Biochemistry, 2002 Mar 12, 41(10), 3302 - 10 The two-domain NK1 fragment of plasminogen: folding, ligand binding, and thermal stability profile; Douglas JT et al.; The two-domain fragment N+K1 (rNK1) {Glu(1)-Glu(163)} of human plasminogen was expressed in E . coli as a hexahistidine-tagged fusion protein and chromatographically purified . The (1)H NMR spectrum supports proper folding of the K1 component within the refolded rNK1 construct (rNK1/K1) . The functional properties of rNK1/K1 were investigated via intrinsic fluorescence titration with kringle-specific omega-aminocarboxylic acid ligands . The affinities closely match those previously measured for the isolated K1, which indicates that the N-domain does not significantly affect the interaction of ligands with the lysine binding site of K1 . Far-UV CD spectra recorded for the N-domain suggest conformational plasticity and flexibility for the module . Two classes of spectra, referred to as types A and B, were identified with the type A spectrum reflecting a higher secondary structure content than that estimated for the type B spectrum . Subtracting the CD spectrum of rK1 from that of rNK1 yields a spectrum (Delta) which reflects the conformation of the N-domain within the rNK1 construct (rNK1/N) . Delta resembles the type A spectrum, suggesting that rNK1/N adopts a relatively more ordered conformation, stabilized by the adjacent rNK1/K1 domain . In contrast, thermal unfolding curves determined via CD indicate that the rNK1/N slightly lowers the melting temperature (T(m)) of rNK1/K1 . Independence of the two domains within rNK1 was tested by monitoring the thermal unfolding of rNK1/K1 when in the presence of the kringle-specific ligand AMCHA, which left the rNK1/N T(m) essentially unaffected, while increasing that of the rNK1/K1 by approximately 10 degrees C. Nature, 2002 Feb 28, 415(6875), 1039 - 42 MEC-2 regulates C . elegans DEG/ENaC channels needed for mechanosensation; Goodman MB et al.; Touch sensitivity in animals relies on nerve endings in the skin that convert mechanical force into electrical signals . In the nematode Caenorhabditis elegans, gentle touch to the body wall is sensed by six mechanosensory neurons that express two amiloride-sensitive Na+ channel proteins (DEG/ENaC) . These proteins, MEC-4 and MEC-10, are required for touch sensation and can mutate to cause neuronal degeneration . Here we show that these mutant or 'd' forms of MEC-4 and MEC-10 produce a constitutively active, amiloride-sensitive ionic current when co-expressed in Xenopus oocytes, but not on their own . MEC-2, a stomatin-related protein needed for touch sensitivity, increased the activity of mutant channels about 40-fold and allowed currents to be detected with wild-type MEC-4 and MEC-10 . Whereas neither the central, stomatin-like domain of MEC-2 nor human stomatin retained the activity of full-length MEC-2, both produced amiloride-sensitive currents with MEC-4d . Our findings indicate that MEC-2 regulates MEC-4/MEC-10 ion channels and raise the possibility that similar ion channels may be formed by stomatin-like proteins and DEG/ENaC proteins that are co-expressed in both vertebrates and invertebrates . Some of these channels may mediate mechanosensory responses. J Biol Chem, 2002 May 10, 277(19), 16782 - 90 Epub 2002 Mar 01. Genetic fusions of globular proteins to the epsilon subunit of the Escherichia coli ATP synthase: Implications for in vivo rotational catalysis and epsilon subunit function; Cipriano DJ et al.; The rotational mechanism of ATP synthase was investigated by fusing three proteins from Escherichia coli, the 12-kDa soluble cytochrome b(562), the 20-kDa flavodoxin, and the 28-kDa flavodoxin reductase, to the C terminus of the epsilon subunit of the enzyme . According to the concept of rotational catalysis, because epsilon is part of the rotor a large domain added at this site should sterically clash with the second stalk, blocking rotation and fully inhibiting the enzyme . E . coli cells expressing the cytochrome b(562) fusion in place of wild-type epsilon grew using acetate as the energy source, indicating their capacity for oxidative phosphorylation . Cells expressing the larger flavodoxin or flavodoxin reductase fusions failed to grow on acetate . Immunoblot analysis showed that the fusion proteins were stable in the cells and that they had no effect on enzyme assembly . These results provide initial evidence supporting rotational catalysis in vivo . In membrane vesicles, the cytochrome b(562) fusion caused an increase in the apparent ATPase activity but a minor decrease in proton pumping . Vesicles bearing ATP synthase containing the larger fusion proteins showed reduced but significant levels of ATPase activity that was sensitive to inhibition by dicyclohexylcarbodiimide (DCCD) but no proton pumping . Thus, all fusions to epsilon generated an uncoupled component of ATPase activity . These results imply that a function of the C terminus of epsilon in F(1)F(0) is to increase the efficiency of the enzyme by specifically preventing the uncoupled hydrolysis of ATP . Given the sensitivity to DCCD, this uncoupled ATP hydrolysis may arise from rotational steps of gammaepsilon in the inappropriate direction after ATP is bound at the catalytic site . It is proposed that the C-terminal domain of epsilon functions to ensure that rotation occurs only in the direction of ATP synthesis when ADP is bound and only in the direction of hydrolysis when ATP is bound. J Biol Chem, 2002 May 17, 277(20), 17428 - 37 Epub 2002 Mar 01. Structural characterization of the M* partly folded intermediate of wild type and P138A aspartate aminotransferase from Escherichia coli; Birolo L et al.; A combination of spectroscopic techniques, hydrogen/deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M* partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A) . Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E . coli, it seems to coincide with an on-pathway folding intermediate for the P138A mutant . Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M* intermediates, with P138A-M* being conceivably more compact than WT-M* . Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138 . These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process . Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N(2) forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposable. Genome Res, 2002 Mar, 12(3), 482 - 6 High-density cell microarrays for parallel functional determinations; Xu CW; Whole-genome sequencing projects have generated a wealth of gene sequences from a variety of organisms . A major challenge is to rapidly uncover gene regulatory circuits and their functional manifestations at the cellular level . Here we report the coupled fabrication of nanocraters ranging in size from 100 pL to 1.5 nL on permeable membranes for culturing cells . Using this approach, we developed bacterial and yeast cell microarrays that allowed phenotypic determinations of gene activities and drug targets on a large scale . Cell microarrays will therefore be a particularly useful tool for studying phenotypes of gene activities on a genome-wide scale. Genome Res, 2002 Mar, 12(3), 470 - 81 Extraction of functional binding sites from unique regulatory regions: the Drosophila early developmental enhancers; Papatsenko DA et al.; The early developmental enhancers of Drosophila melanogaster comprise one of the most sophisticated regulatory systems in higher eukaryotes . An elaborate code in their DNA sequence translates both maternal and early embryonic regulatory signals into spatial distribution of transcription factors . One of the most striking features of this code is the redundancy of binding sites for these transcription factors (BSTF) . Using this redundancy, we explored the possibility of predicting functional binding sites in a single enhancer region without any prior consensus/matrix description or evolutionary sequence comparisons . We developed a conceptually simple algorithm, Scanseq, that employs an original statistical evaluation for identifying the most redundant motifs and locates the position of potential BSTF in a given regulatory region . To estimate the biological relevance of our predictions, we built thorough literature-based annotations for the best-known Drosophila developmental enhancers and we generated detailed distribution maps for the most robust binding sites . The high statistical correlation between the location of BSTF in these experiment-based maps and the location predicted in silico by Scanseq confirmed the relevance of our approach . We also discuss the definition of true binding sites and the possible biological principles that govern patterning of regulatory regions and the distribution of transcriptional signals. Plant J, 2002 Mar, 29(5), 595 - 606 Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis; Jensen AB et al.; Jasmonates induce plant-defence responses and act to regulate defence-related genes including positive feedback of the lipoxygenase 2 (LOX2) gene involved in jasmonate synthesis . To identify jasmonate-signalling mutants, we used a fusion genetic strategy in which the firefly luciferase (FLUC) and Escherichia coli beta-glucuronidase (GUS) reporters were expressed under control of the jasmonate-responsive LOX2 promoter . Spatial and temporal patterns of reporter expression were determined initially, and revealed that JA-responsive expression from the LOX2 promoter required de novo protein synthesis . Reporter activity was also induced by the protein kinase inhibitor staurosporine and antagonized by the protein phosphatase inhibitor okadaic acid . FLUC bio-imaging, RNA gel-blot analysis and progeny analyses identified three recessive mutants that underexpress the FLUC reporter, designated jue1, 2 and 3, as well as two recessive mutants, designated joe1 and 2, that overexpress the reporter . Genetic analysis indicated that reporter overexpression in the joe mutants requires COI . joe1 responded to MeJA with increased anthocyanin accumulation, while joe2 responded with decreased root growth inhibition . In addition, reporter induction and endogenous LOX2 expression by staurosporine was absent in joe2. Plant J, 2002 Mar, 29(5), 545 - 53 A peptide methionine sulfoxide reductase highly expressed in photosynthetic tissue in Arabidopsis thaliana can protect the chaperone-like activity of a chloroplast-localized small heat shock protein; Gustavsson N et al.; The oxidation of methionine residues in proteins to methionine sulfoxides occurs frequently and protein repair by reduction of the methionine sulfoxides is mediated by an enzyme, peptide methionine sulfoxide reductase (PMSR, EC 1.8.4.6), universally present in the genomes of all so far sequenced organisms . Recently, five PMSR-like genes were identified in Arabidopsis thaliana, including one plastidic isoform, chloroplast localised plastidial peptide methionine sulfoxide reductase (pPMSR) that was chloroplast-localized and highly expressed in actively photosynthesizing tissue (Sadanandom A et al., 2000) . However, no endogenous substrate to the pPMSR was identified . Here we report that a set of highly conserved methionine residues in Hsp21, a chloroplast-localized small heat shock protein, can become sulfoxidized and thereafter reduced back to methionines by this pPMSR . The pPMSR activity was evaluated using recombinantly expressed pPMSR and Hsp21 from Arabidopsis thaliana and a direct detection of methionine sulfoxides in Hsp21 by mass spectrometry . The pPMSR-catalyzed reduction of Hsp21 methionine sulfoxides occurred on a minute time-scale, was ultimately DTT-dependent and led to recovery of Hsp21 conformation and chaperone-like activity, both of which are lost upon methionine sulfoxidation (Harndahl et al., 2001) . These data indicate that one important function of pPMSR may be to prevent inactivation of Hsp21 by methionine sulfoxidation, since small heat shock proteins are crucial for cellular resistance to oxidative stress. Lett Appl Microbiol, 2002, 34(3), 227 - 31 EU Drinking Water Directive reference methods for enumeration of total coliforms and Escherichia coli compared with alternative methods; Schets FM et al.; AIMS: The reference methods for enumeration of total coliforms and Escherichia coli as stated in the European Drinking Water Directive were compared with alternative methods . METHODS AND RESULTS: Laboratories used the reference method on Lactose TTC agar (LTTC), the Colilert/18 system, Laurysulphate Agar (LSA), Chromocult Coliform Agar and the E . coli Direct Plating (DP) method . They enumerated more total coliforms on LTTC than on LSA . CONCLUSIONS: LTTC is suitable for analysis of very clean water samples only, due to heavy background growth . Colilert/18 is a good alternative but it enumerates a broader group of total coliforms, resulting in higher counts . The DP method appeared to be the best choice for enumeration of E . coli because Colilert/18 produces lower counts and false-negative results . SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the limitations of the EU reference method on LTTC due to lack of selectivity and suggests alternative methods for the enumeration of total coliforms