Escherichia coli

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-ATPase: effects of mutation, Mg2+, and pH on Kd; Weber J et al.; To study the stator function in ATP synthase, a fluorimetric assay has been devised for quantitative determination of binding affinity of delta-subunit to Escherichia coli F(1)-ATPase . The signal used is that of the natural tryptophan at residue delta28, which is enhanced by 50% upon binding of delta-subunit to alpha(3)beta(3)gammaepsilon complex . K(d) for delta binding is 1.4 nm, which is energetically equivalent (50.2 kJ/mol) to that required to resist the rotor strain . Only one site for delta binding was detected . The deltaW28L mutation increased K(d) to 4.6 nm, equivalent to a loss of 2.9 kJ/mol binding energy . While this was insufficient to cause detectable functional impairment, it did facilitate preparation of delta-depleted F(1) . The alphaG29D mutation reduced K(d) to 26 nm, equivalent to a loss of 7.2 kJ/mol binding energy . This mutation did cause serious functional impairment, referable to interruption of binding of delta to F(1) . Results with the two mutants illuminate how finely balanced is the stator resistance function . delta' fragment, consisting of residues delta1-134, bound with the same K(d) as intact delta, showing that, at least in absence of F(o) subunits, the C-terminal domain of delta contributes zero binding energy . Mg(2+) ions had a strong effect on increasing delta binding affinity, supporting the possibility of bridging metal ion involvement in stator function . High pH environment greatly reduced delta binding affinity, suggesting the involvement of protonatable side-chains in the binding site.

J Biol Chem, 2002 May 10, 277(19), 16928 - 35 Epub 2002 Feb 25.
The high specificities of Phaseolus vulgaris erythro- and leukoagglutinating lectins for bisecting GlcNAc or beta 1-6-linked branch structures, respectively, are attributable to loop B; Kaneda Y et al.; Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities . While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E(4)- and L(4)-PHA) are unique in recognizing larger structures . E(4)- and L(4)-PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a beta1,6-linked branch, respectively . However, the detailed mechanisms of molecular recognition are poorly understood . In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E(4)- and L(4)-PHA . We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A . (1997) J . Mol . Biol . 267, 433-445) . The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance . These comparisons indicate that the high specificities of E(4)- and L(4)-PHA toward bisecting GlcNAc and beta1,6-linked branch structures are almost solely attributable to loop B . The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible . Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus . Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E(4)- and L(4)-PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a beta1,6-linked branch.

J Biol Chem, 2002 May 10, 277(19), 16538 - 46 Epub 2002 Feb 25.
Differential characteristics and subcellular localization of two starch-branching enzyme isoforms encoded by a single gene in Phaseolus vulgaris L; Hamada S et al.; Starch-branching enzymes (SBE) have a dominant role for amylopectin structure as they define chain length and frequency of branch points . We have previously shown that one of the SBE isoforms of kidney bean (Phaseolus vulgaris L.), designated PvSBE2, has a molecular mass (82 kDa) significantly smaller than those reported for isologous SBEs from pea (SBEI), maize (BEIIb), and rice (RBE3) . Additionally, in contrast to the dual location of the pea SBEI in both the soluble and starch granule fractions, PvSBE2 was found only in the soluble fraction during seed development . Analysis of a pvsbe2 cDNA suggested that PvSBE2 is generated from a larger precursor with a putative plastid targeting sequence of 156 residues . Here we describe the occurrence of a larger 100-kDa form (LF-PvSBE2) of PvSBE2 found both in the soluble and starch granule fractions of the developing seeds . The determined N-terminal sequence, VKSSHDSD, of LF-PvSBE2 corresponded to a peptide sequence located 111 amino acids upstream from the N terminus of purified PvSBE2, suggesting that LF-PvSBE2 and PvSBE2 are products of the same gene . Analysis of the products by 5'-RACE (rapid amplification of cDNA ends) and reverse transcription PCR indicated that the two transcripts for pre-LF-PvSBE2 and pre-PvSBE2 are generated by alternative splicing . Recombinant LF-PvSBE2 (rLF-PvSBE2) was purified from Escherichia coli and the kinetic properties were compared with those of recombinant PvSBE2 (rPvSBE2) . rLF-PvSBE2 had much higher affinity for amylopectin (K(m) = 4.4 mg/ml) than rPvSBE2 (18.4 mg/ml), whereas the V(max) of rLF-PvSBE2 (135 units/mg) for this substrate was much lower than that of rPvSBE2 (561 units/mg) . These results suggest that the N-terminal extension of LF-PvSBE2 plays a critical role for localization in starch granules by altering its enzymatic properties.

Trends Immunol, 2002 Mar, 23(3), 135 - 9
Does the shape of lipid A determine the interaction of LPS with Toll-like receptors?
Netea MG, van Deuren M, Kullberg BJ, Cavaillon JM, Van der Meer JW.
Lipopolysaccharide (LPS) triggers the activation of the immune system through the induction of cytokine release . Although it was assumed initially that LPS molecules from different bacteria are similar, recent evidence suggests that structural and functional differences between LPS species are the rule rather than the exception . It has been proposed that the shape of the lipid A component determines the bioactivity of LPS, with lipid A that adopts a conical conformation being more active than lipid A that adopts a cylindrical shape . The mechanism linking the molecular conformation with the biological activity of LPS has not been elucidated . We propose that LPS with a conical shape (e.g . from Escherichia coli) induces cytokine production through Toll-like receptor 4 (TLR4), whereas more cylindrical LPS (e.g . from Porphyromonas gingivalis) induces expression of a different set of cytokines through TLR2 . Strictly cylindrical LPS molecules (e.g . the lipid A precursor Ia or from Rhodobacter sphaeroides) have antagonistic properties at the level of TLRs.

Free Radic Biol Med, 2002 Mar 1, 32(5), 481 - 4
Role of inducible nitrogen oxide synthase in benzene-induced oxidative DNA damage in the bone marrow of mice; Vestergaard S et al.; We investigated the interaction of BZ and lipolysaccharide (LPS), a well-known inflammation-promoting agent, in wild-type and inducible nitrogen oxide synthase (iNOS) knockout mice . BZ generated DNA strand breaks (SB) in the liver of both wild-type and iNOS-deficient mice . In the bone marrow (BM) BZ and LPS generated SB only in wild-type mice . The effects were additive, suggesting that both a redox cycling and an iNOS-dependent pathway may be involved . Formamidopyrimidine DNA glycosylase sensitive sites were elevated by BZ in the BM in both types of mice, whereas endonuclease III sensitive sites were not affected by any treatment . Since BZ is associated with leukemia in humans, it suggests that oxidative DNA base damage rather than SB may be important in the development of leukemia.

Virus Res, 2002 Feb 26, 83(1-2), 159 - 67
Specificity analysis of the conserved hexanucleotides for the replication of bamboo mosaic potexvirus RNA; Chiu WW et al.; In order to investigate the possible function of the potexviral conserved hexanucleotide sequence (ACc/uUAA) found in the 3' untranslated region of bamboo mosaic potexvirus (BaMV) genomic RNA, each nucleotide in the hexamer motif was substituted . Transcripts derived from wild-type and mutants with a loop deletion or a single-nucleotide substitution were inoculated into protoplasts . The accumulation levels of viral coat protein and RNAs detected from Western and Northern blots of each inoculation were examined after a 48-h incubation . Our data revealed that the nucleotides at positions 4-6 of the hexamer motif cannot be replaced by other nucleotides; the first position of this hexamer is purine specific, and the second position is restricted to pyrimidine . Substitution at the third position has less effect on viral accumulation in protoplasts . In addition to the results reported previously that the E . coli over-expressed BaMV RNA-dependent RNA polymerase could specifically interact with the hexamer motif, the hexanucleotides were thought to serve as a recognition site of viral replicase and the specificity may be derived from the functional groups of each nucleotide of this hexamer motif.

Mol Cell, 2002 Feb, 9(2), 353 - 62
Mutational separation of two pathways for editing by a class I tRNA synthetase; Hendrickson TL et al.; Aminoacyl tRNA synthetases (aaRSs) catalyze the first step in protein biosynthesis, establishing a connection between codons and amino acids . To maintain accuracy, aaRSs have evolved a second active site that eliminates noncognate amino acids . Isoleucyl tRNA synthetase edits valine by two tRNA(Ile)-dependent pathways: hydrolysis of valyl adenylate (Val-AMP, pretransfer editing) and hydrolysis of mischarged Val-tRNA(Ile) (posttransfer editing) . Not understood is how a single editing site processes two distinct substrates--an adenylate and an aminoacyl tRNA ester . We report here distinct mutations within the center for editing that alter adenylate but not aminoacyl ester hydrolysis, and vice versa . These results are consistent with a molecular model that shows that the single editing active site contains two valyl binding pockets, one specific for each substrate.

Mol Cell, 2002 Feb, 9(2), 241 - 51
Direct rescue of stalled DNA replication forks via the combined action of PriA and RecG helicase activities; Gregg AV et al.; The PriA protein of Escherichia coli plays a key role in the rescue of replication forks stalled on the template DNA . One attractive model of rescue relies on homologous recombination to establish a new fork via PriA-mediated loading of the DnaB replicative helicase at D loop intermediates . We provide genetic and biochemical evidence that PriA helicase activity can also rescue a stalled fork by an alternative mechanism that requires manipulation of the fork before loading of DnaB on the lagging strand template . This direct rescue depends on RecG, which unwinds forks and Holliday junctions and interconverts these structures . The combined action of PriA and RecG helicase activities may thus avoid the potential dangers of rescue pathways involving fork breakage and recombination.

Mol Cell, 2002 Feb, 9(2), 206 - 7
FtsK: Maxwell's Demon?
Donachie WD.
FtsK, which links chromosome segregation and cell division in E . coli, has now been shown to be an ATP-dependent DNA translocase . It also activates XerCD-dependent recombination, converting chromosome dimers into monomers, by switching the order of strand cleavage by the recombinase subunits.

Curr Biol, 2002 Feb 19, 12(4), 335 - 9
Replication-associated repair of adenine:8-oxoguanine mispairs by MYH; Hayashi H et al.; Cellular DNA is constantly exposed to the risk of oxidation . 8-oxoguanine (8-oxoG) is one of the major DNA lesions generated by oxidation, which is primarily corrected by base excision repair . When it is not repaired prior to replication, replicative DNA polymerases yield misinsertion of an adenine (A) opposite the 8-oxoG on the template strand, generating an A:8-oxoG mispair . MYH, a mammalian homolog of Escherichia coli MutY, is a DNA glycosylase responsible for initiating base excision repair of such a mispair by excising the adenine opposite 8-oxoG . Here, using an in vivo repair system, we show that DNA replication enhances the repair of the A:8-oxoG mispair . Repair efficiency was lower in MYH-deficient murine cells than in MYH-proficient cells . Transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:8-oxoG repair, indicating that a significant part of this replication-associated repair depends on MYH . Expression of a mutant MYH in which the PCNA binding motif was disrupted did not increase the repair efficiency, thus suggesting that the interaction between PCNA and MYH is critical for MYH-initiated repair of A:8-oxoG.

Clin Microbiol Infect, 1998 Feb, 4(12), 682 - 688
Prevalence of diarrheagenic Escherichia coli strains detected by PCR in patients with travelers' diarrhea; Vargas M et al.; OBJECTIVE: To determine the prevalence of the different categories of diarrheagenic Escherichia coli, enterotoxigenic E . coli (ETEC), enteroinvasive E . coli (EIEC), verotoxin-producing E . coli (VTEC), enteroaggregative E . coli (EAggEC), diffusely adherent E . coli (DAEC), and enteropathogenic E . coli (EPEC), associated with travelers' diarrhea . METHODS: Stool specimens from 350 patients with travelers' diarrhea were collected between 1994 and 1996 . The virulence factors of the diarrheagenic E . coli isolated were detected by PCR technique, in subcultures of single colonies of all morphotypes of E . coli observed in culture on MacConkey agar . RESULTS: ETEC (15.7%), EAggEC (13.4%) and DAEC (9.14%) are significantly more prevalent than EIEC (3.4%), EPEC (2.86%) and VTEC (0.86%) (p<0.05; z-test) . The prevalence of ETEC and EAggEC was similar in all geographic areas visited . CONCLUSIONS: PCR is a rapid and specific technique to use in the identification of the different categories of diarrheagenic E . coli and greatly increases the yield of potential enteropathogens from cases of travelers' diarrhea . Not only ETEC but also EAggEC and DAEC strains play a major role in the etiology of travelers' diarrhea, whereas EIEC, EPEC, and VTEC strains play a minor role, leading to the question of whether it is necessary to search routinely for these three types of E . coli in diagnostic laboratories.

Biochemistry, 2002 Mar 5, 41(9), 3262 - 9
Converting a maltose receptor into a nascent binuclear copper oxygenase by computational design; Benson DE et al.; Computational protein design methods were used to identify mutations that are predicted to introduce a binuclear copper center coordinated by six histidines, replacing the maltose-binding site in Escherichia coli maltose-binding protein (MBP) with an oxygen-binding site . A small family of five candidate designs consisting of 9 to 10 mutations each was constructed by oligonucleotide-directed mutagenesis . These mutant proteins were expressed and purified, and their stability, copper- and cobalt-binding properties, and interactions of the resulting metalloprotein complexes with azide, hydrogen peroxide, and dioxygen were characterized . We identified one 10-fold mutant, MBP.Hc.E, that can form Cu(II)(2) and Co(II)(2) complexes that interact with H(2)O(2) and O(2) . The Co(II)(2) protein reacts with H(2)O(2) to form a complex that is spectroscopically similar to a synthetic model that structurally mimics the oxy-hemocyanin core, whereas the Cu(II)(2) protein reacted with O(2) or H(2)O(2) does not . We postulate that the equilibrium between the open and closed conformations of MBP allows species with variable Cu-Cu distances to form, and that such species can bind ligands in geometries that are not observed in natural type III centers . Introduction of one additional mutation in the hinge region of MBP, I329F, known to favor formation of the closed state, results in a binuclear copper center that when reacted with low concentrations of H(2)O(2) mimics the spectroscopic signature of oxy-hemocyanin.

Biochemistry, 2002 Mar 5, 41(9), 3243 - 53
Interaction of the membrane-inserted diphtheria toxin T domain with peptides and its possible implications for chaperone-like T domain behavior; Hammond K et al.; The T domain of diphtheria toxin is believed to aid the low-pH-triggered translocation of the partly unfolded A chain (C domain) through cell membranes . Recent experiments have suggested the possibility that the T domain aids translocation by acting as a membrane-inserted chaperone {Ren, J., et al . (1999) Science 284, 955-957} . One prediction of this model is that the membrane-inserted T domain should be able to interact with sequences that mimic unfolded proteins . To understand the basis of interaction of the membrane-inserted T domain with unfolded polypeptides, its interaction with water-soluble peptides having different sequences was studied . The membrane-inserted T domain was able to recognize helix-forming 23-residue Ala-rich peptides . In the presence of such peptides, hydrophobic helix 9 of the T domain underwent the previously characterized conformational change from a state exhibiting shallow membrane insertion to one exhibiting deep insertion . This conformational change was more readily induced by the more hydrophobic peptides that were tested . It did not occur at all in the presence a hydrophilic peptide in which alternating Ser and Gly replaced Ala or in the presence of unfolded hydrophilic peptides derived from the A chain of the toxin . Interestingly, a peptide with a complex sequence (RKE(3)KE(2)LMEW(2)KM(2)SETLNF) also interacted with the T domain very strongly . We conclude that the membrane-inserted T domain cannot recognize every unfolded amino acid sequence . However, it does not exhibit strong sequence specificity, instead having the ability to recognize and interact with a variety of amino acid sequences having moderate hydrophobicity . This recognition was not strictly correlated with the strength of peptide binding to the lipid, suggesting that more than just hydrophobicity is involved . Although it does not prove that the T domain functions as a chaperone, T domain recognition of hydrophobic sequences is consistent with it having polypeptide recognition properties that are chaperone-like.

Biochemistry, 2002 Mar 5, 41(9), 3207 - 25
Alkaline phosphatase revisited: hydrolysis of alkyl phosphates; O'Brien PJ et al.; Escherichia coli alkaline phosphatase (AP) is the prototypical two metal ion catalyst with two divalent zinc ions bound approximately 4 A apart in the active site . Studies spanning half a century have elucidated many structural and mechanistic features of this enzyme, rendering it an attractive model for investigating the potent catalytic power of bimetallic centers . Unfortunately, fundamental mechanistic features have been obscured by limitations with the standard assays . These assays generate concentrations of inorganic phosphate (P(i)) in excess of its inhibition constant (K(i) approximately 1 muM) . This tight binding by P(i) has affected the majority of published kinetic constants . Furthermore, binding limits k(cat)/K(m) for reaction of p-nitrophenyl phosphate, the most commonly employed substrate . We describe a sensitive (32)P-based assay for hydrolysis of alkyl phosphates that avoids the complication of product inhibition . We have revisited basic mechanistic features of AP with these alkyl phosphate substrates . The results suggest that the chemical step for phosphorylation of the enzyme limits k(cat)/K(m) . The pH-rate profile and additional results suggest that the serine nucleophile is active in its anionic form and has a pK(a) of < or = 5.5 in the free enzyme . An inactivating pK(a) of 8.0 is observed for binding of both substrates and inhibitors, and we suggest that this corresponds to ionization of a zinc-coordinated water molecule . Counter to previous suggestions, inorganic phosphate dianion appears to bind to the highly charged AP active site at least as strongly as the trianion . The dependence of k(cat)/K(m) on the pK(a) of the leaving group follows a Bronsted correlation with a slope of beta(lg) = -0.85 +/- 0.1, differing substantially from the previously reported value of -0.2 obtained from data with a less sensitive assay . This steep leaving group dependence is consistent with a largely dissociative transition state for AP-catalyzed hydrolysis of phosphate monoesters . The new (32)P-based assay employed herein will facilitate continued dissection of the AP reaction by providing a means to readily follow the chemical step for phosphorylation of the enzyme.

Biochemistry, 2002 Mar 5, 41(9), 3109 - 18
Intrastrand DNA cross-links as tools for studying DNA replication and repair: two-, three-, and four-carbon tethers between the N(2) positions of adjacent guanines; Kowalczyk A et al.; A general protocol for preparation of oligonucleotides containing intrastrand cross-links between the exocyclic amino groups of adjacent deoxyguanosines has been developed . A series of 2, 3, and 4 methylene cross-links was incorporated site-specifically into an 11-mer (5'-GGCAGGTGGTG-3', cross-linked positions are underlined) via a reaction between oligonucleotide containing 2-fluoro-O(6)-trimethylsilylethyl deoxyinosines and the appropriate diamine (ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane) . These cross-linked-oligonucleotides were studied for their ability to bend DNA by the method of Koo and Crothers {Koo, H . S., and Crothers, D . M . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 1763-1767} in which the mobility of ligated oligomers in nondenaturing polyacrylamide gels is evaluated . It was found that all cross-links induced bending (2-carbon cross-link, 30.0 +/- 4.0 deg/turn; 3-carbon cross-link, 11.7 +/- 1.6 deg/turn; 4-carbon cross-link, 7.4 +/- 1.0 deg/turn) . Despite the differing extent of helical distortion exhibited by the cross-links, all appeared to be equally blocking to replication by the Escherichia coli polymerases, pol I, pol II, and pol III . In contrast, when incision of the cross-links by the E . coli UvrABC nucleotide incision complex was studied, the extent of incision of the cross-link was found to correlate closely with the degree of bending measured in the gel mobility assay, i.e., the efficiency of incision was 2-carbon >> 3-carbon > 4-carbon.

Biochemistry, 2002 Mar 5, 41(9), 3018 - 24
Structure of FAD-bound L-aspartate oxidase: insight into substrate specificity and catalysis; Bossi RT et al.; L-Aspartate oxidase (Laspo) catalyzes the conversion of L-Asp to iminoaspartate, the first step in the de novo biosynthesis of NAD(+) . This bacterial pathway represents a potential drug target since it is absent in mammals . The Laspo R386L mutant was crystallized in the FAD-bound catalytically competent form and its three-dimensional structure determined at 2.5 A resolution in both the native state and in complex with succinate . Comparison of the R386L holoprotein with the wild-type apoenzyme {Mattevi, A., Tedeschi, G., Bacchella, L., Coda, A., Negri, A., and Ronchi, S . (1999) Structure 7, 745-756} reveals that cofactor incorporation leads to the ordering of two polypeptide segments (residues 44-53 and 104-141) and to a 27 degree rotation of the capping domain . This motion results in the formation of the active site cavity, located at the interface between the capping domain and the FAD-binding domain . The structure of the succinate complex indicates that the cavity surface is decorated by two clusters of H-bond donors that anchor the ligand carboxylates . Moreover, Glu121, which is strictly conserved among Laspo sequences, is positioned to interact with the L-Asp alpha-amino group . The architecture of the active site of the Laspo holoenzyme is remarkably similar to that of respiratory fumarate reductases, providing strong evidence for a common mechanism of catalysis in Laspo and flavoproteins of the succinate dehydrogenase/fumarate reductase family . This implies that Laspo is mechanistically distinct from other flavin-dependent amino acid oxidases, such as the prototypical D-amino acid oxidase.

Biochemistry, 2002 Mar 5, 41(9), 2982 - 9
The crystal structure of HpcE, a bifunctional decarboxylase/isomerase with a multifunctional fold; Tame JR et al.; The structure of the bifunctional enzyme HpcE (OPET decarboxylase/HHDD isomerase) from Escherichia coli shows that the protein consists of highly similar N and C terminal halves . Sequence matches suggest that this fold is widespread among different species, including man . Many of these homologues are uncharacterized but apparently connected with the metabolism of aromatic compounds . The domain shows similar topology to the C terminal domain of fumarylacetoacetate hydrolase (FAH), a functionally related enzyme, despite lacking significant overall sequence similarity . HpcE is known to catalyze two rather different reactions, and comparisons with FAH allow some tentative conclusions to be drawn about the active sites . Key mutations within the active site apparently allow enzymes with this fold to carry out a variety chemical processes.

Biochemistry, 2002 Mar 5, 41(9), 2921 - 31
Structure and spectroscopy of the periplasmic cytochrome c nitrite reductase from Escherichia coli; Bamford VA et al.; The crystal structure and spectroscopic properties of the periplasmic penta-heme cytochrome c nitrite reductase (NrfA) of Escherichia coli are presented . The structure is the first for a member of the NrfA subgroup that utilize a soluble penta-heme cytochrome, NrfB, as a redox partner . Comparison to the structures of Wolinella succinogenes NrfA and Sulfospirillum deleyianum NrfA, which accept electrons from a membrane-anchored tetra-heme cytochrome (NrfH), reveals notable differences in the protein surface around heme 2, which may be the docking site for the redox partner . The structure shows that four of the NrfA hemes (hemes 2-5) have bis-histidine axial heme-Fe ligation . The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand . Analysis of NrfA in solution by magnetic circular dichroism (MCD) suggested that the oxygen ligand arose from water . Electron paramagnetic resonance (EPR) spectra were collected from electrochemically poised NrfA samples . Broad perpendicular mode signals at g similar 10.8 and 3.5, characteristic of weakly spin-coupled S = 5/2, S = 1/2 paramagnets, titrated with E(m) = -107 mV . A possible origin for these are the active site Lys-OH(2) coordinated heme (heme 1) and a nearby bis-His coordinated heme (heme 3) . A rhombic heme Fe(III) EPR signal at g(z) = 2.91, g(y) = 2.3, g(x) = 1.5 titrated with E(m) = -37 mV and is likely to arise from bis-His coordinated heme (heme 2) in which the interplanar angle of the imidazole rings is 21.2 . The final two bis-His coordinated hemes (hemes 4 and 5) have imidazole interplanar angles of 64.4 and 71.8 . Either, or both, of these hemes could give rise to a "Large g max" EPR signal at g(z)() = 3.17 that titrated at potentials between -250 and -400 mV . Previous spectroscopic studies on NrfA from a number of bacterial species are considered in the light of the structure-based spectro-potentiometric analysis presented for the E . coli NrfA.

Mol Ther, 2002 Mar, 5(3), 220 - 2
Escherichia coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ; Koponen JK et al.; Real-time PCR is a powerful method for the quantification of gene expression in biological samples . This method uses TaqMan chemistry based on the 5' -exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product . Many gene therapy studies use lacZ, encoding Escherichia coli beta-galactosidase, as a marker gene . Our results demonstrate that E . coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies . In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used . We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase.

Genomics, 2002 Mar, 79(3), 445 - 50
cDNA cloning, genomic structure, chromosomal mapping, and functional expression of a novel human alanine aminotransferase; Yang RZ et al.; Alanine aminotransferase (ALT) catalyzes the reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate, and thereby has a key role in the intermediary metabolism of glucose and amino acids . Two ALT isoenzymes are known to exist, but only one ALT gene has been cloned, GPT . In this study, we cloned a homolog of GPT and named it GPT2, and the corresponding protein ALT2 . GPT2 shares 69% identity and 78% similarity at the protein level to the previously cloned GPT . The human gene GPT2 encodes a 3.9-kb mRNA, consists of 12 exons, spanning approximately 50 kb of the genome, and maps to chromosome 16q12.1 . GPT2 and GPT differ in mRNA expression in that GPT2 is highly expressed in muscle, fat, and kidney, whereas GPT is mainly expressed in kidney, liver, and heart . In addition, GPT2 seems to be the predominant form of GPT at the mRNA level in these tissues . Expression of ALT2 protein in Escherichia coli produced a functional recombinant enzyme that catalyzes alanine transamination, confirming that the enzyme is an ALT . The more abundant expression of GPT2 than GPT, especially in muscle and fat, suggests a unique and previously unrecognized role of this gene product in glucose, amino acid, and fatty acid metabolism and homeostasis.

Genomics, 2002 Mar, 79(3), 285 - 96
An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22; Jarmuz A et al.; The cytidine (C) to uridine (U) editing of apolipoprotein (apo) B mRNA is mediated by tissue-specific, RNA-binding cytidine deaminase APOBEC1 . APOBEC1 is structurally homologous to Escherichia coli cytidine deaminase (ECCDA), but has evolved specific features required for RNA substrate binding and editing . A signature sequence for APOBEC1 has been used to identify other members of this family . One of these genes, designated APOBEC2, is found on chromosome 6 . Another gene corresponds to the activation-induced deaminase (AID) gene, which is located adjacent to APOBEC1 on chromosome 12 . Seven additional genes, or pseudogenes (designated APOBEC3A to 3G), are arrayed in tandem on chromosome 22 . Not present in rodents, this locus is apparently an anthropoid-specific expansion of the APOBEC family . The conclusion that these new genes encode orphan C to U RNA-editing enzymes of the APOBEC family comes from similarity in amino acid sequence with APOBEC1, conserved intron/exon organization, tissue-specific expression, homodimerization, and zinc and RNA binding similar to APOBEC1 . Tissue-specific expression of these genes in a variety of cell lines, along with other evidence, suggests a role for these enzymes in growth or cell cycle control.

