Biomacromolecules, 2001 Fall, 2(3), 1023 - 8 Poly(amidoamine)s as potential nonviral vectors: ability to form interpolyelectrolyte complexes and to mediate transfection in vitro; Richardson SC et al.; Poly(amidoamine)s (PAAs) are water-soluble polymers that display pH-dependent membrane activity . PAAs have the potential to act as a synthetic alternative to fusogenic peptides and thus promote endosomal escape . The purpose of this study was to investigate for the first time whether PAA have the ability to complex DNA, protect it from nuclease degradation and to promote transfection in vitro . PAAs ISA 1 (Mn 6900) and ISA 23 (Mn 10,500) and their 2-phenylethylamine containing analogues ISA 4 and ISA 22 (Mn approximately 8000) were studied . All PAAs retarded the electrophoretic mobility of lambda Hind III DNA demonstrating interpolyelectrolyte complex (IPEC) formation and toroids of 80-150 nm in diameter (10:1 polymer excess) were visible using TEM . DNase II inhibition was observed . At a polymer:DNA ratio of 10:1, this was ISA 1(89.6 +/- 6.1%), ISA 4 (92.2 +/- 11.2%), ISA 22 (69.4 +/- 3.7%), and ISA 23 (58.0 +/- 10.0%) . PAAs demonstrated the ability to mediate pSV beta-galactosidase transfection of HepG2 cells . At a vector:DNA mass ratio of 5:1, ISA 23 showed equivalent transfection ability compared with polyethylenimine and LipofectIN and was more effective than LipofectACE . These properties suggest that PAAs warrant further development as endosomolytic vectors. J Biol Chem, 2002 Jan 25, 277(4), 2992 - 6 Epub 2001 Nov 14. Holliday junction resolution is modulated by archaeal chromatin components in vitro; Kvaratskhelia M et al.; The Holliday junction-resolving enzyme Hjc is conserved in the archaea and probably plays a role analogous to that of Escherichia coli RuvC in the pathway of homologous recombination . Hjc specifically recognizes four-way DNA junctions, cleaving them without sequence preference to generate recombinant DNA duplex products . Hjc imposes an X-shaped global conformation on the bound DNA junction and distorts base stacking around the point of cleavage, three nucleotides 3' of the junction center . We show that Hjc is autoinhibitory under single turnover assay conditions and that this can be relieved by the addition of either competitor duplex DNA or the architectural double-stranded DNA-binding protein Sso7d (i.e . by approximating in vivo conditions more closely) . Using a combination of isothermal titration calorimetry and fluorescent resonance energy transfer, we demonstrate that multiple Hjc dimers can bind to each synthetic four-way junction and provide evidence for significant distortion of the junction structure at high protein:DNA ratios . Analysis of crystal packing interactions in the crystal structure of Hjc suggests a molecular basis for this autoinhibition . The wider implications of these findings for the quantitative study of DNA-protein interactions is discussed. J Biol Chem, 2002 Feb 22, 277(8), 5858 - 65 Epub 2001 Nov 14. Why OrfY? Characterization of MMOD, a long overlooked component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath); Merkx M et al.; Soluble methane monooxygenase (sMMO) has been studied intensively to understand the mechanism by which it catalyzes the remarkable oxidation of methane to methanol . The cluster of genes that encode for the three characterized protein components of sMMO (MMOH, MMOB, and MMOR) contains an additional open reading frame (orfY) of unknown function . In the present study, MMOD, the protein encoded by orfY, was overexpressed as a fusion protein in Escherichia coli . Pure MMOD was obtained in high yields after proteolytic cleavage and a two-step purification procedure . Western blot analysis of Methylococcus capsulatus (Bath) soluble cell extracts showed that MMOD is expressed in the native organism although at significantly lower levels than the other sMMO proteins . The cofactorless MMOD protein is a potent inhibitor of sMMO activity and binds to the hydroxylase protein (MMOH) with an affinity similar to that of MMOB and MMOR . The addition of up to 2 MMOD per MMOH results in changes in the optical spectrum of the hydroxylase that suggest the formation of a (micro-oxo)diiron(III) center in a fraction of the MMOH-MMOD complexes . Possible functions for MMOD are discussed, including a role in the assembly of the MMOH diiron center similar to that suggested for DmpK, a protein that shares some properties with MMOD. Antimicrob Agents Chemother, 2001 Dec, 45(12), 3660 - 2 Mechanism of action of the des-F(6) quinolone BMS-284756 measured by supercoiling inhibition and cleavable complex assays; Wu P et al.; BMS-284756 (T-3811ME), a novel des-F(6) quinolone, was tested in the supercoiling inhibition and cleavable complex assays against Escherichia coli DNA gyrase, a target of quinolones . The results suggest that BMS-284756 has the same mechanism of action against DNA gyrase as other quinolones and a similar level of potency. Antimicrob Agents Chemother, 2001 Dec, 45(12), 3293 - 303 Dicyclic and tricyclic diaminopyrimidine derivatives as potent inhibitors of Cryptosporidium parvum dihydrofolate reductase: structure-activity and structure-selectivity correlations; Nelson RG et al.; A structurally diverse library of 93 lipophilic di- and tricyclic diaminopyrimidine derivatives was tested for the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of Cryptosporidium parvum (J . R . Vasquez et al., Mol . Biochem . Parasitol . 79:153-165, 1996) . In parallel, the library was also tested against human DHFR and, for comparison, the enzyme from Escherichia coli . Fifty percent inhibitory concentrations (IC(50)s) were determined by means of a standard spectrophotometric assay of DHFR activity with dihydrofolate and NADPH as the cosubstrates . Of the compounds tested, 25 had IC(50)s in the 1 to 10 microM range against one or both C . parvum enzymes and thus were not substantially different from trimethoprim (IC(50)s, ca . 4 microM) . Another 25 compounds had IC(50)s of <1.0 microM, and 9 of these had IC(50)s of <0.1 microM and thus were at least 40 times more potent than trimethoprim . The remaining 42 compounds were weak inhibitors (IC(50)s, >10 microM) and thus were not considered to be of interest as drugs useful against this organism . A good correlation was generally obtained between the results of the spectrophotometric enzyme inhibition assays and those obtained recently in a yeast complementation assay (V . H . Brophy et al., Antimicrob . Agents Chemother . 44:1019-1028, 2000; H . Lau et al., Antimicrob . Agents Chemother . 45:187-195, 2001) . Although many of the compounds in the library were more potent than trimethoprim, none had the degree of selectivity of trimethoprim for C . parvum versus human DHFR . Collectively, the results of these assays comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents . The compounds in the library were also tested as inhibitors of the proliferation of intracellular C . parvum oocysts in canine kidney epithelial cells cultured in folate-free medium containing thymidine (10 microM) and hypoxanthine (100 microM) . After 72 h of drug exposure, the number of parasites inside the cells was quantitated by indirect immunofluorescence microscopy . Sixteen compounds had IC(50)s of <3 microM, and five of these had IC(50)s of <0.3 microM and thus were comparable in potency to trimetrexate . The finding that submicromolar concentrations of several of the compounds in the library could inhibit in vitro growth of C . parvum in host cells in the presence of thymidine (dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in combination with leucovorin, may find use in the treatment of intractable C . parvum infections. Microbes Infect, 2001 Nov, 3(13), 1101 - 9 Priming of human neutrophils by mycobacterial lipoarabinomannans: role of granule mobilisation; Faldt J et al.; Lipoarabinomannans (LAMs) from mycobacteria were investigated concerning their effect on human neutrophils . Two types of LAM, the mannose-capped ManLAM from the virulent Mycobacterium tuberculosis H37Rv and the mannose-lacking AraLAM from a rapidly growing mycobacterial strain were used . Neither AraLAM nor ManLAM induced any significant direct activation of the NADPH-oxidase . Both LAMs, however, primed the neutrophils so that subsequent stimulation with the peptide chemoattractants fMet-Leu-Phe (fMLF), Trp-Lys-Tyr-Met-Val-DMet (WKYMVm) and the mammalian lactose-binding lectin galectin-3 resulted in a markedly enhanced oxidative response . The LAM-induced priming was accompanied by an increased exposure of complement receptors 1 and 3 as well as the formyl peptide receptor on the neutrophil surface, suggesting that the enhanced oxidative response could be due to upregulation of receptors on the cell surface as a result of granule mobilisation . Since LAM-primed neutrophils released 65% of the cell content of gelatinase but showed no increased release of vitamin B(12)-binding protein, mobilisation of the gelatinase granules rather than the specific granules is concluded to be responsible for the priming effects . This is in agreement with the subcellular localisation of receptors for fMLF, WKYMVm, as well as galectin-3, which are stored in the secretory vesicles and gelatinase granules . The priming effect appeared very similar to that of Escherichia coli lipopolysaccharide, and since no differences in activity could be detected between AraLAM and ManLAM, we hypothesize that the lipid anchor of the LAM is responsible for the priming effects. Structure (Camb), 2001 Nov, 9(11), 1117 - 25 Escherichia coli GlpE is a prototype sulfurtransferase for the single-domain rhodanese homology superfamily; Spallarossa A et al.; BACKGROUND: Rhodanese domains are structural modules occurring in the three major evolutionary phyla . They are found as single-domain proteins, as tandemly repeated modules in which the C-terminal domain only bears the properly structured active site, or as members of multidomain proteins . Although in vitro assays show sulfurtransferase or phosphatase activity associated with rhodanese or rhodanese-like domains, specific biological roles for most members of this homology superfamily have not been established . RESULTS: Eight ORFs coding for proteins consisting of (or containing) a rhodanese domain bearing the potentially catalytic Cys have been identified in the Escherichia coli K-12 genome . One of these codes for the 12-kDa protein GlpE, a member of the sn-glycerol 3-phosphate (glp) regulon . The crystal structure of GlpE, reported here at 1.06 A resolution, displays alpha/beta topology based on five beta strands and five alpha helices . The GlpE catalytic Cys residue is persulfurated and enclosed in a structurally conserved 5-residue loop in a region of positive electrostatic field . CONCLUSIONS: Relative to the two-domain rhodanese enzymes of known three-dimensional structure, GlpE displays substantial shortening of loops connecting alpha helices and beta sheets, resulting in radical conformational changes surrounding the active site . As a consequence, GlpE is structurally more similar to Cdc25 phosphatases than to bovine or Azotobacter vinelandii rhodaneses . Sequence searches through completed genomes indicate that GlpE can be considered to be the prototype structure for the ubiquitous single-domain rhodanese module. Structure (Camb), 2001 Nov, 9(11), 1095 - 106 Structure of Thermotoga maritima stationary phase survival protein SurE: a novel acid phosphatase; Zhang RG et al.; BACKGROUND: The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth . rpoS codes for the stationary-phase RNA polymerase sigma subunit, and nlpD codes for a lipoprotein . The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls . The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E . coli subjected to high-temperature growth . The pcm and surE genes are highly conserved in bacteria, archaea, and plants . RESULTS: The structure of SurE from Thermotoga maritima was determined at 2.0 A . The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold . Monomers form a dimer that assembles into a tetramer . Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2 . The active site was identified in the N-terminal domain through analysis of conserved residues . Structure-based site-directed point mutations abolished phosphatase activity . T . maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability . CONCLUSIONS: The structure of SurE provided information about the protein's fold, oligomeric state, and active site . The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified . The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis. Structure (Camb), 2001 Nov, 9(11), 1083 - 93 The mechanism of glycerol conduction in aquaglyceroporins; Jensen MO et al.; BACKGROUND: The E . coli glycerol facilitator, GlpF, selectively conducts glycerol and water, excluding ions and charged solutes . The detailed mechanism of the glycerol conduction and its relationship to the characteristic secondary structure of aquaporins and to the NPA motifs in the center of the channel are unknown . RESULTS: Molecular dynamics simulations of GlpF reveal spontaneous glycerol and water conduction driven, on a nanosecond timescale, by thermal fluctuations . The bidirectional conduction, guided and facilitated by the secondary structure, is characterized by breakage and formation of hydrogen bonds for which water and glycerol compete . The conduction involves only very minor changes in the protein structure, and cooperativity between the GlpF monomers is not evident . The two conserved NPA motifs are strictly linked together by several stable hydrogen bonds and their asparagine side chains form hydrogen bonds with the substrates passing the channel in single file . CONCLUSIONS: A complete conduction of glycerol through the GlpF was deduced from molecular dynamics simulations, and key residues facilitating the conduction were identified . The nonhelical parts of the two half-membrane-spanning segments expose carbonyl groups towards the channel interior, establishing a curve-linear pathway . The conformational stability of the NPA motifs is important in the conduction and critical for selectivity . Water and glycerol compete in a random manner for hydrogen bonding sites in the protein, and their translocations in single file are correlated . The suggested conduction mechanism should apply to the whole family. Structure (Camb), 2001 Nov, 9(11), 1071 - 81 Insights into the structure, solvation, and mechanism of ArsC arsenate reductase, a novel arsenic detoxification enzyme; Martin P et al.; BACKGROUND: In Escherichia coli bearing the plasmid R773, resistance to arsenite, arsenate, antimonite, and tellurite is conferred by the arsRDABC plasmid operon that codes for an ATP-dependent anion pump . The product of the arsC gene, arsenate reductase (ArsC), is required to efficiently catalyze the reduction of arsenate to arsenite prior to extrusion . RESULTS: Here, we report the first X-ray crystal structures of ArsC at 1.65 A and of ArsC complexed with arsenate and arsenite at 1.26 A resolution . The overall fold is unique . The native structure shows sulfate and sulfite ions binding in the active site as analogs of arsenate and arsenite . The covalent adduct of arsenate with Cys-12 in the active site of ArsC, which was analyzed in a difference map, shows tetrahedral geometry with a sulfur-arsenic distance of 2.18 A . However, the corresponding adduct with arsenite binds as a hitherto unseen thiarsahydroxy adduct . Finally, the number of bound waters (385) in this highly ordered crystal structure approaches twice the number expected at this resolution for a structure of 138 ordered residues . CONCLUSIONS: Structural information from the adduct of ArsC with its substrate (arsenate) and with its product (arsenite) together with functional information from mutational and biochemical studies on ArsC suggest a plausible mechanism for the reaction . The exceptionally well-defined water structure indicates that this crystal system has precise long-range order within the crystal and that the upper limit for the number of bound waters in crystal structures is underestimated by the structures in the Protein Data Bank. Structure (Camb), 2001 Nov, 9(11), 1051 - 60 Crystal structure of transcription factor MalT domain III: a novel helix repeat fold implicated in regulated oligomerization; Steegborn C et al.; BACKGROUND: MalT from Escherichia coli, the best-studied member of the MalT family of ATP-dependent transcriptional activators, regulates the genes for malto-oligosaccharide utilization . The active form of this 4 domain protein is a homooligomer, and its multimerization is induced by the binding of maltotriose . Domains II and III of MalT were suggested to mediate the oligomerization process, but its molecular mechanism and the specific functions of these domains remain to be identified . RESULTS: We solved the crystal structure of MalT domain III at 1.45 A resolution by multiple isomorphous replacement phasing . The structure reveals eight copies of a two-helix bundle motif arranged in a novel, right-handed superhelix fold with closed walls, followed by a small C-terminal subdomain . The MalT superhelix contains a potential maltotriose binding site and forms a large hydrophobic protein-protein interaction interface that mediates the contact between two MalT domain III molecules . Structure-based analysis of the two-helix bundle motifs revealed a novel degenerated sequence pattern, and repeats of this pattern could be identified in other regulator proteins . CONCLUSIONS: MalT domain III contains a novel superhelix fold . Its protein-protein interaction interface, however, resembles protein binding sites of other superhelical proteins, suggesting a model with domain III mediating MalT oligomerization . Maltotriose seems to modulate the interaction interface and MalT oligomerization by occupying the ligand binding site inside the superhelix . Similar structural and mechanistic features in other MalT protein-family members and unrelated regulator proteins are indicated by the reappearance of a novel sequence motif derived from the MalT domain III structure. Structure (Camb), 2001 Nov, 9(11), 999 - 1004 Intricacies in ATP-dependent clamp loading: variations across replication systems; Trakselis MA et al.; DNA replication requires the coordinated effort of many proteins to create a highly processive biomachine able to replicate entire genomes in a single process . The clamp proteins confer on replisomes this property of processivity but in turn require clamp loaders for their functional assembly onto DNA . A more detailed view of the mechanisms for holoenzyme assembly in replication systems has been obtained from the advent of novel solution experiments and the appearance of low- and high-resolution structures for the clamp loaders. Biochem Soc Trans, 2001 Nov, 29(Pt 6), 806 - 11 Uncoupling protein, H+ transport and regulation; Klingenberg M et al.; The biochemical functions of uncoupling proteins (UCPs) are discussed with the view of UCP1 as a paradigm . In contrast with UCP1, the heterologous expression of UCP3 in yeast is found to result primarily in extra-mitochondrial deposits and thus is unsuitable for studying UCP3 function . On expression in Escherichia coli inclusion bodies, UCPs extracted and incorporated into vesicles showed no H(+) transport, only Cl(-) transport . Only after addition of coenzyme Q was fully nucleotide-sensitive high-H(+) transport reconstituted, with UCP1 as well as with UCP2 and UCP3 . The newly discovered cofactor role of coenzyme Q in H(+) transport is proposed to imply co-operation with fatty acids for the injection of H(+) into the UCP channel. J Theor Biol, 2001 Nov 7, 213(1), 9 - 19 Gene arrangements and phylogeny in the class Proteobacteria; Kunisawa T; A simple method is presented for reconstructing phylogenetic trees on the basis of gene transposition . It is shown that differences in gene arrangements among genomes could allow us to determine whether a gene transposition event has occurred before or after species divergence from parsimonious considerations . The method is applied to evolutionary relationships among the bacterial class Proteobacteria, for which complete genomic sequences most densely accumulate and comprehensive gene order comparisons are possible . We were able to infer the emergence order of proteobacterial subclasses as epsilon-->beta-->gamma . This order is consistent with sequence-based inferences, which conversely confirms the usefulness of the approach presented here . Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 299 - 304 Design of an artificial light-harvesting unit by protein engineering: cytochrome b(562)-green fluorescent Protein chimera; Takeda S et al.; We have generated a novel model protein for an artificial light-harvesting complex composed of two proteins, cytochrome b(562) (cytb(562)) and enhanced green fluorescent protein (EGFP), in which two chromophores are fixed in each protein matrix . Cytb(562) was appended to the N-terminus of EGFP via a Gly-Ser linker and the resultant fusion protein was successfully expressed in Escherichia coli as a mixture of the apo- and the holo-forms as to the cytb(562) moiety . The fluorescence of EGFP was substantially quenched when the apo-form was reconstituted with hemin . Based on the fluorescence lifetime measurements, it appeared that light energy entrapped by EGFP is transferred to the heme of cytb(562) by resonance energy transfer (energy transfer yield: 65%) . Spatial organization of two chromophores using small and stable protein matrices will be promising toward the construction of an artificial light-harvesting complex by protein engineering . Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 264 - 8 NC1 domain of human type VIII collagen (alpha 1) inhibits bovine aortic endothelial cell proliferation and causes cell apoptosis; Xu R et al.; Endostatin, a natural angiogenesis inhibitor, had been identified for years . It opened a new approach for cancer therapy . Sequence analysis revealed that endostatin is the NC1 domain (non-triple-helical domain) of collagen XVIII . In this report, the cDNA of NC1 domain of type VIII collagen (alpha 1) was cloned and expressed as soluble form in Escherichia coli . The recombinant protein was purified with Ni-NTA agarose column and named as vastatin . It inhibited the proliferation of bovine aortic endothelial (BAE) cell stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner . The ED(50) of vastatin was 0.6 microg/ml, while the ED(50) of endostatin was 0.5 microg/ml . Treatment of BAE cell with vastatin caused G(0)-G(1) arrest and cell apoptosis . It is interesting that sequence analysis showed that there was only about 12% amino acid sequence homology between vastatin and endostatin . The structure-function relationship of these angiogenesis molecules remains to be elucidated . Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 252 - 6 A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coli using biotinylated phages; Nakamura M et al.; Direct visualization of filamentous phage infection in Escherichia coli (E . coli) was attempted using biotinylated phages (BIO-phages) . The biotinylation of the phages did not influence their infectivity into E . coli . E . coli infected with BIO-phages could be detected by using fluorescein-conjugated avidin with confocal laser scanning microscopy, and BIO-phages and BIO-phage-derived proteins in E . coli could be directly observed by using the avidin-biotin-peroxidase complex method with electron microscopy . This is the first report of direct visualization of phage infection and phage-derived proteins in the host cell using a biotin-avidin interaction . This simple and powerful method is applicable to the study of infection by various viruses . Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 161 - 6 Production and characterization of the recombinant Sphingomonas chlorophenolica pentachlorophenol 4-monooxygenase; Wang H et al.; Pentachlorophenol 4-monooxygenase (PCP4MO) from Sphingomonas chlorophenolica is a flavoprotein that hydroxylates PCP in the presence of NADPH and oxygen . In order to investigate the structure and function of active site, recombinant PCP4MO (rePCP4MO) was produced in Escherichia coli as a glutathione S-transferase (GST) fusion protein . Moreover, a tobacco etch virus (TEV) protease cleavage site (EKLYFQG) was introduced into GST-PCP4MO and a his-tagged TEV protease was employed . Hence, a two-step purification protocol was developed which allowed obtaining 15-20 mg of rePCP4MO from 1 L culture . The rePCP4MO revealed identity with native enzyme by SDS-PAGE and N-terminal sequence analyses . Furthermore, a polyclonal PCP4MO antibody was produced with GST-PCP4MO and purified by immunoaffinity chromatography, where both the native and recombinant forms of PCP4MO showed interaction . However, rePCP4MO was identified as apoprotein with no evidence for a typical flavoprotein spectrum . The catalytic activity could be detected in the presence of FAD . The K(m) and V(max) values for PCP were 50 microM and 30 nmol/min/mg, respectively . Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 125 - 9 Thermal stability of human ferritin: concentration dependence and enhanced stability of an N-terminal fusion mutant; Kim SW et al.; Though human L-chain ferritin has been known to be more resistant to physical denaturation than H-type ferritin, its stability characteristics and kinetic information have not been reported in detail . Overexpressed recombinant ferritin (FTN) in Escherichia coli formed inclusion bodies through noncovalent molecular interaction and easily dissolved with regaining the iron-uptake activity by a simple pH-shift process at high protein concentration (>600 mg l(-1)) . FTN was relatively thermostable at low protein concentration (0.2 g l(-1)), but it became extremely thermolabile at high protein concentration (1.3 g l(-1)), i.e., more than 80% of FTN was coprecipitated within 5 min under the same heat-induced denaturation condition . Aggregation rate constant for initial 5 min at high protein concentration was 6.04 x 10(-3) s(-1) for FTN . Surprisingly, glucagon . ferritin mutant (GFTN), consisting of an N-terminus fusion partner, human glucagon (29-residue alpha-helical peptide), showed significantly enhanced thermal stability even at high protein concentration . That is, in spite of 40-min heat treatment, more than 50% of GFTN the still remained soluble with maintaining the same functional properties . The aggregation rate constants were 2.75 x 10(-4) and 2.80 x 10(-4) s(-1) at low and high concentration, respectively, for GFTN . These results suggest a critical participation of the N-terminal domain of ferritin in the temperature-induced aggregation pathway . Presumably, partially denatured amino terminus of FTN is involved in nonspecific molecular interaction resulting in the off-pathway aggregation . It is notable that the purified GFTN showed the same molar capacity of iron (Fe(+3)) storage as standard ferritin . From the analysis of fluorescence emission spectrum, the physical stability of GFTN was also very comparable to that of standard ferritin under the various denaturation conditions induced by GdnHCl . Transgenic Res, 2001 Oct, 10(5), 409 - 22 Selection and orientation of adjacent genes influences DAM-mediated male sterility in transformed maize; Unger E et al.; Anther-targeted expression of E . coli DNA (Adenosine-N6-)-Methyltransferase (DAM) in maize was tested as a means to produce male-sterile plants . A high frequency of male-sterile plants with reduced anther size was observed when DAM was regulated by the maize anther-specific promoter 5126 (5126:DAM) and placed upstream of the herbicide resistance gene, pat, regulated by the cauliflower mosaic virus (CaMV) 35S promoter (35S:PAT) . In contrast, placement of 5126:DAM upstream of a pat gene regulated by either the maize ubiquitin (UBI:PAT) or rice actin (rACTIN:PAT) promoters resulted in male-fertile plants . Based on these observed differences, DAM-mediated sterility was used as a phenotypic marker to assess the contribution of factors affecting gene expression such as orientation of the transcription units, choice of regulatory sequences mediating expression of adjacent genes, and effects of varying the anther-specific promoter regulating DAM . Constructs that place a portion of the CaMV 35S promoter, including the native AS-1 sequences, between 5126:DAM and UBI:PAT yielded a high frequency of male-sterile plants with reduced anther size . Significant differences in the frequency of male-sterile events and the associated anther size were also observed when the position of 35S:PAT was changed relative to 5126:DAM . These data provide evidence that gene expression in transformed maize plants can be impacted by simply altering the order, orientation or regulatory sequences of adjacent genes. Protein Eng, 2001 Sep, 14(9), 699 - 704 Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution; Sun L et al.; We have used directed evolution methods to express a fungal enzyme, galactose oxidase (GOase), in functional form in Escherichia coli . The evolved enzymes retain the activity and substrate specificity of the native fungal oxidase, but are more thermostable, are expressed at a much higher level (up to 10.8 mg/l of purified GOase), and have reduced negative charge compared to wild type, all properties which are expected to facilitate applications and further evolution of the enzyme . Spectroscopic characterization of the recombinant enzymes reveals a tyrosyl radical of comparable stability to the native GOase from Fusarium. Protein Eng, 2001 Sep, 14(9), 669 - 74 N-terminal portion acts as an initiator of the inactivation of pepsin at neutral pH; Tanaka T et al.; Porcine pepsin, an aspartic protease, is unstable at neutral pHs where it rapidly loses activity, however, its zymogen, pepsinogen, is stable at neutral pHs . The difference between the two is the presence of the prosegment in pepsinogen . In this study, possible factors responsible for instability were investigated and included: (i) the distribution of positively charged residues on the surface, (ii) an insertion of a peptide in the C-terminal domain and (iii) the dissociation of the N-terminal fragment of pepsin . Mutations to change the number and the distribution of positive charges on the surface had a minor effect on stability . No effect on stability was observed for the deletion of a peptide from the C-terminal domain . However, mutations on the N-terminal fragment had a major impact on stability . At pH 7.0, the N-fragment mutant was inactivated 5.8 times slower than the wild-type . The introduction of a disulfide bond between the N-terminal fragment and the enzyme body prevented the enzyme from denaturing . The above results showed that the inactivation of pepsin was initiated by the dissociation of the N-fragment and that the sequence of this portion was a major determinant for enzyme stability . Through this study, we have created porcine pepsin with increased pH stability at neutral pHs. Protein Eng, 2001 Sep, 14(9), 647 - 54 Generation of protein lineages with new sequence spaces by functional salvage screen; Kim GJ et al.; A variety of different methods to generate diverse proteins, including random mutagenesis and recombination, are currently available and most of them accumulate the mutations on the target gene of a protein, whose sequence space remains unchanged . On the other hand, a pool of diverse genes, which is generated by random insertions, deletions and exchange of the homologous domains with different lengths in the target gene, would present the protein lineages resulting in new fitness landscapes . Here we report a method to generate a pool of protein variants with different sequence spaces by employing green fluorescent protein (GFP) as a model protein . This process, designated functional salvage screen (FSS), comprises the following procedures: a defective GFP template expressing no fluorescence is first constructed by genetically disrupting a predetermined region(s) of the protein and a library of GFP variants is generated from the defective template by incorporating the randomly fragmented genomic DNA from Escherichia coli into the defined region(s) of the target gene, followed by screening of the functionally salvaged, fluorescence-emitting GFPs . Two approaches, sequence-directed and PCR-coupled methods, were attempted to generate the library of GFP variants with new sequences derived from the genomic segments of E.coli . The functionally salvaged GFPs were selected and analyzed in terms of the sequence space and functional properties . The results demonstrate that the functional salvage process not only can be a simple and effective method to create protein lineages with new sequence spaces, but also can be useful in elucidating the involvement of a specific region(s) or domain(s) in the structure and function of protein. Protein Eng, 2001 Sep, 14(9), 633 - 8 A cross-section of the fitness landscape of dihydrofolate reductase; Aita T et al.; In vitro molecular evolution is regarded as a hill-climbing on a fitness landscape in sequence space, where the 'fitness' is a quantitative measure of a certain physicochemical property of a biopolymer . We analyzed a 'cross-section' of the enzymatic activity landscape of dihydrofolate reductase (DHFR) by using a method of analysis of a fitness landscape . We limited the sequence space of interest to the five-dimensional sequence space, where the coordinate corresponds to the 1st, 16th, 20th, 42nd and 92nd site in the DHFR sequence . Thirty six mutants mapped into the limited sequence space were taken in the analysis . As a result, the cross-section is of the rough Mt Fuji type based on the mutational additivity . The ratio of the mean slope to the roughness is 2.8 and the Z-score of the original ratio against a distribution of random references is 7.0, which indicates a large statistical significance . The existence of such a cross-section was discussed in terms of the occurrence probability of sets of five sites distantly separated from each other on the DHFR 3D structure . Our results support the effectiveness of the evolution strategy which exploits the accumulation of advantageous single point mutations in such a cross-section. Protein Eng, 2001 Sep, 14(9), 609 - 14 Similarity of phylogenetic trees as indicator of protein-protein interaction; Pazos F et al.; Deciphering the network of protein interactions that underlines cellular operations has become one of the main tasks of proteomics and computational biology . Recently, a set of bioinformatics approaches has emerged for the prediction of possible interactions by combining sequence and genomic information . Even though the initial results are very promising, the current methods are still far from perfect . We propose here a new way of discovering possible protein-protein interactions based on the comparison of the evolutionary distances between the sequences of the associated protein families, an idea based on previous observations of correspondence between the phylogenetic trees of associated proteins in systems such as ligands and receptors . Here, we extend the approach to different test sets, including the statistical evaluation of their capacity to predict protein interactions . To demonstrate the possibilities of the system to perform large-scale predictions of interactions, we present the application to a collection of more than 67 000 pairs of E.coli proteins, of which 2742 are predicted to correspond to interacting proteins. J Cell Sci, 2001 Oct, 114(Pt 20), 3727 - 36 Nuclear localization of neutral sphingomyelinase 1: biochemical and immunocytochemical analyses; Mizutani Y et al.; To examine the intracellular localization of neutral sphingomyelinase 1 (nSMase 1), a rabbit polyclonal antibody was raised against a recombinant form of the enzyme expressed in E . coli . It has been reported that, in rat liver or in ascites hepatoma AH7974, high activity of neutral sphingomyelinase (SMase) is found at the plasma membrane, with a lesser but significant amount in nucleus and cytoplasm . The biochemical properties, dithiothreitol requirement and high salt concentration dependency, of cloned and expressed nSMase 1 resemble those of previously described nuclear neutral SMase of AH7974 . The present study was therefore focused on the nuclear localization of this enzyme . Western blotting of subcellular fractions using anti-rat nSMase 1 antibody revealed most nSMase 1 to be associated with the nuclei and some with microsomes, but not with plasma membranes . Consistently, neutral SMase activity in nuclear extract was immunoprecipitated by the antibody, while that of plasma membranes was not . The results indicate that nSMase 1 mainly resides in the nucleus and may thus differ from neutral SMase in plasma membrane . On gel-filtration column chromatography of nuclear extract, the profile of neutral SMase activity corresponded well with immunoreactive protein bands on western blotting, suggesting that a large part of nuclear neutral SMase may be nSMase 1 . Removal of the nuclear envelope by treatment with Triton X-100 did not significantly decrease the amount of nuclear nSMase 1, and western blotting of subnuclear fractions (i.e . nuclear envelope, chromatin, and nuclear matrix) revealed nSMase 1 signal exclusively in the nuclear matrix . Immunocytochemistry with AH7974, as well as rat fibroblast cell line 3Y1, demonstrated nSMase 1 to be localized mainly in the nucleus, with some in the cytoplasm . Moreover, immuno-electron microscopy clearly showed the signal of nSMase 1 to be more dense in the nucleus than in the cytoplasm of AH7974. EMBO J, 2001 Nov 15, 20(22), 6443 - 52 Regulation of nuclear poly(A) addition controls the expression of immunoglobulin M secretory mRNA; Phillips C et al.; B-cell differentiation is accompanied by a dramatic increase in cytoplasmic accumulation and stability of the IgM heavy chain (mu) secretory mRNA . Despite considerable effort, the mechanism is unknown . We have identified three short motifs upstream of the secretory poly(A) site, which, when mutated in the mu heavy chain gene, significantly increase the accumulation of the secretory form of poly(A)(+) mRNA relative to the membrane form and regulate the expression of the secretory poly(A) site in a developmental manner . We show that these motifs bind U1A and inhibit polyadenylation in vitro and in vivo . Overexpression of U1A in vivo results in the selective inhibition of the secretory form . Thus, this novel mechanism selectively controls post-cleavage expression of the mu secretory mRNA . We present evidence that this mechanism is used to regulate alternative expression of other genes. EMBO J, 2001 Nov 15, 20(22), 6371 - 82 Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint; Sironi L et al.; Mad2 is a key component of the spindle checkpoint, a device that controls the fidelity of chromosome segregation in mitosis . The ability of Mad2 to form oligomers in vitro has been correlated with its ability to block the cell cycle upon injection into Xenopus embryos . Here we show that Mad2 forms incompatible complexes with Mad1 and Cdc20, neither of which requires Mad2 oligomerization . A monomeric point mutant of Mad2 can sustain a cell cycle arrest of comparable strength to that of the wild-type protein . We show that the interaction of Mad2 with Mad1 is crucial for the localization of Mad2 to kinetochores, where Mad2 interacts with Cdc20 . We propose a model that features the kinetochore as a 'folding factory' for the formation of a Mad2-Cdc20 complex endowed with inhibitory activity on the anaphase promoting complex. EMBO J, 2001 Nov 15, 20(22), 6297 - 305 Co-chaperones Bag-1, Hop and Hsp40 regulate Hsc70 and Hsp90 interactions with wild-type or mutant p53; King FW et al.; Using highly purified proteins, we have identified intermediate reactions that lead to the assembly of molecular chaperone complexes with wild-type or mutant p53R175H protein . Hsp90 possesses higher affinity for wild-type p53 than for the conformational mutant p53R175H . The presence of Hsp90 in a complex with wild-type p53 inhibits the binding of Hsp40 and Hsc70 to p53, consequently preventing the formation of wild-type p53-multiple chaperone complexes . The conformational mutant p53R175H can form a stable heterocomplex with Hsp90 only in the presence of Hsc70, Hsp40, Hop and ATP . The anti-apoptotic factor Bag-1 can dissociate Hsp90 from a pre- assembled complex wild-type p53 protein, but it cannot dissociate a pre-assembled p53R175H-Hsp40- Hsc70-Hop-Hsp90 heterocomplex . The results presented here provide