and E . coli. Lett Appl Microbiol, 2002, 34(3), 182 - 8 Involvement of RNA and DNA in the staining of Escherichia coli by SYTO 13; Guindulain T et al.; AIMS: To assess the extent to which DNA and RNA bacterial content contributes to fluorescent response of SYTO 13 . METHODS AND RESULTS: RNA and DNA of Escherichia coli 536 cells were extracted and fluorimetrically quantified to compare the different contents, throughout a 24 h culture, with their SYTO 13 fluorescence emission when analysed by the cytometer . SYTO 13 fluorescence varied depending on the stage of bacterial growth and in accordance with both DNA and RNA content . RNA content accounted for at least two-thirds of the total fluorescence of a cell . Escherichia coli cells were treated with chloramphenicol to improve their RNA content . With this treatment, both nucleic acids remained constant but there was a clear improvement in fluorescent emission . SYTO 13 fluorescence was also studied in E . coli X-1488 minicells . CONCLUSIONS: Although both nucleic acids are implicated, RNA accounts for a major part of SYTO 13 fluorescence . The fluorescence cannot be considered as a direct reflection of nucleic acid content . Other factors, such as topology or supercoiling, need to be considered . SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm the efficacy of SYTO 13 for labelling bacteria and for assessing the distinct physiological status . A better knowledge of the parameters implicated in its fluorescence emission has been achieved. Eur J Biochem, 2002 Mar, 269(5), 1579 - 86 Introducing Wilson disease mutations into the zinc-transporting P-type ATPase of Escherichia coli . The mutation P634L in the 'hinge' motif (GDGXNDXP) perturbs the formation of the E2P state; Okkeri J et al.; ZntA, a bacterial zinc-transporting P-type ATPase, is homologous to two human ATPases mutated in Menkes and Wilson diseases . To explore the roles of the bacterial ATPase residues homologous to those involved in the human diseases, we have introduced several point mutations into ZntA . The mutants P401L, D628A and P634L correspond to the Wilson disease mutations P992L, D1267A and P1273L, respectively . The mutations D628A and P634L are located in the C-terminal part of the phosphorylation domain in the so-called hinge motif conserved in all P-type ATPases . P401L resides near the N-terminal portion of the phosphorylation domain whereas the mutations H475Q and P476L affect the heavy metal ATPase-specific HP motif in the nucleotide binding domain . All mutants show reduced ATPase activity corresponding 0-37% of the wild-type activity . The mutants P401L, H475Q and P476L are poorly phosphorylated by both ATP and P(i) . Their dephosphorylation rates are slow . The D628A mutant is inactive and cannot be phosphorylated at all . In contrast, the mutant P634L six residues apart in the same domain shows normal phosphorylation by ATP . However, phosphorylation by P(i) is almost absent . In the absence of added ADP the P634L mutant dephosphorylates much more slowly than the wild-type, whereas in the presence of ADP the dephosphorylation rate is faster than that of the wild-type . We conclude that the mutation P634L affects the conversion between the states E1P and E2P so that the mutant favors the E1 or E1P state. Eur J Biochem, 2002 Mar, 269(5), 1525 - 33 Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae; Ingleston SM et al.; The RuvA, RuvB and RuvC proteins of Escherichia coli act together to process Holliday junctions formed during recombination and DNA repair . RuvA has a well-defined DNA binding surface that is sculptured specifically to accommodate a Holliday junction and allow subsequent loading of RuvB and RuvC . A negatively charged pin projecting from the centre limits binding of linear duplex DNA . The amino-acid sequences forming the pin are highly conserved . However, in certain Mycoplasma and Ureaplasma species the structure is extended by four amino acids and two acidic residues forming a crucial charge barrier are missing . We investigated the significance of these differences by analysing RuvA from Mycoplasma pneumoniae . Gel retardation and surface plasmon resonance assays revealed that this protein binds Holliday junctions and other branched DNA structures in a manner similar to E . coli RuvA . Significantly, it binds duplex DNA more readily . However it does not support branch migration mediated by E . coli RuvB and when bound to junction DNA is unable to provide a platform for stable binding of E . coli RuvC . It also fails to restore radiation resistance to an E . coli ruvA mutant . The data presented suggest that the modified pin region retains the ability to promote junction-specific DNA binding, but acts as a physical obstacle to linear duplex DNA rather than as a charge barrier . They also indicate that such an obstacle may interfere with the binding of a resolvase . Mycoplasma species may therefore process Holliday junctions via uncoupled branch migration and resolution reactions. Eur J Biochem, 2002 Mar, 269(5), 1418 - 27 CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster; Kalmykova AI et al.; An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2 . Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract . Expression of a CK2(beta)tes-beta-galactosidase fusion protein driven by the CK2(beta)tes promoter was found in transgenic flies at postmitotic stages of spermatogenesis . Examination of biochemical characteristics of a recombinant CK2(beta)tes protein expressed in Escherichia coli revealed properties similar to those of CK2beta: (a) CK2(beta)tes protein stimulates CK2alpha catalytic activity toward synthetic peptide; (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2alpha by polylysine; (c) it is able to form (CK2(beta)tes)2 dimers, as well as (CK2alpha)2(CK2(beta)tes)2 tetramers . Using the yeast two-hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha . Northern-analysis has shown that another regulatory (beta') subunit found recently in D . melanogaster genome is also testis-specific . Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis. Tohoku J Exp Med, 2001 Nov, 195(3), 153 - 61 Activation of mitochondrial ATP-dependent protease by peptides and proteins; Watabe S et al.; We examined the effect of peptides or protein on the proteolytic and ATPase activities of mitochondrial ATP-dependent LON protease purified from bovine adrenal cortex . Peptides/proteins including angiotensin I which stimulated ATPase activity without hydrolysis of any peptide bonds stimulated proteolysis of 125I-labeled substrates at low concentrations; whereas at high concentrations they competitively inhibited proteolysis, thus displaying a biphasic activity profile . All peptides and proteins thus examined stimulated degradation of 125I-labeled substrates . When an ATP analog was substituted for ATP, only inhibition; i.e., no stimulation, of proteolysis by unlabeled peptides was observed . Without activator peptides, degradation of {125I} peptides was higher in the presence of an ATP analog than that in the presence of ATP . ADP, a product of the ATPase reaction, inhibited the proteolytic activity in the absence of an activator peptide but not in its presence . From analogy to E . coli ATP-dependent protease La (LON), we suggest that the activator peptides stimulated the proteolysis by releasing enzyme-bound ADP. J Mol Microbiol Biotechnol, 2002 Mar, 4(2), 163 - 9 Characterization of the ves gene, which is expressed at a low temperature in Escherichia coli; Yamada M et al.; A gene, designated ves, that is expressionally responsive to temperature was found in Escherichia coli . Experiments with a single-copy lacZ operon fusion and primer extension analysis revealed that ves was expressed at a low temperature with a peak around 25 degrees C but was hardly expressed at 42 degrees C . After a temperature downshift, the mRNA level increased until 6 to 12 h and then decreased . Consistently, an A + T-rich sequence similar to UP elements seen in cold-shock inducible cold-shock protein (Csp) genes was found up-stream of the ves promoter, and its 5'-untranslated region was found to share similarity with those of the cold-shock inducible and cold-adaptive cspA and cspB genes . Additionally, a putative down-stream box, which also exists in cold-inducible proteins, was found . The ves product was identified by overproduction and determination of its N-terminal sequence . Similarity of the C-terminal portion of Ves to the CspA family suggests that Ves belongs to this family . The results of gene-disruption experiments suggest that ves is not essential for E . coli. J Mol Microbiol Biotechnol, 2002 Mar, 4(2), 127 - 31 A vector with transcriptional terminators increases efficiency of cloning of an RNA virus by reverse transcription long polymerase chain reaction; Cameron-Wilson CL et al.; Full-length cDNA clones of RNA viruses are advantageous for maintaining the genomic sequence without the generation of diversity by accumulation of sequence mutations during productive virus replication . They permit in vitro manipulation of the genomic clone to test the effect of sequence changes on the phenotype of reactivated virus . Infectious cDNA clones have been produced by ligation of subgenomic clones but are sometimes difficult to generate in a single cloning operation . We used reverse-transcription to synthesize full-length cDNA from genomic RNA of Coxsackievirus B3 of the Picornavirus family and enzymatically amplified this by long PCR . Five different cloning vectors were used to clone the long PCR product, including the vector Lorist6 which contains transcriptional terminators on either side of the cloning site to prevent transcription of inserts in E . coli . No recombinant colonies were obtained from any of the vectors lacking transcriptional terminators but three full-length clones were obtained using Lorist6 . The results suggest that transcriptional terminators increase the recovery of cDNA clones of the 7.4 kb Coxsackie virus genome in this cosmid vector, without resort to phage packaging, representing an advance over previous methods and advantages in the molecular manipulation of these viruses. Poult Sci, 2002 Feb, 81(2), 149 - 59 Antibody responses and morbidity following infection with infectious bronchitis virus and challenge with Escherichia coli, in lines divergently selected on antibody response; Yunis R et al.; We evaluated the association between antibody (Ab) production and disease resistance . A controlled-challenge protocol was developed to mimic natural infection and to yield a higher rate of mortality following Escherichia coli (EC) challenge . Chicks were first infected with infectious bronchitis virus (IBV) by injecting a high dose of vaccine (attenuated virus) into their air sacs and then were infected with pathogenic EC introduced intratracheally . The experimental population consisted of lines divergently selected for high (HH) or low (LL) Ab response to EC vaccination, an HH x LL cross (HL), and commercial broilers (CC) . When chicks were vaccinated with EC vaccine, mean Ab titer 15 d post-EC challenge was threefold higher in HH than LL lines, but both lines exhibited very low mortality (approximately 2%) . When chicks were not vaccinated prior to EC challenge, high mortality (8 to 20%) occurred in the slow-growing HH, LL, and HL lines, and much higher mortality (approximately 40%) occurred among the CC broilers that were 38% heavier than the HH, LL, and HL lines . Mean level of Ab to EC, 7 d after EC challenge, was about twofold higher in HH vs . LL chicks and intermediate in HL and CC chicks . Within each line, Ab levels were higher in chicks exhibiting colibacillosis than in healthy ones, suggesting that these Ab were produced as a result of ongoing infection but were too late to fully prevent morbidity and mortality . These results indicate that rapid growth rate substantially reduces broiler viability, whereas Ab levels produced in response to acute pathogenic challenge without prior vaccination do not contribute to disease resistance . Among the relatively slow-growing lines, mortality was about twofold higher in HH than in LL lines . This finding may confirm previous reports that without prior vaccination, high Ab response to acute challenge increases consequent mortality; alternatively, the LL line may be superior in nonspecific defense mechanisms. Vet Res, 2002 Jan-Feb, 33(1), 1 - 12 Potential mechanism of action of J5 vaccine in protection against severe bovine coliform mastitis; Dosogne H et al.; Coliform mastitis is one of the most difficult diseases to treat in the modern dairy industry . Curative therapy with antibiotics remains only moderately effective and depends on the stage at which the disease is treated . The most successful strategies for combating coliform mastitis appear to be prevention by hygienic management or prophylactic immunization . The severity of clinical symptoms of coliform mastitis has been shown to be reduced by immunization with the Escherichia coli J5 vaccine . However, although the J5 vaccine has been licensed in the United States for about 10 years, the immunological basis of its mechanism of action is still unknown . Until