Mol Biol (Mosk), 2002 Jan-Feb, 36(1), 27 - 36
{Streptomyces rimosus aminoglycoside-3'-phosphotransferase VIII: comparisons with aminoglycoside-3'-phosphotransferases of aminoglycoside-producing strains with eukaryotic protein kinases}; Sizova IA et al.; The nucleotide sequence was established for the aphVIII aminoglycoside phosphotransferase gene of an oxytetracycline-producing Streptomyces rimosus strain . The gene is 804 bp in size and possibly codes for APHVIII of 267 residues . Heterologous expression of aphVIII was studied in Escherichia coli and Chlamydomonas reinhardtii . The deduced APHVIII sequence was compared with known sequences of aminoglycoside phosphotransferases of aminoglycoside-producing actinomycete strains and of eukaryotic protein kinases . A local homology of 38 residues was found between APHVIII and actinomycete serine-threonine protein kinases in the conserved region possibly involved in ATP binding . APHVIII differed from aminoglycoside 3'-phosphotransferases of aminoglycoside-producing actinomycete strains and of clinical isolates, and can be classed to a separate group.

J Biol Inorg Chem, 2002 Jan, 7(1-2), 83 - 93 Epub 2001 Jul 11.
The iron-sulfur center of biotin synthase: site-directed mutants; Hewitson KS et al.; Biotin synthase contains an essential {4Fe-4S}+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation . The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the {4Fe-4S} cluster . These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component . To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity . Our results show that six cysteines are important for biotin formation . Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation . The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a {4Fe-4S} cluster, as shown by Mossbauer spectroscopy . The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the {4Fe-4S}+ state, as shown by EPR and Mossbauer spectroscopy . On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well . Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.

J Biol Inorg Chem, 2002 Jan, 7(1-2), 74 - 82 Epub 2001 Jul 27.
Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli; Sahlin M et al.; Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed . Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals . A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical . Reconstitution of other mutants of protein R2 (i.e . Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct . The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen . However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical . We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine . The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants . Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F . Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.

Cancer Chemother Pharmacol, 2002 Feb, 49(2), 149 - 54 Epub 2001 Nov 16.
PEG-asparaginase (Oncaspar) 2500 U/m(2) BSA in reinduction and relapse treatment in the ALL/NHL-BFM protocols; Muller HJ et al.; PURPOSE: As previous data had shown that only two-thirds of patients had the predicted activity time courses when PEG-asparaginase 1000 U/m(2) was used in reinduction after native E . coli asparaginase in induction treatment of acute lymphoblastic leukaemia (ALL), drug monitoring was performed with the use of a higher dose . METHODS: Because one-third of patients had insufficient serum asparaginase activity time courses after a single dose of 1000 U/m(2) PEG-asparaginase during reinduction treatment, a dose of 2500 U/m(2) PEG-asparaginase, which is the approved dosage in Germany, was used in 39 reinduction and 20 relapse patients to determine whether prolongation of the activity time course may be possible with this higher dose, and to look for significant differences between reinduction and relapse patients . RESULTS: After 1, 2 and 3 weeks, the mean activities were 1113 +/- 699 U/l, 231 +/- 259 U/l, and 13 +/- 35 U/l in the reinduction patients, and 1078 +/- 649 U/l, 165 +/- 195 U/l and 19 +/- 28 U/l in the relapse patients, respectively . There were a considerable number of patients with a substantially shortened activity time course in both groups . In 10 of 39 reinduction patients and in 7 of 24 doses during relapse treatment, only activities <100 U/l were found after 1 week with a further fast decline . No statistically significant differences between the two patient groups could be shown at any time-point . CONCLUSIONS: Comparison of these data with activities after 1000 U/m(2) PEG-asparaginase showed no prolongation of the time with activity in the therapeutic range with the higher dose . Therefore, for a longer duration of therapeutic activity, administration of further doses is mandatory.

Cancer Immunol Immunother, 2002 Feb, 50(12), 653 - 62 Epub 2001 Dec 06.
Induction of intratumoral tumor necrosis factor by a synthetic lipid A analog, ONO-4007, with less tolerance in repeated administration and its implication in potent antitumor effects with low toxicity; Satoh M et al.; To evaluate the anti-tumor characteristics of ONO-4007, a synthetic analog of lipid A, the authors examined its acute toxicity and anti-tumor activity in a mouse MM46 mammary tumor system in comparison with LA-15-PP, an E . coli-type synthetic lipid A and LPS . Systemic and local (tumor site) induction of tumor necrosis factor (TNF) by a single i.v . shot of ONO-4007 and LA-15-PP correlated with manifestation of their toxicity, showing that ONO-4007 is 100-fold less effective than LA-15-PP . However, a protocol of repeated administration (3 shots twice a week) exhibited about 10 times more therapeutic potency of ONO-4007 for cancer therapy than expected in the above experiments . In a dose inducing submaximal systemic and intratumoral TNF production, repeated injections (twice a week) of ONO-4007 (10 mg/kg), LA-15-PP (0.1 mg/kg) and LPS (0.1 mg/kg) commonly generated a tolerant state in the systemic response (serum and liver) to subsequent stimulation . The intratumoral response was retained with this repeated administration of ONO-4007, but was not with LA-15-PP or LPS . TIM (tumor-infiltrating macrophages) isolated from mice pre-injected with ONO-4007 and LA-15-PP were found to lose their response to both substances, but the response was rapidly recovered until 72 h after injection and virtually no difference was observed in their response to either drug . The in vitro treatment of naive TIM with ONO-4007 or LA-15-PP for 2 h depressed the response to both substances and the depression continued for 72 h even in culture with fresh medium . The relatively high efficacy of ONO-4007 in cancer therapy likely depends on the retraction of the tolerant state, especially at the tumor site where the response to ONO-4007 is recovered much more efficiently than that to lipid A . While constant recruitment of macrophages to tumor tissue might be involved in the difference of tolerance recovery between this region and others, selective response to ONO-4007 may not be explained simply by the sensitivity of recruited TIM . Pharmacokinetical experiments revealed that repeated injections of LA-15-PP enhanced its clearance from blood circulation, while the clearance of ONO-4007 was stable after repeated injections . Thus, pharmacokinetical properties of ONO-4007 may also possibly be implicated in this event.

Cornea, 2002 Mar, 21(2), 203 - 5
Development and application of an in vitro susceptibility test for Acanthamoeba species isolated from keratitis to polyhexamethylene biguanide and chlorhexidine; Narasimhan S et al.; PURPOSE: To develop a reliable in vitro drug susceptibility test against Acanthamoeba isolates and to determine the minimum cysticidal concentration (MCC) of the drug . METHODS: Doubling dilutions of polyhexamethylene biguanide (PHMB) from 3,200 microg/mL to 3.125 microg/mL and chlorhexidine from 3,200 microg/mL to 1.5625 microg/mL were made in Durham tubes and tested against cysts of 19 Acanthamoeba isolates from keratitis . After the exposure to the drugs for 48 hours, the cysts were washed free of drugs by centrifugation . The deposit (cysts) was cultured on nonnutrient agar plates seeded with heat-killed Escherichia coli . The growth of trophozoites from cysts exposed to each of the dilution was recorded by microscopy to estimate the MCC of the drug . RESULTS: The minimum cysticidal concentration of PHMB varied from 25 microg/mL to 100 microg/mL and that of chlorhexidine varied from 1.56 microg/mL to 100 microg/mL . The mean MCC value for PHMB was 55.26 microg/mL and that for chlorhexidine 32.81 microg/mL . Minimum cysticidal concentration 50 (MCC50) of PHMB and chlorhexidine on Acanthamoeba isolates was 50.0 microg/mL and 25.0 microg/mL, respectively . Anti-Acanthamoeba activity of chlorhexidine was higher than that of PHMB and this was statistically significant (p = 0.036) . The end point of the results of this method was the detection of the viable cysts undergoing excystment and multiplication of trophozoites with a reproducible and clear-cut estimation of the MCC of PHMB and chlorhexidine . CONCLUSION: The in vitro method described can be used as a standard test for assay of MCC of drugs on Acanthamoeba isolates and to study the susceptibility pattern of newer water-soluble anti-Acanthamoeba drugs.

Nucleic Acids Res . 2002 Mar 1;30(5):e18.
An improved helper phage system for efficient isolation of specific antibody molecules in phage display; Baek H et al.; Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin . However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules . We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage . Ex-phage has a mutant pIII gene that produces a functional wild-type pIII in suppressing Escherichia coli strains but does not make any pIII in non-suppressing E.coli strains . Packaging phagemids encoding antibody-pIII fusion in F+ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13KO7 helper phage . Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.

Nucleic Acids Res, 2002 Mar 1, 30(5), 1176 - 81
Directionality of lambda plasmid DNA replication carried out by the heritable replication complex; Baranska S et al.; There are two 'pathways' of replication of lambda plasmids in Escherichia coli . One pathway requires the assembly of a new replication complex before replication and the second pathway is based on the activity of the replication complex inherited by one of two daughter plasmid copies after a preceding replication round . Such a phenomenon was postulated to occur also in other replicons, including Saccharomyces cerevisiae autonomously replicating sequences . Here we investigated directionality of lambda plasmid replication carried out by the heritable and newly assembled replication complexes . Using two-dimensional agarose gel electrophoresis and electron microscopy we demonstrated that in both normal growth conditions and during the relaxed response to amino acid starvation (when only replication carried out by the heritable complex is possible), bidirectionally and undirectionally replicating plasmid molecules occurred in host cells in roughly equal proportions . The results are compatible with the hypothesis that both complexes (heritable and newly assembled) are equivalent.

J Virol, 2002 Mar, 76(6), 2770 - 9
Analysis of an Autographa californica nucleopolyhedrovirus lef-11 knockout: LEF-11 is essential for viral DNA replication; Lin G et al.; The Autographa californica nucleopolyhedrovirus (AcMNPV) lef-11 gene was previously identified by transient late expression assays as a gene important for viral late gene expression . The lef-11 gene was not previously identified as necessary for DNA replication in transient origin-dependent plasmid DNA replication assays . To examine the role of lef-11 in the context of the infection cycle, we generated a deletion of the lef-11 gene by recombination in an AcMNPV genome propagated as a BACmid in Escherichia coli . The resulting AcMNPV lef-11-null BACmid (vAc(lef11KO)) was unable to propagate in cell culture, although a "repair" AcMNPV BACmid (vAc(lef11KO-REP)), which was generated by transposition of the lef-11 gene into the polyhedrin locus of the vAc(lef11KO) BACmid, was able to replicate in a manner similar to wild-type or control AcMNPV viruses . Thus, the lef-11 gene is essential for viral replication in Sf9 cells . The vAc(lef11KO) BACmid was examined to determine if the defect in viral replication resulted from a defect in DNA replication or from a defect in late transcription . The lef-11-null BACmid and control BACmids were transfected into Sf9 cells, and viral DNA replication was monitored . The viral DNA genome of the lef-11-null BACmid (vAc(lef11KO)) was not amplified, whereas replication and amplification of the genomes of the repair BACmid (vAc(lef11KO-REP)), wild-type AcMNPV, and a nonpropagating gp64-null control BACmid (vAc(GUSgp64KO)) were readily detected . Northern blot analysis of transcripts from selected early, late, and very late genes showed that late and very late transcription was absent in cells transfected with the lef-11-null BACmid . Thus, in contrast to prior studies using transient replication and late expression assays, studies of a lef-11-null BACmid indicate that LEF-11 is required for viral DNA replication during the infection cycle.

J Virol, 2002 Mar, 76(6), 2654 - 66
Repression of African swine fever virus polyprotein pp220-encoding gene leads to the assembly of icosahedral core-less particles; Andres G et al.; African swine fever virus (ASFV) polyprotein pp220, encoded by the CP2475L gene, is an N-myristoylated precursor polypeptide that, after proteolytic processing, gives rise to the major structural proteins p150, p37, p34, and p14 . These proteins localize at the core shell, a matrix-like virus domain placed between the DNA-containing nucleoid and the inner envelope . In this study, we have examined the role of polyprotein pp220 in virus morphogenesis by means of an ASFV recombinant, v220i, containing an inducible copy of the CP2475L gene regulated by the Escherichia coli repressor-operator system . Under conditions that repress pp220 expression, the virus yield of v220i was about 2.6 log units lower than that of the parental virus or of the recombinant grown under permissive conditions . Electron microscopy revealed that pp220 repression leads to the assembly of icosahedral particles virtually devoid of the core structure . Analysis of recombinant v220i by immunoelectron microscopy, immunoblotting, and DNA hybridization showed that mutant particles essentially lack, besides the pp220-derived products, a number of major core proteins as well as the viral DNA . On the other hand, transient expression of the CP2475L gene in COS cells showed that polyprotein pp220 assembles into electron-dense membrane-bound coats, whereas a mutant nonmyristoylated version of pp220 does not associate with cellular membranes but forms large cytoplasmic aggregates . Together, these findings indicate that polyprotein pp220 is essential for the core assembly and suggest that its myristoyl moiety may function as a membrane-anchoring signal to bind the developing core shell to the inner viral envelope.

Genetics, 2002 Feb, 160(2), 727 - 40
Assembly of two transgenes in an artificial chromatin domain gives highly coordinated expression in tobacco; Mlynarova L et al.; The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation . We here present the first direct experimental support for this model in plants . Two reporter genes, the E . coli beta-glucuronidase gene and the firefly luciferase gene, driven by different promoters, were placed between copies of the chicken lysozyme A element, a member of the matrix-associated region (MAR) group of chromatin boundary elements, and introduced in tobacco (Nicotiana tabacum) . In plants carrying A elements, quantitative enzyme activities and mRNA levels of both genes show high correlations compared to control plants . The A element thus creates an artificial chromatin domain that yields coordinated expression . Surprisingly, enzyme activities correlated poorly with their respective mRNA levels . We hypothesize that this indicates the occurrence of "error pipelines" in data generation: systematic errors of a given analytical method will point in the same direction and cancel out in correlation analysis, resulting in better correlations . In combining different methods of analysis, however, such errors do not cancel out and as a result relevant correlations can be masked . Such error pipelines will have to be taken into account when different types of (e.g., whole-genome) data sets are combined in quantitative analyses.

Development, 2002 Feb, 129(4), 1037 - 47
Cross-repressive interactions of identity genes are essential for proper specification of cardiac and muscular fates in Drosophila; Jagla T et al.; In Drosophila embryos, founder cells that give rise to cardiac precursors and dorsal somatic muscles derive from dorsally located progenitors . Individual fates of founder cells are thought to be specified by combinatorial code of transcription factors encoded by identity genes . To date, a large number of identity genes have been identified; however, the mechanisms by which these genes contribute to cell fate specification remain largely unknown . We have analysed regulatory interactions of ladybird (lb), msh and even skipped (eve), the three identity genes specifying a subset of heart and/or dorsal muscle precursors . We show that deregulation of each of them alters the number of cells that express two other genes, thus changing the ratio between cardiac and muscular cells, and the ratio between different cell subsets within the heart and within the dorsal muscles . Specifically, we demonstrate that mutation of the muscle identity gene msh and misexpression of the heart identity gene lb lead to heart hyperplasia with similar cell fate modifications . In msh mutant embryos, the presumptive msh-muscle cells switch on lb or eve expression and are recruited to form supernumerary heart or dorsal muscle cells, thus indicating that msh functions as a repressor of lb and eve . Similarly, overexpression of lb represses endogenous msh and eve activity, hence leading to the respecification of msh and eve positive progenitors, resulting in the overproduction of a subset of heart cells . As deduced from heart and muscle phenotypes of numb mutant embryos, the cell fate modifications induced by gain-of-function of identity genes are not lineage restricted . Consistent with all these observations, we propose that the major role of identity genes is to maintain their restricted expression by repressing other identity genes competent to respond positively to extrinsic signals . The cross-repressive interactions of identity genes are likely to ensure their localised expression over time, thus providing an essential element in establishing cell identity.

Development, 2002 Feb, 129(4), 853 - 62
The C . elegans even-skipped homologue, vab-7, specifies DB motoneurone identity and axon trajectory; Esmaeili B et al.; Locomotory activity is defined by the specification of motoneurone subtypes . In the nematode, C . elegans, DA and DB motoneurones innervate dorsal muscles and function to induce movement in the backwards or forwards direction, respectively . These two neurone classes express separate sets of genes and extend axons with oppositely directed trajectories; anterior (DA) versus posterior (DB) . The DA-specific homeoprotein UNC-4 interacts with UNC-37/Groucho to repress the DB gene, acr-5 (nicotinic acetylcholine receptor subunit) . We show that the C . elegans even-skipped-like homoedomain protein, VAB-7, coordinately regulates different aspects of the DB motoneurone fate, in part by repressing unc-4 . Wild-type DB motoneurones express VAB-7, have posteriorly directed axons, express ACR-5 and lack expression of the homeodomain protein UNC-4 . In a vab-7 mutant, ectopic UNC-4 represses acr-5 and induces an anteriorly directed DB axon trajectory . Thus, vab-7 indirectly promotes DB-specific gene expression and posteriorly directed axon outgrowth by preventing UNC-4 repression of DB differentiation . Ectopic expression of VAB-7 also induces DB traits in an unc-4-independent manner, suggesting that VAB-7 can act through a parallel pathway . This work supports a model in which a complementary pair of homeodomain transcription factors (VAB-7 and UNC-4) specifies differences between DA and DB neurones through inhibition of the alternative fates . The recent findings that Even-skipped transcriptional repressor activity specifies neurone identity and axon guidance in the mouse and Drosophila motoneurone circuit points to an ancient origin for homeoprotein-dependent mechanisms of neuronal differentiation in the metazoan nerve cord.

Blood, 2002 Mar 1, 99(5), 1638 - 45
Antithrombin prevents endotoxin-induced hypotension by inhibiting the induction of nitric oxide synthase in rats; Isobe H et al.; Antithrombin (AT) prevents Escherichia coli-induced hypotension in animal models of sepsis, and it further reduces the mortality of patients with septic shock . In the present study, we examined whether AT may prevent the endotoxin (ET)-induced hypotension by promoting the endothelial release of prostacyclin (PGI(2)) in rats . Intravenous administration of AT (250 U/kg) prevented both hypotension and the increases in plasma levels of NO(2)(-)/NO(3)(-) in rats given ET . Lung expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha) was transiently increased after ET administration, followed by the increases in lung tissue levels of TNF-alpha . Both the lung activity of the inducible form of nitric oxide synthase (iNOS) and the lung expression of iNOS mRNA in animals administered ET were gradually increased after the TNF-alpha mRNA expression had peaked . Administration of AT significantly inhibited these increases . Neither DEGR-F.Xa, a selective inhibitor of thrombin generation, nor Trp(49)-modified AT, which is not capable of promoting the endothelial release of PGI(2), showed any effects on these changes induced by ET . Administration of antirat TNF-alpha antibody produced effects similar to those induced by AT . Indomethacin pretreatment abrogated the effects induced by AT . Iloprost, a stable derivative of PGI(2), produced effects similar to those of AT . These findings suggested that AT prevents the ET-induced hypotension by inhibiting the induction of iNOS through inhibiting TNF-alpha production . These effects of AT could be mediated by the promotion of endothelial release of PGI(2) and might at least partly explain the therapeutic effects for septic shock.

J Biotechnol, 2002 Apr 11, 94(3), 287 - 98
Phage antibody fragments library combining a single human light chain variable region with immune mouse heavy chain variable regions; Rojas G et al.; We describe the construction of a phage antibody fragments library which combines, in a single cloning step, a synthetic human light chain variable region (V(L)) with a diverse set of heavy chain variable regions, from a mouse immunized with the prostate specific antigen (PSA) . Despite V(L) restriction, selection from this library rendered two different single chain Fv antibody fragments, specifically recognizing PSA . The human V(L), used as a general partner for mouse heavy chains, was constructed by linking the germline A27 gene and the J(K)1 minigene segment, both of which are prominently involved in human antibody responses . Our approach offers a fast and simple way to produce half-human molecules, while keeping the advantage of immunizing animals for high affinity antibodies.

J Biotechnol, 2002 Apr 11, 94(3), 277 - 85
Expression of human plasminogen kringle 5 as fusion protein with truncated hIFNgamma gene in Escherichia coli; Lu H et al.; The human interferon gamma (hIFNgamma) gene was used as a fusion partner to mediate the expression of heterologous proteins and the effect of the fusion partner length on the expression of the heterologous protein was researched . Plasminogen kringle 5 (pk5), an inhibitor of angiogenesis, was fused to hIFNgamma and its serially truncated fragments, respectively, and the expression of fusion proteins was determined by SDS-Page gel . The pk5 protein was obtained readily by the introduction of sequences recognized by protease factor Xa at the fusion site and ion-exchange chromatography was employed to purify pk5 . The recovery of the biological activities of pk5 was studied using the orthogonal experimental design L9 (3(4)) (four factors, three levels, nine experiments) and evaluated by measurement of anti-endothelial cell proliferation in vitro.

J Biotechnol, 2002 Apr 11, 94(3), 235 - 44
A unique approach for high level expression and production of a recombinant cobra neurotoxin in Escherichia coli; Wang Y et al.; In this report, we describe a simple approach to produce a large quantity of a recombinant cobra neurotoxin containing four pairs of disulfide bonds . A cDNA encoding the toxin was fused, in frame, to the carboxyl termini of thioredoxin via a linker sequence encoding two amino acids, Asp and Pro . Due to the presence of thioredoxin, a soluble form of the fusion protein was expressed in a compartment, sensitive to osmotic pressure, in Escherichia coli . The fusion protein was released into the solution with low ionic strength under an osmotic shock treatment, and purified in a single step using an ion exchange chromatography column . The purified protein was treated in diluted hydrochloric acid to induce hydrolysis of the protein at the Asp-Pro linker site . Then, the recombinant neurotoxin was purified by gel filtration of the acid-treated sample . When the biological activity of the purified toxin was assayed, it was as potent as the natural toxin . Using this protocol, approximately 12 mg of pure recombinant neurotoxin can be produced from one liter of bacterial culture . More importantly, this protocol can be easily used for the production of the toxin at a larger scale with low cost . The approach outlined in this report will be suitable for the production of other recombinant proteins especially those of the 'three-finger' family.

J Immunol Methods, 2002 Mar 1, 261(1-2), 199 - 211
Construction and characterization of affibody-Fc chimeras produced in Escherichia coli; Ronnmark J et al.; Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG . Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture . Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment . Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding . Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects . In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates . One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E . coli lysate . The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed . Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.

J Immunol Methods, 2002 Mar 1, 261(1-2), 65 - 72
Phage library panning against cytosolic fraction of cells using quantitative dot blotting assay: application of selected VH to histochemistry; Nakamura M et al.; Comprehensive preparations of antibodies against various kinds of proteins in cells would be useful in proteome research and antibody-based research . Here we report the panning of a human antibody heavy chain variable domain (VH) phage library against a cytosolic fraction of rat liver to obtain antibodies specific for certain cytoplasmic proteins . Rat liver specimens were homogenized and subjected to differential centrifugation . A 125000 x g supernatant (rat liver cytosol, RLC) was immobilized onto a nitrocellulose membrane and subjected to phage VH library panning . For efficient assessment of binding phages, we established a system that was a combination of monoclonal phage ELISA and quantitative dot blotting of phages . The VH genes of the binding phages were selected and expressed as VH--bacterial alkaline phosphatase (PhoA) conjugates (VH/RLC--PhoAs) in Escherichia coli . One of the VH/RLC--PhoAs stained one major band on Western blotting of RLC and also stained the cytoplasm of hepatocytes histochemically . This is the first report of phage library panning against the cytosolic fraction of cells to obtain human VH fragments, and the application of those human VH fragments to histochemical study.

Acta Pharmacol Sin, 2002 Jan, 23(1), 16 - 22
Mechanism of growth hormone insensitivity induced by endotoxin; Wang P et al.; AIM: To investigate the mechanism of growth hormone (GH) insensitivity induced by endotoxin at receptor and post-receptor levels . METHODS: Sprague-Dawley rats were injected endotoxin along with or without GH administration . The liver expression of insulin-like growth factor I (IGF-I), GH receptor (GHR), and suppresor of cytokine signaling (SOCS)-3 mRNA were detected by reverse transcriptase polymerase cha in reaction, the GH levels were measured by radioimmunoassay, the levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay . RESULTS: Serum GH levels had no significant difference compared with control rats after endotoxin injection, however, liver IGF-I mRNA expression was obviously down-regulated in endotoxemic rats . Liver GHR mRNA expression was predominantly down-regulated after LPS injection; although SOCS-3 mRNA was weakly expressed in control rats, it was strongly up-regulated in endotoxemic rats . Endotoxine stimulated the production of TNF-alpha and IL-6, and the elevated IL-6 levels showed a positive correlation with increased SOCS-3 mRNA expression . Exogenous GH could enhance IGF-I mRNA expression in control rats, but it did fail to prevent the decline in IGF-I mRNA expression in endotoxemic rats . Two different LPS dosages (7.5 mg/kg and 5.0 mg/kg) produced the same down-regulation of IGF-I mRNA expression, however, the higher LPS dosage induced more GHR mRNA down-regulation and more SOCS-3 mRNA up-regulation . CONCLUSION: The mechanism of growth hormone insensitivity induced by endotoxin was associated with down-regulated GHR mRNA expression at receptor level and up-regulated SOCS-3 mRNA expression at post-receptor level . The inhibition at post-receptor level had close relationship with the increased IL-6 secretion.

