimental error, but the potentials of D14C/C56S and D14S variants were more negative by 49 and 78 mV, respectively . Since the VTMCD spectra indicate the presence of a valence-delocalized Fe(2 . 5+)Fe(2.5+) pair in all five variants, the midpoint potentials are interpreted in terms of Cys11 and Cys17 ligating the nonreducible valence-delocalized pair in D14C . Only the D14S variant exhibited a pH-dependent redox potential over the range of 3.5-10, and this is attributed to protonation of the serinate ligand to the reduced cluster (pK(a) = 4.75) . All five variants had similar K(m) and V(m) values in a coupled assay in which Fd was reduced by pyruvate ferredoxin oxidoreductase (POR) and oxidized by ferredoxin NADP oxidoreductase (FNOR), both purified from P . furiosus . Hence, the mode of ligation at each Fe atom in the {Fe(4)S(4)} cluster appears to have little effect on the interaction and the electron transfer between Fd and FNOR. Biochemistry, 1999 Aug 10, 38(32), 10543 - 51 Regulatory properties of recombinant tropomyosins containing 5-hydroxytryptophan: Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site; Farah CS et al.; We have introduced tryptophan codons at different positions of the chicken alpha-tropomyosin cDNA (Monteiro, P . B., Lataro, R . C., Ferro, J . A., and Reinach, F . C . (1994) J . Biol . Chem . 269, 10461-10466) and employed a trp auxotrophic Escherichia coli strain to express the proteins in media containing either normal tryptophan, 5-hydroxytrptophan, or 7-azatryptophan . The fluorescence of these latter two tryptophan analogues is excitable at 312-315 nm at which the natural fluorescence of other thin filament proteins (actin, troponin) is not excited . The recombinant tropomyosins have tryptophans or analogues located at amino acid positions 90, 101, 111, 122, or 185 of the protein, all on the external surface of the tropomyosin coiled-coil (positions "c" or "f" of the hydrophobic heptad repeat) . The first four mutations are located within the third actin-binding zone of tropomyosin, a region not expected to interact directly with troponin or with neighboring tropomyosin molecules in muscle thin filaments, while position 185 is located in a region that has been implicated in interactions with the globular domain of troponin . The fluorescence intensity of the mutant containing 5-hydroxytryptophan at position 122 (5OH122W) is sensitive to actin binding and sensitive to Ca2+-binding to thin filaments reconstituted with troponin . Assuming that the globular domain of troponin binds to a site between residues 150 and 190 of tropomyosin, the distance between the troponin-binding site and the fluorescent probes at position 122 can be estimated to be 4.2-10.2 nm . While X-ray diffraction and electron micrograph reconstitution studies have provided evidence of Ca2+-induced changes in tropomyosin's interactions in the thin filament, their resolution was not sufficient to distinguish between changes involving the whole tropomyosin molecule or only that region directly interacting with troponin . Here we provide a clear demonstration that Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site which is probably associated with a changed interaction with actin. Biochemistry, 1999 Aug 10, 38(32), 10480 - 8 Spectral and kinetic studies of the oxidation of monosubstituted phenols and anilines by recombinant Synechocystis catalase-peroxidase compound I; Regelsberger G et al.; A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Synechocystis PCC 6803 is reported . Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the first examination of second-order rate constants for the oxidation of a series of aromatic donor molecules (monosubstituted phenols and anilines) by a bifunctional catalase-peroxidase compound I using the sequential-mixing stopped-flow technique . Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation . A >/=50-fold excess of peroxoacetic acid is required to obtain a spectrum of relatively pure and stable compound I which is characterized by about 40% hypochromicity, a Soret maximum at 406 nm, and isosbestic points between the native enzyme and compound I at 357 and 430 nm . The apparent second-order rate constant for formation of compound I from ferric enzyme and peroxoacetic acid is (8.74 +/- 0.26) x 10(3) M(-)(1) s(-)(1) at pH 7 . 0 . Reduction of compound I by aromatic donor molecules is dependent upon the substituent effect on the benzene ring . The apparent second-order rate constants varied from (3.6 +/- 0.1) x 10(6) M(-)(1) s(-)(1) for p-hydroxyaniline to (5.0 +/- 0.1) x 10(2) M(-)(1) s(-)(1) for p-hydroxybenzenesulfonic acid . They are shown to correlate with the substituent constants in the Hammett equation, which suggests that in bifunctional catalase-peroxidases the aromatic donor molecule donates an electron to compound I and loses a proton simultaneously . The value of rho, the susceptibility factor in the Hammett equation, is -3.4 +/- 0.4 for the phenols and -5.1 +/- 0.8 for the anilines . The pH dependence of compound I reduction by aniline exhibits a relatively sharp maximum at pH 5 . The redox intermediate formed upon reduction of compound I has spectral features which indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme. Biochemistry, 1999 Aug 10, 38(32), 10272 - 86 Secondary structure forming propensity coupled with amphiphilicity is an optimal motif in a peptide or protein for association with chaperonin 60 (GroEL); Preuss M et al.; The interactions of GroEL with six dansyl peptides were investigated by means of our previously established fluorescence binding assay {Hutchinson, J . P., Oldham, T . C., El-Thaher, T . S . H., and Miller, A . D . (1997) J . Chem . Soc., Perkin Trans . 2, 279-288} . Three peptides (AMPH series) were constructed with a hierarchy of alpha-helix-forming propensities and amphiphilic characteristics . The remaining three peptides (NON-AMPH series) were prepared with a reordered amino acid sequence designed to form peptides of differing non-amphiphilic alpha-helix-forming propensity . Of these six peptides, two (AMPH(+) and NON-AMPH(+)) were N-capped with an S-form alpha-helix-inducing template (Ro 47-1615, Hoffmann-La Roche), two (AMPH(-) and NON-AMPH(-)) were N-capped with an R-form non-inducing template (Ro 47-1614, Hoffmann-La Roche), and two (AMPH(R) and NON-AMPH(R)) were without N-cap modification . This paper describes how the known strength of interaction of an unfolded protein substrate with the molecular chaperone GroEL (K(d) micromolar to nanomolar) may be emulated with a single peptide (AMPH(+)) (apparent K(d) 5 nM) which has a high propensity to form an amphiphilic alpha-helical structure in solution . Secondary structure forming propensity is not, in and of itself, an important contributor to the strength of interaction with GroEL . However, secondary structure forming propensity coupled with amphiphilicity may be sufficient to account for most, if not all, of the interaction strength between GroEL and an unfolded peptide or protein substrate. Biochemistry, 1999 Aug 10, 38(32), 10247 - 55 The solution structure of the Sac7d/DNA complex: a small-angle X-ray scattering study; Krueger JK et al.; Small-angle X-ray scattering has been used to study the structure of the multimeric complexes that form between double-stranded DNA and the archaeal chromatin protein Sac7d from Sulfolobus acidocaldarius . Scattering data from complexes of Sac7d with a defined 32-mer oligonucleotide, with poly{d(GC)}, and with E . coli DNA indicate that the protein binds along the surface of an extended DNA structure . Molecular models of fully saturated Sac7d/DNA complexes were constructed using constraints from crystal structure and solution binding data . Conformational space was searched systematically by varying the parameters of the models within the constrained set to find the best fits between the X-ray scattering data and simulated scattering curves . The best fits were obtained for models composed of repeating segments of B-DNA with sharp kinks at contiguous protein binding sites . The results are consistent with extrapolation of the X-ray crystal structure of a 1:1 Sac7d/octanucleotide complex {Robinson, H., et al . (1998) Nature 392, 202-205} to polymeric DNA . The DNA conformation in our multimeric Sac7d/DNA model has the base pairs tilted by about 35 degrees and displaced 3 A from the helix axis . There is a large roll between two base pairs at the protein-induced kink site, resulting in an overall bending angle of about 70 degrees for Sac7d binding . Regularly repeating bends in the fully saturated complex result in a zigzag structure with negligible compaction of DNA . The Sac7d molecules in the model form a unique structure with two left-handed helical ribbons winding around the outside of the right-handed duplex DNA. J Mol Recognit, 1999 Jul-Aug, 12(4), 258 - 66 Construction and characterization of two anti-sweetener single chain antibodies using radioligand binding, fluorescence and circular dichroism spectroscopy; Pledger DW et al.; Two single-chain antibodies (scFv) that bind the superpotent sweetener ligand, NC-174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, kappa) and NC10.14 (IgG, lambda) . These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25-amino-acid polypeptide linker, and expressing them in E . coli using the pET-11a system . The recombinant proteins were purified using Ni(2+)-NTA-agarose by virtue of a hexahistidine sequence introduced to the C-terminus of the heavy chain variable region during the cloning process . The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy . The far-UV CD spectra of both scFv possessed predominantly beta character, as did those of the Fab, and the near-UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding . The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent . Nature, 1999 Jul 29, 400(6743), 425 - 30 Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction; Corrie JE et al.; A new method is described for measuring motions of protein domains in their native environment on the physiological timescale . Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine . Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs . This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells . The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle. Lancet, 1999 Jul 24, 354(9175), 307 - 8 Fatal septicaemia after fibroid embolisation; Vashisht A et al.; Uterine artery embolisation is a new technique for the treatment of uterine fibroids . We report a death after this procedure. Curr Probl Pediatr, 1999 Aug, 29(7), 208 - 16 Diarrheogenic Escherichia coli; Noguera-Obenza M et al.; The diarrheogenic E coli are currently difficult to diagnose and treat . For physicians in the United States, they are primarily a concern in children returning from international travel . The exception to this generalization is STEC, which, because of the low inoculum, ease of transmission, and serious consequences, are important pathogens in the United States. Biofizika, 1999 May-Jun, 44(3), 483 - 7 {Cu2+-induced permeability of the Escherichia coli cytoplasmic membrane}; Lebedev VS et al.; Cu(2+)-induced permeability of cytoplasmic membranes of Escherichia coli for different cations and neutral molecules of saccharose was estimated by studying their effect on cell plasmolysis during uncharged exchange of cytoplasmic K+ ions by periplasmic space cations . The addition of copper resulted in the exchange of K+ ions by periplasmic Na+, Tris+, streptomycin2+, Cu2+, Ca2+, Mg2+, Cd2+, and Mn2+ . It is concluded that Cu(2+)-induced conducting pathways in bacterial membranes are hydrophilic channels with a radius of approximately 0.5 nm and a nonselective permeability for different cations. J Biol Inorg Chem, 1999 Jun, 4(3), 348 - 53 The metal function in the reactions of bovine serum amine oxidase with substrates and hydrazine inhibitors; De Matteis G et al.; Bovine serum amine oxidase (BSAO) reacts with 2-hydrazinopyridine, which binds the organic co-factor 2,4,5-trihydroxyphenylalanine quinone, forming a band at 435 nm . The band shifts to 526 nm around 60 degrees C, to 415 nm upon denaturation, but only shifts to 429 nm upon Cu2+ depletion . Its wavelength and intensity suggest that the adduct has the azo conformation, whilst the same adduct of crystalline Escherichia coli amine oxidase (ECAO) shows the hydrazone conformation in the X-ray structure . The steady state kinetics of aminomethyl- and aminoethylpyridines confirm that the formation of the product Schiff base, analogous to the azo form of the 2-hydrazinopyridine adduct, is not hindered in solution . The structural stability of the adduct in the absence of Cu2+ is taken to imply hydrogen bonding of the pyridyl nitrogen to a conserved aspartate, as in the ECAO adduct . Thus the ECAO adduct provides a good model for a transient intermediate leading to formation of the BSAO azo adduct . On the basis of this model and of the catalytic competence of Co(2+)-substituted BSAO, confirmed by the present data, a catalytic reaction scheme is proposed. J Dent Res, 1999 Aug, 78(8), 1401 - 9 Structure/function analysis of human cystatin SN and comparison of the cysteine proteinase inhibitory profiles of human cystatins C and SN; Hiltke TR et al.; Cystatins are reversible, competitive inhibitors of cysteine proteinases . Their inhibitory profiles, as well as their affinities for target enzymes, vary with different cysteine proteinases . Human cystatin C and salivary cystatin SN are 120- and 121-amino-acid (a.a.) proteins, respectively, and both contain 2 disulfide bonds . In this study, we examined the structure/function relationship of cystatin SN with respect to the inhibition of papain, with particular emphasis on the role of cystatin SN's cysteine residues, and addressed the inhibitory profiles of these two human cystatins on several cysteine proteinases (papain, clostripain, and calpain II) . The full-length recombinant cystatin C and cystatin SN, and cystatin SN variants (C-truncated {C-tr; a.a . 1-102}, delta 56-60 deletion, cysteine 74-->serine {C74S}, cys 84-->serine {C84S}, cysteine 98-->serine {C98S}, and cysteine 118-->serine {C118S}) were cloned, expressed, and produced in the pET30(b) and pGEX2T Escherichia coli expression systems . All recombinant proteins were tested for the inhibition of papain, and the full-length proteins were also tested for the inhibition of clostripain and calpain II . The secondary structures of the cystatins were also determined and compared . The results showed that the full-length cystatin C and cystatin SN, and the cystatin SN variants C98S and C118S inhibited the activity of papain . However, cystatin SN C-tr and delta 56-60 variants exhibited no inhibitory activity toward papain, while the cystatin SN variants C74S and C84S exhibited slight inhibition at higher concentrations . These results suggested that in the inhibition of papain by cystatin SN, the first disulfide loop is more important than the second . In addition, cystatin C, but not cystatin SN, inhibited calpain II, while neither cystatin inhibited clostripain, and these results, in conjunction with those from other studies, indicated that cystatin C is a broader-spectrum inhibitor of cysteine proteinases than cystatin SN. J Virol, 1999 Sep, 73(9), 7780 - 6 Phase 1 evaluation of intranasal virosomal influenza vaccine with and without Escherichia coli heat-labile toxin in adult volunteers; Gluck U et al.; Virosomal vaccines were prepared by extracting hemagglutinin (HA) and neuraminidase from influenza virus and incorporating it in the membranes of liposomes composed of phosphatidylcholine . Two intranasal spray vaccine series were prepared: one series comprised 7.5 micrograms of HA of each of three strains recommended by the World Health Organization and 1 microgram of Escherichia coli heat-labile toxin (HLT), and the other contained the HA without HLT . In addition, a third vaccine preparation contained 15 micrograms of HA and 2 micrograms of HLT . The parenteral virosomal vaccine contained 15 micrograms of HA without additional adjuvant . The immunogenicity of a single spray vaccination (15 micrograms of HA and 2 micrograms of HLT) was compared with that of two vaccinations (7.5 micrograms of HA with or without 1 microgram of HLT) with an interval of 1 week in 60 healthy working adults . Twenty volunteers received one parenteral virosomal vaccine . Two nasal spray vaccinations with HLT-adjuvanted virosomal influenza vaccine induced a humoral immune response which was comparable to that with a single parenteral vaccination . A significantly higher induction of influenza virus-specific immunoglobulin A was noted in the saliva after two nasal applications . The immune response after a single spray vaccination was significantly lower . It could be shown that the use of HLT as a mucosal adjuvant is necessary to obtain a humoral immune response comparable to that with parenteral vaccination . All vaccines were well tolerated. J Virol, 1999 Sep, 73(9), 7703 - 9 Fish rhabdovirus cell entry is mediated by fibronectin; Bearzotti M et al.; Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus . Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs . Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses . Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa . Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses . (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli . (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV . To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family. J Virol, 1999 Sep, 73(9), 7574 - 81 Antibody-independent protection against rotavirus infection of mice stimulated by intranasal immunization with chimeric VP4 or VP6 protein; Choi AH et al.; This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model . BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP) . One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured . When three 9-microg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein . Following EDIM challenge, shedding was significantly (P = 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only . Three 9-micrograms doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E . coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody . No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice . However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P = <0.001) . Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6 . When MBP::VP6 was administered intranasally to B-cell-deficient microMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls . These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody. J Virol, 1999 Sep, 73(9), 7565 - 73 Antibody-dependent and -independent protection following intranasal immunization of mice with rotavirus particles; McNeal MM et al.; The ability to elicit protective immune responses after intranasal immunization with rotavirus particles, either with or without the attenuated Escherichia coli heat-labile enterotoxin LT(R192G) as an adjuvant, was examined in the adult mouse model . BALB/c mice were administered one or two inoculations of psoralen/UV-inactivated, triple-layered (tl) or double-layered (dl) purified rotavirus particles . Four weeks after immunization, mice were challenged with the murine rotavirus strain EDIM, and the shedding of rotavirus antigen was quantified . Rotaviruses used for immunization included EDIM and heterotypic simian (RRV), bovine (WC3), and human (89-12) strains . tl EDIM stimulated both systemic and intestinal rotavirus antibody responses and complete protection with as little as one 1-microgram dose . Inclusion of LT(R192G) (10 micrograms) significantly increased rotavirus antibody responses and reduced antigen concentrations needed for full protection . Both dl EDIM and heterotypic dl and tl particles stimulated protection, but they did so less than tl EDIM at comparable concentrations, either with or without LT(R192G) . When B-cell-deficient microMt mice were immunized with tl EDIM particles, protection was reduced to levels similar to those induced with dl EDIM and heterotypic particles in BALB/c mice . However, dl EDIM particles induced similar levels of protection in both mouse strains . The protection stimulated by tl or dl EDIM particles was not diminished by CD8 cell depletion prior to immunization in either strain of mice . These results indicate that tl EDIM induced immunity at least partially through responses to its outer capsid proteins, presumably by stimulation of serotype-specific neutralizing antibody . In contrast, the other particles stimulated protection primarily by an antibody-independent mechanism . Finally, depletion of CD8 cells had no effect on protection by either mechanism. J Virol, 1999 Sep, 73(9), 7308 - 16 Characterization of the oriI and oriII origins of replication in phage-plasmid P4; Tocchetti A et al.; In the Escherichia coli phage-plasmid P4, two partially overlapping replicons with bipartite ori sites coexist . The essential components of the oriI replicon are the alpha and cnr genes and the ori1 and crr sites; the oriII replicon is composed of the alpha gene, with the internal ori2 site, and the crr region . The P4 alpha protein has primase and helicase activities and specifically binds type I iterons, present in ori1 and crr . Using a complementation test for plasmid replication, we demonstrated that the two replicons depend on both the primase and helicase activities of the alpha protein . Moreover, neither replicon requires the host DnaA, DnaG, and Rep functions . The bipartite origins of the two replicons share the crr site and differ for ori1 and ori2, respectively . By deletion mapping, we defined the minimal ori1 and ori2 regions sufficient for replication . The ori1 site was limited to a 123-bp region, which contains six type I iterons spaced regularly close to the helical periodicity, and a 35-bp AT-rich region . Deletion of one or more type I iterons inactivated oriI . Moreover, insertion of 6 or 10 bp within the ori1 region also abolished replication ability, suggesting that the relative arrangement of the iterons is relevant . The ori2 site was limited to a 36-bp P4 region that does not contain type I iterons . In vitro, the alpha protein did not bind ori2 . Thus, the alpha protein appears to act differently at the two origins of replication. J Virol, 1999 Sep, 73(9), 7287 - 96 Tyrosine phosphorylation of A17 during vaccinia virus infection: involvement of the H1 phosphatase and the F10 kinase; Derrien M et al.; Vaccinia virus encodes two protein kinases (B1 and F10) and a dual-specificity phosphatase (VH1), suggesting that phosphorylation and dephosphorylation of substrates on serine/threonine and tyrosine residues are important in regulating diverse aspects of the viral life cycle . Using a recombinant in which expression of the H1 phosphatase can be regulated experimentally (vindH1), we have previously demonstrated that repression of H1 leads to the maturation of noninfectious virions that contain several hyperphosphorylated substrates (K . Liu et al., J . Virol . 69:7823-7834) . In this report, we demonstrate that among these is a 25-kDa protein that is phosphorylated on tyrosine residues in H1-deficient virions and can be dephosphorylated by recombinant H1 . We demonstrate that the 25-kDa phosphoprotein represents the product of the A17 gene and that A17 is phosphorylated on serine, threonine, and tyrosine residues during infection . Detection of phosphotyrosine within A17 is abrogated when Tyr(203) (but not Tyr(3), Tyr(6), or Tyr(7)) is mutated to phenylalanine, suggesting strongly that this amino acid is the site of tyrosine phosphorylation . Phosphorylation of A17 fails to occur during nonpermissive infections performed with temperature-sensitive mutants defective in the F10 kinase . Our data suggest that this enzyme, which was initially characterized as a serine/threonine kinase, might in fact have dual specificity . This hypothesis is strengthened by the observation that Escherichia coli induced to express F10 contain multiple proteins which are recognized by antiphosphotyrosine antiserum . This study presents the first evidence for phosphotyrosine signaling during vaccinia virus infection and implicates the F10 kinase and the H1 phosphatase as the dual-specificity enzymes that direct this cycle of reversible phosphorylation. J Bacteriol, 1999 Aug, 181(16), 5131 - 3 Purification and ligand binding of EmrR, a regulator of a multidrug transporter; Brooun A et al.; EmrR, the repressor of the emrRAB operon of Escherichia coli, was purified to 95% homogeneity . EmrR was found to bind putative ligands of the EmrAB pump-2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and carbonyl cyanide p-(trifluoro-methoxy)phenylhydrazone-with affinities in the micromolar range . Equilibrium dialysis experiments suggested one bound ligand per monomer of the dimeric EmrR. J Bacteriol, 1999 Aug, 181(16), 5123 - 5 Escherichia coli gene ydeA encodes a major facilitator pump which exports L-arabinose and isopropyl-beta-D-thiogalactopyranoside; Carole S et al.; Inactivation of the Escherichia coli gene ydeA, which encodes a member of the major facilitator superfamily, decreased the efflux of L-arabinose, thereby affecting the expression of AraC-regulated genes . In addition, overexpression of ydeA decreased the expression of genes regulated by isopropyl-beta-D-thiogalactopyranoside. J Bacteriol, 1999 Aug, 181(16), 5081 - 4 In vitro analysis of the butyrolactone autoregulator receptor protein (FarA) of Streptomyces lavendulae FRI-5 reveals that FarA acts as a DNA-binding transcriptional regulator that controls its own synthesis; Kitani S et al.; FarA of Streptomyces lavendulae FRI-5 is a specific receptor protein for IM-2, a butyrolactone autoregulator that controls the production of a blue pigment and the nucleoside antibiotics showdomycin and minimycin . Gel shift assays demonstrated that FarA binds to the farA upstream region and that this binding is abolished in the presence of IM-2 . The FarA binding sequence was localized by DNase I footprinting to a 28-bp sequence located approximately 70 bp upstream of the farA translational start site . High-resolution S1 nuclease mapping of farA transcripts revealed a putative transcription start site, located at an A residue positioned 64 bp upstream from the farA translation start codon and 4 bp downstream from an Escherichia coli sigma(70)-like -10 recognition region . The FarA-binding sequence overlaps this -10 region and contains the farA transcription initiation site, suggesting that FarA acts as a repressor that, in the absence of IM-2, represses transcription of farA. J Bacteriol, 1999 Aug, 181(16), 4914 - 8 The pur7 gene from the puromycin biosynthetic pur cluster of Streptomyces alboniger encodes a nudix hydrolase; Espinosa JC et al.; Pur7 is the product of a gene from the puromycin biosynthetic pur cluster of Streptomyces alboniger . It was expressed in Escherichia coli as a recombinant protein fused to a His tag and then was highly purified through a Ni(2+) column . It showed a 3'-amino-3'-dATP pyrophosphohydrolase (nudix) activity which produced 3'-amino-3'-dAMP and pyrophosphate . This is consistent with the presence of a nudix box in its amino acid sequence . As observed with other nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement for Mg(2+) . Among a large variety of other nucleotides tested, only 3'-amino-3'-dTTP was a Pur7 substrate, although at lower reaction rates than 3'-amino-3'-dATP . These findings suggest that Pur7 has a high specificity for the 3' amino group at the ribofuranoside moiety of these two substrates . The K(m) and V(max) values for these dATP and dTTP derivatives were 120 microM and 17 microM/min and 3.45 mM and 12.5 microM/min, respectively . Since it is well known that 3'-amino-3'-dATP is a strong inhibitor of DNA-dependent RNA polymerase, whereas 3'-amino-3'-dAMP is not, Pur7 appears to be similar to other nudix enzymes in terms of being a housecleaning agent that permits puromycin biosynthesis to proceed through nontoxic intermediates . Finally, the identification of this activity has allowed a revision of the previously proposed puromycin biosynthetic pathway. J Bacteriol, 1999 Aug, 181(16), 4879 - 89 The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii; Hales LM et al.; To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E . coli rpoS mutation . An open reading frame that is approximately 60% identical to the E . coli rpoS gene was identified . Western blot analysis showed that the level of L . pneumophila RpoS increased in stationary phase . An insertion mutation was constructed in the rpoS gene on the chromosome of L . pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain . Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth . This finding indicates that L . pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress . Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii . These data suggest that L . pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa . Our data indicate that the role of rpoS in L . pneumophila is very different from what has previously been reported for E . coli rpoS. J Bacteriol, 1999 Aug, 181(16), 4834 - 41 Antigen-43-mediated autoaggregation of Escherichia coli is blocked by fimbriation; Hasman H et al.; Antigen 43 (Ag43), the product of the flu gene, is a surface-displayed autotransporter protein of Escherichia coli . Ag43 is responsible for the autoaggregation and flocculation of static liquid cultures of many E . coli strains . The expression of Ag43 has been reported to be phase variable and controlled by the product of the oxyR gene . Type 1 fimbriae are thin adhesive thread-like surface organelles responsible for bacterial receptor recognition and tissue colonization . Like that of Ag43, the expression of type 1 fimbriae is phase variable . Interestingly, previous results have suggested that the expression of type 1 fimbriae and the expression of Ag43 are mutually exclusive . In the present report, we show, by use of well-defined mutants, that fimbriation abolishes Ag43-mediated autoaggregation but does not affect Ag43 expression . Autoaggregation is shown to require an intercellular Ag43-Ag43 interaction, and the physical presence of fimbriae on the cells seems to abrogate this interaction . The Ag43 or OxyR status does not appear to influence fimbria expression, and our results suggest that the expression of Ag43 and the expression of fimbriae are independent processes. J Mol Biol, 1999 Aug 13, 291(2), 463 - 79 Context-dependence of amino acid residue pairing in antiparallel beta-sheets; Zaremba SM et al.; In an effort to understand the driving forces behind antiparallel beta-sheet assembly, we have investigated the mutational tolerance of four pairs of residues in CspA, the major cold shock protein of E . coli . Two buried pairs and two exposed pairs of neighboring amino acids were separately randomized and the corresponding effects on protein stability were assessed using a protein expression screen . The thermal denaturation of a subset of the recovered proteins was measured by circular dichroism spectroscopy in order to determine the range of stabilities sampled by the expressed mutants . As anticipated, buried sites are substantially less tolerant of substitutions than exposed sites with more than half of the exposed residue combinations giving rise to stably folded proteins . The two exposed residue pairs, however, display different degrees of tolerance to substitution and accept different residue pair combinations . Except for the prohibition of proline from interior strand positions, no obvious correlations of mutant stability with any single parameter such as beta-sheet propensity or hydrophobicity can be detected . Mutant combinations recovered in both orientations (e.g . XY and YX) at a given exposed pair site often show markedly different stabilities, indicating that the local environment plays a substantial role in modulating the pairing preferences of residues in beta-sheets . J Mol Biol, 1999 Aug 13, 291(2), 375 - 92 Characterisation and enzymatic properties of tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) from Pyrococcus furiosus; Constantinesco F et al.; The structural gene TRM1 encoding tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) of the hyperthermophilic archaeon Pyrococcus furiosus was cloned and expressed in Escherichia coli . The corresponding recombinant enzyme (pfTrm1p) with a His6-tag at the N terminus was purified to homogeneity in three steps . The enzyme has a native molecular mass of 49 kDa (as determined by gel filtration) and is very stable to heat denaturation (t1/2at 95 degrees C is two hours) . pfTrm1p is a monomer and forms a one to one complex with T7 transcripts of yeast tRNA(Phe) . It methylates a single guanine residue at position 26 using S -adenosyl- l -methionine as donor of the methyl groups . Depending on the incubation temperature, the type of tRNA transcript and the ratio of enzyme to tRNA, m(2)G26 or m(2)2G26 was the main product . The addition of the second methyl group to N (2)guanine 26 takes place in vitro through a monomethylated intermediate, and the enzyme dissociates from its tRNA substrate between the two consecutive methylation reactions . Identity elements in tRNA for mono- and dimethylation reactions by the recombinant pfTrm1p were identified using in vitro T7 transcripts of 33 variants of tRNA(Asp)and tRNA(Phe)from yeast . The efficient dimethylation of G26 requires the presence of base-pairs C11.G24 and G10.C25 and a variable loop of five bases within a correct 3D-core of the tRNA molecule . These identity elements probably ensure the correct presentation of monomethylated m(2)G26 to the enzyme for the attachment of the second methyl group . In contrast, the structural requirements for monomethylation of the same guanine 26 are much more relaxed and tolerate variations in the base-pairs of the D-stem, in the size of the variable loop or distortions of the 3D-architecture of the tRNA molecule . J Mol Biol, 1999 Aug 13, 291(2), 363 - 74 Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52; Baumann P et al.; Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair . Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein . Joint molecule formation required the presence of a 3' or 5'-overhang on the duplex DNA substrate and initiated preferentially at the 5'-end of the complementaryx strand . These results are consistent with a preference for strand transfer in the 3'-5' direction relative to the single-stranded DNA . The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E . coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions . Indeed, E . coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions . Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions . The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA . J Mol Biol, 1999 Aug 13, 291(2), 347 - 61 The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions; Glasfeld A et al.; The interaction of the dimeric Escherichia coli purine repressor (PurR) with its cognate sequences leads to a 45 degrees to 50 degrees kink at a central CpG base step towards the major groove, as dyad-related leucine side-chains interdigitate between these bases from the minor groove . The resulting broadening of the minor groove increases the accessibility of the six central base-pairs towards minor groove interactions with residues from PurR . It has been shown that lysine 55 of PurR makes a direct contact with the adenine base (Ade8) directly 5' to the central CpG base-pair step in the high-affinity purF operator sequence . We have investigated the importance of this interaction in the specificity and affinity of wild-type PurR (WT) for its operators and we have studied a mutant of PurR in which Lys55 is replaced with alanine (K55A) . Complexes of WT and K55A with duplex DNA containing pur operator sequences varied at position 8 were investigated crystallographically, and binding studies were performed using fluorescence anisotropy . The structures of the protein-DNA complexes reveal a relatively unperturbed global conformation regardless of the identity of the base-pair at position 8 or residue 55 . In all structures the combination of higher resolution and a palindromic purF operator site allowed several new PurR.DNA interactions to be observed, including contacts by Thr15, Thr16 and His20 . The side-chain of Lys55 makes productive, though varying, interactions with the adenine, thymine or cytosine base at position 8 that result in equilibrium dissociation constants of 2.6 nM, 10 nM and 35 nM, respectively . However, the bulk of the lysine side-chain apparently blocks high-affinity binding of operators with guanine at position 8 (Kd620 nM) . Also, the high-affinity binding conformation appears blocked, as crystals of WT bound to DNA with guanine at position 8 could not be grown . In complexes containing K55A, the alanine side-chain is too far removed to engage in van der Waals interactions with the operator, and, with the loss of the general electrostatic interaction between the phosphate backbone and the ammonium group of lysine, K55A binds each operator weakly . However, the mutation leads to a swap of specificity of PurR for the base at position 8, with K55A exhibiting a twofold preference for guanine over adenine . In addition to defining the role of Lys55 in PurR minor groove binding, these studies provide structural insight into the minor groove binding specificities of other LacI/GalR family members that have either alanine (e.g . LacI, GalR, CcpA) or a basic residue (e.g . RafR, ScrR, RbtR) at the comparable position . J Mol Biol, 1999 Jul 30, 290(5), 1009 - 18 Induced fit on sugar binding activates ribokinase; Sigrell JA et al.; The enzyme ribokinase phosphorylates ribose at O5* as the first step in its metabolism . The original X-ray structure of Escherichia coli ribokinase represented the ternary complex including ribose and ADP . Structures are presented here for the apo enzyme, as well as the ribose-bound state and four new ternary complex forms . Combined, the structures suggest that large and small conformational changes play critical roles in the function of this kinase . An initially open apo form can allow entry of the ribose substrate . After ribose binding, the active site lid is observed in a closed conformation, with the sugar trapped underneath . This closure and associated changes in the protein appear to assist ribokinase in recognition of the co-substrate ATP as the next step . Binding of the nucleotide brings about further, less dramatic adjustments in the enzyme structure . Additional small movements are almost certainly required during the phosphoryltransfer reaction . Evidence is presented that some types of movements of the lid are allowed in the ternary complex, which may be critical to the creation and breakdown of the transition state . Similar events are likely to take place during catalysis by other related carbohydrate kinases, including adenosine kinase . J Mol Biol, 1999 Jul 30, 290(5), 983 - 96 The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum reveals its substrate and reaction specificity; Steegborn C et al.; Cystathionine gamma-synthase catalyses the committed step of de novo methionine biosynthesis in micro-organisms and plants, making the enzyme an attractive target for the design of new antibiotics and herbicides . The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum has been solved by Patterson search techniques using the structure of Escherichia coli cystathionine gamma-synthase . The model was refined at 2.9 A resolution to a crystallographic R -factor of 20.1 % (Rfree25.0 %) . The physiological substrates of the enzyme, L-homoserine phosphate and L-cysteine, were modelled into the unliganded structure . These complexes support the proposed ping-pong mechanism for catalysis and illustrate the dissimilar substrate specificities of bacterial and plant cystathionine gamma-synthases on a molecular level . The main difference arises from the binding modes of the distal substrate groups (O -acetyl/succinyl versusO -phosphate) . Central in fixing the distal phosphate of the plant CGS substrate is an exposed lysine residue that is strictly conserved in plant cystathionine gamma-synthases whereas bacterial enzymes carry a glycine residue at this position . General insight regarding the reaction specificity of transsulphuration enzymes is gained by the comparison to cystathionine beta-lyase from E . coli, indicating the mechanistic importance of a second substrate binding site for L-cysteine which leads to different chemical reaction types . J Biol Chem, 1999 Aug 13, 274(33), 23673 - 8 Oligomerization of a MutS mismatch repair protein from Thermus aquaticus; Biswas I et al.; The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases . We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures . Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution . Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM . The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer . Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding. J Biol Chem, 1999 Aug 13, 274(33), 23647 - 58 Membrane topology of Alzheimer's disease-related presenilin 1 . Evidence for the existence of a molecular species with a seven membrane-spanning and one membrane-embedded structure; Nakai T et al.; A significant member of early-onset familial type of Alzheimer's disease cases has been shown to be caused by dominant mutations in either of the two genes encoding presenilin 1 (PS1) and presenilin 2 (PS2) . These two proteins are highly homologous to each other and have been reported to be mainly localized to the membranes of intracellular compartments such as the endoplasmic reticulum . Information about the membrane topological structures of these proteins is indispensable for understanding their physiological and pathological roles . Although several models have been proposed previously, their precise membrane topologies remain unknown . In this study, we examined this issue in detail by expressing a series of C-terminally deleted PS1 mutants fused to the hydrophilic portion of Escherichia coli leader peptidase in vitro using a reticulocyte lysate in the presence of microsomal membranes . Our results predict that PS1 exists mainly in a seven membrane-spanning structure with its C-terminal end exposed to the luminal space . This was also confirmed by expressing these fusion proteins in cultured cells . We further showed that a ninth hydrophobic segment is tightly bound to the membrane without spanning it . Based on the above observations, we propose a novel "seven membrane-spanning and one membrane-embedded" topological model for presenilins. J Biol Chem, 1999 Aug 13, 274(33), 23468 - 79 Genetic instabilities in (CTG.CAG) repeats occur by recombination; Jakupciak JP et al.; The expansion of triplet repeat sequences (TRS) associated with hereditary neurological diseases is believed from prior studies to be due to DNA replication . This report demonstrates that the expansion of (CTG.CAG)(n) in vivo also occurs by homologous recombination as shown by biochemical and genetic studies . A two-plasmid recombination system was established in Escherichia coli with derivatives of pUC19 (harboring the ampicillin resistance gene) and pACYC184 (harboring the tetracycline resistance gene) . The derivatives contained various triplet repeat inserts ((CTG.CAG), (CGG.CCG), (GAA.TTC), (GTC.GAC), and (GTG.CAC)) of different lengths, orientations, and extents of interruptions and a control non-repetitive sequence . The availability of the two drug resistance genes and of several unique restriction sites on the plasmids enabled rigorous genetic and biochemical analyses . The requirements for recombination at the TRS include repeat lengths >30, the presence of CTG.CAG on both plasmids, and recA and recBC . Sequence analyses on a number of DNA products isolated from individual colonies directly demonstrated the crossing-over and expansion of the homologous CTG.CAG regions . Furthermore, inversion products of the type {(CTG)(13)(CAG)(67)}.{(CTG)(67)(CAG)(13)} were isolated as the apparent result of "illegitimate" recombination events on intrahelical pseudoknots . This work establishes the relationships between CTG.CAG sequences, multiple fold expansions, genetic recombination, formation of new recombinant DNA products, and the presence of both drug resistance genes . Thus, if these reactions occur in humans, unequal crossing-over or gene conversion may also contribute to the expansions responsible for anticipation associated with several hereditary neurological syndromes. J Biol Chem, 1999 Aug 13, 274(33), 23451 - 5 Active site residues of human beta-glucuronidase . Evidence for Glu(540) as the nucleophile and Glu(451) as the acid-base residue; Islam MR et al.; Human beta-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans . Amino acid sequence and structural homology of hGUSB and Escherichia coli beta-galactosidase active sites led us to propose that residues Glu(451), Glu(540), and Tyr(504) in hGUSB are involved in catalysis, Glu(451) being the acid-base residue and Glu(540) the nucleophile . To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts . The extremely low activity in cells expressing Glu(451), Glu(540), and Tyr(504) hGUSBs supported their roles in catalysis . For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography . The k(cat)/K(m) values (mM(-1).s(-1)) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively . High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu(451) as the acid-base catalyst . We conclude that, like their homologues in E . coli beta-galactosidase, Glu(540) is the nucleophilic residue, Glu(451) the acid-base catalyst, and Tyr(504) is also important for catalysis, although its role is unclear . All three residues are located in the active site cavity previously determined by structural analysis of hGUSB. J Biol Chem, 1999 Aug 13, 274(33), 23378 - 86 Mapping interactions of Escherichia coli GreB with RNA polymerase and ternary elongation complexes; Loizos N et al.; Escherichia coli GreA and GreB modulate transcription elongation by interacting with the ternary elongation complex (containing RNA polymerase, DNA template, and RNA transcript) to induce hydrolytic cleavage of the transcript and release of the 3'-terminal fragment . Hydroxyl radical protein footprinting and alanine-scanning mutagenesis were used to investigate the interactions of GreB with RNA polymerase alone and in a ternary elongation complex . A major determinant for binding GreB to both RNA polymerase and the ternary elongation complex was identified . In addition, the hydroxyl radical footprinting indicated major conformational changes of GreB, in terms of reorientations of the N- and C-terminal domains with respect to each other, particularly upon interactions with the ternary elongation complex. J Biol Chem, 1999 Aug 13, 274(33), 22985 - 92 Protein kinase-mediated regulation of the Na(+)/H(+) exchanger in the rat myocardium by mitogen-activated protein kinase-dependent pathways; Moor AN et al.; We examined regulation of the Na(+)/H(+) exchanger isoform 1 by phosphorylation in the rat myocardium . We utilized cell extracts from adult rat hearts, adult rat extracts fractionated by fast performance liquid chromatography, and extracts from cultured neonatal cardiac myocytes . The carboxyl-terminal 178 amino acids of the Na(+)/H(+) exchanger were expressed in Escherichia coli fused with glutathione S-transferase . The purified protein was used as a substrate for in vitro phosphorylation and in-gel kinase assays . Unfractionated extracts from neonatal myocytes or adult hearts phosphorylated the COOH-terminal domain of the antiporter . Western blot analysis revealed that mitogen-activated protein (MAP) kinase (44 and 42 kDa) and p90(rsk) (90 kDa) were present in specific fractions of cardiac extracts that phosphorylated the COOH-terminal protein . In-gel kinase assays confirmed that protein kinases of approximately 44 and 90 kDa could phosphorylate this domain . MAP kinase and p90(rsk)-dependent phosphorylation of the antiporter could be demonstrated by immunoprecipitation of these kinases from extracts of neonatal cardiac myocytes . PD98059, a mitogen-activated protein kinase kinase inhibitor, decreased MAP kinase and p90(rsk) phosphorylation of the antiporter and abolished serum and endothelin 1-stimulated increases in steady-state pH(i) . These results confirm the presence of MAP kinase-dependent phosphorylation in the regulation of the Na(+)/H(+) exchanger in the rat myocardium and suggest an important role for p90(rsk) phosphorylation in regulation of the protein by endothelin-mediated stimulation of the antiporter. Vet Immunol Immunopathol, 1999 May, 68(2-4), 113 - 30 Bovine monocytoid cells transformed to proliferate cease to exhibit lineage-specific functions; Sager H et al.; Bovine cell lines of the monocyte-Mphi lineage were tested for surface marker expression and were characterized with respect to functions . Cell lines tested encompassed an SV40-transformed cell line (Bo-Mac), a spontaneously emerging monocytoid cell line (M617), and T . annulata-transformed lines derived from bovine Mphi . All lines failed to express surface markers expressed by 1 degrees Mphi, with the exception of CD44, WC9 and the DH59 myleoid cell marker . T . annulata-derived lines expressed, in addition, CD45 and MHC-class-II molecules . Except for nonspecific esterase staining, none of the typical macrophage functions were expressed by any of the cell lines . These included phagocytosis of opsonized E . coli bacteria and of IgG-treated erythrocytes, eliciting of an oxidative burst, the ability to express type-I-interferon (IFN) and to respond to lipopolysaccharide, as determined by four different effector functions (nitric oxide synthesis, tumor necrosis factor (TNF) secretion, IFN production and procoagulant activity upregulation) . When transformation induced by T . annulata was reversed by chemical elimination of the parasite, cells ceased to proliferate but started to acquire some of the phenotypic characteristics of Mphi . This suggests that regardless of their origin, exponentially growing bovine cells of the monocyte-Mphi lineage poorly represent a lineage-specific phenotype and should be used with caution in immunological studies. Biochem Cell Biol, 1999, 77(2), 109 - 18 Stabilities of uncomplemented and complemented M15 beta-galactosidase (Escherichia coli) and the relationship to alpha-complementation; Gallagher CN et al.; M15 beta-galactosidase (Escherichia coli) is a mutant form of beta-galactosidase having residues 11-41 deleted . It is an inactive dimer but can be complemented to the active tetrameric form by the addition of a peptide containing the deleted residues . The activities of uncomplemented and complemented M15 beta-galactosidases decreased starting at 42 degrees C--uncomplemented over a narrow temperature range, complemented over a broad range . This is because uncomplemented protein is a simple dimer while complemented is a mix of interacting oligomers at high temperatures . The effects of added components on stability and alpha-complementation are best explained by binding effects on equilibria between native forms and forms susceptible to inactivation . Mg2+ stabilized complemented protein but destabilized uncomplemented protein (10x less Mg2+ was needed for complemented protein) . Alpha-complementation increased somewhat at low Mg2+ but decreased at high Mg2+ . These effects can be explained by differential Mg2+ binding to the native and susceptible forms . The enhancement of both stability and alpha-complementation by Na+ can be explained by preferential binding of Na+ to the native forms of both the uncomplemented and complemented proteins . Low 2-mercaptoethanol concentrations stabilized uncomplemented M15 beta-galactosidase, but high concentrations destabilized it . All concentrations destabilized complemented M15 beta-galactosidase . Alpha-complementation was enhanced by 2-mercaptoethanol . Thus, there is a correlation between stability of the uncomplemented protein and alpha-complementation at low 2-mercaptoethanol owing to interactions with native forms . The lack of correlation at higher 2-mercaptoethanol probably results from precipitation by 2-mercaptoethanol . In contrast to irreversible thermal inactivation, differences in reversible stability in urea were small . This suggests that quaternary structure and Mg2+ and Na+ sites are lost at low urea concentrations and are unimportant at the urea concentrations that result in reversible denaturation. Biochem Cell Biol, 1999, 77(2), 101 - 8 Molecular cloning of a centrin homolog from Marsilea vestita and evidence for its translational control during spermiogenesis; Hart PE et al.; Spermiogenesis in the water fern Marsilea vestita is a process that reaches completion 11 h after dry microspores are immersed in an aqueous medium at 20 degrees C . Each microspore produces 32 spermatozoids and each spermatozoid has a coiled cell body and approximately 140 cilia . The spermatids make basal bodies de novo, from a structure known as a blepharoplast . From the onset of development, the spores contain a large quantity of protein and stored mRNA . We have found previously that centrin, a protein involved in the function of microtubule organizing centers and present in association with basal bodies in motile cells, is made in large quantity approximately 4 h after the microspores are placed into liquid medium . In this paper, we show that a centrin cDNA (MvCen1) we isolated from M . vestita closely resembles centrin cDNAs from other eukaryotic organisms . MvCen1, synthesized in Escherichia coli as a GST-fusion protein, reacted with anti-centrin monoclonal antibodies on immunoblots . Northern blot analysis demonstrates that centrin mRNA is present in the dry microspore at the time of imbibition, at levels that remain constant over 10 h of development and are unaffected by treatment of spores with alpha-amanitin . The centrin transcripts, stored in dry microspores, cannot be translated in vitro for at least 30 min after imbibition. Plant Mol Biol, 1999 Jun, 40(3), 507 - 21 Phycoerythrins of the oxyphotobacterium Prochlorococcus marinus are associated to the thylakoid membrane and are encoded by a single large gene cluster; Hess WR et al.; An intrinsic divinyl-chlorophyll a/b antenna and a particular form of phycobiliprotein, phycoerythrin (PE) III, coexist in the marine oxyphotobacterium Prochlorococcus marinus CCMP 1375 . The genomic region including the cpeB/A operon of P . marinus was analysed . It encompasses 10,153 nucleotides that encode three structural phycobiliproteins and at least three (possibly five) different polypeptides analogous to cyanobacterial or red algal proteins involved either in the linkage of subunits or the synthesis and attachment of chromophoric groups . This gene cluster is part of the chromosome and is located within a distance of less than 110 kb from a previously characterized region containing the genes aspA-psbA-aroC . Whereas the Prochlorococcus phycobiliproteins are characterized by distinct deletions and amino acid replacements with regard to analogous proteins from other organisms, the gene arrangement resembles the organization of phycobiliprotein genes in some other cyanobacteria, in particular marine Synechococcus strains . The expression of two of the Prochlorococcus polypeptides as recombinant proteins in Escherichia coli allowed the production of individual homologous antisera to the Prochlorococcus alpha and beta PE subunits . Experiments using these sera show that the Prochlorococcus PEs are specifically associated to the thylakoid membrane and that the protein level does not significantly vary as a function of light irradiance or growth phase. Plant Mol Biol, 1999 Jun, 40(3), 479 - 86 Isolation and characterization of cDNAs encoding mitochondrial phosphate transporters in soybean, maize, rice, and Arabidopis; Takabatake R et al.; cDNA clones encoding mitochondrial phosphate transporters were isolated from four herbaceous plants . The cDNAs for the soybean, maize and rice transporters contained entire coding regions, whereas the Arabidopsis cDNA lacked the 5' portion . The hydropathy profiles of the deduced amino acid sequences predicted the existence of six membrane-spanning domains which are highly conserved in the mitochondrial transporter family . In soybeans, the mRNA level for the transporter was high in tissues containing dividing cells . It was suggested that there are multiple copies of transporter genes in both dicots and monocots . The soybean transporter was expressed as inclusion bodies in Escherichia coli, solubilized with detergents, and then reconstituted into liposomes . The resulting proteoliposomes exhibited high phosphate transport activity . The activity was inhibited by N-ethylmaleimide, like those of mammalian phosphate transporters. Plant Mol Biol, 1999 Jun, 40(3), 409 - 18 Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, and copper binding under various salinity conditions; Hibino T et al.; Previously, it was found that the dnaK1 gene of the halotolerant cyanobacterium Aphanothece halophytica encodes a polypeptide of 721 amino acids which has a long C-terminal region rich in acidic amino acid residues . To understand whether the A . halophytica DnaK1 possesses chaperone activity at high salinity and to clarify the role of the extra C-terminal amino acids, a comparative study examined three kinds of DnaK molecules for ATPase activity as well as the refolding activity of other urea-denatured proteins under various salinity conditions . DnaK1s from A . halophytica and Synechococcus sp . PCC 7942 and the C-terminal deleted A . halophytica DnaK1 were expressed in Escherichia coli and purified . The ATPase activity of A . halophytica DnaK1 was very high even at high salinity ( 1.0 M NaCl or KCl), whereas this activity in Synechococcus PCC 7942 DnaK1 decreased with increasing concentrations of NaCl or KCl . The salt dependence on the refolding activity of urea-denatured lactate dehydrogenase by DnaK1s was similar to that of ATPase activity of the respective DnaK1s . The deletion of the C-terminal amino acids of A . halophytica DnaK had no effect on the ATPase activity, but caused a significant decrease in the refolding activity of other denatured proteins . These facts indicate that the extra C-terminal region of A . halophytica DnaK1 plays an important role in the refolding of other urea-denatured proteins at high salinity . Furthermore, it was shown that DnaK1 could assist the copper binding of precursor apo-plastocyanin as well as that of mature apo-plastocyanin during the folding of these copper proteins. FEBS Lett, 1999 Jul 23, 455(3), 355 - 8 Interaction between a mutant release factor one and P-site peptidyl-tRNA is influenced by the identity of the two bases downstream of the stop codon UAG; Zhang S et al.; Termination efficiency of a mutant form of RF (release facor) 1, as compared to the wild-type enzyme, is influenced by the P-site peptidyl-tRNA if the termination signal is UAGA . This effect is weaker at the stronger termination signal UAGU . Similarly, low efficiency of the mutant RF1, together with certain peptidyl-tRNAs, can be increased by changing the second base of the 3'-flanking codon from C to G . The data suggest that the mutant RF1 interacts with the P-site peptidyl-tRNA in conjunction with the context at the 3'-side of the termination codon. FEBS Lett, 1999 Jul 23, 455(3), 349 - 54 Introduction of a new branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium protoporphyrin methyltransferase; Jensen PE et al.; The genes encoding the three Mg chelatase subunits, ChlH, ChlI and ChlD, from the cyanobacterium Synechocystis PCC6803 were all cloned in the same pET9a-based Escherichia coli expression plasmid, forming an artificial chlH-I-D operon under the control of the strong T7 promoter . When a soluble extract from IPTG-induced E . coli cells containing the pET9a-ChlHID plasmid was assayed for Mg chelatase activity in vitro, a high activity was obtained, suggesting that all three subunits are present in a soluble and active form . The chlM gene of Synechocystis PCC6803 was also cloned in a pET-based E . coli expression vector . Soluble extract from an E . coli strain expressing chlM converted Mg-protoporphyrin IX to Mg-protoporphyrin monomethyl ester, demonstrating that chlM encodes the Mg-protoporphyrin methyltransferase of Synechocystis . Co-expression of the chlM gene together with the chlH-I-D construct yielded soluble protein extracts which converted protoporphyrin IX to Mg-protoporphyrin IX monomethyl ester without detectable accumulation of the Mg-protoporphyrin IX intermediate . Thus, active Mg chelatase and Mg-protoporphyrin IX methyltransferase can be coupled in E . coli extracts . Purified ChlI, -D and -H subunits in combination with purified ChlM protein were subsequently used to demonstrate in vitro that a molar ratio of ChlM to ChlH of 1 to 1 results in conversion of protoporphyrin IX to Mg-protoporphyrin monomethyl ester without significant accumulation of Mg-protoporphyrin. Zhongguo Yao Li Xue Bao, 1999 Feb, 20(2), 171 - 4 Effects of dexamethasone, cyproheptadine, anisodamine, and dinoprostone on TNF alpha production in endotoxic shock; Wang LZ et al.; AIM: To study the effects of dexamethasone (Dex), cyproheptadine (Cyp), anisodamine (Ani), and dinoprostone (Din) on lipopolysaccharides (LPS)-induced tumor necrosis factor alpha (TNF alpha) gene expression and antishock effects of inhibiting TNF alpha production . METHODS: Endotoxic shock in rats was produced by i.v . injection of LPS (E coli O111B4, 5 mg.kg-1) . TNF alpha mRNA accumulation was assessed by Northern blot . Plasma TNF alpha contents were determined by radioimmunoassay . RESULTS: The TNF alpha mRNA levels in rat liver at 2 h after LPS challenge was increased obviously (autoradiograms analyzed by scanning were 38 +/- 10 vs saline control 11 +/- 8, P < 0.01) . The plasma TNF alpha contents were markedly increased {(22 +/- 3) micrograms.L-1 vs saline control (2.2 +/- 1.0) micrograms.L-1, P < 0.01} . Dex 5, Cyp 5, Ani 10, or Din 2 mg.kg-1 immediately injected after i.v . LPS markedly decreased the TNF alpha mRNA levels in rat liver and plasma TNF alpha contents . The Dex, Cyp, Ani, and Din improved the mouse survival rate 24 h after LPS 20 mg.kg-1 challenge . CONCLUSION: Dex, Cyp, Ani, and Din strongly inhibit LPS-induced TNF alpha gene expression, and have a beneficial antishock effects. FEMS Microbiol Lett, 1999 Aug 1, 177(1), 123 - 30 Identification of novel immunogenic Mycobacterium tuberculosis peptides that stimulate mononuclear cells from immune donors; Moran AJ et al.; Proteins which are secreted or associated with the cell envelope of Mycobacterium tuberculosis may contain protective T-cell epitopes . Prior to this study, a recombinant clone bank of enzymatically active M . tuberculosis-alkaline phosphatase fusions, were screened for immunogenicity in a murine T-cell model . Five of these were selected for further study, and the IFN-gamma secretion and proliferation of human PBMC from purified protein derivative- (PPD)-positive and PPD-negative donors were measured in response to oligopeptides, Mtb-PhoA fusions and one full-length protein . Epitopes from four of the five selected antigens were immunoreactive in the human model and corresponded to cytochrome d ubiquinol oxidase, cytochrome c oxidase subunit II, MTV005.02 and MTV033.08 . Thus, this strategy identified novel human immunogenic peptides as possible candidates for a subunit vaccine. Rev Med Chil, 1999 Feb, 127(2), 131 - 7 {Metalloproteinase activity in myocardium of rats exposed to endotoxin and its inhibition with doxycycline}; Ruiz S et al.; BACKGROUND: The ventricular dysfunction of endotoxic shock could be secondary to the activity of myocardial metalloproteinases that degrade collagenous matrix . Metalloproteinase activity can be inhibited with doxycycline in some tissues . AIM: To study if the effect of endotoxemia on myocardial metalloproteinase activity can be inhibited with doxycycline . MATERIAL AND METHODS: Left ventricular metalloproteinase activity was studied in four groups of rats . Group 1 received intraperitoneal dextrose in water, group 2 received 8 mg/kg intraperitoneal E coli endotoxin, group 3 received 60 mg/kg/day doxycycline for three days and group 4 received doxycycline and E coli endotoxin . Enzymatic activity was measured by Western Blot and zymography . RESULTS: Zymography showed a higher metalloproteinase 2 (49%) and 9 (100%) activity in rats treated with endotoxin, when compared with control rats . In group 4, doxycycline reduced the activity of metalloproteinases 2 and 9 by 71% and 63% respectively, as compared with group 3 . Western blot showed a 50% increase in the expression of metalloproteinase 1 in rats treated with endotoxin, that was reduced by 64% with the use of doxycycline . CONCLUSIONS: Endotoxin administration increases myocardial metalloproteinases and doxycyclin inhibits this activation . Therefore, doxycyclin could reduce the degradation of myocardial fibrillar collagen and ventricular dysfunction of endotoxic shock. Zentralbl Chir, 1999, 124(6), 530 - 4 {Cryopreserved arterial homografts . A treatment alternative for infected vascular reconstructions}; Reber PU et al.; Deep wound infection or prosthetic vascular graft infection is one of the most challenging complications in vascular surgery with a substantial early and late morbidity and mortality . Surgical treatment usually consists of complete removal of infected vessels or prosthetic vascular grafts followed by extraanatomic bypass procedures . However, this method is associated with significant mortality and amputation rates . Herein, we report two patients with deep wound and prosthetic vascular graft infection who underwent successful in situ reconstruction with cryopreserved arterial homografts . Although the long-term results are missing, this approach may offer a possible treatment alternative for this potentially life-threatening complication. Cytokine, 1999 Aug, 11(8), 561 - 70 The human intracellular interleukin 1 receptor antagonist promoter appropriately regulates gene expression in keratinocytes and gastrointestinal epithelial cells in vivo; Gabay C et al.; The 4555-bp promoter fragment for intracellular interleukin 1 receptor antagonist (4555-bp icIL-1Ra) has recently been demonstrated to regulate gene expression in a cell-type specific manner in vitro in transient transfection studies . To examine the activity of this promoter in vivo, transgenic mice possessing the 4555-bp promoter coupled to the E . coli lacZ reporter gene were created . Expression of endogenous icIL-1Ra and E . coli lacZ mRNA were examined in different tissues by RT-PCR, RNase protection assay and in situ hybridization . In transgenic mice both endogenous icIL-1Ra and E . coli lacZ were co-expressed by keratinocytes and by epithelial cells in different organs of the digestive system . The transgene was also expressed in the brain in four out of five lines, whereas endogenous icIL-1Ra was not detected in this organ . In contrast, only icIL-1Ra mRNA, but not E . coli lacZ mRNA, was detected in lipopolysaccharide (LPS)-stimulated resident peritoneal macrophages from icIL-1Ra promoter transgenic mice . These results indicate that a 4555-bp promoter fragment of human icIL-1Ra appropriately regulates gene transcription in keratinocytes and gastrointestinal epithelial cells in vivo . However, other as yet unidentified regulatory regions influence icIL-1Ra gene expression in macrophages following LPS stimulation . Protein Eng, 1999 Jul, 12(7), 623 - 30 Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42 . Determination of binding constants with wild-type and mutant HPrs; Smallshaw JE et al.; The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system . The binding domain (Fv) has been produced as a single chain Fv (scFv) . The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail . The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II . This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352 . The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated . The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved . Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM . However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively . The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment . It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay. Protein Eng, 1999 Jul, 12(7), 613 - 21 Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface; Daugherty PS et al.; Polypeptide library screening technologies are critically dependent upon the characteristics of the expression system employed . A comparative analysis of the lpp-lac, tet and araBAD promoters was performed to determine the importance of tight regulation and expression level in library screening applications . The surface display of single-chain antibody (scFv) in Escherichia coli as an Lpp-OmpA' fusion was monitored using a fluorescently tagged antigen in conjunction with flow cytometry . In contrast to the lpp-lac promoter, both tet and araBAD promoters could be tightly repressed . Tight regulation was found to be essential for preventing rapid depletion of library clones expressing functional scFv and thus for maintaining the initial library diversity . Induction with subsaturating inducer concentrations yielded mixed populations of uninduced and fully induced cells for both the tet and araBAD expression systems . In contrast, homogeneous expression levels were obtained throughout the population using saturating inducer concentrations and could be adjusted by varying the induction time and plasmid copy number . Under optimal induction conditions for the araBAD system, protein expression did not compromise either cell viability or library diversity . This expression system was used to screen a library of random scFv mutants specific for digoxigenin for clones exhibiting improved hapten dissociation kinetics . Thus, an expression system has been developed which allows library diversity to be preserved and is generally applicable to the screening of E . coli surface displayed libraries. Protein Eng, 1999 Jul, 12(7), 605 - 11 The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli; Wall JG et al.; In a systematic study of the periplasmic folding of antibody fragments in Escherichia coli, we have analysed the expression of an aggregation-prone and previously non-functional anti-phosphorylcholine antibody, T15, as a model system and converted it to a functional molecule . Introduction of heavy chain framework mutations previously found to improve the folding of a related antibody led to improved folding of T15 fragments and improved physiology of the host E.coli cells . Manipulation of the complementarity determining regions (CDR) of the framework-mutated forms of T15 further improved folding and bacterial host physiology, but no improvement was seen in the wild type, suggesting the existence of a hierarchy in sequence positions leading to aggregation . Rational mutagenesis of the T15 light chain led to the production of functional T15 fragments for the first time, with increased levels of functional protein produced from V(H) manipulated constructs . We propose that a hierarchical analysis of the primary amino acid sequence, as we have described, provides guidelines on how correctly folding, functional antibodies might be achieved and will allow further delineation of the decisive structural factors and pathways favouring protein aggregation. Protein Eng, 1999 Jul, 12(7), 581 - 7 Stability, activity and flexibility in alpha-lactalbumin; Greene LH et al.; alpha-Lactalbumins and the type-c lysozymes are homologues with similar folds that differ in function and stability . To determine if the lower stability of alpha-lactalbumin results from specific substitutions required for its adaptation to a new function, the effects of lysozyme-based and other substitutions on thermal stability were determined . Unblocking the upper cleft in alpha-lactalbumin by replacing Tyr103 with Ala, perturbs stability and structure but Pro, which also generates an open cleft, is compatible with normal structure and activity . These effects appear to reflect alternative enthalpic and entropic forms of structural stabilization by Tyr and Pro . Of 23 mutations, only three, which involve substitutions for residues in flexible substructures adjacent to the functional site, increase stability . Two are lysozyme-based substitutions for Leu110, a component of a region with alternative helix and loop conformations, and one is Asn for Lys114, a residue whose microenvironment changes when alpha-lactalbumin interacts with its target enzyme . While all substitutions for Leu110 perturb activity, a Lys114 to Asn mutation increases T(m) by more than 10 degrees C and reduces activity, but two other destabilizing substitutions do not affect activity . It is proposed that increased stability and reduced activity in Lys114Asn result from reduced flexibility in the functional site of alpha-lactalbumin. Curr Eye Res, 1999 Jun, 18(6), 408 - 16 Effects of hypotonic swelling on the cellular distribution and expression of pI(Cln) in human nonpigmented ciliary epithelial cells; Sanchez-Torres J et al.; PURPOSE: It has been proposed that pI(Cln), a highly acidic protein, is a candidate gene product related to the swelling-activated chloride (Cl-) channel Icl.swell in mammalian cells . However, no consensus has been reached as to whether this relationship is direct or indirect . Recently the cDNA for pI(Cln) was isolated from human ciliary epithelial cells . To learn more about the structure-function of pI(Cln) we attempted to: i) overexpress pI(Cln) as a fusion protein in bacteria; ii) carry out its purification; iii) generate polyclonal antibodies to study its expression and cellular localization in the ciliary epithelial cells; and iv) determine whether cell-swelling affects pI(Cln) expression in ciliary epithelial cells . METHODS: The open reading frame (ORF) of human pI(Cln) was subcloned in the pET-20b(+) plasmid and established as a recombinant vector in E . coli BL21(DE3)pLysS cells . Upon induction with iso-propyl-beta-thio-galactopyranoside (IPTG), pI(Cln) was isolated as a His-Tag fusion protein and purified to homogeneity . Polyclonal antibodies were raised in rabbits after immunization with pI(Cln) purified protein, and its expression and cellular distribution in ciliary epithelial cells determined by Western blot, immunoprecipitation and indirect immunofluorescence respectively . Cell-swelling effect on ciliary epithelial cells was carried out upon treatment of cultured cells with hypotonic solution up to 60 min and pI(Cln) expression measured by Northern and Western blot analysis . RESULTS: By Western blot analysis or immunoprecipitation, pI(Cln) antisera recognized a main band of 37-kDa in total cell extracts from ciliary body or metabolically labeled ciliary epithelial cells . By indirect immunofluorescence, pI(Cln) antibodies stained the cytoplasm of NPE in the intact tissue, and the perinuclear region of cultured ciliary epithelial cells . When subjected to hypotonic treatment, NPE cells did not induce translocation of pI(Cln) protein from the cytoplasm into the plasma membrane, nor changes in pI(Cln) expression at the protein level, but did down regulate up to 30% the level of pI(Cln) mRNA in continued hypotonic treatment . CONCLUSIONS: These observations indicate that, contrary to previous suggestions, the pI(Cln) protein is not likely to be in contact with the plasma membrane of ciliary epithelial cells, and its influence on Cl- -channel activity is more likely to be expressed indirectly, (i.e . through cytoskeletal restructuring). Vet Parasitol, 1999 Jul, 84(1-2), 65 - 73 Antibodies produced by mice immunized with recombinant vaccinia viruses expressing two different types of a major Theileria sergenti surface antigen (p32) react with the native surface antigen; Takasima Y et al.; A 32 kDa major surface antigen, p32, of Theileria sergenti at the piroplasm stage is the main target of the host immune response . The immunogenic property of the p32 varies in some strains among the population of Theileria sergenti in Japan where the Chitose type and the Ikeda type are the most common varieties . We have constructed vaccinia virus recombinants vv/p32C and vv/p32I which harbor the Chitose and Ikeda types of p32 gene, respectively . It was found that vv/p32C and vv/p32I produced type-specific p32 which did not cross react with the monoclonal antibodies (mAbs) against the other type of p32 . When mice were immunized with vv/p32C and vv/p32I, antibodies against p32 were detectable 2 weeks after the immunization, and these antibodies reacted with the native surface antigen in purified T . sergenti merozoite. Gene Ther, 1999 Feb, 6(2), 271 - 81 Stabilized plasmid-lipid particles: construction and characterization; Wheeler JJ et al.; A detergent dialysis procedure is described which allows encapsulation of plasmid DNA within a lipid envelope, where the resulting particle is stabilized in aqueous media by the presence of a poly(ethyleneglycol) (PEG) coating . These 'stabilized plasmid-lipid particles' (SPLP) exhibit an average size of 70 nm in diameter, contain one plasmid per particle and fully protect the encapsulated plasmid from digestion by serum nucleases and E . coli DNase I . Encapsulation is a sensitive function of cationic lipid content, with maximum entrapment observed at dioleoyldimethylammonium chloride (DODAC) contents of 5 to 10 mol% . The formulation process results in plasmid-trapping efficiencies of up to 70% and permits inclusion of 'fusigenic' lipids such as dioleoylphosphatidylethanolamine (DOPE) . The in vitro transfection capabilities of SPLP are demonstrated to be strongly dependent on the length of the acyl chain contained in the ceramide group used to anchor the PEG polymer to the surface of the SPLP . Shorter acyl chain lengths result in a PEG coating which can dissociate from the SPLP surface, transforming the SPLP from a stable particle to a transfection-competent entity . It is suggested that SPLP may have utility as systemic gene delivery systems for gene therapy protocols. Gene Ther, 1999 Feb, 6(2), 190 - 7 Cationic liposome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon; Murray KD et al.; We have examined the potential of cationic liposomes as a tool for approaches to gene therapy in the CNS . Our previous work has shown that cationic liposomes formulated from 3 beta-{N-(N',N'-dimethylaminoethane)carbamoyl} cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) could achieve high transfection levels in a neuronal cell line (McQuillin et al . Neuroreport 1997; 8: 1481-1484) . We therefore wished to assess transfection efficiencies in organotypic cultures from the brain with a reporter plasmid expressing E . coli beta-galactosidase in order to mimic an in vivo model . Explant cultures were generated according to the method of Stoppini et al (J Neurosci Meth 1991; 37: 173-182) with slight modifications . Brain slices were maintained on transparent porous membranes and were observed to maintain their intrinsic connectivity and cytoarchitecture to a large degree over periods of up to 6 weeks in culture . CNS tissue was obtained from rats at birth or 5 days after birth . After transfection beta-galactosidase expression was detected in cells of both neuronal and non-neuronal morphology . Control cultures were exposed to liposome alone and a plasmid that had the beta-galactosidase gene insert removed . Only low levels of endogenous beta-galactosidase reactivity were seen in these control cultures . DC-Chol/DOPE-mediated transfection was confirmed using a RT-PCR protocol capable of differentiating between untranscribed plasmid DNA and RNA generated from the transfected vector . These results suggest that cationic liposomes, particularly DC-Chol/DOPE liposomes, will be useful as delivery agents for gene transfer to CNS cells in vitro and possibly in vivo. Hiroshima J Med Sci, 1999 Jun, 48(2), 71 - 7 Effect of FC43se on endotoxin-induced disseminated intravascular coagulation in rats; Ochikubo H et al.; Perfluorotributylamine/Pluronic F68 Stem-Emulsion (FC43se), which is a blood substitute, was assessed for its effectiveness on disseminated intravascular coagulation (DIC) in the rat model . Rats were infused intravenously with 2.5 mg/kg of Escherichia coli lipopolysaccharide (Escherichia coli 055:B5 lipopolysaccharide B) for four hours . At the same time, FC43se or normal physiological saline was infused at 2.5 ml/kg/hr . The white blood cell and platelet counts, prothrombin time (PT), activated partial thromboplastin time (APTT), and the plasma levels of interleukin-1 beta (IL-1 beta), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF alpha) were determined at 4 hr . The infusion of FC43se markedly prevented a decrease in platelet counts (p = 0.0004) and a prolongation of both PT and APTT (p < 0.05 and p < 0.03 each) . The serum level of IL-1 beta and IL-4 showed no significant change . The serum level of IL-6, IL-10 and TNF alpha increased significantly (p = 0.0007, p = 0.0004 and p < 0.05 each) with infusion of FC43se in rats treated with bacterial endotoxin . FC43se has beneficial effects on endotoxin-induced DIC as an anticoagulant and anti-inflammatory cytokine induced agent. Philos Trans R Soc Lond B Biol Sci, 1999 Jun 29, 354(1386), 991 - 4 Aggregation of truncated GST-HD exon 1 fusion proteins containing normal range and expanded glutamine repeats; Hollenbach B et al.; We have shown previously by electron microscopy that the purified glutathione S-transferase (GST)-Huntington's disease (HD) exon 1 fusion protein with 51 glutamine residues (GST-HD51) is an oligomer, and that site-specific proteolytic cleavage of this fusion protein results in the formation of insoluble more highly ordered protein aggregates with a fibrillar or ribbon-like morphology (E . Scherzinger et al . (1997) Cell 90, 549-558) . Here we report that a truncated GST HD exon 1 fusion protein with 51 glutamine residues, which lacks the proline-rich region C-terminal to the polyglutamine (polyQ) tract (GST-HD51 delta P) self-aggregates into high-molecular-mass protein aggregates without prior proteolytic cleavage . Electron micrographs of these protein aggregates revealed thread-like fibrils with a uniform diameter of ca . 25 nm . In contrast, proteolytic cleavage of GST-HD51 delta P resulted in the formation of numerous clusters of high-molecular-mass fibrils with a different, ribbon-like morphology . These structures were reminiscent of prion rods and beta-amyloid fibrils in Alzheimer's disease . In agreement with our previous results with full-length GST-HD exon 1, the truncated fusion proteins GST-HD20 delta P and GST-HD30 delta P did not show any tendency to form more highly ordered structures, either with or without protease treatment. Mutat Res, 1999 Aug 11, 429(1), 37 - 44 Effect of interaction between 5-azacytidine and DNA (cytosine-5) methyltransferase on C-to-G and C-to-T mutations in Escherichia coli; Doiron KM et al.; The purpose of this study was to determine the effect of the Dcm cytosine methyltransferase on 5-azacytidine (5-azaC) mutagenesis in Escherichia coli . We used a Lac reversion assay to measure C-to-G and C-to-T mutations at a single, methylatable cytosine in the lacZ gene, in the presence and absence of Dcm . C-to-G mutations are stimulated by 5-azaC but are largely independent of Dcm . In contrast, C-to-T mutations are not stimulated by 5-azaC in either wild type or dcm cells . However, in cells which contain Dcm but are defective in very short patch repair, the normally high frequency of spontaneous C-to-T mutations is decreased by the analog in a dose-dependent manner . Mutat Res, 1999 Aug 11, 429(1), 27 - 35 The presence of traces of iron and copper ions during gamma-irradiation does not result in clear mutational hot spots in the lacI gene; Wijker CA et al.; Oxidative radicals, which are produced during ionizing irradiation of DNA in water, damage the DNA and may result in mutations, which are in general randomly distributed . Alternatively, the addition of transition metal ions, like iron or copper, to DNA in combination with H(2)O(2) and a reducing agent also results in the production of oxidative radicals . Due to binding of the transition metal ions to DNA, the production of these radicals is very local, and results in a mutational spectrum in which the mutations are not randomly distributed . If transition metal ions are complexed to the DNA during irradiation, and react with radiation-induced species such as hydrogen peroxide, site-specific formation of.OH radicals on these sites may occur, leading to the formation of mutational hot spots . This study examines the influence of the presence of traces of iron or copper ions during gamma-irradiation of plasmid DNA in water, on the possible formation of mutational hot spots in the lacI gene . Comparison of the mutational spectra, after irradiation in the presence or in the absence of transition metal ions, shows that there are indeed relatively more positions in the lacI gene where more than one mutation occurs, suggesting formation of mutational hot spots in the presence of transition metal ions . However, the appearance of these hot spots is rather weak . Although in all three mutational spectra G:C to A:T mutations are predominant, there are also some differences between the types of mutations in these spectra . These differences in mutational spectra might reflect the different preferences of iron and copper ions to bind specific sites in the DNA . Indeed, there appears to be a high association of mutations at CC or GG sites in the mutational spectrum in the presence of copper ions, confirming the observation that copper binds preferably at two adjacent guanines in the DNA . It can be concluded from this study that the presence of small amounts of transition metal ions during gamma-irradiation influences the types and distribution of gamma-radiation-induced mutations, although no major mutational hot spots can be observed . Development, 1999 Sep, 126(17), 3795 - 809 Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5; Acampora D et al.; The Dlx5 gene encodes a Distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of the mouse embryo . In later developmental stages, it appears in the branchial arches, the otic and olfactory placodes and their derivatives, in restricted brain regions, in all extending appendages and in all developing bones . We have created a null allele of the mouse Dlx5 gene by replacing exons I and II with the E . coli lacZ gene . Heterozygous mice appear normal . Beta-galactosidase activity in Dlx5+/- embryos and newborn animals reproduces the known pattern of expression of the gene . Homozygous mutants die shortly after birth with a swollen abdomen . They present a complex phenotype characterised by craniofacial abnormalities affecting derivatives of the first four branchial arches, severe malformations of the vestibular organ, a delayed ossification of the roof of the skull and abnormal osteogenesis . No obvious defect was observed in the patterning of limbs and other appendages . The defects observed in Dlx5-/- mutant animals suggest multiple and independent roles of this gene in the patterning of the branchial arches, in the morphogenesis of the vestibular organ and in osteoblast differentiation. Biochemistry, 1999 Aug 3, 38(31), 10205 - 14 Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: an amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein; Zitzewitz JA et al.; The alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli is a 268-residue 8-stranded beta/alpha barrel protein . Two autonomous folding units, comprising the first six strands (residues 1-188) and the last two strands (residues 189-268), have been previously identified in this single structural domain protein by tryptic digestion {Higgins, W., Fairwell, T., and Miles, E . W . (1979) Biochemistry 18, 4827-4835} . The larger, amino-terminal fragment, alphaTS(1-188), was overexpressed and independently purified, and its equilibrium and kinetic folding properties were studied by absorbance, fluorescence, and near- and far-UV circular dichroism spectroscopies . The native state of the fragment unfolds cooperatively in an apparent two-state transition with a stability of 3.98 +/- 0.19 kcal mol(-1) in the absence of denaturant and a corresponding m value of 1.07 +/- 0.05 kcal mol(-1) M(-1) . Similar to the full-length protein, the unfolding of the fragment shows two kinetic phases which arise from the presence of two discrete native state populations . Additionally, the fragment exhibits a significant burst phase in unfolding, indicating that a fraction of the folded state ensemble under native conditions has properties similar to those of the equilibrium intermediate populated at 3 M urea in full-length alphaTS . Refolding of alphaTS(1-188) is also complex, exhibiting two detectable kinetic phases and a burst phase that is complete within 5 ms . The two slowest isomerization phases observed in the refolding of the full-length protein are absent in the fragment, suggesting that these phases reflect contributions from the carboxy-terminal segment . The folding mechanism of alphaTS(1-188) appears to be a simplified version of the mechanism for the full-length protein {Bilsel, O., Zitzewitz, J . A., Bowers, K.E, and Matthews, C . R.