now, protection by J5 vaccination has often been explained by a straightforward mechanism of enhanced antibody production resulting in increased opsonization of coliform bacteria and lipopolysaccharides (LPS) . The possibility that J5 vaccination could decrease risk factors for coliform mastitis such as impaired blood polymorphonuclear neutrophil leukocyte (PMN) diapedesis has never been investigated . This review provides arguments to support the hypothesis that J5 vaccination may reduce the severity of coliform mastitis by inducing a condition of mammary gland hyper-responsiveness, characterized by a T helper 1 (Th1) response and mediated by memory cells inside the mammary gland, finally resulting in enhanced PMN diapedesis upon an intramammary infection. Acta Gastroenterol Latinoam, 2001, 31(5), 399 - 402 {Disseminated infection due to strongyloides stercoralis in AIDS patients . A report of 2 cases}; Trione N et al.; Strongyloides stercoralis is an intestinal nematode that infects humans worldwide . Infected patients with severe involvement of cellular immunity may develop a syndrome characterized by the dissemination of larvae throughout the body . Extraintestinal strongyloidiasis has been infrequently reported and despite the prevalence of the helminth in tropical and developing countries there are few cases reported in AIDS patients . Most patients with disseminated strongyloidiasis present with fever, cough, diarrhea and shortness of breath . Chest radiographs usually show diffuse infiltrates . The diagnosis has been made by finding the helminth in respiratory secretions or stool . Enteric organisms like Escherichia coli can often be isolated in the blood or cerebrospinal fluid . We report two cases of disseminated strongyloidiasis in AIDS patients, in which stercoralis larvae were detected in sputum and stool samples. Indian J Med Res, 2001 Sep, 114, 95 - 8 A study on some phenotypic virulence markers of enteropathogenic Escherichia coli; Prasannan M et al.; BACKGROUND & OBJECTIVES: The problem of enteropathogenic Escherichia coli (EPEC) causing diarrhoea in infants exists in India . But often the enteropathogenic status is not based on adequate characterization . Hence there is a need for evaluating the serotyping being used to identify EPEC for its validity in the light of recent knowledge on phenotypic markers of virulence . This study was done to evaluate the EPEC isolates for two potential virulence factors namely entero-adhesiveness with subsequent actin accumulation and verotoxin production . METHODS: Fifty consecutive EPEC strains identified by serotyping from stool samples of children with diarrhoea during January 1997 to June 1999 were studied for HEp-2 cell adherence, the fluorescent actin staining (FAS) characteristics of Hep-2 cells and vero cytotoxin production . RESULTS: Serotypes O55, O125 and O126 accounted for most of the isolates . In the Hep-2 assay, 72 per cent of the strains showed localised pattern of adherence and 22 per cent showed a mixed pattern of localised and diffuse adherence . In the FAS test 96 per cent strains showed typical staining while none of the strains produced verotoxin . INTERPRETATION & CONCLUSION: 'O' serogrouping appears to be still the simplest and an useful test for presumptive identification of EPEC . The FAS test for confirmation of EPEC was found to be very consistent in indicating EPEC. Science, 2002 Mar 1, 295(5560), 1715 - 9 Structural basis of gating by the outer membrane transporter FecA; Ferguson AD et al.; Siderophore-mediated acquisition systems facilitate iron uptake . We present the crystallographic structure of the integral outer membrane receptor FecA from Escherichia coli with and without ferric citrate at 2.5 and 2.0 angstrom resolution . FecA is composed of three distinct domains: the barrel, plug, and NH2-terminal extension . Binding of ferric citrate triggers a conformational change of the extracellular loops that close the external pocket of FecA . Ligand-induced allosteric transitions are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm . These data establish the structural basis of gating for receptors dependent on the cytoplasmic membrane protein TonB . By compiling available data for this family of receptors, we propose a mechanism for the energy-dependent transport of siderophores. Science, 2002 Mar 1, 295(5560), 1658 - 9 Close before opening; Postle K; As bacteria need iron from the environment to survive, they have evolved active iron transporter proteins in their outer membranes . In her Perspective, Postle discusses new insights into iron transport revealed by the crystal structure of the iron transporter FecA in E . coli (Ferguson et al.). J Biol Chem, 2002 May 10, 277(19), 16648 - 54 Epub 2002 Feb 28. Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3; Johansson AS et al.; Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known . The activity with Delta(5)-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione . Removal of these groups caused an 1.1 x 10(5)-fold decrease in k(cat); the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione . The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene . Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate . Mutating residue 111 into phenylalanine caused a 25-fold decrease in k(cat)/K(m) for the steroid isomerization . The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutation k(cat)/K(m) was reduced to 0.8% of that of the wild-type value . In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S . K(i) values for Delta(5)-androstene-3,17-dione and Delta(4)-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S . The pK(a) of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F . The pK(a) of the active-site Tyr-9 was 7.9 for the wild-type enzyme . The pH dependence of k(cat)/K(m) of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK(a) values, 6.2 and 8.1 . The basic limb of the pH dependence of k(cat) and k(cat)/K(m) disappears in the Y9F mutant . Therefore, the higher kinetic pK(a) reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione . We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate. J Bacteriol, 2002 Mar, 184(6), 1796 - 800 Site-directed mutagenesis studies of selected motif and charged residues and of cysteines of the multifunctional tetracycline efflux protein Tet(L); Jin J et al.; All of the transmembrane glutamates of Tet(L) are essential for tetracycline (TET) resistance, and E397 has been shown to be essential for all catalytic modes, i.e., TET-Me(2+) and Na(+) efflux and K(+) uptake . Loop residues D74 and G70 are essential for TET flux but not for Na(+) or K(+) flux . A cysteineless Tet(L) protein exhibits all activities. J Bacteriol, 2002 Mar, 184(6), 1794 - 5 Lack of regulation of the modification-dependent restriction enzyme McrBC in Escherichia coli; Murphy M et al.; Restriction alleviation (RA) by the type I restriction enzyme EcoKI is caused by treatments that damage DNA . RA is due to proteolysis of the EcoKI HsdR subunit by the ClpXP ATP-dependent protease . Here we show that the modification-dependent enzyme McrBC is not subject to RA, although it is moderately sensitive to ClpAP. J Bacteriol, 2002 Mar, 184(6), 1685 - 92 Oxygen-mediated regulation of porphobilinogen formation in Rhodobacter capsulatus; Biel AJ et al.; A Rhodobacter capsulatus hemC mutant has been isolated and used to show that oxygen regulates the intracellular levels of porphobilinogen . Experiments using a hemB-cat gene fusion demonstrated that oxygen does not transcriptionally regulate hemB transcription . Porphobilinogen synthase activity is not regulated by oxygen nor is the enzyme feedback inhibited by hemin or protoporphyrin IX . It was demonstrated that less than 20% of {(14)C}aminolevulinate was incorporated into bacteriochlorophyll, suggesting that the majority of the aminolevulinate is diverted from the common tetrapyrrole pathway . Porphobilinogen oxygenase activity was not observed in this organism; however, an NADPH-linked aminolevulinate dehydrogenase activity was demonstrated . The specific activity of this enzyme increased with increasing oxygen tension . The results presented here suggest that carbon flow over the common tetrapyrrole pathway is regulated by a combination of feedback inhibition of aminolevulinate synthase and diversion of aminolevulinate from the pathway by aminolevulinate dehydrogenase. J Bacteriol, 2002 Mar, 184(6), 1661 - 8 TrwD, the hexameric traffic ATPase encoded by plasmid R388, induces membrane destabilization and hemifusion of lipid vesicles; Machon C et al.; TrwD, a hexameric ATP hydrolase encoded by plasmid R388, is a member of the PulE/VirB11 protein superfamily of traffic ATPases . It is essential for plasmid conjugation, particularly for expression of the conjugative W pilus . In the present study, we analyzed the effects that TrwD produced on unilamellar vesicles consisting of cardiolipin and phosphatidylcholine in equimolar amounts . TrwD induced dose-dependent vesicle aggregation and intervesicular mixing of the lipids located in the outer monolayers in the presence of calcium . It also induced extensive leakage of the vesicular aqueous contents . A point mutant of TrwD with a mutation in the P loop of the nucleotide-binding region (K203Q) that lacks both ATPase activity and the ability to support conjugation showed the same behavior as native TrwD in all of these processes, which were independent of the presence of ATP . Structure prediction methods revealed a close similarity to Helicobacter pylori protein HP0525, another member of the PulE/VirB11 family, whose crystal structure is known . The interpretation of our data in the light of this structure is that TrwD interacts with the lipid bilayer through hydrophobic regions in its N-terminal domain, which leads to a certain degree of membrane destabilization . TrwD appears to be a part of the conjugation machinery that interacts with the membranous systems in order to facilitate DNA transfer in bacteria. J Bacteriol, 2002 Mar, 184(6), 1640 - 8 TonB interacts with nonreceptor proteins in the outer membrane of Escherichia coli; Higgs PI et al.; The Escherichia coli TonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B(12) across the outer membrane . Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes . The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail . Here we begin to examine the nature of TonB interactions with the outer membrane . Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane . Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane . This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors . Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB . Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous . Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking . These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously . Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes . These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors. J Bacteriol, 2002 Mar, 184(6), 1607 - 16 Osmoregulation of dimer resolution at the plasmid pJHCMW1 mwr locus by Escherichia coli XerCD recombination; Pham H et al.; Xer-mediated dimer resolution at the mwr site of plasmid pJHCMW1 is osmoregulated in Escherichia coli . Whereas under low-salt conditions, the site-specific recombination reaction is efficient, under high-salt conditions, it proceeds inefficiently . Regulation of dimer resolution is independent of H-NS and is mediated by changes in osmolarity rather than ionic effects . The low level of recombination at high salt concentrations can be overcome by high levels of PepA or by mutating the ARG box to a sequence closer to the E . coli ARG box consensus . The central region of the mwr core recombination site plays a role in regulation of site-specific recombination by the osmotic pressure of the medium. J Bacteriol, 2002 Mar, 184(6), 1565 - 70 The histone-like protein HU does not obstruct movement of T7 RNA polymerase in Escherichia coli cells but stimulates its activity; Morales P et al.; In vivo, RNA polymerases (RNAPs) do not transcribe naked DNA but do transcribe protein-associated DNA . Studies with the model enzyme T7 RNAP have shown that, in eukaryotic cells or in vitro, nucleosomes can inhibit both transcription initiation and elongation . We examine here whether the presence of HU, one of the major histone-like proteins in Escherichia coli cells (the genuine milieu for T7 RNAP) affects its activity . An engineered lac operon fused to the T7 late promoter was introduced into the chromosome of T7 RNAP-producing strains that either overexpress HU or lack it . The flows of RNAP that enter and exit this operon were compared with regard to the content of HU . We found that the fraction of T7 RNAP molecules that do not reach the end of the lac operon (ca . 