Methods, 2001 Nov, 25(3), 344 - 50
Comparison of rRNA cleavage by complementary 1,10-phenanthroline-Cu(II)- and EDTA-Fe(II)-derivatized oligonucleotides; Bowen WS et al.; The chemical nucleases 1,10-phenanthroline-Cu(II) and EDTA-Fe(II), have proven to be valuable tools for structural analysis of nucleic acids . Both have found applications in footprinting and directed proximity studies of DNA and RNA . Derivatives of each that provide for tethering to nucleic acid or protein are commercially available, allowing their widespread use for structural analysis of macromolecules . Although their applications are somewhat overlapping, differences in their cleavage mechanisms and chemical properties allow them to provide distinct and complementary structural information . The purpose of this study is to compare directly the cleavage patterns of tethered 1,10-phenanthroline-Cu(II) and EDTA-Fe(II) complexes within a similar experimental system . Here, the region surrounding nucleotide 1400 of 16S rRNA from Escherichia coli serves as a substrate for chemical cleavage directed by a derivatized complementary oligonucleotide . This region of rRNA is known to be involved in the decoding of mRNA during translation . The results of this study provide evidence in support of the mechanistic differences previously established for EDTA-Fe(II) and 1,10-phenathroline-Cu(II) . The delocalized cleavage envelope produced by EDTA-Fe(II) cleavage suggests the involvement of a diffusible reactive species . On the other hand, rRNA cleavage induced by the tethered 1,10-phenanthroline-Cu(II) complex appears localized to the proximity of the chemical nuclease under normal conditions, although the production of an unknown diffusible species appears to occur during long reaction times .

Life Sci, 2002 Jan 18, 70(9), 1013 - 21
Corticotropin-releasing hormone affects cytokine production in human HaCaT keratinocytes; Zbytek B et al.; CRH cutaneous expression is significantly enhanced after exposure to various stimuli (Physiol Rev 2000, 80;979-1020) . We evaluated the effect of CRH on cytokine production in HaCaT keratinocytes, a cell line shown to express CRH receptors coupled to cAMP activation and calcium-dependent transmission pathways . It is demonstrated for the first time that exogenously added CRH stimulates production of IL-6 and IL-11 . It also inhibits production of IL-1beta and does not affect TNF-alpha production . Our results indicate that CRH function(s) during cutaneous stress may be mediated by differential effects on cytokine production.

Nature, 2002 Feb 21, 415(6874), 937 - 41
Structural basis for recognition of acidic-cluster dileucine sequence by GGA1; Shiba T et al.; GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-interacting proteins) are critical for the transport of soluble proteins from the trans-Golgi network (TGN) to endosomes/lysosomes by means of interactions with TGN-sorting receptors, ADP-ribosylation factor (ARF), and clathrin . The amino-terminal VHS domains of GGAs form complexes with the cytoplasmic domains of sorting receptors by recognizing acidic-cluster dileucine (ACLL) sequences . Here we report the X-ray structure of the GGA1 VHS domain alone, and in complex with the carboxy-terminal peptide of cation-independent mannose 6-phosphate receptor containing an ACLL sequence . The VHS domain forms a super helix with eight alpha-helices, similar to the VHS domains of TOM1 and Hrs . Unidirectional movements of helices alpha6 and alpha8, and some of their side chains, create a set of electrostatic and hydrophobic interactions for correct recognition of the ACLL peptide . This recognition mechanism provides the basis for regulation of protein transport from the TGN to endosomes/lysosomes, which is shared by sortilin and low-density lipoprotein receptor-related protein.

Nature, 2002 Feb 21, 415(6874), 933 - 7
Structural basis for acidic-cluster-dileucine sorting-signal recognition by VHS domains; Misra S et al.; Specific sorting signals direct transmembrane proteins to the compartments of the endosomal-lysosomal system . Acidic-cluster-dileucine signals present within the cytoplasmic tails of sorting receptors, such as the cation-independent and cation-dependent mannose-6-phosphate receptors, are recognized by the GGA (Golgi-localized, gamma-ear-containing, ADP-ribosylation-factor-binding) proteins . The VHS (Vps27p, Hrs and STAM) domains of the GGA proteins are responsible for the highly specific recognition of these acidic-cluster-dileucine signals . Here we report the structures of the VHS domain of human GGA3 complexed with signals from both mannose-6-phosphate receptors . The signals bind in an extended conformation to helices 6 and 8 of the VHS domain . The structures highlight an Asp residue separated by two residues from a dileucine sequence as critical recognition elements . The side chains of the Asp-X-X-Leu-Leu sequence interact with subsites consisting of one electropositive and two shallow hydrophobic pockets, respectively . The rigid spatial alignment of the three binding subsites leads to high specificity.

J Biol Chem, 2002 May 3, 277(18), 15558 - 65 Epub 2002 Feb 21.
The FabR (YijC) transcription factor regulates unsaturated fatty acid biosynthesis in Escherichia coli; Zhang YM et al.; Unsaturated fatty acid biosynthesis is a vital facet of Escherichia coli physiology and requires the expression of two genes, fabA and fabB, in the type II fatty acid synthase system . This study links the FabR (YijC) transcription factor to the regulation of unsaturated fatty acid content through the regulation of fabB gene expression . The yijC (fabR) gene was deleted by replacement with a selectable cassette, and the resulting strains (fabR::kan) possessed significantly elevated levels of unsaturated fatty acids, particularly cis-vaccenate, in their membrane phospholipids . The altered fatty acid composition was observed in the fabR::kan fabF1 double mutant pinpointing fabB as the condensing enzyme responsible for the increased cis-vaccenate production . The fabR::kan strains had 4- to 8-fold higher levels of fabB and a 2- to 3-fold increase in fabA transcripts as judged by Northern blotting, Affymetrix array analysis, and real-time PCR . FabR did not regulate the enzymes of fatty acid beta-oxidation . The elevated level of fabB mRNA was reflected by higher condensing enzyme activity in fabR::kan fabF1 double mutants . Thus, FabR functions as a repressor that potently controls the expression of the fabB gene, which in turn, modulates the physical properties of the membrane by altering the level of unsaturated fatty acid production.

J Biol Chem, 2002 May 3, 277(18), 15385 - 92 Epub 2002 Feb 21.
Probing the limits of electrostatic catalysis by uracil DNA glycosylase using transition state mimicry and mutagenesis; Jiang YL et al.; The DNA repair enzyme uracil DNA glycosylase (UDG) hydrolyzes the glycosidic bond of deoxyuridine in DNA by a remarkable mechanism involving formation of a positively charged oxacarbenium ion-uracil anion intermediate . We have proposed that the positively charged intermediate is stabilized by being sandwiched between the combined negative charges of the anionic uracil leaving group and a conserved aspartate residue that are located on opposite faces of the sugar ring . Here we establish that a duplex DNA oligonucleotide containing a cationic 1-aza-deoxyribose (I) oxacarbenium ion mimic is a potent inhibitor of UDG that binds tightly to the enzyme-uracil anion (EU(-)) product complex (K(D) of EU(-) = 110 pm) . The tight binding of I to the EU(-) complex results from its extremely slow off rate (k(off) = 0.0008 s(-1)), which is 25,000-fold slower than substrate analogue DNA . Removal of Asp(64) and His(187), which are involved in stabilization of the cationic sugar and the anionic uracil leaving group, respectively, specifically weakens binding of I to the UDG-uracil complex by 154,000-fold, without significantly affecting substrate or product binding . These results suggest that electrostatic effects can effectively stabilize such an intermediate by at least -7 kcal/mol, without leading to anticatalytic stabilization of the substrate and products.

J Biol Chem, 2002 May 10, 277(19), 16517 - 27 Epub 2002 Feb 21.
Topography of helices 5-7 in membrane-inserted diphtheria toxin T domain: identification and insertion boundaries of two hydrophobic sequences that do not form a stable transmembrane hairpin; Rosconi MP et al.; The T domain of diphtheria toxin undergoes a low pH-induced conformational change that allows it to penetrate cell membranes . T domain hydrophobic helices 8 and 9 can adopt two conformations, one close to the membrane surface (P state) and a second in which they apparently form a transmembrane hairpin (TM state) . We have now studied T domain helices 5-7, a second cluster of hydrophobic helices, using Cys-scanning mutagenesis . After fluorescently labeling a series of Cys residues, penetration into a non-polar environment, accessibility to externally added antibodies, and relative depth in the bilayer were monitored . It was found that helices 5-7 insert shallowly in the P state and deeply in the TM state . Thus, the conformational changes in helices 5-7 are both similar and somehow linked to those in helices 8 and 9 . The boundaries of deeply inserting sequences were also identified . One deeply inserted segment was found to span residues 270 to 290, which overlaps helix 5, and a second spanned residues 300 to 320, which includes most of helix 6 and all of helix 7 . This indicates that helices 6 and 7 form a continuous hydrophobic segment despite their separation by a Pro-containing kink . Additionally, it is found that in the TM state some residues in the hydrophilic loop between helices 5 and 6 become more highly exposed than they are in the P state . Their exposure to external solution in the TM state indicates that helices 5-7 do not form a stable transmembrane hairpin . However, helix 5 and/or helices 6 plus 7 could form transmembrane structures that are in equilibrium with non-transmembrane states, or be kinetically prevented from forming a transmembrane structure . How helices 5-7 might influence the mechanism by which the T domain aids translocation of the diphtheria toxin A chain across membranes is discussed.

Vaccine, 2002 Feb 22, 20(11-12), 1660 - 9
Immunogenicity of the Brucella melitensis recombinant ribosome recycling factor-homologous protein and its cDNA; Cassataro J et al.; A study was conducted to evaluate the immunogenicity of the Brucella melitensis ribosome recycling factor (RRF)-homologous protein (CP24) . The CP24 gene was cloned, expressed in Escherichia coli and purified . The resulting purified recombinant protein (rCP24) produced delayed-type hypersensitivity (DTH) reactions in B . melitensis-infected mice but not in naive controls . Thus, we decided to characterise the immune responses generated with DNA vaccination (pcDNACP24) or immunisation with the rCP24 in adjuvant . Animals injected with pcDNACP24 exhibited a dominance of IgG2a to IgG1 while mice injected with rCP24 developed a higher response of IgG1 than IgG2a . Both immunisation protocols were capable of eliciting CP24-specific gamma interferon (IFN-gamma) producing cells . Spleen cells from pcDNACP24-immunised mice did not produce interleukin (IL)-4, IL-10 or up-regulation of IL-2 mRNA . Cells from rCP24-immunised mice produced IL-10, up-regulated IL-2 mRNA but did not produce IL-4 . Neither immunisation with purified CP24 nor injection of pcDNACP24 protected mice against challenge with live smooth B . melitensis . However, the potential of CP24 for a Brucella diagnostic test based on an in vitro antigen (Ag)-specific IFN-gamma production or DTH test would be worth testing.

Protein Expr Purif, 2002 Mar, 24(2), 292 - 301
Expression of ayu (Plecoglossus altivelis) Pit-1 in Escherichia coli: its purification and immunohistochemical detection using monoclonal antibody; Chiu CC et al.; The pituitary-specific transcription factor Pit-1 belongs to the family of POU-domain proteins and is known to play an important role in the differentiation of pituitary cells . Here we report the complete nucleotide sequence of cDNA encoding Pit-1 from the brackish water fish, ayu (Plecoglossus altivelis) . Nucleotide sequence analysis of 1910 bp of ayu Pit-1 cDNA revealed an open reading frame of 1074 bp that encodes a protein of 358 amino acids containing a POU-specific domain, POU homeodomain, and an STA (Ser/Thr-rich activation) transactivation domain . We inserted the coding region of Pit-1 cDNA, obtained by PCR, into a pET-20b(+) plasmid to produce recombinant Pit-1 in Escherichia coli BL21 (DE3) pLysS cells . Upon induction with isopropyl beta-D-thiogalactopyranoside, Pit-1 was expressed and accumulated as inclusion bodies in E . coli . The protein was then purified in one step by affinity chromatography on a nickel-nitrilotriacetic acid agarose column under denaturing conditions . This method yielded 0.7 mg of highly pure and stable protein per 200 ml of bacterial culture . A band of 40 kDa, resolved as recombinant ayu Pit-1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, agrees well with the molecular mass calculated from the translated cDNA sequence . The purified recombinant Pit-1 was confirmed in vitro through Western blot analysis, using its monoclonal antibody . This monoclonal antibody detected Pit-1 in the nuclei of ayu developing pituitary by immunohistochemical reaction . It serves as a good reagent for the detection of ayu Pit-1 in situ .

Protein Expr Purif, 2002 Mar, 24(2), 282 - 91
Affinity purification and characterization of the Escherichia coli molecular chaperones; Nam SH et al.; The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well-documented affinity for proteins lacking tertiary structure . The molecular chaperones were induced from lon(-) Escherichia coli mutants, affinity purified with an immobilized beta-casein column, and assayed for refolding activity with thermally and chemically denatured carbonic anhydrase B (CAB) . Chaperones were induced with three treatments: heat shock at 39 degrees C, heat shock 42 degrees C, and alcohol shock with 3% ethanol (v/v) . Lysates were applied to an immobilized beta-casein (30 mg/g beads) column . After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water or 1 mM Mg-ATP . The cold water and Mg-ATP eluates were analyzed by SDS-PAGE . Western analysis identified five E . coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES . The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP . Refolding denatured CAB in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured CAB and a 68% recovery of chemically denatured CAB . The use of affinity matrices for the chaperone purification which are effective as in vitro folding aids will be presented .

Protein Expr Purif, 2002 Mar, 24(2), 242 - 54
Identification of in vitro folding conditions for procathepsin S and cathepsin S using fractional factorial screens; Tobbell DA et al.; Human procathepsin S and cathepsin S were expressed as inclusion bodies in Escherichia coli . Following solubilization of the inclusion body proteins, fractional factorial protein folding screens were used to identify folding conditions for procathepsin S and cathepsin S . A primary folding screen, including eight factors each at two levels, identified pH and arginine as the main factors affecting procathepsin S folding . In a second simple screen, the yields were further improved . The in vitro folding of mature cathepsin S has never been reported previously . In this study we used a series of fractional factorial screens to identify conditions that enabled the active enzyme to be generated without the prodomain although the yields were much lower than achieved with procathepsin S . Our data show the power of fractional factorial screens to rapidly identify folding conditions even for a protein that does not easily fold into its active conformation .

Protein Expr Purif, 2002 Mar, 24(2), 173 - 80
Methods for preparation of recombinant cytokine proteins V . mutant analogues of human interferon-gamma with higher stability and activity; Pechenov SE et al.; Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared . Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant . Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein . The main idea of this scheme is to remove cellular impurities before recombinant protein renaturation . Folding kinetics of IFN-gamma was studied by reversed-phase HPLC . IFN-gamma and mutant proteins were characterized by their thermal stability and biological activity . Introduction of the intramolecular disulfide bond together with C-terminal shortening and replacement of C-terminal residue was shown to result in increasing the thermal stability by 19 degrees C and four times enhancement of biological activity compared with intact IFN-gamma molecule .

J Struct Biol, 2001 Oct, 136(1), 53 - 66
Polymer-mediated compaction and internal dynamics of isolated Escherichia coli nucleoids; Cunha S et al.; Nucleoids of Escherichia coli were isolated by osmotic shock under conditions of low salt in the absence of added polyamines or Mg(2+) . As determined by fluorescence microscopy, the isolated nucleoids in 0.2 M NaCl are expanded structures with an estimated volume of about 27 microm(3) according to a procedure based on a 50% threshold for the fluorescence intensity . The nucleoid volume is measured as a function of the concentration of added polyethylene glycol . The collapse is a continuous process, so that a coil-globule transition is not witnessed . The Helmholtz free energy of the nucleoids is determined via the depletion interaction between the DNA helix and the polyethylene glycol chains . The resulting compaction relation is discussed in terms of the current theory of branched DNA supercoils and it is concluded that the in vitro nucleoid is crosslinked in a physical sense . Despite the congested and crosslinked state of the nucleoid, the relaxation rate of its superhelical segments, as monitored by dynamic light scattering, turns out to be purely diffusional . At small scales, the nucleoid behaves as a fluid . (C) 2001 Elsevier Science (USA).

Can J Vet Res, 2002 Jan, 66(1), 19 - 25
Recombinant equine interleukin-1beta induces putative mediators of articular cartilage degradation in equine chondrocytes; Tung JT et al.; Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research . Equine recombinant interleukin-1beta was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized . Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1beta mRNA, which was cloned into an expression vector, expressed in E . coli, and purified using a Ni2+ chromatographic method . The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2) . Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1beta-treated equine cartilage explant cultures . A recombinant peptide of approximately 21 kd was obtained . Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes . Furthermore, cartilage explants exposed to reIL-1beta had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media . The development of a biologically active, species-specific IL-1beta provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses.

J Exp Zool, 2002 Mar 1, 292(4), 376 - 83
Selective enhanced phosphorylation of shrimp beta-tubulin by PKC-delta with PEP(taxol), a synthetic peptide encoding the taxol binding region; Chen WY et al.; Beta-tubulin cDNA from the shrimp Penaeus japonicus was isolated by homology cloning . Expression of cDNA in Escherichia coli yielded a 55 kDa polypeptide, positive for monoclonal antibodies against mammalian beta-tubulin . Autoradiography demonstrated the bacterially expressed hepatopancreas beta-tubulin of P . japonicus is specifically phosphorylated by the delta isoenzyme of protein kinase C (PKC-delta) purified from the plasma membrane of the shrimp heart, in the presence of the receptor for activated PKC (RACK), but not in its absence . Purified shrimp heart PKC-delta is able to phosphorylate bacterially expressed shrimp beta-tubulin without the presence of Ca(++), but requires Mg(++) . The kinase activity of purified PKC-delta on bacterially expressed beta-tubulin was enhanced by incubation with PEP(taxol), a synthetic peptide encoding the taxol-binding region of beta-tubulin . In other words, PEP(taxol) modulates the kinase activity of PKC-delta through RACK .

Eur J Immunol, 2002 Mar, 32(3), 597 - 605
Ligands for murine NKG2D display heterogeneous binding behavior; Carayannopoulos LN et al.; NKG2D transmits stimulatory signals to natural killer cells and other hematopoietic cells, leading to enhanced proliferation, cytokine secretion and target killing . Murine and human NKG2D each recognize five known class I-related molecules with distinct primary structures . Here, we used surface plasmon resonance to examine the binding of murine NKG2D to its cognate ligands: RAE-1B6 (a newly described C57BL/6J variant of RAE-1), RAE-1 delta (common to BALB and C57BL6/J), and H60 (expressed in BALB, but not C57BL/6J) . While RAE-1B6 and H60 display relatively high affinities for NKG2D with K(D) in the 20-30 nM range and k(off )in the 0.03s(-1) to 0.06s(-1) range (t(1/2) approximately 10-20s); the RAE-1 delta variant binds with a lower affinity: K(D) of approximately 750 nM . Furthermore, RAE-1 delta displays biphasic kinetics with dominant k(off) of approximately 0.2s(-1) (t(1/2) approximately 3s), partially explaining the lower affinity . Thus, H60 and RAE-1B6 bind NKG2D with almost identical kinetics while sharing only 20% amino acid sequence identity; whereas other RAE-1 molecules demonstrate faster dissociation and lower affinities than RAE-1B6 despite sharing 90% sequence identity . C57BL/6J mice, although not expressing the H60 gene product, retain a high-affinity ligand for NKG2D in the form of RAE-1B6.

Gene Ther, 2002 Jan, 9(2), 127 - 34
Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinical-grade adenoviral vectors . Evidence of cirrhosis reversion; Garcia-Banuelos J et al.; Adenoviral vectors efficiently target normal liver cells; however, a clear-cut description of the safety boundaries for using adenovectors in hepatic cirrhosis has not been settled . With this in mind, we used a first-generation, replication-deficient adenoviral vector carrying the E . coli lacZ gene (Ad5betaGal) to monitor therapeutic range, biodistribution, toxicity and transduction efficiency in Wistar rats made cirrhotic by two different experimental approaches resembling alcoholic cirrhosis and biliary cirrhosis in humans . Further, we show proof of concept on fibrosis reversion by a 'therapeutic' Ad-vector (AdMMP8) carrying a gene coding for a collagen-degrading enzyme . Dose-response experiments with Ad5betaGal ranging from 1 x 10(8)-3 x 10(12) viral particles (vp) per rat (250 g), demonstrated that adenovirus-mediated gene transfer via iliac vein at 3 x 10(11 )vp/rat, resulted in an approximately 40% transduction in livers of rats made cirrhotic by chronic intoxication with carbon tetrachloride, compared with approximately 80% in control non-cirrhotic livers . In rats made cirrhotic by bile-duct obstruction only, 10% efficiency of transduction was observed . Biodistribution analyses showed that vector expression was detected primarily in liver and at a low level in spleen and kidney . Although there was an important increase in liver enzymes between the first 48 h after adenovirus injection in cirrhotic animals compared to non-transduced cirrhotic rats, this hepatic damage was resolved after 72-96 h . Then, the cDNA for neutrophil collagenase, also known as Matrix Metalloproteinase 8 (MMP8), was cloned in an Ad-vector and delivered to cirrhotic rat livers being able to reverse fibrosis in 44% . This study demonstrates the potential use of adenoviral vectors in safe transient gene therapy strategies for human liver cirrhosis.

Exp Physiol, 2002 Mar, 87(2), 153 - 62
Regional expression of inducible nitric oxide synthase in the kidney stimulated by lipopolysaccharide in the rat; Chou DE et al.; The renal medulla contains more mRNA of the inducible isoform of nitric oxide synthase (iNOS) than the cortex, which may be important in preventing ischaemic injury, since blood flow and tissue oxygen tension are normally low in this region . We examined the effects of the bacterial endotoxin E . coli lipopolysaccharide (LPS) on renal function and regional expression of iNOS in male Sprague-Dawley rats . In six rats, glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were 0.95 +/- 0.09 ml min(-1) g(-1) and 3.36 +/- 0.20 ml min(-1) g(-1), respectively, and decreased significantly to 0.35 +/- 0.09 and 1.74 +/- 0.54 ml min(-1) g(-1), respectively, 1 h after administration of LPS . In an additional seven rats, GFR and ERPF were 0.91 +/- 0.07 and 2.97 +/- 0.30 ml min(-1) g(-1), respectively, 18 h after LPS administration; these values were similar to those in control rats . In all rats, arterial pressure was stable throughout all study periods . In control rats, immunoblot analysis revealed expression of the iNOS protein in the cortex and more pronounced expression in the medulla . In rats studied 18 h after LPS treatment, there was a striking increase in the iNOS expression in the outer medulla . Immunohistochemical examination in the LPS-treated rats showed limited iNOS immunostaining in the cortex, localised to the vascular endothelium and macula densa; however, intense and widespread staining was noted in the tubular and vascular structures of the outer medulla . These findings demonstrated a differential constitutive expression of iNOS protein in different regions of the rat kidney, and marked augmentation of iNOS expression in the outer medulla by LPS.

Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 564 - 6 Epub 2002 Feb 21.
Crystallization and preliminary X-ray crystallographic analysis of SEDL; Jang SB et al.; SEDL (known also as sedlin) is a 140 amino-acid protein with a putative role in endoplasmic reticulum-to-Golgi transport . Several missense mutations and deletion mutations in the SEDL gene, which result in protein truncation by frame shift, are responsible for spondyloepiphyseal dysplasia tarda, a progressive skeletal disorder . The protein is identical to MIP-2A, which was shown to interact physically with c-myc promotor-binding protein 1 (MBP-1) and relieve the regulatory role of MBP-1 as a general transcription repressor . In order to gain insights into the function of SEDL by structural analysis, the protein was overexpressed and crystallized as a first step . SEDL was overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method at 298 K . The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 46.69, b = 101.30, c = 66.15 A . The unit cell is likely to contain one molecule of SEDL, with a crystal volume per protein mass (V(M)) of 2.36 A(3)Da(-1) and a solvent content of about 47.9% by volume . A native data set to 2.8 A resolution was obtained from a flash-cooled crystal using synchrotron radiation.

Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 549 - 52 Epub 2002 Feb 21.
Crystallization of diaminopimelate decarboxylase from Escherichia coli, a stereospecific D-amino-acid decarboxylase; Momany C et al.; The final step in lysine biosynthesis in bacteria, the conversion of meso-diaminopimelate to L-lysine, is catalyzed by the only known D-amino-acid decarboxylase, diaminopimelate decarboxylase (DDC) . The Escherichia coli DDC has been cloned, overexpressed in E . coli with a carboxy-terminal polyhistidine purification tag and crystallized from lithium sulfate . The protein is intensely yellow, owing to the pyridoxal-5'-phosphate cofactor, and is enzymatically active . Large well ordered crystals, belonging to space group P6(1)22 with unit-cell parameters a = b = 98.6, c = 177 A, make high-resolution X-ray diffraction studies possible to characterize the residues important in stereospecific decarboxylation and reprotonation during catalytic turnover.

Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 539 - 41 Epub 2002 Feb 21.
Crystallization and preliminary X-ray analysis of the ATP-binding domain of the ABC transporter haemolysin B from Escherichia coli; Kranitz L et al.; Haemolysin B (HlyB) is a transmembrane protein which belongs to the superfamily of ABC transporters . In vivo, it mediates the non-classical translocation of the 107 kDa toxin HlyA across both membranes of Escherichia coli together with haemolysin D and the outer membrane protein TolC . The cytosolic ATP-binding domain of HlyB has been overexpressed and purified as an N-terminal His-tag fusion protein . Here, the crystallization of the ATPase domain of HlyB in the presence of ATP is described . A native data set has been obtained at a resolution of 2.8 A . Crystals belong to the primitive tetragonal space group P4(x)2(1)2, where x is very likely to be 1 or 3, with unit-cell parameters a = b = 104.6, c = 125.8 A, alpha = beta = gamma = 90 degrees.

Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 536 - 8 Epub 2002 Feb 21.
Expression, purification, refolding and crystallization of the carbohydrate-recognition domain of p58/ERGIC-53, an animal C-type lectin involved in export of glycoproteins from the endoplasmic reticulum; Velloso LM et al.; p58/ERGIC-53 is a mammalian calcium-dependent lectin that serves as a glycoprotein-sorting receptor between the endoplasmic reticulum (ER) and the Golgi complex . It is a type I transmembrane protein with two lumenal domains, one of which is a carbohydrate-recognition domain (CRD) and homologous to leguminous lectins . The CRD of p58, the rat homologue of human ERGIC-53, was overexpressed in insect cells and Escherichia coli, purified and crystallized using Li(2)SO(4) as a precipitant . The crystals belong to space group I222, with unit-cell parameters a = 49.6, b = 86.1, c = 128.1 A, and contain one molecule per asymmetric unit, corresponding to a packing density of 2.4 A(3)Da(-1) . Knowledge of the structure of p58/ERGIC-53 will provide a starting model for understanding receptor-mediated glycoprotein sorting between the ER and the Golgi.

Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 519 - 21 Epub 2002 Feb 21.
Cloning, purification, crystallization and preliminary X-ray studies of RFC boxes II-VIII of replication factor C from Methanococcus jannaschii; Lee I et al.; Replication factor C (RFC) is the accessory protein required to load the proliferating cell nuclear antigen (PCNA) onto DNA in replication process . RFC is composed of several subunits and each subunit contains the highly conserved sequences RFC boxes II-VIII . RFC boxes II-VIII of the large subunit of replication factor C from Methanococcus jannaschii has been overexpressed in Escherichia coli, purified and crystallized at 295 K using ammonium sulfate as precipitant . Crystals belong to the space group R32, with unit-cell parameters a = b = 238.23 (5), c = 73.17 (12) A . Native data were collected at 100 K to a resolution of 3.2 A using a synchrotron-radiation source.

Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 511 - 2 Epub 2002 Feb 21.
Crystallization of the Oct-1/SNAP190 peptide/DNA complex; Hovde S et al.; Crystals of the Oct-1 POU/SNAP190 peptide/DNA tertiary complex have been obtained by hanging-drop vapor diffusion at 293K in 20% 2-propanol, 20% PEG 4000 and 0.1M sodium citrate pH 5.6 . The Oct-1 POU protein has two domains, one a homeodomain and the other a POU domain, which are connected by a flexible linker . The DNA used in the complex is slightly different in the octamer region compared with the two previously crystallized Oct-1 POU/DNA complexes . The DNA is 14 base pairs, with an octamer sequence of 5'-ATGTAGAT-3' and an overhang of one base on both strands . The SNAP190 peptide is 27 amino acids long (residues 884-910) . The crystals diffract to 2.3 A (94.1% completeness) at the synchrotron under cryogenic (123K) conditions . The crystals are triclinic, space group P1, with unit-cell parameters a = 36.4, b = 54.9, c = 77.6A, alpha = 94.9, beta = 99.6, gamma = 109.2 degrees . This structure will provide insight into how Oct-1 interacts with SNAP190, a critical component of the small nuclear RNA-activating protein complex (SNAPc) . Transcription of human snRNA genes is activated by these direct protein-protein interactions.

Acta Crystallogr D Biol Crystallogr, 2002 Mar, 58(Pt 3), 392 - 7 Epub 2002 Feb 21.
Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding protein; Bucher MH et al.; Pyrococcus furiosus maltodextrin-binding protein readily forms large orthorhombic crystals that diffract to high resolution . This protein was used as a model system to investigate the influence of five short affinity tags (His(6), Arg(5), Strep tag II, FLAG tag and the biotin acceptor peptide) on the formation of protein crystals and their ability to diffract X-rays . The results indicate that the amino-acid sequence of the tag can have a profound effect on both of these parameters . Consequently, the ability to obtain diffracting crystals of a particular protein may depend as much on which affinity tag is selected as it does on whether an affinity tag is used at all.

J Biol Chem, 2002 May 3, 277(18), 15807 - 12 Epub 2002 Feb 20.
Assessing the metabolic function of the MutT 8-oxodeoxyguanosine triphosphatase in Escherichia coli by nucleotide pool analysis; Tassotto ML et al.; In Escherichia coli the mutT gene is one of several that acts to minimize mutagenesis by reactive oxygen species . The bacterial MutT protein and its mammalian homolog have been shown to catalyze in vitro the hydrolysis of the oxidized deoxyguanosine nucleotide, 8-oxo-dGTP, to its corresponding monophosphate . Thus, the protein is thought to "sanitize" the nucleotide pool by ridding the cell of a nucleotide whose incorporation into DNA would be intensely mutagenic . However, because others have shown mutT mutations to be mutagenic under some conditions of anaerobic growth, and have shown 8-oxo-dGTP to be a poor DNA polymerase substrate, there is reason to question this model . We have devised an assay for 8-oxo-dGTP in bacterial extracts . Using this assay, which involves reversed-phase high-performance liquid chromatography and electrochemical detection, we have been unable to detect 8-oxo-dGTP in extracts of three different mutT mutants of E . coli, even after growth of the bacteria in the presence of hydrogen peroxide . Our estimated upper limit for 8-oxo-dGTP content of these bacteria is about 200 molecules/cell, corresponding to a concentration of about 0.34 microm . When 8-oxo-dGTP was added at 0.34 microm to an in vitro DNA replication system primed with a DNA template that permits scoring of replication errors and with the four normal dNTPs at their estimated intracellular concentrations, there was no detectable effect upon the frequency of replication errors . These findings lead us to question the conclusion that 8-oxo-dGTP is the most significant physiological substrate for the MutT protein.

J Biol Chem, 2002 May 3, 277(18), 16002 - 10 Epub 2002 Feb 20.
Disulfide bond formation promotes the cis- and trans-dimerization of the E-cadherin-derived first repeat; Makagiansar IT et al.; Cadherin is a cell adhesion molecule crucial for epithelial and endothelial cell monolayer integrity . The previously solved x-ray crystallographic structure of the E-CAD12 cis-dimer displayed an unpaired Cys(9), which protruded away from the Cys(9) on the other protomer . To investigate the possible biological function of Cys(9) within the first repeat (the E-cadherin-derived N-terminal repeat), E-CAD1 was overexpressed and secreted into the periplasmic space of Escherichia coli cells . Recombinant E-CAD1 produced a mixed monomer and dimer in an equilibrium fashion . The dimer was linked by a disulfide through Cys(9) pairing . Analysis by high pressure liquid chromatography and electron microscopy suggested the existence of oligomeric complexes . Mutation at Trp(2) appears to indicate that these oligomeric complexes trans-dimerize . Interestingly, mutation of Cys(9) affected not only the cis-dimerization, but also the trans-oligomerization of E-CAD1 . Accordingly, it is plausible that, under oxidative stress, the homophilic interactions of E-cadherin through E-CAD1 may be promoted and stabilized by this disulfide bond.

J Biol Chem, 2002 May 3, 277(18), 15530 - 8 Epub 2002 Feb 20.
Tight binding inhibition of protein phosphatase-1 by phosphatidic acid . Specificity of inhibition by the phospholipid; Jones JA et al.; Phosphatidic acid (PA) has been identified as a bioactive lipid second messenger, yet despite extensive investigation, no cellular target has emerged as a mediator of its described biological effects . In this study, we identify the gamma isoform of the human protein phosphatase-1 catalytic subunit (PP1c gamma) as a high affinity in vitro target of PA . PA inhibited the enzyme dose-dependently with an IC(50) of 15 nm . Mechanistically, PA inhibited the enzyme noncompetitively with the kinetics of a tight binding inhibitor and a K(i) value of 0.97 +/- 0.24 nm . Together, these data describe one of the most potent in vitro effects of PA . To further elucidate the interaction between PA and PP1c gamma, structure/function analysis of the lipid was carried out using commercially available and synthetically generated analogs of PA . These studies disclosed that the lipid-protein interaction is dependent on the presence of the lipid phosphate as well as the presence of the fatty acid side chains, because lipids lacking either of these substituents resulted in complete loss of inhibition . However, the specific composition of the fatty acid side chains was not important for inhibition . Using 1-O-hexadecyl,2-oleoyl-PA, it was also shown that the carbonyl group of the sn-1 acyl linkage is not required for the lipid-protein interaction . Finally, using a lipid-protein overlay assay, it was demonstrated that PP1c gamma specifically and directly interacts with phosphatidic acid while not significantly binding other phospholipids . These results identify PA as a tight binding and specific inhibitor of PP1, and they raise the hypothesis that PP1c gamma may function as a mediator of PA action in cells . They also argue for the existence of a specific high affinity PA-binding domain on the enzyme.

J Biol Chem, 2002 May 3, 277(18), 15345 - 53 Epub 2002 Feb 20.
Yeast ribosomal protein L12 is a substrate of protein-arginine methyltransferase 2; Chern MK et al.; Type III protein-arginine methyltransferase from the yeast Saccharomyces cerevisiae (RMT2) was expressed in Escherichia coli and purified to apparent homogeneity . The cytosolic, ribosomal, and ribosome salt wash fractions from yeast cells lacking RMT2 were used as substrates for the recombinant RMT2 . Using S-adenosyl-l-methionine as co-substrate, RMT2 methylated a protein in the ribosome salt wash fraction . The same protein in the ribosomal fraction was also methylated by RMT2 after pretreating the sample with endonuclease . Amino acid analysis affirmed that the labeling products were delta-N-monomethylarginines . The methylated protein from the ribosomal or the ribosome salt wash fraction was isolated by two-dimensional gel electrophoresis and identified as ribosomal protein L12 by mass spectrometry . Using synthetic peptides, recombinant L12, and its mutant as substrates, we pinpointed Arg(67) on ribosomal protein L12 as the methyl acceptor . L12 was isolated from wild type yeast cells that have been grown in the presence of S-adenosyl-l-{methyl-(3)H}methionine and subjected to amino acid analysis . The results indicate that L12 contains delta-N-monomethylarginines.

J Biol Chem, 2002 Apr 12, 277(15), 12495 - 8 Epub 2002 Feb 20.
ADP-dependent glucokinase/phosphofructokinase, a novel bifunctional enzyme from the hyperthermophilic archaeon Methanococcus jannaschii; Sakuraba H et al.; A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing . The gene encoded a protein of 462 amino acids with a molecular weight of 53,361 . The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively . The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized . To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase . A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric . The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate . The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively . The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively . The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase . A gene duplication event might lead to the two enzymatic activities.

Vet Microbiol, 2002 Apr 2, 85(4), 333 - 42
An ELISA for antibodies against infectious bronchitis virus using an S1 spike polypeptide; Wang CH et al.; Using the whole infectious bronchitis virus (IBV) for detecting the antibody against IBV by enzyme-linked immunosorbent assay (ELISA) is a routine work in poultry industry . To prepare virus is time consuming and tedious . Furthermore, the whole viral antigen detects all antibodies against the viral structural proteins, including spike (S), nucleocapsid, matrix, and other proteins . Among those, S protein is related to neutralization . Thus, to develop and express protein fragment from S gene and to use the protein as a coating antigen for antibody detection against IBV are the purposes of this experiment . A partial S gene fragment (n.t . 1143-1665) was cloned into pRSET vectors and transformed into competent Escherichia coli (E . coli) BL21 (DE3) . A 27.5 kDa fusion protein (S-fg, containing S1-F and partial S2-G antigenic sites) was successfully expressed, affinity-purified and detected specifically with chicken anti-IBV serum by Western blot . The expressed S-fg protein was used as a coating antigen for developing an ELISA (S-fg ELISA) for serum antibody detection in anti-IBV antisera from different IBV serotypes and in field sera . The results show that the S-fg fusion protein is highly cross-reactive among different IBV serotypes, and the S-fg ELISA is found to be a convenient, economical, and efficient method for antibody detection against IBV.

J Neurosci Methods, 2002 Mar 15, 114(2), 205 - 12
A monoclonal antibody to tryptophan hydroxylase: applications and identification of the epitope; Haycock JW et al.; Recombinant rabbit tryptophan hydroxylase (TPH) was expressed in Escherichia coli and purified from inclusion bodies by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . A mouse monoclonal antibody and rabbit and sheep polyclonal antibodies were generated . In immunohistochemical studies of formaldehyde-fixed primate brain, the monoclonal strongly labeled not only cell bodies in the raphe nuclei but also fibers in the cerebral cortex . Truncation mutants and peptide pre-competition were used to localize the epitope to E103SVPWFP109 . Although the primary sequences of TPH encoded by mRNAs from brain and pineal gland are identical, differences in the immunoreactivity of TPH protein from these two sources were observed in blot immunolabeling studies . TPH immunoreactivity migrated as an M(r) approximately equal 56000 band in each of the tissues except human pineal glands, in which the TPH reactivity was approximately 3 kDa lower . In addition, the relative intensities of TPH immunolabeling across the four tissues differed among these antibodies and a previously described monoclonal antibody against phenylalanine hydroxylase (PH8), which cross-reacts with TPH . Whereas PH8 exhibited roughly equivalent TPH reactivity per protein in both tissues from both species, TPH from human and rat raphe nuclei was preferentially recognized by the present monoclonal . By contrast, the affinity-purified sheep polyclonal antibody reacted preferentially with TPH from human and rat pineal gland, and the affinity-purified rabbit polyclonal antibody appeared to selectively recognize TPH from human pineal gland.

Zhonghua Gan Zang Bing Za Zhi, 2002 Feb, 10(1), 28 - 30
{Construction and expression of humanized anti-HBsAg scFv targeting interferon-alpha in escherichia coli}; Xia X et al.; OBJECTIVE: To develop a bacteria expression system to produce the fusion protein of humanized anti-HBsAg scFV and interferon-alpha . METHODS: The expression vector was constructed after cleaving the plasmids harboring the humanized anti-HBsAg scFv and interferon alpha respectively and ligating to linearized pET22b subsequence . The expression of fusion protein in E.coli was analyzed by SDS-PAGE . The binding activity and antiviral activity of the fusion protein was characterized by competing inhibition test and cytopathic effect reduction . RESULTS: The plasmid harboring the in frame arranged fusion gene was constructed and identified . After induction for 12h, a new band close to 4.5 10(4) was observed using SDS-PAGE . Results of competing ELISA and cytopathic effect reduction showed the fusion protein retained its specific binding activity and antiviral activities . CONCLUSIONS: The construction and expression of the fusion gene of humanized anti-HBsAg scFv and interferon in E.coli are successful.

Genes Cells, 2002 Jan, 7(1), 49 - 58
The H1 and H2 regions of the activation domain of herpes simplex virion protein 16 stimulate transcription through distinct molecular mechanisms; Ikeda K et al.; BACKGROUND: The Herpes Simplex Virion Protein 16 (VP16) contains a strong activation domain which can be subdivided into two regions, H1 and H2, both of which independently activate transcription in vivo . Several components of the basal transcription machinery have been shown to interact with the activation domain of VP16, mostly through the H1 region . RESULTS: We show that the H2 region binds directly to histone acetyltransferase, CBP (CREB (cAMP Responsive Element Binding Protein) Binding Protein) both in vivo and in vitro . The sites of interaction with the H2 region were mapped to both the amino- and carboxy-terminal segments of CBP . A mutation in the H2 region disrupts the interaction with CBP and abolishes the ability of VP16 to mediate in vitro transactivation from chromatin templates in an acetyl-CoA dependent manner . In contrast, human Mediator, another co-activator complex, binds specifically to both the H1 and H2 regions . CONCLUSION: The H1 and H2 regions of the VP16 activation domain activate transcription via distinct pathways . The H2 requires CBP for activation, whereas the H1 may function through Mediator and general transcription factors.

Eur J Biochem, 2002 Feb, 269(4), 1304 - 15
Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A ligase gene family; Lindermayr C et al.; 4-Coumarate:CoA ligase (4CL) is involved in the formation of coenzyme A thioesters of hydroxycinnamic acids that are central substrates for subsequent condensation, reduction, and transfer reactions in the biosynthesis of plant phenylpropanoids . Previous studies of 4CL appear to suggest that many isoenzymes are functionally equivalent in supplying substrates to various subsequent branches of phenylpropanoid biosyntheses . In contrast, divergent members of a 4CL gene family were identified in soybean (Glycine max L.) . We isolated three structurally and functionally distinct 4CL cDNAs encoding 4CL1, 4CL2, and 4CL3 and the gene Gm4CL3 . A fourth cDNA encoding 4CL4 had high similarity with 4CL3 . The recombinant proteins expressed in Escherichia coli possessed highly divergent catalytic efficiency with various hydroxycinnamic acids . Remarkably, one isoenzyme (4CL1) was able to convert sinapate; thus the first cDNA encoding a 4CL that accepts highly substituted cinnamic acids is available for further studies on branches of phenylpropanoid metabolism that probably lead to the precursors of lignin . Surprisingly, the activity levels of the four isoenzymes and steady-state levels of their transcripts were differently affected after elicitor treatment of soybean cell cultures with a beta-glucan elicitor of Phytophthora sojae, revealing the down-regulation of 4CL1 vs . up-regulation of 4CL3/4 . A similar regulation of the transcript levels of the different 4CL isoforms was observed in soybean seedlings after infection with Phytophthora sojae zoospores . Thus, partitioning of cinnamic acid building units between phenylpropanoid branch pathways in soybean could be regulated at the level of catalytic specificity and the level of expression of the 4CL isoenzymes.

Eur J Biochem, 2002 Feb, 269(4), 1267 - 77
Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves; Markham JE et al.; A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells . The insoluble protein expressed in E . coli was purified and used to raise antibodies . Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa . Soluble protein was purified to homogeneity from S . frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA . The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa . The purified protein had activity of both 6-phosphofructo-2-kinase specific activity 10.4-15.9 nmol min(-1) x mg protein (-1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmol x mol(-1) mg protein(-1) . The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate . In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP . Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed . These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.

Eur J Biochem, 2002 Feb, 269(4), 1145 - 53
Stabilization of a (betaalpha)8-barrel protein by an engineered disulfide bridge; Ivens A et al.; The aim of this study was to increase the stability of the thermolabile (betaalpha)8-barrel enzyme indoleglycerol phosphate synthase from Escherichia coli by the introduction of disulfide bridges . For the design of such variants, we selected two out of 12 candidates, in which newly introduced cysteines potentially form optimal disulfide bonds . These variants avoid short-range connections, substitutions near catalytic residues, and crosslinks between the new and the three parental cysteines . The variant linking residues 3 and 189 fastens the N-terminus to the (betaalpha)8-barrel . The rate of thermal inactivation at 50 degrees C of this variant with a closed disulfide bridge is 65-fold slower than that of the reference dithiol form, but only 13-fold slower than that of the parental protein . The near-ultraviolet CD spectrum, the reactivity of parental buried cysteines with Ellman's reagent as well as the decreased turnover number indicate that the protein structure is rigidified . To confirm these data, we have solved the X-ray structure to 2.1-A resolution . The second variant was designed to crosslink the terminal modules betaalpha1 and betaalpha8 . However, not even the dithiol form acquired the native fold, possibly because one of the targeted residues is solvent-inaccessible in the parental protein.

Eur J Biochem, 2002 Jan, 269(2), 719 - 27
Functional site of endogenous phospholipase A2 inhibitor from python serum; Thwin MM et al.; The functional site of 'phospholipase A2 inhibitor from python' (PIP) was predicted based on the hypothesis of proline brackets . Using different sources of secretory phospholipase A2 (sPLA2s) as enzyme, and {3H}arachidonate-labelled Escherichia coli as substrate, short synthetic peptides representing the proposed site were examined for their secretory phospholipase A2 (sPLA2) inhibitory activity . A decapeptide P-PB.III proved to be the most potent of the tested peptides in inhibiting sPLA2 enzymatic activity in vitro, and exhibited striking anti-inflammatory effects in vivo in a mouse paw oedema model . P-PB.III inhibited the enzymatic activity of class I, II and III PLA2s, including that of human synovial fluid from arthritis patients . When tested by ELISA, biotinylated P-PB.III interacted positively with various PLA2s, suggesting that the specific region of PIP corresponding to P-PB.III, is likely to be involved in the PLA2-PLI interaction . The effect of P-PB.III on the peritoneal inflammatory response after surgical trauma in rats was also examined . P-PB.III effectively reduced the extent of postsurgical peritoneal adhesions as compared to controls . sPLA2 levels at seventh postoperative day in the peritoneal tissue of P-PB.III-treated rats were also significantly reduced (P < 0.05) in comparison to those of the untreated controls . The present results shed additional insight on the essential structural elements for PLA2 binding, and may be useful as a basis for the design of novel therapeutic agents.

Eur J Biochem, 2002 Jan, 269(2), 680 - 7
Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana; Wajant H et al.; The nitrilase AtNIT1 from Arabidopsis thaliana was overexpressed in Escherichia coli with an N-terminal His6 tag and purified by zinc chelate affinity chromatography in a single step almost to homogeneity in a 68% yield with a specific activity of 34.1 U.mg-1 . The native enzyme (approximately 450 kDa) consists of 11-13 subunits (38 kDa) . The temperature optimum was determined to be 35 degrees C and a pH optimum of 9 was found . Thus, recombinant AtNIT1 resembles in its properties the native enzyme and the nitrilase from Brassica napus . The stability of AtNIT1 could be significantly improved by the addition of dithiothreitol and EDTA . The substrate range of AtNIT1 differs considerably from those of bacterial nitrilases . Aliphatic nitriles are the most effective substrates, showing increasing rates of hydrolysis with increasing size of the residues, as demonstrated in the series butyronitrile, octanenitrile, phenylpropionitrile . In comparison with 3-indolylacetonitrile, the rate of hydrolysis of 3-phenylpropionitrile is increased by a factor of 330, and the Km value is reduced by a factor of 23 . With the exception of fluoro, substituents in the alpha position to the nitrile function completely inhibit the hydrolysis.

Eur J Biochem, 2002 Jan, 269(2), 451 - 7
The unique sites in SulA protein preferentially cleaved by ATP-dependent Lon protease from Escherichia coli; Nishii W et al.; SulA protein is known to be one of the physiological substrates of Lon protease, an ATP-dependent protease from Escherichia coli . In this study, we investigated the cleavage specificity of Lon protease toward SulA protein . The enzyme was shown to cleave approximately 27 peptide bonds in the presence of ATP . Among them, six peptide bonds were cleaved preferentially in the early stage of digestion, which represented an apparently unique cleavage sites with mainly Leu and Ser residues at the P1, and P1' positions, respectively, and one or two Gln residues in positions P2-P5 . They were located in the central region and partly in the C-terminal region, both of which are known to be important for the function of SulA, such as inhibition of cell growth and interaction with Lon protease, respectively . The other cleavage sites did not represent such consensus sequences, though hydrophobic or noncharged residues appeared to be relatively preferred at the P1 sites . On the other hand, the cleavage in the absence of ATP was very much slower, especially in the central region, than in the presence of ATP . The central region was predicted to be rich in alpha helix and beta sheet structures, suggesting that the enzyme required ATP for disrupting such structures prior to cleavage . Taken together, SulA is thought to contain such unique cleavage sites in its functionally and structurally important regions whose preferential cleavage accelerates the ATP-dependent degradation of the protein by Lon protease.

Clin Microbiol Infect, 1999 Aug, 5(8), 457 - 461
Molecular typing of fluoroquinolone-resistant and fluoroquinolone-susceptible Escherichia coli isolated from blood of neutropenic cancer patients in a single center; Tascini C et al.; OBJECTIVE: To investigate the molecular epidemiology of fluoroquinolone-resistant (FQ-R) and fluoroquinolone-susceptible (FQ-S) bacteremic Escherichia coli isolates from neutropenic patients by pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis . METHODS: Nineteen FQ-R and 27 FQ-S isolates of E . coli, obtained from patients on a hematologic ward over a 7-year period, were genotyped by PFGE and RAPD using two different random primers (1247 and 1283) . RESULTS: PFGE analysis was able to type all FQ-S isolates and most (17/19, 89%) FQ-R isolates of E . coli . All isolates were genotypically unrelated, with the exception of two indistinguishable FQ-R isolates from different patients in the same period . RAPD analysis typed all isolates, including those FQ-R isolates untypable by PFGE, but was unable to distinguish between some isolates that were different by PFGE . Using primer 1247, RAPD analysis identified six pairs and one triad, while primer 1283 identified seven pairs and one triad of indistinguishable isolates . CONCLUSIONS: No spread of epidemic FQ-R or FQ-S E . coli isolates was documented among neutropenic patients . RAPD analysis is a powerful genotyping method, but appeared to be less reproducible and discriminatory than PFGE for investigating E . coli isolates.

Cell Microbiol, 2002 Jan, 4(1), 19 - 28
Requirement of Rho-family GTPases in the invasion of Type 1-piliated uropathogenic Escherichia coli; Martinez JJ et al.; Bladder infections caused by uropathogenic Escherichia coli (UPEC) depends on the ability of E . coli to express type 1 pili . The adhesive component of the pilus, FimH, mediates the invasion of E . coli into the bladder epithelium, a mechanism that facilitates the survival and persistence of E . coli in the bladder . The invasion mechanism requires actin polymerization, focal adhesion kinase phosphorylation and PI 3-kinase activation as well as the formation of FAK/PI 3-kinase and downstream vinculin/alpha-actinin complexes . In this study, we report a role for Rho-GTPase family members, namely RhoA, Cdc42 and Rac1, in the invasion process . Internalization of type 1-piliated E . coli (fimH+) and FimH-coated micro-spheres was inhibited by compactin, a pan-Rho-GTPase inhibitor and dominant negative isoforms of Rac1 and Cdc42 . Expression of active Rac1 induced an internalization of E . coli that was insensitive to wortmannin and genistein . Expression of constitutively active Cdc42 induced the formation of FAK/PI 3-kinase and vinculin/alpha-actinin complexes whereas active Rac1 induced only a vinculin/alpha-actinin complex . Taken together, these data suggest that FimH-mediated invasion is dependent on GTP-binding protein activity that involves Cdc42 and PI 3-kinase activation probably upstream of Rac1.

J Med Chem, 2002 Feb 28, 45(5), 1146 - 9
Synthesis and biological evaluation of a new furo{2,3-h}quinolin-2(1H)-one; Chilin A et al.; A new furoquinolinone derivative, namely 4-hydroxymethyl-1,6,8-trimethylfuro{2,3-h}quinolin-2(1H)-one (HOFQ), was synthesized and its biological activity studied . By UVA activation, HOFQ induced strong antiproliferative effects in Ehrlich ascite cells, which lost their ability to transmit the tumor by transplantation . HOFQ exhibited poor genotoxicity and absence of skin phototoxicity . Actually, HOFQ sensitization forms DNA-protein cross-linkages but not interstrands cross-links . Therefore, HOFQ appears to be a new promising drug for PUVA photochemotherapy and photopheresis.