(1999) Biochemistry 38, 1018-1029} . Four parallel channels in the full-length protein are reduced to a pair of channels that most likely reflect a cis/trans proline isomerization reaction in the amino-terminal fragment . The off- and on-pathway intermediates that exist for both full-length alphaTS and alphaTS(1-188) may reflect the preponderance of local interactions in the beta/alpha barrel motif. Biochemistry, 1999 Aug 3, 38(31), 10178 - 86 Secondary structure and fold homology of the ArsC protein from the Escherichia coli arsenic resistance plasmid R773; Stevens SY et al.; Resistance to several toxic anions in Escherichia coli is conferred by the ars operon carried on plasmid R773 . The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells . In this paper, we report the determination of the overall fold for ArsC, a 16 kDa protein of the ars operon involved in the reduction of arsenate to arsenite, using multidimensional, multinuclear NMR . The protein is found to contain large regions of extensive mobility, particularly in the active site . A model fold, computed on the basis of a preliminary set of NOEs, was found to be structurally homologous to E . coli glutaredoxin, thiol transferases, and glutathione S-transferase . Some kinship to the structure of low molecular weight tyrosine phosphatases, based on rough topological similarity but more so on the basis of a common anion-binding-loop motif H-CX(n)R, was also detected . Although functional, secondary, and tertiary structural homology is observed with these molecules, no significant homology in primary structure was detected . The mobilities of the active site of ArsC and of other enzymes are discussed. Biochemistry, 1999 Aug 3, 38(31), 10052 - 8 Role of individual cysteine residues and disulfide bonds in the structure and function of Aspergillus ribonucleolytic toxin restrictocin; Nayak SK et al.; Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins . It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis . Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147 . These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131 . In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides . The mutants were expressed and purified from Escherichia coli . Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro . However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities . The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion . Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin . For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required. Biochemistry, 1999 Aug 3, 38(31), 10024 - 31 Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies; Shim JH et al.; Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2) . The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7 . Asp144 also plays a key role in the preparation of the active site geometry for catalysis . The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2 . The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH . The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate . The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme {Su, Y., Yamashita, M . M., Greasley, S . E . , Mullen, C . A., Shim, J . H., Jennings, P . A., Benkovic, S . J., and Wilson, I . A . (1998) J . Mol . Biol . 281, 485-499}, permitting a more exact formulation of the probable catalytic mechanism. Biochemistry, 1999 Aug 3, 38(31), 9964 - 70 Divalent metal derivatives of the hamster dihydroorotase domain; Huang DT et al.; Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides . The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified . The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0 . The apoenzyme was reconstituted with different divalent cations at pH 7.4 . Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity . Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound . The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II) . These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced . The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site . The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site . The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored. Biochemistry, 1999 Aug 3, 38(31), 9831 - 9 Investigation of the ATP binding site of Escherichia coli aminoimidazole ribonucleotide synthetase using affinity labeling and site-directed mutagenesis; Mueller EJ et al.; Aminoimidazole ribonucleotide (AIR) synthetase (PurM) catalyzes the conversion of formylglycinamide ribonucleotide (FGAM) and ATP to AIR, ADP, and P(i), the fifth step in de novo purine biosynthesis . The ATP binding domain of the E . coli enzyme has been investigated using the affinity label {(14)C}-p-fluorosulfonylbenzoyl adenosine (FSBA) . This compound results in time-dependent inactivation of the enzyme which is accelerated by the presence of FGAM, and gives a K(i) = 25 microM and a k(inact) = 5.6 x 10(-)(2) min(-)(1) . The inactivation is inhibited by ADP and is stoichiometric with respect to AIR synthetase . After trypsin digestion of the labeled enzyme, a single labeled peptide has been isolated, I-X-G-V-V-K, where X is Lys27 modified by FSBA . Site-directed mutants of AIR synthetase were prepared in which this Lys27 was replaced with a Gln, a Leu, and an Arg and the kinetic parameters of the mutant proteins were measured . All three mutants gave k(cat)s similar to the wild-type enzyme and K(m)s for ATP less than that determined for the wild-type enzyme . Efforts to inactivate the chicken liver trifunctional AIR synthetase with FSBA were unsuccessful, despite the presence of a Lys27 equivalent . The role of Lys27 in ATP binding appears to be associated with the methylene linker rather than its epsilon-amino group . The specific labeling of the active site by FSBA has helped to define the active site in the recently determined structure of AIR synthetase {Li, C., Kappock, T . J., Stubbe, J., Weaver, T . M., and Ealick, S . E . (1999) Structure (in press)}, and suggests additional flexibility in the ATP binding region. Biochemistry, 1999 Aug 3, 38(31), 9798 - 803 Membrane protein-ligand interactions in Escherichia coli vesicles and living cells monitored via a biosynthetically incorporated tryptophan analogue; Broos J et al.; This paper presents a deceptively straightforward experimental approach to monitoring membrane protein-ligand interactions in vesicles and in living Escherichia coli cells . This is achieved via the biosynthetic incorporation of 7-azatryptophan, a tryptophan analogue with a red-shifted absorption spectrum, allowing collection of the emission signal of the target protein in a high tryptophan background via red-edge excitation . The approach is demonstrated for the mannitol permease of E . coli (EII(mtl)), an integral membrane protein of 637 amino acids, including four tryptophans, and single-tryptophan mutants of EII(mtl) . By using a tryptophan auxotroph, a high level of 7-azatryptophan incorporation in EII(mtl) was achieved . The change in emission signal of the purified enzyme upon mannitol binding (-28%) was 4-fold larger than with EII(mtl) containing tryptophan, demonstrating the known higher sensitivity of this analogue for changes in the microenvironment {Schlesinger, R . (1968) J . Biol . Chem . 243, 3877-3883} . Changes in emission signal could also be monitored (-5%) when the enzyme was situated in vesicles, although it constituted only 10-15% of the total cytoplasmic membrane fraction . Of the five single-tryptophan mutants, the emission signal of the mutant with 7-azatryptophan at position 198 was the most sensitive for mannitol binding . Changes in emission signal not only were observed in vesicles (-18%) but also could be monitored in viable cells (-5%) . The fact that only modest expression levels and no protein purification are needed makes this a useful approach for the characterization of numerous protein systems under in vitro and in vivo conditions. J Interferon Cytokine Res, 1999 Jun, 19(6), 589 - 99 Identification by two-dimensional gel electrophoresis of vaccinia virus and cellular phosphoproteins modified after inducible expression of the dsRNA-activated protein kinase; Pavon M et al.; The interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) is a serine/threonine kinase that plays an important role in the biology of IFN, exerting antiviral and anticellular actions . These effects have been correlated with phosphorylation of the eukaryotic initiation factor eIF-2alpha and the NF-kappaB inhibitor IkappaB, although it has not been demonstrated that IkappaB is a direct target of PKR in vivo . In view of the various biological effects of PKR, it is likely that other cellular substrates are involved in PKR action . To identify novel substrates of PKR, we have carried out a systematic study of the phosphorylated proteins from cultured cells following PKR activation using high-resolution two-dimensional gel electrophoresis (2D-PAGE) . We have used metabolic labeling with {32P}orthophosphate of HeLa cells infected with vaccinia virus (VV) recombinants expressing wild type (wt) or the catalytically inactive mutant form (K296R) of PKR under regulation of the Escherichia coli lacI operator/repressor system . Upon induction of PKR in the presence of isopropyl-beta-D-thiogalactoside (IPTG), the 68-kDA wt enzyme and eIF-2alpha are phosphorylated . These events lead to changes in the phosphorylation state of viral and cellular proteins . A distinct set of VV-induced phosphoproteins remained phophorylated, while the labeling of other viral proteins decreased markedly, probably as a result of a PKR-dependent translational block . Five proteins of unknown origin (68, 26, 20, 19, 15-16 kDA) appeared to be newly phosphorylated after PKR activation . Expression of the catalytically inactive K296R mutant form of PKR did not induce changes in the phosphorylation of VV proteins . Thus, by 2D-PAGE, we identified cellular and VV-induced phosphoproteins modified after PKR activation . Some or all of the phosphoproteins appearing or increasing in amount after PKR activation might not be direct targets of PKR, but rather indirect consequences of PKR activation. Biochimie, 1999 Jun, 81(6), 619 - 29 Do plastid envelope membranes play a role in the expression of the plastid genome? Sato N, Rolland N, Block MA, Joyard J. A unique biochemical machinery is present within the two envelope membranes surrounding plastids (Joyard et al., Plant Physiol . 118 (1998) 715-723) that reflects the stage of development of the plastid and the specific metabolic requirements of the various tissues . Envelope membranes are the site for the synthesis and metabolism of specific lipids . They are also the site of transport of metabolites, proteins and information between plastids and surrounding cellular compartments . For instance, a complex machinery for the import of nuclear-encoded plastid proteins is rapidly being elucidated . The functional studies of plastid envelope membranes result in the characterization of an increasing number of envelope proteins with unexpected functions . For instance, recent experiments have demonstrated that envelope membranes bind specifically to plastid genetic systems, the nucleoids surrounded by plastid ribosomes . At early stages of plastid differentiation, the inner envelope membrane contains a unique protein (named PEND protein) that binds specifically to plastid DNA . This tight connection suggests that the PEND protein is at least involved in partitioning the plastid DNA to daughter plastids during division . The PEND protein can also provide a physical support for replication and transcription . In addition, factors involved in the control of plastid protein synthesis can become associated to envelope membranes . This was shown for a protein homologous to the E . coli ribosome recycling factor and for the stabilizing factors of some specific chloroplast mRNAs encoding thylakoid membrane proteins . In fact, the envelope membranes together with the plastid DNA are the two essential constituents of plastids that confer identity to plastids and their interactions are becoming uncovered through molecular as well as cytological studies . In this review, we will focus on these recent observations (which are consistent with the endosymbiotic origin of plastids) and we discuss possible roles for the plastid envelope in the expression of plastid genome. Nucleosides Nucleotides, 1999 Apr-May, 18(4-5), 1113 - 7 Synthesis of a new analog of thymidine for in vivo non-radioactive labeling of DNA; Rodriguez-Tanty C et al.; The introduction of 6-(p-bromobenzoylamino)caproyl radical in the methyl group of 2'-O-deoxythymidine is described . In vivo incorporation of this nucleoside to DNA was determined using a monoclonal antibody that recognized the radical. FEBS Lett, 1999 Jul 9, 454(3), 341 - 4 Primary structure and expression analysis of human UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, the bifunctional enzyme in neuraminic acid biosynthesis; Lucka L et al.; N-Acetylneuraminic acid is a main constituent of glycoproteins and gangliosides . In many membrane-bound receptors it is the target for external stimuli . The key enzyme for its biosynthesis is the bifunctional enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, catalysing the first two steps of the biosynthesis in the cytosol . The rat enzyme was previously isolated and characterised . In this report we present the corresponding human cDNA sequence, compare it with the primary structure of the rodent enzyme, and report the analysis of its expression in different human tissues and cell lines. FEBS Lett, 1999 Jul 9, 454(3), 307 - 11 Disordered N-terminal residues affect the folding thermodynamics and kinetics of maltose binding protein; Ganesh C et al.; Maltose binding protein (MBP) exhibits a slow phase of folding at pH 7.4, 298 K . The kinetics of this phase has been characterized as a function of denaturant concentration and temperature . Denaturant double-jump experiments and the activation energy for folding indicate that the slow phase involves processes other than proline isomerization . Although the first five N-terminal residues are disordered in the MBP crystal structure, mutations in this region slow down folding and destabilize the native structure . This is the first report showing that disordered N-terminal residues can affect folding kinetics and stability. FEBS Lett, 1999 Jul 9, 454(3), 303 - 6 Role of the C-terminal extremities of the smooth muscle myosin heavy chains: implication for assembly properties; Quevillon-Cheruel S et al.; The two light meromyosin isoforms from rabbit smooth muscle were prepared as recombinant proteins in Escherichia coli . These species which differed only by their C-terminal extremity showed the same circular dichroism spectra and endotherms in measurements of differential scanning calorimetry . Their solubility properties were different at pH 7.0 in the absence of monovalent salts . Their paracrystals formed at low pH differed by their aspect and number . These data suggest a role for the C-terminal extremity of myosin heavy chains in the assembly of myosin molecules in filaments and consequently in the contractility of smooth muscles. FEBS Lett, 1999 Jul 9, 454(3), 262 - 6 Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis; Haruki M et al.; Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family . We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis . Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif . We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme . We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity . The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding . This residue is probably structurally important to make the conformation of the active-site functional. FEBS Lett, 1999 Jul 9, 454(3), 220 - 4 A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation; Djajanegara I et al.; The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists . Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids . To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively . The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine . In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories {Rhoads et al . (1998) J . Biol . Chem . 273, 30750-30756; Vanlerberghe et al . (1998) Plant Cell 10, 1551-1560} . However, the mutated protein is instead activated specifically by succinate . Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism . Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them. FEBS Lett, 1999 Jul 9, 454(3), 177 - 80 Induction of the SOS DNA repair response in Escherichia coli by nitric oxide donating agents: dinitrosyl iron complexes with thiol-containing ligands and S-nitrosothiols; Lobysheva II et al.; The ability of nitric oxide (NO) donor compounds to induce the SOS DNA repair response in Escherichia coli is reported . Dinitrosyl iron complexes with glutathione and cysteine (DNIC) are the most potent SOS-inducers . S-Nitrosothiols (RSNO) mediate a similar response at 10-100 microM, but the response decreases sharply at concentrations above 0.5 mM . Pretreatment of the cells with the chelating agent o-phenanthroline (OP) prevents induction of the SOS response by all agents used . On the other hand, the toxicity of S-nitrosothiols is higher than that of DNIC . The EPR study shows the appearance of an EPR DNIC-type signal after incubation of the cells with S-nitrosoglutathione because of mutual transformation between RSNO and DNIC in the presence of accessible iron inside the cells . Pretreatment of the cells with OP leads to a decrease in this signal . Analysis of NO donor effects reveals a dual role of the iron ions in reactivity and toxicity of the compounds studied, i.e . (i) stabilization of the cytotoxic RSNO and (ii) generation of the SOS signal. Naunyn Schmiedebergs Arch Pharmacol, 1999 Jun, 359(6), 493 - 9 Role of nitric oxide and K+-channels in vascular hyporeactivity induced by endotoxin; Chen SJ et al.; This study was to investigate possible mechanisms associated with vascular hyporeactivity to vasoconstrictor agents in rats with endotoxaemia . Wistar-Kyoto rats were anaesthetised and injected with endotoxin {E . coli lipopolysaccharide (LPS); 10 mg/kg, i.v.} for 4 h . Pressor responses to noradrenaline (NA; 1 microg/kg, i.v.) were determined prior to and at every hour after LPS injection . After the in vivo experiment, rat thoracic aortas were excised and prepared as rings 3-4 mm in width . The endothelium was mechanically removed to evaluate K(+)-channel activity and the effects of nitric oxide (NO) on the vascular smooth muscle . Our results demonstrated that: (1) injection of LPS caused a significant fall in blood pressure and a severe vascular hyporeactivity to NA in the anaesthetised rat, (2) the relaxation induced by the K(+)channel opener cromakalim was greater in rings obtained from endotoxaemic rats and this enhanced relaxation was partially inhibited by pretreatment of these rings with 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ), an inhibitor of the NO/cGMP pathway, (3) endotoxaemia for 4 h was also associated with a profound vascular hyporeactivity to NA ex vivo and this vascular hyporesponsiveness was partially inhibited by ODQ, tetraethylammonium (TEA, a non-selective inhibitor of K(+)-channels) and charybdotoxin {CTX, a selective inhibitor of large conductance calcium-activated K(+)- channels (BK(Ca))}, but not by apamin, and (4) the combination of TEA or CTX with ODQ completely restored that vascular responsiveness to normal . These results suggest that activation of BK(Ca) and overproduction of NO in the vascular smooth muscle simultaneously contribute to vascular hyporeactivity to vasoconstrictor agents in endotoxaemia. Acta Physiol Hung, 1997-98, 85(4), 291 - 302 Changes in transthoracic electrical impedance during endotoxemia in dogs; Adamicza A et al.; Our aim was to investigate the role of hematocrit (H) and respiration in transthoracic electrical impedance during endotoxemia . Transthoracic electrical impedance at end-expiratory apnea (Z0) and at end-inspiration (Zmax), H values, and extravascular lung water level (EVLW), estimated by means of gravimetric analysis and the impedance method, were measured in splenectomized and mechanically ventilated dogs . In endotoxemia, there were increases in Z0, Zmax, H and the respiratory frequency . In the splenectomized dogs, both impedances slightly increased without any significant change in H . In the ventilated dogs, Z0, and Zmax increased similarly, while H increased . In the splenectomized, ventilated dogs, no changes were found in the impedances or H . The EVLW values showed that there was no serious edema in the endotoxemic groups . The results suggest that Z0 increased mainly in association with the increase in H . We conclude that the noninvasive measurements of the changes in impedance can be used for continuous monitoring of the fluid and gas shifts in the thorax. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9269 - 76 Intragenic suppressors of Hsp70 mutants: interplay between the ATPase- and peptide-binding domains; Davis JE et al.; ATP hydrolysis and polypeptide binding, the two key activities of Hsp70 molecular chaperones, are inherent properties of different domains of the protein . The coupling of these two activities is critical because the bound nucleotide determines, in part, the affinity of Hsp70s for protein substrate . In addition, cochaperones of the Hsp40 (DnaJ) class, which stimulate Hsp70 ATPase activity, have been proposed to play an important role in promoting efficient Hsp70 substrate binding . Because little is understood about this functional interaction between domains of Hsp70s, we investigated mutations in the region encoding the ATPase domain that acted as intragenic suppressors of a lethal mutation (I485N) mapping to the peptide-binding domain of the mitochondrial Hsp70 Ssc1 . Analogous amino acid substitution in the ATPase domain of the Escherichia coli Hsp70 DnaK had a similar intragenic suppressive effect on the corresponding I462T temperature-sensitive peptide-binding domain mutation . I462T protein had a normal basal ATPase activity and was capable of nucleotide-dependent conformation changes . However, the reduced affinity of I462T for substrate peptide (and DnaJ) is likely responsible for the inability of I462T to function in vivo . The suppressor mutation (D79A) appears to partly alleviate the defect in DnaJ ATPase stimulation caused by I462T, suggesting that alteration in the interaction with DnaJ may alter the chaperone cycle to allow productive interaction with polypeptide substrates . Preservation of the intragenic suppression phenotypes between eukaryotic mitochondrial and bacterial Hsp70s suggests that the phenomenon studied here is a fundamental aspect of the function of Hsp70:Hsp40 chaperone machines. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9224 - 9 A phenotype for enigmatic DNA polymerase II: a pivotal role for pol II in replication restart in UV-irradiated Escherichia coli; Rangarajan S et al.; DNA synthesis in Escherichia coli is inhibited transiently after UV irradiation . Induced replisome reactivation or "replication restart" occurs shortly thereafter, allowing cells to complete replication of damaged genomes . At the present time, the molecular mechanism underlying replication restart is not understood . DNA polymerase II (pol II), encoded by the dinA (polB) gene, is induced as part of the global SOS response to DNA damage . Here we show that pol II plays a pivotal role in resuming DNA replication in cells exposed to UV irradiation . There is a 50-min delay in replication restart in mutant cells lacking pol II . Although replication restart appears normal in DeltaumuDC strains containing pol II, the restart process is delayed for >90 min in cells lacking both pol II and UmuD'(2)C . Because of the presence of pol II, a transient replication-restart burst is observed in a "quick-stop" temperature-sensitive pol III mutant (dnaE486) at nonpermissive temperature . However, complete recovery of DNA synthesis requires the concerted action of both pol II and pol III . Our data demonstrate that pol II and UmuD'(2)C act in independent pathways of replication restart, thereby providing a phenotype for pol II in the repair of UV-damaged DNA. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 8979 - 84 High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5'-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase; Lawrence CC et al.; Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2 . The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y.) cofactor and R1 is the site of the conversion of nucleoside diphosphates to 2'-deoxynucleoside diphosphates . A mutant in the R1 subunit of Escherichia coli RNR, E441Q, was generated in an effort to define the function of E441 in the nucleotide-reduction process . Cytidine 5'-diphosphate was incubated with E441Q RNR, and the reaction was monitored by using stopped-flow UV-vis spectroscopy and high-frequency (140 GHz) time-domain EPR spectroscopy . These studies revealed loss of the Y . and formation of a disulfide radical anion and present experimental mechanistic insight into the reductive half-reaction catalyzed by RNR . These results support the proposal that the protonated E441 is required for reduction of a 3'-ketodeoxynucleotide by a disulfide radical anion . On the minute time scale, a second radical species was also detected by high-frequency EPR . Its g values suggest that this species may be a 4'-ketyl radical and is not on the normal reduction pathway . These experiments demonstrate that high-field time-domain EPR spectroscopy is a powerful new tool for deconvolution of a mixture of radical species. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 8973 - 8 Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA; O'Connor M et al.; The downstream box (DB) is a sequence element that enhances translation of several bacterial and phage mRNAs . It has been proposed that the DB enhances translation by base pairing transiently to bases 1469-1483 of 16S rRNA, the so-called anti-DB, during the initiation phase of translation . We have tested this model of enhancer action by constructing mutations in the anti-DB that alter its mRNA base-pairing potential and examining expression of a variety of DB-containing mRNAs in strains expressing the mutant anti-DB 16S rRNA . We found that the rRNA mutant was viable and that expression of all tested DB-containing mRNAs was completely unaffected by radical alterations in the proposed anti-DB . These findings lead us to conclude that enhancement of translation by the DB does not involve mRNA-rRNA base pairing. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 8955 - 60 Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms; Osakabe K et al.; A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms . To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem . Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H) . COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde . Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate . Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, k(cat)/K(m) of CAld5H for coniferyl aldehyde was approximately 140 times greater than that for ferulate . More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (K(i) = 0.59 microM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate . In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation . 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol . Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde . Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 8919 - 24 UmuD'(2)C is an error-prone DNA polymerase, Escherichia coli pol V; Tang M et al.; The damage-inducible UmuD' and UmuC proteins are required for most SOS mutagenesis in Escherichia coli . Our recent assay to reconstitute this process in vitro, using a native UmuD'(2)C complex, revealed that the highly purified preparation contained DNA polymerase activity . Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD'(2)C . E . coli dinB has recently been shown to have DNA polymerase activity (pol IV) . We suggest that UmuD'(2)C, the fifth DNA polymerase discovered in E . coli, be designated as E . coli pol V . In the presence of RecA, beta sliding clamp, gamma clamp loading complex, and E . coli single-stranded binding protein (SSB), pol V's polymerase activity is highly "error prone" at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE) . Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE . In accordance with the "A-rule," dAMP is preferentially incorporated opposite the lesion . A pol V mutant, UmuD'(2)C104 (D101N), has no measurable lesion bypass activity . A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3'-OH template-primer ends . We show, however, that despite competition for primer-3'-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 8890 - 4 Domain structure and lipid interaction of recombinant yeast Tim44; Weiss C et al.; Tim44 is an essential component of the machinery that mediates the translocation of nuclear-encoded proteins across the mitochondrial inner membrane . It functions as a membrane anchor for the ATP-driven protein import motor whose other subunits are the mitochondrial 70-kDa heat-shock protein (mhsp70) and its nucleotide exchange factor, mGrpE . To understand how this motor is anchored to the inner membrane, we have overexpressed Tim44 in Escherichia coli and studied the properties of the pure protein and its interaction with model lipid membranes . Limited proteolysis and analytical ultracentrifugation indicate that Tim44 is an elongated monomer with a stably folded C-terminal domain . The protein binds strongly to liposomes composed of phosphatidylcholine and cardiolipin but only weakly to liposomes containing phosphatidylcholine alone . Studies with phospholipid monolayers suggest that Tim44 binds to phospholipids of the mitochondrial inner membrane both by electrostatic interactions and by penetrating the polar head group region. J Neuroimmunol, 1999 Aug 3, 98(2), 112 - 20 Monoclonal antibodies raised against human acetylcholine receptor bind to all five subunits of the fetal isoform; Jacobson L et al.; The human muscle acetylcholine receptor (AChR) is an oligomeric membrane protein consisting of (alpha1)2,beta,delta,epsilon subunits in the adult form and (alpha 1)2,beta,gamma,delta in the fetal form . The adult AChR is the target for autoantibodies in myasthenia gravis (MG), and antibodies that block the function of fetal AChR can cross the placenta and paralyse the developing baby causing joint contractures . Monoclonal antibodies (mAbs) raised against purified AChR were characterised previously in terms of binding to five regions, three of which appeared to partially overlap, but the subunit localisation of the regions was not clearly established and they were assumed to be mainly on the immunodominant alpha subunits . We have studied binding of the mAbs to AChR subunit extracellular fragments expressed in E . coli, and to AChRs derived from TE671 cells and from fibroblast cell lines expressing human/Torpedo and Torpedo/mouse hybrid receptors . Using a combination of Western blotting and immunoprecipitation experiments, we demonstrate the subunit specificity of each mAb . The results confirm our previous observations but importantly show that only two of the regions are on the alpha subunit, the three others being on the beta, gamma and delta subunits of human AChR . Thus these mAbs should be useful in studies of AChR subunit expression in normal and diseased tissue, and to define further the binding sites of antibodies in MG patients. Surg Endosc, 1999 Aug, 13(8), 792 - 6 Peritoneal response to a septic challenge . Comparison between open laparotomy, pneumoperitoneum laparoscopy, and wall lift laparoscopy; Balague C et al.; BACKGROUND: Laparoscopic surgery has a lower incidence of surgical infection than open surgery . Differential factors that may modify the bacterial biology and explain this finding to some extent include CO(2) atmosphere, less desiccation of intraabdominal structures, fewer temperature changes, and a better preserved peritoneal and systemic immune response . Previous data suggest that the immune response and acute phase response are better preserved after laparoscopy . Therefore, we designed a study to evaluate the early peritoneal response to sepsis in an experimental peritonitis model comparing open surgery with CO(2) and abdominal wall lift laparoscopy . METHODS: The study subjects comprised 360 mice distributed into the following four groups: group 1, n = 72 (controls); group 2, n = 96 (open surgery), 2-3 cm laparotomy, with abdominal cavity exposed to the air for 30 min; group 3, n = 96, CO(2) laparoscopy (5 mmHg pneumoperitoneum) for 30 min; group 4, n = 96, wall lift laparoscopy for 30 min . Intraabdominal contamination in the four groups was induced with 1 ml of E . coli suspension (1 x 10(4) CFU/ml) 10 min before abdomen closure . Peritoneal fluid and blood samples were obtained 1.5, 3, 24, and 72 h after surgery, and TNF, IL-1, and IL-6 were measured (via ELISA), as well as quantitative culture . RESULTS: The number of CFU (colony-forming units) obtained in peritoneal fluid and positive blood culture rates were significantly lower in the laparoscopic groups than in the open group . IL-1 peritoneal levels were significantly lower after 24 h and 72 h in the laparoscopy groups . IL-6 levels decreased sharply in the laparoscopy groups at 24 h and 72 h . There were no differences between the two types of laparoscopy models (CO(2) and wall lift) . CONCLUSIONS: Peritoneal response to sepsis is better preserved after laparoscopy than after open surgery . CO(2) does not seem to influence bacterial growth . According to these findings, laparoscopy entails less local trauma and better preserved intraabdominal conditions. J Clin Invest, 1999 Aug, 104(3), 253 - 62 Pathogenic Escherichia coli increase Cl- secretion from intestinal epithelia by upregulating galanin-1 receptor expression; Hecht G et al.; Galanin is widely distributed in enteric nerve terminals lining the human gastrointestinal (GI) tract . We have shown previously that galanin-1 receptors (Gal1-R) are expressed by epithelial cells lining the human GI tract, and upon activation cause Cl- secretion . Because expression of this receptor is transcriptionally regulated by nuclear factor-kappa B (NF-kappa B), which is activated by enteric pathogens as a part of the host epithelial response to infection, we investigated whether such bacterial pathogens could directly increase Gal1-R expression in the T84-cell model system . Pathogenic Escherichia coli, but not nonpathogenic E . coli, activate a p50/p65 NF-kappa B complex that binds to oligonucleotides corresponding to a recognition site located within the 5' flanking region of the human GAL1R gene . Pathogenic E . coli, but not normal commensal organisms, increase Gal1-R mRNA synthesis and {(125)I}galanin binding sites . Whereas galanin increases short-circuit current (Isc) approximately 5-fold in uninfected T84 cells, exposure to pathogenic, but not nonpathogenic, E . coli results in galanin increasing Isc approximately 20-fold . To confirm the validity of these in vitro observations, we also studied C57BL/6J mice infected with enterohemorrhagic E . coli (EHEC) by gavage . Infection caused a progressive increase in both NF-kappa B activation and Gal1-R expression, with maximal levels of both observed 3 days after gavage . Ussing chamber studies revealed that colons infected with EHEC, but not those exposed to normal colonic flora, markedly increased Isc in response to galanin . These data indicate that pathogen-induced increases in Gal1-R expression by epithelial cells lining the colon may represent a novel unifying pathway responsible for at least a portion of the excessive fluid secretion observed during infectious diarrhea. Genetics, 1999 Aug, 152(4), 1439 - 47 Expression vectors for Methanococcus maripaludis: overexpression of acetohydroxyacid synthase and beta-galactosidase; Gardner WL et al.; A series of integrative and shuttle expression vectors was developed for use in Methanococcus maripaludis . The integrative expression vectors contained the Methanococcus voltae histone promoter and multiple cloning sites designed for efficient cloning of DNA . Upon transformation, they can be used to overexpress specific homologous genes in M . maripaludis . When tested with ilvBN, which encodes the large and small subunits of acetohydroxyacid synthase, transformants possessed specific activity 13-fold higher than that of the wild type . An expression shuttle vector, based on the cryptic plasmid pURB500 and the components of the integrative vector, was also developed for the expression of heterologous genes in M . maripaludis . The beta-galactosidase gene from Escherichia coli was expressed to approximately 1% of the total cellular protein using this vector . During this work, the genes for the acetohydroxyacid synthase (ilvBN) and phosphoenolpyruvate synthase (ppsA) were sequenced from a M . maripaludis genomic library. Genetics, 1999 Aug, 152(4), 1429 - 37 Isolation of acetate auxotrophs of the methane-producing archaeon Methanococcus maripaludis by random insertional mutagenesis; Kim W et al.; To learn more about autotrophic growth of methanococci, we isolated nine conditional mutants of Methanococcus maripaludis after transformation of the wild type with a random library in pMEB.2, a suicide plasmid bearing the puromycin-resistance cassette pac . These mutants grew poorly in mineral medium and required acetate or complex organic supplements such as yeast extract for normal growth . One mutant, JJ104, was a leaky acetate auxotroph . A plasmid, pWDK104, was recovered from this mutant by electroporation of a plasmid preparation into Escherichia coli . Transformation of wild-type M . maripaludis with pWDK104 produced JJ104-1, a mutant with the same phenotype as JJ104, thus establishing that insertion of pWDK104 into the genome was responsible for the phenotype . pWDK104 contained portions of the methanococcal genes encoding an ABC transporter closely related to MJ1367-MJ1368 of M . jannaschii . Because high levels of molybdate, tungstate, and selenite restored growth to wild-type levels, this transporter may be specific for these oxyanions . A second acetate auxotroph, JJ117, had an absolute growth requirement for either acetate or cobalamin, and wild-type growth was observed only in the presence of both . Cobinamide, 5', 6'-dimethylbenzimidazole, and 2-aminopropanol did not replace cobalamin . This phenotype was correlated with tandem insertions in the genome but not single insertions and appeared to have resulted from an indirect effect on cobamide metabolism . Plasmids rescued from other mutants contained portions of ORFs denoted in M . jannaschii as endoglucanase (MJ0555), transketolase (MJ0681), thiamine biosynthetic protein thiI (MJ0931), and several hypothetical proteins (MJ1031, MJ0835, and MJ0835.1). Genetics, 1999 Aug, 152(4), 1417 - 28 Genetic identification of three ABC transporters as essential elements for nitrate respiration in Haloferax volcanii; Wanner C et al.; More than 40 nitrate respiration-deficient mutants of Haloferax volcanii belonging to three different phenotypic classes were isolated . All 15 mutants of the null phenotype were complemented with a genomic library of the wild type . Wild-type copies of mutated genes were recovered from complemented mutants using two different approaches . The DNA sequences of 13 isolated fragments were determined . Five fragments were found to overlap; therefore nine different genomic regions containing genes essential for nitrate respiration could be identified . Three genomic regions containing genes coding for subunits of ABC transporters were further characterized . In two cases, genes coding for an ATP-binding subunit and a permease subunit were clustered and overlapped by four nucleotides . The third gene for a permease subunit had no additional ABC transporter gene in proximity . One ABC transporter was found to be glucose specific . The mutant reveals that the ABC transporter solely mediates anaerobic glucose transport . Based on sequence similarity, the second ABC transporter is proposed to be molybdate specific, explaining its essential role in nitrate respiration . The third ABC transporter is proposed to be anion specific . Genome sequencing has shown that ABC transporters are widespread in Archaea . Nevertheless, this study represents only the second example of a functional characterization. Genetics, 1999 Aug, 152(4), 1353 - 61 Extragenic pleiotropic mutations that repress glycosyl hydrolase expression in the hyperthermophilic archaeon Sulfolobus solfataricus; Haseltine C et al.; The hyperthermophilic archaeon Sulfolobus solfataricus employs a catabolite repression-like regulatory system to control enzymes involved in carbon and energy metabolism . To better understand the basis of this system, spontaneous glycosyl hydrolase mutants were isolated using a genetic screen for mutations, which reduced expression of the lacS gene . The specific activities of three glycosyl hydrolases, including an alpha-glucosidase (malA), a beta-glycosidase (lacS), and the major secreted alpha-amylase, were measured in the mutant strains using enzyme activity assays, Western blot analysis, and Northern blot analysis . On the basis of these results the mutants were divided into two classes . Group I mutants exhibited a pleiotropic defect in glycosyl hydrolase expression, while a single group II mutant was altered only in lacS expression . PCR, Southern blot analysis, comparative heterologous expression in Escherichia coli, and DNA sequence analysis excluded cis-acting mutations as the explanation for reduced lacS expression in group I mutants . In contrast lacS and flanking sequences were deleted in the group II mutant . Revertants were isolated from group I mutants using a lacS-specific screen and selection . These revertants were pleiotropic and restored glycosyl hydrolase activity either partially or completely to wild-type levels as indicated by enzyme assays and Western blots . The lacS mutation in the group II mutant, however, was nonrevertible . The existence of group I mutants and their revertants reveals the presence of a trans-acting transcriptional regulatory system for glycosyl hydrolase expression. Genetics, 1999 Aug, 152(4), 1277 - 83 The archaeal molecular chaperone machine: peculiarities and paradoxes; Macario AJ et al.; A major finding within the field of archaea and molecular chaperones has been the demonstration that, while some species have the stress (heat-shock) gene hsp70(dnaK), others do not . This gene encodes Hsp70(DnaK), an essential molecular chaperone in bacteria and eukaryotes . Due to the physiological importance and the high degree of conservation of this protein, its absence in archaeal organisms has raised intriguing questions pertaining to the evolution of the chaperone machine as a whole and that of its components in particular, namely, Hsp70(DnaK), Hsp40(DnaJ), and GrpE . Another archaeal paradox is that the proteins coded by these genes are very similar to bacterial homologs, as if the genes had been received via lateral transfer from bacteria, whereas the upstream flanking regions have no bacterial markers, but instead have typical archaeal promoters, which are like those of eukaryotes . Furthermore, the chaperonin system in all archaea studied to the present, including those that possess a bacterial-like chaperone machine, is similar to that of the eukaryotic-cell cytosol . Thus, two chaperoning systems that are designed to interact with a compatible partner, e.g., the bacterial chaperone machine physiologically interacts with the bacterial but not with the eucaryal chaperonins, coexist in archaeal cells in spite of their apparent functional incompatibility . It is difficult to understand how these hybrid characteristics of the archaeal chaperoning system became established and work, if one bears in mind the classical ideas learned from studying bacteria and eukaryotes . No doubt, archaea are intriguing organisms that offer an opportunity to find novel molecules and mechanisms that will, most likely, enhance our understanding of the stress response and the protein folding and refolding processes in the three phylogenetic domains. Neuroscience, 1999, 93(1), 393 - 400 Immune challenge-stimulated hypophysiotropic corticotropin-releasing hormone messenger RNA expression is associated with an induction of neurotensin messenger RNAs without alteration of vasopressin messenger RNAs; Juaneda C et al.; The corticotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus are the final common pathway of the neuroendocrine adaptative response to a variety of stressors . To meet varied homeostatic needs, corticotropin-releasing hormone neurons exhibit a marked phenotypical plasticity, enabling them to rapidly modify their neuroendocrine output . In particular, they synthesize the neuropeptides vasopressin and neurotensin . Under many experimental circumstances, it is observed that corticotropin-releasing hormone and vasopressin are regulated in parallel, whereas the expression of neurotensin seems dissociated, in these neurons, evoking different transcriptional control over the co-existing neuropeptides depending on the adaptative response required . Using radioactive and dual-label in situ hybridization techniques, we have studied the respective expression of paraventricular corticotropin-releasing hormone, vasopressin and neurotensin messenger RNAs in the context of an immune challenge . A single intraperitoneal injection of the endotoxin lipopolysaccharide was administered to adult male rats that were killed 8 h later . Compared to control animals, lipopolysaccharide-injected rats showed elevated plasma corticosterone (614+/-65 vs 185+/-40 ng/ml in control) and increased expression of paraventricular corticotropin-releasing hormone messenger RNA (+200%); expression of neurotensin messenger RNA was induced in about one-third of corticotropin-releasing hormone neurons, whereas vasopressin messenger RNA expression remained unchanged . Therefore, in this experimental context and at the time-point examined, co-existing corticotropin-releasing hormone and vasopressin appeared differentially expressed, and an additional stimulus (inflammation) is demonstrated to result in neurotensin expression in neuroendocrine corticotropin-releasing hormone neurons . Neurotensin may be released in the pituitary portal blood to trigger pituitary response associated with mobilization of the immune system. Biol Chem, 1999 Jun, 380(6), 717 - 21 Human Fcgamma receptor IIb expressed in Escherichia coli reveals IgG binding capability; Sondermann P et al.; Fcgamma receptors (FcgammaR) are expressed on immunologically active cells where they trigger B and T cell responses and are responsible for the clearance of immunocomplexes . They occur as type I transmembrane proteins and also in soluble forms (sFcR) comprising only the ecto domains of the receptors . State-of-the-art research has generated demand for highly pure and homogeneous sFcgammaR preparations: first, studies of the immunoregulative potential of the soluble FcgammaRs have been hampered by co-purified growth factors . Second, they are needed for crystallographic analyses to solve questions such as the exact location of the binding site for IgG on the receptor, and the graded affinities of the receptors for different IgG subclasses . This has been unsuccessful due to limitations in availability and homogeneity of sFcgammaR expressed in eukaryotic cells . In order to address these problems we expressed the extracellular part of the human FcgammaRIIb in E . coli . The protein was refolded, purified in a three-step procedure and characterized by SDS-PAGE, mass spectrometry as well as N-terminal sequencing . The unglycosylated FcgammaRIIb is active because it binds immobilized antibody as well as the IgG Fc-fragment in solution . Finally, the receptor was crystallized in orthorhombic, tetragonal and hexagonal crystal forms that diffracted X-rays to resolutions of 1.7 A, 2.7 A and 3.8 A respectively. Eur J Biochem, 1999 Jul, 263(1), 212 - 21 Characterization of recombinant Arabidopsis thaliana threonine synthase; Laber B et al.; Threonine synthase (TS) catalyses the last step in the biosynthesis of threonine, the pyridoxal 5'-phosphate dependent conversion of L-homoserine phosphate (HSerP) into L-threonine and inorganic phosphate . Recombinant Arabidopsis thaliana TS (aTS) was characterized to compare a higher plant TS with its counterparts from Escherichia coli and yeast . This comparison revealed several unique properties of aTS: (a) aTS is a regulatory enzyme whose activity was increased up to 85-fold by S-adenosyl-L-methionine (SAM) and specifically inhibited by AMP; (b) HSerP analogues shown previously to be potent inhibitors of E . coli TS failed to inhibit aTS; and (c) aTS was a dimer, while the E . coli and yeast enzymes are monomers . The N-terminal region of aTS is essential for its regulatory properties and protects against inhibition by HSerP analogues, as an aTS devoid of 77 N-terminal residues was neither activated by SAM nor inhibited by AMP, but was inhibited by HSerP analogues . The C-terminal region of aTS seems to be involved in dimer formation, as the N-terminally truncated aTS was also found to be a dimer . These conclusions are supported by a multiple amino-acid sequence alignment, which revealed the existence of two TS subfamilies . aTS was classified as a member of subfamily 1 and its N-terminus is at least 35 residues longer than those of any nonplant TS . Monomeric E . coli and yeast TS are members of subfamily 2, characterized by C-termini extending about 50 residues over those of subfamily 1 members . As a first step towards a better understanding of the properties of aTS, the enzyme was crystallized by the sitting drop vapour diffusion method . The crystals diffracted to beyond 0.28 nm resolution and belonged to the space group P222 (unit cell parameters: a = 6.16 nm, b = 10.54 nm, c = 14.63 nm, alpha = beta = gamma = 90 degrees). Eur J Biochem, 1999 Jul, 263(1), 163 - 9 Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartic acid (cyanophycin): molecular cloning of the gene of Synechocystis sp . PCC 6803, expression in Escherichia coli, and biochemical characterization of the purified enzyme; Richter R et al.; The branched polypeptide multi-L-arginyl-poly-L-aspartic acid, also called cyanophycin, is a water-insoluble reserve material of cyanobacteria . The polymer is degraded by a specific hydrolytic enzyme called cyanophycinase . By heterologous expression in Escherichia coli, a gene encoding cyanophycinase has been identified in the sequenced genome of Synechocystis sp . PCC 6803 . The gene, designated cphB, codes for a protein of 29.4 kDa . The high level of expression of active cyanophycinase in E . coli from the Synechocystis gene allowed for its purification to electrophoretic homogeneity . The enzyme, which appears to be specific for cyanophycin, hydrolysed the polymer to a dipeptide consisting of aspartic acid and arginine . Based on inhibitor sensitivity and primary sequence, cyanophycinase appears to be a serine-type exopeptidase related to dipeptidase E {Conlin, C.A., Haakensson, K., Liljas, A . & Miller, C.G . (1994) J . Bacteriol . 176, 166-172}. Eur J Biochem, 1999 Jul, 263(1), 128 - 36 Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from sea anemone: topology in lipid membranes; Anderluh G et al.; Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina . It readily creates pores in membranes containing sphingomyelin . Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis . At approximately every tenth residue, a cysteine was introduced . Nineteen single cysteine mutants were produced in Escherichia coli and purified . The accessibility of the thiol groups in lipid-embedded cysteine mutants was studied by reaction with biotin maleimide . Most of the mutants were modified, except those with cysteines at positions 105 and 114 . Mutants R144C and S160C were modified only at high concentrations of the probe . Similar results were obtained if membrane-bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C . Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding . These results were further confirmed by labeling the mutants with acrylodan, a polarity-sensitive fluorescent probe . When labeled mutants were combined with vesicles, the following mutants exhibited blue-shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C . The overall results suggest that at least two regions are embedded within the lipid membrane: the N-terminal 13-20 region, probably forming an amphiphilic helix, and the tryptophan-rich 105-120 region . Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups . The association with the membrane appears to be unique and different from that of bacterial pore-forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel-forming toxins. EMBO J, 1999 Aug 2, 18(15), 4149 - 56 Molecular interactions in ribose transport: the binding protein module symmetrically associates with the homodimeric membrane transporter; Park Y et al.; The Escherichia coli high-affinity ribose transporter is composed of the periplasmic ribose-binding protein (RBP or RbsB), the membrane component (RbsC) and the ATP-binding protein (RbsA) . In order to dissect the molecular interactions initiating the transport process, RbsC suppressors for transport-defective rbsB mutations were isolated . These suppressors are localized in two regions of RbsC, which are allele-specific to N- or C-terminal domain mutations of RBP, suggesting that there are two distinct regions of RbsC, each interacting with one of the two domains of RBP . To demonstrate that these two regions provide a homodimeric binding surface for RBP we constructed a dimeric rbsC in which two genes are joined tandemly from head to tail with the addition of a linker . The dimeric RbsC protein is stable and functional in growth and ribose uptake . By exploiting the allele specificity between the domain-specific mutations and their suppressors, we generated all mutation-suppressor combinations in a single rbsB plus the dimeric rbsC genes . Their phenotypes are consistent with the proposal that the binding protein module interacts symmetrically with homodimeric RbsC . The mode of association proposed here for the ribose transport components could be extended to other ABC transporters with similar structural organizations. EMBO J, 1999 Aug 2, 18(15), 4118 - 27 ATP synthesis by F-type ATP synthase is obligatorily dependent on the transmembrane voltage; Kaim G et al.; ATP synthase is the universal enzyme that manufactures cellular ATP using the energy stored in a transmembrane ion gradient . This energy gradient has two components: the concentration difference (DeltapH or DeltapNa(+)) and the electrical potential difference DeltaPsi, which are thermodynamically equivalent . However, they are not kinetically equivalent, as the mitochondrial and bacterial ATP synthases require a transmembrane potential, DeltaPsi, but the chloroplast enzyme has appeared to operate on DeltapH alone . Here we show that, contrary to the accepted wisdom, the 'acid bath' procedure used to study the chloroplast enzyme develops not only a DeltapH but also a membrane potential, and that this potential is essential for ATP synthesis . Thus, for the chloroplast and other ATP synthases, the membrane potential is the fundamental driving force for their normal operation . We discuss the biochemical reasons for this phenomenon and a model that is consistent with these new experimental facts. Antimicrob Agents Chemother, 1999 Aug, 43(8), 2063 - 5 Identification of functional amino acids in the macrolide 2'-phosphotransferase II; Taniguchi K et al.; Macrolide 2'-phosphotransferase {MPH(2')} transfers the gamma phosphate of ATP to the 2'-OH group of macrolide antibiotics . The role of aspartic acids in the putative ATP-binding site of MPH(2')II was investigated through the substitution of alanine for aspartate by site-directed mutagenesis . D200A, D209A, D219A, and D231A mutant strains were unable to inactivate the substrate oleandomycin, while a D227A mutant retained 7% of the activity of the original enzyme. J Biol Chem, 1999 Aug 6, 274(32), 22839 - 46 Stimulated activity of human topoisomerases IIalpha and IIbeta on RNA-containing substrates; Wang Y et al.; Eukaryotic topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics . Central to the activities of the enzyme is its ability to introduce transient double-stranded breaks in the DNA helix, where the two subunits of the enzyme become covalently attached to the generated 5'-ends through phosphotyrosine linkages . Here, we demonstrate that human topoisomerases IIalpha and IIbeta are able to cleave ribonucleotide-containing substrates . With suicide substrates, which are partially double-stranded molecules containing a 5'-recessed strand, cleavage of both strands was stimulated approximately 8-fold when a ribonucleotide rather than a deoxyribonucleotide was present at the scissile phosphodiester of the recessed strand . The existence of a ribonucleotide at the same position in a normal duplex substrate also enhanced topoisomerase II-mediated cleavage, although to a lesser extent . The enzyme covalently linked to the 5'-ribonucleotide in the cleavage complex efficiently performed ligation, and ligation occurred equally well to acceptor molecules terminated by either a 3'-ribo- or deoxyribonucleotide . Besides the enhanced topoisomerase II-mediated cleavage of ribonucleotide-containing substrates, cleavage of such substrates could be further stimulated by ATP or antitumor drugs . In conclusion, the observed in vitro activities of the human topoisomerase II isoforms indicate that the enzymes can operate on RNA or RNA-containing substrates and thus might possess an intrinsic RNA topoisomerase activity, as has previously been demonstrated for Escherichia coli topoisomerase III. J Biol Chem, 1999 Aug 6, 274(32), 22502 - 7 The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase; Thoden JB et al.; Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate, which is subsequently employed in both the pyrimidine and arginine biosynthetic pathways . The reaction mechanism is known to proceed through at least three highly reactive intermediates: ammonia, carboxyphosphate, and carbamate . In keeping with the fact that the product of CPS is utilized in two competing metabolic pathways, the enzyme is highly regulated by a variety of effector molecules including potassium and ornithine, which function as activators, and UMP, which acts as an inhibitor . IMP is also known to bind to CPS but the actual effect of this ligand on the activity of the enzyme is dependent upon both temperature and assay conditions . Here we describe the three-dimensional architecture of CPS with bound IMP determined and refined to 2.1 A resolution . The nucleotide is situated at the C-terminal portion of a five-stranded parallel beta-sheet in the allosteric domain formed by Ser(937) to Lys(1073) . Those amino acid side chains responsible for anchoring the nucleotide to the polypeptide chain include Lys(954), Thr(974), Thr(977), Lys(993), Asn(1015), and Thr(1017) . A series of hydrogen bonds connect the IMP-binding pocket to the active site of the large subunit known to function in the phosphorylation of the unstable intermediate, carbamate . This structural analysis reveals, for the first time, the detailed manner in which CPS accommodates nucleotide monophosphate effector molecules within the allosteric domain. J Biol Chem, 1999 Aug 6, 274(32), 22131 - 4 Evidence for 4-hydroxyproline in viral proteins . Characterization of a viral prolyl 4-hydroxylase and its peptide substrates; Eriksson M et al.; 4-Hydroxyproline, the characteristic amino acid of collagens and collagen-like proteins in animals, is also found in certain proline-rich proteins in plants but has been believed to be absent from viral and bacterial proteins . We report here on the cloning and characterization from a eukaryotic algal virus, Paramecium bursaria Chlorella virus-1, of a 242-residue polypeptide, which shows distinct sequence similarity to the C-terminal half of the catalytic alpha subunits of animal prolyl 4-hydroxylases . The recombinant polypeptide, expressed in Escherichia coli, was found to be a soluble monomer and to hydroxylate both (Pro-Pro-Gly)(10) and poly(L-proline), the standard substrates of animal and plant prolyl 4-hydroxylases, respectively . Synthetic peptides such as (Pro-Ala-Pro-Lys)(n), (Ser-Pro-Lys-Pro-Pro)(5), and (Pro-Glu-Pro-Pro-Ala)(5) corresponding to proline-rich repeats coded by the viral genome also served as substrates . (Pro-Ala-Pro-Lys)(10) was a particularly good substrate, with a K(m) of 20 microM . The prolines in both positions in this repeat were hydroxylated, those preceding the alanines being hydroxylated more efficiently . The data strongly suggest that P . bursaria Chlorella virus-1 expresses proteins in which many prolines become hydroxylated to 4-hydroxyproline by a novel viral prolyl 4-hydroxylase. FEBS Lett, 1999 Jul 16, 455(1-2), 162 - 4 Redox control of hydrogenase activity in the green alga Scenedesmus obliquus by thioredoxin and other thiols; Wunschiers R et al.; The activity of the NiFe-hydrogenase from the green alga Scenedesmus obliquus is inhibited by both algal thioredoxins f and I+II, and by Escherichia coli thioredoxin . The strongest inhibition was observed with homologous chloroplastic thioredoxin f (I50 = 21 nM) and E . coli thioredoxin (I50 = 83 nM) . For the homologous cytoplasmic thioredoxins I+II an I50 of 667 nM was determined . Glutathione shows a similar but much less pronounced inhibitory effect whereas dithiothreitol had no effect . In addition to glucose-6-phosphate dehydrogenase, NiFe-hydrogenase is only the second enzyme known to be inhibited by reduced thioredoxin. FEBS Lett, 1999 Jul 16, 455(1-2), 79 - 82 Existence of uncoupling protein-2 antigen in isolated mitochondria from various tissues; Jezek P et al.; Antibodies against Escherichia coli-expressed uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) were raised by operating the blotted proteins into the spleen of minipigs . The antisera reacted more intensively with the recombinant UCP2 and UCP3 than with uncoupling protein-1 (UCP1) isolated from brown adipose tissue . Moreover, anti-UCP2 and cross-reacting anti-UCP3 antibodies identified the presence of the UCP2/3 antigen in isolated mitochondria from rat heart, rat kidney, rat brain, rabbit epididymal white adipose tissue, hamster brown adipose tissue, and rabbit skeletal muscle . It has been concluded that UCP2 is expressed in these tissues (UCP3 in skeletal muscle); however their existence in mitochondria had not previously been demonstrated. FEBS Lett, 1999 Jul 16, 455(1-2), 63 - 9 Identification of the DNA binding surface of H-NS protein from Escherichia coli by heteronuclear NMR spectroscopy; Shindo H et al.; The DNA binding domain of H-NS protein was studied with various N-terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D- and 3D-NMR spectroscopies . It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H-NS falls in the range from 1/6 to 1/25 for H-NS(60-137), H-NS(70-137) and H-NS(80-137), whereas it was much weaker for H-NS(91-137) . Thus, the DNA binding domain was defined to be the region from residue A80 to the C-terminus . Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60-R93 in mutant protein H-NS(60-137) forms a long stretch of disordered, flexible chain, and also showed that the structure of the C-terminal region (residues A95-Q137) in mutant H-NS(60-137) was nearly identical to that of H-NS(91-137) . 1H and 15N chemical shift perturbations induced by complex formation of H-NS(60-137) with an oligonucleotide duplex 14-mer demonstrated that two loop regions, i.e . residues A80-K96 and T110-A117, play an essential role in DNA binding. FEBS Lett, 1999 Jul 16, 455(1-2), 49 - 54 Inhibition of Escherichia coli DNA polymerase-I by the anti-cancer drug cis-diaminedichloroplatinum(II): what roles do polymerases play in cis-platin-induced cytotoxicity? Duman RK, Heath RT, Bose RN. Activities of Escherichia coli DNA polymerase-I were examined in the presence of the anti-tumor drug cis-diaminedichloroplatinum(II) and its inactive geometric isomer trans-diaminedichloroplatinum(II) . The trans-isomer did not inhibit the enzyme activity . The anti-tumor drug, on the other hand, retarded the enzyme in its ability to extend the primer strand of DNA . Two alternative mechanisms of inhibition, covalent binding of cis-diaminedichloroplatinum(II) to the polymerase and to the template DNA, were explored . Selective preincubations of the platinum drug with the polymerase and DNA reveal that the inhibition is primarily due to covalent binding to the enzyme . The rates of inhibition were found to be first order in enzyme and zeroth order in platinum in the concentration range 0.05-3.0 mM . A mechanism that deals with the formation of an initial platinum-polymerase-I complex with a binding constant > 10(5) M(-1) followed by a further reaction to form an inhibitory complex is consistent with the kinetic data . The rate limiting first order rate constant for the formation of the inhibitory complex is comparable to that observed for the thiol coordination of peptides containing cysteine residues . Analyses of known structures and functions of catalytic domains of various polymerases point to the direction that the inhibition is perhaps due to the distortion of the DNA binding domain of the enzyme due to platinum coordination. Cell, 1999 Jul 23, 98(2), 217 - 27 Oxidative protein folding is driven by the electron transport system; Bader M et al.; Disulfide bond formation is catalyzed in vivo by DsbA and DsbB . Here we reconstitute this oxidative folding system using purified components . We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism . We find that disulfide bond formation and the electron transport chain are directly coupled . DsbB uses quinones as electron acceptors, allowing various choices for electron transport to support disulfide bond formation . Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions . Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron acceptors such as fumarate . This flexibility reflects the vital nature of the disulfide catalytic system. Plant Cell Physiol, 1999 May, 40(5), 504 - 14 Identification of clp genes expressed in senescing Arabidopsis leaves; Nakabayashi K et al.; Clp protease is a highly selective protease in E . coli, which consists of two types of subunits, the regulatory subunit with ATPase activity, ClpA, and the catalytic subunit, ClpP . In order to examine the possible association of plant Clp protease with the degradation of protein in senescing chloroplasts, we isolated a cDNA clone for ClpC which is a plant homologue of ClpA from Arabidopsis thaliana in addition to ERD1 which we had isolated earlier {Kiyosue et al . (1993) Biochem . Biophys . Res . Commun . 196: 1214} . We also isolated a clone for the plastidic gene, clpP (pclpP) and cDNA clones for putative nuclear clpP genes (nclpP1-6) . We analyzed the expression of these clp genes in Arabidopsis leaves after various dark periods and during natural senescence . The expression of erd1 was increased by dark-induced and by natural senescence, as reported earlier {Nakashima et al . (1997) Plant J . 12: 851}, while that of AtclpC was decreased . Two catalytic subunits nclpPs (nclpP3 and nclpP5) showed high expression in naturally senescing leaves, but the expression of pclpP and the other nclpPs was not changed . Immunoblot analysis of chloroplast protein and in vitro import analysis demonstrated that both nucleus-encoded regulatory subunits as well as nClpP5 were localized in the chloroplast stroma . These observations suggest that chloroplast Clp protease is composed of very complicated combinations of subunits, and that ERD1, nClpP5 and pClpP have a role in the concerted degradation of protein in senescing chloroplasts. Plant Cell Physiol, 1999 May, 40(5), 477 - 81 Molecular cloning and expression of Arabidopsis fatty acid hydroperoxide lyase; Matsui K et al.; Fatty acid hydroperoxide lyase (HPOL), an enzyme of the octadecanoid pathway that forms carbon-6 aldehydes such as n-hexanal or (Z)-3-hexenal, was cloned from Arabidopsis thaliana as a full-length cDNA . The HPOL activity obtained by expressing the cDNA in Escherichia coli formed n-hexanal from linoleic acid 13-hydroperoxide, whereas linoleic acid 9-hydroperoxide was not a substrate for the enzyme . The HPOL mRNA is expressed at low level in leaves; however, its accumulation can be found in the inflorescence . Wounding or methyl jasmonate treatments increase the mRNA level in leaves . These results indicate that the HPOL gene is up-regulated in leaves in response to wounding and that the enzyme may be an active component of the octadecanoid defense response. J Chromatogr A, 1999 Jul 2, 848(1-2), 131 - 8 Heparin elution of transcription factors from DNA-Sepharose columns; Gadgil H et al.; A novel method using heparin for eluting transcription factors from DNA-Sepharose columns was characterized . CAAT enhancer binding protein (C/EBP) or lac repressor fusion proteins were both eluted with heparin from columns containing specific DNA sequences coupled to cyanogen bromide activated Sepharose . The amount of the lac repressor chimera which eluted from the column was shown to increase with increases in the mobile phase heparin concentration . The elution of the protein was also shown to be dependent on the amount of DNA coupled to the column and more protein eluted from columns containing lesser amounts of DNA . These data suggest that heparin and DNA compete for binding to the protein; this competition causes elution . Comparison of heparin- and salt-eluted protein demonstrated the heparin-eluted fraction was significantly purer and comparable to that obtained by elution with isopropyl beta-D-thiogalactopyranoside, a lactose analog . Heparin elution represents an important new tool in the purification of transcription factors and other DNA-binding proteins by DNA affinity chromatography. J Biomol NMR, 1999 Jun, 14(2), 149 - 55 Refinement of the structure of protein-RNA complexes by residual dipolar coupling analysis; Bayer P et al.; The main limitation in NMR-determined structures of nucleic acids and their complexes with proteins derives from the elongated, non-globular nature of physiologically important DNA and RNA molecules . Since it is generally not possible to obtain long-range distance constraints between distinct regions of the structure, long-range properties such as bending or kinking at sites of protein recognition cannot be determined accurately nor precisely . Here we show that use of residual dipolar couplings in the refinement of the structure of a protein-RNA complex improves the definition of the long-range properties of the RNA . These features are often an important aspect of molecular recognition and biological function; therefore, their improved definition is of significant value in RNA structural biology. J Biomol NMR, 1999 Jun, 14(2), 105 - 14 Improved segmental isotope labeling of proteins and application to a larger protein; Otomo T et al.; A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein {Yamazaki et al . (1998) J . Am . Chem . Soc., 120, 5591-5592} . This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain . However, there is a problem with the yield of the segmentally labeled protein . In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13C/15N-labeled protein samples . This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation . The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased . Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins . The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids . All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples. FEMS Microbiol Lett, 1999 Jul 15, 176(2), 489 - 93 Regulation of copy number and stability of phage lambda derived pTC lambda 1 plasmid in the light of the dimer/multimer catastrophe hypothesis; Herman-Antosiewicz A et al.; The dimer catastrophe hypothesis has been proposed previously to explain instability of multicopy plasmids whose partitioning is random, contrary to low copy number plasmids which are stably maintained and actively partitioned . Until now, this hypothesis has been investigated using multicopy ColE1 plasmids . However, for more detailed testing of the dimer/multimer catastrophe hypothesis, one should use a plasmid which can be maintained at either low or high copy number and still possesses the same mechanism of replication regulation . Here we used a modified lambda plasmid, pTC lambda 1 . The advantage of this plasmid is that it can be maintained at different copy numbers depending on the concentration of an inducer which stimulates the initiation of plasmid replication . Results obtained with this plasmid in recombination proficient and deficient cells generally support the dimer/multimer catastrophe hypothesis, but also suggest some modification in the model. FEMS Microbiol Lett, 1999 Jul 15, 176(2), 443 - 8 The role of a conserved histidine residue, His324, in Trigonopsis variabilis D-amino acid oxidase; Lin LL et al.; To investigate the functional role of an invariant histidine residue in Trigonopsis variabilis D-amino acid oxidase (DAAO), a set of mutant enzymes with replacement of the histidine residue at position 324 was constructed and their enzymatic properties were examined . Wild-type and mutant enzymes have been purified to homogeneity using the His-bound column and the molecular masses were determined to be 39.2 kDa . Western blot analysis revealed that the in vivo synthesized mutant enzymes are immuno-identical with that of the wild-type DAAO . The His324Asn and His324Gln mutants displayed comparable enzymatic activity to that of the wild-type enzyme, while the other mutant DAAOs showed markedly decreased or no detectable activity . The mutants, His324/Asn/Gln/Ala/Tyr/Glu, exhibited 38-181% increase in Km and a 2-10-fold reduction in kcat/Km . Based on the crystal structure of a homologous protein, pig kidney DAAO, it is suggested that His324 might play a structural role for proper catalytic function of T . variabilis DAAO. FEMS Microbiol Lett, 1999 Jul 15, 176(2), 395 - 401 OmpF changes and the complexity of Escherichia coli adaptation to prolonged lactose limitation; Zhang E et al.; The disaccharide lactose has no specific diffusion pathway across the outer membrane of Escherichia coli . At least three classes of spontaneous mutation affecting outer membrane permeability arose with each of three independent E . coli populations adapting to prolonged lactose limitation in chemostats . Both structural and regulatory mutations affecting OmpF porin predominated in isolates after 210-280 generations of culture . Six types of ompF mutation were found, including in-frame deletions and substitutions at Arg82 and Asp113, all affecting the channel constriction residues of OmpF . Isolates had increased susceptibility to antibiotics and were affected in the quantity of OmpF, LamB and OmpA proteins . A minimum of three or four mutations was evident in all isolates after 280 generations in a lactose-limited environment, in addition to lac mutations defined in previous studies. Biosci Biotechnol Biochem, 1999 Jun, 63(6), 1051 - 5 Cloning and expression in Escherichia coli of a gene coding for a secondary alcohol dehydrogenase from Candida parapsilosis; Yamamoto H et al.; A gene encoding a stereo-specific secondary alcohol dehydrogenase (CpSADH) that catalyzed the oxidation of (S)-1,3-BDO to 4-hydroxy-2-butanone was cloned from Candida parapsilosis . This CpSADH-gene consisted of 1,009 nucleotides coding for a protein with M(r) 35,964 . A recombinant Escherichia coli JM109 strain harboring the expression plasmid, pKK-CPA1, produced (R)-1,3-BDO (93.5% ee., 94.7% yield) from the racemate without any additive to regenerate NAD+ from NADH. Biosci Biotechnol Biochem, 1999 Jun, 63(6), 1045 - 50 Role of carbohydrate moiety in carboxypeptidase Y: structural study of mutant enzyme lacking carbohydrate moiety; Shimizu H et al.; To study the roles of the carbohydrate moiety in the function of carboxypeptidase Y, asparagine residues at 13, 87, 168, and 368, the four-consensus N-linked glycosylation sites, were altered to alanine with site-directed mutagenesis . The mutant enzyme of 51 kDa completely lost the carbohydrate moiety which was present in the 61-kDa wild-type enzyme . Structural studies of the mutant enzyme showed that it maintained the native-like structure; hydrolytic activity, and substrate specificity of the mutant enzyme analogous to those of the wild-type enzyme . Susceptibility of the mutant enzyme toward proteolysis and pressure denaturation was reduced by 10-20% . It is concluded that the carbohydrate moiety functions to maintain the structural integrity of the enzyme under stressed. Growth Factors, 1999, 16(4), 293 - 303 Characterization of fibroblast growth factor-6 expressed by Chinese hamster ovary cells as a glycosylated mitogen for human vascular endothelial cells; Asada M et al.; The gene for fibroblast growth factor (FGF)-6/hst-2 was originally identified by its close homology with the FGF-4/hst-1 gene . Aside from its ability to transform cultured fibroblasts, the characteristics of FGF-6 protein have only been studied using a simple preparation from E . coli . In the present study, we expressed FGF-6 cDNA in CHO cells and characterized the resultant protein . We found that CHO cells secreted several forms of the FGF-6 polypeptide, and that there were multiple N-terminal modifications . The longest form (18-kDa) contained the sequence, SerProAlaGlyAlaArg, as its N-terminus, which was consistent with the signal peptide cleavage site predicted from its primary structure . The core polypeptide was primarily modified by heterogeneous N-glycans that were sialylated to a small degree; among them, biantennary structures were found to predominate . Moreover, possible O-glycosylation was also detected . N-glycosylated FGF-6 potently induced DNA synthesis and proliferation of human vascular endothelial cells, whereas in the absence of N-glycosylation, FGF-6 mitogenicity was substantially diminished . The results clearly indicate that FGF-6 expressed by mammalian cells is a glycosylated mitogen for vascular endothelial cells and further suggests that N-glycosylation plays a key role in determining the mitogenicity of FGF-6. Appl Environ Microbiol, 1999 Aug, 65(8), 3304 - 11 Nanomolar levels of dimethylsulfoniopropionate, dimethylsulfonioacetate, and glycine betaine are sufficient to confer osmoprotection to Escherichia coli; Cosquer A et al.; We combined the use of low inoculation titers (300 +/- 100 CFU/ml) and enumeration of culturable cells to measure the osmoprotective potentialities of dimethylsulfoniopropionate (DMSP), dimethylsulfonioacetate (DMSA), and glycine betaine (GB) for salt-stressed cultures of Escherichia coli . Dilute bacterial cultures were grown with osmoprotectant concentrations that encompassed the nanomolar levels of GB and DMSP found in nature and the millimolar levels of osmoprotectants used in standard laboratory osmoprotection bioassays . Nanomolar concentrations of DMSA, DMSP, and GB were sufficient to enhance the salinity tolerance of E . coli cells expressing only the ProU high-affinity general osmoporter . In contrast, nanomolar levels of osmoprotectants were ineffective with a mutant strain (GM50) that expressed only the low-affinity ProP osmoporter . Transport studies showed that DMSA and DMSP, like GB, were taken up via both ProU and ProP . Moreover, ProU displayed higher affinities for the three osmoprotectants than ProP displayed, and ProP, like ProU, displayed much higher affinities for GB and DMSA than for DMSP . Interestingly, ProP did not operate at substrate concentrations of 200 nM or less, whereas ProU operated at concentrations ranging from 1 nM to millimolar levels . Consequently, proU(+) strains of E . coli, but not the proP(+) strain GM50, could also scavenge nanomolar levels of GB, DMSA, and DMSP from oligotrophic seawater . The physiological and ecological implications of these observations are discussed. Science, 1999 Jul 30, 285(5428), 751 - 3 Detecting protein function and protein-protein interactions from genome sequences; Marcotte EM et al.; A computational method is proposed for inferring protein interactions from genome sequences on the basis of the observation that some pairs of interacting proteins have homologs in another organism fused into a single protein chain . Searching sequences from many genomes revealed 6809 such putative protein-protein interactions in Escherichia coli and 45,502 in yeast . Many members of these pairs were confirmed as functionally related; computational filtering further enriches for interactions . Some proteins have links to several other proteins; these coupled links appear to represent functional interactions such as complexes or pathways . Experimentally confirmed interacting pairs are documented in a Database of Interacting Proteins. Nat Struct Biol, 1999 Aug, 6(8), 729 - 34 Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ; Tomomori C et al.; Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system . Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions . Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions . The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase. Protein Expr Purif, 1999 Aug, 16(3), 417 - 23 Preparation and characterization of recombinant dolphin fish (Coryphaena hippurus) growth hormone; Paduel A et al.; Dolphin fish (Coryphaena hippurus) growth hormone (dfGH) cDNA encoding the mature protein was cloned in a pET11a expression vector and expressed in Escherichia coli BL21 cells upon induction with isopropyl-1-thio-beta-d-galactopyranoside as an insoluble protein . The expressed protein, contained within the inclusion-body pellet, was solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine, and purified to homogeneity, as evidenced by SDS-PAGE . Gel filtration on a Superdex column under nondenaturing conditions and amino-terminal analysis showed the purified protein to be monomeric methionyl-dfGH . Binding assays of the (125)I-labeled dfGH to dolphin fish liver microsomal fraction resulted in high specific binding characterized by a K(a) of 0.77 nM(-1) and a B(max) of 285 fmol/mg microsomal fraction protein . The purified dfGH was capable of stimulating proliferation of FDC-P1-B9 cells transfected with rabbit growth hormone (GH) receptor . The maximal effect of dfGH was identical to that of human GH but their respective EC(50) values were 28 nM versus 0.095 nM . Carcinogenesis, 1999 Aug, 20(8), 1417 - 24 Inefficient in vivo repair of mismatches at an oncogenic hotspot correlated with lack of binding by mismatch repair proteins and with phase of the cell cycle; Matton N et al.; Repair rates of mismatched nucleotides located at an activating hotspot of mutation, H-ras codon 12, have been analyzed in vivo in mammalian cells . Repair rates at codon 12 are significantly improved in cells synchronized to the G(1) stage of the mammalian cell cycle as compared with non-synchronous cells, demonstrating that mismatch repair mechanisms are active in G(1) . Repair rates in non-synchronous cells for the same mismatches at a nearby non-hotspot of mutation, H-ras codon 10, are also significantly improved over repair rates at codon 12 in non-synchronous cells, demonstrating that DNA mismatch repair rates can differ depending on the sequence context . These results suggest that inefficiencies in mismatch repair are responsible, at least in part, for the well documented hotspot of mutation at codon 12 . Further experiments involving gel-shift analysis demonstrate a mismatch-specific binding factor for which the degree of binding correlates with in vivo repair rates for each mismatch tested at the codon 12 location . This binding factor appears to be the hMutSalpha heterodimer as identified by monoclonal antibody assay and inhibition of binding by ATP . Furthermore, a lack of binding is observed only for G:A mismatches at the codon 12 location . This lack of binding correlates with the low rate of repair observed in vivo for G:A mismatches at codon 12 versus the improved repair rates for G:A mismatches at codon 10 . This may have biological relevance in that G:C-->T:A tranversions are a common mutation at this location in naturally occurring human tumors . These results suggest that there is lowered efficiency in the kinetics of mismatch repair at codon 12 . Mismatches at this location are therefore more likely to be replicated before repair, thus resulting in a mutation. Diabetes, 1999 Aug, 48(8), 1645 - 51 Characterization of glucokinase mutations associated with maturity-onset diabetes of the young type 2 (MODY-2): different glucokinase defects lead to a common phenotype; Miller SP et al.; Glucokinase (GK) is expressed in the pancreatic beta-cells and liver, and plays a key role in the regulation of glucose homeostasis . The enzymatic activity and thermal stability of wild-type (WT) GK and several mutant forms associated with maturity-onset diabetes of the young type 2 (MODY-2) were determined by a steady-state kinetic analysis of the purified expressed proteins . The eight MODY-2 mutations studied were Ala53Ser, Val367Met, Gly80Ala, Thr168Pro, Arg36Trp, Thr209Met, Cys213Arg, and Val226Met . These missense mutations were shown to have variable effects on GK kinetic activity . The Gly80Ala and Thr168Pro mutations resulted in a large decrease in Vmax and a complete loss of the cooperative behavior associated with glucose binding . In addition, the Gly80Ala mutation resulted in a sixfold increase in the half-saturating substrate concentration (S0.5) for ATP, and Thr168Pro resulted in eight- and sixfold increases in the S0.5 values for ATP and glucose, respectively . The Thr209Met and Val226Met mutations exhibited three- and fivefold increases, respectively, in the S0.5 for ATP, whereas the Cys213Arg mutation resulted in a fivefold increase in the S0.5 for glucose . These mutations also led to a small yet significant reduction in Vmax . Of all the mutations studied, only the Cys213Arg mutation had reduced enzymatic activity and decreased thermal stability . Two mutants, Ala53Ser and Val367Met, showed kinetic and thermal stability properties similar to those of WT . These mutants had increased sensitivities to the known negative effectors of GK activity, palmitoyl-CoA, and GK regulatory protein . Taken together, these results illustrate that the MODY-2 phenotype may be linked not only to kinetic alterations but also to the regulation of GK activity. Structure Fold Des, 1999 Jul 15, 7(7), 733 - 44 Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase; Leppanen VM et al.; BACKGROUND: Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by converting pyruvate and CoA to formate and acetylCoA . The PFL mechanism involves an unusual radical cleavage of pyruvate, involving an essential C alpha radical of Gly734 and two cysteine residues, Cys418 and Cys419, which may form thiyl radicals required for catalysis . We undertook this study to understand the structural basis for catalysis . RESULTS: The first structure of a fragment of PFL (residues 1-624) at 2.8 A resolution shows an unusual barrel-like structure, with a catalytic beta finger carrying Cys418 and Cys419 inserted into the centre of the barrel . Several residues near the active-site cysteines can be ascribed roles in the catalytic mechanism: Arg176 and Arg435 are positioned near Cys419 and may bind pyruvate/formate and Trp333 partially buries Cys418 . Both cysteine residues are accessible to each other owing to their cis relationship at the tip of the beta finger . Finally, two clefts that may serve as binding sites for CoA and pyruvate have been identified . CONCLUSIONS: PFL has striking structural homology to the aerobic ribonucleotide reductase (RNR): the superposition of PFL and RNR includes eight of the ten strands in the unusual RNR alpha/beta barrel as well as the beta finger, which carries key catalytic residues in both enzymes . This provides the first structural proof that RNRs and PFLs are related by divergent evolution from a common ancestor. Mol Divers, 1998-99, 4(2), 75 - 89 The limits of specificity: an experimental analysis with RNA aptamers to MS2 coat protein variants; Hirao I et al.; It has been hypothesized that selections for aptamers with high affinity for a given target molecule will of necessity identify aptamers that have high specificity for that target . We have attempted to assess this hypothesis by selecting aptamers that can bind to MS2 coat protein or to single- or double-substitution variants of the coat protein . Some aptamers selected to bind MS2 coat protein or its variants were mildly specific for their cognate targets, discriminating by two- to fourfold against closely related proteins . Specificity determinants on both the coat proteins and the aptamers could be identified . However, many aptamers could readily bind to each of the different coat proteins . The identification of such aptamer 'generalists' belies the proposed relationship between the affinities and specificities of selected RNA ligands . These results imply that, while aptamers may not finely discriminate between closely related targets, neither will their binding be negated by mutations in targets . Aptamer pharmaceuticals may therefore better resist the evolution of resistance. Mutat Res, 1999 Jul, 437(1), 51 - 60 Mismatch repair is diminished during stationary-phase mutation; Harris RS et al.; This paper is an invited Response to a recent Commentary {P.L . Foster, Rev . Mut . Res . 