15%) is the same whether the host cells overexpressed HU or lacked it: thus, the enzyme either freely displaces HU or transcribes through it . However, in these cells, the transcript yield was increased when HU is overexpressed and decreased in the hup mutants, presumably reflecting changes in DNA supercoiling . Thus, in contrast to eukaryotic nucleosomes, HU does not impair T7 RNAP activity but has a stimulatory effect . Finally, our results suggest that HU can also influence mRNA stability in vivo. J Bacteriol, 2002 Mar, 184(6), 1556 - 64 Involvement of superoxide dismutases in the response of Escherichia coli to selenium oxides; Bebien M et al.; Selenium can provoke contrasting effects on living organisms . It is an essential trace element, and low concentrations have beneficial effects, such as the reduction of the incidence of cancer . However, higher concentrations of selenium salts can be toxic and mutagenic . The bases for both toxicity and protection are not clearly understood . To provide insights into these mechanisms, we analyzed the proteomic response of Escherichia coli cells to selenate and selenite treatment under aerobic conditions . We identified 23 proteins induced by both oxides and ca . 20 proteins specifically induced by each oxide . A striking result was the selenite induction of 8 enzymes with antioxidant properties, particularly the manganese and iron superoxide dismutases (SodA and SodB) . The selenium inductions of sodA and sodB were controlled by the transcriptional regulators SoxRS and Fur, respectively . Strains with decreased superoxide dismutase activities were severely impaired in selenium oxide tolerance . Pretreatment with a sublethal selenite concentration triggered an adaptive response dependent upon SoxRS, conferring increased selenite tolerance . Altogether, our data indicate that superoxide dismutase activity is essential for the cellular defense against selenium salts, suggesting that superoxide production is a major mechanism of selenium toxicity under aerobic conditions. Appl Environ Microbiol, 2002 Mar, 68(3), 1280 - 9 Reversal of flagellar rotation is important in initial attachment of Escherichia coli to glass in a dynamic system with high- and low-ionic-strength buffers; McClaine JW et al.; The attachment rates of wild-type, smooth-swimming, tumbly, and paralyzed Escherichia coli to glass was measured at fluid velocities of 0.0044 and 0.044 cms(-1) (corresponding to shear rates of 0.34 and 3.4 s(-1), respectively), in 0.02 and 0.2 M buffer solutions . At the highest ionic strength, we did not observe a significant difference in the attachment rate of wild-type and paralyzed cells at either fluid velocity . However, when the ionic strength was reduced, paralyzed bacteria attached at rates 4 and 10 times lower than that of the wild type under fluid velocities of 0.0044 and 0.044 cms(-1), respectively . This suggested that the rotation of the flagella assisted in attachment . We then compared the attachment rates of smooth-swimming (counterclockwise rotation only) and tumbly (clockwise rotation only) cells to the wild type to determine whether the direction of rotation was important to cell attachment . At 0.0044 cms(-1), the smooth-swimming cells attached at rates similar to that of the wild type in both buffer solutions but significantly less at the higher fluid velocity . Tumbly cells attached at much lower rates under all conditions . Thus, the combination of clockwise and counterclockwise flagellar rotation and their coupling appeared to be important in cell attachment . We considered a number of hypotheses to interpret these observations, including a residence time analysis and a comparison of traditional Derjaguin-Landau-Verwey-Overbeek (DLVO) theory to soft-particle theory. J Biochem (Tokyo), 2002 Mar, 131(3), 445 - 52 A cytosolic form of aminopeptidase P from Drosophila melanogaster: molecular cloning and characterization; Kulkarni GV et al.; Using a functional genomic approach, we have identified and characterized a cytosolic form of aminopeptidase P from Drosophila melanogaster . This study represents the first characterization of an insect aminopeptidase P . The complete sequence of a 12.5 kbp genomic clone from D . melanogaster showed the presence of a 1,839 bp ORF, encoding a protein of 613 amino acids with a calculated molecular mass of 68.5 kDa . The deduced amino acid sequence was 48% identical and 66% similar to rat and human cytosolic aminopeptidase P . Amino acids important for catalytic activity and the metal binding ligands were found to be conserved between Drosophila AP-P and its mammalian homologues . The recombinant enzyme expressed in Escherichia coli hydrolyzed the amino terminal Xaa-Pro bond of substance P and bradykinin, revealing its functional identity . Further enzyme characterization showed the enzyme to be a manganese-dependent metallopeptidase . Immunoblot analysis showed that DAP-P is located exclusively in the cytosol and is temporally regulated during Drosophila development . In the adult fly, the protein could be detected in gut, testis and ovary, with a high level of expression in brain. J Biochem (Tokyo), 2002 Mar, 131(3), 419 - 25 Sequence-independent DNA binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli; Makise M et al.; The DnaA protein specifically binds to the origin of chromosomal DNA replication and initiates DNA synthesis . In addition to this sequence-specific DNA binding, DnaA protein binds to DNA in a sequence-independent manner . We here compared the two DNA binding activities . Binding of ATP and ADP to DnaA inhibited the sequence-independent DNA binding, but not sequence-specific binding . Sequence-independent DNA binding, but not sequence-specific binding, required incubation at high temperatures . Mutations in the C-terminal domain affected the sequence-independent DNA binding activity less drastically than they did the sequence-specific binding . On the other hand, the mutant DnaA433, which has mutations in a membrane-binding domain (K327 to I344) was inert for sequence-independent binding, but could bind specifically to DNA . These results suggest that the two DNA binding activities involve different domains and perform different functions from each other in Escherichia coli cells. J Biochem (Tokyo), 2002 Mar, 131(3), 313 - 7 Crystal structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase complexed with cofactors: implications of a flexible loop movement upon substrate binding; Yajima S et al.; The key enzyme in the nonmevalonate pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be an effective target of antimalarial drugs . Here we report the crystal structure of DXR complexed with NADPH and a sulfate ion from Escherichia coli at 2.2 A resolution . The structure showed the presence of an extra domain, which is absent from other NADPH-dependent oxidoreductases, in addition to the conformation of catalytic residues and the substrate binding site . A flexible loop covering the substrate binding site plays an important role in the enzymatic reaction and the determination of substrate specificity. J Org Chem, 2002 Mar 8, 67(5), 1480 - 9 One-pot two-step enzymatic coupling of pyrimidine bases to 2-deoxy-D-ribose-5-phosphate . A new strategy in the synthesis of stable isotope labeled deoxynucleosides; Ouwerkerk N et al.; The enzymatic synthesis of thymidine from 2-deoxy-D-ribose-5-phosphate is achieved, in a one-pot two-step reaction using phosphoribomutase (PRM) and commercially available thymidine phosphorylase (TP) . In the first step the sugar-5-phosphate is enzymatically rearranged to alpha-2-deoxy-D-ribose-1-phosphate . Highly active PRM is easily obtained from genetically modified overproducing E . coli cells (12,000 units/84 mg protein) and is used without further purification . In the second step thymine is coupled to the sugar-1-phosphate . The thermodynamically unfavorable equilibrium is shifted to the product by addition of MnCl(2) to precipitate inorganic phosphate . In this way the overall yield of the beta-anomeric pure nucleoside increases from 14 to 60% . In contrast to uracil, cytosine is not accepted by TP as a substrate . Therefore, 2'-deoxy-cytidine is obtained by functional group transformations of the enzymatically prepared 2'-deoxy-uridine . The method has been demonstrated by the synthesis of {2',5'-(13)C(2)}- and {1',2',5'-(13)C(3)}thymidine as well as {1',2',5'-(13)C(3)}2'-deoxyuridine and {3',4'-(13)C(2)}2'-deoxycytidine . In addition the nucleoside bases thymine and uracil are tetralabeled at the (1,3-(15)N(2),2,4-(13)C(2))-atomic positions . All compounds are prepared without any scrambling or dilution of the labeled material and are thus obtained with a very high isotope enrichment (96-99%) . In combination with the methods that have been developed earlier it is concluded that each of the (13)C- and (15)N-positions and combination of positions of the pyrimidine deoxynucleosides can be efficiently labeled starting from commercially available and highly (13)C- or (15)N-enriched formaldehyde, acetaldehyde, acetic acid, potassium cyanide, methylamine hydrochloride, and ammonia. Lancet Infect Dis, 2001 Dec, 1(5), 304 - 13 Enteroaggregative Escherichia coli; Okeke IN et al.; Enteroaggregative Escherichia coli (EAEC) are an increasingly important cause of diarrhoea . E . coli belonging to this category cause watery diarrhoea, which is often persistent and can be inflammatory . EAEC have been implicated in sporadic diarrhoea in children and adults, in both developing and developed countries, and have been identified as the cause of several outbreaks worldwide . EAEC are defined by their ability to adhere to epithelial cells in a characteristic "stacked-brick" pattern but are otherwise highly heterogeneous . Genes that could contribute to the pathogenicity of EAEC encode adhesins, toxins, and other factors, all of which are only partially conserved . Practicable tools are needed to improve diagnosis and identify risk factors . EAEC-infected individuals can be treated with fluoroquinolones but there is a need to examine alternative treatment protocols. RNA, 2002 Jan, 8(1), 97 - 109 RNase E plays an essential role in the maturation of Escherichia coli tRNA precursors; Li Z et al.; Conversion of tRNA precursors to their mature forms requires the action of both endo- and exoribonucleases . Although studies over many years identified the endoribonuclease, RNase P, and several exoribonucleases as the enzymes responsible for generating the mature 5' and 3' termini, respectively, of Escherichia coli tRNAs, relatively little is known about how tRNAs are separated from long multimeric or multifunction transcripts, or from long leader and trailer sequences . To examine this question, the tRNA products that accumulate in mutant strains devoid of multiple exoribonucleases plus one or several endoribonucleases were analyzed by northern analysis . We find that the multifunction tyrT transcript, which contains two tRNA(Tyr)1 sequences separated by a 209-nt spacer region plus a downstream mRNA, is cleaved at three sites in the spacer region by the endoribonuclease, RNase E . When both RNase E and RNase P are absent, a product containing both tRNAs accumulates . Two multimeric tRNA transcripts, those for tRNA Arg-His-Leu-Pro and tRNA Gly-Cys-Leu also require RNase E for maturation . For the former transcript, products with long 3' extensions on tRNA(Arg), tRNA(His), and tRNA(Pro), as well as the primary transcript, accumulate in the absence of RNase E . For the latter transcript, RNase E cleaves downstream of each tRNA . Little processing of either multimeric transcript occurs in the absence of both RNase E and RNase P . These data indicate that RNase E is a major contributor to the initial processing of E . coli tRNA transcripts, providing substrates for final maturation by RNase P and the 3' exoribonucleases . Based on this new information, a detailed model for tRNA maturation is proposed. Rinsho Byori, 2002 Jan, 50(1), 13 - 9 {Production of recombinant human CRP and its application on clinical testing}; Matuo Y et al.; Recently, changes in the serum CRP level 1/10 the concentration range ordinarily used as a marker of acute inflammation has received attention in relation to cardiovascular injury at the AACC/CDC joint forum at Atlanta held on March 13, 2001 . We have succeeded in the development of recombinant human CRP(rCRP) by inserting the cloned CRP gene into expression vector pTRP, followed by transformation of E . coli . Genes encoding the signal peptide of E . coli alkaline phosphatase and kil gene were additionally inserted, so that rCRP can be secreted into the culture supernatant . Five grams of rCRP was purified from 180 L culture supernatant by affinity chromatography . The purified rCRP was indistinguishable from native rCRP with respect to Ca(2+)-dependent binding activity to phosphorylcholine, electrophoretic behavior in the presence or absence of SDS, N-terminal amino acid, and immunochemical properties . rCRP was found to have a potential as a reference material and/or calibrator for hsCRP assay. J Mol Recognit, 2002 Jan-Feb, 15(1), 6 - 18 Functional characterization of an anti-estradiol antibody by site-directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the V(L) domain in estradiol recognition; Coulon S et al.; The high-affinity monoclonal anti-estradiol antibody 9D3 presents a specificity defect towards estradiol-3-sulphate and 3-glucuronide conjugates incompatible with use in direct immunoassays . The corresponding single-chain variable fragment (scFv), cloned and