J Surg Res, 2002 Mar, 103(1), 1 - 7
Effect of creatine monohydrate on cardiac function in a rat model of endotoxemia; Vona-Davis L et al.; BACKGROUND: Reports have attributed cardiac failure during acute models of endotoxemia to a lack of high-energy phosphates . This study was undertaken to investigate whether creatine (Cr) administered during perfusion could enhance myocardial protection and improve recovery of cardiac function in a rat model of endotoxemia . METHODS: Acute endotoxemia was induced in rats by a bolus injection of Escherichia coli endotoxin (LPS: 4 mg/kg, ip) while control rats were injected with an equal volume of 0.9% normal saline . To assess the adequacy of energy metabolism, freeze-clamped hearts were obtained from animals to study the concentrations of endogenous ATP, phosphocreatine (PCr), inorganic phosphate (P(i)), and intracellular pH by (31)P-cryomagnetic resonance spectroscopy . In a separate experiment, isolated hearts were perfused via a Langendorff column with Krebs-Henseleit buffer containing different concentrations of creatine monohydrate (1, 3, or 10 mM) . Cardiac performance was evaluated via a paced (300 bpm) isovolumetric balloon preparation . Measurements of cardiac function including left ventricular developed pressure (LVDP), the maximum rates of ventricular pressure rise (LV +dP/dt) and fall (LV -dP/dt), and coronary flow were made for both LPS and saline-treated animals . RESULTS: High-energy phosphate ratios of PCr/ATP and PCr/P(i) in hearts declined significantly at 4 h after endotoxin treatment . As anticipated, LVDP and LV +dP/dt(max) at a given preload and heart rate were significantly (P < 0.05) lower at 4 h when measured at the same time point . The functional recovery of these parameters was not improved by the addition of creatine monohydrate to the perfusion buffer . Creatine produced a significant (P < 0.05) negative inotropic effect in hearts from saline-treated animals . The LVDP was reduced by 30% at the lowest concentration and by 50% at the highest concentration of creatine monohydrate . Furthermore, creatine significantly (P < 0.05) reduced LV -dP/dt(max) in both saline and LPS-treated rats . These data demonstrate that exogenous creatine does not contribute to myocardial preservation in endotoxemia . CONCLUSIONS: Energy stores in the rat heart decline early in endotoxemia accompanied by reduced myocardial performance, suggesting that the ability of the heart to perform mechanical work is impaired . Cardiac dysfunction in an acute model of endotoxemia was not improved with exogenous creatine during perfusion . Creatine's effects were primarily lusitropic by delaying the onset of myocardial relaxation in all hearts . The deleterious effects of exogenous creatine monohydrate in normal hearts should be examined in future experimental studies.

Plant Mol Biol, 2002 Feb 1, 48(3), 299 - 308
Molecular cloning, expression and characterization of tropinone reductase II, an enzyme of the SDR family in Solanum tuberosum (L.); Keiner R et al.; Calystegines are nortropane alkaloids that are found in Solanaceae containing the classical tropane alkaloids hyoscyamine and scopolamine, and in other Solanaceae such as potato, Solanum tuberosum (L.) . Calystegines are assumed to derive from the classical tropane alkaloid pathway . We isolated a cDNA from S . tuberosum with high homology to the pseudotropine-forming tropinone reductase (TRII), which presents as the first putative metabolite specific to calystegines . The equivalent amino acid sequence shows typical motifs of a short-chain dehydrogenase (SDR) . The recombinant TRII protein expressed in Escherichia coli catalyzes pseudotropine formation from tropinone with a Km value, a pH optimum, substrate and co-substrate preferences similar to those reported for the TRII enzymes from other Solanaceae species . The gene is expressed in roots, tubers and aerial parts of potato . The distribution of the TRII transcript in comparison with the calystegine concentrations in the tissues suggests transport of calystegines or their precursors between potato organs.

Plant Mol Biol, 2002 Feb 1, 48(3), 277 - 85
cDNA cloning and expression of carotenogenic genes during flower development in Gentiana lutea; Zhu C et al.; All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library . They encode a geranylgeranyl pyrophosphate synthase, a phytoene synthase, a phytoene desaturase and a zeta-carotene desaturase . The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli . The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants . Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family . Tissue-specific expression of the genes and expression during flower development was investigated . The expression of the phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting . Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), phytoene desaturase (pds) and zeta-carotene desaturase (zds) were detected in flowers and leaves but not in stems . Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids . Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G . lutea . For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.

Plant Mol Biol, 2002 Feb 1, 48(3), 255 - 65
Cloning and characterization of a cDNA encoding a cobalamin-independent methionine synthase from potato (Solanum tuberosum L.); Zeh M et al.; A potato cDNA clone, StMS1, that encodes a methionine synthase was isolated . This protein was identified on the basis of both structural and functional evidence . The predicted sequence of the protein encoded by StMS1 shows a high degree of similarity to methionine synthases from other organisms and the expression of StMS1 in bacterial mutant strains restored the mutant's ability to synthesize methionine . Genomic organization and expression analyses suggest that StMS1 is a low-copy gene and is differentially expressed in potato organs . StMS1 expression was found in all tissues, but at elevated levels in flowers, basal levels in sink and source leaves, roots and stolons, and low levels in stems and tubers . RNA expression data were confirmed by western blot analysis except that the protein content in leaves was less than expected from the RNA data . Western blot analysis of subcellular fractions revealed that the protein is located in the cytosol . However, the changing pattern of gene expression during the day/night period implied a light-dependent control of MS transcription normally seen for enzymes localized in plastids . The expression of MS was shown to be light-inducible with its highest expression at midday . These RNA data were not confirmed at the protein level since protein content levels remained constant over the whole day . Feeding experiments of detached leaves revealed that sucrose or sucrose-derived products are responsible for StMS1 induction . This induction can be blocked by treatment with DCMU during the light period . Western analysis revealed that the amount of StMS1 is not affected by either treatment . This experiment confirmed the presence of a day/night rhythm . Methionine synthase expression is regulated by photoassimilates but this seems not to detectably alter protein levels.

Planta, 2002 Jan, 214(3), 476 - 83
Overexpression of violaxanthin de-epoxidase: properties of C-terminal deletions on activity and pH-dependent lipid binding; Hieber AD et al.; Violaxanthin de-epoxidase (VDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of violaxanthin to form antheraxanthin and zeaxanthin . VDE is predicted to be a lipocalin protein with a central barrel structure flanked by a cysteine-rich N-terminal domain and a glutamate-rich C-terminal domain . A full-length Arabidopsis thaliana (L.) Heynh . VDE and deletion mutants of the N- and C-terminal regions were expressed in Escherichia coli and tobacco (Nicotiana tabacum L . cv . Xanthi) plants . High expression of VDE in E . coli was achieved after adding the argU gene that encodes the E . coli arginine AGA tRNA . However, the specific activity of VDE expressed in E . coli was low, possibly due to incorrect folding . Removal of just 4 amino acids from the N-terminal region abolished all VDE activity whereas 71 C-terminal amino acids could be removed without affecting activity . The difficulties with expression in E . coli were overcome by expressing the Arabidopsis VDE in tobacco . The transformed tobacco exhibited a 13- to 19-fold increase in VDE specific activity, indicating correct protein folding . These plants also demonstrated an increase in the initial rate of nonphotochemical quenching consistent with an increased initial rate of de-epoxidation . Deletion mutations of the C-terminal region suggest that this region is important for binding of VDE to the thylakoid membrane . Accordingly, in vitro lipid-micelle binding experiments identified a region of 12 amino acids that is potentially part of a membrane-binding domain . The transformed tobacco plants are the first reported example of plants with an increased level of VDE activity.

Planta, 2002 Jan, 214(3), 446 - 55
Characterization of three new members of the Arabidopsis thaliana calmodulin gene family: conserved and highly diverged members of the gene family functionally complement a yeast calmodulin null; Zielinski RE; Three genes encoding members of the EF-hand family of Ca2+-binding proteins were identified from Arabidopsis thaliana (L.) Heynh . sequences deposited in the expressed sequence tag and genomic sequence databases . Full-length cDNAs for each of the genes, Cam7, Cam8, and Cam9, were sequenced . Cam7 encodes a conventional 16.8-kDa, 148-amino-acid calmodulin protein (CaM) . In contrast, Cam8 and 9 encode highly diverged isoforms of the protein that share 73 and 49% amino acid sequence identity, respectively, with CaM7 . RNA gel blot and reverse transcription-polymerase chain reaction experiments revealed that each of the genes is expressed in leaves, flowers and siliques . To test the functional properties of the polypeptides encoded by these genes, they were expressed in Escherichia coli and the yeast Saccharomyces cerevisiae . Each was purified by Ca2+-dependent hydrophobic affinity chromatography . CaM7, but neither CaM8 nor CaM9, formed a complex with a basic amphiphilic helical peptide in the presence of Ca2+ that could be identified by gel electrophoresis . In spite of these in vitro differences, each of the sequences functionally substituted for yeast CMD1 to maintain viability . Isolation of yeast strains complemented by Cam9 required selection against the plasmid harboring wild-type yeast sequences, whereas complementation by Cam7 and Cam8 did not . These results suggest that the mechanism of action of CaM8 and CaM9 is similar to that of more conventional CaM sequences . CaM9, and to a lesser degree CaM8, however, appear to represent Ca2+-binding sensor proteins that interact with a more limited set of target proteins than do more conventional CaM isoforms.

World J Gastroenterol, 2001 Dec, 7(6), 841 - 5
Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells; Li X et al.; AIM: To clone the cDNA of UGT1A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGT1A9 . METHODS: cDNA of UGT1 A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E.Coli DH5(alpha) . The inserted fragment, verified by DNA sequencing, was subcloned into the Hind III /Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9 . CHL cells were transfected with the resultant recombinants, pREP9-UGT1A9, and selected by G418 (400 mg x L(-1)) for one month . The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples forUGT1A9 assays . The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC . RESULTS: The sequence of the cDNA segment cloned, which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region . The recombinant constructed, pREP9-UGT1A9, contains the entire coding region, along with 18 bp of the 5' and 55 bp of the 3' untranslated region of theUGT1A9 cDNA, respectively . The cell lines established expressed the protein of UGT1A9, and the enzyme activity towards propranolol in S9 protein was found to be 101+/- 24 pmol x min(-1) x mg(-1) protein (n=3), but was not detectable in parental CHL cells . CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL cells . The CHL-UGT1 A9 cell lines established efficiently expressed the protein ofUGT1A9 for the further enzyme study of drug glucuronidation.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 2072 - 7
Specificity and robustness in transcription control networks; Sengupta AM et al.; Recognition by transcription factors of the regulatory DNA elements upstream of genes is the fundamental step in controlling gene expression . How does the necessity to provide stability with respect to mutation constrain the organization of transcription control networks? We examine the mutation load of a transcription factor interacting with a set of n regulatory response elements as a function of the factor/DNA binding specificity and conclude on theoretical grounds that the optimal specificity decreases with n . The predicted correlation between variability of binding sites (for a given transcription factor) and their number is supported by the genomic data for Escherichia coli . The analysis of E . coli genomic data was carried out using an algorithm suggested by the biophysical model of transcription factor/DNA binding . Complete results of the search for candidate transcription factor binding sites are available at http://www.physics.rockefeller.edu/~boris/public/search_ecoli.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1894 - 8
The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to oris, an origin of viral DNA replication; Eom CY et al.; We have identified cellular proteins that interact with the herpes simplex virus type 1 (HSV-1) origin-binding protein (UL9 protein) by screening a HeLa cell complementary DNA library by using the yeast two-hybrid system . Approximately 7 x 10(5) colonies were screened . Five of the 48 positive clones contained cDNAs that encoded the p150(Glued) component of the dynactin complex, three contained cDNAs for the neural F Box 42-kDa protein (NFB42), which is highly enriched in neural tissue, and three contained hTid-1, a human homologue of the bacterial DnaJ protein . We have focused in this report on the interaction of the viral UL9 protein with the cellular hTid-1 . In vitro immunoprecipitation experiments confirmed that hTid-1 interacts with the UL9 protein . Electrophoretic mobility-shift assays indicated that the hTid-1 enhances the binding of UL9 protein to an HSV-1 origin, ori(s), and facilitates formation of the multimer from the dimeric UL9 protein . hTid-1 had no effect on the DNA-dependent ATPase or helicase activities associated with the UL9 protein . These findings implicate hTid-1 in HSV-1 DNA replication, and suggest that this cellular protein may provide a chaperone function analogous to the DnaJ protein in Escherichia coli DNA replication.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1888 - 93
The crystal structure and mutational analysis of a novel RNA-binding domain found in the human Tap nuclear mRNA export factor; Ho DN et al.; The Tap protein mediates the sequence nonspecific nuclear export of cellular mRNAs as well as the sequence-specific export of retroviral mRNAs bearing the constitutive transport element (CTE) . Previously, the structures of individual Tap subdomains, including ribonucleoprotein and leucine-rich repeat domains, have been described . Here, we report the crystal structure of a functional CTE RNA-binding domain of human Tap, including the N-terminal arm of the ribonucleoprotein domain and interdomain linking polypeptide . To identify residues that interact with the CTE, we have introduced 38 alanine substitutions for surface residues in the Tap CTE-binding domain and tested these mutants for their ability to support CTE-dependent nuclear RNA export and CTE binding . Four residues that cluster on a concave surface in the leucine-rich repeat domain were found to be critical for CTE binding and define a CTE-interacting surface on this domain . The second critical CTE-interacting surface on Tap is defined by three previously identified residues on the surface of the ribonucleoprotein domain . The structural and mutational data define a novel RNA-binding site on the Tap protein.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1847 - 52
Energetics by NMR: site-specific binding in a positively cooperative system; Tochtrop GP et al.; Proteins with multiple binding sites exhibit a complex behavior that depends on the intrinsic affinities for each site and the energetic communication between the sites . The contributions from intrinsic affinity and cooperativity are difficult to deconvolute using conventional binding experiments that lack information about the occupancies of individual sites . Here, we report the concerted use of NMR and isothermal titration calorimetry to determine the intrinsic and cooperative binding free energies for a ligand-protein complex . The NMR measurements provided the site-specific information necessary to resolve the binding parameters . Using this approach, we observed that human ileal bile acid binding protein binds two molecules of glycocholic acid with low intrinsic affinity but an extraordinarily high degree of positive cooperativity . The highly cooperative nature of the binding provides insights into the protein's biological mechanism . With ongoing improvements in sensitivity and resolution, NMR methods are becoming more amenable to dissecting the complex binding energetics of multisite systems.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1825 - 30
An NMR approach to structural proteomics; Yee A et al.; The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale . Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy . Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR . Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination . NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis . Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1819 - 24
Polypeptide release at sense and noncognate stop codons by localized charge-exchange alterations in translational release factors; Uno M et al.; The mechanism of stop codon recognition during translation has long been a puzzle . Only recently has it been established that a tripeptide in the bacterial release factors (RFs) 1 and 2 serves as the "anticodon" in deciphering stop codons in mRNA . However, the molecular basis of the accuracy of stop codon recognition is unknown . Although specific tripeptides in the RFs are primarily responsible for selective reading of cognate stop codons, charge-flip variant RF proteins, altered at conserved Glu residues adjacent to the tripeptide-anticodon, are shown here to be crucial to codon recognition . Changes of these Glu residues are capable of triggering polypeptide release at noncognate stop codons and also at sense codons . These changes also reverse the growth inhibition by RFs containing "harmful" tripeptide-anticodon changes . These findings suggest that electrostatic interactions involving negative charges in domain C of the RFs mediate their accurate docking in the ribosome . Our results also establish that the charge flipping creates a phenotype/translation termination by "codon bypassing" via relaxed positioning of the RF tripeptide-anticodon in the decoding pocket of the ribosome.

Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 3171 - 5 Epub 2002 Feb 19.
Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching; Stricker J et al.; FtsZ, the major cytoskeletal component of the bacterial cell-division machine, assembles into a ring (the Z-ring) that contracts at septation . FtsZ is a bacterial homolog of tubulin, with similar tertiary structure, GTP hydrolysis, and in vitro assembly . We used green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching to show that the E . coli Z-ring is extremely dynamic, continually remodeling itself with a half-time of 30 s . ZipA, a membrane protein involved in cell division that colocalizes with FtsZ, was equally dynamic . The Z-ring of the mutant ftsZ84, which has 1/10 the guanosine triphosphatase activity of wild-type FtsZ in vitro, showed a 9-fold slower turnover in vivo . This finding implies that assembly dynamics are determined primarily by GTP hydrolysis . Despite the greatly reduced assembly dynamics, the ftsZ84 cells divide with a normal cell-cycle time.

Mol Pharmacol, 2002 Mar, 61(3), 495 - 506
Midazolam oxidation by cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding sites and of the metabolic pathway that leads to enzyme inactivation; Khan KK et al.; Midazolam (MDZ) oxidation by recombinant CYP3A4 purified from Escherichia coli and 30 mutants generated at 15 different substrate recognition site positions has been studied to determine the role of individual residues in regioselectivity and to investigate the possible existence of multiple binding sites . Initial results showed that oxidation of MDZ by CYP3A4 causes time- and concentration-dependent enzyme inactivation with K(I) and k(inact) values of 5.8 microM and 0.15 min(-1), respectively . The different time courses of MDZ hydroxylation by mutants that predominantly formed 1'-OH MDZ as opposed to 4-OH MDZ provided strong evidence that the 1'-OH MDZ pathway leads to CYP3A4 inactivation . Correlational analysis of 1'-OH formation versus 4-OH formation by the mutants supports the inference that the two metabolites result from the binding of MDZ at two separate sites . Thus, substitution of residues Phe-108, Ile-120, Ile-301, Phe-304, and Thr-309 with a larger amino acid caused an increase in the ratio of 1'-OH/4-OH MDZ formation, whereas substitution of residues Ser-119, Ile-120, Leu-210, Phe-304, Ala-305, Tyr-307, and Thr-309 with a smaller amino acid decreased this ratio . Kinetic analyses of nine key mutants revealed that the alteration in regioselectivity is caused by a change in kinetic parameters (V(max) and K(M)) for the formation of both metabolites in most cases . The study revealed the role of various active-site residues in the regioselectivity of MDZ oxidation, identified the metabolic pathway that leads to enzyme inactivation, and provided an indication that the two proposed MDZ binding sites in CYP3A4 may be partially overlapping.

J Biol Chem, 2002 May 10, 277(19), 16484 - 8 Epub 2002 Feb 19.
Mechanism of interaction between leucine-based sorting signals from the invariant chain and clathrin-associated adaptor protein complexes AP1 and AP2; Kongsvik TL et al.; The cytoplasmic tail of the invariant chain contains two leucine-based sorting signals, and each of those seems sufficient to route the invariant chain to its intracellular destination in either normal or polarized cells . It is believed that the intracellular routing of the invariant chain is mediated by its interactions with the clathrin-associated adaptor protein complexes AP1 and AP2 . We () have previously demonstrated the in vitro interactions between the cytoplasmic tail of the invariant chain and AP1/AP2 complexes . These interactions were specific and depended on the critical leucine residues in the invariant chain's sorting signals . In the present study, we decided to investigate the molecular mechanism of these interactions . To this end, we constructed a set of glutathione S-transferase fusion proteins that contained the intact cytoplasmic tail of the invariant chain and its various mutants to define residues important for its interactions with AP1 and AP-2 . Our results demonstrated the importance of several residues other than the critical leucine residues for such interactions . A strong correlation between in vitro binding of AP2 to the invariant chain and in vivo internalization of the invariant chain was observed, confirming the primary role of AP2 in recognition of endocytic signals . In addition, we demonstrated different requirements for AP1 and AP2 binding to cytoplasmic tail of the invariant chain, which may reflect that the different sorting pathways mediated by AP1 and AP2 involve their recognition of the primary structure of the sorting signal.

J Biol Chem, 2002 Apr 26, 277(17), 14717 - 23 Epub 2002 Feb 19.
Mapping of the dimer interface of the Escherichia coli mannitol permease by cysteine cross-linking; van Montfort BA et al.; A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease . This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts . A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline)(3)-catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length . The engineered cysteines were at the borders of the predicted membrane-spanning alpha-helices . Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides . Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys(73)) . Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys(134)), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer . Upon mannitol binding, the Cys(134) becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain . In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for alpha-helix packing in the mannitol permease.

J Biol Chem, 2002 May 3, 277(18), 15407 - 12 Epub 2002 Feb 19.
Generality of the branched pathway in transcription initiation by Escherichia coli RNA polymerase; Susa M et al.; Transcription initiation has been assumed to be a multi-step sequential process, although additional steps could exist . Initiation from the T7A1 promoter, in particular, apparently behaves in vitro in a manner that can be fully explained by the sequential pathway . However, initiation from the lambda P(R)AL promoter has been shown to follow a branched pathway from which a part of the enzyme-promoter complex is arrested at the promoter raising the question as to which mechanism is general . We found that a moribund complex, characteristic of the arrested branch, is formed at the T7A1 promoter, especially in low salt condition indicating that the initiation mechanism for this promoter is also branched . The results of DNA footprinting suggested that holoenzyme in the moribund complex is dislocated on DNA from the position of productive complex . However, only a small fraction of the binary complex becomes arrested at this promoter, and the interconversion between subspecies of binary complex is apparently more reversible than at the lambda P(R)AL promoter, which explains why the reaction pathway appears to be sequential . These findings suggest a generality of the branched pathway mechanism, which would resolve contradictory observations that have been reported for various promoters.

J Biol Chem, 2002 May 3, 277(18), 15971 - 8 Epub 2002 Feb 19.
Molecular determinants of the mechanism underlying acceleration of the interaction between antithrombin and factor Xa by heparin pentasaccharide; Quinsey NS et al.; The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis . Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases . A specific pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition . Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versus thrombin . We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin . To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5 . fXa(Q61A) interacts with antithrombin alone with a nearly normal k(ass); however, we observe only a 4-fold increase in k(ass) in the presence of H5 . The x-ray crystal structure of fXa reveals that Gln(61) forms part of the S1' and S3' pocket, suggesting that the P' region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.

J Biol Chem, 2002 May 10, 277(19), 16985 - 92 Epub 2002 Feb 19.
The human prepro-orexin gene regulatory region that activates gene expression in the lateral region and represses it in the medial regions of the hypothalamus; Moriguchi T et al.; Prepro-orexin is a precursor of the neuropeptides orexin-A and -B, which are localized in the neuronal population of the lateral hypothalamic area (LHA) . We wished to elucidate the mechanisms by which the prepro-orexin gene is specifically activated in orexin neurons in the LHA . The 3.2-kb 5'-flanking region of the human prepro-orexin gene is sufficient for the specific expression of an Escherichia coli lacZ reporter gene in orexin neurons . Therefore, we examined a series of reporter constructs harboring this 3.2-kb regulatory region or its deletion in a reporter transgenic mouse assay . There are two phylogenetically conserved regions located 287 bp (orexin regulatory element (OE) 1) and 2.5 kb (OE2) upstream of the transcription initiation site of the human prepro-orexin gene . In transgenic mice, both OE1 and OE2 are necessary for expressing the human prepro-orexin gene in the LHA and for repressing its expression in the medial regions of the hypothalamus . Through serial deletion analysis of OE1, we found that the 57-bp core region of OE1 is critical for its spatial gene regulatory function in vivo . Mutation analysis further demonstrated that without contribution from the OE1 core region, the lacZ reporter is expressed ectopically in the medial regions of the hypothalamus . Thus, OE1 contains crucial cis-acting elements regulating prepro-orexin gene expression specifically in the LHA.

Infect Immun, 2002 Mar, 70(3), 1631 - 4
Induction of dnaK through its native heat shock promoter is necessary for intramacrophagic replication of Brucella suis; Kohler S et al.; The heat shock protein DnaK is essential for intramacrophagic replication of Brucella suis . The replacement of the stress-inducible, native dnaK promoter of B . suis by the promoter of the constitutively expressed bla gene resulted in temperature-independent synthesis of DnaK . In contrast to a dnaK null mutant, this strain grew at 37 degrees C, with a thermal cutoff at 39 degrees C . However, the constitutive dnaK mutant, which showed high sensitivity to H(2)O(2)-mediated stress, failed to multiply in murine macrophage-like cells and was rapidly eliminated in a mouse model of infection, adding strong arguments to our hypothesis that stress-mediated and heat shock promoter-dependent induction of dnaK is a crucial event in the intracellular replication of B . suis.

Infect Immun, 2002 Mar, 70(3), 1193 - 201
Membrane localization of the S1 subunit of pertussis toxin in Bordetella pertussis and implications for pertussis toxin secretion; Farizo KM et al.; Pertussis toxin is secreted from Bordetella pertussis with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters . The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known . To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins . In wild-type B . pertussis, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes . In mutants of B . pertussis that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes . Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of B . pertussis . First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100 . Second, recombinant S1 previously has been shown to localize to the outer membrane of Escherichia coli (J . T . Barbieri, M . Pizza, G . Cortina, and R . Rappuoli, Infect . Immun . 58:999-1003, 1990) . Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins . By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit . Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of B . pertussis . Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system.

Infect Immun, 2002 Mar, 70(3), 1106 - 12
Identification of a second Arcanobacterium pyogenes neuraminidase and involvement of neuraminidase activity in host cell adhesion; Jost BH et al.; Arcanobacterium pyogenes, a common inhabitant of the upper respiratory and urogenital tracts of economically important animals, such as cattle and swine, is also an opportunistic pathogen associated with suppurative infections in these animals . A . pyogenes expresses neuraminidase activity encoded by the nanH gene, and previously, construction of a nanH mutant of A . pyogenes BBR1 indicated that a second neuraminidase is present in this strain . A 5,112-bp gene, nanP, was cloned and sequenced, and this gene conferred neuraminidase activity on an Escherichia coli host strain . The predicted 186.8-kDa NanP protein exhibited similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases . As expected, insertional inactivation of the nanP gene in A . pyogenes BBR1 resulted in a mutant with reduced neuraminidase activity . However, insertional inactivation of the nanP gene in the nanH mutant strain resulted in a complete lack of neuraminidase activity . Like NanH, NanP was localized to the A . pyogenes cell wall . However, unlike the nanH gene, which was present in 100% of the strains examined, nanP was present in only 64.2% of the isolates (n = 53) . A . pyogenes adheres to HeLa cells, and a nanP mutant displayed a wild-type adhesion phenotype with these cells . In contrast, the ability of a nanH nanP double mutant to bind to HeLa cells was reduced by 53% . The wild-type adhesion phenotype was restored by providing nanP in trans . These data indicate that the neuraminidases of A . pyogenes play a role in adhesion of this organism to host epithelial cells.

Infect Immun, 2002 Mar, 70(3), 1056 - 68
Transcutaneous immunization using colonization factor and heat-labile enterotoxin induces correlates of protective immunity for enterotoxigenic Escherichia coli; Yu J et al.; Enterotoxigenic Escherichia coli (ETEC) diarrheal disease is a worldwide problem that may be addressed by transcutaneous delivery of a vaccine . In several human settings, protective immunity has been associated with immune responses to E . coli colonization factors and to the heat-labile toxin that induces the diarrhea . In this set of animal studies, transcutaneous immunization (TCI) using recombinant colonization factor CS6 and cholera toxin (CT) or heat-labile enterotoxin (LT) as the adjuvant induced immunoglobulin G (IgG) and IgA anti-CS6 responses in sera and stools and antibody responses that recognized CS6 antigen in its native configuration . The antitoxin immunity induced by TCI was also shown to protect against enteric toxin challenge . Although immunization with LT via the skin induced mucosal secretory IgA responses to LT, protection could also be achieved by intravenous injection of the immune sera . Finally, a malaria vaccine antigen, merzoite surface protein 1(42) administered with CT as the adjuvant, induced both merzoite surface protein antibodies and T-cell responses while conferring protective antitoxin immunity, suggesting that both antiparasitic activity and antidiarrheal activity can be obtained with a single vaccine formulation . Overall, our results demonstrate that relevant colonization factor and antitoxin immunity can be induced by TCI and suggest that an ETEC traveler's diarrhea vaccine could be delivered by using a patch.