possible molecular mechanisms that can help to explain the observed in vivo role of molecular chaperones in the stabilization and cellular localization of wild-type and mutant p53 protein. EMBO J, 2001 Nov 15, 20(22), 6246 - 55 Apparent genetic redundancy facilitates ecological plasticity for nitrate transport; Unkles SE et al.; Aspergillus nidulans possesses two high-affinity nitrate transporters, encoded by the nrtA and the nrtB genes . Mutants expressing either gene grew normally on 1-10 mM nitrate as sole nitrogen source, whereas the double mutant failed to grow on nitrate concentrations up to 200 mM . These genes appear to be regulated coordinately in all growth conditions, growth stages and regulatory genetic backgrounds studied . Flux analysis of single gene mutants using 13NO3(-) revealed that K(m) values for the NrtA and NrtB transporters were approximately 100 and approximately 10 microM, respectively, while V(max) values, though variable according to age, were approximately 600 and approximately 100 nmol/mg dry weight/h, respectively, in young mycelia . This kinetic differentiation may provide the necessary physiological and ecological plasticity to acquire sufficient nitrate despite highly variable external concentrations . Our results suggest that genes involved in nitrate assimilation may be induced by extracellular sensing of ambient nitrate without obligatory entry into the cell. Gene, 2001 Oct 31, 278(1-2), 115 - 24 Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis; Kaps I et al.; The goal of this study was to establish a two-plasmid co-expression system for Mycobacterium smegmatis . Two vectors with compatible origins of replication and a polylinker, which allows modular cloning of promoters and genes, were constructed and used to clone genes encoding a blue fluorescent protein (BFP) and a green fluorescent protein (GFP) . A 160-fold variation of GFP expression levels in M . smegmatis was achieved by combining three promoters with different copy numbers of the vectors . An efficient energy transfer between BFP and GFP in M . smegmatis was observed by fluorescence measurements and demonstrated that these genes were simultaneously expressed from both vectors . Thus, these vectors will be valuable for all strategies where co-expression of proteins in M . smegmatis is needed, e.g . for constructing a two-hybrid system or for deleting essential genes. FEBS Lett, 2001 Nov 9, 508(1), 103 - 6 SecDFyajC is not required for the maintenance of the proton motive force; Nouwen N et al.; SecDFyajC of Escherichia coli is required for efficient export of proteins in vivo . However, the functional role of SecDFyajC in protein translocation is unclear . We evaluated the postulated function of SecDFyajC in the maintenance of the proton motive force . As previously reported, inner membrane vesicles (IMVs) lacking SecDFyajC are defective in the generation of a stable proton motive force when energized with succinate . This phenomenon is, however, not observed when NADH is used as an electron donor . Moreover, the proton motive force generated in SecDFyajC-depleted vesicles stimulated translocation to the same extent as seen with IMVs containing SecDFyajC . Further analysis demonstrates that the reduced proton motive force with succinate in IMVs lacking SecDFyajC is due to a lower amount of the enzyme succinate dehydrogenase . The expression of this enzyme complex is repressed by growth on glucose media, the condition used to deplete SecDFyajC . These results demonstrate that SecDFyajC is not required for proton motive force-driven protein translocation. J Am Chem Soc, 2001 Nov 21, 123(46), 11353 - 9 Biosynthesis of vitamin B(6) in yeast: incorporation pattern of glucose; Gupta RN et al.; Two yeasts, Saccharomyces cerevisiae ATCC 7752 and Candida utilis ATCC 9256, were incubated in the presence of variously multiply (13)C-labeled samples of D-glucose . The (13)C incorporation pattern within pyridoxamine dihydrochloride, established by (13)C NMR spectroscopy, differed from that which had previously been found within pyridoxine, isolated from Escherichia coli . Thus, the origin of the carbon skeleton of vitamin B(6) in yeast differs substantially from its origin in E . coli . In particular, in yeast the distribution of (13)C within the C(5) chain C-2',2,3,4,4' of pyridoxamine corresponds to the distribution of (13)C within the C(5) chain C-1,2,3,4,5 of the ribose component of cytidine . It follows that the C(5) chains of pyridoxamine and of ribose originate from a common glucose-derived pentulose or pentose intermediate . By contrast, in E . coli the C(5) chain of pyridoxine is derived from 1-deoxy-D-xylulose 5-phosphate which, in turn, originates by condensation of pyruvic acid with glyceraldehyde 3-phosphate. Plant Physiol, 2001 Nov, 127(3), 1299 - 309 Isolation and characterization of a new peroxiredoxin from poplar sieve tubes that uses either glutaredoxin or thioredoxin as a proton donor; Rouhier N et al.; A sequence coding for a peroxiredoxin (Prx) was isolated from a xylem/phloem cDNA library from Populus trichocarpa and subsequently inserted into an expression plasmid yielding the construction pET-Prx . The recombinant protein was produced in Escherichia coli cells and purified to homogeneity with a high yield . The poplar Prx is composed of 162 residues, a property that makes it the shortest plant Prx sequence isolated so far . It was shown that the protein is monomeric and possesses two conserved cysteines (Cys) . The Prx degrades hydrogen peroxide and alkyl hydroperoxides in the presence of an exogenous proton donor that can be either thioredoxin or glutaredoxin (Grx) . Based on this finding, we propose that the poplar protein represents a new type of Prx that differs from the so-called 2-Cys and 1-Cys Prx, a suggestion supported by the existence of natural fusion sequences constituted of a Prx motif coupled to a Grx motif . The protein was shown to be highly expressed in sieve tubes where thioredoxin h and Grx are also major proteins. Plant Physiol, 2001 Nov, 127(3), 1243 - 55 Arabidopsis dynamin-like 2 that binds specifically to phosphatidylinositol 4-phosphate assembles into a high-molecular weight complex in vivo and in vitro; Kim YW et al.; Arabadopsis dynamin-like (ADL) 2, a member of the high-molecular weight (M(r)) dynamin family found in Arabidopsis, has been shown to be targeted to the plastid . In the chloroplast, most of the ADL2 was present in the fraction containing the envelope membranes when analyzed by suborganellar fractionation . Sucrose gradient and gel filtration experiments showed that when associated with membranes, ADL2 existed as a high-M(r) complex, whereas the soluble form existed as a monomer . The recombinant ADL2 expressed in Escherichia coli was present as a high-M(r) form and showed higher GTPase activity at a low NaCl concentration, whereas ADL2 existed as a low-M(r) form with a low level of GTPase activity at a high NaCl concentration . Electron microscopy studies revealed that the purified recombinant ADL2 formed spiral-coiled structures or rings . In the presence of guanosine-5'-O-(3-thio)triphosphate, these structures were transformed into a long rod structure . In contrast, in the presence of GDP, these structures disassembled into oligomers that were shown to be tetramer with 4-fold symmetry . Finally, a lipid-binding assay revealed that recombinant ADL2 purified from E . coli bound specifically to phosphatidylinositol 4-phosphate . Together, these results demonstrated that the biochemical properties of ADL2 were very similar to those of dynamin and other related proteins . Based on this similarity, we propose that ADL2 may be involved in vesicle formation at the chloroplast envelope membrane. Plant Physiol, 2001 Nov, 127(3), 1224 - 33 Biochemical characterization of the Arabidopsis biotin synthase reaction . The importance of mitochondria in biotin synthesis; Picciocchi A et al.; Biotin synthase, encoded by the bio2 gene in Arabidopsis, catalyzes the final step in the biotin biosynthetic pathway . The development of radiochemical and biological detection methods allowed the first detection and accurate quantification of a plant biotin synthase activity, using protein extracts from bacteria overexpressing the Arabidopsis Bio2 protein . Under optimized conditions, the turnover number of the reaction was >2 h(-1) with this in vitro system . Purified Bio2 protein was not efficient by itself in supporting biotin synthesis . However, heterologous interactions between the plant Bio2 protein and bacterial accessory proteins yielded a functional biotin synthase complex . Biotin synthase in this heterologous system obeyed Michaelis-Menten kinetics with respect to dethiobiotin (K(m) = 30 microM) and exhibited a kinetic cooperativity with respect to S-adenosyl-methionine (Hill coefficient = 1.9; K(0.5) = 39 microM), an obligatory cofactor of the reaction . In vitro inhibition of biotin synthase activity by acidomycin, a structural analog of biotin, showed that biotin synthase reaction was the specific target of this inhibitor of biotin synthesis . It is important that combination experiments using purified Bio2 protein and extracts from pea (Pisum sativum) leaf or potato (Solanum tuberosum) organelles showed that only mitochondrial fractions could elicit biotin formation in the plant-reconstituted system . Our data demonstrated that one or more unidentified factors from mitochondrial matrix (pea and potato) and from mitochondrial membranes (pea), in addition to the Bio2 protein, are obligatory for the conversion of dethiobiotin to biotin, highlighting the importance of mitochondria in plant biotin synthesis. Plant Physiol, 2001 Nov, 127(3), 1102 - 12 A novel phospholipase D of Arabidopsis that is activated by oleic acid and associated with the plasma membrane; Wang C et al.; Oleate-dependent phospholipase D (PLD; EC 3.1.4.4) has been reported in animal systems, but its molecular nature is unkown . Multiple PLDs have been characterized in plants, but none of the previously cloned PLDs exhibits the oleate-activated activity . Here, we describe the biochemical and molecular identification and characterization of an oleate-activated PLD in Arabidopsis . This PLD, designated PLDdelta, was associated tightly with the plasma membrane, and its level of expression was higher in old leaves, stems, flowers, and roots than in young leaves and siliques . A cDNA encoding the oleate-activated PLD was identified, and catalytically active PLDdelta was expressed from its cDNA in Escherichia coli . PLDdelta was activated by free oleic acid in a dose-dependent manner, with the optimal concentration being 0.5 mM . Other unsaturated fatty acids, linoleic and linolenic acids, were less effective than oleic acid, whereas the saturated fatty acids, stearic and palmitic acids, were totally ineffective . Phosphatidylinositol 4,5-bisphosphate stimulated PLDdelta to a lesser extent than oleate . Mutation at arginine (Arg)-611 led to a differential loss of the phosphatidylinositol 4,5-bisphosphate-stimulated activity of PLDdelta, indicating that separate sites mediate the oleate regulation of PLDdelta . Oleate stimulated PLDdelta's binding to phosphatidylcholine . Mutation at Arg-399 resulted in a decrease in oleate binding by PLDdelta and a loss of PLDdelta activity . However, this mutation bound similar levels of phosphatidylcholine as wild type, suggesting that Arg-399 is not required for PC binding . These results provide the molecular information on oleate-activated PLD and also suggest a mechanism for the oleate stimulation of this enzyme. J Biol Chem, 2002 Feb 22, 277(8), 6073 - 9 Epub 2001 Nov 12. Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve . Binding specificity and role in PIKfyve . Endomenbrane localization; Sbrissa D et al.; PIKfyve is a phosphatidylinositol (PtdIns) 3-phosphate (P)-metabolizing enzyme, which, in addition to a C-terminally positioned catalytic domain, harbors several evolutionarily conserved domains, including a FYVE finger . The FYVE finger domains are thought to direct the protein localization to intracellular membrane PtdIns 3-P . Recent studies with several FYVE domain proteins challenge this general concept . Here we have examined the binding of PIKfyve's FYVE domain to PtdIns 3-P in vitro and in vivo and a plausible contribution of this binding mechanism for the intracellular localization of the full-length protein . We document now a specific and high affinity interaction of a recombinantly produced PIKfyve FYVE domain peptide fragment with PtdIns 3-P-containing liposomes that requires the presence of the conservative core of basic residues within the FYVE domain . PIKfyve localization to membranes of the late endocytic pathway was found to be absolutely dependent on the presence of an intact FYVE finger . Cell treatment with PI 3-kinase inhibitor wortmannin dissociated endosome-bound PIKfyve, indicating that the protein targeted the membrane PtdIns 3-P . An enzymatically inactive peptide fragment of the PIKfyve catalytic domain was found to also specifically bind to PtdIns 3-P-containing liposomes, with residue Lys-1999 being critical in the interaction . This binding, however, was of relatively low affinity and, in the cellular context, was found ineffective in directing the molecule to PtdIns 3-P-enriched endosomes . Collectively, these results demonstrate that interaction of the FYVE domain with PtdIns 3-P is absolutely necessary for PIKfyve targeting to the membranes of the late endocytic pathway and determine PIKfyve as a downstream effector of PtdIns 3-P. J Biol Chem, 2002 Jan 25, 277(4), 2547 - 53 Epub 2001 Nov 12. Complex formation of cytochrome P450cam with Putidaredoxin . Evidence for protein-specific interactions involving the proximal thiolate ligand; Unno M et al.; We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450(cam) and investigated the effects of diprotein complex formation with reduced putidaredoxin . We have found that the Fe-NO stretching mode of NO-P450(cam) can be resolved into two peaks at 551 and 561 cm(-1), and the binding of putidaredoxin increases the intensity of the high frequency component . Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond . In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation . On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds . We also find that cytochrome b(5) minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b(5) bind to the same or similar site on P450(cam) . These observations suggest that there is a key specific interaction between P450(cam) and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S --> Fe electron donation . These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine. J Biol Chem, 2002 Feb 15, 277(7), 4747 - 54 Epub 2001 Nov 12. Molecular characterization of the homo-phytochelatin synthase of soybean Glycine max: relation to phytochelatin synthase; Oven M et al.; The phytochelatin homologs homo-phytochelatins are heavy metal-binding peptides present in many legumes . To study the biosynthesis of these compounds, we have isolated and functionally expressed a cDNA GmhPCS1 encoding homo-phytochelatin synthase from Glycine max, a plant known to accumulate homo-phytochelatins rather than phytochelatins upon the exposure to heavy metals . The catalytic properties of GmhPCS1 were compared with the phytochelatin synthase AtPCS1 from Arabidopsis thaliana . When assayed only in the presence of glutathione, both enzymes catalyzed phytochelatin formation . GmhPCS1 accepted homoglutathione as the sole substrate for the synthesis of homo-phytochelatins whereas AtPCS1 did not . Homo-phytochelatin synthesis activity of both recombinant enzymes was significantly higher when glutathione was included in the reaction mixture . The incorporation of both glutathione and homoglutathione into homo-phytochelatin, n = 2, was demonstrated using GmhPCS1 and AtPCS1 . In addition to bis(glutathionato)-metal complexes, various other metal-thiolates were shown to contribute to the activation of phytochelatin synthase . These complexes were not accepted as substrates by the enzyme, thereby suggesting that a recently proposed model of activation cannot fully explain the catalytic mechanism of phytochelatin synthase (Vatamaniuk, O . K., Mari, S., Lu, Y . P., and Rea, P . A . (2000) J . Biol . Chem . 275, 31451-31459). J Biol Chem, 2002 Feb 1, 277(5), 3210 - 8 Epub 2001 Nov 12. Priming of macrophages with lipopolysaccharide potentiates P2X7-mediated cell death via a caspase-1-dependent mechanism, independently of cytokine production; Le Feuvre RA et al.; ATP stimulation of cell surface P2X7 receptors results in cytolysis and cell death of macrophages . Activation of this receptor in bacterial lipopolysaccharide (LPS)-activated macrophages or monocytes also stimulates processing and release of the cytokine interleukin-1beta(IL-1beta) through activation of caspase-1 . The cytokine interleukin 18 (IL-18) is also cleaved by caspase-1 and shares pro-inflammatory characteristics with IL-1beta . The objective of the present study was to test the hypothesis that IL-1beta, IL-18, and/or caspase-1 activation contribute directly to macrophage cell death induced by LPS and ATP . Macrophages were cultured from normal mice or those in which genes for the P2X7 receptor, IL-1beta, IL-1alpha, IL-18, or caspase-1 had been deleted . Our data confirm the importance of the P2X7 receptor in ATP-stimulated cell death and IL-1beta release from LPS-primed macrophages . We demonstrate that prolonged stimulation with ATP leads to cell death, which is partly dependent on LPS priming and caspase-1, but independent of cytokine processing and release . We also provide evidence that LPS priming of macrophages makes them highly susceptible to the toxic effects of brief exposure to ATP, which leads to rapid cell death by a mechanism that is dependent on caspase-1 but, again, independent of cytokine processing and release. J Biol Chem, 2002 Feb 1, 277(5), 3202 - 9 Epub 2001 Nov 08. Neither Reb1p nor poly(dA*T) elements are responsible for the highly specific chromatin organization at the ILV1 promoter; Moreira JM et al.; Analysis of the chromatin structure at the yeast ILV1 locus revealed highly positioned nucleosomes covering the entire locus except for a hypersensitive site in the promoter region . All previously identified cis-acting elements required for GCN4-independent ILV1 basal level transcription, including a binding site for the REB1 protein (Reb1p), and a poly(dA*dT) element (26 As out of 32 nucleotides) situated 15 base pairs downstream of the Reb1p-binding site, reside within this hypersensitive site . The existence of a second A*T-rich element (25 As out of 33 nucleotides) present six base pairs upstream of the Reb1p-binding site, suggested that nucleosome exclusion from the hypersensitive site in the ILV1 promoter region might be dictated by synergistic action of the two poly(dA*dT) elements . Replacing one or both of them had, however, no effect on the chromatin structure of the ILV1 promoter, although drastically reduced basal transcription . Similarly, deletion of the Reb1p-binding site, albeit affecting ILV1 expression, had no detectable effect on chromatin at the ILV1 promoter . The absence of a good correlation between effects of these elements on gene activity and on chromatin structure at the ILV1 promoter indicates that the chromatin organization present at the ILV1 promoter is independent of the known regulatory elements and most likely dictated directly by the DNA sequence. J Biol Chem, 2002 Jan 11, 277(2), 1255 - 60 Epub 2001 Nov 08. MutS preferentially recognizes cisplatin- over oxaliplatin-modified DNA; Zdraveski ZZ et al.; Loss of mismatch repair leads to tumor resistance by desensitizing cells to specific DNA-damaging agents, including the anticancer drug cisplatin . Cisplatin analogs with a diamminocyclohexane (DACH) carrier ligand, such as oxaliplatin and Pt(DACH)Cl(2), do not elicit resistance in mismatch repair-deficient cells and therefore present promising therapeutic agents . This study compared the interactions of the purified Escherichia coli mismatch repair protein MutS with DNA modified to contain cisplatin and DACH adducts . MutS recognized the cisplatin-modified DNA with 2-fold higher affinity in comparison to the DACH-modified DNA . ADP stimulated the binding of MutS to cisplatin-modified DNA, whereas it had no effect on the MutS interaction with DNA modified by DACH or EN adducts . In parallel cytotoxicity experiments, methylation-deficient E . coli dam mutants were 2-fold more sensitive to cisplatin than DACH compounds . A panel of recombination-deficient mutants showed striking sensitivity to both compounds, indicating that both types of adducts are strong replication blocks . The differential affinity of MutS for DNA modified with the different platinum analogs could provide the molecular basis for the distinctive cellular responses to cisplatin and oxaliplatin. Infect Immun, 2001 Dec, 69(12), 7941 - 5 Characterization of the adhesin of Escherichia coli F18 fimbriae; Smeds A et al.; Previous research has suggested that the adhesin encoded by the F18 fimbrial operon in Escherichia coli is either the FedE or FedF protein . In this work, we show that anti-FedF antibodies, unlike anti-FedE serum, were able to inhibit E . coli adhesion to porcine enterocytes . Moreover, specific adhesion to enterocytes was shown with purified FedF-maltose binding protein. Infect Immun, 2001 Dec, 69(12), 7793 - 9 Dynamic changes in neutrophil defensins during endotoxemia; Klut ME et al.; Bacterial endotoxin or lipopolysaccharide (LPS) is an important causative agent of sepsis . This study determines the expression of defensins NP-2 and NP-5 and the function of polymorphonuclear leukocytes (PMN) in rabbits treated with LPS . PMN functional activity was assessed by measuring CD18 expression and H(2)O(2) production and by examining the lungs . NP-2 and, to a minor extent, NP-5 of circulating PMN increase during endotoxemia . This early increase is concomitant with neutrophilia and elevated CD18 expression and H(2)O(2) production, as well as with enhanced NP-2 immunoreactivity in pulmonary microvessels . A decline in defensins, shortly after the last LPS treatment, is associated with a decrease in the circulating activated PMN and enhanced immunoreactivity in the inflammatory cells, as well as with lung tissue damage . This study shows that LPS-induced changes in the defensins of circulating PMN correlate with the number and activated condition of these cells and suggests that PMN-derived products implement the inflammatory reaction that leads to lung injury and sepsis. Infect Immun, 2001 Dec, 69(12), 7616 - 24 Molecular characterization of thermoinduced immunogenic proteins Q1p42 and Hsp15 of Leptospira interrogans; Nally JE et al.; Leptospira interrogans is a mammalian pathogen which must adapt to a range of new environmental conditions including temperature change when it infects new hosts . In vitro studies of organisms cultured at 30 degrees C and shifted to 37 degrees C for 5 to 7 days have confirmed that synthesis of several proteins involved in equine infection is regulated in response to temperature change (J . E . Nally, J . F . Timoney, and B . Stevenson, Infect . Immun . 69:400-404, 2001) . In order to specifically identify antigenic proteins upregulated at 37 degrees C, groups of three ponies were immunized with organisms shifted to 37 degrees C for 5 to 7 days or with organisms maintained at 30 degrees C . A lambda ZAP II genomic DNA library was screened with the pool of antisera to organisms shifted to 37 degrees C . Clones reactive with this pool but unreactive with the pool of pony antisera to organisms cultured at 30 degrees C were selected for further analysis . Sequence analysis of the first two clones identified open reading frames for proteins designated Qlp42 and Hsp15 . Qlp42 is predicted to be an outer membrane lipoprotein . Its synthesis was upregulated when cultures were shifted from 30 to 37 degrees C and downregulated when cultures were shifted from 37 to 30 degrees C . Although the predicted molecular mass of Qlp42 is 39.8 kDa for the mature protein, Qlp42-specific equine antiserum was reactive with two bands of 30 and 29.5 kDa . Hsp15 is a stress response protein and a member of the Hsp20/alpha-crystallin family . PCR detected homologues of qlp42 and hsp15 in pathogenic serovars of L . interrogans but not in the nonpathogenic Leptospira biflexa . Enzyme-linked immunosorbent assays of antibody in convalescent sera from mares naturally infected with L . interrogans suggest that Qlp42 is expressed during leptospiral infection. Infect Immun, 2001 Dec, 69(12), 7356 - 64 Induction of epithelial cell death including apoptosis by enteropathogenic Escherichia coli expressing bundle-forming pili; Abul-Milh M et al.; Infection with enteropathogenic Escherichia coli (EPEC) is a major cause of severe infantile diarrhea, particularly in parts of the developing world . The bundle-forming pilus (BFP) of EPEC is an established virulence factor encoded on the EPEC adherence factor plasmid (EAF) and has been implicated in both localized adherence to host cells and bacterial autoaggregation . We investigated the role of BFP in the ability of EPEC binding to kill host epithelial cells . BFP-expressing strains killed all three cell lines tested, comprising HEp-2 (laryngeal), HeLa (cervical), and Caco-2 (colonic) cells . Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA, and morphological changes detected by electron microscopy indicated evidence of apoptosis . The extent of cell death was significantly greater for BFP-expressing strains, including E2348/69, a wild-type clinical isolate, as well as for a laboratory strain, HB101, transformed with a bfp-carrying plasmid . Strains which did not express BFP induced significantly less cell death, including a bfpA disruptional mutant of E2348/69, EAF plasmid-cured E2348/69, HB101, and HB101 complemented with the locus of enterocyte effacement pathogenicity island . These results indicate a direct correlation between BFP expression and induction of cell death, including apoptosis, an event which may involve the targeting of host cell membrane phosphatidylethanolamine. Infect Immun, 2001 Dec, 69(12), 7205 - 12 Characterization of receptor-mediated signal transduction by Escherichia coli type IIa heat-labile enterotoxin in the polarized human intestinal cell line T84; Wimer-Mackin S et al.; Escherichia coli type IIa heat-labile enterotoxin (LTIIa) binds in vitro with highest affinity to ganglioside GD1b . It also binds in vitro with lower affinity to several other oligosialogangliosides and to ganglioside GM1, the functional receptor for cholera toxin (CT) . In the present study, we characterized receptor-mediated signal transduction by LTIIa in the cultured T84 cell model of human intestinal epithelium . Wild-type LTIIa bound tightly to the apical surface of polarized T84 cell monolayers and elicited a Cl(-) secretory response . LTIIa activity, unlike CT activity, was not blocked by the B subunit of CT . Furthermore, an LTIIa variant with a T14I substitution in its B subunit, which binds in vitro to ganglioside GM1 but not to ganglioside GD1b, was unable to bind to intact T84 cells and did not elicit a Cl(-) secretory response . These findings show that ganglioside GM1 on T84 cells is not a functional receptor for LTIIa . The LTIIa receptor on T84 cells was inactivated by treatment with neuraminidase . Furthermore, LTIIa binding was blocked by tetanus toxin C fragment, which binds to gangliosides GD1b and GT1b . These findings support the hypothesis that ganglioside GD1b, or possibly a glycoconjugate with a GD1b-like oligosaccharide, is the functional receptor for LTIIa on T84 cells . The LTIIa-receptor complexes from T84 cells were associated with detergent-insoluble membrane microdomains (lipid rafts), extending the correlation between toxin binding to lipid rafts and toxin function that was previously established for CT . However, the extent of association with lipid rafts and the magnitude of the Cl(-) secretory response in T84 cells were less for LTIIa than for CT . These properties of LTIIa and the previous finding that enterotoxin LTIIb binds to T84 cells but does not associate with lipid rafts or elicit a Cl(-) secretory response may explain the low pathogenicity for humans of type II enterotoxin-producing isolates of E . coli. Am J Physiol Gastrointest Liver Physiol, 2001 Dec, 281(6), G1423 - 31 Hepatic and extrahepatic factors critical for liver injury during lipopolysaccharide exposure; Moulin F et al.; Bacterial endotoxin {lipopolysaccharide (LPS)} causes liver injury in vivo that is dependent on platelets, neutrophils {polymorphonuclear leukocytes (PMNs)}, and several inflammatory mediators, including thrombin . We tested the hypothesis that thrombin contributes to LPS-induced hepatocellular injury through direct interactions with platelets and/or PMNs in vitro . Perfusion of isolated livers from LPS-treated rats with buffer containing thrombin resulted in a significant increase in alanine aminotransferase (ALT) activity in the perfusion medium, indicating hepatocellular damage . This effect was completely abolished by prior depletion of PMNs from the LPS-treated donor rats but not by depletion of platelets, suggesting interaction between thrombin and PMNs in the pathogenesis . Thrombin did not, however, enhance degranulation of rat PMNs in vitro, and it was not directly toxic to isolated rat hepatocytes in the presence of PMNs even after LPS exposure, suggesting that hepatocellular killing by the PMN-thrombin combination requires the intervention of an additional factor(s) within the liver . In livers from naive donors perfused with buffer containing PMNs and LPS, no injury occurred in the absence of thrombin . Addition of thrombin (10 nM) to the medium caused pronounced ALT release . These results indicate that thrombin and PMNs are sufficient extrahepatic requirements for LPS-induced hepatocellular damage in intact liver. Free Radic Biol Med, 2001 Nov 15, 31(10), 1170 - 8 Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione-platinum complex; Arner ES et al.; Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum (II) (cisplatin, CDDP) and inhibited intracellular activity of the thioredoxin system, i.e., thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH . Conversely, increased cellular activity of the Trx system confers resistance to CDDP . In this study, we have analyzed the interaction of CDDP with Trx and TrxR in order to clarify the mechanism . The inhibition with time-dependent kinetics by CDDP of NADPH-reduced (but not oxidized) TrxR was irreversible, strongly suggesting covalent modification of the reduced selenocysteine-containing active site . Assuming second order kinetics, the rate constant of TrxR inhibition by CDDP was 21 +/- 3 M(-1) x s(-1) . Transplatin was found to be an even more efficient inhibitor, with a second order rate constant of 84 +/- 22 M(-1) x s(-1), whereas carboplatin (up to 1 mM) gave no inhibition of the enzyme under the same conditions . Escherichia coli Trx or human or bacterial glutaredoxin (Grx) activities were in comparison only slightly or not at all inhibited by either CDDP, transplatin, or carboplatin . However, glutaredoxins were found to be inhibited by the purified glutathione adduct of cisplatin, bis-(glutathionato)platinum(II) (GS-Platinum complex, GS-Pt), with an IC50 = 350 microM in the standard beta-hydroxyethyl disulfide-coupled assay for human Grx . Also the mammalian Trx system was inhibited by GS-Pt with similar efficiency (IC(50) = 325 microM), whereas neither the E . coli Trx system nor glutathione reductase were inhibited . Formation of GS-Pt is a major route for cellular elimination of CDDP . The fact that GS-Pt inhibits the mammalian Trx as well as Grx systems shows that CDDP may exert effects at several stages of its metabolism, including after conjugation with GSH, which are intimately linked with the cellular disulfide/dithiol redox regulatory systems. Vet Parasitol, 2001 Dec 3, 102(1-2), 35 - 44 Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1; Kimbita EN et al.; The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T . gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E . coli) as a glutathione-S-transferase (GST) fusion protein . The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis . The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein . The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T . gondii and sera from normal cats or mice . The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N . caninum) . Some 193 cat sera were tested for antibodies to T . gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay . Both positive and negative sera were confirmed by Western blot analysis . The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter. Biochemistry, 2001 Nov 20, 40(46), 14098 - 105 Substrate recognition by a yeast 2'-phosphotransferase involved in tRNA splicing and by its Escherichia coli homolog; Steiger MA et al.; The final step of tRNA splicing in Saccharomyces cerevisiae requires 2'-phosphotransferase (Tpt1) to transfer the 2'-phosphate from ligated tRNA to NAD, producing mature tRNA and ADP ribose-1' '-2' '-cyclic phosphate . To address how Tpt1 protein recognizes substrate RNAs, we measured the steady-state kinetic parameters of Tpt1 protein with 2'-phosphorylated ligated tRNA and a variety of related substrates . Tpt1 protein has a high apparent affinity for ligated tRNA (K(m,RNA), 0.35 nM) and a low turnover rate (k(cat), 0.3 min(-1)) . Tpt1 protein recognizes both tRNA and the internal 2'-phosphate of RNAs . Steady-state kinetic analysis reveals that as RNAs lose structure and length, K(m,RNA) and k(cat) both increase commensurately . For a 2'-phosphorylated octadecamer derived from the anticodon stem-loop of ligated tRNA, K(m,RNA) and k(cat) are 5- and 8-fold higher, respectively, than for ligated tRNA, whereas for a simple substrate like pApA(p)pA, K(m,RNA) and k(cat) are 430- and 150-fold higher, respectively . Tpt1 is not detectably active on a trimer with a terminal 5'- or 3'-phosphate and is very inefficient at removal of a terminal 2'-phosphate unless there is an adjacent 3'-phosphate or phosphodiester . The K(m,NAD) for Tpt1 is substrate dependent: K(m,NAD) is 10 microM with ligated tRNA, 200 microM with pApA(p)pA, and 600 microM with pApApA(p) . Preliminary analysis of KptA, a functional Tpt1 protein homologue from Escherichia coli, reveals that KptA protein is strikingly similar to yeast Tpt1 in its kinetic parameters, although E . coli is not known to have a 2'-phosphorylated RNA substrate. Biochemistry, 2001 Nov 20, 40(46), 14069 - 80 IscA, an alternate scaffold for Fe-S cluster biosynthesis; Krebs C et al.; An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity . Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form . Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, Mossbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies . Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins . Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile {4Fe-4S}(2+) cluster per (Nif)IscA homodimer, via a transient {2Fe-2S}(2+) cluster intermediate . The cluster assembly process was monitored temporally using UV-vis absorption and Mossbauer spectroscopy, and the intermediate {2Fe-2S}(2+)-containing species was additionally characterized by resonance Raman spectroscopy . The Mossbauer and resonance Raman properties of the {2Fe-2S}(2+) center are consistent with complete cysteinyl ligation . The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one {2Fe-2S}(2+) or one {4Fe-4S}(2+) per homodimer suggest that both cluster types are subunit bridging . In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur . The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions . On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed . It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly. Biochemistry, 2001 Nov 20, 40(46), 13964 - 71 Transport-defective mutations alter the conformation of the energy-coupling motif of an outer membrane transporter; Coggshall KA et al.; The bacterial outer membrane transporter for vitamin B(12), BtuB, derives its energy for transport by interacting with the trans-periplasmic membrane protein TonB . This interaction with TonB occurs in part through an N-terminal segment in the BtuB sequence called the Ton box . In the present study, site-directed spin labeling of intact outer membrane preparations was used to investigate the conformation of the Ton box in wild-type BtuB and in two transport-defective mutants, L8P and V10P . In the wild-type protein, the Ton box is folded into the barrel of the transporter . The conformation of this segment is dramatically different in the transport-defective mutants L8P and V10P, where the Ton box is found to be flexible, and undocked from the transporter barrel with a greater exposure to the periplasm . In the wild-type protein, vitamin B(12) induces an undocking of the Ton box, but its addition to these transport defective mutants produces little or no change in the conformation of the Ton box . Proline substitutions at positions that do not alter transport do not alter the wild-type conformation of the Ton box; thus, the effect of substituting proline at positions 8 and 10 on the docked state of the Ton box appears to be unique . The failure of these mutants to execute the B(12) transport cycle may be a result of the altered conformation of the Ton box. Biochemistry, 2001 Nov 20, 40(46), 13925 - 32 Differential regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase isoforms; Tokumitsu H et al.; We have previously demonstrated that the alpha isoform of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KKalpha) is strictly regulated by an autoinhibitory mechanism and activated by the binding of Ca(2+)/CaM {Tokumitsu, H., Muramatsu, M., Ikura, M., and Kobayashi, R . (2000) J . Biol . Chem . 275, 20090-20095} . In this study, we find that rat brain extract contains Ca(2+)/CaM-independent CaM-KK activity . This result is consistent with an enhanced Ca(2+)/CaM-independent activity (60-70% of total activity) observed with the recombinant CaM-KKbeta isoform . By using various truncation mutants of CaM-KKbeta, we have identified a region of 23 amino acids (residues 129-151) located at the N-terminus of the catalytic domain as an important regulatory element of the autonomous activity . A CaM-KKbeta deletion mutant of this domain shows a significant increase of Ca(2+)/CaM dependency for the CaM-KK activity as well as for the autophosphorylation activity . The activities of CaM-KKalpha and CaM-KKbeta chimera, in which autoinhibitory sequences were replaced by each other, were completely dependent on Ca(2+)/CaM, suggesting that the autoinhibitory regions of CaM-KKalpha and CaM-KKbeta are functional . These results establish for the first time that residues 129-151 of CaM-KKbeta participate in the release of the autoinhibitory domain from its catalytic core, resulting in generation of autonomous activity. Biochemistry, 2001 Nov 20, 40(46), 13876 - 87 Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli; Hirschman A et al.; We investigated the functional roles of putative active site residues in Escherichia coli CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokinase activity and binding affinity for ATP . We designed these mutations to alter key positions in sequence motifs conserved in the protein histidine kinase family, including the N box (H376 and N380), the G1 box (D420 and G422), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the "GT block" (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases . Four of the mutant CheA proteins exhibited no detectable autokinase activity (Kin(-)) . Of these, three (N380D, D420N, and G422A) exhibited moderate decreases in their affinities for ATP in the presence or absence of Mg(2+) . The other Kin(-) mutant (G470A/G472A/G474A) exhibited wild-type affinity for ATP in the absence of Mg(2+), but reduced affinity (relative to that of wild-type CheA) in the presence of Mg(2+) . The other five mutants (Kin(+)) autophosphorylated at rates slower than that exhibited by wild-type CheA . Of these, three mutants (H376Q, D420E, and F455Y/F459Y) exhibited severely reduced k(cat) values, but preserved K(M)(ATP) and K(d)(ATP) values close to those of wild-type CheA . Two mutants (T499S and T499A) exhibited only small effects on k(cat) and K(M)(ATP) . Overall, these results suggest that conserved residues in the N box, G1 box, G2 box, and F box contribute to the ATP binding site and autokinase active site in CheA, while the GT block makes little, if