produced in E . coli, exhibited a 10-fold lower affinity for estradiol (K(a)=1.2 x 10(9) M (-1)) and a slightly increased specificity defect for the 3-position . Site-directed mutagenesis revealed critical residues involved in estradiol recognition and produced mutants exhibiting up to a 3-fold increase of the binding affinity for estradiol and up to a 2-fold decrease of the cross-reactivity with estradiol-3-sulphate . A comparative model of the antibody 9D3-estradiol complex was built in which the estradiol D-ring is buried into the binding pocket while the 3-, 6- and 7-positions are solvent exposed, agreeing with the lack of specificity for these three positions . Two potential alternative orientations of the A-ring, one close to CDR H3 and L2 loops, and the other one close to CDR H2 and L3 loops, have been considered for the docking of estradiol, none of which could be unambiguously privileged taking into account data from cross-reactivity measurements, photolabelling and mutagenesis studies . For both orientations, estradiol is stabilized by hydrogen bonding of the 17beta-OH group with TyrL36, His89 and GlnH35 in the first case, or TyrL36, only, in the second case and by van der Waals contacts from TyrL91 with alpha- or beta-face of estradiol, respectively, and from ValH95 and GlyH97 with the opposite face . To elucidate the molecular basis of antibody 9D3 specificity, as compared with that of another anti-estradiol antibody 15H11, single variable domains (V(H) and V(L)) and scFv hybrids have been constructed . The binding activity of V(L)9D3 as well as the specificity of the V(L)9D3/V(H)15H11 hybrid, both similar to antibody 9D3, revealed a prominent role of V(L) in estradiol recognition . These findings establish premises for antibody engineering to reduce cross-reactivity, especially with estradiol-3-conjugates . Rapid Commun Mass Spectrom, 2002, 16(6), 616 - 26 Hydrogen/deuterium exchange for higher specificity of protein identification by peptide mass fingerprinting; Bienvenut WV et al.; Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences . Such amounts of material continuously increase protein database sizes . At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT . One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product . Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically . Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E . coli proteins . This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results . Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results . In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement . The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides . This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure . This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides . Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments . Electrophoresis, 2002 Feb, 23(4), 640 - 6 Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis; Werhahn W et al.; We report a subdivision of the mitochondrial proteome into defined sets of proteins, which is based on the combination of three different gel electrophoresis procedures . First, Blue-native polyacrylamide gel electrophoresis is employed to separate mitochondrial protein complexes . The protein complexes are electroeluted and completely detached from Coomasssie blue . Subsequently the subunits of the protein complexes are separated by isoelectric focusing and finally by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . The resolution capacity of the procedure is demonstrated for the ATP synthase complex, the cytochrome c reductase complex and the preprotein translocase of the outer mitochondrial membrane (the TOM complex) . The method allows the separation of isoforms of subunits forming part of protein complexes, whose occurrence seems to be rather a rule than an exception in higher eukaryotes . Furthermore, extremely hydrophobic proteins are detectable on the gels. J Microbiol Methods, 2002 May, 49(3), 321 - 3 A simple and rapid assay of collagen-like polymer in crude lysate from Escherichia coli; Yin J et al.; An assay for the quantification of collagen-like polymer (CLP) in Escherichia coli cells utilizing the specific reaction between collagenase and CLP is presented . It involves thermal treatment to precipitate non-CLP proteins, digestion of CLP by collagenase and detection of the absorbance of the liberated amino acids and peptides from CLP by a ninhydrin-based method . CLP concentration is determined from the absorbance measurement. Zhonghua Bing Li Xue Za Zhi, 1999 Oct, 28(5), 352 - 5 {Cloning whole length cDNA of related genes responsible for smooth muscle cells proliferation in atherogenesis and study on its function}; Zhao G et al.; OBJECTIVE: To clone whole length cDNA of the related genes responsible for vascular smooth muscle cell (SMC) proliferation in atherogenesis, and to study its function . METHODS: ox-LDL was added as a stimulant to the SMC culture medium . Subtractive library was established using subtractive hybridization technique in order to clone the related genes fragments . With the whole length cDNA library established, the whole length cDNA of the related gene was cloned . The protein expressed was studied . RESULTS: 4 new gene fragments and one whole length cDNA were cloned . The new cloned gene is able to express a protein of about 44000 daltons and closely related to the activity of ox-LDL . CONCLUSIONS: The new cloned gene is considered responsible for SMC proliferation. Clin Sci (Lond), 2002 Mar, 102(3), 337 - 44 Hepatocyte mitochondrial metabolism is inhibited in neonatal rat endotoxaemia: effects of glutamine; Markley MA et al.; Glutamine has beneficial effects on enterocytes and the immune system in sepsis, but its effects on hepatic metabolism remain unknown . The aim of the present study was to determine the effects of glutamine on hepatocyte energy metabolism under conditions of neonatal endotoxaemia . Suckling Wistar rats were injected intraperitoneally with 200 microg/kg lipopolysaccharide . Oxygen consumption was measured polarographically in hepatocytes respiring on either palmitate (0.5 mM) or palmitate plus glutamine (10 mM) . Total hepatocyte oxygen consumption was similar in hepatocytes from control and endotoxic rats, but this was due to a decrease in intramitochondrial and an increase in extramitochondrial oxygen consumption in the cells from endotoxic animals . The addition of glutamine to hepatocytes from endotoxic rats restored intramitochondrial oxygen consumption to control levels . Although glutamine did not reverse the inhibition of the thermogenic proton leak observed in endotoxaemia, it significantly increased oxygen consumption due to mitochondrial ATP synthesis (P=0.03) . Glutamine significantly increased the hepatocyte ATP/ADP ratio (P=0.02 compared with hepatocytes from endotoxic rats) . Electron microscopy revealed morphological damage to the mitochondria of hepatocytes from endotoxic rats, and a return to a normal appearance with the addition of glutamine . We conclude that glutamine reverses the inhibition of mitochondrial metabolism that is observed in endotoxaemia . The effect is primarily at the level of ATP synthesis. Eur Surg Res, 2002 Jan-Apr, 34(1-2), 68 - 72 Endotoxin inactivation by enterally applied colostrum of different composition; Seifert J et al.; BACKGROUND AND PURPOSE: Enteral applied bovine colostrum can significantly reduce endotoxin concentration in plasma . Since colostrum is a mixture of biological active ingredients 3 possible substances which are able to influence the endotoxin elimination were concentrated in 3 different colostrum products . Immunoglobulin-, lactoferrin- and casein-enriched colostra and lactoferrin alone were orally administered to endotoxinaemic rats . METHODS: Endotoxinaemia was induced to rats by enteral application of 10(10) E . coli together with 40 mg Nebacetin . Control animals received albumin . From all rats plasma samples were taken over the time of 5 h and endotoxin concentration determined with limulus lysate and chromogenic substrate . RESULTS: Whereas in control animals as well as in animals treated with casein-enriched colostrum a marked increase of endotoxin values to over 130 EU/dl could be observed after 5 h, the oral application of gammaglobulin-enriched and especially lactoferrin-enriched colostrum decreased endotoxin values by more than 50% . The most effective endotoxin elimination was seen with lactoferrin alone . CONCLUSIONS: From this results it can be concluded that not only gammaglobulin but especially lactoferrin seems to be responsible for the elimination of endotoxin with regard to enterally applied colostrum preparations . Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2766 - 71 Epub 2002 Feb 26. Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli; Roberts SA et al.; CueO (YacK), a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli . The crystal structure of CueO has been determined to 1.4-A resolution by using multiple anomalous dispersion phasing and an automated building procedure that yielded a nearly complete model without manual intervention . This is the highest resolution multicopper oxidase structure yet determined and provides a particularly clear view of the four coppers at the catalytic center . The overall structure is similar to those of laccase and ascorbate oxidase, but contains an extra 42-residue insert in domain 3 that includes 14 methionines, nine of which lie in a helix that covers the entrance to the type I (T1, blue) copper site . The trinuclear copper cluster has a conformation not previously seen: the Cu-O-Cu binuclear species is nearly linear (Cu-O-Cu bond angle = 170 degrees) and the third (type II) copper lies only 3.1 A from the bridging oxygen . CueO activity was maximal at pH 6.5 and in the presence of >100 microM Cu(II) . Measurements of intermolecular and intramolecular electron transfer with laser flash photolysis in the absence of Cu(II) show that, in addition to the normal reduction of the T1 copper, which occurs with a slow rate (k = 4 x 10(7) M(-1)x (-1)), a second electron transfer process occurs to an unknown site, possibly the trinuclear cluster, with k = 9 x 10(7) M(-1) x (-1), followed by a slow intramolecular electron transfer to T1 copper (k approximately 10 s(-1)) . These results suggest the methionine-rich helix blocks access to the T1 site in the absence of excess copper. Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2702 - 7 Epub 2002 Feb 26. Cloning of nitroalkane oxidase from Fusarium oxysporum identifies a new member of the acyl-CoA dehydrogenase superfamily; Daubner SC et al.; The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes with production of nitrite and hydrogen peroxide . The sequences of several peptides from the fungal enzyme were used to design oligonucleotides for the isolation of a portion of the NAO gene from an F . oxysporum genomic DNA preparation . This sequence was used to clone the cDNA for NAO from an F . oxysporum cDNA library . The sequence of the cloned cDNA showed that NOA is a member of the acyl-CoA dehydrogenase (ACAD) superfamily . The members of this family share with NAO a mechanism that is initiated by proton removal from carbon, suggesting a common chemical reaction for this superfamily . NAO was expressed in Escherichia coli and the recombinant enzyme was characterized . Recombinant NAO has identical kinetic parameters to enzyme isolated from F . oxysporum but is isolated with oxidized FAD rather than the nitrobutyl-FAD found in the fungal enzyme . NAO purified from E . coli or from F . oxysporum has no detectable ACAD activity on short- or medium-chain acyl CoAs, and medium-chain acyl-CoA dehydrogenase and short-chain acyl-CoA dehydrogenase are unable to catalyze oxidation of nitroalkanes. Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2690 - 5 Epub 2002 Feb 26. Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis; Drew D et al.; We present an approach that allows rapid determination of the topology of Escherichia coli inner-membrane proteins by a combination of topology prediction and limited fusion-protein analysis . We derive new topology models for 12 inner-membrane proteins: MarC, PstA, TatC, YaeL, YcbM, YddQ, YdgE, YedZ, YgjV, YiaB, YigG, and YnfA . We estimate that our approach should make it possible to arrive at highly reliable topology models for roughly 10% of the approximately 800 inner-membrane proteins thought to exist in E . coli. Plant Cell Physiol, 2002 Feb, 43(2), 152 - 8 Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon; Kang HG et al.; We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases . This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively . The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function . RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP) . In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development . Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells . In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low . This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway . We also analyzed endogenous GAs from seeds of the parthenocarpic fruits . The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development. J Biol Chem, 2002 May 10, 277(19), 16614 - 23 Epub 2002 Feb 26. Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a component of an intronic splicing enhancer complex that activates the splicing of the alternative exon 6A from chicken beta-tropomyosin pre-mRNA; Expert-Bezancon A et al.; Splicing of the chicken beta-tropomyosin exon 6A is stimulated, both in vivo and in vitro, by an intronic pyrimidine-rich element (S4) located 37 nucleotides downstream of exon 6A . Several pyrimidine-rich sequences are able to substitute for the natural S4 enhancer with various stimulatory effects . We show that the different enhancer sequences recruit U1 small nuclear ribonucleoprotein (SnRNP) to the exon 6A 5' splice site, with an efficiency that correlates with the splicing activation . By using RNA affinity and two-dimensional gel electrophoresis, we characterized several proteins that bind to the different enhancer sequences . Heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP I (polypyrimidine track-binding protein, PTB) exhibit a higher level of interaction with the strong enhancer sequences (S4) than with the weakest enhancers . Functional analysis shows that hnRNP K is a component of the enhancer complex that promotes exon 6A splicing through the wild-type S4 sequence . The addition of recombinant hnRNP K to nuclear extracts preincubated with poly(rC) RNA competitor completely restores splicing efficiency to the original level . hnRNP I (PTB) was also found associated with the strong enhancer sequences . Its function in the splicing of exon 6A is discussed. J Biol Chem, 2002 May 10, 277(19), 16606 - 13 Epub 2002 Feb 26. The Aes protein directly controls the activity of MalT, the central transcriptional activator of the Escherichia coli maltose regulon; Joly N et al.; MalT, the transcriptional activator of the maltose regulon from Escherichia coli, is the prototype of a new family of transcription factors . Its activity is controlled by multiple regulatory signals . ATP and maltotriose (the inducer) are two effectors of the activator that positively control its multimerization, a critical step in promoter binding . In addition, MalK, the ABC component of the maltodextrin transport system, and the two enzymes MalY and Aes down-regulate MalT activity in vivo . By using a biochemical approach, we demonstrate here that (i) Aes controls MalT activity through direct protein-protein interaction, (ii) Aes competes with maltotriose for MalT binding, (iii) ATP and ADP differentially affect the competition between Aes and the inducer, and (iv) part, if not all, of the Aes binding site is located in DT1, the N-terminal domain of the activator, which also contains the ATP binding site . All of these characteristics point toward an identical mode of action for MalY and Aes . However, we have identified an amino acid substitution in MalT that suppresses MalT inhibition by Aes without interfering with its inhibition by MalY, suggesting that the binding sites of the two inhibitory proteins do not coincide . The differential effects of ATP and ADP on the competition between the inducer and Aes (or MalY) suggest that the ATPase activity displayed by MalT plays a role in the negative control of its activity. J Biol Chem, 2002 May 17, 277(20), 17548 - 55 Epub 2002 Feb 26. Catalytic properties, thiol pK value, and redox potential of Trypanosoma brucei tryparedoxin; Reckenfelderbaumer N et al.; The dithiol protein tryparedoxin is a component of the unique trypanothione/trypanothione reductase metabolism of trypanosomatids and is involved in the parasite synthesis of deoxyribonucleotides and the detoxication of hydroperoxides . Tryparedoxin is a highly abundant protein in all life stages of Trypanosoma brucei, the causative agent of African sleeping sickness . As shown here, its functional properties are intermediate between those of classical thioredoxins and glutaredoxins . The redox potential of T . brucei tryparedoxin of -249 mV was determined by protein-protein redox equilibration with Escherichia coli thioredoxin . The trypanothione/tryparedoxin couple is probably the most significant factor determining the cytosolic redox potential of the parasites . The pK value of Cys(40), the first thiol in the WCPPC motif, is 7.2 as derived from the thiolate absorption at 240 nm and the rate of carboxymethylation . Alteration of the active site into that of thioredoxin (CGPC) did not affect the pK value . In contrast, in the mutant with the glutaredoxin motif (CPYC) the pK dropped to < or =4.0 . The fact that the pK value of tryparedoxin coincides with the intracellular pH of the parasite may contribute to the reactivity of tryparedoxin in thiol disulfide exchange reactions. Invest Ophthalmol Vis Sci, 2002 Mar, 43(3), 656 - 61 Molecular properties of wild-type and mutant betaIG-H3 proteins; Kim JE et al.; PURPOSE: BetaIG-H3 is a TGF-beta-induced cell adhesion molecule, the mutations of which are responsible for a group of 5q31-linked corneal dystrophies . The characteristic findings in these diseases are accumulation of protein deposits of different ultrastructures . To understand the mechanisms of protein deposits in 5q31-linked corneal dystrophies, the molecular properties of betaIG-H3 and the effects of mutation on these properties were studied in vitro . METHODS: Substitution mutations were generated by two-step PCR . Wild-type and mutant recombinant betaIG-H3 proteins were raised in Escherichia coli . For structural study, nondenaturing gel electrophoresis, cross-linking experiments, and electron microscopy examination were performed . A solid-phase interaction assay was performed for the interaction of betaIG-H3 with other matrix proteins . Wild-type and mutant betaIG-H3 cDNAs were cloned into a mammalian expression vector and overexpressed in the corneal epithelial cells by transient transfection . Immunoprecipitation and immunoblot analysis were performed with an antibody against human betaIG-H3 . Cell adhesion was assayed by measuring enzyme activities of N-acetyl-beta-D-glucosaminidase . RESULTS: The recombinant betaIG-H3 protein self-assembled to form multimeric bands and appeared to have a fibrillar structure . Solid-phase in vitro interaction assay showed that it bound strongly to type I collagen, fibronectin, and laminin; moderately to collagen type II and VI; and minimally to collagen type IV . Five recombinant mutant forms of betaIG-H3 (R124C, R124H, R124L, R555W, and R555Q) commonly found in 5q31-linked corneal dystrophies did not significantly affect the fibrillar structure, interactions with other extracellular matrix proteins, or adhesion activity in cultured corneal epithelial cells . In addition, the mutations apparently produced degradation products similar to those of wild-type betaIG-H3 . CONCLUSIONS: BetaIG-H3 polymerizes to form a fibrillar structure and strongly interacts with type I collagen, laminin, and fibronectin . Mutations found in the 5q31-linked corneal dystrophies do not significantly affect these properties . The results suggest that mutant forms of betaIG-H3 may require other cornea-specific factors, to form the abnormal accumulations in 5q31-linked corneal dystrophies. EMBO J, 2002 Mar 1, 21(5), 1139 - 47 MRM2 encodes a novel yeast mitochondrial 21S rRNA methyltransferase; Pintard L et al.; Mitochondria of the yeast Saccharomyces cerevisiae assemble their ribosomes from ribosomal proteins, encoded by the nuclear genome (with one exception), and rRNAs of 15S and 21S, encoded by the mitochondrial genome . Unlike cytoplasmic rRNA, which is highly modified, mitochondrial rRNA contains only three modified nucleotides: a pseudouridine (Psi(2918)) and two 2'-O-methylated riboses (Gm(2270) and Um(2791)) located at the peptidyl transferase centre of 21S rRNA . We demonstrate here that the yeast nuclear genome encodes a mitochondrial protein, named Mrm2, which is required for methylating U(2791) of 21S rRNA, both in vivo and in vitro . Deletion of the MRM2 gene causes thermosensitive respiration and leads to rapid loss of mitochondrial DNA . We propose that Mrm2p belongs to a new class of three eukaryotic RNA-modifying enzymes and is the orthologue of FtsJ/RrmJ, which methylates a nucleotide of the peptidyl transferase centre of Escherichia coli 23S rRNA that is homologous to U(2791) of 21S rRNA . Our data suggest that this universally conserved modified nucleotide plays an important function in vivo, possibly by inducing conformational rearrangement of the peptidyl transferase centre. EMBO J, 2002 Mar 1, 21(5), 1132 - 8 RNA quality control: degradation of defective transfer RNA; Li Z et al.; The distinction between stable (tRNA and rRNA) and unstable (mRNA) RNA has been considered an important feature of bacterial RNA metabolism . One factor thought to contribute to the difference between these RNA populations is polyadenylation, which promotes degradation of unstable RNA . However, the recent discovery that polyadenylation also occurs on stable RNA led us to examine whether poly(A) might serve as a signal for eliminating defective stable RNAs, and thus play a role in RNA quality control . Here we show that a readily denaturable, mutant tRNA(Trp) does not accumulate to normal levels in Escherichia coli because its precursor is rapidly degraded . Degradation is largely dependent on polyadenylation of the precursor by poly(A) polymerase and on its removal by polynucleotide phosphorylase . Thus, in the absence of these two enzymes large amounts of tRNA(Trp) precursor accumulate . We propose that defective stable RNA precursors that are poorly converted to their mature forms may be polyadenylated and subsequently degraded . These data indicate that quality control of stable RNA metabolism in many ways resembles normal turnover of unstable RNA. EMBO J, 2002 Mar 1, 21(5), 995 - 1003 The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure; Bessonneau P et al.; Escherichia coli preprotein translocase comprises a membrane-embedded trimeric complex of SecY, SecE and SecG . Previous studies have shown that this complex forms ring-like assemblies, which are thought to represent the preprotein translocation channel across the membrane . We have analyzed the functional state and the quaternary structure of the SecYEG translocase by employing cross-linking and blue native gel electrophoresis . The results show that the SecYEG monomer is a highly dynamic structure, spontaneously and reversibly associating into dimers . SecG-dependent tetramers and higher order SecYEG multimers can also exist in the membrane, but these structures form at high SecYEG concentration or upon overproduction of the complex only . The translocation process does not affect the oligomeric state of the translocase and arrested preproteins can be trapped with SecYEG or SecYE dimers . Dissociation of the dimer into a monomer by detergent induces release of the trapped preprotein . These results provide direct evidence that preproteins cross the bacterial membrane, associated with a translocation channel formed by a dimer of SecYEG. EMBO J, 2002 Mar 1, 21(5), 876 - 84 The morphogenic linker peptide of HBV capsid protein forms a mobile array on the interior surface; Watts NR et al.; Many capsid proteins have peptides that influence their assembly . In hepatitis B virus capsid protein, the peptide STLPETTVV, linking the shell-forming 'core' domain and the nucleic acid-binding 'protamine' domain, has such a role . We have studied its morphogenic properties by permuting its sequence, substituting it with an extraneous peptide, deleting it to directly fuse the core and protamine domains and assembling core domain dimers with added linker peptides . The peptide was found to be necessary for the assembly of protamine domain-containing capsids, although its size-determining effect tolerates some modifications . Although largely invisible in a capsid crystal structure, we could visualize linker peptides by cryo-EM difference imaging: they emerge on the inner surface and extend from the capsid protein dimer interface towards the adjacent symmetry axis . A closely sequence-similar peptide in cellobiose dehydrogenase, which has an extended conformation, offers a plausible prototype . We propose that linker peptides are attached to the capsid inner surface as hinged struts, forming a mobile array, an arrangement with implications for morphogenesis and the management of encapsidated nucleic acid. Biophys J, 2002 Mar, 82(3), 1667 - 76 Imaging the electrostatic potential of transmembrane channels: atomic probe microscopy of OmpF porin; Philippsen A et al.; The atomic force microscope (AFM) was used to image native OmpF porin and to detect the electrostatic potential generated by the protein . To this end the OmpF porin trimers from Escherichia coli was reproducibly imaged at a lateral resolution of approximately 0.5 nm and a vertical resolution of approximately 0.1 nm at variable electrolyte concentrations of the buffer solution . At low electrolyte concentrations the charged AFM probe not only contoured structural details of the membrane protein surface but also interacted with local electrostatic potentials . Differences measured between topographs