Drug Metab Dispos, 2002 Mar, 30(3), 349 - 53
The alkaloid rutaecarpine is a selective inhibitor of cytochrome P450 1A in mouse and human liver microsomes; Ueng YF et al.; Rutaecarpine, evodiamine, and dehydroevodiamine are quinazolinocarboline alkaloids isolated from a traditional Chinese medicine, Evodia rutaecarpa . The in vitro effects of these alkaloids on cytochrome P450 (P450)-catalyzed oxidations were studied using mouse and human liver microsomes . Among these alkaloids, rutaecarpine showed the most potent and selective inhibitory effect on CYP1A-catalyzed 7-methoxyresorufin O-demethylation (MROD) and 7-ethoxyresorufin O-deethylation (EROD) activities in untreated mouse liver microsomes . The IC(50) ratio of EROD to MROD was 6 . For MROD activity, rutaecarpine was a noncompetitive inhibitor with a K(i) value of 39 +/- 2 nM . In contrast, rutaecarpine had no effects on benzo{a}pyrene hydroxylation (AHH), aniline hydroxylation, and nifedipine oxidation (NFO) activities . In human liver microsomes, 1 microM rutaecarpine caused 98, 91, and 77% decreases of EROD, MROD, and phenacetin O-deethylation activities, respectively . In contrast, less than 15% inhibition of AHH, tolbutamide hydroxylation, chlorzoxazone hydroxylation, and NFO activities were observed in the presence of 1 microM rutaecarpine . To understand the selectivity of inhibition of CYP1A1 and CYP1A2, inhibitory effects of rutaecarpine were studied using liver microsomes of 3-methylcholanthrene (3-MC)-treated mice and Escherichia coli membrane expressing bicistronic human CYP1A1 and CYP1A2 . Similar to the CYP1A2 inhibitor furafylline, rutaecarpine preferentially inhibited MROD more than EROD and had no effect on AHH in 3-MC-treated mouse liver microsomes . For bicistronic human P450s, the IC(50) value of rutaecarpine for EROD activity of CYP1A1 was 15 times higher than the value of CYP1A2 . These results indicated that rutaecarpine was a potent inhibitor of CYP1A2 in both mouse and human liver microsomes.

Int J Biochem Cell Biol, 2002 Apr, 34(4), 414 - 26
Characterization of platelet-derived growth factor-C (PDGF-C): expression in normal and tumor cells, biological activity and chromosomal localization; Dijkmans J et al.; The predicted platelet-derived growth factor-C (PDGF-C) polypeptide contains an N-terminal CUB-like domain and a C-terminal domain with homology to members of the PDGF/vascular endothelial growth factor (VEGF) family . PDGF-C mRNA is widely expressed in normal tissues and does not appear to be up-regulated in the tumor cell lines tested . The PDGF-C gene was mapped to human chromosome 4q31-32 . PDGF-C protein and the CUB domain of PDGF-C expressed in Escherichia coli, were able to stimulate proliferation of human artery smooth muscle cells, but were inactive on umbilical vein endothelial cells, osteoblasts, fibroblasts, skeletal muscle cells (SkMC), bovine chondrocytes, and rat myocardium cells . Although the mitogenic activity of PDGF-C and the CUB domain was only observed at concentrations ranging from 1 to 10 microg/ml, substitution of Cys(124) by Ser or deletion of Cys(124) significantly reduced the mitogenic activity . Our data suggest a possible role of the CUB domain of PDGF-C in addition to its role in maintaining latency of the PDGF domain.

Biochem J, 2002 Mar 1, 362(Pt 2), 453 - 8
Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372; Makise M et al.; The DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli . Acidic phospholipids decrease its affinity for adenine nucleotides, and re-activate the ADP-bound form to the ATP-bound form . We have previously reported that two mutant forms, DnaAR328E and DnaAK372E, have decreased affinity for cardiolipin (CL) . In the present study, we constructed a mutant DnaA protein, DnaA435, with both R328E and K372E, and compared its biochemical characteristics with those of DnaAR328E and DnaAK372E . DnaA435 could bind to oriC DNA, but did not bind ATP or ADP . In DnaA435, compared with DnaAR328E and DnaAK372E, CL caused less inhibition of oriC DNA binding, suggesting that amino acids R328 and K372 are involved in the interaction of DnaA with acidic phospholipids . DnaA435 could initiate DNA synthesis on oriC both in vivo and in vitro . Based on these results, we propose that ATP activates DnaA protein by changing its higher order structure around R328 and K372.

Biochem J, 2002 Mar 1, 362(Pt 2), 443 - 51
Two mutations in troponin I that cause hypertrophic cardiomyopathy have contrasting effects on cardiac muscle contractility; Burton D et al.; We investigated the effects of two mutations in human cardiac troponin I, Arg(145)-->Gly and Gly(203)-->Ser, that are reported to cause familial hypertrophic cardiomyopathy . Mutant and wild-type troponin I, overexpressed in Escherichia coli, were used to reconstitute troponin complexes in vanadate-treated guinea pig cardiac trabeculae skinned fibres, and thin filaments were reconstituted with human cardiac troponin and tropomyosin along with rabbit skeletal muscle actin for in vitro motility and actomyosin ATPase assays . Troponin containing the Arg(145)-->Gly mutation inhibited force in skinned trabeculae less than did the wild-type, and had almost no inhibitory function in the in vitro motility assay . There was an enhanced inhibitory function with mixtures of 10-30% {Gly(145)}troponin I with the wild-type protein . Skinned trabeculae reconstituted with troponin I containing the Gly(203)-->Ser mutation and troponin C produced less Ca(2+)-activated force (64+/-8% of wild-type) and demonstrated lower Ca(2+) sensitivity {Delta(p)Ca(50) (log of the Ca(2+) concentration that gave 50% of maximal activation) 0.25 unit (P<0.05)} compared with wild-type troponin I, but thin filaments containing {Ser(203)}-troponin I were indistinguishable from those containing the wild-type protein in in vitro motility and ATPase assays . Thus these two mutations each result in hypertrophic cardiomyopathy, but have opposite effects on the overall contractility of the muscle in the systems we investigated, indicating either that we have not yet identified the relevant alteration in contractility for the Gly(203)->Ser mutation, or that the disease does not result directly from any particular alteration in contractility.

Biochem J, 2002 Mar 1, 362(Pt 2), 383 - 8
A novel human small subunit of calpains; Schad E et al.; Typical calpains are heterodimeric cysteine proteases which have distinct large catalytic subunits (80 kDa) but share a common small regulatory subunit (30 kDa; css1) . Here we report the identification, cloning and characterization of a novel human small subunit (css2) encoded by an intronless gene, capns2, located on chromosome 16 . This new protein displays 73% sequence identity within the Ca(2+)-binding region but lacks two oligo-Gly stretches characteristic of the N-terminal domain of the conventional small subunit . css2 appears to be the functional equivalent of the conventional small subunit in vitro in that it helps the large subunit fold into the active conformation of similar Ca(2+) sensitivity when the two proteins are co-expressed in Escherichia coli . The purification of various chimaeric rat 80 kDa-human css2 constructs, on the other hand, shows that css2 binds the large subunit much more weakly than css1 . Further, it does not undergo the autolytic conversion typical of the classical small subunit . The expression of this protein in vivo, as assessed from its appearance in expressed sequence tag clones, is rather limited, making it an example of a tissue-specific, rather than ubiquitous, small subunit.

Biochem J, 2002 Mar 1, 362(Pt 2), 375 - 82
Phosphorylation of Xenopus transcription factor IIIA by an oocyte protein kinase CK2; Westmark CJ et al.; Transcription factor IIIA (TFIIIA), isolated from the cytoplasmic 7 S ribonucleoprotein complex of Xenopus oocytes, is phosphorylated when incubated with {gamma-(32)P}ATP . This modification is due to a trace kinase activity that remains associated with the factor through several steps of purification . The kinase can use either ATP or GTP, and will phosphorylate casein and phosvitin to the exclusion of TFIIIA . The kinase is reactive with a ten-amino-acid peptide that is a specific substrate for protein kinase CK2 (CK2; formerly casein kinase II) . In addition, inhibition of phosphorylation by heparin and stimulation by spermidine indicate that the activity can be ascribed to CK2 . Phospho amino acid analysis established that serine is the sole phosphoryl acceptor in TFIIIA . There are four consensus sites for CK2 in TFIIIA; all contain serine residues at the putative site of phosphorylation . TFIIIA immunoprecipitated from oocytes, which were incubated with {(32)P}orthophosphate, is also phosphorylated exclusively on serine residues . Only the cyanogen bromide fragment, which was derived from the N-terminal end of TFIIIA, is labelled in vivo . A recognition sequence for CK2, located at Ser(16) in the beta-turn of the first zinc-finger domain, is the only protein kinase consensus sequence present in this peptide . Assays in vitro with site-specific mutants of TFIIIA established that Ser(16) is the preferred site of phosphorylation, with some secondary modification at Ser(314).

Biochem J, 2002 Mar 1, 362(Pt 2), 329 - 37
Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens; Vontas JG et al.; A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens {Vontas, Small and Hemingway (2001) Biochem . J . 357, 65-72} . To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N . lugens were partially purified by anion exchange and affinity chromatography . The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects . A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain . Northern analysis showed that nlgst1-1 was overexpressed in resistant insects . nlgst1-1 was expressed in Escherichia coli, purified and characterized . The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N . lugens GSTs . Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids . Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N . lugens.

Biochem J, 2002 Mar 1, 362(Pt 2), 317 - 27
Molecular interactions between desmosomal cadherins; Syed SE et al.; Desmocollins (Dscs) and desmogleins (Dsgs) are cell-adhesion molecules involved in the formation of desmosome cell-cell junctions and share structural similarities to classical cadherins such as E-cadherin . In order to identify and provide quantitative information on the types of protein-protein interactions displayed by the type 2 isoforms and investigate the role of Ca(2+) in this process, we have developed an Escherichia coli expression system to generate recombinant proteins containing the first two extracellular domains, namely Dsg2(1-2) and Dsc2(1-2) . Analytical ultracentrifugation, chemical cross-linking, CD, fluorescence and BIAcore have been used to provide the first direct evidence of Ca(2+) binding to desmosomal cadherins . These studies suggest that Dsc2(1-2) not only exhibits homophilic interactions in solution, but can also form heterophilic interactions with Dsg2(1-2) . The latter, on the other hand, shows much weaker homophilic association . Our results further demonstrate that heterophilic interactions are Ca(2+)-dependent, whereas the Ca(2+)-dependence of homophilic association is less clear . Our data indicate that the functional properties of Dsc2(1-2) are more similar to those of classical cadherins, consistent with the observation that Dsc shares a higher level of sequence homology with classical cadherins than does Dsg . In addition to corroborating the conclusions of previously reported transfection studies which suggest the formation of lateral heterodimers and homodimers, our results also provide direct quantitative information on the strength of these interactions which are essential for understanding the adhesion mechanism.

Biochem J, 2002 Mar 1, 362(Pt 2), 265 - 71
The transfer of transthyretin and receptor-binding properties from the plasma retinol-binding protein to the epididymal retinoic acid-binding protein; Sundaram M et al.; Members of the lipocalin superfamily share a common structural fold, but differ from each other with respect to the molecules with which they interact . They all contain eight beta-strands (A-H) that fold to form a well-defined beta-barrel, which harbours a binding pocket for hydrophobic ligands . These strands are connected by loops that vary in size and structure and make up the closed and open ends of the pocket . In addition to binding ligands, some members of the family interact with other macromolecules, the specificity of which is thought to be associated with the variable loop regions . Here, we have investigated whether the macromolecular-recognition properties can be transferred from one member of the family to another . For this, we chose the prototypical lipocalin, the plasma retinol-binding protein (RBP) and its close structural homologue the epididymal retinoic acid-binding protein (ERABP) . RBP exhibits three molecular-recognition properties: it binds to retinol, to transthyretin (TTR) and to a cell-surface receptor . ERABP binds retinoic acid, but whether it interacts with other macromolecules is not known . Here, we show that ERABP does not bind to TTR and the RBP receptor, but when the loops of RBP near the open end of the pocket (L-1, L-2 and L-3, connecting beta-strands A-B, C-D and E-F, respectively) were substituted into the corresponding regions of ERABP, the resulting chimaera acquired the ability to bind TTR and the receptor . L-2 and L-3 were found to be the major determinants of the receptor- and TTR-binding specificities respectively . Thus we demonstrate that lipocalins serve as excellent scaffolds for engineering novel biological functions.

Vopr Virusol, 2002 Jan-Feb, 47(1), 21 - 5
{Characterization of a panel of monoclonal antibodies to hepatitis C NS3 recombinant protein }; Abdulmedzhidova AG et al.; Recombinant protein rNS3 imitating helicase region (1356-1459 amino acid residues) of hepatitis C virus (HCV) was expressed in E . coli cells and used for BALB/c mice immunization . Seven hybrydoma clones producing monoclonal antibodies (MAbs) to rHS3 were obtained . All MAbs reacted in ELISA with NS3 protein from Murex anti-HCV Version III and in immunoblotting from RIBA 3 . These MAbs detect 5 individual epitopes, 4 of which were conformational and 1 discontinuous . All MAbs could compete for rNS3 binding with serum antibodies from patients with chronic hepatitis C, which suggests that these MAbs can recognize the natural HCV NS3 protein.

Vet Microbiol, 2002 Mar 22, 85(3), 275 - 84
Replicon typing of F18 fimbriae encoding plasmids of enterotoxigenic and verotoxigenic Escherichia coli strains from porcine postweaning diarrhoea and oedema disease; Fekete PZ et al.; The presence of fimbrial adhesin F18 is frequently found in enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E . coli (VTEC) strains responsible for diarrhoea and oedema disease of weaned pigs . The F18 adhesin occurs in two antigenic variants: F18ab is characteristic of VTEC while F18ac is more typical for ETEC . F18 encoding plasmids of 17 phenotypically characterized porcine E . coli isolates (10 ETEC, 6 VTEC and 1 ETEC/VTEC) were tested with a DNA probe for F18 fimbrial adhesin and with replicon probes for the RepFIa, RepFIb and for the RepFIc family of basic replicons . In all the cases, the F18 probe hybridized to only one plasmid band of size higher than 42MDa . All F18 plasmids were determined to be unireplicon plasmids belonging to the RepFIc replicon family of the F incompatibility complex . There was no difference between F18ac plasmids of ETEC and F18ab plasmids of VTEC strains in terms of replicon type or subtype . However, the size of F18ab plasmids of the VTEC strains varied between 42 and 98MDa, in contrast to F18ac plasmids of ETEC strains (constantly approximately 98MDa).

Vet Microbiol, 2002 Mar 22, 85(3), 251 - 7
Development of a sandwich ELISA and comparison with PCR for the detection of F11 and F165 fimbriated Escherichia coli isolates from septicaemic disease in farm animals; Bell CJ et al.; The P fimbriae F11 and F165 that have been demonstrated on Escherichia coli septicaemic strains in poultry and calves, respectively, possess a nearly identical major subunit that demonstrates a serological cross-reaction . A polyclonal antibody-based sandwich ELISA (sELISA) that was specific for both F11 and F165 fimbriated strains was compared with a PCR method to detect F11/F165 fimbriated strains, in a collection of E . coli strains isolated from diseased animals . Of 298 isolates tested, 36 were positive by PCR of which only 14 were sELISA positive . There were no sELISA positive but PCR negative results . The 36 PCR positive isolates comprised 11 avian strains of which 10 were sELISA positive, 20 bovine strains of which 4 were sELISA positive and 3 ovine strains, 1 porcine strain and 1 equine strain all of which were sELISA negative . The F11/F165 incidence of 10.7% in 103 poultry and 18.3% in 109 bovine isolates demonstrates a moderate level of these factors in E . coli septicaemic cases in Northern Ireland.

FEBS Lett, 2002 Feb 13, 512(1-3), 341 - 4
Insertion and glycosylation of Pf3-derived membrane proteins in microsomes; Ridder A et al.; To get insight into the insertion mechanism of small newly synthesized single-spanning membrane proteins, Pf3 coat protein mutants were constructed with potential glycosylation sites in the N-terminus . Some of these proteins, when synthesized in vitro in the presence of microsomes, became efficiently glycosylated, proving that they insert into the membrane and translocate their N-terminus to the lumenal side . Such Pf3 constructs also insert efficiently into Escherichia coli vesicles and even in pure lipid vesicles, suggesting a common mechanism, which might be spontaneous . Glycosylation was sensitive to changes in the amino acid sequence of the N-terminus, suggesting that it depends on the structure of the protein and/or its positioning with respect to the lipid-water interface.

FEBS Lett, 2002 Feb 13, 512(1-3), 323 - 8
Property comparison of recombinant amphibian and mammalian allantoicases; Vigetti D et al.; Allantoicase is an enzyme involved in uric acid degradation . Although it is commonly accepted that allantoicase is lost in mammals, birds and reptiles, we have recently identified its transcripts in mice and humans . The mouse mRNA seems capable of encoding a functional allantoicase, therefore we expressed the Xenopus and mouse allantoicases (MAlc and XAlc, respectively) in Escherichia coli and characterized the recombinant enzymes . The two recombinant allantoicases show a similar temperature and pH stability but, although XAlc and MAlc share a 54% amino acid identity, they differ in sensitivity to bivalent cations, in substrate affinity and in the level of expression in tissues (as revealed by means of Western blot analysis) . We propose that the loss of allantoicase activity in mouse is due to a low substrate affinity and to a reduced expression level of the enzyme.

FEBS Lett, 2002 Feb 13, 512(1-3), 209 - 12
Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro; Ramachandiran V et al.; The coding sequence for chloramphenicol acetyl transferase (CAT) contains several rare codons; three of them are ATA encoding isoleucine in positions 13, 84 and 119 of the amino acid sequence . Expression of CAT on Escherichia coli ribosomes in vitro results in mostly full-length product but also distinct smaller polypeptides from less than 3 kDa to over 20 kDa . As reported earlier, the smaller polypeptides are the predominant products, if translation is initiated with fluorophore-Met-tRNA(f) . All this translational pausing is eliminated when the first ATA codon is mutated to ATC, a frequently used codon for isoleucine in E . coli . Addition of large amounts of E . coli tRNA to the coupled transcription/translation reaction does not reduce the number of pause-site peptides seen in the expression of wild-type CAT . Thus we hypothesize that the mRNA structure may be an important determinant for translational pausing.

FEBS Lett, 2002 Feb 13, 512(1-3), 191 - 8
Heterologous expression and topography of the main intrinsic protein (MIP) from rat lens; Drake KD et al.; Wild type rat lens main intrinsic protein (MIP) and MIP mutated (F73I, F75L) to resemble the glycerol facilitator of Escherichia coli in the region of the NPA1 box were used to investigate the topology of MIP in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and expression in mouse erythroid leukaemia cells (MEL C88) . Differential fixation for staining was used, with paraformaldehyde for externally exposed antigenic sites, and acetone for both externally and internally exposed protein antigenic sites . Immunofluorescence using antibodies to synthetic MIP peptides showed that wild type MIP had a six transmembrane topography . The N- and C-termini were intracellular in both expression systems, and both NPA boxes were found to be extracellular . These results show that residues around the NPA1 box can influence the folding of the MIP in the membrane, and provide structural evidence for the poor water transport properties of MIP, as the NPA boxes lie outside the plane of the membrane.

FEBS Lett, 2002 Feb 13, 512(1-3), 185 - 90
Molecular simulations of the large conductance mechanosensitive (MscL) channel under mechanical loading; Bilston LE et al.; The MscL channel is a mechanosensitive channel which is gated by membrane stress or tension . Here, we describe a series of simulations which apply simulated mechanical stress to a molecular model of the MscL channel using two methods - direct force application to the transmembrane segments, and anisotropic pressure coupling . In the latter simulations, pressures less than that equivalent to a bilayer tension of 12 dyn/cm did not cause the channel to open, while pressures in excess of this value resulted in the channel opening . These results are in approximate agreement with experimental findings.

FEBS Lett, 2002 Feb 13, 512(1-3), 149 - 51
8-Chloro-dGTP, a hypochlorous acid-modified nucleotide, is hydrolyzed by hMTH1, the human MutT homolog; Fujikawa K et al.; The human mutT homolog, hMTH1, suppresses spontaneous mutations by degrading the endogeneous mutagen, 8-hydroxy-dGTP . We previously reported the broad substrate specificity of hMTH1, which also degrades the oxidatively damaged purine nucleotides, 2-hydroxy-dATP, 8-hydroxy-dATP, 2-hydroxy-ATP, and 8-hydroxy-GTP, in addition to 8-hydroxy-dGTP . In this paper, we describe the hMTH1 activity for 8-chloro-dGTP, which could be formed in inflamed tissue by the reaction of dGTP with hypochlorous acid, a product of myeloperoxidase from activated human neutrophils . The hMTH1 protein was mixed with 1-20 microM of 8-chloro-dGTP and 8-hydroxy-dGTP, and the reaction products were quantified by anion-exchange HPLC to measure the pyrophosphatase reaction rate . The kinetic parameters revealed that 8-chloro-dGTP was degraded by hMTH1 with 50% efficiency as compared with that of 8-hydroxy-dGTP . This result suggests that 8-chloro-dGTP is an intrinsic substrate for hMTH1.

Biochemistry, 2002 Feb 26, 41(8), 2876 - 83
Conformation of fork junction DNA in a complex with Escherichia coli RNA polymerase; Heyduk E et al.; Escherichia coli RNA polymerase is able to bind fork junction DNA containing a conserved -10 promoter element in a sequence-specific manner, and it is believed that polymerase-fork junction DNA interaction mimics those between the enzyme and the promoter DNA in the open complex . In this report we determined the conformation of polymerase-bound fork junction DNA in solution . A series of distances between sites in the fork junction DNA in complex with polymerase were determined using luminescence and fluorescence resonance energy transfer . A series of fork junction DNAs were prepared containing the luminescent or fluorescent donor probe at the upstream or at the downstream end of the fork DNA and acceptor probes at nine positions within the fork junction DNA . The measured distances were compared with analogous distances in a model reference DNA duplex, and the observed distance differences were used to build a model of the fork junction DNA in a complex with the polymerase . The obtained model revealed an insignificant perturbation of the duplex part of the fork DNA in a complex with the polymerase whereas a sharp kink of DNA was observed at the ds/ss DNA boundary of the fork junction DNA.

Biochemistry, 2002 Feb 26, 41(8), 2805 - 13
Chemically mediated site-specific proteolysis . Alteration of protein-protein interaction; Wang B et al.; The design and synthesis of a novel iodine-labile serine protease inhibitor was realized by the use of an ecotin analogue containing allylglycine at position 84 in lieu of methionine . Allylglycine-containing ecotins were synthesized by in vitro translation of the ecotin gene containing an engineered nonsense codon (TAG) at the positions of interest . A misacylated suppressor tRNA activated with the unnatural amino acid allylglycine was employed for the suppression of the nonsense codons in a cell-free protein biosynthesizing system, permitting the elaboration of ecotin analogues containing allyglycine at the desired sites . The derived ecotin analogues were capable of inhibiting bovine trypsin with inhibitory constants (K(i)s) comparable to that of wild-type ecotin . Iodine treatment of ecotin analogue Met84(A)Gly resulted in the deactivation of ecotin, caused by peptide backbone cleavage at its P1 reactive site . Upon iodine treatment, active trypsin could be released from the protein complex with ecotin analogue Met84(A)Gly . This constitutes a novel strategy for modulation of serine protease activity and more generally for alteration of protein-protein interaction by a simple chemical reagent.

Biochemistry, 2002 Feb 26, 41(8), 2797 - 804
Concerted and stepwise dehydration mechanisms observed in wild-type and mutated Escherichia coli dTDP-glucose 4,6-dehydratase; Hegeman AD et al.; The conversion of dTDP-glucose into dTDP-4-keto-6-deoxyglucose by Escherichia coli dTDP-glucose 4,6-dehydratase (4,6-dehydratase) takes place in the active site in three steps: dehydrogenation to dTDP-4-ketoglucose, dehydration to dTDP-4-ketoglucose-5,6-ene, and rereduction of C6 to the methyl group . The 4,6-dehydratase makes use of tightly bound NAD(+) as the coenzyme for transiently oxidizing the substrate, activating it for the dehydration step . Dehydration may occur by either of two mechanisms, enolization of the dTDP-4-ketoglucose intermediate, followed by elimination {as proposed for beta-eliminations by Gerlt, J . A., and Gassman, P . G . (1992) J . Am . Chem . Soc . 114, 5928-5934}, or a concerted 5,6-elimination of water from the intermediate . To assign one of these two mechanisms, a simultaneous kinetic characterization of glucosyl C5((1)H/(2)H) solvent hydrogen and C6((16)OH/(18)OH) solvent oxygen exchange was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . The reaction of the wild-type enzyme is shown to proceed through a concerted dehydration mechanism . Interestingly, mutation of Asp135, the acid catalyst, to Asn or Ala alters the mechanism, allowing enolization to occur to varying extents . While aspartic acid 135 is the acid catalyst for dehydration in the wild-type enzyme, the differential enolization capabilities of D135N and D135A dehydratases suggest an additional role for this residue . We postulate that the switch from a concerted to stepwise dehydration mechanism observed in the aspartic acid variants is due to the loss of control over the glucosyl C5-C6 bond rotation in the active site.