any, contribution . We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to binding interactions that contribute to the stability of the complex between CheA and Mg(2+)-bound ATP in both the ground state and the transition state for the CheA autophosphorylation reaction. Biochemistry, 2001 Nov 20, 40(46), 13833 - 9 A GCN4 variant with a C-terminal basic region binds to DNA with wild-type affinity; Hollenbeck JJ et al.; Basic-region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a leucine zipper dimerization domain and a basic region that directly contacts DNA . In all naturally occurring bZip proteins, the basic region is positioned N-terminal to the leucine zipper . We have designed a series of model bZip peptides in which the basic region of the yeast transcriptional activator GCN4 is placed C-terminal to its leucine zipper . DNA-binding studies demonstrate that the optimal reverse GCN4 (rGCN4) peptide is able to bind specifically and with wild-type affinity to DNA despite this unnatural arrangement of the two subdomains . These results suggest that a thermodynamic basis for the observed N-terminal positioning of the basic region relative to the dimerization domain is unlikely. Oncogene, 2001 Oct 25, 20(48), 7096 - 7 Induction of endogenous beta-galactosidase by ionizing radiation complicates the analysis of p53-LacZ transgenic mice; Coates PJ et al.; Studies of the response of p53-lacZ transgenic mice have uncovered an unexpected induction of endogenous acid-beta-galactosidase activity following whole body irradiation . Strong induction of endogenous enzyme activity is seen in a variety of mouse strains commonly used in the production of transgenes . The induction of endogenous enzyme activity therefore complicates the analysis of p53-lacZ transgenes and may also influence the analysis of radiation responses in other lacZ-reporter mice. J Biol Chem, 2002 Jan 18, 277(3), 1749 - 54 Epub 2001 Nov 09. Miscoordination of the iron-sulfur clusters of the anaerobic transcription factor, FNR, allows simple repression but not activation; Scott C et al.; The FNR protein of Escherichia coli regulates target genes in response to anaerobiosis . Environmental oxygen is sensed by the acquisition of oxygen-labile {4Fe-4S} clusters that promote dimerization, DNA binding, and productive interactions with RNA polymerase . Three N-terminal cysteine residues (Cys(20), Cys(23), and Cys(29)) and Cys(122) act as ligands for the FNR iron sulfur clusters . An FNR variant, FNR-C20S, that retains only trace activity in vivo can acquire {4Fe-4S} clusters in vitro that enhance site-specific DNA binding . Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivo activity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively . Here we show that FNR-C20S can repress a simple FNR-regulated promoter in vivo and that it can form productive heterodimers with an FNR variant with altered DNA binding specificity, FNR-E209V . Transcription studies with FNR-E209V.FNR-C20S heterodimers indicate that the presence of a miscoordinated iron-sulfur cluster (FNR-C20S) in the downstream (but not the upstream) subunit of the FNR dimer impairs activation from a class II promoter and that this impairment can be overcome by amino acid substitutions known to unmask AR2 or improve AR3 in the affected subunit. Vox Sang, 2001 Oct, 81(3), 194 - 8 Isolation of human scFv antibody fragments against ABO blood group antigens from a phage display library; Santos-Esteban E et al.; BACKGROUND AND OBJECTIVES: Phage display has proven very useful for the isolation of antibodies against a number of antigens . We used this technology to isolate scFv antibody fragments against A and B red blood cell antigens . MATERIALS AND METHODS: Phages from a phage display library were selected using unmodified red blood cells as a source of antigen . Bound phages were absorbed onto cells lacking the antigen of interest and used to infect Escherichia coli cells . Phages were rescued and assayed for specificity by enzyme-linked immunosorbent assay (ELISA) . RESULTS: After several rounds of panning and subtraction on red blood cells, one anti-A and one anti-B human scFv antibody fragments were isolated . CONCLUSION: Isolation of anti-A and anti-B scFv antibody fragments on whole cells is an alternative method of obtaining antibodies against native cell-surface antigens. Mol Microbiol, 2001 Oct, 42(2), 519 - 26 The RepA protein of plasmid pSC101 controls Escherichia coli cell division through the SOS response; Ingmer H et al.; Although plasmid copy number varies widely among different plasmid species, normally copy number is maintained within a narrow range for any given plasmid . Such copy number control has been shown to occur by regulation of the rate of plasmid DNA replication . Here we report a novel mechanism by which the pSC101 plasmid also can detect an imbalance between the cellular level of its replication protein, RepA, and plasmid-borne RepA binding sites to inhibit bacterial DNA replication and delay host cell division when RepA is in relative excess . We show that delayed cell division occurs by RepA-mediated induction of the SOS response and can be reversed by over-expression of the host DNA primase, DnaG . The effects of RepA excess are prevented by introducing a surfeit of RepA binding sites . The mechanism reported here may help to limit variation in plasmid copy number and allow repopulation of cells with plasmids when copy number falls--potentially pre-empting plasmid loss in cultures of dividing cells. Mol Microbiol, 2001 Oct, 42(2), 483 - 94 Orientational control of fimE expression in Escherichia coli; Sohanpal BK et al.; Phase-variable expression of type 1 fimbriae is, in part, controlled by site-specific DNA inversion of the fim switch in Escherichia coli . Of the two fim recombinases (FimB and FimE) that catalyse the inversion reaction, FimE exhibits a strong bias for phase switching from the ON to the OFF orientation . The specificity associated with fimE is the result of two different mechanisms: (i) FimE exhibits a preference for the invertible element in the ON orientation as substrate for recombination; (ii) the invertible element in the OFF orientation acts in cis to inhibit recombinase activity (orientational control) . We show here that the invertible element negatively regulates fimE, even though expression of a fimE-lacZYA transcriptional fusion is unaffected by orientational control . The fimE transcript extends into the invertible region and hence switch ON-specific and switch OFF-specific mRNA contain different sequences . Furthermore, we show that orientational control is suppressed by the insertion of a structured RNA (tRNA(Gly)) between fimE and the fim switch, indicating that the switch OFF-specific mRNA is inactivated by 3' to 5' degradation . Analysis of the fim switch reveals that it contains two inhibitory elements that exert orientational control independently. Mol Microbiol, 2001 Oct, 42(2), 331 - 44 Import of colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasm; Journet L et al.; Several proteins of the Tol/Pal system are required for group A colicin import into Escherichia coli . Colicin A interacts with TolA and TolB via distinct regions of its N-terminal domain . Both interactions are required for colicin translocation . Using in vivo and in vitro approaches, we show in this study that colicin A also interacts with a third component of the Tol/Pal system required for colicin import, TolR . This interaction is specific to colicins dependent on TolR for their translocation, strongly suggesting a direct involvement of the interaction in the colicin translocation step . TolR is anchored to the inner membrane by a single transmembrane segment and protrudes into the periplasm . The interaction involves part of the periplasmic domain of TolR and a small region of the colicin A N-terminal domain . This region and the other regions responsible for the interaction with TolA and TolB have been mapped precisely within the colicin A N-terminal domain and appear to be arranged linearly in the colicin sequence . Multiple contacts with periplasmic-exposed Tol proteins are therefore a general principle required for group A colicin translocation. Biochemistry (Mosc), 2001 Sep, 66(9), 984 - 8 Activation of the Escherichia coli SoxRS-regulon by nitric oxide and its physiological donors; Vasil'eva SV et al.; Activation of the Escherichia coli SoxRS-regulon by nitric oxide (NO) and its physiological donors (S-nitrosothiol (GS-NO) and dinitrosyl iron complexes with glutathione (DNIC(glu)) and cysteine (DNIC(cys)) ligands) has been studied . To elucidate the molecular mechanisms of signal transduction via nitrosylation of Fe-S-centers in SoxR, the ability of pure NO and NO-producing agents to activate the SoxRS-regulon in E . coli cells bearing a soxS::lacZ operon (promoter) fusion has been compared . EPR spectroscopy of whole cells has been used to monitor the formation of inducible protein-DNIC complexes . DNIC(cys), GS-NO, and pure NO appeared to be potent inducers of soxS expression, whereas DNIC(glu) was considerably less efficient . Thus, lower in vitro stability of DNIC(cys) was in contrast with its higher biological activity . Pretreatment of the cells with o-phenanthroline, a chelating agent for iron, prevented soxS expression by GS-NO . Treatment of intact E . coli cells with DNIC, GS-NO, and NO at equimolar concentration 150 microM resulted in formation of a single EPR-detectable DNIC-type signal with g = 2.03 . The initial stage in the SoxR transcription activity is supposed to include two steps: first, DNIC primers are formed from exogenous NO and free iron, and then these DNIC disintegrate SoxR {2Fe-2S} clusters and thus activate SoxRS-regulon transcription. Biochemistry (Mosc), 2001 Sep, 66(9), 973 - 8 Role of glutathione in the response of Escherichia coli to osmotic stress; Smirnova GV et al.; The growth of Escherichia coli mutants deficient in glutathione synthesis (gshA) and in glutathione reductase (gor) was suppressed in medium of elevated osmolarity . A mutant in gamma-glutamyl transpeptidase (ggt) displayed better ability for osmoadaptation than the parental strain . The unfavorable effect of the gsh mutation on osmoadaptation of growing E . coli cells was more pronounced at low concentrations of K+ in the medium . An increase in osmolarity caused an increase in the intracellular content of glutathione . Changes in the extracellular glutathione level were biphasic: the glutathione level rapidly decreased during the first stage of the response and increased during the second stage . The changes in glutathione levels suggest that under hyperosmotic shock the glutathione transport from the medium into the cell can contribute to the intracellular glutathione accumulation . Changes in the level of intracellular K+ were similarly biphasic: a rapid increase in the K+ level during the first stage of the response to hyperosmotic shock changed to a gradual decrease during the second stage . In mutant gshA cells adapted to osmotic shock, the intracellular K+ level was markedly higher than in the parental strain cells . The possible role of glutathione in the response of E . coli to osmotic shock is discussed. Dev Cell, 2001 Aug, 1(2), 187 - 99 Two zinc finger proteins, OMA-1 and OMA-2, are redundantly required for oocyte maturation in C . elegans; Detwiler MR et al.; Oocytes are released from meiotic prophase I arrest through a process termed oocyte maturation . We present here a genetic characterization of oocyte maturation, using C . elegans as a model system . We show that two TIS11 zinc finger-containing proteins, OMA-1 and OMA-2, express specifically in maturing oocytes and function redundantly in oocyte maturation . Oocytes in oma-1;oma-2 mutants initiate but do not complete maturation and arrest at a defined point in prophase I . Two maturation signal-induced molecular events, including the maintenance of activated MAP kinase, do not occur in Oma oocytes . The Oma prophase arrest is released by inactivation of a MYT-1-like kinase, suggesting that OMA-1 and OMA-2 function upstream of MYT-1 as positive regulators of prophase progression during meiotic maturation. DNA Seq, 2001 Jul, 12(1), 39 - 51 Molecular cloning, sequencing analysis and expression of the catalase-peroxidase gene from Halobacterium salinarum; Long S et al.; The gene encoding catalase-peroxidase was cloned from chromosomal DNA from the Archaea, Halobacterium salinarum . The nucleotide sequence of a 3.5 kb fragment, containing the catalase-peroxidase gene and its flanking regions was determined . A 2.16 kb open reading frame was obtained, encoding the enzyme which was comprised of 720 amino acid residues with a calculated molecular weight of 80 kDa . The deduced amino acid sequence of the H . salinarum catalase-peroxidase showed a high degree of identity to other bifunctional catalase-peroxidases . A transcriptional start site was identified 183 bp upstream of the translational start codon . Southern blot analysis indicated that catalase-peroxidase was a single copy gene . The Archaeal catalase-peroxidase gene was expressed in Escherichia coli, and the expressed fusion protein exhibited both catalase and peroxidase activities. Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 406 - 9 {Constructing and expression of three zinc-fingers peptide with specific DNA recognition property in Escherichia coli}; Zhang SX et al.; For investigating the DNA binding property of classical zinc finger protein Zif268, an in vivo transcription interference experiment was once utilized to develop a genetic selection assay . By screening a library in which the key amino acids of the third zinc finger from Zif268 were randomized, some single fingers with new binding specificity were obtained . In this study, by combining the single fingers, two three-finger peptides cDNA ZF123 and 2ZF123 were constructed by an over-lap PCR technique using the DNA binding domain of Zif268 as the template . After three times PCR, the products were inserted into pUC18 for cloning . The ZF123 and 2ZF123 cDNA were also inserted into pGEX-2T for expression in Escherichia coli after sequencing confirmation . The result showed that the three-finger peptides were expressed at a high level in E . coli JM109 . The fusion protein GST-ZF123/2ZF123 have the relative molecular weight of 34.0 kD and consisted about 20% of the total soluble cell protein as detected by SDS-PAGE . After supersonic treatment, the soluble part of the bacterial extract was purified . After two additional thrombin cleavage and Sepharose 4B affinity purification steps, the free three-fingers peptide proteins were also obtained . The construction and obtaining of the three-fingers peptide cDNA and its products will facilitate the in vivo and in vitro DNA binding specificity study and the design of the hybrid transcription factors. Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 396 - 9 {Cloning and expression of MGMT cDNA and analysis of the DNA repair activity of the recombinant protein}; Zhu YW et al.; A cDNA encoding human O6-methylguanine-DNA methyltranferase (MGMT) was cloned from Hela S3 cells and the sequence is identical with the published data . The MGMT cDNA was inserted into the expression vector pET-21a and transformed into E . coli BL21 (DE3) . A 24 kD protein was expressed after IPTG induction . Essays using lethal dose of alkylating agents indicate that expression of MGMT protein can repair the DNA mutations of the recombinant bacteria produced by alkylating agents. Arch Microbiol, 2001 Nov, 176(5), 370 - 6 Expression of the pho regulon interferes with induction of the uhpT gene in Escherichia coli K-12; Hoffer SM et al.; The uptake of hexose 6-phosphates in Escherichia coli is mediated by the transporter UhpT, the synthesis of which is induced by the presence of glucose 6-phosphate (glucose 6-P) in the medium . Since this protein functions as an anion exchanger, it is generally assumed to be geared for the use of sugar phosphates as a carbon source . However, the question was unresolved whether this transporter can also provide the cells with glucose 6-P as a phosphate source . It is demonstrated in this work that UhpT-mediated glucose 6-P uptake does not allow the cells to grow on glucose 6-P as phosphate source . Hence, the expression of UhpT under phosphate limitation would not be particularly advantageous and some form of interaction between the uhp system and the Pi-limitation-inducible pho regulon, the products of which are involved in phosphate acquisition, may be anticipated . Indeed, the use of an uhpT-lacZ fusion revealed that much higher concentrations of the inducer glucose 6-P were required to elevate uhpT transcription when the pho regulon was expressed . This interference was the result of degradation of glucose 6-P by one of the products of the pho regulon, the periplasmic enzyme alkaline phosphatase . The specific form of interaction between the Pho and Uhp systems is designated inducer degradation. Circ Res, 2001 Nov 9, 89(10), 874 - 81 Interaction between PEVK-titin and actin filaments: origin of a viscous force component in cardiac myofibrils; Kulke M et al.; The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes . Here, human N2-B-cardiac PEVK was expressed in Escherichia coli and tested-along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains-for a possible interaction with actin filaments . In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin . The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments . High {Ca(2+)} reversed the PEVK effect . PEVK concentrations >/=10 microgram/mL caused actin bundling . Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength . In cosedimentation assays, PEVK-titin interacted weakly with actin at 0 degrees C, but more strongly at 30 degrees C, suggesting involvement of hydrophobic interactions . To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period . The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude . The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca(2+)-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap . Steady-state passive force dropped only after longer exposure to gelsolin . We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils . The interaction could also oppose shortening during contraction. Trends Plant Sci, 2001 Nov, 6(11), 510 - 3 An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana; Leyman B et al.; Trehalose accumulation has been documented in many organisms, such as bacteria and fungi, where it serves a storage and stress-protection role . Although conspicuously absent in most plants, trehalose biosynthesis genes were discovered recently in higher plants . We have uncovered a family of 11 TPS genes in Arabidopsis thaliana, one of which encodes a trehalose-6-phosphate (Tre6P) synthase, and a subfamily of which might encode the still elusive Tre6P phosphatases . A regulatory role in carbon metabolism is likely but might not be restricted to the TPS control of hexokinase activity as documented for yeast . Incompatibility between high trehalose levels and chaperone-assisted protein folding might be a reason why plants have evolved to accumulate some alternative stress-protection compounds to trehalose. J Org Chem, 2001 Nov 16, 66(23), 7770 - 5 Biosynthesis of terpenoids: efficient multistep biotransformation procedures affording isotope-labeled 2C-methyl-D-erythritol 4-phosphate using recombinant 2C-methyl-D-erythritol 4-phosphate synthase; Hecht S et al.; This paper describes the recombinant expression of the ispC gene of Escherichia coli specifying 2C-methyl-D-erythritol 4-phosphate synthase in a modified form that can be purified efficiently by metal-chelating chromatography . The enzyme was used for the preparation of isotope-labeled 2C-methyl-D-erythritol 4-phosphate employing isotope-labeled glucose and pyruvate as starting materials . The simple one-pot methods described afford numerous isotopomers of 2C-methyl-D-erythritol 4-phosphate carrying (3)H, (13)C, or (14)C from commercially available precursors . The overall yield based on the respective isotope-labeled starting material is approximately 50%. J Org Chem, 2001 Nov 16, 66(23), 7561 - 7 Synthesis and biological activity of N-sulfonylphosphoramidates: probing the electrostatic preferences of alkaline phosphatase; Burlingham BT et al.; N-Sulfonylphosphoramidates have been synthesized to investigate the electrostatic requirements for binding to alkaline phosphatase . Alkyl- and aryl N-benzylated sulfonamides were phosphorylated with bromophosphates or synthesized via phosphoramidite chemistry in moderate yields (44-77%.) The resulting tribenzylated N-sulfonylphosphoramidates may be deprotected in one step to give the free acids in quantitative yields . Physical data of N-sulfonylphosphoramidates, including pK(a)'s and stability toward hydrolysis, were determined . Inhibition data suggests that AP does not bind trianionic N-sulfonylphosphoramidates better than dianionic N-sulfonylphosphoramidates, although N-sulfonylphosphoramidates are bound tighter than N-phenylphosphoramidate . k(cat) for the hydrolysis of N-sulfonylphosphoramidates by bovine and E . coli alkaline phosphatases is 10-60% that of p-nitrophenyl phosphate. Anal Biochem, 2001 Nov 15, 298(2), 314 - 21 Distribution of B6 vitamers in Escherichia coli as determined by enzymatic assay; Fu TF et al.; An enzymatic method for determination of B6 vitamers is presented . In this method pyridoxal 5'-phosphate is used to activate aposerine hydroxymethyltransferase to form the catalytically active holoenzyme . The active serine hydroxymethyltransferase, and two other enzymes that form a metabolic cycle, convert serine to glycine and CO2 with the concomitant production of two equivalents of NADPH . The rate of the cycle is directly proportional to the amount of active holoserine hydroxymethyltransferase, which is a measure of the amount of pyridoxal 5'-phosphate in the original sample . The cycle operates about 50 times per minute giving a 100-fold enhancement of NADPH production with respect to original pyridoxal 5'-phosphate content . Other B6 vitamers are converted to pyridoxal 5'-phosphate by a preincubation with a combination of pyridoxal kinase and pyridoxine 5'-phosphate oxidase . A complete analysis of B6 vitamers can be completed in less than 1 h and the assay is linear in the 2- to 50-pmol range of pyridoxal 5'-phosphate . The method is applied to the determination of the B6 vitamer pools in extracts of Escherichia coli . The results show that the pool of pyridoxal 5'-phosphate that is not bound to proteins is large enough to account for product inhibition of both pyridoxal kinase and pyridoxine 5'-phosphate oxidase . Genome Inform Ser Workshop Genome Inform, 2000, 11, 185 - 95 Two-phase partition method for simulating a biological system at an extremely high speed; Kurata H et al.; To accelerate the calculation speed for simulating a biological system, we proposed a novel simulation method, the two-phase partition method, which calculated molecular processes at a higher speed than any other proposed method . This method divides a biological system, which can be described by chemical reaction equations, into two-phases: the binding and reaction phases . We demonstrated the capability of the two-phase partition method to simulate a complex biological system at an extremely high speed and clarified the accuracy of the simulation . The two-phase partition method is very useful for simulating complex interactions among proteins and DNAs. Genome Inform Ser Workshop Genome Inform, 2000, 11, 141 - 50 Protein sequence-structure alignment based on site-alignment probabilities; Miyazawa S; A protein sequence-structure alignment method for database searches is examined on how effectively this method together with a simple scoring function previously developed can identify compatibilities between sequences and structures of proteins . The scoring function consists of pairwise contact energies, repulsive packing potentials of residues for overly dense arrangement and short-range potentials for secondary structures . Pairwise contact interactions in a sequence-structure alignment are evaluated in a mean field approximation on the basis of probabilities of site pairs to be aligned . Gap penalties are assumed to be proportional to the number of contacts at each residue position, and as a result gaps will be more frequently placed on protein surfaces than in cores . In addition to minimum energy alignments, we use probability alignments made by successively aligning site pairs in order by pairwise alignment probabilities . Results show that the present energy function and alignment method can detect well both folds compatible with a given sequence and, inversely, sequences compatible with a given fold . Probability alignments consisting of most reliable site pairs only can yield small root mean square deviations, and including less reliable pairs increases the deviations . Remarkably, by this method some individual sequence-structure pairs are detected having only 5-20% sequence identity. Crit Care Med, 2001 Nov, 29(11), 2185 - 93 Effects of levosimendan, a novel inotropic calcium-sensitizing drug, in experimental septic shock; Oldner A et al.; OBJECTIVE: Levosimendan is a novel inodilator that improves cardiac contractility by sensitizing troponin C to calcium . This drug has proved to be effective in treating advanced congestive heart failure but has not been evaluated in septic settings . The purpose of the present study was to study the effects of this drug in a porcine model of endotoxemia . DESIGN: Prospective experimental study . SUBJECTS: Fourteen landrace pigs . INTERVENTIONS: All animals were anesthetized and catheterized for measurement of central and pulmonary hemodynamics . Ultrasonic flow probes were placed around the renal artery and portal vein to measure blood flow . A tonometer was placed in the ileum to measure mucosal pH . Levosimendan was given to six animals as a bolus (200 microg x kg(-1)) followed by a continuous infusion (200 microg x kg(-1) x hr(-1)) . Thirty minutes after onset of levosimendan treatment, all animals received endotoxin (20 microg x kg(-1) x hr(-1) for 3 hrs) . MEASUREMENTS AND MAIN RESULTS: At baseline, levosimendan induced a systemic vasodilation with a reduction in blood pressure and an increase in heart rate . A tendency to an increase in cardiac index did not reach statistical significance (p =.055) . Cardiac index and systemic oxygen delivery were markedly improved in the levosimendan group during endotoxemia . Systemic vascular resistance and blood pressure were reduced in the levosimendan group . The latter parameter, however, was only different from the control group during the initial phase of endotoxin shock but not at the late, most pronounced phase of shock . Levosimendan also efficiently attenuated endotoxin-induced pulmonary hypertension . Portal venous blood flow and gut oxygen delivery were improved, but no concomitant reduction in endotoxin-induced intestinal mucosal acidosis was observed . Renal blood flow was unaffected, as was the endotoxin-induced increase in plasma endothel