436 (1999) 179-184} entitled "Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking" . The Commentary argues that no evidence exists supporting the idea that mismatch repair is limiting specifically during stationary-phase mutation . A primary concern of the author is to question the method that we used previously to measure growth-dependent mutation . In this method, mutation rates are calculated using counts of mutant colonies taken at times when those colonies arise, rather than at a predetermined, fixed time . Here we show further data that illustrate why this must be done to ensure accurate mutation measurements . Such accuracy was necessary for our published determination that mismatch repair proteins are not limiting during growth-dependent mutation, but become so during stationary-phase mutation . We review the evidence supporting the idea that stationary-phase reversion of a lac frameshift mutation occurs in an environment of decreased mismatch repair capacity . Those data are substantial . The data presented in the Commentary, in apparent contradiction to this idea, do not justify the conclusion presented there . Mutat Res, 1999 Jul, 437(1), 11 - 20 Principal causes of hot spots for cytosine to thymine mutations at sites of cytosine methylation in growing cells . A model, its experimental support and implications; Lutsenko E et al.; In Escherichia coli and human cells, many sites of cytosine methylation in DNA are hot spots for C to T mutations . It is generally believed that T.G mismatches created by the hydrolytic deamination of 5-methylcytosines (5meC) are intermediates in the mutagenic pathway . A number of hypotheses have been proposed regarding the source of the mispaired thymine and how the cells deal with the mispairs . We have constructed a genetic reversion assay that utilizes a gene on a mini-F to compare the frequency of occurrence of C to T mutations in different genetic backgrounds in exponentially growing E . coli . The results identify at least two causes for the hot spot at a 5meC: (1) the higher rate of deamination of 5meC compared to C generates more T.G than uracil.G (U.G) mismatches, and (2) inefficient repair of T.G mismatches by the very short-patch (VSP) repair system compared to the repair of U . G mismatches by the uracil-DNA glycosylase (Ung) . This combination of increased DNA damage when the cytosines are methylated coupled with the relative inefficiency in the post-replicative repair of T.G mismatches can be quantitatively modeled to explain the occurrence of the hot spot at 5meC . This model has implications for mutational hot and cold spots in all organisms . Protein Expr Purif, 1999 Aug, 16(3), 454 - 62 Expression of a zinc-binding domain of boar spermatidal transition protein 2 in Escherichia coli; Sato H et al.; Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal 34 region . Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner . However, there was some nonspecific recognition of the oligonucleotide . Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1-103) in Escherichia coli . Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d . TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol . TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO(-)(3) and HPLC on Nucleosil 300 7C18 and on Diol-120 . Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2 . Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc . Protein Expr Purif, 1999 Aug, 16(3), 440 - 7 An uniquely purified HCV NS3 protease and NS4A(21-34) peptide form a highly active serine protease complex in peptide hydrolysis; Sardana VV et al.; The N-terminal domain of the hepatitis C virus (HCV) polyprotein containing the NS3 protease (residues 1027 to 1206) was expressed in Escherichia coli as a soluble protein under the control of the T7 promoter . The enzyme has been purified to homogeneity with cation exchange (SP-Sepharose HR) and heparin affinity chromatography in the absence of any detergent . The purified enzyme preparation was soluble and remained stable in solution for several weeks at 4 degrees C . The proteolytic activity of the purified enzyme was examined, also in the absence of detergents, using a peptide mimicking the NS4A/4B cleavage site of the HCV polyprotein . Hydrolysis of this substrate at the expected Cys-Ala scissile bond was catalyzed by the recombinant protease with a pseudo second-order rate constant (k(cat)/K(M)) of 205 and 196,000 M(-1) s(-1), respectively, in the absence and presence of a central hydrophobic region (sequence represented by residues 21 to 34) of the NS4A protein . The rate constant in the presence of NS4A peptide cofactor was two orders of magnitude greater than reported previously for the NS3 protease domain . A significantly higher activity of the NS3 protease-NS4A cofactor complex was also observed with a substrate mimicking the NS4B/5A site (k(cat)/K(M) of 5180 +/- 670 M(-1) s(-1)) . Finally, the optimal formation of a complex between the NS3 protease domain and the cofactor NS4A was critical for the high proteolytic activity observed . Protein Expr Purif, 1999 Aug, 16(3), 432 - 9 Overexpression in Escherichia coli and characterization of the chloroplast triosephosphate isomerase from spinach; Tang GL et al.; An important Calvin cycle enzyme, chloroplast triosephosphate isomerase (cpTPI) from spinach, has been cloned and expressed in up to 15% of the total cell protein using the P(L) expression vector in Escherichia coli . An even higher level expression, up to 36% of the total protein, was achieved by replacing the nucleotide sequence between the ribosomal binding site and the initial codon, ATG, with an AT-rich sequence . Computer modeling revealed that the moderate change in the standard free energy (5'-DeltaG degrees ) of mRNA secondary structure in the translation initial region might be the major factor which led to the later high-level expression . The overexpressed spinach cpTPI was soluble and fully active and was able to be purified beyond 95% purity by DEAE-Sepharose and Sephadex G-75, and around 55 mg of purified enzymes was obtained from 1 liter of cultured bacteria . With d-glyceraldehyde 3-phosphate as substrate, K(m (D-3-P)) is 0 . 68 mM, V(max (G-3-P)) is 3.16 x 10(4) micromol/min . mg, and K(cat (G-3-P)) is 4.51 x 10(3)/s; with dihydroxyacetone phosphate as substrate, the corresponding values are 7.27 mM, 1.04 x 10(3) micromol/min . mg, and 1.16 x 10(2)/s, respectively . J Med Chem, 1999 Jul 29, 42(15), 2936 - 45 Synthesis and biological activity of (hydroxymethyl)- and (diethylaminomethyl)benzopsoralens; Chilin A et al.; Some benzopsoralens, carrying a hydroxymethyl or a diethylaminomethyl group at the 3, 5, 8, and 11 positions, were prepared, and their biological activity was compared with that of 4-(hydroxymethyl)benzopsoralen (BP) . 5-(Hydroxymethyl)benzopsoralen (7b), 11-(hydroxymethyl)benzopsoralen (7c), and 11-(diethylaminomethyl)benzopsoralen (8c) induced marked antiproliferative effects in mammalian cells by simple incubation in the dark; this activity appeared to be related to their ability to inhibit topoisomerase II . Benzopsoralens appeared to be more active, especially BP and 7c, upon UVA activation . Compounds carrying a methyl group at the 4 position together with a hydroxymethyl or diethylaminomethyl at the 8 position (7d and 8d, respectively) were also effective, although to a lower extent; instead, a substituent at the 3 position canceled all activity . Benzopsoralens did not induce interstrand cross-links in DNA in vitro, as seen in the induction of cytoplasmic <<petite>> mutations and double-strand breaks in yeast . This behavior is also compatible with their low mutagenic activity in E . coli WP2 and with the absence of any phototoxicity on the skin . For these features, benzopsoralens seem to be interesting potential drugs for PUVA photochemotherapy and photopheresis . The activity shown in the dark is not sufficient for their possible use as antitumor drugs, but it does offer a new model for the study of topoisomerase inhibitors. Electrophoresis, 1999 Jun, 20(7), 1469 - 75 Elimination of contaminant Escherichia coli chromosomal DNA from preparations of P1 artificial chromosome recombinants facilitates directed subcloning; Davidson H et al.; The subcloning of large inserts (>50 kbp) from P1-derived artificial chromosomes (PACs) was found to be hindered by the presence of contaminating Escherichia coli chromosomal fragments which, because of their smaller median size, are recovered preferentially as unwanted subclones . A significant fraction of contaminating DNA was seen to persist after conventional plasmid purification methods . We describe a rigorous protocol for eliminating the bulk of contamination that involves plasmid isolation on commercially available silica-based columns followed by three pulsed field gel electrophoresis steps . Using this, we were able to subclone 55, 85 and 90 kbp PAC inserts but failed to subclone a 195 kbp PAC insert . After surveying a range of DNA purification methods, we devised an optimised protocol that allowed us to subclone the 195 kbp insert . The optimised protocol, which reliably yields DNA with essentially no contaminating material, consists of plasmid isolation on silica-based columns followed by treatment with highly purified DNaseI and retrieval by electroelution of restriction-digested DNA electrophoresed on a single pulsed field gel . By inference it is applicable to the purification of large inserts from other single-copy plasmid vectors such as bacterial artificial chromosomes (BACs). Biochemistry (Mosc), 1999 Jul, 64(7), 832 - 8 Modeling of irreversible thermal protein denaturation at varying temperature . II . The complete kinetic model of Lumry and Eyring; Lyubarev AE et al.; The model for thermal denaturation of proteins involving consecutive reversible and irreversible steps (Lumry and Eyring model) has been analyzed . The most general case, when equilibrium in the first step is established slowly in comparison with the rate of the second step and the heat effect value for the second step is either greater than or less than zero, has been considered . The theoretical dependences of excess heat capacity on temperature have been constructed . The variation of the shape of the theoretical curves with varied values of the enthalpy change for the second step, Arrhenius equation parameters for both steps, and the scanning rate has been studied. J Physiol Pharmacol, 1999 Jun, 50(2), 287 - 97 Role of thromboxane A2 and platelet activating factor in early haemodynamic response to lipopolysaccharide in rats; Bochenski J et al.; The mechanism of early pulmonary and systemic haemodynamic response to intravenous infusion of LPS from Escherichia coli was investigated in anesthetised Wistar rats . 10 mg of LPS given at a rate of 4 mg/kg/min but not at a rate of 1 mg/kg/min induced an increase in pulmonary arterial pressure (PAP) and a fall in systemic arterial pressure (SAP) . Pretreatment with a PAF receptor antagonist; WEB 2170 (5 and 25 mg/kg) inhibited both PAP and SAP responses to LPS (4 mg/kg/min) while an inhibitor of thromboxane synthesis; Camonagrel (10 and 20 mg/kg) abolished PAP response without a major effect on SAP response to LPS . In conclusion, both PAF and TXA2 mediate LPS induced rise in pulmonary arterial pressure while LPS-induced fall in systemic arterial pressure is mediated by PAF. Urol Res, 1999 Apr, 27(2), 97 - 102 A novel surgical organ perfusion method for effective ex vivo and in vivo gene transfer into renal glomerular cells; Parpala-Sparman T et al.; In an attempt to develop gene therapy for Alport syndrome, we have evaluated surgical methods for gene transfer into pig kidneys . For gene transfer we used an adenovirus expressing the Escherichia coli beta-galactosidase gene as a reporter gene . The viral preparation was first infused in vivo into the porcine renal artery . Then explanted kidneys were perfused ex vivo at body temperature for 12 hours with the viral solution and, finally the kidney perfusions were carried out in vivo via laparotomy for 60 and 120 minutes . Gene transfer was determined visually on histological cryosections after 5-bromo-4-chloro-3-indoyl-beta-galactopyranoside (X-gal) and periodic acid-Schiff (PAS) staining . Perfusion of whole porcine kidneys ex vivo resulted in strong expression in about 80% of glomeruli . The in vivo kidney perfusion via laparotomy for 120 minutes resulted in reporter gene expression of about 75% of the glomeruli examined after 4 days . Expression was observed almost exclusively in glomeruli, while little if any expression was found in other renal structures . The present results suggest that operatively performed kidney perfusion may be used for gene transfer in treatment of glomerular disease . This surgical approach may also prove useful for somatic gene therapy of other organs. Acta Biotheor, 1998-99, 46(4), 379 - 81 Ideas in theoretical biology . Origin of cancerous cells from tumours; Niu DK et al.; With a previous paper (Niu & Wang, 1995), a general, hypothetical outline of the mechanism of carcinogenesis was proposed . With reference to the fact of starvation-induced hypermutation in micro-organisms, we propose that the hypoxia commonly seen in the cells at the centre of solid tumours might also result in hypermutation, and then p53-dependent programmed cell death . Like the apparently adaptive mutations in micro-organisms, only those genes (e.g . p53) that enable the cells to escape from apoptosis may be selected. J Biochem (Tokyo), 1999 Aug, 126(2), 445 - 8 The Escherichia coli pldC gene encoding lysophospholipase L(1) is identical to the apeA and tesA genes encoding protease I and thioesterase I, respectively; Karasawa K et al.; We deduced the amino acid sequence of Escherichia coli lysophospholipase L(1) by determining the nucleotide sequence of the pldC gene encoding this enzyme . The translated protein was found to contain 208 amino acid residues with a hydrophobic leader sequence of 26 amino acid residues . The molecular weight of the purified enzyme (20,500) was in good agreement with the predicted size (20,399) of the processed protein . A search involving a data bank showed that the nucleotide sequence of the pldC gene was identical to those of the apeA and tesA genes encoding protease I and thioesterase I, respectively . Consistent with the identity of the pldC gene with these two genes, the enzyme purified from E . coli overexpressing the pldC gene showed both protease I and thioesterase I activities. J Biochem (Tokyo), 1999 Aug, 126(2), 387 - 94 Determination of the solution structure of the N-domain plus linker of Antarctic eel pout antifreeze protein RD3; Miura K et al.; RD3, a new antifreeze protein (AFP) extracted from antarctic eel pout is a single polypeptide divided into homologous N-terminal (residues Asn(1)-Glu(64)) and C-terminal (residues Ser(74)-Glu(134)) domains, each of which has a high sequence identity with Type III AFP . A 9-residue linker (-D(65)GTTSPGLK(73)-) connects these two domains in tandem and is thought to play a significant role in defining the nature of the intact molecule . The present paper shows for the first time the solution structure and preliminary (15)N-NMR backbone dynamics data of the N-domain plus the linker of recombinant RD3 protein (RD3-Nl: residues 1-73) by employing homo- and heteronuclear multidimensional NMR spectroscopy . Forty converged structures of RD3-Nl were successfully calculated by using a total of 958 NMR-derived structural restraints . It was found that the N-domain of RD3-Nl has a globular form comprising six beta-strands, three type III turns, and several loops, which stabilize a flat, ice-binding site formed on one side of this domain . Further, the linker portion appears to have a definitive structure, which is independent of the globular N-domain . This definitive linker is roughly divided into two short strands, -D(65)GTTSP(70)- and -G(71)LK(73)-, which are bent around -T(67)TSPG(71)- at an angle of approximately 60 degrees . This bending motif of the linker may function to orient the two ice-binding sites of the N- and C-domains of RD3 in the same direction, leading to their simultaneous interactions with the ice crystal surface. J Biochem (Tokyo), 1999 Aug, 126(2), 354 - 60 Repression of the gene encoding succinate dehydrogenase in response to glucose is mediated by the EIICB(Glc) protein in Escherichia coli; Takeda S et al.; The Escherichia coli sdhCDAB operon encodes succinate dehydrogenase, an enzyme complex involved in the tricarboxylic acid (TCA) cycle . Expression of this operon is under complex transcriptional regulation in response to growth conditions, such as anaerobiosis and carbon sources . Typically, the expression of sdhCDAB is known to be subjected to "an aerobic repression" and "a glucose repression." The molecular mechanism underlying the anaerobic repression has been well documented, involving both the ArcB-ArcA two-component system and the Fnr global anaerobic regulator . However, the mechanism underlying the glucose repression is not yet clear, because the involvement of the general catabolite regulators such as CRP and CRA has been dismissed . In this study, we conducted a series of genetic analyses to identify the regulator gene(s) involved in the glucose repression of sdh . The results demonstrate that the EIICB(Glc) protein (the ptsG gene product), a component of the major glucose transporter, acts as a crucial mediator in glucose repression . These results support the view that the EIICB(Glc) protein functions not only as a glucose transporter, but also as a glucose-sensing signal transducer that modulates the glucose repression of the sdhCDAB operon. J Biochem (Tokyo), 1999 Aug, 126(2), 347 - 53 A new UV method for serum gamma-glutamyltransferase assay using recombinant 4-aminobenzoate hydroxylase as a coupling enzyme; Mizutani Y et al.; 4-aminobenzoate hydroxylase (4ABH) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate to 4-hydroxyaniline . For use as a clinical reagent, the gene encoding 4ABH from Agaricus bisporus was cloned by the RACE method . Also, the cDNA encoding 4ABH was expressed in Escherichia coli cells as a fusion protein with glutathione S-transferase (GST) . The expressed GST-4ABH fusion protein (recombinant 4ABH) in the soluble fraction exhibits decarboxylative hydroxylation and additional NADH oxidation activities.We investigated a new ultraviolet spectrometric method for determining serum gamma-glutamyltransferase (gamma-GT) using recombinant 4ABH as a coupling enzyme . The principle of the method is as follows . Using gamma-glutamyl-3-choloro-4-aminobenzoate (L-gamma-glu-PAClBA) and glycylglycine as the donor and acceptor substrates, 3-choloro-4-aminobenzoate (PAClBA) is formed by the catalysis of serum gamma-GT . PAClBA is stoichiometrically converted to 3-choloro-4-hydroxyaniline (PHClA) and NAD(+) by 4ABH and NADH . However, NADH oxidation results in a high reagent blank, which is considered as a drawback for use as a clinical reagent.Using recombinant 4ABH, we examined the effects of pH and detergents on these two activities, and found that several detergents suppress the additional NADH oxidation activity with little or no effect on hydroxylation activity . The results indicate a promising approach to establishing an ultraviolet spectrophotometric method for determining serum gamma-GT activity using L-gamma-glu-PAClBA as the donor substrate and recombinant 4ABH as a coupling enzyme. J Biochem (Tokyo), 1999 Aug, 126(2), 271 - 7 Sulfating-activity and stability of cDNA-expressed allozymes of human phenol sulfotransferase, ST1A3*1 ((213)Arg) and ST1A3*2 ((213)His), both of which exist in Japanese as well as Caucasians; Ozawa S et al.; We recently found single amino acid substitutions ((213)Arg/His and (223)Met/Val) in polymorphic human phenol-sulfating phenol sulfotransferase (SULT: cDNAs encoding ST1A3, P PST or HAST1/2) among Caucasians and African-Americans . In a Japanese population (n = 143), allele frequencies of (213)Arg and (213)His were 83.2 and 16 . 8%, respectively, but the (223)Val allele was not found . (213)His homozygosity was reportedly associated with both very low (>7-fold) sulfating activities of p-nitrophenol (at 4 microM) and low thermostability in platelets . Sulfating-activity determinations using recombinant (213)Arg- and (213)His-forms (ST1A3*1 and ST1A3*2, respectively) did not, however, reveal appreciable deficiency in {(35)S}3'-phosphoadenosine 5'-phosphosulfate (PAPS)-dependent sulfation of p-nitrophenol (4 microM) by ST1A3*2 (7.5 vs . 10.2 nmol/min/nmol SULT for ST1A3) . Kinetic parameters for p-nitrophenol for p-nitrophenol sulfation supported the slight decrease in sulfating activities at 4 microM (K(m), 0.82 vs . 1.75 microM; V(max), 13.2 vs . 13.1 nmol/min/nmol SULT, respectively, for ST1A3*1 and *2) . p-Nitrophenyl sulfate-dependent 2-naphthol sulfation by ST1A3*2 was 69% of that by ST1A3*1 (p<0.05) . However, ST1A3*2 was remarkably unstable at 45 and 37 degrees C as compared to ST1A3*1 . The lower p-nitrophenol sulfating activity of ST1A3*2 may explain the lower platelet p-nitrophenol sulfation in ST1A3*2 homozygotes . Protein instability and ST1A3 gene regulation may be both involved in the polymorphism of p-nitrophenol sulfation in human tissues. Biochemistry, 1999 Jul 27, 38(30), 9752 - 7 Purification and properties of the Streptomyces peucetius DpsC beta-ketoacyl:acyl carrier protein synthase III that specifies the propionate-starter unit for type II polyketide biosynthesis; Bao W et al.; Biosynthesis of the polyketide-derived carbon skeleton of daunorubicin (DNR) begins with propionate rather than acetate, which is the starter unit for most other aromatic polyketides . The dpsCgene has been implicated in specifying the unique propionate-starter unit, and it encodes a protein that is very similar to the Escherichia coli beta-ketoacyl:acyl carrier protein (ACP) synthase III (FabH or KS III) enzyme of fatty acid biosynthesis . Purified DpsC was found to use propionyl-coenzyme A as substrate and to be acylated by propionate at the Ser-118 residue . DpsC exhibits KS III activity in catalyzing the condensation of propionyl-CoA and malonyl-ACP, and also functions as an acyltransferase in the transfer of propionate to an ACP . The DpsC enzyme has a high-substrate specificity, utilizing only propionyl-CoA, and not malonyl-CoA, 2-methylmalonyl-CoA or acetyl-CoA, as the starter unit of DNR biosynthesis. Biochemistry, 1999 Jul 27, 38(30), 9617 - 25 Spectroscopic characterization of active mutants of manganese peroxidase: mutations on the proximal side affect calcium binding of the distal side; Banci L et al.; The mutants at position 242 of manganese peroxidase (MnP), where the native Asp has been substituted with a Ser or a Glu, have been shown to be active, and are here characterized by electronic, EPR, and NMR spectroscopies . We have also mutated another residue on the proximal side, Phe 190 to Val and Leu, yielding active mutants . When studied by the above-mentioned spectroscopies, the mutants at both positions 242 and 190 exhibit three pH-dependent transitions . In contrast to the transitions observed at low and high pH, the spectroscopic studies reveal that the transition at intermediate pH has pK(a) values up to 2 units lower for the mutants at D242E and -S and F190V than for the wild type . This process is due to the ionization of a group that affects the transition to the bis-histidine coordination at the iron . The observed changes in the pK(a) values are related to the altered affinity of the calcium-binding site in the distal pocket . Other variations are observed in the other two pK(a) values . Characterization of the cyanide derivatives indicates that the location and orientation of the distal and proximal His residues are essentially identical to that in the wild type . Our results indicate that mutations on the proximal side residues can affect changes in the distal side . In particular, deprotonation of a group, whose pK(a) is influenced by the nature of the residues in the proximal side, produces a movement of helix B, which in turn induces the coordination of the distal His and the loss of the distal calcium ion. Biochem Pharmacol, 1999 Jul 15, 58(2), 325 - 8 Characterization of an antineoplastic glucuronide prodrug; Cheng TL et al.; The specificity of tumor therapy may be improved by preferentially activating antineoplastic prodrugs at tumor cells pretargeted with antibody-enzyme conjugates . In this study, the conditions required for the efficient activation of p-hydroxyaniline mustard glucuronide (BHAMG) to p-hydroxyaniline mustard (pHAM) were investigated . pHAM induced cross-links in linearized double-stranded DNA at about 180-fold lower concentrations than BHAMG, indicating that the nucleophilicity of pHAM was decreased by the presence of a glucuronide group . The partition coefficient of BHAMG was about 1890 times lower than pHAM in an octanol-water two-phase system, suggesting that the reduced toxicity of BHAMG was due to both hindered diffusion across the lipid bilayer of cells and decreased reaction with nuclear DNA . BHAMG was significantly less toxic to BHK cells that expressed cytosolic