Biochemistry, 2002 Feb 26, 41(8), 2675 - 83
Time-resolved step-scan Fourier transform infrared spectroscopy of the CO adducts of bovine cytochrome c oxidase and of cytochrome bo(3) from Escherichia coli; Bailey JA et al.; We have used cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared (TR-FTIR) spectroscopies to explore the redox-linked proton-pumping mechanism of heme-copper respiratory oxidases . These techniques are used to probe the structure and dynamics of the heme a(3)-Cu(B) binuclear center and the coupled protein structures in response to the photodissociation of CO from heme Fe and its subsequent binding to and dissociation from Cu(B) . Previous cryogenic (80 K) FTIR CO photodissociation difference results were obtained for cytochrome bo(3), the ubiquinol oxidase of Escherichia coli {Puustinen, A., et al . (1997) Biochemistry 36, 13195-13200} . These data revealed a connectivity between Cu(B) and glutamic acid E286, a residue which has been implicated in proton pumping . In the current work, the same phenomenon is observed using the CO adduct of bovine cytochrome aa(3) under cryogenic conditions, showing a perturbation of the equivalent residue (E242) to that in bo(3) . Furthermore, using time-resolved (5 micros resolution) step-scan FTIR spectroscopy at room temperature, we observe the same spectroscopic perturbation in both cytochromes aa(3) and bo(3) . In addition, we observe evidence for perturbation of a second carboxylic acid side chain, at higher frequency in both enzymes at room temperature . The high-frequency feature does not appear in the cryogenic difference spectra, indicating that the perturbation is an activated process . We postulate that the high-frequency IR feature is due to the perturbation of E62 (E89 in bo(3)), a residue near the opening of the proton K-channel and required for enzyme function . The implications of these results with respect to the proton-pumping mechanism are discussed . Finally, a fast loss of over 60% of the Cu(B)-CO signal in bo(3) is observed and ascribed to one or more additional conformations of the enzyme . This fast conformer is proposed to account for the uninhibited reaction with O(2) in flow-flash experiments.

J Mol Biol, 2002 Feb 15, 316(2), 395 - 406
Hybridization of alpha class subunits generating a functional glutathione transferase A1-4 heterodimer; Gustafsson A et al.; Within the Alpha class of the mammalian glutathione transferases two variants of subunit interfaces exist . One is conserved among the A4 subunits, whereas the second one is found in all other members of the Alpha class . The ability of the two Alpha class subunit interfaces to adopt a functional heterodimeric structure has been investigated here.The heterodimer GST A1-4 was obtained by co-expression of the two human Alpha class subunits A1 and A4 in Escherichia coli . A histidine tail was added to the N terminus of the A1 subunit to facilitate the purification of the heterodimer . The heterodimer was formed in a small proportion implying that the efficiency of the hybridization between subunit A1 and A4 is less than the propensity for homodimer formation . The hybrid enzyme was stable at low temperatures, but the two subunits dissociated and reassociated into homodimers at 40 degrees C.Three different substrates were used for subunit-selective kinetic characterization of the GST A1-4 heterodimer: 1-chloro-2,4-dinitrobenzene, nonenal and Delta(5)-androstene-3,17-dione . Both subunit A1 and subunit A4 were active in GST A1-4, but the specific activities and k(cat) values were lower than the average values of the two parental isoenzymes . However, at high temperatures the subunits of the hybrid enzyme dissociated and formed homodimers, and the activities increased to expected values . Hence, the low activities of the individual subunits in the heterodimer were reversible . The non-additive kinetic properties of the subunits in the heterodimer therefore highlight the importance of fine-tuned subunit interactions for optimal catalytic efficiency of GST A1-1 and GST A4-4 .

Arch Tierernahr, 2001, 54(2), 117 - 26
The effectiveness of an Escherichia coli phytase in improving phosphorus and calcium bioavailabilities in poultry and young pigs; Igbasan FA et al.; The effectiveness of an Escherichia coli phytase in comparison with a commercially available Aspergillus phytase in improving the bioavailability of phosphorus in broilers, layers and young pigs was studied in three separate experiments . Three basal diets, marginally deficient in dietary P mainly provided as phytate, were formulated . Both phytases were added to the diets at the rate of 500 U/kg diet . The phytases significantly (P < or = 0.05) improved the availability of phytate P to broilers, layers and young pigs . Aspergillus and E . coli phytases enhanced the pre-caecal digestibility of P by 11 and 29% for broilers and 18 and 25% for layers, respectively . Total tract digestibility of P (P balance) was also enhanced but with smaller magnitude . In pigs, total tract digestibility of P was improved by 33 and 34% by Aspergillus and E . coli phytases, respectively . Under the conditions of this study, it was observed that E . coli consistently, though with small magnitude in layers and pigs, enhanced the availability of phytate P at the same range or slightly better than Aspergillus phytase . It was only in pigs that the availability of Ca was significantly (P < or = 0.05) improved by addition of both phytases . It can be concluded that E . coli phytase is highly effective in improving the bioavailability of phytate P to broilers, layers and young pigs . This seems to be based on the high proteolytic stability of the enzyme in the digestive tract, as shown recently.

J Biol Chem, 2002 May 3, 277(18), 16124 - 30 Epub 2002 Feb 15.
Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site; Iverson TM et al.; The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor . The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region . The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region . The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal (Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits . It has not been established whether both of these sites are mechanistically significant . Co-crystallization studies of the E . coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-{1-(p-chlorophenyl)ethyl} 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site . In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D) . A comparison of the Q(P) site of the E . coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved . This acidic residue, Glu-C29, in the E . coli enzyme may act as a proton shuttle from the quinol during enzyme turnover.

EMBO Rep, 2002 Mar, 3(3), 261 - 7 Epub 2002 Feb 15.
GyrI: a counter-defensive strategy against proteinaceous inhibitors of DNA gyrase; Chatterji M et al.; DNA gyrase is the target of two plasmid-encoded toxins CcdB and microcin B17, which ensure plasmid maintenance . These proteins stabilize gyrase-DNA covalent complexes leading to double-strand breaks in the genome . In contrast, the physiological role of chromosomally encoded inhibitor of DNA gyrase (GyrI) in Escherichia coli is unclear and its mechanism of inhibition has not been established . We demonstrate that the mode of inhibition of GyrI is distinct from all other gyrase inhibitors . It inhibits DNA gyrase prior to, or at the step of, binding of DNA by the enzyme . GyrI reduces intrinsic as well as toxin-stabilized gyrase-DNA covalent complexes . Furthermore, GyrI reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin, substantiating the in vitro results . Thus, GyrI is an antidote to DNA gyrase-specific proteinaceous poisons encoded by plasmid addiction systems.

Am J Respir Crit Care Med, 2002 Feb 15, 165(4), 521 - 6
Acute hyperoxic lung injury does not impede adenoviral-mediated alveolar gene transfer; Factor P et al.; The transfer of protective genes to the alveolar epithelium can attenuate lung injury if accomplished before its onset . The pathobiology of acute lung injury (ALI) includes formidable hurdles to gene transfer, including alveoli filled with fluid, inflammatory cells, and cytokines, all of which may impair gene transfer after the onset of injury . We tested the hypothesis that adenovectors could efficiently transduce injured alveoli by exposing adult, male Sprague-Dawley rats to 100% oxygen for 48 or 60 h before endotracheal instillation of either 1 x 10(9) or 4 x 10(9) plaque-forming units of an adenovirus that expresses an Escherichia coli lac Z gene (adbeta-gal) in a surfactant-based vehicle (Survanta) . X-gal staining 72 h postinfection revealed transgene expression in all segments of room air control and hyperoxic lungs infected with either dose of adbeta-gal . Net transgene expression in hyperoxic lungs was not different from room air controls despite the presence of pulmonary edema and severe histologic injury . These findings show that adenovectors can efficiently transduce the alveoli of acutely injured, edematous lungs . The data indicate that the pathophysiologic processes of ALI do not impair adenoviral-mediated alveolar gene transfer and provide support for the development of gene therapies for ALI.

Am J Respir Crit Care Med, 2002 Feb 15, 165(4), 463 - 9
Intratracheal endotoxin causes systemic inflammation in ventilated preterm lambs; Kramer BW et al.; Intratracheal endotoxin causes acute inflammation in the adult lung, and injurious styles of mechanical ventilation can result in systemic inflammation derived from the lungs . We asked how ventilated premature and near-term lungs responded to intratracheal endotoxin and if systemic inflammation occurred . Lambs delivered at 130 d gestational age (GA) were treated with surfactant or surfactant plus endotoxin (0.1 mg/kg or 10 mg/kg) (Escherichia coli, serotype O55:B5) and were ventilated for 6 h . Both endotoxin doses resulted in impaired gas exchange and systemic inflammation in the preterm lambs . Lambs at 141 d GA (term 146 d) were given either 10 mg/kg intratracheal endotoxin, 10 mg/kg endotoxin plus high tidal volume ventilation for the first 30 min of life, or 5 microg/kg endotoxin given intravenously . Endotoxin alone (10 mg/kg) caused lung inflammation but no systemic effects after 6 h of ventilation . Lambs given 10 mg/kg endotoxin plus high tidal volume ventilation or 5 microg/kg endotoxin intravenously had decreased gas exchange and systemic inflammation . Endotoxin was detected in the plasma of lambs at 130 d GA but not at 141 d GA . Inflammation in the lungs was more severe in preterm animals . Mechanical ventilation of the endotoxin-exposed preterm lung resulted in systemic effects at a low endotoxin dose and without high tidal volume ventilation.

Brain Res Bull, 2002 Jan 15, 57(2), 179 - 85
Effects of selective cyclooxygenase enzyme inhibitors on lipopolysaccharide-induced dual thermoregulatory changes in rats; Dogan MD et al.; The effects of selective cyclooxygenase-1 and cyclooxygenase-2 inhibitors (valeryl salicylate and SC-58236, respectively) on Escherichia coli O111:B4 lipopolysaccharide (LPS)-induced dual thermoregulatory changes and serum tumor necrosis factor-alpha elevation were investigated in rats . LPS (50 microg/kg, intraperitoneal) produced an initial hypothermia that was then followed by fever . Serum tumor necrosis factor-alpha levels elevated at the initial phase of hypothermia . Valeryl salicylate injections (20, 40, and 80 mg/kg, subcutaneous {s.c.}) completely inhibited hypothermia without any effect on the elevated serum tumor necrosis factor-alpha levels and on the subsequent fever . On the other hand, SC-58236 injections (10, 20, and 40 mg/kg, s.c.) only partially abolished the hypothermia . SC-58236 had no effect on the initiation of fever, however completely inhibited the maintenance of fever . The serum tumor necrosis factor-alpha elevation was not reduced by SC-58236 treatment . The combination of valeryl salicylate and SC-58236 also failed to inhibit the initiation of fever . These findings suggest that cycloxygenase-1 may have a predominant role for the development of LPS-induced hypothermia, but cyclooxygenase-1 does not seem to be involved in the mediation of LPS-induced fever . Meanwhile, cyclooxgenase-2 may be critical for the late phase rather than the initiation of the fever response in rats.

Mol Biochem Parasitol, 2002 Mar, 120(1), 61 - 72
Novel antifolate resistant mutations of Plasmodium falciparum dihydrofolate reductase selected in Escherichia coli; Chusacultanachai S et al.; A simple and effective system has been developed from which a number of Plasmodium falciparum dihydrofolate reductase (pfDHFR) mutants conferring resistance to antifolates were randomly generated and characterized . The system exploited error-prone PCR to generate random mutations in the pfDHFR . Using the synthetic gene encoding for wild-type and quadruple mutant (N51I+C59R+S108N+I164L) pfDHFRs as templates, mutants resistant to pyrimethamine (Pyr), m-Cl analogue of Pyr (SO3) and WR99210 were selected by bacterial complementation system in which the endogenous DHFR activity of bacterial host cells, but not of Plasmodium, is selectively inhibited by trimethoprim (Tmp) . Mutants conferring resistance to antimalarial antifolates were selected under the condition that inhibited the growth of the wild-type pfDHFR . All obtained Pyr resistant mutants possessed S108 mutation, in combination with common mutations of N51I, C59R and I164L previously found in the field . New Pyr resistant mutants with novel mutations (K27T, N121D, N144K and V213E) not found in the field were also identified . Exposure of the randomly mutated pfDHFR libraries to WR99210 or SO3 resulted in selection of novel single and multiple mutants including D54N, F58L and a combination of C50R, K181R, T219P and K227E, which exhibited 2- to over 2000-fold increase in resistance against antifolates . Kinetic analysis of these mutants suggested that apart from the active site residues that are crucial for DHFR activity, residues remote from the binding pocket also play essential roles in substrate and inhibitor binding.

J Virol Methods, 2002 Mar, 101(1-2), 95 - 103
Quantitation of cucurbit yellow stunting disorder virus in Bemisia tabaci (Genn.) using digoxigenin-labelled hybridisation probes; Ruiz L et al.; A cost-efficient hybridisation assay was developed to estimate the amount of cucurbit yellow stunting disorder virus (CYSDV) in Bemisia tabaci (Gennadius) whiteflies infesting protected cucumber crops . cDNA from the coat protein (cp) gene and the hsp70 homologue protein gene from CYSDV were obtained by reverse transcriptase-PCR from viruliferous whiteflies and cloned into plasmids . Digoxigenin (DIG)-labelled cDNA probes reacted with extracts from these whiteflies applied on nylon membranes . Precision and linear ranges were established in a hybridisation analysis using known concentrations of unlabelled homologue cDNA . Extracts from non-viruliferous B . tabaci showed a concentration-dependent effect on the assay with cp-specific probes but not with hsp70-specific probes . The hsp70 probe was used to evaluate natural B . tabaci populations in commercial cucumber crops, and the estimated amounts of CYSDV per whitefly were found ranging from 5.6 fg to approximately 2.5 pg of corresponding hsp70-cDNA.

Plant J, 2001 Dec, 28(5), 555 - 67
Novel ABA- and dehydration-inducible aldehyde dehydrogenase genes isolated from the resurrection plant Craterostigma plantagineum and Arabidopsis thaliana; Kirch HH et al.; In order to identify genes that are critical for the ABA-dependent stress response in the resurrection plant Craterostigma plantagineum, a gene was isolated with homology to class 3 variable substrate aldehyde dehydrogenases (ALDH) . The C . plantagineum gene Cp-ALDH constitutes a novel class of plant ALDHs . In a search for corresponding genes from Arabidopsis thaliana, Ath-ALDH3 and Ath-ALDH4 were isolated, showing 70% and 80% similarity to Cp-ALDH . Phylogenetically, the Cp- and Ath-ALDH3 and -ALDH4 proteins are closely related to aldehyde dehydrogenases from bacteria and mammalian species and are separated from known plant ALDHs and betaine-aldehyde dehydrogenases (BADH) . Cp-ALDH transcript and polypeptide are up-regulated in vegetative tissues and callus in response to dehydration or ABA-treatment . Ath-ALDH3 expression was induced in response to dehydration and ABA treatment, while Ath-ALDH4 is constitutively expressed at a low level . Recombinant Cp-ALDH protein oxidizes nonanal, propionaldehyde and acetaldehyde, with Km values of 2.2 microm, 0.27 mm and 3.23 mm, respectively, in an NAD-dependent manner . Immunogold electron microscopy shows that Cp-ALDH is localized in plastids.

Mol Microbiol, 2002 Jan, 43(1), 217 - 26
Post-transcriptional enhancement of Escherichia coli bgl operon silencing by limitation of BglG-mediated antitermination at low transcription rates; Dole S et al.; The silent bgl operon of Escherichia coli is activated by spontaneous mutations that derepress its promoter . In addition, expression depends on specific transcriptional antitermination within the operon by the antiterminator protein BglG . Here, we show that BglG-mediated antitermination limits expression of the bgl operon when the cellular transcription rate is low . The expression levels of chromosomally encoded activated bgl operon alleles are low but increase significantly when BglG protein is provided in trans or when the expression is rendered independent of BglG-mediated antitermination by mutation of the terminator . Plasmid-encoded activated bgl operon alleles are expressed at high levels . Moreover, a moderate (threefold) further increase in the transcription rate of chromosomally encoded activated bgl operon alleles in an rpoS mutant can result in high (up to 50-fold increased) expression levels . These data show that the expression of the bgl operon does not correlate linearly with its cellular transcription rate . Moderate differences in the transcription initiation rate are amplified post-transcriptionally into large changes in the expression level of the operon by the requirement of a threshold for BglG-mediated antitermination . Implications for bgl operon regulation by global regulators H-NS, RpoS and others are discussed.

Mol Microbiol, 2002 Jan, 43(1), 195 - 205
Mutational analysis of F-pilin reveals domains for pilus assembly, phage infection and DNA transfer; Manchak J et al.; The F-pilus has been implicated in recipient cell recognition during the establishment of a stable mating pair before conjugation as well as forming part of the conjugative pore for DNA transfer . The F-pilus is the site of attachment of the filamentous phages (M13, f1 and fd), which attach to the F-pilus tip, and the RNA phages, R17 and Qbeta, which attach to different sites exposed on the sides of the pilus . R17 has been shown to undergo eclipse, or capsid release, outside the cell on pili attached to cells . New and existing mutants of traA combined with natural variants of F-pilin were assayed for pilin stability and processing, pilus elongation, transfer, phage sensitivity and R17 eclipse . Phenotypes of these mutants indicated that the F-pilin subunit contains specific regions that can be associated with pilus assembly, phage sensitivity and DNA transport . Mutations involving lysines and phenylalanines within residues 45-60 suggest that these residues might participate in transmitting a signal down the length of the pilus that initiates DNA transfer or R17 eclipse.

Mol Microbiol, 2002 Jan, 43(1), 173 - 86
Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue; Kirby C et al.; Plasmids have been described in almost all bacterial species analysed and have proven to be essential genetic tools . In many bacteria these extrachromosomal DNAs are cryptic with no known markers or function, which makes their characterization and genetic exploitation extremely difficult . Here we describe a system that will allow the rescue of any circular DNA (plasmid or phage) using an in vitro transposition system to deliver both a selectable marker (kanamycin) and an Escherichia coli plasmid origin of replication . In this study, we demonstrate the rescue of four cryptic plasmids from the opportunistic pathogen Mycobacterium avium . To evaluate the host range of the rescued plasmids, we have examined their ability to be propagated in Mycobacterium smegmatis and Mycobacterium bovis BCG, and their compatibility with other mycobacterial plasmids . In addition, we use a library of transposon insertions to sequence one plasmid, pVT2, and to begin a genetic analysis of plasmid genes . Using this approach, we identified a putative conjugative relaxase, suggesting this myco-bacterial plasmid is transferable, and three genes required for plasmid establishment and replication.

Mol Microbiol, 2002 Jan, 43(1), 159 - 71
RNase E levels in Escherichia coli are controlled by a complex regulatory system that involves transcription of the rne gene from three promoters; Ow MC et al.; The rne gene of Escherichia coli encodes RNase E, an essential endoribonuclease that is involved in both mRNA decay and rRNA processing . Here we present evidence that the gene is transcribed from three promoters: p1, p2 and p3 . The p2 and p3 promoters map 34 and 145 nt upstream from the previously characterized rne promoter, p1, generating unusually long 5' UTRs of 395 and 506 nt respectively . Based on promoter-lacZ transcriptional fusions, p1 is a more efficient promoter than either p2 or p3 . Low copy number or single copy number vectors carrying rne transcribed from either p1, p2 or p3 alone complement the rne 1018::bla deletion mutation at 30 degrees C, 37 degrees C and 44 degrees C . However, normal autoregulation requires the presence of all three promoters . A comparison among intracellular levels of RNase E, the half-lives of the rpsO, rpsT and rne mRNAs, and growth rates, indicates that the cell contains a considerable excess of RNase E protein . In addition, when the rne transcript is stabilized at low RNase E levels, it is not efficiently translated.

Mol Microbiol, 2002 Jan, 43(1), 107 - 17
The universal stress protein paralogues of Escherichia coli are co-ordinately regulated and co-operate in the defence against DNA damage; Gustavsson N et al.; We have cloned, characterized and inactivated genes encoding putative UspA paralogues in Escherichia coli . The yecG (uspC), yiiT (uspD) and ydaA (uspE) genes were demonstrated to encode protein pro-ducts and these were mapped to spots in the E . coli proteomic database . Expression analysis using chromosomal transcriptional lacZ fusions and two-dimensional gels revealed that all usp genes analysed are regulated in a similar fashion . Thus, uspC, D and E are all induced in stationary phase and by a variety of stresses causing growth arrest of cells . Induction is independent of rpoS but is abolished in a deltarelA deltaspoT (ppGpp0) background and rescued by suppressor mutations rendering the beta-subunit of RNA polymerase to behave like a stringent polymerase . Ectopic elevation of ppGpp levels in growing cells, by overproducing the RelA protein, triggered the induction of all usp genes . The expression of all usp genes was also elevated by a mutation in the ftsK cell division gene, and this super-induction could be suppressed by inactivating recA indicating that the usp paralogues are involved in the management of DNA . Indeed, uspC, uspD and uspE deletion mutants were all found to be sensitive to UV exposure . Overexpression of UspD could compensate for the lack of a chromosomal uspD gene but not a uspA gene . Similarly, UspA overproduction could only compensate for the lack of chromosomal uspA . Moreover, combination of usp mutations had no additive effect on UV sensitivity indicating that they are all co-operating and required in the same pathway, which could explain the co-ordinated regulation of the genes.

Lett Appl Microbiol, 2002, 34(2), 130 - 3
In-well cell lysis technique reveals two new megaplasmids of 103.0 and 212.6 MDa in the multiple plasmid-containing strain V517 of Escherichia coli; Pedraza RO et al.; AIMS: Identification of two new plasmids in the multiple plasmid-containing strain V517 of Escherichia coli . METHOD AND RESULTS: By using an in-well mild cell lysis technique suitable for megaplasmids observation, two plasmids of 103.0 and 212.6 MDa were detected in the multiplasmid-containing E . coli V517 . CONCLUSIONS: The two new megaplasmids that were completely overlooked when standard disruptive procedures were used, can now be added to the list of eight plasmids with molecular size from 1.36 to 35.84 MDa reported earlier . SIGNIFICANCE AND IMPACT OF THE STUDY: This finding allows to use the strain V517 not only as a size reference of small and moderately large plasmids but as a size reference of megaplasmids as well.

J Appl Microbiol, 2002, 92(2), 261 - 8
Characterization of a cationic surfactant-resistant mutant isolated spontaneously from Escherichia coli; Ishikawa S et al.; AIMS: In order to investigate the mechanism of bacterial resistance to surfactants, a spontaneous mutant of Escherichia coli, OW66, resistant to a cationic surfactant cetyltrimethylammonium bromide (CTAB), was isolated and its physiological properties analysed . METHODS AND RESULTS: Strain OW66 grew in M9 medium containing CTAB at 45 micromol l(-1), whereas its parent strain, OW6, did not, even at 15 micromol l(-1) . The mutant was also resistant to some other surfactants, antibiotics, heavy metals, organic solvents and oxidants examined . To determine the differences in physiology between strains OW66 and OW6, the compositions of their cell surface structures were analysed . In strain OW66, the relative content of OmpC in particular was higher than that of OmpF, whereas a reverse situation was seen in OW6 strain . The lipopolysaccharide (LPS) profile was different between these strains, and altered LPS in strain OW66 was suggested to be involved in the resistance to CTAB . CONCLUSIONS: A CTAB-resistant E . coli isolate possesses an altered outer membrane . SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment with a relatively low concentration of CTAB was found to introduce multi-drug resistance into bacterial cells . This acquired resistance should be taken into account with the frequent use of surfactants in industries and various environments.

Chem Res Toxicol, 2002 Feb, 15(2), 165 - 9
Mutagenesis by O(6)-{4-oxo-4-(3-pyridyl)butyl}guanine in Escherichia coli and human cells; Pauly GT et al.; Site-specific mutagenesis by O(6)-{4-oxo-4-(3-pyridyl)butyl}guanine (O(6)-pobGua), a product of DNA pyridyloxobutylation by metabolites of the tobacco-specific nitrosamines N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was studied in Escherichia coli strain DH10B and human kidney cells (293) when the modified base was incorporated in either a double-stranded or a gapped shuttle vector . In the repair-competent E . coli strain, less than 3% of the colonies produced by double-stranded vectors harboring the modified base were mutant whereas 96% were mutant when DH10B cells were transformed with modified gapped vectors . By contrast, transformation of DH10B cells with plasmids derived from O(6)-pobGua-containing double-stranded and gapped vectors previously replicated in 293 cells produced 7 and 16% mutant colonies, respectively . These percentages increased to 42 and 82%, respectively, when the 293 cells were pretreated with O(6)-benzylguanine to inactivate the O(6)-alkylguanine-DNA alkyltransferase protein . These findings confirm that the adduct is readily repaired by the human O(6)-alkylguanine-DNA alkyltransferase in both double-stranded and gapped vectors and suggest that it is also highly mutagenic in both human cells and E . coli . In the E . coli strain, the adduct produced exclusively G --> A transition mutations although in human 293 cells it also produced G --> T transversions and more complex mutations in addition to G --> A transitions . These data suggest that O(6)-{4-oxo-4-(3-pyridyl)butyl}guanine can contribute significantly to the mutagenic risk posed by exposure to both NNN and NNK in tobacco smoke.

Biotechniques, 2002 Feb, 32(2), 422 - 4, 426, 428-30
Epitope tagging genomic DNA using a CD-tagging Tn10 minitransposon; Telmer CA et al.; Here, we describe an efficient system for epitope tagging cloned genes by CD tagging using a mini-Tn10 transposon delivery vector . The system was tested against a lambdaFIX genomic clone of the human nucleolin gene . Transfection of HeLa cells with the tagged gene led to the expression of both the appropriately spliced tagged transcript and the appropriately localized tagged protein.

Braz J Med Biol Res, 2002 Feb, 35(2), 181 - 90
Effects of lipopolysaccharide on low- and high-density cultured rabbit vascular smooth muscle cells: differential modulation of nitric oxide release, ERK1/ERK2 MAP kinase activity, protein tyrosine phosphatase activity, and DNA synthesis; Barbosa de Oliveira LC et al.; Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation . In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E . coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis . Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin . NO release was increased by 86.6% (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls . Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC . DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger . Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities . Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation . Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells . LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases.

Protein Sci, 2002 Mar, 11(3), 680 - 7
The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain; Frank BS et al.; Small autonomously folding proteins are of interest as model systems to study protein folding, as the same molecule can be used for both experimental and computational approaches . The question remains as to how well these minimized peptide model systems represent larger native proteins . For example, is the core of a minimized protein tolerant to mutation like larger proteins are? Also, do minimized proteins use special strategies for specifying and stabilizing their folded structure? Here we examine these questions in the 35-residue autonomously folding villin headpiece subdomain (VHP subdomain) . Specifically, we focus on a cluster of three conserved phenylalanine (F) residues F47, F51, and F58, that form most of the hydrophobic core . These three residues are oriented such that they may provide stabilizing aromatic-aromatic interactions that could be critical for specifying the fold . Circular dichroism and 1D-NMR spectroscopy show that point mutations that individually replace any of these three residues with leucine were destabilized, but retained the native VHP subdomain fold . In pair-wise replacements, the double mutant that retains F58 can adopt the native fold, while the two double mutants that lack F58 cannot . The folding of the double mutant that retains F58 demonstrates that aromatic-aromatic interactions within the aromatic cluster are not essential for specifying the VHP subdomain fold . The ability of the VHP subdomain to tolerate mutations within its hydrophobic core indicates that the information specifying the three dimensional structure is distributed throughout the sequence, as observed in larger proteins . Thus, the VHP subdomain is a legitimate model for larger, native proteins.