recorded at variable ionic strength allowed mapping of the electrostatic potential of OmpF porin . The potential map acquired by AFM showed qualitative agreement with continuum electrostatic calculations based on the atomic OmpF porin embedded in a lipid bilayer at the same electrolyte concentrations . Numerical simulations of the experimental conditions showed the measurements to be reproduced quantitatively when the AFM probe was included in the calculations . This method opens a novel avenue to determine the electrostatic potential of native protein surfaces at a lateral resolution better than 1 nm and a vertical resolution of approximately 0.1 nm. J Hepatol, 2002 Mar, 36(3), 385 - 94 Overexpression of the mouse Fas gene in human Hep3B hepatoma cells overcomes their resistance to Fas-mediated apoptosis; Lamboley C et al.; BACKGROUND/AIMS: Fas-induced apoptosis is one of the main forms of apoptosis occurring in hepatocytes . We have previously demonstrated that the human hepatoma cell line Hep3B is resistant to Fas-mediated apoptosis . In this study, we investigated whether the human Fas receptor itself, or the Fas transduction pathway was responsible for the resistant phenotype . METHODS: Clones of Hep3B cells overexpressing the mouse Fas gene (Hep3B(mfas)) were generated by transfection, and apoptosis was studied by (i) chromatin condensation and nuclear fragmentation, (ii) flow cytometry, (iii) DNA fragmentation and (iv) poly (ADP-ribose) polymerase cleavage . RESULTS: Use of the species-specific and agonistic anti-mFas monoclonal antibody (JO2), showed that the mFas receptor was correctly routed to the plasma membrane of Hep3B(mfas) cells . Using the four above-mentioned criteria, we demonstrated that JO2 triggered mFas-mediated apoptosis of Hep3B(mfas), but not of Hep3B(pCi) cells (transfected with an empty vector) . CONCLUSIONS: Our data show (i) that the Fas signaling pathway can be completed when a functional mFas receptor is expressed in Hep3B cells, and thus, (ii) that the death-inducing signaling complex components and the effector caspases are functional in Hep3B cells . Moreover, they suggest that the Fas subunits are not pre-assembled at the cell membrane before receptor-ligand interaction. Acta Pharmacol Sin, 2002 Feb, 23(2), 143 - 51 Expression and purification of catalytic domain of human macrophage elastase for high throughput inhibitor screening; Cheng DH et al.; AIM: To obtain a catalytically active human macrophage elastase catalytic domain (hMECD) and to establish an efficient high-throughput method for screening macrophage elastase inhibitors . METHODS: Catalytic domain of human macrophage elastase was expressed in E coli and characterized to establish a high-throughput screening assay using a colorimetric method . A set of 8560 pure compounds and mixtures were screened . RESULTS: We have constructed an efficient E coli system for this human protein expression, and the recombinant hMECD protein was purified to homogeneity using anion-exchange chromatography after in vitro refolding from inclusion bodies . The yield of active hMECD protein was 23 mg from one liter of E coli culture after purification . Calcium and zinc ions were required both in refolding and enzymatic activity, but high concentration of zinc inhibited the refolding and activity . The hMECD cleaved several synthetic substrates including a chromogenic thiopeptolide and fluorogenic peptides with optimal activity around pH 8.0 . Screening of 8560 compounds and mixtures led to identify 27 pure compounds and 14 natural products with inhibitory activity higher than 80 % at 20 mg/L . CONCLUSION: An efficient expression and purification method for hMECD protein has been established, and the assay is effective, reliable, and fast in identifying the recombinant protein inhibitors. Acta Pharmacol Sin, 2002 Feb, 23(2), 117 - 23 A new model for random screening inhibitors of vascular endothelial growth factor receptor 1 kinase; Zhuang SF et al.; AIM: To establish a 96-well plate based kinase assay using a recombinant vascular endothelial growth factor (VEGF) receptor 1 kinase domain protein . METHODS: A human VEGF receptor 1 kinase domain protein was expressed in E coli, and its activity was monitored by its ability of phosphorylating the polyE4Y substrate coated on the walls of 96-well plates with antibody recognition and a colorimetric readout . A random screening of a sample organic compound library was carried out, and the hits were characterized with a transformed cell line stably expressing VEGF receptor 1 protein . RESULTS: An efficient E coli expression system for human VEGF receptor 1 kinase domain protein was constructed, and the purified recombinant protein was used to establish a practical screening assay for kinase inhibitors in vitro . Two thousand eight hundred organic compounds were screened, and two disubstituted furans (A1 and A5) with new structure showed inhibition of VEGF receptor 1 kinase . Compound A1 inhibited only phosphorylation of substrate, while compound A5 inhibited both autophosphorylation and substrate phosphorylation . Both inhibitors affected phosphorylation in the transformed cells . CONCLUSION: The recombinant receptor kinase based assay is simple and effective in identifying kinase inhibitors. Clin Microbiol Infect, 1995 Sep, 1(1), 31 - 34 Inhibition of Mononuclear Cell Procoagulant Activity by Lipophosphoglycan of Leishmania donovani; Del Prete R et al.; BACKGROUND: Since fibrin formation is an expression of the response of the host to parasite spread, the lipophosphoglycan (LPG) of Leishmania donovani and its carbohydrate fragment (PG) were examined for their capacity to inhibit procoagulant activity (PCA) production by human mononuclear cells stimulated with Escherichia coli endotoxin in vitro . METHODS: the putative inhibitory effect of LPG and its PG fragment was evaluated on the basis of their in vitro capacity to prolong significantly the time required for coagulation induced by endotoxin-stimulated mononuclear cells . RESULTS: LPG exhibited the most inhibitory activity, whereas the carbohydrate domain was not effective . These results are in agreement with the notion that LPG (but not PG) has an inhibitory effect on protein kinase C activity which plays a key role in the production of PCA by human monocytes . CONCLUSIONS: From a pathophysiological point of view, these data suggest the possibility that Leishmania avoids fibrin entrapment in the host through this inhibitory mechanism. J Am Chem Soc, 2002 Mar 6, 124(9), 1852 - 3 An effective method for the discrimination of motional anisotropy and chemical exchange; Kneller JM et al.; Analysis of the ratio of transverse and longitudinal relaxation rates (R2/R1) is an approach commonly used for estimation of overall correlation time and identification of chemical exchange in biological macromolecules . However, this analysis fails to distinguish between chemical exchange and motional anisotropy . We describe a simple method for identifying chemical exchange and motional anisotropy using the product, R1R2 . In the slow tumbling regime, the R1R2 product results in a constant value that is independent of overall correlation time and motional anisotropy . This analysis provides a simple method for rapidly estimating and dissociating the effects of motional anisotropy and chemical exchange in NMR heteronuclear spin relaxation data . We demonstrate the utility of the method with 15N relaxation data collected on the proteins E . coli ribonuclease H and the trimeric E . coli membrane associated lipoprotein lpp. J Mol Biol, 2002 Feb 22, 316(3), 853 - 66 Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III; Hadi MZ et al.; Abasic sites and non-conventional 3'-ends, e.g . 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors . To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair . Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing . Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities . Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function . We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity . These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold . Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair . J Mol Biol, 2002 Feb 22, 316(3), 829 - 37 Reaction mechanism of GTP cyclohydrolase I: single turnover experiments using a kinetically competent reaction intermediate; Schramek N et al.; GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3'-triphosphate, which is the first committed precursor of tetrahydrofolate and tetrahydrobiopterin . The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry . The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320 . This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain . Deconvolution of the optical spectra suggested four linearly independent processes . A fifth reaction step was attributed to photodecomposition of the enzyme product . Pre-steady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the final product, dihydroneopterin triphosphate . Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein . The data obtained with the wild-type and mutant protein in conjunction with earlier quenched flow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate . J Mol Biol, 2002 Feb 22, 316(3), 799 - 805 Motifs of serine and threonine can drive association of transmembrane helices; Dawson JP et al.; Known sequence motifs containing key glycine residues can drive the homo-oligomerization of transmembrane helices . To find other motifs, a randomized library of transmembrane interfaces was generated in which glycine was omitted . The TOXCAT system, which measures transmembrane helix association in the Escherichia coli inner membrane, was used to select high-affinity homo-oligomerizing sequences in this library . The two most frequently occurring motifs were SxxSSxxT and SxxxSSxxT . Isosteric mutations of any one of the serine and threonine residues to non-polar residues abolished oligomerization, indicating that the interaction between these positions is specific and requires an extended motif of serine and threonine hydroxyl groups . Computational modeling of these sequences produced several chemically plausible structures that contain multiple hydrogen bonds between the serine and threonine residues . While single serine or threonine side-chains do not appear to promote helix association, motifs can drive strong and specific association through a cooperative network of interhelical hydrogen bonds . J Mol Biol, 2002 Feb 22, 316(3), 517 - 29 The C-terminal domains of the RNA polymerase alpha subunits: contact site with Fis and localization during co-activation with CRP at the Escherichia coli proP P2 promoter; McLeod SM et al.; Fis is a versatile transactivator that functions at many different promoters . Fis activates transcription at the RpoS-dependent proP P2 promoter when bound to a site that overlaps the minus sign35 hexamer by a mechanism that requires the C-terminal domain of the alpha subunit of RNA polymerase (alphaCTD) . The region on Fis responsible for activating transcription through the alphaCTD has been localized to a short beta-turn near the DNA-binding determinant on one subunit of the Fis homodimer . We report here that Fis-dependent activation of proP P2 transcription requires two discrete regions on the alphaCTD . One region, consisting of residues 264-265 and 296-297, mediates DNA binding . A second patch, comprising amino acid residues 271-273, forms a ridge on the surface of the alphaCTD that we propose interacts with Fis . The accompanying paper shows that these same regions on alphaCTD are utilized for transcriptional activation at the rrnB and rrnE P1 promoters by Fis bound to a site upstream of the core promoter (centered at minus sign71/minus sign72) . In addition to stimulation of proP P2 transcription by Fis, CRP co-activates this promoter when bound to a remote site upstream from the promoter (centered at -121.5) . RNA polymerase preparations lacking one alphaCTD of the alpha dimer were employed to demonstrate that the beta'-associated alpha(II)CTD was utilized preferentially by Fis at proP P2 in the presence and absence of CRP . These experiments define the overall architecture of the proP P2 initiation complex where Fis and CRP each function through a different alphaCTD . J Mol Biol, 2002 Feb 22, 316(3), 501 - 16 Architecture of Fis-activated transcription complexes at the Escherichia coli rrnB P1 and rrnE P1 promoters; Aiyar SE et al.; The transcription factor Fis activates the Escherichia coli rRNA promoters rrnB P1 and rrnE P1 by binding to sites centered at -71 and -72, respectively, and interacting with the C-terminal domain of the alpha subunit of RNA polymerase (RNAP alphaCTD) . To understand the mechanism of activation by Fis at these promoters, we used oriented alpha-heterodimeric RNAPs and heterodimers of Fis to determine whether one or both subunits of alpha and Fis participate in the alphaCTD-Fis interaction . Our results imply that only one alphaCTD in the alpha dimer and only one activation-proficient subunit in the Fis dimer are required for activation by Fis . A library of alanine substitutions in alpha was used to identify the alphaCTD