Escherichia coli-derived beta-glucuronidase (betaG) compared with cells that were engineered to secrete betaG, demonstrating that extracellular localization of betaG was required for optimal activation of BHAMG . The extended retention of mAb RH1 on the surface of AS-30D cells was also consistent with extracellular activation of BHAMG . Taken together, our results indicate that the low toxicity of BHAMG was due to hindered cellular uptake and low alkylating activity . BHAMG must be enzymatically activated outside of tumor cells for maximum cytotoxicity, and non-internalizing antibodies are preferred for human tumor therapy by targeted antibody-enzyme activation of BHAMG. J Gen Virol, 1999 Jul, 80 ( Pt 7), 1777 - 88 Expression, assembly competence and antigenic properties of hepatitis B virus core gene deletion variants from infected liver cells; Preikschat P et al.; Previous studies have shown that the progression of hepatitis B virus-related liver disease in long-term immunosuppressed kidney transplant recipients is associated with the accumulation of virus variants carrying in-frame deletions in the central part of the core gene . A set of naturally occurring core protein variants was expressed in Escherichia coli in order to investigate their stability and assembly competence and to characterize their antigenic and immunogenic properties . In addition, a library of core gene variants generated in vitro with deletions including the major immunodominant region (MIR) of the core protein was investigated . The position and length of deletions determined the behaviour of mutant core proteins in E . coli and their assignment to one of the three groups: (i) assembly-competent, (ii) stable but assembly-incompetent and (iii) unstable proteins . In vivo core variants with MIR deletions between amino acids 77 and 93 belong to the first group . Only proteins with the shortest deletion (amino acids 86-93) showed stability and self-assembly at the same level as wild-type cores, and they showed reduced antigenicity and immunogenicity . Mutants with deletions extending N-terminally beyond residue G73 or C-terminally beyond G94 were found to be assembly-incompetent . We suggest that G73 and G94 are involved in the folding and the native assembly of core molecules, whereas the intervening sequence determines the antibody response . Depending on their ability to form stable proteins or to assemble into particles, core mutants could contribute to liver cell pathogenesis in different ways. Protein Sci, 1999 Jul, 8(7), 1530 - 5 Effects of tryptophan to phenylalanine substitutions on the structure, stability, and enzyme activity of the IIAB(Man) subunit of the mannose transporter of Escherichia coli; Markovic-Housley Z et al.; The hydrophilic subunit of the mannose transporter (IIAB(Man)) of Escherichia coli is a homodimer that contains four tryptophans per monomer, three in the N-terminal domain (Trp12, Trp33, and Trp69) and one in the C-terminal domain (Trp182) . Single and double Trp-Phe mutants of IIABMan and of the IIA domain were produced . Fluorescence emission studies revealed that Trp33 and Trp12 are the major fluorescence emitters, Trp69 is strongly quenched in the native protein and Trp182 strongly blue shifted, indicative of a hydrophobic environment . Stabilities of the Trp mutants of dimeric IIA(Man) and IIAB(Man) were estimated from midpoints of the GdmHCl-induced unfolding transitions and from the amount of dimers that resisted dissociation by SDS (sodium dodecyl sulfate), respectively . W12F exhibited increased stability, but only 6% of the wild-type phosphotransferase activity, whereas W33F was marginally and W69F significantly destabilized, but fully active . Second site mutations W33F and W69F in the background of the W12F mutation reduced protein stability and suppressed the functional defect of W12F . These results suggest that flexibility is required for the adjustments of protein-protein contacts necessary for the phosphoryltransfer between the phosphorylcarrier protein HPr, IIA(Man), IIB(Man), and the incoming mannose bound to the transmembrane IIC(Man)-IID(Man) complex. Protein Sci, 1999 Jul, 8(7), 1475 - 83 A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase; Armstrong N et al.; Production of folded and biologically active protein from Escherichia coli derived inclusion bodies can only be accomplished if a scheme exists for in vitro naturation . Motivated by the need for a rapid and statistically meaningful method of determining and evaluating protein folding conditions, we have designed a new fractional factorial protein folding screen . The screen includes 12 factors shown by previous experiments to enhance protein folding and it incorporates the 12 factors into 16 different folding conditions . By examining a 1/256th fraction of the full factorial, multiple folding conditions were determined for the ligand binding domains from glutamate and kainate receptors, and for lysozyme and carbonic anhydrase B . The impact of each factor on the formation of biologically active material was estimated by calculating factor main effects . Factors and corresponding levels such as pH (8.5) and L-arginine (0.5 M) consistently had a positive effect on protein folding, whereas detergent (0.3 mM lauryl maltoside) and nonpolar additive (0.4 M sucrose) were detrimental to the folding of these four proteins . One of the 16 conditions yielded the most folded material for three out of the four proteins . Our results suggest that this protein folding screen will be generally useful in determining whether other proteins will fold in vitro and, if so, what factors are important . Furthermore, fractional factorial folding screens are well suited to the evaluation of previously untested factors on protein folding. Bioorg Khim, 1999 Apr, 25(4), 270 - 4 {Heterologous expression of murine lymphotoxins in Escherichia coli and preparation of antibodies}; Korobko VG et al.; Genes encoding fragments of polypeptide chains of murine lymphotoxins (LT), namely, LT-alpha truncated from the N-terminus and the LT-beta extracellular domain, containing N-terminal hepta- and hexahistidine epitopes, respectively, were expressed in E . coli cells . The recombinant proteins purified by metallochelate chromatography were used to obtain polyclonal antibodies that specifically recognize murine LT. Hokkaido Igaku Zasshi, 1999 May, 74(3), 173 - 88 {Analysis of transcript mutations due to transcriptional slippage in rat p53 tumor suppressor gene with the use of yeast functional assay}; Ba Y; Transcriptional slippage was previously found in Escherichia coli during RNA elongation at runs of 10 or more As or Ts, resulting in the addition of untemplated A or U residues . To evaluate the incidence of transcriptional slippage in vivo, we employed a yeast functional assay, and analyzed the frequency and spectrum of mutations in mRNA of the tumor suppressor p53 in rat tissues . In this assay, yeast are transfected with p53 PCR products and a gapped p53 expression vector, which allow homologous recombination in vivo and yield a percentage of red colonies which reflects the proportion of mutant PCR products . Insertion mutations of single base of adenine (A) at stretches of 6 As were frequently detected in the liver samples of LEC rats which develop spontaneous hepatitis and hepatocellular carcinoma . For excluding the possibility of artifacts involvement, p53 cDNA was amplified by PCR from plasmids containing wild-type p53 and tested with the yeast functional assay, which resulted in no A insertion after sequencing 23 mutant clones . Furthermore, in vitro transcript of wild-type p53 was synthesized by SP6 RNA polymerase, and then, reverse-transcribed, PCR-amplified, and tested with the yeast functional assay . The overall rate of A insertion was much lower than that in the LEC rat liver . Since A insertions were found predominantly at nucleotides 293-298 in exon 4, an exon 4-specific yeast functional assay was developed . A insertion was detected in 4.8% of the PCR product of mRNA but 0-0.1% from genomic DNA, which suggested that such A insertion was caused by transcriptional slippage in vivo . The A insertion rate abruptly increased in acute hepatitis stage in the LEC rat liver, while the rate slowly increased by aging in control WKAH rat liver . It was suggested that cell damage and aging were primarily responsible for the increased rate of transcriptional slippage. Biosens Bioelectron, 1999 Apr 30, 14(4), 397 - 404 Application of peptide nucleic acid to the direct detection of deoxyribonucleic acid amplified by polymerase chain reaction; Sawata S et al.; Double-stranded DNA amplified by polymerase chain reaction (PCR) was detected by peptide nucleic acid (PNA) using a BIAcore 2000 biosensor based on surface plasmon resonance (SPR) . PNA is an artificial oligo amide that is capable of forming highly stable complexes with complementary oligonucleotides . We succeeded in the direct detection of double-stranded DNA, amplified by PCR with high-sequence specificity . It was shown that the target DNA was available for detection over the range of 40-160 nM . Therefore, the detection limit was 7.5 pmol of the target DNA (143 bases, applied volume 30 microliters) . Our DNA detection system, the combination of BIAcore and the probe PNA, could detect the target DNA with good reproducibility . In this report, we show that our system is a powerful tool for the diagnosis of pathologically significant DNA. Nutrition, 1999 Jul-Aug, 15(7-8), 540 - 5 In vitro influence of parenteral lipid emulsions on the respiratory burst of neutrophils; Heine J et al.; The in vitro effect of a fish oil-derived lipid emulsion (omega-3) on the superoxide anion production during the respiratory burst (RB) of human neutrophils was compared to a LCT lipid (Intralipid), and an LCT/MCT emulsion (Lipofundin MCT) . The effects of two concentrations (60 and 600 micrograms/mL) were evaluated by rhodamine in a flow cytometer . The RB was induced either by stimulation with Escherichia coli (E . coli) or by priming with TNF-alpha and FMLP stimulation . The results (mean +/- SD%, P < 0.05) were compared to positive control responses (RB without lipids) . omega-3 (60 micrograms/mL, -8.2 {9.3}%; 600 micrograms/mL, -9.6 {11.1}%) and LCT (600 micrograms/mL, -8.0 {9.3}%) significantly suppressed the RB after stimulation with E . coli . LCT/MCT increased the RB after E . coli (60 micrograms/mL, 15.7 {15.4}%; 600 micrograms/mL, 42.7 {21.4}%) as well as after TNF-alpha/FMLP stimulation (600 micrograms/mL, 27.4 {23.7}%) . The in vitro influence of parenteral lipid emulsions on the superoxide anion production of human neutrophils is dependent on the length of the fatty acid molecule. Rapid Commun Mass Spectrom, 1999, 13(15), 1586 - 94 Extracting and visualizing matrix-assisted laser desorption/ionization time-of-flight mass spectral fingerprints; Jarman KH et al.; We have developed a method for constructing and extracting matrix-assisted laser desorption/ionization (MALDI) fingerprints . This method is fully automated and statistically based, allowing a large number of spectra to be analyzed at a time in an objective manner . This method can be used to extract the fingerprint of a particular analyte from a spectrum containing multiple analytes . Therefore, this method lends itself well to real-world applications where samples to be analyzed are likely to be impure . We illustrate this method on experimental results from a series of studies of E . coli and B . atrophaeus MALDI time-of-flight mass spectrometry (TOFMS) fingerprints . Chem Biol, 1999 Aug, 6(8), 577 - 84 Polyketide synthase acyl carrier protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis; Zhou P et al.; BACKGROUND: Using an acyl-acyl carrier protein (ACP) as a starter unit, type II polyketide synthases (PKSs) generate a wide range of polyketide products by successive decarboxylative condensations with the two-carbon donor malonyl (ACP) . In vitro experiments have demonstrated that polyketide biosynthesis in reconstituted PKS systems requires the fatty acid synthase (FAS) enzyme malonyl CoA:ACP acyltransferase (FabD) from streptomycetes . It has also been shown that holo-ACPs from a type II PKS can catalyze self-malonylation in the presence of malonyl CoA and negate this FabD requirement . The relative roles of FabD and ACP self-malonylation in PKS biosynthesis in vivo are still not known . RESULTS: We have examined the ACP specificity of the Streptomyces glaucescens FabD and shown that it reacts specifically with monomeric forms of ACP, with comparable k(cat)/K(M) values for ACPs from both type II PKS and FAS systems . Incubations of tetracenomycin ACP (TcmM) with the Escherichia coli FAS ACP (AcpP) unexpectedly revealed that, in addition to the self-malonylation process, TcmM can catalyze the malonylation of AcpP . The k(cat)/K(M) value for the TcmM-catalyzed malonylation of S . glaucescens FAS ACP is two orders of magnitude smaller than that observed for the FabD-catalyzed process . CONCLUSIONS: The ability of a PKS ACP to catalyze malonylation of a FAS ACP is a surprising finding and demonstrates for the first time that PKS ACPs and FabD can catalyze the same reaction . The differences in the catalytic efficiency of these two proteins rationalizes in vitro observations that FabD-independent polyketide biosynthesis proceeds only at high concentrations of a PKS ACP. Chem Biol, 1999 Aug, 6(8), 507 - 17 Identification and characterization of a type II peptidyl carrier protein from the bleomycin producer Streptomyces verticillus ATCC 15003; Du L et al.; BACKGROUND: Nonribosomal peptide synthetases (NRPSs) catalyze the assembly of a structurally diverse group of peptides by the multiple-carrier thiotemplate mechanism . All NRPSs known to date are exclusively type I modular enzymes that consist of domains, such as adenylation (A), peptidyl carrier protein (PCP) and condensation (C) domains, for individual enzyme activities . Although several A and PCP domains have been demonstrated to function independently, aminoacylation in trans has been successful only between PCPs and their cognate A domains . RESULTS: We have identified within the bleomycin-biosynthesis gene cluster from Streptomyces verticillus ATCC15003 the blmI gene that encodes a discrete PCP protein . We overexpressed the blmI gene in Escherichia coli, purified the BlmI protein, and demonstrated that apo-BlmI can be efficiently modified into holo-BlmI either in vivo or in vitro by PCP-specific 4'-phosphopantetheine transferases (PPTases) . Unlike the PCP domains in type I NRPSs, BlmI lacks its cognate A domain and can be aminoacylated by Val-A, an A domain from a completely unrelated type I NRPS . CONCLUSIONS: BlmI represents the first characterized type II PCP . The BlmI type II PCP, like the PCP domains of type I NRPSs, can be 4'-phospho-pantetheinylated by PCP-specific PPTases but is biochemically distinct in that it can be aminoacylated by an A domain from a completely unrelated type I NRPS . Our results provide for the first time the genetic and biochemical evidence to support the existence of a type II NRPS, which might be useful in the combinatorial manipulation of NRPS proteins to generate novel peptides. Chem Biol, 1999 Aug, 6(8), 519 - 29 Structural basis for engineering of retinoic acid receptor isotype-selective agonists and antagonists; Gehin M et al.; BACKGROUND: Many synthetic retinoids have been generated that exhibit a distinct pattern of agonist/antagonist activities with the three retinoic acid receptors (RARalpha, RARbeta and RARgamma) . Because these retinoids are selective tools with which to dissect the pleiotropic functions of the natural pan-agonist, retinoic acid, and might constitute new therapeutic drugs, we have determined the structural basis of their receptor specificity and compared their activities in animal and yeast cells . RESULTS: There are only three divergent amino acid residues in the ligand binding pockets (LBPs) of RARalpha, RARbeta and RARgamma . We demonstrate here that the ability of monospecific (class I) retinoid agonists and antagonists to bind to and induce or inhibit transactivation by a given isotype is directly linked to the nature of these residues . The agonist/antagonist potential of class II retinoids, which bind to all three RARs but depending on the RAR isotype have the potential to act as agonists or antagonists, was also largely determined by the three divergent LBP residues . These mutational studies were complemented by modelling, on the basis of the three-dimensional structures of the RAR ligand-binding domains, and a comparison of the retinoid agonist/antagonist activities in animal and yeast cells . CONCLUSIONS: Our results reveal the rational basis of RAR isotype selectivity, explain the existence of class I and II retinoids, and provide a structural concept of ligand-mediated antagonism . Interestingly, the agonist/antagonist characteristics of retinoids are not conserved in yeast cells, suggesting that yeast co-regulators interact with RARs in a different way than the animal cell homologues do. Acta Crystallogr D Biol Crystallogr, 1999 Aug, 55 ( Pt 8), 1478 - 80 Crystallization and initial X-ray analysis of rabbit mature sterol carrier protein 2; Choinowski T et al.; Sterol carrier protein 2 (SCP2) is a basic intracellular protein which facilitates the in vitro intermembrane transfer of cholesterol, phospholipids and glycolipids . SCP2 was expressed in Escherichia coli, purified to apparent electrophoretic homogeneity and crystallized . Single crystals were obtained by hanging-drop vapour diffusion using ammonium sulfate as precipitant . These crystals belong to space group P4(1)2(1)2 or its enantiomorph, with unit-cell parameters a = b = 57.5, c = 86.5 A, and have one molecule in the crystallographic asymmetric unit . Intensity data to 1.8 A resolution were collected from native SCP2 crystals using synchrotron radiation, were processed and scaled with an R(linear) = 4.9%. Acta Crystallogr D Biol Crystallogr, 1999 Aug, 55 ( Pt 8), 1474 - 7 Crystallization and preliminary crystallographic analysis of the Escherichia coli tyrosine aminotransferase; Ko TP et al.; Tyrosine aminotransferase catalyzes transamination for both dicarboxylic and aromatic amino-acid substrates . The substrate-free Escherichia coli tyrosine aminotransferase (eTAT) bound with the cofactor pyridoxal 5'-phosphate (PLP) was crystallized in the trigonal space group P3(2) . A low-resolution crystal structure of eTAT was determined by molecular-replacement methods . The overall folding of eTAT resembles that of the aspartate aminotransferases, with the two identical subunits forming a dimer in which each monomer binds a PLP molecule via a covalent bond linked to the epsilon-NH(2) group of Lys258 . Comparison of the structure of eTAT with those of the open, half-open or closed form of chicken or E . coli aspartate aminotransferases shows the eTAT structure to be in the open conformation. Acta Crystallogr D Biol Crystallogr, 1999 Aug, 55 ( Pt 8), 1405 - 13 Disorder and twin refinement of RNA heptamer double helices; Mueller U et al.; An RNA helix with seven base pairs which was derived from the acceptor stem of Escherichia coli tRNA(Ala), rGGGGCUA.rUAGCUCC (ALA(wt)), as well as a variant, rGGGGCUA.rUAGCCCC (ALA(C70)), in which the single G.U wobble base pair of ALA(wt) was replaced by G.C, crystallize in space group C2 . Both non-isomorphic crystal forms display a complex packing pattern, which can be described alternatively as disorder or pseudo-merohedral twinning . The structure of ALA(wt) was determined by SIRAS phasing using an isomorphous iodine derivative, rGGGGCi(5)UA.rUAGCUCC (ALA(I)) . All three RNA structures were subsequently subjected to twin refinement in space group P1, using anisotropic thermal displacement parameters at resolutions of 1.16, 1.23 and 1.4 A for ALA(wt), ALA(I) and ALA(C70), respectively . Alternatively, the structure of ALA(wt) was refined in space group C2 assuming twofold disorder of the molecular orientation . The refined structures are of reasonable quality according to all available indicators . There are no systematic differences between the molecular models resulting from twin refinement and disorder refinement. Am J Hum Genet, 1999 Aug, 65(2), 327 - 35 Identification of a mutation cluster in mevalonate kinase deficiency, including a new mutation in a patient of Mennonite ancestry; Hinson DD et al.; Mevalonate kinase (MKase) deficiency (MKD) is a rare autosomal recessive disorder in the pathway of cholesterol and nonsterol isoprenoid biosynthesis . Thus far, two disease-causing missense alleles have been identified, N301T and A334T . We report four additional mutations associated with MKD: L264F, T243I, L265P, and I268T, the last found in a patient of Mennonite ancestry . Electrophoretic analysis of bacterially expressed wild-type and mutant MKase indicated that I268T and T243I mutants produced normal or somewhat reduced amounts of MKase protein; conversely, L264F and L265P mutations resulted in considerably decreased, or absent, MKase protein . Immunoblot analysis of MKase from all patients suggested that the MKase polypeptide was grossly intact and produced in amounts comparable to control levels . Three mutations resulted in significantly diminished MKase enzyme activity (<2%), whereas the I268T allele yielded approximately 20% residual enzyme activity . Our results should allow more-accurate identification of carriers and indicate a mutation "cluster" within amino acids 240-270 of the mature MKase polypeptide. Genes Dev, 1999 Jul 15, 13(14), 1861 - 70 Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of holliday junction resolution; van Gool AJ et al.; Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions . Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process . Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers . The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved . Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration . Within such a structure, DNA isomerization can have little role in determining the orientation of resolution . To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome . We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction . Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration. Circulation, 1999 Jul 27, 100(4), 400 - 6 Matrix metalloproteinase inhibitor prevents acute lung injury after cardiopulmonary bypass; Carney DE et al.; BACKGROUND: Acute lung injury (ALI) after cardiopulmonary bypass (CPB) results from sequential priming and activation of neutrophils . Activated neutrophils release neutral serine, elastase, and matrix metalloproteinases (MMPs) and oxygen radical species, which damage alveolar-capillary basement membranes and the extracellular matrix, resulting in an ALI clinically defined as adult respiratory distress syndrome (ARDS) . We hypothesized that treatment with a potent MMP and elastase inhibitor, a chemically modified tetracycline (CMT-3), would prevent ALI in our sequential insult model of ALI after CPB . METHODS AND RESULTS: Anesthetized Yorkshire pigs were randomized to 1 of 5 groups: control (n=3); CPB (n=5), femoral-femoral hypothermic bypass for 1 hour; LPS (n=7), sham bypass followed by infusion of low-dose Escherichia coli lipopolysaccharide (LPS; 1 microgram/kg); CPB+LPS (n=6), both insults; and CPB+LPS+CMT-3 (n=5), both insults plus intravenous CMT-3 dosed to obtain a 25-micromol/L blood concentration . CPB+LPS caused severe lung injury, as demonstrated by a significant fall in PaO(2) and an increase in intrapulmonary shunt compared with all groups (P<0.05) . These changes were associated with significant pulmonary infiltration of neutrophils and an increase in elastase and MMP-9 activity . CONCLUSIONS: All pathological changes typical of ALI after CPB were prevented by CMT-3 . Prevention of lung dysfunction followed an attenuation of both elastase and MMP-2 activity . This study suggests that strategies to combat ARDS should target terminal neutrophil effectors. Curr Biol, 1999 Jul 15, 9(14), R518 - 20 Genetic recombination: Helicases and topoisomerases link up; Wu L et al.; RecQ helicases and topoisomerase III are both required for genome stability, particularly to prevent 'promiscuous' genetic recombination . A recent study demonstrates that, together, these enzymes can catalyse the interlinking of plasmid DNA, and suggests a novel mechanism for the control of recombination. Biometals, 1999 Mar, 12(1), 47 - 52 Influence of metal ions and temperature on the conformation of Escherichia coli K1 capsular polysaccharide; Koenig MK et al.; Escherichia coli K1 secretes a homopolymer capsular polysaccharide (CPS) consisting of alpha 2, 8 linked N-acetylneuraminic acid (poly alpha 2,8NeuNAc) . Typically poly alpha 2,8NeuNAc is arranged in low and high order alpha helices with carboxyl and hydroxyl groups extending from the helices . Several properties of CPS such as antigenicity and metal binding can be influenced by its structural conformation . We examined the influences of metal ions and temperature on the secondary structure of poly alpha 2,8NeuNAc . Conformation alteration was detected by ultraviolet (UV) spectroscopy and circular dichroism (CD) . The majority of metal ions tested had no detectable influence on poly alpha 2,8NeuNAc structure . In contrast, Yb3+, Hg2+, and Cu2+ ions greatly altered the UV and CD spectra, which suggests that these ions had disrupted the alpha helical structure of poly alpha 2,8NeuNAc . These changes were influenced by the metal ion concentration . When poly alpha 2,8NeuNAc was incubated at temperatures ranging from 20-60 degrees C, alterations in its UV absorption spectra were also seen . The most significant change occurred between 35 and 40 degrees C . In summary, this study suggests that the higher order structure and function of bacterial CPS may be influenced by environmental factors. J R Army Med Corps, 1999 Jun, 145(2), 95 - 101 Travellers' diarrhoea: a military problem? Connor P, Farthing MJ. One hundred years ago, apart from the treatment of war injuries, the prevention and treatment of diarrhoea was the dominant preoccupation of the deployed military doctor . Since, then our understanding of the pathogenesis and pathophysiology of enteric disease has developed exponentially and our armamentarium for the treatment of enteric diseases has expanded considerably . However, diarrhoea continues to be the dominant military medical concern in deployed units . Here, we examine the evidence for this, discuss the reasons why and critically evaluate current modes of prevention and treatment that are now available to the military medical officer. J Microencapsul, 1999 Jul-Aug, 16(4), 489 - 99 Physical properties and heavy metal uptake of encapsulated Escherichia coli expressing a metal binding gene (NCP); Bang SS et al.; A recombinant Escherichia coli expressing the Neurospora crassa metallothionein gene (NCP) has previously been shown to remove low levels of Cd and other metals from solution . For further development as a biosorbent, the encapsulation of the NCP is investigated by various matrices . The NCP was encapsulated in alginate, chitosan-alginate or kappa-carrageenan, and its physical properties characterized . Results indicated that encapsulation in alginate resulted in fragile beads, whereas encapsulation in kappa-carrageenan or chitosan-alginate provided more physical and chemical integrity to the beads . Maximal heavy metal removal by cells encapsulated in carrageenan occurred within 3 h, while a gradual increase in removal was observed up to 24 h for cells encapsulated in chitosan-alginate . Metal removal by cells encapsulated in alginate beads was lower than those encapsulated in carrageenan or chitosan-alginate. Genetika, 1999 Apr, 35(4), 450 - 8 {Formation of the heterozygous tandem duplication in the process of conjugation recombination in Escherichia coli: study of the effect of mutations for the recQ, uvrD, and recJ genes}; Sukhodolets VV; Stable tandem duplications were shown to originate from conjugational recombination between Escherichia coli HfrH strains carrying mutations for the deo operon . The duplications deoC deoD/deoA deoB::Tn5 usually constitute approximately 5% of the Deo+ offspring . The effect of mutations for the recQ, uvrD, and recJ genes on the frequency of duplications was studied . The CM1563 strain carrying the recQ mutation was shown to give, as a recipient, 20% of duplications in the Deo+ offspring . However, this property of CM1563 seems to depend on the presence of a spontaneous mutation of unknown nature, which also increased UV sensitivity of bacteria . The recQ mutation itself increased the frequency of duplications by less than 50% . The recJ mutation did not virtually affect the frequency of duplications . The uvrD mutation possessing the recombinogenic effect was shown to increase the frequency of deo+ recombinants and simultaneously decrease the frequency of duplications . Tandem duplications are assumed to be normal intermediates of multi-stage conjugational recombination initiated by the integration of the proximal region of the Hfr chromosome into different nonhomologous regions of the recipient chromosome.
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