Protein Sci, 2002 Mar, 11(3), 659 - 68
The distinctive functions of the two structural calcium atoms in bovine pancreatic deoxyribonuclease; Chen CY et al.; The two amino acid residues, Asp 99 and Asp 201, involved in the coordination of the two calcium atoms in the X-ray structure of bovine pancreatic (bp) DNase, were individually changed by site-directed mutagenesis . The two altered proteins, brDNase(D99A) and brDNase(D201A) were expressed in Escherichia coli and purified by anion exchange chromatography . Equilibrium dialysis showed that mutation destroyed one Ca(2+)-binding site each in brDNase(D99A) and brDNase(D201A) . Compared with bpDNase, the Vmax value for brDNase(D99A) remained unchanged and that for brDNase(D201A) was decreased, whereas the K(m) values for the two variants were increased two- to threefold when the DNA hydrolytic hyperchromicity assay was used . Like bpDNase, brDNase(D99A) was able to make double scission on duplex DNA with Mg(2+) plus Ca(2+) and was effectively protected by Ca(2+) from the trypsin inactivation . But under the same conditions, brDNase(D201A) lost the double-scission ability and was not protected by Ca(2+) . Nevertheless, the two variant proteins retained the characteristics of the Ca(2+)-induced conformational changes and the Ca(2+) protection against the beta-mercaptoethanol disruption of the essential disulfide bond, suggesting that other weaker Ca(2+)-binding sites not found in the X-ray structure were responsible for these properties . Therefore, the two structural calcium atoms are not for maintaining the overall conformation of the active DNase, as it has been indicated in the X-ray analysis, but rather play the role in the fine-tuning of the DNase activity.

Protein Sci, 2002 Mar, 11(3), 601 - 13
Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein-protein cooperative interactions; Mou TC et al.; The gene 5 protein (g5p) of the Ff virus contains five Tyr, individual mutants of which have now all been characterized by CD spectroscopy . The protein has a dominant tyrosyl 229-nm L(a) CD band that is shown to be approximately the sum of the five individual Tyr contributions . Tyr41 is particularly important in contributing to the high cooperativity with which the g5p binds to ssDNA, and Y41F and Y41H mutants are known to differ in dimer-dimer packing interactions in crystal structures . We compared the solution structures and binding properties of the Y41F and Y41H mutants using CD spectroscopy . Secondary structures of the mutants were similar by CD analyses and close to those derived from the crystal structures . However, there were significant differences in the binding properties of the two mutant proteins . The Y41H protein had an especially low binding affinity and perturbed the spectrum of poly{d(A)} in 2 mM Na(+) much less than did Y41F and the wild-type gene 5 proteins . Moreover, a change in the Tyr 229 nm band, assigned to the perturbation of Tyr34 at the dimer-dimer interface, was absent in titrations with the Y41H mutant under low salt conditions . In contrast, titrations with the Y41H mutant in 50 mM Na(+) exhibited typical CD changes of both the nucleic acid and the Tyr 229-nm band . Thus, protein-protein and g5p-ssDNA interactions appeared to be mutually influenced by ionic strength, indicative of correlated changes in the ssDNA binding and cooperativity loops of the protein or of indirect structural constraints.

Protein Sci, 2002 Mar, 11(3), 546 - 57
Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1; Ma C et al.; Vpu is an 81-residue accessory protein of HIV-1 . Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein . The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain . Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu . To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2-81) . Vpu(2-37) encompasses the N-terminal transmembrane alpha-helix; Vpu(2-51) spans the N-terminal transmembrane helix and the first cytoplasmic alpha-helix; Vpu(28-81) includes the entire cytoplasmic domain containing the two C-terminal amphipathic alpha-helices without the transmembrane helix . Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E . coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy . The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy . Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides . The biological activities of the constructs of Vpu were evaluated . The ion channel activity is confined to the transmembrane alpha-helix . The C-terminal alpha-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation.

Protein Sci, 2002 Mar, 11(3), 522 - 8
Propagation of a single destabilizing mutation throughout the Escherichia coli ribonuclease HI native state; Spudich G et al.; A point mutation (I53A) in the core of Escherichia coli RNase H* is known to destabilize both the native conformation (DeltaG(UN)) and the kinetic intermediate (DeltaG(UI)) by 2 kcal/mole . Here, we have used native-state hydrogen deuterium exchange to ask how this destabilization is propagated throughout the molecule . Stability parameters were obtained for individual residues in I53A and compared with those from the wild-type protein . A destabilization of 2 kcal/mole was observed in residues in the core but was not detected in the periphery of the molecule . These results are consistent with the localized destabilization of the core observed in the early intermediate of the kinetic folding pathway, supporting the resemblance of this kinetic intermediate to the partially unfolded form detected in the native state at equilibrium . A thermodynamic cycle also shows no interaction between Ile 53 and a residue in the periphery . There is, however, an increase in the number of denaturant-independent exchange events in the periphery of I53A, showing that effects of the point mutation are communicated to regions outside the core, although perhaps not through changes in stability . In sum, this work shows that localized regions within a protein can be destabilized independently . Furthermore, it implies a correspondence between the kinetic intermediate and the equilibrium PUF, as the magnitude and localization of the destabilization are the same in both.

J Exp Bot, 2002 Mar, 53(368), 565 - 7
PARF-1: an Arabidopsis thaliana FYVE-domain protein displaying a novel eukaryotic domain structure and phosphoinositide affinity; Heras B et al.; A full-length cDNA encoding a novel protein named PARF-1 was isolated from Arabidopsis thaliana . PARF-1 is the first eukaryotic protein to be identified that displays a domain structure which includes a FYVE-finger domain, a Pleckstrin Homology (PH) domain, as well as multiple Regulator of Chromosome Condensation-1 (RCC1) repeats . Northern blot analysis revealed that PARF-1 mRNA is present at high levels in flowers, but only at very low levels in other tissues . Recombinant PARF-1 fusion proteins expressed in E . coli were found to display unique binding specificities for monophosphorylated phosphoinositide lipids . The unusual domain structure of PARF-1 combined with its phosphoinositide specificity suggests that it may fulfil unexpected functions in higher plants.

J Exp Bot, 2002 Mar, 53(368), 407 - 13
Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?
Sugawara H, Shibuya K, Yoshioka T, Hashiba T, Satoh S.
Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene . CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals . The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers . In the present study, DC-CPIn cDNA was cloned and expressed in E . coli . The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain . Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence . Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.

J Biol Chem, 2002 Apr 19, 277(16), 14172 - 6 Epub 2002 Feb 14.
Self-assembly of human MxA GTPase into highly ordered dynamin-like oligomers; Kochs G et al.; Human MxA protein is a member of the interferon-induced Mx protein family and an important component of the innate host defense against RNA viruses . The Mx family belongs to a superfamily of large GTPases that also includes the dynamins and the interferon-regulated guanylate-binding proteins . A common feature of these large GTPases is their ability to form high molecular weight oligomers . Here we determined the capacity of MxA to self-assemble into homo-oligomers in vitro . We show that recombinant MxA protein assembles into long filamentous structures with a diameter of about 20 nm at physiological salt concentration as demonstrated by sedimentation assays and electron microscopy . In the presence of guanosine nucleotides the filaments rearranged into rings and more compact helical arrays . Our data indicate that binding and hydrolysis of GTP induce conformational changes in MxA that may be essential for viral target recognition and antiviral activity.

J Biol Chem, 2002 May 3, 277(18), 15286 - 92 Epub 2002 Feb 14.
Guanine nucleotide exchange factor-like factor (Rlf) induces gene expression and potentiates alpha 1-adrenergic receptor-induced transcriptional responses in neonatal rat ventricular myocytes; Post GR et al.; Expression of constitutively active Ras (V12Ras) in cultured neonatal rat ventricular myocytes or targeted cardiac expression of V12Ras in transgenic mice induces myocardial cell growth and expression of genes that are markers of cardiac hypertrophy including atrial natriuretic factor (ANF) and myosin light chain-2 . However, the signaling pathways that modulate the effects of Ras on acquisition of the various features of cardiac hypertrophy are not known . We identified the Ral guanine nucleotide exchange factor-like factor (Rlf) in a yeast two-hybrid screen of human heart cDNA library using Ras as bait, suggesting that Ras signaling in the heart may involve Rlf . We demonstrate here that Rlf is expressed in human heart . Expression of wild type Rlf or Rlf-CAAX, a membrane-targeted mutant of Rlf, transactivated ANF and myosin light chain-2 promoters but did not activate canonical cAMP responsive elements or phorbol ester responsive elements, suggesting that Rlf expression does not lead to a generalized increase in transcription . Transfection of mutant ANF promoter-reporter gene constructs demonstrated that the proximal serum response element is both necessary and sufficient for Rlf-inducible ANF expression . Rlf-induced ANF promoter activation required Ral and Cdc42 but not RhoA, Rac1, ERK, or p38 kinase activation . In addition, Rlf potentiated alpha(1)-adrenergic receptor (alpha(1)-AR)-induced ANF expression . Prolonged activation of the alpha(1)-AR increases RalGTP levels in neonatal rat ventricular myocytes, further emphasizing a role for Ral guanine nucleotide exchange factors in alpha(1)-AR signaling . Overall, this study supports the concept that Rlf and Ral are important previously unrecognized signaling components that regulate transcriptional responses in myocardial cells.

EMBO J, 2002 Feb 15, 21(4), 789 - 800
Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate; Zharkov DO et al.; Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA . Nei shares with formamidopyrimidine-DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential beta-elimination steps . However, Nei differs significantly from Fpg in substrate specificity . We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 A resolution . The crosslink is derived from a Schiff base intermediate that precedes beta-elimination and is stabilized by reduction with NaBH(4) . Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains . DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site . Amino acids involved in substrate binding and catalysis are identified . Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base . Based on structural features of the complex and site-directed mutagenesis studies, we propose a catalytic mechanism for Nei.

EMBO J, 2002 Feb 15, 21(4), 769 - 78
The hemK gene in Escherichia coli encodes the N(5)-glutamine methyltransferase that modifies peptide release factors; Heurgue-Hamard V et al.; Class 1 peptide release factors (RFs) in Escherichia coli are N(5)-methylated on the glutamine residue of the universally conserved GGQ motif . One other protein alone has been shown to contain N(5)-methylglutamine: E.coli ribosomal protein L3 . We identify the L3 methyltransferase as YfcB and show that it methylates ribosomes from a yfcB strain in vitro, but not RF1 or RF2 . HemK, a close orthologue of YfcB, is shown to methylate RF1 and RF2 in vitro . hemK is immediately downstream of and co-expressed with prfA . Its deletion in E.coli K12 leads to very poor growth on rich media and abolishes methylation of RF1 . The activity of unmethylated RF2 from K12 strains is extremely low due to the cumulative effects of threonine at position 246, in place of alanine or serine present in all other bacterial RFs, and the lack of N(5)-methylation of Gln252 . Fast-growing spontaneous revertants in hemK K12 strains contain the mutations Thr246Ala or Thr246Ser in RF2 . HemK and YfcB are the first identified methyltransferases modifying glutamine, and are widely distributed in nature.

EMBO J, 2002 Feb 15, 21(4), 715 - 24
Transcriptional regulation of fis operon involves a module of multiple coupled promoters; Nasser W et al.; The transcription of the Escherichia coli fis gene is strongly activated during the outgrowth of cells from stationary phase . The high activity of the promoter of the fis operon requires the transcription factor IHF . Previously, we identified a divergent promoter, div, located upstream of the fis promoter . In this study we demonstrate that at least two additional promoters, designated fis P2 and fis P3, are located in the control region of the fis operon . The fis P2 and div promoters overlap completely, whereas fis P3 and div P are arranged as face-to-face divergent promoters . We show that the div and the tandem fis promoters counterbalance each other, such that their activity is kept on a lower than potentially attainable level . Furthermore, we demonstrate an unusual activation mechanism by IHF, involving a coordinated shift in the balance of promoter activities . We infer that these coupled promoters represent a regulatory module and propose a novel "dynamic balance" mechanism involved in the transcriptional control of the fis operon.

EMBO J, 2002 Feb 15, 21(4), 685 - 93
Unique and overlapping roles for ZipA and FtsA in septal ring assembly in Escherichia coli; Pichoff S et al.; ZipA and FtsA are essential division proteins in Escherichia coli that are recruited to the division site by interaction with FtsZ . Utilizing a newly isolated temperature-sensitive mutation in zipA we have more fully characterized the role of ZipA . We confirmed that ZipA is not required for Z ring formation; however, we found that ZipA, like FtsA, is required for recruitment of FtsK and therefore all downstream division proteins . In the absence of FtsA or ZipA Z rings formed; however, in the absence of both, new Z rings were unable to form and preformed Z rings were destabilized . Consistent with this, we found that an FtsZ mutant unable to interact with both ZipA and FtsA was unable to assemble into Z rings . These results demonstrate that ZipA and FtsA are both required for recruitment of additional division proteins to the Z ring, but either one is capable of supporting formation and stabilization of Z rings.

EMBO J, 2002 Feb 15, 21(4), 536 - 45
Membrane sequestration of the signal transduction protein GlnK by the ammonium transporter AmtB; Coutts G et al.; The Amt proteins are ammonium transporters that are conserved throughout all domains of life, being found in bacteria, archaea and eukarya . In bacteria and archaea, the Amt structural genes (amtB) are invariably linked to glnK, which encodes a member of the P(II) signal transduction protein family, proteins that regulate enzyme activity and gene expression in response to the intracellular nitrogen status . We have now shown that in Escherichia coli and Azotobacter vinelandii, GlnK binds to the membrane in an AmtB-dependent manner and that GlnK acts as a negative regulator of the transport activity of AmtB . Membrane binding is dependent on the uridylylation state of GlnK and is modulated according to the cellular nitrogen status such that it is maximal in nitrogen-sufficient situations . The membrane sequestration of GlnK by AmtB represents a novel form of signal transduction in which an integral membrane transport protein functions to link the extracellular ammonium concentration to the intracellular responses to nitrogen status . The results also offer new insights into the evolution of P(II) proteins and a rationale for their trigonal symmetry.

Plant J, 2002 Feb, 29(4), 417 - 26
Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana; Taji T et al.; Raffinose family oligosaccharides (RFO) accumulating during seed development are thought to play a role in the desiccation tolerance of seeds . However, the functions of RFO in desiccation tolerance have not been elucidated . Here we examine the functions of RFO in Arabidopsis thaliana plants under drought- and cold-stress conditions, based on the analyses of function and expression of genes involved in RFO biosynthesis . Sugar analysis showed that drought-, high salinity- and cold-treated Arabidopsis plants accumulate a large amount of raffinose and galactinol, but not stachyose . Raffinose and galactinol were not detected in unstressed plants . This suggests that raffinose and galactinol are involved in tolerance to drought, high salinity and cold stresses . Galactinol synthase (GolS) catalyses the first step in the biosynthesis of RFO from UDP-galactose . We identified three stress-responsive GolS genes (AtGolS1, 2 and 3) among seven Arabidopsis GolS genes . AtGolS1 and 2 were induced by drought and high-salinity stresses, but not by cold stress . By contrast, AtGolS3 was induced by cold stress but not by drought or salt stress . All the GST fusion proteins of GST-AtGolS1, 2 and 3 expressed in Escherichia coli had galactinol synthase activities . Overexpression of AtGolS2 in transgenic Arabidopsis caused an increase in endogenous galactinol and raffinose, and showed reduced transpiration from leaves to improve drought tolerance . These results show that stress-inducible galactinol synthase plays a key role in the accumulation of galactinol and raffinose under abiotic stress conditions, and that galactinol and raffinose may function as osmoprotectants in drought-stress tolerance of plants.

Eur J Biochem, 2002 Feb, 269(3), 1022 - 32
Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii; Happe T et al.; Chlamydomonas reinhardtii, a unicellular green alga, contains a hydrogenase enzyme, which is induced by anaerobic adaptation of the cells . Using the suppression subtractive hybridization (SSH) approach, the differential expression of genes under anaerobiosis was analyzed . A PCR fragment with similarity to the genes of bacterial Fe-hydrogenases was isolated and used to screen an anaerobic cDNA expression library of C . reinhardtii . The cDNA sequence of hydA contains a 1494-bp ORF encoding a protein with an apparent molecular mass of 53.1 kDa . The transcription of the hydrogenase gene is very rapidly induced during anaerobic adaptation of the cells . The deduced amino-acid sequence corresponds to two polypeptide sequences determined by sequence analysis of the isolated native protein . The Fe-hydrogenase contains a short transit peptide of 56 amino acids, which routes the hydrogenase to the chloroplast stroma . The isolated protein belongs to a new class of Fe-hydrogenases . All four cysteine residues and 12 other amino acids, which are strictly conserved in the active site (H-cluster) of Fe-hydrogenases, have been identified . The N-terminus of the C . reinhardtii protein is markedly truncated compared to other non-algal Fe-hydrogenases . Further conserved cysteines that coordinate additional Fe-S-cluster in other Fe-hydrogenases are missing . Ferredoxin PetF, the natural electron donor, links the hydrogenase from C . reinhardtii to the photosynthetic electron transport chain . The hydrogenase enables the survival of the green algae under anaerobic conditions by transferring the electrons from reducing equivalents to the enzyme.

Eur J Biochem, 2002 Feb, 269(3), 969 - 76
Kinetic study of sn-glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1; Han JS et al.; A gene having high sequence homology (45-49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) . This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon . The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer . The optimum reaction temperature of this enzyme was observed to be 94-96 degrees C at near neutral pH . This enzyme was subjected to two-substrate kinetic analysis . The enzyme showed substrate specificity for NAD(P)H-dependent dihydroxyacetone phosphate reduction and NAD(+)-dependent glycerol-1-phosphate (Gro1P) oxidation . NADP(+)-dependent Gro1P oxidation was not observed with this enzyme . For the production of Gro1P in A . pernix cells, NADPH is the preferred coenzyme rather than NADH . Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H . However, NAD(P)(+) acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate . This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A . pernix follows a ordered bi-bi mechanism.

Eur J Biochem, 2002 Feb, 269(3), 933 - 42
Functional analysis of a small heat shock/alpha-crystallin protein from Artemia franciscana . Oligomerization and thermotolerance; Crack JA et al.; Oviparously developing embryos of the brine shrimp, Artemia franciscana, synthesize abundant quantities of a small heat shock/alpha-crystallin protein, termed p26 . Wild-type p26 functions as a molecular chaperone in vitro and is thought to help encysted Artemia embryos survive severe physiological stress encountered during diapause and anoxia . Full-length and truncated p26 cDNA derivatives were generated by PCR amplification of p26-3-6-3, then cloned in either pET21(+) or pRSETC and expressed in Escherichia coli BL21(DE3) . All constructs gave a polypeptide detectable on Western blots with either p26 specific antibody, or with antibody to the His(6) epitope tag encoded by pRSETC . Full-length p26 in cell-free extracts of E . coli was about equal in mass to that found in Artemia embryos, but p26 lacking N- and C-terminal residues remained either as monomers or small multimers . All p26 constructs conferred thermotolerance on transformed E . coli, although not all formed oligomers, and cells expressing N-terminal truncated derivatives of p26 were more heat resistant than bacteria expressing p26 with C-terminal deletions . The C-terminal extension of p26 is seemingly more important for thermotolerance than is the N-terminus, and p26 protects E . coli against heat shock when oligomer size and protein concentration are low . The findings have important implications for understanding the functional mechanisms of small heat shock/alpha-crystallin proteins.

Eur J Biochem, 2002 Feb, 269(3), 893 - 901
Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis; Bokma E et al.; Hevamine is a chitinase from the rubber tree Hevea brasiliensis . Its active site contains Asp125, Glu127, and Tyr183, which interact with the -1 sugar residue of the substrate . To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli . After refolding and purification they were characterized by both structural and enzyme kinetic studies . Mutation of Tyr183 to phenylalanine produced an enzyme with a lower k(cat) and a slightly higher K(m) than the wild-type enzyme . Mutating Asp125 and Glu127 to alanine gave mutants with approximately 2% residual activity . In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower k(cat) and an approximately twofold higher K(m) than the wild-type enzyme . More interestingly, it showed activity to higher pH values than the other variants . The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N-acetyl group of the -1 sugar residue has rotated away from the C1 atom of that residue . The combined structural and kinetic data show that Asp125 and Tyr183 contribute to catalysis by positioning the carbonyl oxygen of the N-acetyl group near to the C1 atom . This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis.

Regul Toxicol Pharmacol, 2002 Feb, 35(1), 56 - 71
Preclinical safety evaluation of recombinant human interleukin-10; Rosenblum IY et al.; Escherichia coli-derived recombinant human interleukin-10 (rhuIL-10) has been evaluated in an extensive series of in vivo and in vitro nonclinical safety studies, including genetic toxicology, single- and repeat-dose systemic toxicity and toxicokinetics, reproductive toxicity, and specialized irritation studies . The primary test species in the toxicology studies were the mouse and monkey based on rhuIL-10 activity in receptor binding and ex vivo cytokine assays . Supported by a detailed preclinical program of therapeutic and prophylactic animal models in autoimmune diseases, the initial clinical development program has focused on investigating the therapeutic potential of rhuIL-10 (Tenovil) in Crohn's disease and rheumatoid arthritis . The results of the subcutaneous toxicity studies, up to 3 months dosing duration in mice and 6 months dosing duration in monkeys, support the development of rhuIL-10 for present and future clinical indications by the subcutaneous route of administration . (c) 2002 Elsevier Science (USA).

J Mol Biol, 2001 Nov 30, 314(3), 633 - 45
The structures of Escherichia coli inorganic pyrophosphatase complexed with Ca(2+) or CaPP(i) at atomic resolution and their mechanistic implications; Samygina VR et al.; Two structures of Escherichia coli soluble inorganic pyrophosphatase (EPPase) complexed with calcium pyrophosphate (CaPP(i)-EPPase) and with Ca(2+) (Ca(2+)-EPPase) have been solved at 1.2 and 1.1 A resolution, respectively . In the presence of Mg(2+), this enzyme cleaves pyrophosphate (PP(i)) into two molecules of orthophosphate (P(i)) . This work has enabled us to locate PP(i) in the active site of the inorganic pyrophosphatases family in the presence of Ca(2+), which is an inhibitor of EPPase.Upon PP(i) binding, two Ca(2+) at M1 and M2 subsites move closer together and one of the liganded water molecules becomes bridging . The mutual location of PP(i) and the bridging water molecule in the presence of inhibitor cation is catalytically incompetent . To make a favourable PP(i) attack by this water molecule, modelling of a possible hydrolysable conformation of PP(i) in the CaPP(i)-EPPase active site has been performed . The reasons for Ca(2+) being the strong PPase inhibitor and the role in catalysis of each of four metal ions are the mechanistic aspects discussed on the basis of the structures described .

J Mol Biol, 2001 Nov 30, 314(3), 387 - 99
Role of the ATP-binding site of SopA protein in partition of the F plasmid; Libante V et al.; SopA belongs to a large family of bacterial partition protein ATPases . It helps stabilize the F plasmid by acting as the primary repressor of transcription of the sopAB operon, preventing the destabilizing effects of Sop protein excess . It is also thought to act directly in the F partition mechanism . We have examined the role of SopA in partition and repression by observing the consequences of replacing an invariant ATP-binding site lysine, K120, by glutamine or arginine . Circular dichroism studies of the purified mutant proteins revealed no major differences from wild-type, but in the presence of ADP or ATP each protein showed a characteristic spectrum which suggested a distinct conformational change . The K120Q mutant retained most of the wild-type ATPase activity while the K120R mutant lost it . In neither case was the residual activity stimulated by SopB, as occurs for wild-type SopA . The strength of sop promoter repression by the mutant SopA proteins alone was comparable to that resulting from SopB-enhancement of wild-type SopA, but SopB enhanced repression by the mutant SopA proteins either slightly (K120R) or not at all (K120Q) . Mini-Fs in which the sop operon was controlled by a constitutive promoter were destabilized by the mutations, demonstrating the need for SopA and its ATP-binding site in the partition process . The K120R mini-F was lost at the same rate as a mini-F lacking the sopC centromere, the K120Q mutant was lost faster . SopAK120R at high levels was more effective than SopA(+) in disrupting the partition complex, whereas SopAK120Q did not disrupt it at all . These results suggest that one function of SopA in the partition mechanism is to break the paired plasmid structure to allow F molecules to segregate to daughter cells .

J Mol Biol, 2001 Nov 30, 314(3), 365 - 74
Homology between O-linked GlcNAc transferases and proteins of the glycogen phosphorylase superfamily; Wrabl JO et al.; The O-linked GlcNAc transferases (OGTs) are a recently characterized group of largely eukaryotic enzymes that add a single beta-N-acetylglucosamine moiety to specific serine or threonine hydroxyls . In humans, this process may be part of a sugar regulation mechanism or cellular signaling pathway that is involved in many important diseases, such as diabetes, cancer, and neurodegeneration . However, no structural information about the human OGT exists, except for the identification of tetratricopeptide repeats (TPR) at the N terminus . The locations of substrate binding sites are unknown and the structural basis for this enzyme's function is not clear . Here, remote homology is reported between the OGTs and a large group of diverse sugar processing enzymes, including proteins with known structure such as glycogen phosphorylase, UDP-GlcNAc 2-epimerase, and the glycosyl transferase MurG . This relationship, in conjunction with amino acid similarity spanning the entire length of the sequence, implies that the fold of the human OGT consists of two Rossmann-like domains C-terminal to the TPR region . A conserved motif in the second Rossmann domain points to the UDP-GlcNAc donor binding site . This conclusion is supported by a combination of statistically significant PSI-BLAST hits, consensus secondary structure predictions, and a fold recognition hit to MurG . Additionally, iterative PSI-BLAST database searches reveal that proteins homologous to the OGTs form a large and diverse superfamily that is termed GPGTF (glycogen phosphorylase/glycosyl transferase) . Up to one-third of the 51 functional families in the CAZY database, a glycosyl transferase classification scheme based on catalytic residue and sequence homology considerations, can be unified through this common predicted fold . GPGTF homologs constitute a substantial fraction of known proteins: 0.4% of all non-redundant sequences and about 1% of proteins in the Escherichia coli genome are found to belong to the GPGTF superfamily . Copyright 2001 Academic Press.

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