determinants required for Fis-dependent transcription at rrnB P1 and rrnE P1 . We propose that the transcriptional activation region of the promoter-proximal subunit of the Fis dimer interacts with a determinant that includes E273 of one alphaCTD to activate transcription . We further suggest that the Fis contact to alphaCTD results in alphaCTD interactions with DNA that differ somewhat from those that occur at UP elements in the absence of Fis . The accompanying paper shows that the 273 determinant on alphaCTD is also targeted by Fis at the proP P2 promoter where the activator binds overlapping the -35 hexamer . Thus, similar Fis-alphaCTD interactions are used for activation of transcription when the activator is bound at very different positions on the DNA . Biochem Biophys Res Commun, 2002 Mar 8, 291(4), 1045 - 51 Nuclear factor-kappa B-independent regulation of lipopolysaccharide-mediated interleukin-6 biosynthesis; Haddad JJ et al.; The possible involvement of nuclear factor (NF)-kappa B in mediating the regulation of interleukin (IL)-6 biosynthesis in response to E . coli-derived lipopolysaccharide-endotoxin (LPS) was investigated in vitro . In alveolar epithelial cells, irreversible inhibition of the proteasome complex by carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132; 1-50 muM) did not affect LPS-mediated IL-6 secretion . Whereas the selective inhibition of the NF-kappa B pathway by the action of caffeic acid phenyl ethyl ester (CAPE; 1-100 microM) attenuated LPS-dependent IL-6 production at 100 muM, sulfasalazine (SSA; 0.1--10 mM), a potent and irreversible inhibitor of NF-kappa B, did not inhibit LPS-dependent IL-6 secretion . Incorporation of a selectively permeant inhibitor of NF-kappa B, SN-50 (1-20 microM), a peptide which contains the nuclear localization sequence (NLS) for the p50 NF-kappa B subunit and the amino-terminal sequence of Kaposi fibroblast growth factor to promote cell permeability, did not reduce LPS-mediated release of IL-6 . These data indicate a NF-kappa B-independent pathway mediating LPS-dependent regulation of IL-6 biosynthesis in the airway epithelium. Biochem Biophys Res Commun, 2002 Mar 8, 291(4), 979 - 86 Evidence for "pre-recruitment" as a new mechanism of transcription activation in Escherichia coli: the large excess of SoxS binding sites per cell relative to the number of SoxS molecules per cell; Griffith KL et al.; In response to the oxidative stress imposed by redox-cycling compounds like paraquat, Escherichia coli induces the synthesis of SoxS, which then activates the transcription of approximately 100 genes . The DNA binding site for SoxS-dependent transcription activation, the "soxbox," is highly degenerate, suggesting that the genome contains a large number of SoxS binding sites . To estimate the number of soxboxes in the cell, we searched the E . coli genome for SoxS binding sites using as query sequence the previously determined optimal SoxS binding sequence . We found approximately 12,500 sequences that match the optimal binding sequence under the conditions of our search; this agrees with our previous estimate, based on information theory, that a random sequence the size of the E . coli genome contains approximately 13,000 soxboxes . Thus, fast-growing cells with 4-6 genomes per cell have approximately 65,000 soxboxes . This large number of potential SoxS binding sites per cell raises the interesting question of how SoxS distinguishes between the functional soxboxes located within the promoters of target genes and the plethora of equivalent but nonfunctional binding sites scattered throughout the chromosome . To address this question, we treated cells with paraquat and used Western blot analysis to determine the kinetics of SoxS accumulation per cell; we also determined the kinetics of SoxS-activated gene expression . The abundance of SoxS reached a maximum of 2,500 molecules per cell 20 min after induction and gradually declined to approximately 500 molecules per cell over the next 1.5 h . Given that activation of target gene expression began almost immediately and given the large disparity between the number of SoxS molecules per cell, 2,500, and the number of SoxS binding sites per cell, 65,000, we infer that SoxS is not likely to activate transcription by the usual "recruitment" pathway, as this mechanism would require a number of SoxS molecules similar to the number of soxboxes . Instead, we propose that SoxS first interacts in solution with RNA polymerase and then the binary complex scans the chromosome for promoters that contain a soxbox properly positioned and oriented for transcription activation . We name this new pathway "pre-recruitment." Biochem Biophys Res Commun, 2002 Mar 8, 291(4), 951 - 8 Identification of AHNAK as a novel autoantigen in systemic lupus erythematosus; Skoldberg F et al.; To identify candidate autoantigens associated with arthritis, a rat chondrocyte cDNA library was immunoscreened with serum from a patient with rheumatoid arthritis . One isolated cDNA encoded part of AHNAK, a 700-kDa phosphoprotein with DNA binding properties, that appears to be involved in several signal transduction pathways . Immunoreactivity against an in vitro translated human AHNAK fragment was detected in 4.6% (5/109) of patients with rheumatoid arthritis, 29.5% (18/61) of patients with systemic lupus erythematosus (SLE), and 1.2% (2/172) of blood donors . Anti-AHNAK antibodies reacted with a recombinant human AHNAK fragment and with native AHNAK from C32 cell lysates . In vitro translated AHNAK fragment could be cleaved by granzyme B and caspase-3 . Anti-AHNAK positive SLE patients had a higher frequency of homogeneous antinuclear antibody staining patterns and a lower frequency of recent mucosal ulcerations . This is the first report that AHNAK can be targeted by the immune system in autoimmune disease. Biochem Biophys Res Commun, 2002 Mar 8, 291(4), 939 - 44 Cloning and expression of a lombricine kinase from an echiuroid worm: insights into structural correlates of substrate specificity; Ellington WR et al.; Phosphagen kinases constitute a large family of enzymes catalyzing the reversible phosphorylation of guanidino acceptor compounds . These guanidino substrates differ substantially in size and chemical properties . In spite of the appearance of X-ray crystal structures for two members of this family, creatine kinase (CK) and arginine kinase (AK), the structural correlates of substrate specificity remain to be fully elucidated . We have determined the cDNA and deduced amino acid sequences for lombricine (guanidinethylphosphoserine) kinase (LK) from the echiuroid worm Urechis caupo and expressed the cDNA in Escherichia coli . The recombinant protein was purified by affinity chromatography and showed high capacity for phosphorylation of lombricine . Phosphagen kinases consist of a small, N-terminal domain and a much larger domain connected by a linker sequence . A key event in catalysis in CK and AK, and certainly all other phosphagen kinases, is a large conformational change involving involving a rotation of the two domains and the movement of two highly conserved flexible loops (one located in the small domain; the other located in the large domain of these enzymes) which clamp down on the substrates . Multiple sequence alignments of Urechis LK with the only other LK sequence available and CK, AK and glycocyamine kinase sequences, confirm the importance of the small flexible loop located in the N-terminal domain of phosphagen kinases as one component of the structural determinants of guanidine specificity . The role of the other flexible loop in the large domain in terms of substrate specificity remains questionable. Biochem Biophys Res Commun, 2002 Mar 8, 291(4), 921 - 4 Catalytic function of a novel protein protochlorophyllide oxidoreductase C of Arabidopsis thaliana; Pattanayak GK et al.; In Arabidopsis thaliana Por C has been identified only on sequence homology to that of por A and por B . To demonstrate its catalytic function Arabidopsis thaliana protochlorophyllide oxidoreductase C gene (por c) that codes for the mature part of POR C protein having 335 amino acids was expressed in Escherchia coli cells . The POR C enzyme in the presence of NADPH and protochlorophyllide when incubated in dark formed a ternary complex . When it was excited at 433 nm, it had a fluorescence emission peak at 636 nm . After illumination with actinic cool white fluorescent light, a peak at 673 nm due to chlorophyllide gradually increased with concomitant decrease of 636 nm emission, demonstrating the gradual phototransformation of protochlorophyllide to chlorophyllide . The significance of differential por gene expression in light and dark among different species is discussed. Biochem Biophys Res Commun, 2002 Mar 8, 291(4), 884 - 9 Identification and characterization of a novel type of membrane-associated prostaglandin E synthase; Tanikawa N et al.; Membrane-associated prostaglandin E synthase (mPGE synthase) was previously purified to apparent homogeneity from the microsomal fraction of bovine heart (Watanabe, K., et al., Biochim . Biophys . Acta 1439, 406--414, 1999) . The N-terminal 22-amino acid sequence of the purified enzyme was identical to that of the 88th to 109th amino acids deduced from the monkey (AB046026) or human (AK024100) cDNA that encodes a hypothetical protein with unknown function . The primary structure has the consensus region of glutaredoxin and of thioredoxin . We constructed an expression plasmid, using the vector (pTrc-HisA) and the monkey cDNA for the 290-amino-acid polypeptide . The recombinant protein with a M(r) of 33 kDa exhibited PGE synthase activity and was purified to apparent homogeneity by nickel-chelating column chromatography . The V(max) and K(m) values for PGH(2) of the purified recombinant mPGE synthase were about 3.3 mumol/min center dot mg of protein and 28 muM, respectively . The recombinant enzyme was activated by various SH-reducing reagents, i.e., dithiothreitol, glutathione (GSH), and beta-mercaptoethanol, in order of decreasing effectiveness . Moreover, the mRNA distribution was high in the heart and brain, but the mRNA was not expressed in the seminal vesicles . These results indicate that the recombinant mPGE synthase is identical to the enzyme purified from the microsomal fraction of bovine heart, and is a novel type of mPGE synthase based on the primary structure, a broad specificity of thiol requirement, and tissue distribution. Biochem Biophys Res Commun, 2002 Mar 8, 291(4), 875 - 83 Biophysical characterization of cyclic nucleotide phosphodiesterases; Hofmann A et al.; We have compared selected biophysical properties of three phosphodiesterases, from Arabidopsis thaliana, Saccharomyces cerevisiae, and Escherichia coli . All of them belong to a recently identified family of cyclic nucleotide phosphodiesterases . Experiments elucidating folding stability, protein fluorescence, oligomerization behavior, and the effects of substrates were conducted, revealing differences between the plant and the yeast protein . According to CD spectroscopy, the latter protein exhibits an (alpha + beta) fold rather than an (alpha/beta) fold as found with CPDase (A . thaliana) . The redox-dependent structural reorganization recently found for the plant protein by X-ray crystallography could not be detected by CD spectroscopy due to its only marginal effect on the total percentage of helical content . However, in the present study a redox-dependent effect was also observed for the yeast CPDase . The enzymatic activity of wild type CPDase (A . thaliana) as well as of four mutants were characterized by isothermal titration calorimetry and the results prove the requirement of all four residues of the previously identified tandem signature motif for the catalytic function . Within the comparison of the three proteins in this study, the PDase Homolog/RNA ligase (E . coli) shares more similarities with the plant than with the yeast protein. Biosci Biotechnol Biochem, 2002 Jan, 66(1), 127 - 34 Substrate specificity at the P1' site of Escherichia coli OmpT under denaturing conditions; Okuno K et al.; Though OmpT has been reported to mainly cleave the peptide bond between consecutive basic amino acids, we identified more precise substrate specificity by using a series of modified substrates, termed PRX fusion proteins, consisting of 184 residues . The cleavage site of the substrate PRR was Arg140-Arg141 and the modified substrates PRX substituted all 19 natural amino acids at the P1' site instead of Arg141 . OmpT under denaturing conditions (in the presence of 4 M urea) cleaved not only between two consecutive basic amino acids but also at the carboxyl side of Arg140 except for the Arg140-Asp141, -Glu141, and -Pro141 pairs . In addition to Arg140 at the P1 site, similar results were obtained when Lys140 was substituted into the P1 site . In the absence of urea, an aspartic acid residue at the P1' site was unfavorable for OmpT cleavage of synthetic decapeptides, the enzyme showed a preference for a dibasic site. J Biol Chem, 2002 May 24, 277(21), 18390 - 6 Epub 2002 Feb 25. Quantitative determination of binding affinity of delta-subunit in Escherichia coli F1